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ZEYNALI, AMIRBAHADOR. "Two-photon assisted direct laser writing of proteinaceous microarchitectures containing plasmonic nanoparticles; characterization and optimization." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2021. http://hdl.handle.net/10281/304319.
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Le nanoparticelle metalliche, grazie alle loro affascinanti proprietà ottiche ed elettrochimiche, attirano l'attenzione di diverse discipline scientifiche e di ricerca ingegneristica. Tra queste proprietà, l'effetto fototermico indotto dalle risonanze plasmoniche, è una caratteristica notevole ed esclusiva delle nanoparticelle di metalli nobili che, ad oggi, vengono già sfruttate per vari scopi, sia per ricerca che, soprattutto, per applicazioni biomediche. Il fenomeno della risonanza plasmonica superficiale, caratterizzato da bande ben definite, fornisce a queste nanoparticelle una notevole la flessibilità nella risposta ottica che si può vantaggiosamente trasferire a matrici polimeriche in cui queste vengano disperse. In particolare, la possibilità di indurre un aumento di temperatura altamente localizzato che può essere attivato tramite un dispositivo di stimolazione esterno come una sorgente di luce, renderebbe tali matrici strumenti preziosi nel campo dei trattamenti cellulari e, in generale, dell'ingegneria dei tessuti
In questa tesi, la tecnica di scrittura (photo-cross-link) laser diretta (DLW), attivata da assorbimento a due fotoni, è stata impiegata per fabbricare micro-architetture con il diverso modulo elastico (da 80kPa a 800kPa) a partire da un inchiostro proteico composto da albumina di siero bovino (BSA), un foto-iniziatore (Rose Bengale o blu di metilene) e nanoparticelle d'oro a simmetria non sferica (GNP). Mostriamo qui che la presenza di queste ultime, se opportunamente schermate dall’interazione con il foto-iniziatore, fornisce alle microstrutture foto-polimerizzate la capacità di generare un aumento della temperatura locale mediante stimolazione nella regione spettrale del vicino infrarosso. L'efficienza fototermica misurata sotto l’effetto di radiazione laser focalizzata a 800 nm (in continua) su microstrutture caricate con una bassa concentrazione di atomi d'oro (1% w/w) ha raggiunto 12.2 pm 0.4 C/W, che costituisce un record di effetto fototermico indotto su una microstruttura a base proteica proteinica stampata tramite DLW.
La funzionalità foto-termica derivante dalle GNP incorporate nelle microstrutture proteiche fabbricate riveste una notevole potenzialità nello studio delle risposte di sistemi viventi, come cellule e colture di batteri, al rilascio di calore altamente localizzato e controllato sia per quanto riguarda il tempo di irraggiamento che la dose rilasciata.Metallic nanoparticles, due to their fascinating optical and electrochemical properties, attract the attention of different science and engineering research disciplines. Among these properties, the plasmonic photothermal effect is a notable and exclusive feature of noble-metal nanoparticles that, by today, are exploited through lots of research activities for various purposes, especially for biomedical applications. This optically-tunable phenomenon uniquely increases the flexibility of the optical response of host matrices, by allowing to induce highly localized temperature increases that can be triggered via simple external stimulation device like a light source. Such matrices can be valuable tools in the field of cell treatments and, in general, tissue engineering. In the present study, the two-photon-assisted direct laser writing (DLW) technique was employed to fabricate microarchitectures with the different elastic modulus (80kPa to 800kPa) from a proteinaceous ink composed of bovine serum albumin (BSA), rose Bengal (RB), or methylene blue (MB), and non-spherical symmetric gold nanoparticles (GNPs), with the ability to generate local temperature increase by stimulation in the near-infrared spectral region. The recorded photothermal efficiency measured using focused continuous wave (CW) laser irradiation at 800nm on microstructures loaded with GNPs at low gold atom concentration (1%w/w) reached 12.2 pm 0.4 C/W, that is a record photothermal effect induced from a printed proteinaceous feature. This photo-thermal functionality arising from the GNPs embedded within the fabricated proteinaceous microstructures can then be applied for studying responses of living systems like cells and bacteria cultures under an externally triggered highly localized heat release.
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Turk, Boris. "Papain-like cysteine proteinases : regulation by proteinase inhibitors and pH /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 1996. http://epsilon.slu.se/avh/1996/91-576-5227-9.gif.
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Zhang, Xiaorong. "The roles of proteinases and proteinase inhibitors in plant-nematode interactions." Diss., Virginia Tech, 1994. http://hdl.handle.net/10919/37260.
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Bridges, Sylvia Shadinger. "Design, synthesis, and evaluation of cysteine protease inhibitors." Diss., Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/29738.
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Proteases are enzymes that cleave protein amide bonds. Proteases are involved in a myriad of biological processes and are considered good targets for drug design. The proteases described herein are cysteine proteases, which utilize a cysteine residue thiol to attack the amide carbonyl, leading to amide bond cleavage. Irreversible inhibitors of cysteine proteases react with the active site cysteine, forming a covalent bond and rendering the enzyme inactive.
The first project involved the design and synthesis of aza-peptide epoxide inhibitors for calpain, a clan CA, ubiquitous, calcium-activated human enzyme involved in neurodegeneration. These inhibitors proved to be poor inactivators of calpain, demonstrating that the aza-peptide epoxide is a warhead specific to clan CD cysteine proteases (caspases, gingipains). Subsequently, a known epoxide inhibitor of calpain was optimized to create a more potent inhibitor. Several of these inhibitors were more potent than the parent, and all were demonstrated to inhibit calpain in a breast cancer cell line which was treated with paclitaxel to spike calpain activity.
The second project involved the design and solid phase synthesis of aza-peptide Michael acceptor caspases inhibitors. The two goals of this project were to develop a solid phase method for synthesis of inhibitors that are tedious to synthesize in solution phase, and to use a variety of amino acid residues to determine the optimal interactions in the P3? position for various caspases. The synthesis was successful, and the optimal P3? residues were determined.
The third project involved the kinetic evaluation of aza-peptide epoxide and Michael acceptor inhibitor designed for the gingipains. Gingipains K and R are virulence factors in the pathology of Porphyromonas gingivalis involved in gingivitis and periodontal disease. These inhibitors proved to be extremely potent inactivators of gingipains, with some of the highest rates of inhibition measured in the Powers laboratory. Gingipain K preferred larger, aromatic moieties in the P1? position, while gingipain R preferred the Michael acceptor inhibitors, with the P1? substituent having less of an impact on potency.
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Souza, Thais Paula de. "Efeito dos inibidores de proteinase de soja no padrão de expressão de proteinases de Spodoptera frugiperda." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-05112013-150120/.
