Thesis (Ph. D.)--Pennsylvania State University, 1992. / Includes bibliographical references.

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published_or_final_version / abstract / toc / Botany / Doctoral / Doctor of Philosophy

Sweet Corn Inhibitor (SCI), a small (11811Da.) protein from the seeds of opaque-2 corn is a potent and specific inhibitor of trypsin and the activated Hageman Factor (Factor βXIIa) of the human blood plasma coagulation system. With the eventual aim of obtaining insight into the structure- function relationships of the selective SCI-pXIIa interaction, a synthetic gene for SCI was cloned into Saccharomyces cerevisiae (yeast) and Escherichia coli (E.coli) expression systems in an attempt to obtain overexpression of the recombinant gene product. The establishment of functional expression, together with an isolation and purification procedure for SCI would provide a system for obtaining selected reactive-site mutants of SCI by cassette- and oligonucleotide-directed mutagenesis. A yeast secretion vector for a truncated form of SCI (tSCI) was constructed by cloning the gene for α-factor prepro-tSCI fusion, downstream to the α-mating factor (MFα1) promoter of yeast. Yeast transformants containing the expression vector failed to express and secrete the desired product. The synthetic gene encoding the complete SCI sequence was cloned into E.coli expression vectors that directed both cytoplasmic and periplasmic expression. In cytoplasmic expression, the SCI gene was cloned directly downstream to the powerful, inducible λ-phage PL- and trc-promoters. No expression was obtained with the latter. With the former, expression levels of up to 3% of the total bacterial protein were obtained. These levels were improved 3- to 4-fold on incorporation of the E.coli dnaY gene product. Solubilisation and refolding of the purified SCI inclusion bodies failed to yield the active, correctly folded product. Failure to obtain an N-terminal sequence indicated an incompletely processed N-terminal methionine. For periplasmic expression, SCI, fused in-frame to the signal sequence of OmpA, a major E.coli outer membrane protein, was cloned into the same λ-phage P_L promoter vector. High levels (=10%) of expression of insoluble SCI were obtained. The nearly homogeneous product was obtained by a two-step procedure, involving ion-exchange chromatography, followed by hydrophobic interaction chromatography. Characterisation by N-terminal sequencing, SDS-PAGE and electrospray mass spectrometry, confirmed the presence of correctly processed SCI in the form of covalently associated dimers. Refolding studies are at present in progress.

Proteinase P-II purified from parasitized Pacific whiting muscle was previously identified to be one form of cathepsin L. It appeared to be present in three isozymatic forms on non-denaturing PAGE gel stained for activity. Its autolytic degradation was observed on SDS-PAGE gel under its optimum condition, 55°C and pH 5.5, in the absence of substrate. Amino acid composition analysis revealed that this enzyme had a considerably greater proportion of hydrophobic amino acids than cathepsin L from other fish species, and monosaccharide analysis showed it was not glycosylated. The N-terminal amino acid sequence of the enzyme was 60-65% identical with cathepsin L from chicken and mammalian species, but only 39% identical with mammalian cathepsin B. The moderate identity of the N-terminal amino acid sequence of P-II with other cathepsin L revealed that this cysteine proteinase from Pacific whiting might be encoded by a cathepsin L-related gene. Two degenerate primers were designed to clone cathepsins cDNA from rainbow trout. The 500-bp PCR product from rainbow trout liver cDNA contained at least three different cysteine proteinase sequences, referred to as SFL2, SFL5, and SFL17. SFL5 was the partial cDNA of trout cathepsin L, which was over 80% identical with chicken cathepsin L amino acid sequence. SFL5 was labeled with Dig-11-dUTP and used to screen a trout liver cDNA library. One positive clone referred to as LC was identified and contained a 700-bp insertion overlapping with SFL5. By combining the two overlapping sequences, a 895-bp cDNA sequence was identified, which included 88% of the mature enzyme and a 307-bp 3' end untranslated part. Its deduced amino acid sequences had 83% identity, 91% similarity with chicken cathepsin L and 73% identity, 86% similarity with human cathepsin L. SFL2 might be the partial cDNA of a novel cathepsin L-related cysteine proteinase. SFL17 may be the partial cDNA of trout cathepsin K. It had 70% identity and 89% similarity with rabbit and human cathepsin K at the amino acid level. / Graduation date: 1996

