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by Ricardo Wong Chi Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 128-138). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Overview of Serine Protease Inhibitors --- p.1 / Chapter 1.2 --- Classification of Serine Protease Inhibitors --- p.2 / Chapter 1.2.1 --- Kunitz Type Serine Protease Inhibitors --- p.7 / Chapter 1.2.2 --- Bowman-Birk Type Serine Protease Inhibitors --- p.11 / Chapter 1.2.3 --- Squash Type Serine Protease Inhibitors --- p.16 / Chapter 1.3 --- Role of Serine Protease Inhibitors in Plants --- p.20 / Chapter 1.4 --- Nutritional Fact of Serine Protease Inhibitors --- p.22 / Chapter 1.5 --- Possible Applications of Serine Protease Inhibitors --- p.25 / Chapter 1.5.1 --- Medical Applications --- p.25 / Chapter 1.5.2 --- Agricultural Applications --- p.29 / Chapter 1.6 --- Rationale of the Present Study --- p.31 / Chapter Chapter 2 --- Screening of Seeds for Inhibitory Activities Against Serine Proteases --- p.33 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and Methods --- p.37 / Chapter 2.2.1 --- Materials --- p.37 / Chapter 2.2.2 --- Extraction Method --- p.37 / Chapter 2.2.3 --- Assays for Proteases Inhibitory Activities --- p.38 / Chapter 2.2.3.1 --- Assay for Chymotrypsin Activity --- p.38 / Chapter 2.2.3.2 --- Assay for Trypsin Activity --- p.38 / Chapter 2.2.3.3 --- Assay for Elastase Activity --- p.39 / Chapter 2.2.3.4 --- Assay for Subtilisin Activity --- p.39 / Chapter 2.2.3.5 --- Assays for Protease Inhibitory Activities --- p.40 / Chapter 2.2.4 --- Determination of Protein Concentration --- p.41 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Extraction --- p.42 / Chapter 2.3.2 --- Serine Proteases Inhibitory Activities --- p.42 / Chapter 2.4 --- Discussion --- p.47 / Chapter Chapter 3 --- Isolation of Chymotrypsin Inhibitor and Trypsin Inhibitors from Momordica cochinchinensis Seeds --- p.49 / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Materials --- p.56 / Chapter 3.2.2 --- Protein Extraction --- p.57 / Chapter 3.2.3 --- SP-Sepharose Chromatography --- p.57 / Chapter 3.2.4 --- Reversed Phase High Pressure Liquid Chromatography --- p.58 / Chapter 3.2.5 --- Assays for Chymotrypsin and Trypsin Inhibitory Activities --- p.60 / Chapter 3.2.6 --- Titration of Chymotrypsin --- p.61 / Chapter 3.2.7 --- Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.62 / Chapter 3.2.8 --- Coupling of Trypsin-Sepharose 4B Affinity Column --- p.63 / Chapter 3.2.9 --- Affinity Chromatography on Trypsin-Sepharose 4B --- p.64 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- SP-Sepharose Chromatography --- p.65 / Chapter 3.3.2 --- Reversed Phase High Pressure Liquid Chromatography --- p.67 / Chapter 3.3.3 --- Summary of Purification --- p.71 / Chapter 3.3.4 --- Titration of Chymotrypsin --- p.74 / Chapter 3.3.5 --- Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.74 / Chapter 3.3.6 --- Affinity Chromatography on Trypsin-Sepharose 4B --- p.78 / Chapter 3.4 --- Discussion --- p.81 / Chapter Chapter 4 --- Characterization of Chymotrypsin Inhibitor and Trypsin Inhibitors --- p.88 / Chapter 4.1 --- Introduction --- p.88 / Chapter 4.2 --- Materials and Methods --- p.90 / Chapter 4.2.1 --- Materials --- p.90 / Chapter 4.2.2 --- Determination of Molecular Weight --- p.90 / Chapter 4.2.3 --- Amino Acid Sequence Analysis --- p.91 / Chapter 4.2.4 --- Surface Plasmon Resonance Measurement --- p.92 / Chapter 4.2.4.1 --- Immobilization of Ligands on the Surface of Optical Biosensors --- p.92 / Chapter 4.2.4.2 --- Determination of Kinetics Constants --- p.93 / Chapter 4.2.4.3 --- pH Dependence of the Inhibition by Chymotrypsin Inhibitor --- p.93 / Chapter 4.2.4.4 --- Data Analysis --- p.94 / Chapter 4.2.5 --- Effect of Chymotrypsin Inhibitor on the Estereolytic Activity and Proteolytic Activity of Chymotrypsin --- p.95 / Chapter 4.2.6 --- Specificities of the Inhibitors % --- p.96 / Chapter 4.2.7 --- Binding Ratio of CI to Different Proteases --- p.97 / Chapter 4.2.8 --- Effects of the Proteases on Their Corresponding Inhibitors --- p.97 / Chapter 4.2.8.1 --- Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.97 / Chapter 4.2.8.2 --- Assay for Chymotrypsin Inhibitory Activity --- p.98 / Chapter 4.3 --- Results --- p.99 / Chapter 4.3.1 --- Molecular Weight of the Inhibitors --- p.99 / Chapter 4.3.2 --- N-terminal Amino Acid Sequence --- p.99 / Chapter 4.3.3 --- Surface Plasmon Resonance Measurement --- p.102 / Chapter 4.3.3.1 --- Kinetics of Chymotrypsin Inhibitor --- p.102 / Chapter 4.3.3.2 --- Kinetics of Trypsin Inhibitors --- p.106 / Chapter 4.3.3.3 --- pH Dependence of the Inhibition by Chymotrypsin Inhibitor --- p.106 / Chapter 4.3.4 --- Effect of Chymotrypsin Inhibitor on the Estereolytic Activity and Proteolytic Activity of Chymotrypsin --- p.106 / Chapter 4.3.5 --- Specificities of the Inhibitors --- p.110 / Chapter 4.3.6 --- Binding Ratio of CI to Different Proteases --- p.112 / Chapter 4.3.7 --- Effects of the Proteases on Their Corresponding Inhibitors --- p.112 / Chapter 4.4 --- Discussion --- p.119 / Chapter Chapter 5 --- Conclusion --- p.125 / References --- p.128

