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1
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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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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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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4
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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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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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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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.
8
Lindmark, Anders. "On the biosynthesis and processing of cathepsin G, leukocyte elactase, and azurocidin neutrophil granule members of a hematopoietic serine protease superfamily /". Lund : Dept. of Hematology, Lund University, 1997. http://catalog.hathitrust.org/api/volumes/oclc/38985787.html.
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Wang, Yudong y 汪玉東. "Neutrophil serine proteases as novel biomarkers for autoimmune diabetes". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/208026.
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Background and Objectives:
Type 1 diabetes (T1D) is an autoimmune disease that results from the immune-mediated destruction of insulin-producing β cells in the islets of Langerhans within the pancreas. A combination of genetic and environmental triggers has been acknowledged to contribute to the development of T1D. However, the detailed mechanisms underlying the initiation and progression of autoimmune diabetes still remain poorly understood. Recent studies have found that the reduction of circulating neutrophils is accompanied by neutrophil infiltration in the pancreas at the onset of T1D, suggesting that neutrophils may be causally involved in the pathogenesis of this disorder. However, further investigations are needed to clarify the precise roles of neutrophils and their cellular components in autoimmune destruction of pancreatic β cells.
The objective of this study was to investigate whether neutrophil elastase (NE) and proteinase 3 (PR3), both neutrophil serine proteases stored in neutrophil primary granules, and NETosis, a unique form of cell death of neutrophils characterized by the release of decondensed chromatin and granular contents to the extracellular space, were involved in the pathogenesis of T1D.
Key findings:
1) We developed several in-house immunoassays for the measurement of circulating levels of NE, PR3 and their endogenous inhibitor alpha-1 antitrypsin (A1AT), and validated the specificity, precision and sensitivity of these assays in clinical samples;
2) We provided the first clinical evidence demonstrating that both circulating protein levels and enzymatic activities of NE and PR3 were dramatically increased in patients with T1D, especially in those with disease duration less than one year. On the contrary, circulating concentrations of A1AT were significantly decreased in these patients;
3) By measuring circulating levels of myeloperoxidase (MPO)-DNA complexes, we demonstrated that NETosis was evidently increased in T1D patients, and positively correlated with the circulating protein levels as well as enzymatic activities of NE and PR3, suggesting that increased circulating NE and PR3 at least in part attributed to augmented NETosis;
4) Circulating NE and PR3 levels increased progressively with the increase in the positive numbers and titers of autoantibodies against pancreatic β cell antigens, but no significant correlation of NE or PR3 with fasting blood glucose levels was observed, suggesting that elevated NE and PR3 might be causally associated with β-cell autoimmunity, but not glycaemic status, in T1D patients. Furthermore, an obvious elevation of NE and PR3 was detected even in those autoantibody-negative patients, suggesting that circulating NE and PR3 may serve as a novel class of biomarkers for the early diagnosis of T1D.
Conclusions:
Our present study demonstrated that the drastic elevation of NE and PR3, accompanied by a decrease in the endogenous inhibitor A1AT and the enhancement of NETosis, are closely associated with the β-cell autoimmunity in patients with T1D. Measurement of circulating protein levels of neutrophil serine proteases and/or their enzymatic activities can be used to assist the differential diagnosis of autoimmune diabetes.published_or_final_version
Medicine
Doctoral
Doctor of Philosophy
10
Huang, Kui. "Structural studies of the interactions between serine proteinases and protein inhibitors". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1995. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq22151.pdf.
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VIALA, VINCENT L. "Análise combinada do transcriptoma de glândula de veneno e do proteoma do veneno da espécie pseudonaja textilis (Elapidae: Serpentes)". reponame:Repositório Institucional do IPEN, 2014. http://repositorio.ipen.br:8080/xmlui/handle/123456789/10630.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
FAPESP:09/10305-8
12
Sin, Suk-fong. "Characterization of proteinase inhibitor II from Solanum Americanum". Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/HKUTO/record/B38628776.
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Fan, Yun-Hua. "Synthesis and studies of prospective hosts for monsaccharides orbital interactions that control an aqueous organic equilibrium and an electrodynamic aspect of serine protease action". Diss., Georgia Institute of Technology, 1998. http://hdl.handle.net/1853/27451.
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Wharry, Thomas Scott. "The synthesis of novel phosphonate and phosphinate inhibitors of proteinase enzymes". Thesis, Queen's University Belfast, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263577.