Alpha1-proteinase inhibitor (alpha1-PI) has been shown to be a useful marker of gastrointestinal protein loss in some species. The objectives of this study were, first, to develop and analytically validate an ELISA for the measurement of alpha1-PI in feces and serum from cats, and, second, to evaluate fecal alpha1-PI concentrations in healthy cats and cats with chronic gastrointestinal disease. The lower detection limits of the ELISA were 0.02 g/L for serum and 0.04 microgram/gram for feces. The observed-to-expected (O/E) ratios for serial dilutions of serum and fecal samples ranged from 100.0 to 129.7% (mean +/- SD: 112.2 +/- 9.9%) and 103.5 to 141.6% (115.6 +/- 12.8%), respectively. The O/E ratios for samples spiked with seven known concentrations of alpha1-PI ranged from 82.3 to 107.8% (94.7 +/- 7.6%) for serum and 78.5 to 148.7% (96.8 +/- 18.2%) for feces. The coefficients of variation for intra-assay and inter-assay variability were <7.9% and &lt;12.1% for serum, and 5.3%, 11.8%, and 14.2% and 7.7%, 10.2%, and 20.4% for feces, respectively. Reference intervals were 0.6 to 1.4 g/L for serum and up to 1.6 microgram/g for feces. We conclude that this ELISA is sufficiently linear, accurate, precise, and reproducible. For the clinical evaluation, twenty cats with clinical signs of chronic gastrointestinal disease and 20 healthy control cats were enrolled. The diseased cats were grouped into two groups: mild to moderate idiopathic inflammatory bowel disease (IBD) (Group A; n=8) and severe IBD or neoplastic disease (Group B; n=12), based on histopathology results of endoscopic biopsies. Fecal alpha1-PI concentrations and serum concentrations of total protein, albumin, globulin, cobalamin, folate, pancreatic lipase immunoreactivity, and trypsin-like immunoreactivity were determined. Nineteen of the 20 diseased cats had increased fecal alpha1-PI concentrations, ranging from 1.9 to 233.6 microgram/g (normal range: <= 1.6 microgram/g). Fecal alpha1-PI concentrations were statistically significantly different between healthy cats and cats of Group A (median: 3.9 microgram/g, range: 1.3 to 9.2 microgram/g, P<0.001) or cats of Group B (median: 20.6 microgram/g, 4.3 to 233.6 microgram/g; P<0.001), and also between cats of Groups A and B (P<0.01). Hypoalbuminemia, hypoproteinemia, and hypocobalaminemia were detected in 88%, 83%, and 56% of the diseased cats, respectively. Our study suggests that increased fecal alpha1-PI concentrations in association with hypoalbuminemia may be a common finding in cats with IBD or GI neoplasia. Furthermore, alpha1-PI concentrations appear to be higher in cats with severe IBD or confirmed GI neoplasia when compared to cats with mild to moderate IBD.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pos-Graduação em Farmacologia, Florianópolis, 2009. / Made available in DSpace on 2012-10-24T20:47:45Z (GMT). No. of bitstreams: 1 275439.pdf: 2261901 bytes, checksum: e2aefcb3dc4f72e77c617b673afe9cc5 (MD5) / O objetivo do presente estudo foi investigar os possíveis mecanismos e mediadores envolvidos no comportamento de coçar causado pela administração intradérmica (i.d.), no dorso de camundongos, de agonistas do receptor ativado por proteinases-2 (PAR-2). Ambos os agonistas do PAR-2, tripsina e SLIGRL-NH2, causaram comportamento de coçar, sugestivo de prurido, de maneira dependente da dose. Esse efeito foi bloqueado pelo co-tratamento com o antagonista seletivo para o receptor PAR-2, FSLLRY-NH2, ou pela dessensibilização prévia deste receptor. A participação do sistema opióide foi verificada pelo tratamento com morfina ou naloxona, agonista e antagonista dos receptores opióides, respectivamente, que inibiram a coceira induzida pela tripsina. Além disso, o