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Dentre as substâncias químicas secretadas pelas plantas, contra os insetos herbívoros, os inibidores de peptidases são de grande interesse. A atenção dada a esses inibidores deve-se ao fato, de eles serem uma boa alternativa no controle de insetos praga, uma vez que não causam danos ao meio ambiente. Contudo, muitas espécies de insetos são capazes de escapar dos efeitos negativos dos inibidores de peptidases das plantas, via diferentes mecanismos adaptativos. Devido a esse fato, é importante compreender os mecanismos desenvolvidos pelos insetos para burlar os efeitos dos inibidores de peptidases das plantas. Diante desse panorama, este trabalho teve como objetivo estudar o mecanismo adaptativo das serina endopeptidases de Spodoptera frugiperda aos inibidores de endopeptidases de soja. Foram realizadas análises do transcriptoma dos intestinos das lagartas mantidas em exposição crônica ao inibidor. Para averiguar os efeitos causados devido à exposição crônica ao inibidor, foram realizadas comparações da expressão relativa, dos genes de tripsinas e quimotripsinas, de intestinos de lagartas de sexto instar. Contudo, para entender o efeito da exposição aguda ao inibidor, lagartas de S. frugiperda foram criadas em dieta artificial controle até o primeiro dia do sexto instar, após esse período elas foram transferidas para dieta artificial acrescida com 0,5 % dos inibidores de endopeptidases de soja, durante 48 horas. Para verificar a ocorrência de um possível controle epigenético na expressão dos genes, as lagartas foram conduzidas até a fase adulta e os adultos, de cada tratamento, foram acasalados entre si, constituindo uma segunda geração. Dados de expressão relativa foram obtidos, de indivíduos da primeira e segunda geração, e foram então comparados. Foram identificados 14 possíveis genes de quimotripsinas e nove possíveis genes de tripsinas. Os genes de tripsina foram divididos em dois grupos distintos em relação a sua sensibilidade aos inibidores de endopepetidases de soja e expressão relativa. Houve uma resposta diferenciada na ativação dos genes de serina endopeptidases de S. frugiperda, a qual dividiu os genes em dois grupos, os responsivos e os não responsivos ao inibidor. A exposição aguda ao inibidor ativou um pequeno grupo de genes, enquanto que a exposição crônica promoveu uma maior amplitude de expressão gênica, sugerindo mecanismos temporalmente regulados. Por último, evidências indicam, pela primeira vez, a possível ocorrência de um mecanismo epigenético, na resposta das enzimas digestivas aos inibidores de serina endopeptidases de soja.Among the chemicals secreted by plants against insect herbivores, peptidase inhibitors (PIs) are of great interest. The attention given to PIs is due to the fact that they are a good alternative to control insect pests since they do not cause damage to human health and the environment. However, many species of insects are able to escape the negative effects of PIs plants via different adaptive mechanisms. Because of this, it is important to understand the mechanisms developed by insects to circumvent the effects of PIs plants. Against this background, this research aimed to study the adaptive mechanism of serine endopeptidases Spodoptera frugiperda to soybean endopeptidase inhibitors. For this purpose, larvae of S. frugiperda were reared on artificial diet and control artificial diet plus 0.5% of endopeptidase inhibitors of soybean. We conducted a transcriptome midgut of worms maintained in chronic ingestion of the inhibitor. The relative expression of the genes, trypsin and chymotrypsin, was also compared the midgut of sixth instar larvae kept in these diets. In another experiment, the larvae were conducted to moth, then the treatments were mated forming a second generation. Relative expression data were obtained for individuals of the first and second generation, and then compared. Identified 14 genes with potential of chymotrypsin and 9 trypsin like genes. The trypsin gene were divided into two groups for both their sensitivity to PI soybean endopeptidase, and for their relative expression pattern. There was a differentiated response on the genes activation of S. frugiperda serina endopeptidases. The genes were clustered in 2 groups, the responsive ones and the non responsive to the inhibitor. The acute exposition to the inhibitor activated a small group of genes and the chronic exposition affected several genes, indicating the existence of temporal regulated mechanism. Besides, there is a possible occurance of an epigenetic mechanism, which is related to the digestive serina endopeptidases inhibitors of soybean.
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Nadalini, Larissa Cristina Deppmann. "Caracterização do mecanismo adaptativo de Spodoptera frugiperda aos inibidores de proteinase de plantas." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-07022008-151221/.
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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.
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Estrada, Sergio. "Cystatin A, a mammalian cysteine proteinase inhibitor : mechanism of inhibition of target proteinases by recombinant cystatin A variants /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 1998. http://epsilon.slu.se/avh/1998/91-576-5448-4.gif.
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Souza, Diego Pereira de. "ProteÃnas inibidoras de fitopatÃgenos em fluidos laticÃferos: atividade e mecanismo de aÃÃo." Universidade Federal do CearÃ, 2010. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=7887.
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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel SuperiorUm relevante nÃmero de espÃcies vegetais à descrito como plantas produtoras de um fluido leitoso comumente denominado de lÃtex. Nestas espÃcies, o lÃtex à sintetizado e armazenado sob pressÃo em um sistema de canais formados por cÃlulas altamente especializadas denominadas de laticÃferas, em cujos citoplasmas estÃo presentes todas as estruturas eucariontes em meio à Ãgua, borracha e inÃmeras molÃculas, muitas das quais especÃficas deste conteÃdo. Muitos estudos tÃm sugerido que molÃculas produzidas nestes fluidos participam da defesa vegetal. Neste trabalho, o lÃtex de 5 espÃcies foi coletado e processado em laboratÃrio para obtenÃÃo de suas fraÃÃes protÃicas e estas foram avaliadas quanto a atividade sobre fungos fitopatogÃnicos atravÃs de ensaios de inibiÃÃo da germinaÃÃo de esporos e crescimento de hifas. ProteÃnas do lÃtex de C. procera (PLCp), Cryptostegia grandiflora (PLCg) e Carica candamarcensis (P1 G10) apresentaram atividade antifÃngica enquanto que Plumeria rubra (PLPr) e Euphorbia tirucalli (PLEt) nÃo apresentaram atividade sobre qualquer dos fungos avaliados (Colletotrichum gloeosporioides, Fusarium oxysporum, Fusarium solani, Rhizoctonia solani, Neurospora sp. e Aspergillus niger). A atividade inibitÃria das fraÃÃes protÃicas se correlacionou diretamente com a presenÃa de atividade proteolÃtica do tipo cisteÃnica presente nas amostras de PLCp, PLCg e P1G10. A atividade antifÃngica foi aumentada na presenÃa de DTT, um ativador destas proteases e foi diminuÃda ou eliminada quanto Ãs amostras foram prÃ-tratadas com iodoacetamida (IAA), um inibidor especÃfico de proteases cisteÃnicas. AlÃm disso, a atividade antifÃngica foi observada quando papaÃna, uma protease cisteÃnica purificada do lÃtex de Carica papaya foi avaliada, mas tripsina e quimotripsina, duas proteases serÃnicas nÃo apresentaram atividade. AtravÃs de cromatografia em coluna de Mono-S Sepharose acoplada ao sistema de FPLC, uma protease cisteÃnica foi isolada de PLCg. A proteÃna purificada (Cg24-I) apresentou massa molecular de 24,118 KDa. A Cg24-I apresentou atividade proteolÃtica mÃxima em pH 8,0 e foi inibida por IAA e E-64, utilizando azocaseÃna e BANA como substratos, respectivamente. Cg24-I inibiu a germinaÃÃo de F. solani e foi capaz de alterar a permeabilidade das membranas dos esporos na concentraÃÃo de 90 ng/ml. Esse conjunto de resultados sugere que proteinases cisteÃnicas de fluidos laticÃferos participam da defesa das plantas contra fungos fitopatogÃnicos e que o provÃvel mecanismo de aÃÃo destas proteÃnas seja a alteraÃÃo da permeabilidade da membrana plasmÃtica destes microrganismos. A descriÃÃo de atividade antifÃngica de proteases cisteÃnicas oriundas de fluidos laticÃferos nÃo à ainda descrita em detalhes na literatura, sendo este um trabalho com carÃter original.
Canal systems containing secretions, such as latex, are widely disseminated in the plant kingdom. These fluids are chemically complex and exhibit intense metabolism. Despite their origin, latex is the cytoplasm of specialized cells growing intrusively into organized tissues and organs, forming an interconnected network allowing latex exudation immediately after tissue damage. Insecticidal effects of latex proteins have been described, however minor studies were devoted to investigate antifungal activities in latex. In this study proteins extracted from latex of Calotropis procera (Ait.) R.Br (PLCp), Plumeria rubra L.(PLPr), Carica candamarcensis Hook F.(P1 G10), Cryptostegia grandiflora (PLCg), and Euphorbia tirucalli L. (PLEt) were tested for antifungal activity against six phytopathogens (Fusarium solani, F. oxysporium, Aspergilus niger, Rhizoctonia solani, Neurospora sp. and Colletrotricum gloerosporioides). PLCp, PLCg and P1G10 exhibited antifungal activity and PLPr and PLEt were not efetive. Inhibitory activity of the protein fractions correlated with the cysteine-type proteolytic activity found in these fractions. The endogenous proteolytic activity and inhibitory activity on fungal growth were both increased when samples were first activated with DTT, a cysteine proteinase activator. Conversely, pre-treatment of samples with iodoacetamide, an inhibitor of these proteases rendered all samples deficient of both, proteolytic and antifungal activities. Antifungal of activity of cysteine proteinases of latex origin was also confirmed when papain, obtained from latex of Caryca papaya was tested while purified trypsin and chemotrysin, two serine-type proteases were not antifungal. A cysteine proteinase was thus, purified form PLCg by ion exchange chromatography on a Mono-S Sepharose matrix monitored by a FPLC system. The protein, named Cg24-I exhibited molecular mass of 26.118 KDa determined by MALDI spectrometry; maximum of proteolysis at pH 8.0 and inhibited by iodoacetamide and E-64 when assayed with azocazein or BANA as substrates. Cg24-I inhibited germination of F. solani and altered membrane permeability of spores at a minimum concentration of 90 ng/ml. Results present here suggest that cysteine proteinases of laticifer fluids are proteins with antifungal activity capable of damaging spore structure and inhibiting hyphae growth. Reports of antifungal activity of latex proteases are still scarce in literature and this work appears as an important contribution to this field. Furthermore, this work gives important evidence for the multiple defensive role of latex in plants.