Orientador: Maria Ligia Rodrigues Macedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T12:29:39Z (GMT). No. of bitstreams: 1 Ramos_VanessadaSilveira_M.pdf: 1587017 bytes, checksum: 9b8d97615e6656343053ce2fd7f519ba (MD5) Previous issue date: 2008 / Resumo: Inibidores de proteinases (IPs) são amplamente distribuídos em animais, microorganismos e plantas. IPs de plantas são geralmente proteínas pequenas que tem sido principalmente descritas como ocorrendo em órgãos de reserva, tais como sementes e tubérculos, mas eles também foram encontrados nas partes aéreas da planta, nas folhas, flores e raízes. O possível papel sugerido para inibidores de proteinase de sementes inclui a função como parte do sistema defensivo da planta contra pragas via inibição de suas enzimas proteolíticas. A atividade dos IPs é devido a sua capacidade de formar complexos estáveis com as proteases alvos, bloqueando, alterando ou prevenindo o acesso ao sítio ativo da enzima. Lepidoptera são insetos que frequentemente atacam uma ampla variedade de culturas, causando alto dano econômico. A. kuehniella é encontrada mundialmente, este inseto ataca grão estocados e produtos de arroz, aveia, centeio, milho e trigo. Neste trabalho, um inibidor purificado das sementes de Plathymenia foliolosa (Mimosoideae) ¿ PFIT foi utilizado em dietas artificiais, e através de bioensaios seu potencial tóxico contra A. kuehniella foi determinado. A ingestão de PFIT resultou em uma redução significativa na sobrevivência e no peso larval (32,3% e 66,1% respectivamente) e também reduziu a viabilidade larval e o peso das pupas (35,7% e 9,1% respectivamente). Os resultados dos experimentos nutricionais realizados com larvas de A. kuehniella apresentaram uma redução na eficiência de conversão do alimento ingerido (ECI) e alimento digerido (ECD), e um aumento na digestibilidade aproximada (AD) e no CM (custo metabólico). Para examinar o efeito da proteína sobre o inseto, a atividade das proteinases intestinais das larvas que se alimentaram em dietas livres do inibidor e alimentadas em dieta contendo o inibidor a 0,7% foi comparada através de ensaios enzimáticos e eletroforese em géis de atividade enzimática. As larvas de quarto instar alimentadas em dieta contendo PFIT apresentaram uma diminuição na atividade tríptica do intestino e um aumento na atividade tríptica das fezes, confirmado por ensaios enzimáticos e na eletroforese de atividade. Além disso, a atividade tríptica das larvas que se alimentaram em dieta com PFIT foi sensível à inibição por PFIT. Estes resultados sugerem que PFIT possui efeitos anti-metabólicos quando ingeridos por A. kuehniella / Abstract: Proteinase inhibitors (PIs) are widely distributed in animals, microorganisms, and plants. Plant PIs are generally small proteins that have mainly been described as occurring in storage tissues, such as tubers and seeds, but they have also been found in the aerial parts of plants, in the leaves, flowers and roots. The possible roles suggested for seed proteinase inhibitors include the function as a part of the plant defensive system against pest via inhibition of their proteolytic enzymes. The activity of PIs is due to their capacity to form stable complexes with target proteases, blocking, altering or preventing access to the enzyme active site. Lepidoptera are often insects which attack a wide range of crops, causing damage high economic value. A. kuehniella is found worldwide, this insect attacks stored grains, and products of rice, oat, rye, corn and wheat. In this report, the pure inhibitor from seeds of Plathymenia foliolosa (Mimosoideae) ¿ PFTI was monitoring by an insect bioassay its toxic activity toward A. kuehniella. The chronic ingestion of PFTI did result in a significant reduction in larval survival and weight (32.3% and 66.1% respectively) and also reduced the larval viability and the weight of pupae (35.7% and 9.1% respectively). The results from nutritional experiments realized with A. kuehniella larvae presented a reduction in efficiency of conversion of ingested food (ECI) and digested food (ECD), and an increase in approximate digestibility (AD) and CM (metabolic cost). To examine the protein effects on insect, the midgut proteinases of A. kuehniella larvae reared on artificial PI-free diet and on a diet containing PFTI at 0.7% were compared by using enzymatic assays and polyacrilamide gel electrophoresis. The fourth instar larvae reared on a diet containing PFTI showed a decrease in tryptic activity of gut and increase in tryptic activity of faeces, as confirmed by BApNA as substrate and by activity in gels. In addition, the tryptic activity in PFTI-fed larvae was sensitive to PFTI. These results suggest that PFTI have a potential antimetabolic effect when ingested by A. kuehniella / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientadores: Maria Lígia Rodrigues Macedo, Ricardo Aparício / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T17:21:54Z (GMT). No. of bitstreams: 1 Oliveira_CaioFernandoRamalhode_M.pdf: 5489630 bytes, checksum: 