comportamento pruriginoso induzido por esta enzima foi previnido pelo tratamento com os antiinflamatórios, celecoxibe (inibidor seletivo para a COX-2) ou dexametasona (corticóide). A participação de mastócitos também foi observada, uma vez que o pré-tratamento com composto 48/80, degranulador de mastócitos, ou com cromoglicato de sódio, estabilizador de mastócitos, reduziu a prurido causado pela tripsina. Ademais, o modelo parece ser dependente da ativação do receptor histaminérgico H4, mas não dos receptores H1, H2 ou H3, ja que o antagonista não-seletivo H3/H4 (tioperamida), mas não os antagonistas seletivos H1 (pirilamina e loratadina), H2 (cimetidina e ranitidina) ou H3 (iodofempropite), bloqueou o efeito pruriginoso da tripsina. O antagonista misto dos receptores histaminérgicos/serotoninérgicos, ciproeptadina, e o inibidor não seletivo de proteases, gabexato mesilato, foram eficazes em reduzir o prurido causado pelos agonistas do PAR-2, sugerindo o envolvimento da serotonina e da liberação de proteases. Interessantemente, o tratamento neonatal com capsaicina, ou o tratamento do adulto com resiníferatoxina, estratégias que promovem desenssibilização e/ou destruição de fibras sensoriais, preveniram o comportamento pruriginoso evocado por ambos os agonistas do PAR-2. Similarmente, o tratamento com o antagonista seletivo para o receptor TRPV1, SB366791, ou a deleção do gene para este receptor, inibiram o prurido induzido pela ativação do PAR-2. Esta reposta também foi dependente dos neuropeptídeos substância P (SP), neurocinina B (NKB) e CGRP, uma vez que o tratamento com os antagonistas seletivos para os receptores NK1 (FK888, para SP), NK3 (SR142801, para NKB), ou CGRP1 (fragmento CGRP8-37, para o CGRP), reduziu a coceira induzida pelos ativadores do PAR-2. Por fim, o prétratamento com antagonistas seletivos para os receptores B2 (Hoe 140 ou FR173657) ou B1 (DALBK ou SSR240612) das cininas, ou a deleção gênica destes receptores, preveniu a coceira induzida por ambos os agonistas do PAR-2. Em conjunto, estes resultados sugerem que a injeção i.d. de agonistas do PAR-2 constitui um modelo experimental reprodutível para o estudo de mecanismos e de possíveis abordagens terapêuticas, relacionados a promoção da coceira mediada pela liberação de proteases e ativação do PAR-2. Diferentes mediadores e tipos celulares liberados ou localizados na pele parecem contribuir para este processo. De relevância, neste sentido ressalta-se o envolvimento de mediadores da inflamação neurogênica, de fibras sensoriais, do receptor TRPV1, e de ambos os receptores B2 e B1 para as cininas. / The aim of this study was to investigate some mechanisms and mediators underlying the scratching behaviour induced by intradermal (i.d.) injection of proteinase activated receptor-2 (PAR-2) agonists on the back of the mouse neck. Both PAR-2 agonists, trypsin and SLIGRL-NH2, were able to evoke a marked and dose-related scratching behaviour in mice. This response was widely reduced by the co-treatment with the selective PAR-2 receptor antagonist, FSLLRY-NH2, or by previous trypsin-induced PAR-2 receptor desensitization. Opioidergic system involvement was evaluated by the treatment with morphine or naloxone, a non-selective opioid receptors agonist and antagonist, respectively, which were both effective against trypsin-induced itch. Moreover, trypsin-induced scratching behaviour was blockaded by the pre-treatment with celecoxib (a selective COX-2 inhibitor) or dexamethasone (a corticoid). Also, contribution of mast cells was evident as the scratching behaviour induced by trypsin was prevented by the pre-treatment with compound 48/80, a mast cell degranulator, or disodium cromoglycate, a mast cell stabilizer. Furthermore, PAR-2 receptor activation-induced scratching behaviour seems to be dependent on histamine H4 receptor activation, but not histamine H1, H2 or H3 receptors, as the pre-treatment with the dual histamine H3/H4 receptors antagonist (thioperamide), but not with the selective histamine H1 (pyrilamine or loratadine), H2 (cimetidine or ranitidine) or H3 (iodophenpropit) receptor antagonists, was able to interfere with trypsin response. Pre-treatment with the mixed serotonergic/histaminergic receptor