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Dacre, Kirstie Jane. "Involvement of mast cells and mast cell serine proteinases in equine heaves". Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/29721.
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Mast cells release potent mediators upon degranulation, including serine proteinases. These proteinases play a pivotal role in the pathogenesis of human asthma. Due to the similarities between human asthma and equine heaves, a similar role for the mast cell in equine heaves is proposed. Clinical heaves horses had significantly increased BALF tryptase concentrations compared to controls or heaves horses in remission, whereas BALF tryptase concentrations of controls and heaves horses in remission did not significantly differ. Horses with other pulmonary diseases also had significantly elevated BALF tryptase concentrations compared to controls. Cloning and sequencing of these proteinases revealed an alanine 216 substitution in equine tryptase, which confers increased arginine substrate specificity and may restrict fibrinogenolysis in vivo. Probing of tryptase mRNA transcript regulation in control and heaves susceptible horses revealed no significant change in airway liminal cell pellet tryptase expression following hay/straw challenge of control or heaves horses. However, bronchiolar tissues from heaves horses in early resolution phase had significantly down-regulated tryptase transcripts compared to controls. Furthermore, immunohistochemistry revealed significant intra-epithelial recruitment of tryptase positive mast cells in heaves horses compared to controls, suggesting involvement of tissue mast cells in response to challenge. In vitro hay dust suspension (HDS) challenge induced significant airway luminal mast cell degranulation in heaves susceptible horses, however a similar dose response trend was also evident in control horses. The increased number of intra-epithelial mast cells in heaves horses may explain the divergent mast cell response to in vivo and in vitro challenges. HDS-induced mast cell degranulation in both control and heaves horses may suggest non-IgE mediated degranulation. Alternatively, both control and heaves horses may have been sensitised to HDS allergens and phenotypic diversity may ultimately determine response to challenge.
16
Quarrell, Rachel E. L. "The inhibition of serine proteinases, and the development of a combinatorial library approach". Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259894.
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Sin, Suk-fong y 冼淑芳. "Characterization of proteinase inhibitor II from Solanum Americanum". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B38628776.
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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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SILVA, JOSE A. A. da. "Medicao dos receptores ativados por proteases (PARs) em atividades biologicas da giroxina". reponame:Repositório Institucional do IPEN, 2009. http://repositorio.ipen.br:8080/xmlui/handle/123456789/9493.
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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
20
Tam, Ka-ho Chris. "Targeting mTOR as a novel therapeutic strategy for hepatocellular carcinoma". Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B41508476.
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Tierney, Marcus John 1973. "Post-transcriptional regulation of plasminogen activator inhibitor type 2". Monash University, Dept. of Medicine, 2002. http://arrow.monash.edu.au/hdl/1959.1/8496.
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Tsiftsoglou, Stefanos Alex. "Structural and functional studies on human complement factor I". Thesis, University of Oxford, 2005. http://ora.ox.ac.uk/objects/uuid:745ea729-07ae-4c15-be83-bb3bb0db99ff.
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The complement system is considered as the chief recognition and effector component of innate immunity; it is involved in inflammation and enhances the adaptive immune response. Factor I (fI) is a heterodimeric serine protease consisting of a heavy (HC) and a light-catalytic (LC) chain; it circulates in an active form regulating complement by selectively cleaving only C3b or C4b in the presence of a cofactor such as factor H (fH), CR1, MCP or C4bp. The cleavage of C3b occurs through a ternary complex formed between fI, C3b and a cofactor like fH and yields iC3b, a major opsonin. The structural and functional properties of fI were investigated. The interchain disulphide bond formed between C309-C435 tnat links the HC and LC of fI as well as the composition of the TV-linked carbohydrates of fI were determined. By using two independent assays, the proteolytic and amidolytic assays, the catalytic properties of human fI were characterised in detail. The catalytic subunit, the SP domain, was shown to have a native conformation that accommodates substrate recognition and cleavage, fI has specificity similar to thrombin, but exhibits lower catalytic activity. fI amidolytic activity reaches optimum at pH 8.25 and is insensitive to ionic strength. This is in contrast to its proteolytic activity within the fI-C3b-fH reaction, in which the pH optimum for C3b cleavage is <5.5 and the reaction rate is highly dependent on ionic strength. The rate of cleavage of tripeptide AMC substrates by fI was unaffected by fH or C3(NH3) at optimum pH. fI and the isolated SP domain were found to have similar amidolytic activities, but strikingly different proteolytic activities on C3(NH 3 ). fl did not cleave C3(NH3) in the absence of fH, but cleaved it rapidly at two sites in its presence. The SP domain however, cleaved C3(NH3) slowly in the absence of fH, at more than two sites. Cleavage by the SP domain was inhibited, not stimulated, by fH. These results suggested that the HC domains and/or the cofactor must orient the natural substrates and restrict cleavage by fI to the two sites which yield iC3b. The implications of these findings are discussed.