6240754a8c7ab9bd7931534cadba14a6 (MD5) Previous issue date: 2011 / Resumo: Diversas pesquisas buscam candidatos para o controle alternativo de pragas agrícolas, uma vez conhecidos os malefícios oriundos do emprego de inseticidas químicos nas lavouras. Neste trabalho, um inibidor de tripsina isolado de sementes de Entada acaciifolia (Mimosoideae) - (EATI) foi purificado e caracterizado sob o ponto de vista funcional e estrutural. O inibidor mostrou-se estável a variações térmicas e de pH, alem de apresentar sua atividade inibitória inalterada tanto na forma oxidada quanto na forma reduzida com 100 mM de DTT. Quando submetido a cromatografia liquida de alta eficiência em coluna C-18, EATI foi resolvido em 4 picos, indicando a presença de 4 isoformas, sendo que a isoforma majoritária apresentou uma massa acurada de 19.725 Daltons, revelada através de espectrometria de massa MALDI-TOF. Outras características como a estequiometria de inibição, a constante de inibição (Ki), a analise da composição global de aminoácidos e o sequenciamento N-terminal permitiram classificar EATI como membro da família Kunitz de inibidores de serinoprotease. Estudos de dicroísmo circular revelaram um alto conteúdo de fohas-? e estruturas não ordenadas alem da aparente ausência de ?-helices, padrão comum a esta classe de proteínas. A incubação em 1 mM de DTT resultou em perturbações em sua cadeia polipeptídica, corroborando os ensaios in vitro. EATI foi resistente a desnaturação por uréia, característica relatada para outros inibidores vegetais. O inibidor apresentou uma interessante atividade inibitória in vitro contra as proteases de diversos insetos-praga. De acordo com estes resultados, apontamos EATI como uma ferramenta promissora no combate a pragas agrícolas / Abstract: Several studies seeking alternative candidates for the control of agricultural pests, once known harm from the use of chemical insecticides in crops. In this work, a trypsin inhibitor isolated from Entada acaciifolia seeds (Mimosoideae) - (EATI) was purified and characterized from the point of view unctional and structural. The inhibitor was stable to thermal heat and pH range, in addition to its inhibitory activity unchanged in both the oxidized and in reduced form with 100 mM DTT. When subjected to high performance liquid chromatography on C-18 column, EATI was resolved in four peaks, indicating the presence of four isoforms, with the major isoform showing an accurate mass of 19,725 Daltons, as revealed by MALDI-TOF mass spectrometry. Other characteristic such as stoichiometry of inhibition, the dissociation constant (Ki), the analysis of global amino acid composition and N-terminal sequencing allowed to classify EATI as member of the Kunitz family of serine protease inhibitors. Circular dichroism studies revealed a high content of ?- sheets and unordered structures beyond the apparent absence of ?-helix, typical of this class of proteins. Incubation in 1 mM DTT resulted in small perturbations in their polypeptide chain, corroborate the in vitro assays. EATI was resistant to denaturation by urea, a characteristic reported for other plant inhibitors. The inhibitor presented an interesting inhibitory activity in vitro against the proteases of several insect pests. According to these results, we focused EATI as a promising tool in the fight against agricultural pests / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Maria Ligia Rodrigues Macedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T03:11:15Z (GMT). No. of bitstreams: 1 Kubo_CarlosEduardoGavira_M.pdf: 1973367 bytes, checksum: afd46afbc30805315577f477defd25c7 (MD5) Previous issue date: 2007 / Resumo: As plantas sintetizam inibidores de proteinases (IPs) como um dos mecanismos de defesa contra o ataque de insetos-praga e patógenos. Os IPs são polipeptídios hábeis em se ligar às enzimas proteolíticas localizadas no intestino médio dos insetos, impedindo sua atividade proteolítica por inibição competitiva. Esse processo leva a uma redução da disponibilidade de aminoácidos para a síntese protéica, e desta maneira, a uma redução no crescimento e desenvolvimento por parte do inseto. Portanto, os IPs são considerados fatores anti-metabólicos. Heliothis virescens (Fabr., 1781) (Lepidoptera: Noctuidae) é uma mariposa, cujas larvas atacam principalmente culturas em campo como: alfafa, algodão, soja e fumo. Entretanto, ocasionalmente podem atacar também culturas como couve, alface, ervilha, pimentão, guandu, abóbora e tomate. Neste trabalho, um inibidor purificado das sementes de Adenanthera pavonina L. (Mimosaceae) ¿ ApTI foi utilizado em dietas artificiais, e através de bioensaios seu potencial inseticida contra H. virescens foi determinado. A inibição in vitro de proteinases do inseto por ApTI sugerem que o inibidor possua efeitos anti-metabólicos quando ingeridos pelas larvas . Entretanto, a ingestão de ApTI não resultou em uma redução significativa no crescimento e desenvolvimento do inseto. Não sendo observadas diferenças na mortalidade larval, no ganho de peso e no tempo de desenvolvimento larval e pupal. A