antagonist, cyproheptadine, or the non-selective protease inhibitor, gabexate mesylate, were capable of inhibiting trypsin-induced pruritus, suggesting the participation of serotoninergic receptors and protease release. Interestingly, both neonatal capsaicin treatment and resiniferatoxin administration to adult mice, to promote sensorial fiber desensitization and/or destruction, were able to suppress the itching response induced by PAR-2 agonists. Similarly, the selective TRPV1 receptor antagonist, SB366791, or the genetic deletion of TRPV1, greatly diminished the PAR-2-induced scratching behaviour. The pruritogenic response induced by PAR-2 agonists in mice is widely dependent on the release of neuropeptides substance P (SP), CGRP and neurokinin B (NKB), as the treatment with the selective NK1 (FK888, for SP), NK3 (SR142801, for NKB) or CGRP1 (fragment CGRP8-37, for CGRP) receptor antagonists caused a marked reduction of PAR-2-induced pruritus. Of note, the pretreatment with both kinin B2 (Hoe 140 or FR173657) and B1 (DALBK or SSR240612) receptor antagonists, or the genetic ablation of kinin receptors, significantly diminished the scratching behaviour evoked by PAR-2 agonists. Take together, the results of the present study suggest that i.d. injection of PAR-2 agonists on the back of the mouse neck seems to be a reproducible model to study the mechanisms and therapeutic strategies, related to itch transmission caused by proteinases release and PAR-2 activation. Besides, this response is widely dependent on mast cell activation and subsequently release of neurokinins, kinins and the TRPV1 receptor activation.

Made available in DSpace on 2015-09-17T15:25:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-03-27. Added 1 bitstream(s) on 2015-09-17T15:46:30Z : No. of bitstreams: 1 000846264.pdf: 4110745 bytes, checksum: 9413f0f4aa0915b943ec1e5d62692439 (MD5) / O objetivo do presente estudo in vitro foi avaliar a interação entre os microorganismos C. albicans, S. aureus sensível (MSSA) e resistente (MRSA) à meticilina com relação a alguns dos seus principais fatores de virulência. Estes fatores foram analisados considerando os micro-organismos isoladamente e em culturas mistas. Foi verificada a interação entre esses micro-organismos através da contagem do número de colônias viáveis (UFC/mL), análise da atividade metabólica por meio do ensaio XTT e quantificação da biomassa total por meio do corante cristal violeta nos tempos de 90 minutos, 12, 24 e 48 horas. Para análise da produção de proteinase (SAP) e fosfolipase (PL-C), kits fluorimétricos foram utilizados. A estrutura dos biofilmes foi avaliada por microscopia eletrônica de varredura (MEV). Culturas simples e mistas dos micro-organismos também foram utilizadas para infectar um epitélio oral humano reconstituído (EOHR). A capacidade de colonização e invasão foi avaliada após 24 horas utilizando a técnica de hibridização in situ (PNA-FISH). A avaliação do dano causado pelos micro-organismos ao tecido foi realizada por quantificação da liberação da enzima lactato desidrogenase (LDH). Técnicas moleculares (RT-qPCR) foram empregadas para quantificação do número de micro-organismos colonizando o epitélio e também para avaliação da expressão de genes de virulência. A expressão gênica foi avaliada em superfície biótica (EOHR) e abiótica (placa de poliestireno de 24 poços) para culturas simples e mistas dos micro-organismos. Os resultados dos ensaios de quantificação foram analisados utilizando ANOVA a dois critérios com teste post-hoc de Tukey (α = 0,05), enquanto os dados de atividade enzimática foram analisados por ANOVA a um critério com correção de Welch seguido pelo teste de Games-Howell. Os resultados da expressão gênica foram... (Resumo completo clicar acesso eletrônico) / The aim of this study was to evaluate, in vitro, the interaction between C. albicans and methicillinsusceptible (MSSA) and resistant (MRSA) S. aureus in relation to some of their major virulence factors. These factors were analyzed considering the three microorganisms in single and dual cultures. The interaction between these microorganisms was evaluated by counting colony-forming units (CFU/mL), analysis of metabolic activity by the XTT