23
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.
24
Marcello, Matthew R. "Analysis of recombinant human prostasin carrying a serine active site mutation". Honors in the Major Thesis, University of Central Florida, 2003. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/325.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
Health and Public Affairs
Molecular Biology and Microbiology
25
Brown, Audra Denise. "α-aminoalkylphosphonate di(chlorophenyl) esters as inhibitors of serine proteases : Part II: A kinetic study of the coupling of the hydrolysis product of the N-tosylalanine ester of 5-phenyl-3-hydroxypyrrole to various diazonium salts : Part III: Rates of thrombin acylation and deacylaton upon reaction with low molecular weight acylating agents, carbamylating agents and carbonylati". Thesis, Georgia Institute of Technology, 1994. http://hdl.handle.net/1853/27552.
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Tam, Ka-ho Chris y 譚家豪. "Targeting mTOR as a novel therapeutic strategy for hepatocellular carcinoma". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B41508476.
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Zeng, Yibo y 曾毅博. "Biophysical characterization hpn-like (HPNL), a histidine- and glutamine-rich protein". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hdl.handle.net/10722/210333.
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Ma, Yan. "Role of the Ca2+ / calmodulin-dependent protein kinase II pathway in the cardioprotective effect of estrogen". Click to view the E-thesis via HKUTO, 2008. http://sunzi.lib.hku.hk/hkuto/record/B41290744.
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Zeng, Yibo. "Biophysical characterization hpn-like (HPNL), a histidine- and glutamine-rich protein". Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B42841185.
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Pekkarinen, Anja. "The serine proteinases of Fusarium grown on cereal proteins and in barley grain and their inhibition by barley proteins /". Espoo : VTT, 2003. http://www.vtt.fi/inf/pdf/publications/2003/P487.pdf.
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Karlson, Ulrika. "Cutting Edge – Cleavage Specificity and Biochemical Characterization of Mast Cell Serine Proteases". Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3529.
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Boucaut, Kerry Jane. "Function and regulation of the human serine protease Testisin". Thesis, Queensland University of Technology, 2002.
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33
Fonseca, Fabiana Vieira. "Isolamento e caracterização de um novo conjunto de serinoproteases com atividade trombina-like e de L-aminoacido oxidase do veneno de Crotalus durissus cascavella". [s.n.], 2006. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314482.
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Orientador: Marcos Hikari ToyamaDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O uso de toxinas isoladas de venenos como ferramentas moleculares na compreensão de diversos eventos fisiológicos e patológicos tem sido comprovadas por vários trabalhos na literatura. A serpente Crotalus durissus cascavella é encontrada nas áreas de caatinga do nordeste do Brasil, desde o Maranhão até norte do estado de Minas Gerais, e apesar de pouco estudada sua picada constitui um importante problema da saúde pública (Martins et al, 1998). A agregação platequetária é bem caracterizada para a convulxina isolada do veneno de Crotalus durissus cascavella e Crotalus durissus terrificus. O objetivo principal do projeto foi avaliar a atividade de agregação plaquetária induzida por outras frações biológicas e farmacologicamente importantes do veneno total de Cotalus durissus cascavella, que neste projeto foram a crotoxina e giroxina. Através de uma combinação de varias metodologias em HPLC como exclusão molecular, troca iônica e de fase reversa conseguimos isolar os principais constituintes da crotoxina (PLA2, crotapotina e as proteínas ¿trombina-like¿) e a L-aminoácido oxidase a partir da giroxina. Durante o fracionamento do veneno total em coluna de HPLC de exclusão molecular foram detectados dois picos de atividade serino protease, um na fração giroxínica e outro na fração crotoxínica, sendo que na fração crotoxínica encontrou-se uma nova protease até então não caracterizada e na fração giroxínica foram monitoradas a atividade L-aminácido oxidase. Através da cromatografia em HPLC de troca iônica em DEAE 5PW obteve-se a fração Laminoácido oxidase cujo grau de homogeneidade molecular foi confirmado por HPLC de fase reversa. Da fração crotoxínica foram obtidos três grupos principais de proteínas (PLA2, crotapotinas e proteases) e da fração proteolítica foram isoladas três isoformas principais denominadas de F201, F202 