fim de estudar a adaptação das lagartas à presença do inibidor, a atividade das proteinases intestinais das larvas que se alimentaram em dietas livres do inibidor e alimentadas em dieta contendo o inibidor a 0,4% foi comparada através de ensaios enzimáticos e eletroforese em géis de atividade enzimática. As larvas de quarto instar alimentadas em dieta contendo ApTI apresentaram um aumento de cerca de duas vezes na atividade tríptica, confirmado por ensaios enzimáticos e na eletroforese de atividade. Além disso, a atividade tríptica das larvas que se alimentaram em dieta com ApTI é menos sensível a inibição por ApTI, indicando que novas enzimas mais resistentes ao inibidor foram produzidas. Estes resultados sugerem que as larvas de H. virescens foram capazes de se adaptarem fisiologicamente ao inibidor pela superprodução de enzimas do tipo tripsina existente e pela produção de um novo tipo de enzima do tipo tripsina que é menos susceptível a ação inibitória de ApTI / Abstract: Proteinase inhibitor proteins (PIs) are one of the defensive chemicals produced by plants against pests and pathogens. 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. Therefore, PIs are known as antinutritional factors. The tobacco budworm, Heliothis virescens (Fabr., 1781) (Lepidoptera: Noctuidae) is a polyphagous insect larvae that is principally a field crop pest, attacking such crops as alfalfa, cotton, soybean and tobacco. However, it sometimes attacks such vegetables as cabbage, lettuce, pea, pepper, pigeon pea, squash, and tomato, especially when cotton or other favored crops are abundant. In this report, the pure inhibitor from seeds of Adenanthera pavonina L. (Mimosaceae) ¿ ApTI was monitoring by an insect bioassay its insecticidal activity toward H. virescens. The in vitro inhibitions of proteinases activities by ApTI suggest that ApTI would have a potential antimetabolic effect when ingested by insect larvae. However, chronic ingestion of ApTI did not result in a significant reduction of growth and development of tobacco budworm. No differences in larval mortality, weight gain, larval and pupal developmental time were observed. To study this adaptation, the midgut proteinases of H. virescens larvae reared on artificial PI-free diet and on a diet containing ApTI at 0.4% were compared by using enzymatic assays and polyacrilamide gel electrophoresis. The fourth instar larvae reared on a diet containing ApTI showed a 2-fold increase in tryptic activity, as confirmed by BApNA as substrate and by activity in gels. In addition, the tryptic activity in ApTI-fed larvae was less sensitive to ApTI, indicating that novel proteolytic form resistant to ApTI was induced in larvae reared on a diet containing this inhibitor. These results suggest that H. virescens larvae were able to physiologically adapt to inhibitor by overproduction of an existing trypsin-like enzyme and production of a new type of trypsin-like enzyme that is less susceptible to inhibitory action of ApTI / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Maria Lígia Rodrigues Macedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T23:17:16Z (GMT). No. of bitstreams: 1 Bezerra_CezardaSilva_M.pdf: 16272882 bytes, checksum: c9bdf2bf628fb555d1bc1ccc47c972c5 (MD5) Previous issue date: 2014 / Resumo: Os inibidores de proteases de plantas têm sido amplamente estudados como uma alternativa para o controle de insetos-praga devido à capacidade de inibir enzimas envolvidas na digestão. Assim sendo, o objetivo deste trabalho foi estudar a atividade biológica do inibidor de tripsina de sementes de Inga vera (IVTI) sobre o desenvolvimento, fisiologia nutricional e atividade enzimática de Anagasta kuehniellla. Larvas neonatas (n=40) foram mantidas em dieta artificial sem inibidor (controle) ou contendo 1% de IVTI (p/p) até atingirem o quarto e quinto instares. As análises realizadas determinaram o efeito sobre o desenvolvimento destas, desde período larval até emergência dos adultos. Através de outras aferições elaboramos uma tabela de parâmetros nutricionais. A atividade proteolítica do homogenato intestinal e fecal foi analisada através de zimograma e ensaios enzimáticos in vitro, utilizando BApNA e Suc-AAPF-pNA como substratos para tripsina e quimotripsina, respectivamente. IVTI foi incubado com o homogenato intestinal larval para verificar a degradação do mesmo. O consumo de IVTI pelas larvas provocou uma redução de cerca de 50% no peso médio larval e uma redução significativa taxa de sobrevivência de 15%, bem como o prolongamento do período larval em 8 dias. A análise dos índices nutricionais revelou uma redução na eficiência de conversão do alimento ingerido e digerido e um aumento no custo metabólico, sugerindo que IVTI apresenta efeito antinutricional para esta espécie. IVTI alterou a atividade proteolítica intestinal das larvas com a redução atividade tríptica e o aumento da atividade quimotríptica. Na análise fecal, os níveis da atividade tríptica foram semelhantes tanto nas fezes das larvas alimentadas em dieta controle quanto contendo inibidor, entretanto foi observado