assay and quantification of the total biomass by crystal violet dye at 90 minutes, 12, 24, and 48 hours. To analyze the production of proteinase (SAP) and phospholipase (PL-C), fluorometric kits were used. The structure of the biofilms was acessed by scanning electron microscopy (SEM). Further, single and dual cultures of microorganisms were used to infect a reconstituted human oral epithelium (RHOE). The pattern of colonization and invasion of the RHOE was evaluated after 24 hours using the in situ hybridization technique (PNA-FISH). The damage caused by microorganisms in the tissue was evaluated by quantification of the lactate dehydrogenase enzyme (LDH) released. Molecular techniques (RT-qPCR) were also used to quantify the number of microorganisms colonizing the epithelium and to evaluate the expression of virulence genes. Gene expression was evaluated in biotic (RHOE) and abiotic (24-well polystyrene microplate) surfaces for single and dual cultures. The results from quantification assays were compared using two-way ANOVA with Tukey post-hoc test, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test. The results of gene expression were analyzed by one-way ANOVA with Tukey for data satisfying the assumptions of normality and homoscedasticity or one-way Welch-ANOVA followed by Games-Howell for heteroscedastic data. LDH results were also...(Complete abstract electronic access below)

Orientadores: Sergio Maramgani, Claudio Augusto Machado Sampaio / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-21T19:45:45Z (GMT). No. of bitstreams: 1 Leme_LucianaDiCieroToledo_D.pdf: 9158581 bytes, checksum: d80e70dba366d1980e84dad1b3b10893 (MD5) Previous issue date: 1996 / Resumo: A presença de inibidores de serinoproteinases foi investigada em sementes de duas variedades de Bauhinia variegata (Legumonosae, Caesalpinoideae). A purificação dos inibidores de Bauhínia variegata variedade cândida e variedade lilás por cromatografia de troca iônica, cromatografias de exclusão molecular e subsequente cromatografia de fase reversa, evidenciaram nestas espécies 3 isoformas (BvcTI-1, 2 e 3; e BvITI-1, 2 e 3) de inibidores de proteinase com cadeia polipeptídica única. A análise global de aminoácidos resultou em 167 e 180 resíduos de aminoácidos para BvcTI-3 e BvITI-3, respectivamente, e em massa molecular calculada de 18529 para BvcTI-3 e de 20018 para BvITI-3, e 4 resíduos de cisteína para BvcTI-3 e 2 resíduos de cisteína para BvITI-3. Estes resultados incluem estes inibidores na Família' de inibidores tipo Kunitz. A coloração para inibidores de tripsina após focalização isoelétrica demonstrou a presença de inibidores com pontos isoelétricos aparentes de 4,85, 5,00 e 5,15. A determinação da estrutura primária completa de BvcTI-3 e da estrutura primária do N-terminal das isoformas de BvcTI e BvITI, confiram alta homologia com inibidores tipo Kunitz. Os inibidores BvcTI e BvlTI atuam seletivamente sobre serinoproteinases. Extratos de sementes e inibidores purificados de Bauhínia variegata var. cândida e varo. lilás foram testados para atividade de inibição contra tripsina e quimotripsina bovina, porcina e humana, sendo que estas enzimas foram fortemente inibidas pelos inibidores em estudo. O teste de edema através do extravasamento de plasma em pele de coelhos, mostrou que BvcTI potenciou a calicreína de pâncreas de porco / Abstract: The presence of serine proteinase inhibitors was investigated in the seeds of Bauhinia variety cândida and Bauhinia variegata variety lilás (Leguminosae, Caesalpinoideae ). The purification of inhibitors from the Bauhinias by ion-exchange chromatography, molecular exclusion chromatography and subsequent reverse phase chromatography, showed the presence of three isoforms (BvcTI-1, 2 and 3 ; BvITI-1, 2 and 3) in the two species studied with single polypeptide chain. The aminoacid analisis of the forms BvcTI-3 and BvITI-3 resulted in 167 and 180 aminoacids residues, respectively, and the calculated molecular masses were 18529 to BvcTI-3 and 20018 to BvITI-3. It showed 4 cysteine residues to BvcTI-3 and 2 cysteine residues to BvITI-3, and no free thiol groups. This results suggest that these proteins belongs to the Kunitz-type plant inhibitors family. Staining