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Macgregor, James Mylne. "Alimentary tract proteinases of the Southern corn rootworm (Diabrotica undecimpunctata howardi) and the potential of potato Kunitz proteinase inhibitors for larval control." Thesis, Durham University, 2001. http://etheses.dur.ac.uk/3808/.
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Proteolytic digestion by larval Diabrotica undecimpunctata howardi (Barber) (D.undecimpunctata) has been investigated with the aim of producing transgenic plants possessing enhanced resistance to this economically important crop pest. Biochemical characterisation in vitro by pH dependent hydrolysis and inhibition assays incorporating E-64, pepstatin A and soybean Kunitz trypsin inhibitor showed the majority of hydrolytic activity occurs at pH 5.5 and is performed by cysteine and aspartic endopeptidases. Cysteine and aspartic proteinase encoding clones were isolated from a larval alimentary tract cDNA library. Four cathepsin L-like cysteine proteinases and two cathepsin D-like aspartic proteinase cDNA clones were identified by codmg homology to known proteinase sequences. Analysis of primary and secondary sequence features revealed D. undecimpunctata aspartic proteinase 1 exhibits features associated with cathepsins E and is proposed to be a D. undecimpunctata cathepsm E-like aspartic proteinase.Cathepsin D-like aspartic proteinase inhibitors of the potato Kunitz protemase inhibitor (PKPI) family have been isolated by PCR and expressed employing the pET expression system (Novagen). In vitro assays demonstrated the inhibitory activity of PKPI-A and PKPl-B inhibitors against larval D. undecimpunctata alimentary tract proteolytic enzymes. To the authors knowledge this work represents the first reporting of the expression and purification of biologically active PKPI proteins. In vitro assays incorporating oryzacystatin I and PKPI proteins resulted in increased inhibition of proteolytic activity compared to single inhibitor and uninhibited control reactions. Inhibition assays provide evidence for the potential of a dual protemase inhibitor strategy to arrest protein hydrolysis by larval D. undecimpunctata, preventing essential amino acid absorption. Further research is necessary to characterise the properties of the digestive enzymes isolated in this work and the inhibitory spectrum of PKPI proteins. Transgenic crops expressing a combination of oryzacystatin and PKPI proteins would be predicted to show enhanced resistance to insect herbivores by virtue of digestive proteolysis inhibition.
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Mistry, Rohit. "Development of a sheep model to investigate the role of neutrophil proteinases and their endogenous inhibitors in acute lung injury : a novel proteinase inhibitor." Thesis, Imperial College London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308934.
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Silva, Walciane da. "Estudo da ação do inibidor de proteinase de Adenanthera pavonina sobre o desenvolvimento e atividade das proteinases intestinais de lagartas de Diatraea suchharalis (FABR., 1794)." [s.n.], 2008. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314519.
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Orientador: Maria Ligia Rodrigues MacedoDissertação (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
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Hvenegaard, Ana Paula Franco do Amaral. "Estudo retrospectivo do tratamento ambulatorial da úlcera indolente em cães da raça Boxer." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/10/10137/tde-02022012-134654/.
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Úlceras indolentes são úlceras corneais superficiais, espontâneas, que apresentam curso prolongado e que tendem a recidivar. Comumente observadas em cães de meia idade, da raça Boxer, provoca dor de início agudo e necessita de tratamento específico, já que este, quando não realizado de forma correta, pode prolongar o curso da lesão por semanas a meses. A doença é explicada por diversas alterações da superfície ocular. Com o objetivo de avaliar a eficácia dos tratamentos ambulatoriais preconizados no Serviço de Oftalmologia do Hospital Veterinário da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo (HOVET-FMVZ-USP), e as principais considerações observadas no levantamento dos prontuários, realizou-se estudo retrospectivo dos casos atendidos entre os anos de 1997 e 2008. Segundo os resultados, observou-se que a maioria dos cães da raça Boxer apresentaram úlcera indolente, distrofia corneal e catarata; que as úlceras indolentes foram mais frequentemente observadas em fêmeas de meia idade e que a maioria dos proprietários demoraram mais de 15 dias para levar seus animais ao HOVET-FMVZ-USP; que as alterações oculares mais frequentemente referidas pelos proprietários foram o blefarospasmo, olho vermelho e a secreção; que as principais características das lesões observadas após o exame oftalmológico foram que a maioria das úlceras eram transparentes, apresentando epitélio não aderido ou com algum grau de vascularização; unilaterais, mais frequentemente observadas no olho direito; de aparecimento espontâneo e localizadas no centro da córnea. Quanto ao tratamento, observou-se que os inibidores das proteinases foram as medicações mais frequentemente prescritas e que sua administração não interferiu no tempo de cicatrização corneal ou na formação de granuloma. Vitamina C, apesar de ter prolongado de maneira significante o tempo de cicatrização corneal, reduziu a inflamação, consideração observada pela diminuição da presença de granuloma. Debridamento/cauterização corneal, além de não interferir na formação de granuloma, acelerou, significativamente, o processo de cicatrização. A antibioticoterapia e a administração de Atropina 1 % não interferiu no tempo de cicatrização, mas se relacionaram diretamente, de forma estatisticamente significante, à presença de granuloma. O uso de anti-inflamatórios tópicos e sistêmicos também não interferiu no tempo de cicatrização, mas diminuíram, de maneira significante, a presença de granuloma nos cães em que foram administrados. Observou-se também que a não administração de atropina 1 %, antibióticos e anti-inflamatórios não interferiu no tempo de cicatrização, nem na formação de granuloma; que o tempo de alteração ocular, antes da primeira consulta e as características das lesões não interferiram, de maneira relevante, no tempo de cicatrização corneal. Portanto, conhecer os diversos tipos de tratamento se mostra fundamental para o sucesso da resolução da doença, já que este deve ser específico, realizado de forma cautelosa e por tempo indeterminado, cuidando para que a lesão não progrida e promovendo o retorno da transparência corneal.Indolent ulcers are superficial corneal ulcers that occurs spontaneously, presents prolonged course and tend to relapse. Commonly observed in middle-aged Boxer dogs, causes pain of acute onset and requires appropriate treatment. The disease is explained by several changes on the corneal surface. Aiming to assess the effectiveness of clinical treatments, recommended by the Ophthalmology Service of the Veterinary Hospital, of the Veterinary College, of the University of São Paulo (HOVET-FMVZ-USP) and to evaluate major considerations registered on its medical records, a retrospective study was conducted (1997 2008). Results demonstrated that, during studied period: most Boxer dogs presented indolent ulcers, corneal dystrophy and cataracts; indolent ulcers were frequently observed in middle-aged female Boxers and most owners took more than 15 days to bring their animals to the hospital; blepharospasm, red eye and ocular discharge were the most owner´s referred ocular alterations at the primary consultation; main features of examined lesions were transparent ulcers presenting non adherent epithelium and/or some degree of vascularization; unilateral, often observed at the right eye, of spontaneous onset and located at the center of the cornea. Regarding treatment, proteinase inhibitors were the most often prescribed medications; its administration did not affect corneal healing or granuloma formation. Vitamin C prolonged, significantly, the corneal healing time, although, its administration reduced its inflammation, observed by the decrement on the granuloma frequency. Corneal debridement / cauterization, did not interfere on granuloma formation and was capable to accelerate, significantly, the healing process. Antibiotics and 1 % atropine did not affect the healing time, but were statistically related to the presence of granuloma. Topical and systemic antiinflammatories did not interfere at the healing time, but decreased, significantly, the presence of granuloma. Not to administer atropine 1%, antibiotics and antiinflammatories, did not interfere at the corneal healing time nor the formation of granuloma. Duration period of ocular alterations before the first consultation and characteristics of the lesions did not interfere at the corneal healing time. Therefore, to know the various types of treatments seems to be fundamental to the resolution of the indolent ulcer, as treatment must be specific, performed cautiously and for indefinitely period, preventing the progression of the lesion, and promoting the return of corneal transparency.