um aumento no nível da atividade quimotríptica nas larvas alimentadas com inibidor. IVTI não foi degradado pelas enzimas intestinais, sendo excretado nas fezes e permanecendo com sua atividade inibitória ativa. O zimograma não revelou nenhuma forma variante de enzima nas larvas alimentadas com inibidor, mas foi possível observar quais sofreram influências pelo mesmo. Com base nesses resultados, IVTI apresentou uma atividade tóxica e antinutricional contra A. kuehniella / Abstract: Plant protease inhibitors have been extensively studied as an alternative for the control of insect pests because of their ability to inhibit enzymes digestive enzymes. In this work, the biological activity of trypsin inhibitor of Inga vera seed (IVTI) on the development, nutritional physiology and enzyme activity of Anagasta kuehniellla was evaluated. Neonate larvae (n=40) were maintained on artificial diet without inhibitor (control) or containing 1% IVTI (w / w) until fourth and fifth instar. This bioassay determined the effect on the development from the larval period to adult emergence. Through other measurements prepared a table of nutritional parameters. The proteolytic activity of intestinal and fecal homogenate was analyzed by zymography and enzymatic assays, in vitro, using BApNA and Suc-AAPF-pNA as substrates for trypsin and chymotrypsin, respectively. IVTI was incubated with the larval gut and check the degradation of the inhibitor. IVTI consumption by the larvae resulted in a reduction of about 50% of larval weight and a significant larval survival rate of 15%, as well as the extension of the larval period to 8 days. Nutritional analyses showed a reduction of efficiency of conversion of food eaten and digested and an increase in metabolic cost, suggesting that IVTI produces an anti-nutritional effect for this specie. IVTI changed the proteolytic activity in the gut of the larvae with decrease of trypsin activity and increase of chymotrypsin activity. Fecal analyses, the levels of trypsin activity were similar in the feces of larvae fed on control diet as containing inhibitor, however there was an increase in the level of chymotrypsin activity in larvae fed with inhibitor. IVTI was not degraded by intestinal enzymes, but it excreted in the feces and their inhibitory activity remained active. Zymogram revealed no variant form of the enzyme in the larvae fed inhibitor, but was observed which were influenced by it. Based on these results, IVTI presented an anti-nutritional and toxic activity against A. kuehniella / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientadores: Maria Lígia Rodrigues Macedo, Odalys Garcia Cabrera, Goran Neshich / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T08:34:15Z (GMT). No. of bitstreams: 1 Ramos_VanessadaSilveira_D.pdf: 9537452 bytes, checksum: 829f959a45ba9dc3fb47b19126255aca (MD5) Previous issue date: 2013 / Resumo: Os inibidores de tripsina têm sido utilizados com sucesso para aumentar a resistência das plantas contra os insetos. No presente trabalho avaliamos a eficiência de um inibidor de tripsina de Inga laurina (ILTI) sobre o desenvolvimento de larvas de Diatraea saccharalis e Heliothis virescens, duas importantes pragas da cana-de-açúcar e tabaco, respectivamente. Para estes fins foram utilizadas diversas estratégias (i) purificação da proteína ILTI nativa a partir de sementes de I. laurina e sua utilização para suplementação de dieta artificial fornecida como alimento às larvas dos insetos em estudo. Isto com o intuito de avaliar os efeitos do inibidor no desenvolvimento destes indivíduos; (ii) obtenção da sequência de DNA do gene ilti, clonagem, expressão e purificação da proteína recombinante assim como avaliação da sua atividade inibitória; (iii) obtenção de plantas transgênicas de cana-de-açúcar e tabaco com o gene ilti obtido a partir do DNA genômico de I. laurina ou do gene ilti-s sintetizado quimicamente usando o uso de códons da cana-de-açúcar. A adição de 0,1% (p/p) de ILTI na dieta de D. saccharalis não alterou a sobrevivência larval, mas resultou em uma redução de 56% no peso das larvas. As larvas de H. virescens que foram alimentadas em uma dieta contendo 0,5% (p/p) de ILTI apresentaram uma redução de 84% no peso. Experimentos in vitro mostraram que ILTI não foi digerido pelas proteinases do intestino médio de ambas as espécies de larvas. Ensaios trípticos permitiram observar que não houve alteração nos níveis de tripsina no intestino médio de ambos os insetos. Entretanto, verificou-se uma redução de 55,3% na atividade tríptica das fezes de D. saccharalis e um aumento de 24,1% desta atividade nas fezes de H. virescens. A atividade da tripsina em ambas as espécies alimentadas com ILTI foi sensível ao inibidor, sugerindo que nenhuma nova proteinase resistente à ILTI foi induzida. Adicionalmente, a proteína recombinante reILTI produzida em Escherichia coli apresentou atividade inibitória similar à proteína nativa em testes de atividade anti-tríptica. Com isso, reILTI pode ser considerada como uma potencial ferramenta biotecnológica. Finalmente, larvas de D. saccharalis