e F203, sendo a fração F202, a fração majoritária. F202 foi obtida com maior homegeneidade molecular com massa molecular de 28kDa e mostrou uma alta quantidade de ácido aspártico, ácido glutâmico e outros aminoácidos importantes como histidina, cisteína e lisina. Esta proteína mostrou alta especificidade para BApNA e mostrou um comportamento Michaelis-Menten com Vmáx estimado em 5,64 µM/min e um Km de 0,58mM para este substrato. Neste trabalho foi investigada a habilidade desta proteína em degradar fibrinogênio e observou-se que F202 clivou ambas as cadeias a e ß. A atividade enzimática assim como a agregação plaquetária foi inibida fortemente com a incubação com TLCK, um inibidor específico para serinoproteases. O N-terminal da seqüência de aminoácidos de F202 mostrou alta homologia com outras proteínas ¿trombina-like¿, mas foi significantemente diferente da ¿trombina-like¿ isolada da fração giroxina. Crotalus durissus cascavella apresenta uma fração menos estudada denominada giroxina que tem sido descrita como uma proteína ¿trombina-like¿ como relatado por Raw et al. (1986) e Alexander et al. (1988). Neste trabalho foi demonstrado que a giroxina é uma fração heterogênea composta de uma ¿trombina-like¿ e proteína LAO, a qual parece estar envolvida em várias atividades
Abstract: The use of the isolated toxins from poisons as molecular tools in the understanding of diverse physiological and pathological events has been proved by some works in literature. The serpent Crotalus durissus cascavella is found in the areas of Caatinga Northeast of Brazil, since Maranhão until North of the state of Minas Gerais, and in spite of it hasn¿t been studied a lot, its bite constitutes an important problem to the public health (Martins et al, 1998). The platelet aggregation is well characterized to the isolated convulxin of the poison of Crotalus durissus cascavella and Crotalus durissus terrificus. The main objective of the project was to evaluate the activity of platelet aggregation induced by gyroxin and crotoxin that are biological and pharmacological important fractions of the total poison of Cotalus durissus cascavella. Through a combination of various methodologies in HPLC -as molecular exclusion, ionic exchange and the reverse phase- we managed to isolate the main constituent of the crotoxin (PLA2, crotapotin and the thrombin-like proteins) and the L-amino acid oxidase from the gyroxin. During the fragmentation of the total poison in column of HPLC of molecular exclusion two peaks of serine protease were found: one in the gyroxin fraction and another one in the crotoxin fraction. In the crotoxin fraction a new protease in not characterized yet and in the gyroxin fraction was found the L-amino acid activity oxidase. Through the chromatography in HPLC of ionic exchange in DEAE 5PW that allowed to the attainment of the fraction L-amino acid oxidase whose degree of molecular homogeneity was confirmed by HPLC of reverse phase. From the crotoxin fraction three main groups of proteins (PLA2, crotapotin and proteases) were isolated, and from the named proteolyitic fraction three isoforms of F201, F202 and F203, noticing that the F202 fraction is the major one. The fraction F202 showed a high quantity of aspartic acid, glutamic acid and others amino acids very important as histidine, cysteine and lysine and so more molecular homogeneity could be obtained and with molecular mass of 28kDa. This protein whose behavior Michaelis-Menten with Vmáx measured in 5,64 µM/min and one Km de 0,58 mM to this substratum showed high specificity to BapNA. In this work was investigated the ability of this protein in degrading the fibrinogen and was observed that the F202 made the cleavage into both chains a and ß. The enzymatic activity as well as the platelet aggregation were strongly inhibited with the incubation with TLCK, a specific inhibitor to serine protease. The N-terminal of the amino acid sequence of F202 showed the high homology with other proteins thrombin-like, but it was significantly different from thrombin-like isolated fro m the gyroxin fraction. Crotalus durissus cascavella presents a fraction less studied named gyroxin that has been described as a protein thrombin-like as related by Raw et al. (1986) and Alexander et al. (1988). In this work was demonstrated that the gyroxin is a composed heterogeneous fraction of one thrombin-like and protein LAO, and this seems to be involved in some activities
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular
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ROUCHON, PASCALE. "Inhibiteurs de masse moleculaire elevee des cysteine et des serine proteinases du muscle squelettique de bovin : purification et caracterisation physicochimique et cinetique". Clermont-Ferrand 2, 1995. http://www.theses.fr/1995CLF21728.