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Tobouti, Priscila Lie. "Avaliação da expressão gênica da proteína aspartil secretada 2, 5 e 9 (SAP-2, SAP-5 e SAP-9) e sua correlação com a invasão epitelial por Candida albicans em modelo experimetal de estomatite protéica in vivo." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/25/25150/tde-27072011-110643/.
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A Estomatite protética associada a Candida (EPC) acomete a mucosa bucal em contato com próteses removíveis e, clinicamente, caracteriza-se por eritema com diferentes graus de inflamação. Esta doença é considerada de etiologia multifatorial, isto é, uma associação de fatores como trauma, falta de higienização, uso contínuo da prótese, hipersensibilidade ao material usado na dentadura, diabetes, terapia imunossupressora e infecção por fungo, como diferentes espécies de Candida. Os principais fatores de virulência deste fungo são a formação de hifas, dimorfismo, alterações fenotípicas, aderência, persistência, sinergismo com as bactérias, interferências com o sistema de defesa do hospedeiro e a produção de enzimas hidrolíticas. Dentre as enzimas hidrolíticas, a proteinase aspartil secretada (SAP) é uma das mais importantes para a patogenia de C. albicans, sendo nociva para o tecido epitelial e para o sistema imune do hospedeiro. Não está totalmente compreendida a real penetração do fungo nos tecidos e sua correlação com a presença da SAP, na doença estomatite protética. Essa dificuldade de avaliação pode ser justificada pelas divergências intrínsecas e extrínsecas observadas em muitos aspectos, como diferentes costumes, hábitos sociais, estado emocional e fisiológico. A utilização de um modelo experimental em animais poderá minimizar essas divergências e fornecer condições mais padronizadas para o experimento. Neste trabalho, foram avaliadas, quantitativamente, a expressão gênica das enzimas SAP-2, SAP-5 e SAP-9, presentes no biofilme formado na superfície interna das placas acrílicas superiores de ratos e, microscopicamente, a invasão do fungo no tecido epitelial do palato. Para isso, foram selecionados 49 ratos Wistar, com 90 dias de vida, pesando em média 300g, os quais foram divididos em 3 grupos: Controle, Placa/Candida e Placa, acompanhados durante 2, 4 e 6 dias. Os resultados demostraram que, em 4 dias de uso da placa acrílica contaminada, houve, em alguns ratos, sinais clínicos de inflamação no palato duro; microscopicamente, hiperplasia epitelial, hiperqueratinização, invasão fúngica nas camadas superficiais do revestimento epitelial, microabscessos de Munro e hiperplasia papilar; e maior percentual de neutrófilos no Grupo Placa/Candida em relação aos Grupos Controle e Placa. Também no quarto dia de uso da placa acrílica superior, no Grupo Placa/Candida, o biofilme formado na sua superfície interna apresentou a mais alta expressão gênica relativa das enzimas SAP-2, SAP-5 e SAP-9 que os períodos de 2 e 6 dias de uso. Assim, a invasão fúngica no revestimento epitelial do palato duro pode estar correlacionada com a alta expressão de RNAm das SAPs -2, -5 e -9.Denture stomatitis (D.S.) affects the oral mucosa in contact with removable dentures, and clinically characterized by erythema with varying degrees of inflammation. This disease is considered a multifactorial etiology, with a combination of factors such as trauma, lack of hygiene, continuous use of stents, hypersensitivity to the material used for dentures, diabetes, immunosuppressive therapy and fungal infection, such as different species of Candida. The main virulence factors of the fungus are the formation of hyphae, dimorphism, phenotypic changes, adherence, persistence, synergism with bacteria, interference with the host defense system and the production of hydrolytic proteins. Among the hydrolytic proteins, the secreted aspartyl proteinase (SAP) is one of the most important in the pathogenesis of C. albicans. SAP is harmful to both the epithelial tissue and to the host\'s immune system. It is not fully understood the real penetration of the fungus in the epithelium tissue and its correlation with the presence of SAP, in denture stomatitis. This difficulty in evaluation can be justified by the intrinsic and extrinsic differences observed in many aspects, different customs, social`s habits, emotional and physiological state. Using an experimental animal model may minimize these differences and provide more standardized conditions for the experiment. In the present work, the aim was to evaluate quantitatively the gene expression of enzymes SAP-2, SAP-5 and SAP-9 of the biofilm formed in internal surface of rat`s upper acrylic plates, and to analysis microscopically, the fungal invasion in palatal epithelial tissue. 49 Wistar rats were selected, 90 days old, weighing on average 300g. They were divided into three groups: Control Group, Plate/Candida and Plate, follow by 2, 4 and 6 days of the use of the plates. The results demonstrated, in four days of use of the acrylic plate, clinical signs of inflammation in the hard palate; microscopically epithelial hyperplasia, hyperkeratinization, fungal invasion in the superficial layers of the epithelial lining, Munro`s microabscess and papillary hyperplasia; and higher percentage of neutrophils in the Plate/Candida Group, compared to Control and Plate Groups. In the period of 4 days, the relative gene expression of the SAPs-2, -5 and -9 in biofilm also showed to be higher in Plate/Candida Group, compared with the periods of 2 and 6 days. Thus, the fungal invasion in the epithelial lining of the hard palate may be correlated with high mRNA expressionn of SAPs -2, -5 e -9.
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Rukamp, Brian John. "Design, synthesis, and evaluation of novel thiobenzyl ester substrates and aza-peptide inhibitors for serine and cysteine proteases." Diss., Available online, Georgia Institute of Technology, 2004:, 2003. http://etd.gatech.edu/theses/available/etd-04072004-180202/unrestricted/rukamp%5Fbrian%5Fj%5F200312%5Fphd.pdf.
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Rukamp, Karrie Eileen Adlington. "Design and synthesis of inhibitors for serine and cysteine proteases." Diss., Available online, Georgia Institute of Technology, 2004:, 2003. http://etd.gatech.edu/theses/available/etd-04082004-180343/unrestricted/rukamp%5Fkarrie%5Fe%5Fa%5F200312%5Fphd.pdf.
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Monteiro, Júnior José Edvar. "Caracterização bioquímica, funcional e estrutural de duas cistatinas recombinantes de feijão-caupi." reponame:Repositório Institucional da UFC, 2012. http://www.repositorio.ufc.br/handle/riufc/18882.
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MONTEIRO JUNIOR, José Edvar. Caracterização bioquímica, funcional e estrutural de duas cistatinas recombinantes de feijão-caupi. 2012. 179 f.Tese (Doutorado em Bioquímica)-Universidade Federal do Ceará, Fortaleza-CE, 2012.Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-20T14:56:24Z No. of bitstreams: 1 2012_tese_jemonteirojunior.pdf: 22956458 bytes, checksum: 4912cb5f52b152df0d6dcc912bb7ccef (MD5)
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mRNA sequences coding for two cystatins were isolated from leaves of hydroponically growing cowpea plants. The sequences were ligated in the expression vector pET302NT-HIS, which was introduced into Escherichia coli, ArcticExpress (DE3) cells. The expression was induced by addition of 0.5 mM IPTG and the recombinant proteins VuCys1 (Vigna unguiculata one-domain cystatin) and VuCys2 (Vigna unguiculata two-domain cystatin), both fused to an N-terminal 6x-His tag, were purified by homogeneity using an affinity matrix containing Ni2+ immobilized to Sepharose. The apparent molecular masses for VuCys1 (yield of 10 mgP.L-1 culture) and VuCys2 (yield of 22 mgP.mL-1 culture) were 14 and 26 kDa, respectively. Both inhibitors were strongly active against the proteinases papain and chymopapain, while bromelain and particularly cathepsin B were less susceptible to in vitro inhibition. No inhibition was detected against the serine proteinase trypsin. Thermal stability tests revealed that both cystatins are thermostable proteins able to achieve a minimum residual proteolytic inhibition activity against papain of 95% (VuCys1) and 85% (VuCys2) when incubated at 100 C for 60 minutes. Similar stability results were also obtained when the proteins were tested to the ability of to inhibit papain activity after incubation in pH values ranging from 2.0 to 11.0. Circular dichroism spectroscopy measurements demonstrated that the secondary structure arrangement of both cystatins undergoes only fewer alterations when both proteins were incubated in temperatures varying from 10 to 90 C and pH values varying from 2.0 to 11.0, as well. These data are in agreement with the thermal and pH stability results previously obtained on papain inhibition assays. Biological assays conducted with different phytopathogenic fungi didn’t show any negative impact on spore germination as well as on mycelial growth of the tested fungi. Different human pathogens, including the pathogenic yeast Candida albicans were shown to be insensible to VuCys1 and VuCys2. Some scientific reports have proposed the use of cystatins as potential molecules in the control and inhibition of the activity of cysteine proteinases related to carcinogenic process. However, cytotoxicity assays performed on three tumor cell lineages revealed no toxic effects of VuCys1 and VuCys2. Inhibitor activities were also tested against digestive enzymes isolated from third instar larvae of the bruchid insects Callosobruchus maculatus and Zabrotes subfasciatus, two major cowpea plagues. Both cystatins were able to cause high inhibition of C. maculatus enzymes; however they were poorly active against Z. subfasciatus counterpart. Feeding tests were conducted in which both cystatins were added to artificial seeds at final concentrations of 0.025, 0.05 and 0.1% and supplied to C. maculatus. Despite the in vitro inhibition of C. maculatus larvae enzymes, the bioassay data suggest that larvae and adult insects appear to develop adaptive mechanisms which can make them insensible to the ingestion of the inhibitors. Crystallographic studies were carried out in order to solve the tridimensional structure of cystatins. 576 different crystallization conditions were tested in which three were favorable to the formation and growth of diffractable crystals of VuCys1 protein. These crystals belong to the orthorhombic space group P212121 and the unit cell dimension was a = 41.48 Å; b = 64.68 Å and c = 87.91 Å, α = β = γ = 90. V. unguiculata one-domain cystatin presents a typical 3D domain swapped dimmer molecular structure, which was solved at 1.95 Å resolution. No crystals were obtained for VuCys2. The physiological importance of this structure to the plant, the structural stability of both inhibitors and the results raised from biological assays are all discussed in this work.