e H. virescens foram alimentadas com plantas transgênicas de cana-de-açúcar e tabaco - respectivamente - expressando os genes ilti e/ou ilti-s. Os resultados mostraram que os transgênicos foram capazes de interferir no metabolismo dos insetos-praga testados sugerindo que esta estratégia pode ser promissora na obtenção de plantas mais resistentes ao ataque de insetos / Abstract: Trypsin inhibitors have been successfully used to increase plant resistance against insects. In this work, we evaluated the efficiency of a trypsin inhibitor from Inga laurina (ILTI) on the development of Diatraea saccharalis and Heliothis virescens, two important pests of sugarcane and tobacco, respectively. For these purposes, several strategies were employed: (i) purification of the native protein ILTI from I. laurina seeds, and its use as a supplement in the artificial diet provided for the insect larvae. This purpose was performed in order to evaluate the effects of ILTI on the development of these individuals, (ii) elucidation of the DNA sequence of the ilti gene; cloning, expression and purification of the recombinant protein (reILTI) as well as evaluation of its inhibitory activity, (iii) production of transgenic plants of sugarcane and tobacco with the gene ilti from I. laurina genomic DNA or the gene ilti-s, which was chemically synthesized using the sugarcane codon usage. The addition of 0.1% (w/w) ILTI in the diet of D. saccharalisdid not alter larval survival but resulted in a reduction of 56% in the weight of the larvae. The H. virescens larvae that were fed a diet containing 0.5% (w/w) ILTI showed an 84% decrease in weight. In vitro experiments showed that ILTI was not digested by the midgut proteinases of either species of larvae. Triptic assays allowed observe that there was no alteration in the trypsin levels in the midgut of either insect. However, there was a reduction of 55.3% in the triptic activity in the feces of D. saccharalis and an increase of 24.1% of this activity in the feces of H. virescens. The trypsin activity in both species fed with ILTI was sensitive to the inhibitor, suggesting that no novel proteinase resistant to ILTI was induced. Additionally, reILTI showed similar inhibitory activity to those of the native protein in the anti-tryptic assays. Therefore, reILTI can be considered a potential biotechnological tool. Finally, larvae of D. saccharalis and H. virescens were fed with transgenic sugarcane and tobacco plants, respectively. The results showed that transgenic plants were able to interfere with the metabolism of these insect pests, suggesting that this is a promising strategy for the production of plants that are more resistant to insect attack / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

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Ho, Sai Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 144-164). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.ii / ABSTRACT --- p.xii / 撮要 --- p.xv / LIST OF ABBREIVIATIONS --- p.xvi / LIST OF TABLES --- p.xvii / LIST OF FIGURES --- p.xix / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Overview of lectins --- p.1 / Chapter 1.1.1 --- History of lectins --- p.1 / Chapter 1.1.2 --- Definitions of lectins --- p.2 / Chapter 1.1.3 --- Classification and nomenclature of lectins based on structure --- p.2 / Chapter 1.1.4 --- Classification and nomenclature of lectins based on carbohydrate-bindingspecificity --- p.4 / Chapter 1.1.5 --- Structure of plant lectins --- p.4 / Chapter 1.1.6 --- Biological function of plant lectins --- p.5 / Chapter 1.1.6.1 --- Anti-viral activity of plant lectiins --- p.5 / Chapter 1.1.6.2 --- Lectins as plant defense proteins --- p.6 / Chapter 1.1.6.3 --- Insecticidal activity of plant lectins --- p.7 / Chapter 1.1.6.4 --- Anti-fungal activity of plant lectins --- p.7 / Chapter 1.1.6.5 --- Mitogenic activity of plant lectins --- p.7 / Chapter 1.1.6.6 --- Anti-tumor and anti-proliferative activity of plant lectins --- p.9 / Chapter 1.1.7 --- Background of legume lectins --- p.11 / Chapter 1.1.7.1 --- Structure of legume lectins --- p.11 / Chapter 1.1.7.2 --- Functions and activities of legume lectins --- p.12 / Chapter 1.2 --- Overview of serine protease inhibitors in plants --- p.14 / Chapter 1.2.1 --- Classification of serine protease inhibitor --- p.15 / Chapter 1.2.2 --- The main functions of plant serine protease inhibitors --- p.17 / Chapter 1.2.3 --- Commercial application of serine protease inhibirtors --- p.19 / Chapter 1.2.3.1 --- Medical application --- p.19 / Chapter 1.2.3.2 --- Transgenic application in agriculture --- p.22 / Chapter 1.3 --- Overview of Pathogenesis-related proteins in plants --- p.25 / Chapter 1.3.1 --- Overview of PR-5 family Thaumatin-like proteins (TLPs) --- p.27 / Chapter 1.3.1.1 --- Structural similarities among TLPs --- p.28 / Chapter 1.3.1.2 --- Antifungal activity of TLP --- p.31 / Chapter 1.3.2 --- Overview of Chinase-like proteins (CLPs) --- p.33 / Chapter 1.3.2.1 --- Classification of chitinase --- p.34 / Chapter 1.3.2.1.1 --- On the basis of amino acid sequence of