for trypsin inhibitors after isoelectric focusing showed the presence of inhibitors with isoeletric point about 4.85, 5.00 and 5.15. The primary structure sequence of BvcTI-3 was determined, confirming the inhibitor as Kunitz type. The inhibitors BvcTI and BvlTI act selectively on serine proteinases. Extracts from seeds and purifieds inhibitors of both Bauhinia variegata variety candida and Bauhinia variegata variety lilas were tested for inhibitory activity against trypsin and chymotrypsin, from cattle, pig and humans. This three enzymes were strongly inhibited by both inhibitors. The oedema test in rabits showed the BvcTI made the kallikrein of porcine pancreas more potent to plasma. extravasation / Doutorado / Bioquimica / Doutor em Ciências Biológicas

Evolution has provided vast genetic diversity, enabling plants to surmount many biotic pressures. Plants have evolved various morphological and biochemical adaptations to cope with herbivores attacks. Despite that, yearly, around 40 % of worldwide crop production is lost due to pests and pathogens, with 13 % due to insects. Tuta absoluta has become a major pest threatening tomato crops worldwide and without the appropriated management it can cause production losses between 80 to 100 %. To cope with this threat, we need to strengthen plant defense arsenals. The incorporation to plants of defensive genes like proteinase inhibitors by means of genetic engineering is a promising alternative. In the first chapter of this work we investigated the inhibitory activity of two trypsin inhibitors from barley; BTI-CMe and BTI-CMc. Besides, we succeeded to increase the BTI-CMc in vitro inhibitory activity by introducing a single mutation in its putative reactive site. In the second chapter, we investigated the in vivo effect of (a serine proteinase inhibitor) BTI-CMe and a (cysteine proteinase inhibitor) Hv-CPI2 isolated from barley on Tuta absoluta and we examined the effect of their expression on the tomato defensive response. We found that larvae fed on the double transgenic plants showed a notable reduction in weight. Moreover, only 56% of the larvae reached the adult stage. The emerged adults showed wings deformities and reduced fertility. We also investigated the effect of proteinase inhibitors ingestion on the insect digestive enzymes. Our results showed a decrease in larval trypsin activity. Proteinase inhibitors had no harmful effect on Nesidiocoris tenuis; a predator of Tuta absoluta, despite transgenic tomato plants attracted the mirid. We investigated whether or not plant defensive mechanisms were activated in the transgenic tomato plants and found that, interestingly, the expression of the barley cysteine proteinase inhibitor promoted plant defense, inducing the tomato endogenous wound inducible proteinase inhibitor 2 (Pin2) gene. Moreover, glandular trichomes production was increased and the emission of volatile organic compounds was altered. Our results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to pests. / La evolución ha proporcionado una gran diversidad genética, permitiendo a las plantas superar muchas presiones bióticas. Las plantas han desarrollado diversas adaptaciones morfológicas y bioquímicas para hacer frente a los ataques de los herbívoros. A pesar de ello, anualmente, alrededor del 40 % de la producción mundial de cultivos se pierde debido a plagas y patógenos, siendo un 13 % debido a insectos. Tuta absoluta se ha convertido en una de las principales plagas que amenazan los cultivos de tomate en todo el mundo y sin la gestión adecuada puede causar pérdidas de producción entre el 80 y el 100 %. Para hacer frente a esta amenaza, necesitamos fortalecer los arsenales de defensa de las plantas. La incorporación a las plantas, mediante ingeniería genética, de genes defensivos como los inhibidores de proteinasas es una alternativa prometedora. En el primer capítulo de este trabajo se investigó la actividad inhibitoria de dos inhibidores de tripsina procedentes de cebada; BTI-CMe y BTI-CMc. Además, se logró aumentar la actividad inhibitoria in vitro de BTI-CMc mediante la introducción de una única mutación en su putativo centro reactivo. En el segundo capítulo, se investigó el efecto in vivo