Sequências de mRNA que codificam para duas cistatinas foram isoladas de folhas de plantas de feijão-caupi cultivadas em sistema hidropônico. As sequências foram ligadas no vetor de expressão pET302NT-His, o qual foi introduzido em células de Escherichia coli, ArcticExpress (DE3). A indução da expressão foi realizada por meio de adição de IPTG (0,5 mM) e as proteínas recombinantes VuCys1 – (cistatina de um domínio de Vigna unguiculata) e VuCys2 – (cistatina de dois domínios de V. unguiculata) ambas fusionadas a uma cauda de histidina N-terminal, foram purificadas por homogeneidade em matriz de afinidade constituída de Ni2+ imobilizado à Sepharose. VuCys1 apresentou uma massa molecular aparente de 14 kDa e um rendimento de 10 mgP.L-1 de meio de cultura, já a proteína VuCys2 mostrou uma massa molecular aparente de 26 kDa e um rendimento de 22 mgP.L-1 de meio de cultura. As duas proteínas foram fortemente ativas contra as proteinases papaína e quimopapaína, moderadamente ativas contra bromelaína e apenas fracamente ativas contra catepsina B, enquanto que nenhuma atividade inibitória foi detectada contra a proteinase serínica tripsina. Ensaios de estabilidade térmica mostraram que as duas cistatinas são proteínas termoestáveis, uma vez que, mesmo após incubação a 100 C por 60 minutos apresentam atividade inibitória residual contra papaína superior a 95% (VuCys1) e superior a 85% (VuCys2). Resultados similares de estabilidade também foram obtidos quando as proteínas foram testadas quanto à capacidade de inibir a atividade de papaína, após incubação em valores de pH variando de 2,0 a 11,0. Análises espectroscópicas de dicroísmo circular revelaram que o padrão de estruturas secundárias de ambos inibidores sofre pouca alteração após incubação em temperaturas variando de 10 a 90 C e em valores de pH variando de 2,0 a 11,0. Estes dados estão de acordo com os resultados de elevada estabilidade térmica e a extremos de pH previamente obtidos nos ensaios de inibição in vitro de papaína. Ensaios biológicos realizados com diferentes espécies de fungos fitopatogênicos não mostraram nenhum efeito negativo das proteínas sobre a germinação de esporos ou crescimento micelial dos fungos testados. Os inibidores também não se mostraram ativos contra diferentes patógenos humanos, incluindo a levedura patogênica Candida albicans. Alguns relatos científicos propõem o uso de cistatinas como agentes em potencial no controle e inibição da atividade de proteinases cisteínicas relacionados a processos carcinogênicos. Contudo, testes de citotoxicidade dos inibidores para três diferentes linhagens de células tumorais não mostraram potencial citotóxico. Os inibidores também foram testados quanto à capacidade de inibir a atividade de enzimas digestivas isoladas do intestino de larvas de terceiro instar dos insetos bruquídeos Callosobruchus maculatus e Zabrotes subfasciatus, duas importantes pragas do feijão-caupi. Ambas as cistatinas apresentaram elevado potencial inibitório contra as enzimas de C. maculatus sendo, porém, fracamente ativas contra as de Z. subfasciatus. Bioensaios foram realizados nos quais as cistatinas foram inseridas em sementes artificiais nas concentrações finais de 0,025; 0,05 e 0,1% e administradas a C. maculatus. A despeito da inibição in vitro das enzimas digestivas das larvas de C. maculatus, os resultados do bioensaio sugerem que, tanto larvas como insetos adultos, parecem desenvolver mecanismos adaptativos à administração dos inibidores que os tornam insensíveis à sua ingestão. Estudos cristalográficos foram realizados na tentativa de solucionar a estrutura tridimensional das cistatinas. 576 condições de cristalização foram testadas das quais três levaram à formação e crescimento de cristais difratáveis de VuCys1. Os cristais pertencem ao grupo espacial ortorrômbico, P212121, e a célula unitária apresentou dimensões de a = 41,48; b = 64,68 e c = 87,91 Å, α = β = γ = 90. VuCys1 apresenta uma estrutura molecular de dímero de domínios trocados a qual foi resolvida a uma resolução de 1,95 Å. Cristais de VuCys2 não foram obtidos nas condições testadas. O significado fisiológico desta estrutura para a planta, a estabilidade estrutural de ambos inibidores e os resultados referentes aos diferentes bioensaios são discutidos no presente trabalho.
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Leung, Donmienne Doen Mun. "Studies of serine and cysteine protease inhibitors /." St. Lucia, Qld, 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16491.pdf.
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Kraunsoe, James A. E. "Inhibitors of serine proteinases." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318814.
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Kingsbury, Oliver William. "The inhibition of cysteine proteinases." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360390.
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Johnstone, Thomas W. "Neutrophil serine proteinases and autoimmunity." Thesis, Queen's University Belfast, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241372.
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Lockwood, B. C. "Proteinases in trichomonads and trichomoniasis." Thesis, University of Stirling, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377537.
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Rodrigues, Janaina Aparecida de Oliveira. "Produção de anticorpos monoclonais para uma proteinase acida extracelular (aspartil proteinase) de Candida spp." [s.n.], 2004. http://repositorio.unicamp.br/jspui/handle/REPOSIP/289363.
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Orientador: Jose Francisco HoflingTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Nycander, Maria. "Interaction of cystatins with cysteine proteinases /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 1998. http://epsilon.slu.se/avh/1998/91-576-5428-X.gif.
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Cullen, Breda M. "The characterisation of breast tumour proteinases." Thesis, Queen's University Belfast, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.317436.
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Jupp, Raymond A. "Substrates and inhibitors of aspartic proteinases." Thesis, Cardiff University, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.238215.
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Valler, M. J. "Substrates and inhibitors of aspartic proteinases." Thesis, Bucks New University, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376428.
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Knolle, Martin Daniel. "Characterisation of proteinases in pulmonary pathology." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.608015.
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Tamaki, Fabio Kendi. "Serina proteinases digestivas de insetos-modelo." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-25052011-145048/.