glycosyl hydrolase --- p.34 / Chapter 1.3.2.1.2 --- On the basis of amino acid sequence of plant chitinase --- p.35 / Chapter 1.3.2.2 --- Antifungal activity of CLP --- p.36 / Chapter 1.3.3 --- Anti-freeze property of PR proteins --- p.38 / Chapter 1.3.4 --- Application of PR proteins in agriculture --- p.40 / Chapter 1.4 --- Rationale of the present study --- p.42 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.43 / Chapter 2.2 --- Preparation of crude extract --- p.44 / Chapter 2.2.1 --- Hokkaido large black soybean --- p.44 / Chapter 2.2.2 --- Emperor banana --- p.45 / Chapter 2.3 --- Purification --- p.45 / Chapter 2.4 --- Chromatography --- p.46 / Chapter 2.4.1 --- DEAE-cellulose chromatography --- p.46 / Chapter 2.4.2 --- Affi-gel Blue gel --- p.47 / Chapter 2.4.3 --- SP-Sepharse --- p.48 / Chapter 2.4.4 --- Mono Q HR 5/5 and Mono S HR 5/5 --- p.49 / Chapter 2.4.5 --- Superdex 75 and superdex 200 --- p.50 / Chapter 2.5 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.50 / Chapter 2.6 --- Protein concentration determination --- p.54 / Chapter 2.7 --- Preparation of rabbit reticulocyte lysate --- p.54 / Chapter 2.8 --- Determination of N-terminal amino acid sequence --- p.56 / Chapter 2.9 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.56 / Chapter 2.10 --- Thermal stability determination assays --- p.57 / Chapter 2.10.1 --- Stability at various temperatures --- p.57 / Chapter 2.10.2 --- Stability at 100°C --- p.57 / Chapter 2.11 --- Assay of pH dependence of hemagglutinating activity --- p.58 / Chapter 2.12 --- Assay of ion dependence of hemagglutinating activity --- p.58 / Chapter 2.13 --- Assay of antifungal activity --- p.58 / Chapter 2.14 --- Assay of trypsin inhibitory activity --- p.60 / Chapter 2.15 --- Assay of antibacterial activity --- p.61 / Chapter 2.16 --- Assay for cytotoxic activity on cancer cell lines --- p.61 / Chapter 2.17 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.62 / Chapter 2.18 --- Assay of mitogenic activity --- p.63 / Chapter Chapter 3 --- Purification and Characterization of Defense-Related Proteins from their Respective Sources / Chapter 3.1 --- Purification and Characterization of a Lectin from the Seeds of Hokkaido large black soybean / Chapter 3.1.1 --- Introduction --- p.65 / Chapter 3.1.2 --- Results --- p.66 / Chapter 3.1.3 --- Purification --- p.68 / Chapter 3.1.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.69 / Chapter 3.1.3.2 --- Anion-exchange chromatography on DEAE-cellulose --- p.70 / Chapter 3.1.3.3 --- Anion-exchange chromatography on Mono Q column --- p.71 / Chapter 3.1.3.4 --- Gel filtration on Superdex 200 column --- p.72 / Chapter 3.1.3.5 --- Hemagglutinating activity at each purification step --- p.73 / Chapter 3.1.4 --- Characterization of Lectin --- p.74 / Chapter 3.1.4.1 --- Molecular mass determination --- p.74 / Chapter 3.1.4.2 --- N-terminal amino acid sequencing --- p.76 / Chapter 3.1.4.3 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.77 / Chapter 3.1.4.4 --- Thermal stability --- p.78 / Chapter 3.1.4.5 --- Assay of pH dependence of hemagglutinating activity --- p.80 / Chapter 3.1.4.6 --- Assay of ion dependence of hemagglutinating activity --- p.81 / Chapter 3.1.4.7 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.82 / Chapter 3.1.4.8 --- Assay of mitogenic activity --- p.83 / Chapter 3.1.4.9 --- Assay of antibacterial activity --- p.84 / Chapter 3.1.5 --- Discussion --- p.86 / Chapter 3.2 --- Purification and Characterization of a Trypsin inhibitor from the Seeds of Hokkaido large black soybean / Chapter 3.2.1 --- Introduction --- p.93 / Chapter 3.2.2 --- Results --- p.94 / Chapter 3.2.3 --- Purification --- p.95 / Chapter 3.2.3.1 --- Anion-exchange chromatography on Mono Q column --- p.96 / Chapter 3.2.3.2 --- Gel filtration on Superdex 75 column --- p.98 / Chapter 3.2.3.3 --- Trypsin inhibitory activity at each purification step --- p.99 / Chapter 3.2.4 --- Characterization of trypsin inhibitory --- p.100 / Chapter 3.2.4.1 --- Molecular mass determination --- p.100 / Chapter 3.2.4.2 --- N-terminal amino acid sequencing --- p.102 / Chapter 3.2.4.3 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.103 / Chapter 3.2.4.4 --- Antiproliferative effect on MCF-7 and Hep G2 cells --- p.104 / Chapter 3.2.4.5 --- pH and thermal stability --- p.105 / Chapter 3.2.5 --- Discussion --- p.106 / Chapter 3.3 --- Purification and Characterization of a Thaumatin-like protein and Chitinase-like protein from Emperor Banana / Chapter 3.3.1 --- Introduction --- p.108 / Chapter 3.3.2 --- Results --- p.109 / Chapter 3.3.3 --- Purification --- p.111 / Chapter 3.3.