de un inhibidor de serin proteinasa (BTI-CMe) y un inhibidor de cisteín proteinasa (Hv-CPI2) aislado de cebada en Tuta absoluta y se examinó el efecto de su expresión en la respuesta defensiva del tomate. Se encontró que las larvas alimentadas con las plantas transgénicas dobles mostraron una notable reducción de peso. Además, sólo el 56 % de las larvas alcanzó la etapa adulta. Los adultos emergentes mostraron deformidades de las alas y reducción de la fertilidad. También se investigó el efecto de la ingesta de inhibidores de proteinasa en las enzimas digestivas de los insectos. Nuestros resultados mostraron una disminución en la actividad tripsina larvaria. Los inhibidores de proteinasas no tuvieron efectos nocivos sobre Nesidiocoris tenuis(depredador de Tuta absoluta) a pesar de que las plantas transgénicas de tomate atrajeron al mirido. Se investigó si los mecanismos defensivos de las plantas se activaban en las plantas de tomate transgénico y se encontró que, curiosamente, la expresión de la cistatina de cebada promovía la defensa de la planta, induciendo el gen del inhibidor de proteasa 2 endógeno del tomate, inducible por herida (Pin2). Además, aumentó la producción de tricomas glandulares y se alteró la emisión de compuestos orgánicos volátiles. Nuestros resultados demuestran la utilidad de la co-expresión de diferentes inhibidores de proteinasas para el aumento de la resistencia de las plantas a plagas. / L'evolució ha proporcionat una gran diversitat genètica, permetent a les plantes superar moltes pressions biòtiques. Les plantes han desenvolupat diverses adaptacions morfològiques i bioquímiques per fer front als atacs dels herbívors. Tot i això, anualment, al voltant del 40 % de la producció mundial de cultius es perd a causa de plagues i patògens, amb un 13 % a causa de insectes. Tuta absoluta s'ha convertit en una de les principals plagues que amenacen els cultius de tomaca a tot el món i sense la gestió adequada pot causar pèrdues de producció entre el 80 i el 100 %. Per fer front a aquesta amenaça, necessitem enfortir els arsenals de defensa de les plantes. La incorporació a les plantes de gens defensius com els inhibidors de proteïnases per mitjà de l'enginyeria genètica és una alternativa prometedora. En el primer capítol d'aquest treball es va investigar l'activitat inhibitòria de dos inhibidors de tripsina aïllats a partir d'ordi; BTI-CMe i BTI-CMC. A més, es va aconseguir augmentar l'activitat inhibitòria in vitro de BTI-CMC mitjançant la introducció d'una única mutació en el seu lloc reactiu putatiu. En el segon capítol, es va investigar l'efecte in vivo d'un inhibidor de serin proteinasa (BTI-CMe) i un inhibidor de cisteïn proteinasa (Hv-CPI2) aïllats d'ordi en Tuta absoluta i es va examinar l'efecte de la seva expressió en la resposta defensiva del tomaca. Es va trobar que les larves alimentades amb les plantes transgèniques dobles van mostrar una notable reducció de pes. A més, només el 56 % de les larves va aconseguir l'etapa adulta. Els adults emergents van mostrar deformitats de les ales i reducció de la fertilitat. També es va investigar l'efecte de la ingesta d'inhibidors de proteinasa en els enzims digestius dels insectes. Els nostres resultats van mostrar una disminució en l'activitat tripsina larvària. Els inhibidors de proteïnases no van tenir efectes nocius sobre Nesidiocoris tenuis, un depredador de Tuta absoluta, tot i les plantes transgèniques de tomaca van atreure al mirid. Es va investigar si els mecanismes defensius de les plantes s'activaven a les plantes de tomaca transgènic i es va trobar que, curiosament, l'expressió de cistatina d'ordi promovia la defensa de la planta, induint el gen de l'inhibidor de proteasa 2 endogen de la tomaca, induïble per ferida (Pin2). A més, va augmentar la producció de tricomes glandulars i es va alterar l'emissió de compostos orgànics volàtils. Els nostres resultats demostren la utilitat de la co-expressió de diferents inhibidors de proteïnases per a l'augment de la resistència de les plantes a plagues. / Hamza, R. (2017). Enhancement of tomato resistance to Tuta absoluta by the expression of two barley proteinase inhibitors [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/92723 / TESIS