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Tripsinas e quimotripsinas, enzimas pertencentes à classe das serina proteinases, são as principais enzimas proteolíticas digestivas presentes no intestino médio de insetos de diversas ordens. Entretanto, enzimas de diferentes insetos possuem propriedades cinéticas distintas, sendo os motivos dessas diferenças especulados. Precipitações por sulfato de amônio das tripsinas de Tenebrio molitor, Diatraea saccharalis e Spodoptera frugiperda mostram que insetos Lepidópteros possuem serina proteinases mais hidrofóbicas, que foi confirmado através de cromatografias de interação hidrofóbica e da análise de acesso do solvente às superfícies protéicas em modelagens tridimensionais de seqüências. Tal fato está relacionado à formação de oligômeros e resistência a defesas de plantas. Inativações por TPCK mostram que quimotripsinas digestivas de S. frugiperda, inseto polífago, reagem duas ordens de grandeza mais lentamente e possui um deslocamento do pH ótimo de modificação em mais de uma unidade quando comparada com dos outros dois organismos, fato relacionado à resistência a cetonas presentes em diversas plantas. A tripsina digestiva de Periplaneta americana foi purificada e microsseqüenciada, resultando na seqüência VSPAFSYGTG e associada a um alérgeno (denominado PaTry), expresso nos cecos e na região anterior do ventrículo. O anticorpo anti-tripsina de M. domestica reconheceu apenas uma banda no intestino de P. americana e foi utilizado para imunocitolocalizar tripsinas nos tecidos epiteliais, demonstrando que esta é secretada por exocitose nos cecos e na região anterior do ventrículo, como esperado. Por último, a atividade majoritária de quimotripsina se localiza surpreendentemente na região posterior do ventrículo de M. domestica. Apesar disso, apenas 28% dessa atividade é perdida através das fezes, pois 31% da atividade enzimática se encontra firmemente aderida à membrana, e 41% na fração celular solúvel (associada ao glicocálice), sendo a atividade solúvel luminal correspondente a apenas 12%, indicando a existência de pelo menos duas espécies moleculares distintas, uma solúvel e uma aderida à membrana, comprovado inativações térmicas das duas atividades (solúvel e aderida à membrana) na presença e na ausência de Triton X-100, sendo que a atividade aderida à membrana apresentou uma maior meia vida com uma cinética de primeira ordem nos dois casos. Ensaio em gel demonstrou que o homogeneizado possui apenas uma banda de atividade quimotríptica de 30 kDa. A atividade solúvel majoritária foi purificada até a homogeneidade, apresentando uma banda de 30 kDa em SDS-PAGE, pH ótimo de 7,4 e é 90% inativada por TPCK 0,1 mM em pH 8,5 em 15 min. Ela prefere substratos contendo Phe em P1, apesar clivar substratos contendo Tyr e Leu. Uma seqüência contígua similar a quimotripsina foi obtida a partir de uma biblioteca de cDNA de intestino médio de M. domestica, formada por 71 ESTs (de 826 seqüências obtidas ao acaso), indicando que esta deve corresponder à atividade majoritária. Essa seqüência, denominada MdChy1, prediz uma proteína madura de 28.639,2 Da e foi clonada e expressa de maneira recombinante em E. coli BL21 (DE-3) Star, sendo utilizada para produção de anticorpos policlonais em coelhos, que reconheceram uma banda de 30 kDa no ventrículo anterior e posterior, mas não no médio. Esses anticorpos foram utilizados para imunomarcações e reconheceram proteínas no lúmem, nas microvilosidades e em pequenas vesículas do epitélio, demonstrando que a quimotripsina é secretada ao lúmem por exocitose e indicando que o MdChy1 corresponde à atividade majoritária de quimotripsina. Análises de expressão em M. domestica indicam a existência de dois conjuntos de serina proteinases digestivas, um expresso na região anterior e um segundo na região posterior do ventrículo. O MdChy1 é expresso na região posterior, local em que se encontra a atividade majoritária de quimotripsina. Uma reconstrução filogenética dos genes similares a quimotripsinas de Drosophila melanogaster e de M. domestica demonstram que a MdChy1 se agrupa com genes expressos no intestino médio, portanto, com função digestiva.Trypsins and chymotrypsins, serine proteinases enzymes, are the major proteolytic activities present in the midgut of insects. However, enzymes obtained from different insects present different kinetic properties, and the reason for the differences are speculated. Trypsin precipitation of Tenebrio molitor, Diatraea saccharalis and Spodoptera frugiperda with ammonium sulfate showed that Lepidopteran insects possess serine proteinases with a higher superficial hydrophobicity than insects belonging to other orders, which may be associated to oligomerization of enzymes and resistance to inhibitors present in the food. This was confirmed by hydrophobic interaction chromatography and analysis of solvent access to serine proteinases surface. Moreover, inactivations of chymotrypsins by TPCK showed that S. frugiperda chymotrypsins react one order slower and has an optimum pH of modification more than 1 unit higher than chymotrypsins of D. saccharalis and T. molitor, which was related with the resistance of the enzyme to the presence of plant ketones, since S. frugiperda is a polyphagous organism. The digestive trypsin from Periplaneta americana midgut was purified microssequenced, resulting in the sequence VSPAFSYGTG, coincident to the MPA3 allergen (named PaTry), which is expressed in the caeca and anterior ventriculus. Western blot using M. domestica trypsin antisera recognized a single band, and immunohistochemical assays using this antisera showed that the P. americana trypsin is secreted by exocitosys in caeca and anterior ventriculus, which is coincident to the expression data. Although the major M. domestica chymotrypsin activity is present in the posterior ventriculus, only 28% of the activity is lost in feces, because 31% of activity is membrane-bound, and 41% is in the cellular soluble fraction (glycocalix-associated), and only 12% of total activity is soluble in the lumen, indicating the existence of at least two molecular species of chymotrypsins. Thermal inactivations of both activities (soluble and membrane-bound) showed that they may represent two different molecular enzymes, since the membrane-bound activity has a higher half-life than the soluble both in the presence and in the absence of Triton X-100. Both activities presented a linear first-order inactivation kinetic. In gel assays showed the presence of only one activity band in the midgut of 30 kDa. The major soluble activity was purified through one affinitychromatography, and active fractions presented a single 30 kDa band, a optimum pH of 7.4 and was 90% modified by TPCK 0.1 mM at pH 8.5 during 15 min. This enzyme preferentially cleaves substrates containing Phe residues in P1, although it cleaves substrates containing Tyr and Leu. A contig of a chymotrypsin-like sequence was randomly obtained from a cDNA library of M. domestica midguts from 71 ESTs (a total of 826 sequences), indicating that this sequence corresponds to the major activity present in the lumen. This sequence, named MdChy1, predicted a protein with 28639.2 Da which was cloned, recombinantly expressed in E. coli BL21 (DE-3) Star, this recombinant MdChy1 was used to raise polyclonal antibodies in rabbit. A western blot using this antisera recognised a single band in the anterior and posterior ventriculus, but not in the middle. Imunno-gold labeling of epithelium marked proteins in the lumen, at the microvilli and inside small vesicles, demonstrating that chymotrypsin is secreted through exocytosis in M. domestica and reinforcing that MdChy1 corresponds to the major chymotryptic activity found in the midgut. A semi-quantitative RT-PCR of M. domestica serine proteinase-like genes demonstrated that there are two set of genes, one expressed in the anterior and another in the posterior ventriculus, as visualized in western blot. MdChy1 is expressed in the posterior ventriculus, where the major chymotryptic activity is found. A phylogenetic reconstruction of Drosophila melanogaster chymotrypsin-like sequences and M. domestica chymotrypsins showed that MdChy1 branched with sequences expressed in midgut, thus coding proteins involved in digestion.
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Pope, Brent Karl. "Factors Affecting Growth of Proteinase Positive and Proteinase Negative Streptococcus cremoris UC310 in Ultrafiltered Milk Retentate." DigitalCommons@USU, 1987. https://digitalcommons.usu.edu/etd/5356.
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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.
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Rawat, Reetika. "Characterization of the promoter of SmCP, the gene encoding Solanum melongena cysteine proteinase." Thesis, Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B34740156.
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Arruda, Ligia Hansen. "Caracterização estrutural da interação de serino proteinases de Spodoptera frugiperda (Lepidoptera: Noctuidae) e inibidores de proteinases de plantas." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-24052011-091301/.