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.112 / Chapter 3.3.3.2 --- Cation exchange chromatography on Mono S column --- p.113 / Chapter 3.3.3.3 --- Gel filtration on Superdex 75 column --- p.114 / Chapter 3.3.3.3.1 --- Fraction MS 2 --- p.114 / Chapter 3.3.3.3.2 --- Fraction MS 4 --- p.115 / Chapter 3.3.3.3.3 --- Fraction MS 5 --- p.118 / Chapter 3.3.4 --- Characterization of the thaumatin-like protein --- p.121 / Chapter 3.3.4.1 --- N-terminal amino acid sequence determination --- p.121 / Chapter 3.3.4.2 --- Assay for antifungal activity --- p.122 / Chapter 3.3.4.3 --- Thermal stability --- p.124 / Chapter 3.3.4.4 --- pH stability --- p.125 / Chapter 3.3.4.5 --- Resistance to trypsin digestion --- p.125 / Chapter 3.3.4.6 --- Anti-HIV-1 reverse transcriptase activity --- p.126 / Chapter 3.3.4.7 --- Discussion --- p.127 / Chapter 3.3.5 --- Characterization of the two chitinase-like protein --- p.131 / Chapter 3.3.5.1 --- N-terminal amino acid sequence determination --- p.131 / Chapter 3.3.5.1.1 --- Emperor banana MS2 CLP --- p.131 / Chapter 3.3.5.1.2 --- Emperor banana MS4 CLP --- p.132 / Chapter 3.3.5.2 --- Assay for antifungal activity --- p.133 / Chapter 3.3.5.3 --- Discussion --- p.136 / Chapter Chapter 4 --- general discussion --- p.138 / References --- p.144

A double sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify the proteinaceous inhibitor of tissue plasminogen activator (t-PA) which occur in the tissue of Erythrina caffra Thunb. Using the ELISA the t-PA inhibitor could be detected in nanogramme quantities on the micro titer plate. The concentration of the t-PA inhibitor was determined in different tissues of Erythrina caffra. t-PA inhibitor concentrations in the order of 1 000 microgrammes per gramme protein were found in the seeds. Relatively small quantities of t - PA inhibitor, in the order of 10 to 50 microgrammes per gramme protein, occurred in root, shoot, leaf and living bark material. The t-PA inhibitor was found to accumulate in a similar way to the storage proteins in developing seeds. The accumulation of the inhibitor is at a relatively low level during the early period of seed development but increases exponentially just before the seeds reach their maximum size. The t-PA inhibitor content of the cotyledons decreased drastically during the process of germination and subsequent seedling development. The disappearance of the inhibitor be the result of total degradation of the molecule can or partial proteolysis with the modified molecule still being present in the tissue. An attempt was made to increase the t-PA inhibitor content of excised leaves of Erythrina caffra with protein inducing substances such as polyamines, precursors of ethylene and phytic acid. The protein inducing compounds included cell wall hydrolysates of Erythrina caffra, the marine alga Ecklonia maxima Osbeck (Papenfuss) as well as Lycopersicon esculentum Mill which induced the, synthesis of proteinase inhibitors suggested to be involved in the defense mechanism of plants. None of the substances used, increased the t-PA inhibitor content of excised leaves or in vitro cultures of Erythrina caffra. It is suggested that the t-PA inhibitor is probably not involved in a defense mechanism of Erythrina caffra. A callus and suspension culture derived from shoot tissue was developed to determine the occurrence of the t-PA inhibitor in vitro. The optimal nutrient medium for the growth of callus was the salts and vitamins of MURASHIGE and SKOOG (1962). The medium was supplemented with 3 % sucrose, 0. 1 gramme per litre meso - inositol, 10 micromoles per litre benzyl adenine and 5 micromoles per litre 2,4- dichlorophenoxyacetic acid . Different auxins and cytokinins had a similar growth stimulatory effect on the growth of callus derived from a number of organs of Erythrina caffra. The callus from different organs did however, grow at different rates on the same nutrient medium. Callus derived from leaf, shoot, and cotyledonary tissue grew at similar rates on the nutrient media of MURASHIGE and SKOOG (1962), SCHENK and HILDEBRANDT (1972) and B5 (GAMBORG, MILLER and OJIMA, 1968) despite large differences in the concentration of the nutrients in the three nutri.ent media. The source of nitrogen and ratio of nitrate to ammonium was critical to the growth of callus cultures . The optimal concentration of nitrate and ammonium was 30 millimoles per litre . The growth of callus from different organs was significantly affected by the concentration of sucrose in the nutrient medium. A concentration of 3% was optimal for callus growth. Temperature had a significant effect on the growth of callus. The optimal temperature for callus growth was 25 °C. A shoot cell suspension culture was established and maintained at the same temperature and on the same medium as the callus cultures but with a ten times lower concentration of growth regulators. A low shake speed was essential for the growth of the suspension culture. Maximum growth was obtained at a shake speed of 60 rpm. Relatively low quantities of t-PA inhibitor, in the order of 1 to 5 microgrammes per gramme protein, was detected in the suspension cultures. An attempt was made to increase the t-PA inhibitor content of the suspension cultures with the pro te in i nduc i ng compounds used on excised leaves, but without success. However, the t-PA inhibitor content of the suspension culture was significantly increased with a ten times increase in the sulphate content of the nutrient medium. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1990.