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As plantas desenvolveram diferentes mecanismos para reduzir o ataque de insetos, incluindo compostos protéicos de defesa, como os inibidores de proteinases (IPs). Os insetos, ao longo da evolução, desenvolveram estratégias para superar as barreiras defensivas das plantas, permitindo a sua alimentação e desenvolvimento, como a super expressão de genes de enzimas digestivas sensíveis e insensíveis aos IPs de plantas. Uma das abordagens desse trabalho foi identificar novas serinoproteinases no intestino de lagartas de Spodoptera frugiperda. Duas novas quimotripsinas e trê novas tripsinas foram identificadas e juntamente com mais 10 genes já conhecidos que codificam estas enzimas foram submetidos à análise de expressão gênica por PCR em tempo real. Entre essas duas famílias de serinoproteinases (SPs) os genes que codificam as quimotripsinas apresentam uma regulação positiva mais ampla do que aqueles que codificam as tripsinas. Estudos de modelagem molecular das quimotripsinas também foram realizados. Foram construídos modelos tridimensionais à partir de modelagem por homologia além de análises de dinâmica molecular e docagem com oito diferentes IPs do tipo Bowman- Birk. Os resultados mostram quais quimotripsinas apresentam as maiores afinidades aos inibidores testados de maneira geral e individual, inferidos à partir da estimativa de energia livre do sistema. Também foi encontrada uma serina extra próxima ao sítio catalítico de três quimotrispsinas modeladas que pode interferir na afinidade dessas enzimas já que este aminoácido apresenta perda de área acessível ao solvente quando complexada ao IP de soja testado. Os resultados de expressão gênica e grau de sensibilidade foram comparados e não se observou qualquer relação entre esses parâmentros. Isso sugere que as lagartas da espécie S. frugiperda combinam diferentes estratégias adaptativas como o aumento de expressão de todas as suas quimotripsinas independentemente do grau de sensibilidade das enzimas.Plants have developed different mechanisms to reduce insect attack, including defence proteins such as proteinase inhibitors (PIs). In turn, insects have evolved strategies to overcome these plant defence mechanisms, such as the hyperexpression of PI-sensitive and insensitive digestive enzymes, allowing the insect to thrive. One of the aims of this work was to identify new serine proteinases (SPs) in the gut of the fall armyworm larvae, Spodoptera frugiperda. Two new chymotrypsins and three new trypsins were identified, and together with 10 previously identified genes, the genes that encode these enzymes were subjected to real-time PCR and gene expression analysis. Between these two families of serine-proteinases the genes that encode chymotrypsins show a greater positive regulation then those encoding the trypsins. Molecular modelling studies of the chymotrypsins were carried out, and 3D models were generated using homology modelling, which were then further refined by dynamic molecular and docking analyses with 8 different Bowman-Birk type PIs. The results demonstrate which chymotrypsins possess the highest affinities to the tested inhibitors in a general and individual manner, inferred from the estimated free energies. A serine residue in very close proximity to the catalytic site was present in three of chymotrypsins investigated, which may be affecting the enzymes affinity since the residue has a reduced accessible area to the solvent when complexed to the soya PI tested. The genetic expression patterns and the degree of PI-sensitivity were also compared and no relation between the parameters was found. This suggests that the larvae of the species S. frugiperda combine different adaptive strategies like the increase in expression of its entire chymotrypsin arsenal regardless of the degree of PI-sensitivity of the enzymes.
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Leisegang, Rena. "Proteinase A und Schaumstabilität biochemische Untersuchungen der Wechselwirkungen von Proteinase A aus Saccharomyces cerevisiae und schaumaktivem Lipidtransferprotein /." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=969247214.
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Schnepel, Jörn. "Klonierung, Expression und Charakterisierung kryptischer Fibronektin-Proteinasen." [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=969330472.
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Turkington, Philip Thomas. "The role of polymorphonuclear proteinases in haemostasis." Thesis, Queen's University Belfast, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.317075.
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Rowan, Andrew D. "The pineapple proteinases : characterization and clinical use." Thesis, Open University, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.290495.
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Cruz, Carlos Eduardo Silva da. "Caracterização de proteinases envolvidas na geração de peptídeos antimicrobianos no intestino de Rhipicephalus (Boophilus) microplus." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-25032010-161909/.
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Sabe-se que a hemoglobina é uma rica fonte de peptídeos antimicrobianos (hemocidinas). A primeira hemocidina derivada da hemoglobina bovina caracterizada em carrapatos foi o peptídeo Hb33-61, que é ativo contra bactérias gram-positivas e fungos. Acredita-se que tais hemocidinas sejam geradas proteoliticamente no intestino do carrapato. Neste trabalho nós caracterizamos bioquimicamente uma catepsina D, designada BmAP. A análise da expressão gênica por qPCR mostrou que ela é expressa predominantemente no intestino. Através de LC-MS/MS, determinamos a especificidade de clivagem da BmAP utilizando Hb bovina, e verificamos que resíduos hidrofóbicos foram preferencialmente clivados nos subsítios P1 e P1. Também investigamos a especificidade de clivagem da catepsina L intestinal BmCL1, utilizando uma biblioteca combinatória de tetrapeptídeos e através de hemoglobinólise in vitro. A BmCL1 preferiu resíduos alifáticos no P2 e polares no P1 e P1. Além disso, hidrolisou a cadeia da Hb bovina entre A63/A64, gerando peptídeos com estrutura primária similar ao Hb 33-61. A hemoglobinólise com a BmAP e/ou BmCL1 resultou na formação de algumas hemocidinas, corroborando a hipótese do seu envolvimento na geração endógena de peptídeos antimicrobianos.It is known that hemoglobin is a rich source of antimicrobial peptides (hemocidins). The first hemoglobin-derived hemocidin characterized in ticks was the peptide Hb33-61, which is active against Gram-positive bacteria and fungi. It is believed that hemocidins are endogenously generated in the tick gut. In this work we biochemically characterized a cathepsin D, designated BmAP. Expression analysis by qRT-PCR showed that it is expressed predominantly in the gut. Through LC-MS/MS, we determined the cleavage specificity of BmAP using bovine hemoglobin, and we verified that hydrophobic residues were preferentially cleaved at the subsites P1 and P1. We also investigated the cleavage specificity of the intestinal cathepsin L BmCL1, using a positional scanning synthetic combinatorial library and through in vitro hemoglobinolysis. BmCL1 preferred aliphatic residues at P2 and polar residues at P1 and P1. Also, it hydrolysed the subunit of bovine hemoglobin at A63/A64, generating peptides with a primary structure similar to Hb 33-61. Hemoglobinolysis with BmAP and/or BmCL1 resulted in the formation of some hemocidins, corroborating the hypothesis that these proteinases are involved in the endogenous generation of antimicrobial peptides
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Nattrass, Gregory Scott. "Molecular and functional characterisation of a system ASC-like neutral amino acid transporter expressed in the wool follicle /." Title page, contents and abstract only, 2000. http://web4.library.adelaide.edu.au/theses/09ANP/09anpn284.pdf.
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Li, Yang 1974. "Characterization of two type II transmembrane serine proteases, hepsin and corin." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=79034.
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Type II transmembrane serine proteases (TTSPs), including hepsin and corin, are a new class of cell surface catalytic enzymes. In the present study, a non-transmembrane isoform of hepsin, named hepsin/-TM that originates from alternative splicing, was identified. Unlike the transmembrane hepsin isoform, this non-transmembrane isoform was distributed within the cytoplasm and likely to be modified after translation. Real-time PCR experiments revealed that hepsin was expressed in all tested human tissues, but hepsin/-TM only in kidney, brain and lung tissues. Significantly, hepsin/-TM was not expressed in liver where hepsin was originally identified. However, hepsin/-TM was highly expressed in brain where hepsin was expressed at a significantly lower level. Moreover, these two isoforms showed different expression patterns in several colon cancer cell lines. Furthermore, ten corin-interacting proteins were identified and a variety of corin mutants were generated for the studies of the relationship of corin structure and function.
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McKeown, Brendan Gerard. "A study of the 2A and 3C mediated cleavage events in the processing of the foot-and-mouth disease virus polyprotein." Thesis, Queen's University Belfast, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318800.
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Sit, Mae-Le. "The role of serine proteases in angiogenesis /." St. Lucia, Qld, 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16412.pdf.
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Du, Bingfan Groutas William C. "Novel inhibitors of human leukocyte proteinase 3." Diss., Click here for available full-text of this thesis, 2006. http://library.wichita.edu/digitallibrary/etd/2006/t013.pdf.
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Thesis (M.S.)--Wichita State University, Dept. of Chemistry."May 2006." Title from PDF title page (viewed on October 19, 2006). Thesis adviser: William C. Groutas. Includes bibliographic references (leaves 56-61).
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Roy, Samir. "Vascular effects of proteinase receptor activating peptides." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape17/PQDD_0011/MQ34991.pdf.
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Bernstein, Nina Khazanovich. "Structural studies of malarial aspartic proteinase activation." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0014/NQ59937.pdf.
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Tehrani, Kamin A. "Synthesis and kinetics of cysteine proteinase inhibitors." Thesis, Georgia Institute of Technology, 1991. http://hdl.handle.net/1853/26967.
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Souto, Maior Ana M. "Proteinase production from an alkalophilic Bacillus species." Thesis, University of Manchester, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315973.
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Chetland, J. "The design and synthesis of proteinase inhibitors." Thesis, University of Liverpool, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.372687.
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Winterburn, Timothy John. "Engineering and design of aspartic proteinase inhibitors." Thesis, Cardiff University, 2005. http://orca.cf.ac.uk/55593/.
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Aspartic proteinases are of considerable interest to the pharmaceutical, agrochemical and food industries. Many small molecule inhibitors have been synthesised chemically to block the action of individual members of this proteinase family. In contrast, only a handful of naturally-occurring, gene-encoded inhibitors of aspartic proteinases are known. One such inhibitor is IA3, an intrinsically unstructured 68-residue cytosolic protein from Saccharomyces cerevisiae, which operates through an unprecedented mode of action. On contacting the target enzyme, residues 2-32 form an amphipathic a-helix that occludes the active site cleft. The potency and specificity of this inhibitor were investigated by producing >80 variants in the form of recombinant proteins in Escherichia coli or as synthetic peptides preliminary attempts were also made to examine the potential of directed evolution by phage display for the generation of further mutants. The inhibitory activity of these variant polypeptides at different pH values was measured against the natural target proteinase from S. cerevisiae and against vacuolar aspartic proteinases from Pichia pastoris and Aspergillus fumigatus, as well as the human lysosomal enzyme, cathepsin D. New IA3-like sequences were also identified in five closely-related yeasts. One, from Saccharomyces castellii, differed substantially from S. cerevisiae IA3 and so was investigated in parallel. Key elements of the inhibitory sequence (i.e. residues 2-34) in the S. castellii IA3 were identified in relation to their counterparts in S. cerevisiae IA3 and the different effects of their respective C-terminal regions (i.e. residues 34 to C-terminus) were unravelled through the production of chimaeric polypeptides. In this way, potent IA3-derived inhibitors of the P. pastoris and A. fumigatus proteinases were produced, and an effective inhibitor of cathepsin D was identified. IA3 is shown to be remarkably adaptable as an inhibitor, and its potency may be re-directed to inhibit more distant enzymes of commercial interest.
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Chin, Ai-Yen. "Proteinase-activated receptor 4 and cell apoptosis." Thesis, University of Hull, 2013. http://hydra.hull.ac.uk/resources/hull:8124.
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In response to injury, the collagen-producing cells fibroblasts and myofibroblasts play an important role in promoting extracellular matrix deposition and release of inflammatory mediators in order to repair the damaged tissue. However, persistence and over-activity of these cells is associated with excessive collagen deposition and hence development of brosis, a pathological scarring process that leads to destruction of organ architecture and impairment of organ function. Removal of fibroblasts and myofibroblasts during fibrogenesis by apoptosis is therefore required for the normal resolution of tissue repair responses. Proteinase-activated protein 4 (PAR4) belongs to a subfamily of multifunctional G protein-coupled receptors which are activated by the proteolytic unmasking of a tethered peptide ligand that resides within their N-terminus. In addition to its well-studied role in platelet function, PAR4, like the other PAR family members, is thought to be involved in pro-inflammatory responses and fibroproliferative processes. However, the role of PAR4 in these processes remains largely unknown. The hypothesis of this thesis is that during injury, PAR4 expression is upregulated and this somehow contributes to apoptosis. To test this, the expression of PAR4 was examined in two types of fibroblasts, cultured from human lung and renal explants. Results indicate that PAR4 is not expressed in these cultured fibroblasts. Surprisingly, contradictory to a previous observation, exposure of lung fibroblasts to lipopolysaccharide did not consistently induce PAR4 expression. As an alternative to the use of PAR4-expressing fibroblasts, a stable PAR4-expressing human cell line (HEK-PAR4) was generated. Using these cells in an in vitro signalling assay, PAR4 was found to confer responsiveness to PAR4 activating peptide but not to a serine protease cathepsin G, which was previously shown to activate PAR4 in platelets. Interestingly, the observation that cathepsin G-treated cells elicit a reduced signalling response to the stimulatory action of PAR4 activating peptide suggests that cathepsin G might cleave PAR4 at the ECL2 domain and cause PAR4 to be unresponsive to the PAR4 AP. Next, serine proteases cathepsin G and trypsin were demonstrated as apoptosis inducers for human lung and renal fibroblasts. They induced pronounced morphologic changes in the fibroblasts (which do not express PAR4). In HEK-PAR4 cells, cathepsin G induced similar apoptotic level in comparison to empty vector control. Together, these findings suggest that cathepsin G can trigger apoptosis independently of PAR4. On the other hand, PAR4 activating peptide triggered higher percentage of apoptosis in HEK-PAR4 cells compared to empty vector control, suggesting that apoptosis is mediated by PAR4 signalling in this case. Epithelium has a role in modulating a variety of inflammatory processes through the production and release of inflammatory cytokines. Apoptosis of damaged epithelium during inflammation has been reported to promote fibroblast growth and collagen deposition. In the last part of this thesis, media derived from confluent epithelial cell cultures were demonstrated to induce proliferation of lung and renal fibroblasts in a concentration dependent manner. This observation preliminarily supports the idea that epithelial cell injury may promote the proliferation of fibroblasts via the secretion of multiple cytokines. In conclusion, this thesis demonstrates that apoptosis can be induced, first, by cathepsin G and trypsin in human primary bronchial fibroblasts and human primary renal fibroblasts independently of PAR4, and second, by activation of PAR4 in a non-fibroblastic PAR4-expressing cell line.
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Ally, Nafisa. "The specificity of proteinase-adhesins from Porphyromonas gingivalis." Monash University, Dept. of Biochemistry and Molecular Biology, 2003. http://arrow.monash.edu.au/hdl/1959.1/9477.
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Odei-Addo, Frank. "Purification and characterization of serine proteinase inhibitors from two South African indigenous plants, Acacia karoo and Acacia schweinfurthii." Thesis, Nelson Mandela Metropolitan University, 2009. http://hdl.handle.net/10948/1291.
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Serine proteases are known to perform a wide range of functions essential to life; however there has to be some form of control mechanism in place. One of the many control mechanisms is their specific inhibition by protein proteinase inhibitors. Proteinase inhibitors in plants, present in their seeds, participate in defense mechanisms and their production is induced by herbivory or wounding. Plant proteinase inhibitors have been reported to inhibit a variety of serine proteinases, including enzymes of the blood coagulation cascade. In this study, various indigenous seed extracts were screened for potential serine proteinase inhibition. Acacia schweinfurthii was selected as a potential inhibitor that inhibited trypsin and factor X. The AS inhibitor was successfully purified to homogeneity by precipitating with 80 percent (v/v) acetone and the sequential chromatographic steps including ion-exchange chromatography, size exclusion chromatography, affinity purification on a trypsin-agarose column and RP-HPLC. Reducing SDS-PAGE conditions revealed an inhibitor of two polypeptide chains A and B of approximate molecular weights 16 and 10 kDa, respectively, and under non-reducing conditions, 25 kDa was observed. The inhibitor was shown to inhibit trypsin, chymotrypsin and factor X indicating the dynamic nature of the reactive site. An enzyme: inhibitor ratio of 1:1, and a Ki of 3.45nM was determined for the AS inhibitor on trypsin, and the inhibitor also weakly inhibit chymotrypsin. AS inhibitor and STI inhibited factor X with a Ki values of 13.7nM and 77.5μM respectively. Amino acid analysis revealed Mmin values of the A- and B- chain of 15,000 and 7,800, respectively. The effect of seed extracts on the activated partial thrombin time (APTT) and prothrombin time (PT) was tested. No prolongation of the PT was obtained. For the crude extracts of AK and AS, IC200 values of 4.6 and 189.62 μg/mL, were respectively obtained. For the purified fractions of STI, AS and AK, IC200 values of 51.5, 114.31 and 893.8 μg/ml were observed, respectively. Keywords: proteinase inhibitors, Acacia species, trypsin inhibitor, FX inhibitor.