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Six inhibiteurs de proteinases ont ete isoles a partir du muscle squelettique de bovin et leurs caracteristiques physico-chimiques ont ete etudies. Trois d'entre eux, nommes pi1, pi2 et pi3 (pour papaine inhibiteur 1, 2, 3) sont specifiques des cysteine proteinases lysosomales. Ils ont des masses moleculaires respectives de 43, 69 et 61 kda. Pi1 et pi2 sont extremement stables au ph et a la temperature alors que l'activite de pi3 diminue significativement aux ph acides (inferieur a 6) et aux temperatures superieures a 50c. Les trois inhibiteurs ont peu d'effet sur la cathepsine b. Par contre ils ont une tres grande affinite pour la papaine et la cathepsine l et a un degre moindre la cathepsine h. Les constantes d'association de l'ordre de 10#+#6 m#-#1. S#-#1 suggerent une interaction relativement lente de ces inhibiteurs proteiques envers les cysteine proteinases testees, conclusion supportee par le fait que la reaction de formation du complexe necessite des preincubations de 10 a 50 minutes avant d'atteindre l'equilibre. A partir de l'etude des differents parametres cinetiques mesures, sur les trois inhibiteurs purifies nous ne pouvons envisager un role in vivo vis a vis des cathepsines l et h que pour pi1 et pi2. Concernant l'identite de ces inhibiteurs, il semblerait qu'ils ne correspondent a aucun inhibiteurs connus. Les trois autres inhibiteurs purifies nommes ti1, ti2 et ti3 (pour trypsine inhibiteur 1, 2, 3) sont specifiques des serine proteinases. Ti2 et ti3 sont stables dans une large gamme de ph (4-12) mais leur activite diminue aux ph tres acides. Leur activite reste inchangee apres chauffage jusqu'a 60c mais decroit ensuite rapidement. Au contraire ti1 apparait tres sensible au ph et particulierement instable a la chaleur. Chaque inhibiteur inhibe fortement la trypsine et la chymotrypsine. Ti2 et ti3 presentent aussi une forte affinite pour l'elastase et a un degre moindre pour la cathepsine g). Au contraire, ti1 ne possede aucune activite inhibitrice vis a vis de ces 2 dernieres enzymes. L'interaction est relativement lente avec la trypsine et la chymotrypsine et plus rapide avec l'elastase et la cathepsine g. Le proteasome 20 s qui possede notamment une activite de type serine proteinase ne semble inhibe par aucun des trois inhibiteurs. La (ou les) proteinase(s) cible(s) de ces inhibiteurs dans la cellule musculaire reste donc inconnue. Les trois inhibiteurs purifies montrent tous quelques similitudes (masse moleculaire et specificite) avec d'autres inhibiteurs isoles de divers tissus et fluides. Cependant aucune conclusion ne peut etre tiree sur leur identite avec ces inhibiteurs
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LE, BARILLEC KARINE. "Effets de l'elastase et de la cathepsine g, deux serine proteinases du polynucleaire neutrophile, sur le cd14 monocytaire, recepteur aux lipopolysaccharides bacteriens". Paris 6, 1999. http://www.theses.fr/1999PA066283.
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Les defenses constitutives ou innees de l'organisme pour combattre l'intrusion d'un pathogene reposent sur le recrutement tissulaire de phagocytes sanguins vers le foyer inflammatoire. Les phagocytes, qu'ils soient mononuclees ou polynucleaires, sont capables d'ingerer et de detruire les micro-organismes dans des vacuoles de phagocytose. C'est en participant au processus de phagocytose que les polynuleaires neutrophiles peuvent liberer dans le milieu extracellulaire le contenu de leurs granules et en particulier deux serine proteinases, l'elastase et la cathepsine g. Ces deux enzymes possedent de tres nombreux substrats polypeptidiques, matriciels, plasmatiques ou exprimes sur des types varies de cellules. Peu d'interactions ont ete decrites entre l'hle, la cg, et des molecules exprimees par les monocytes. Puisqu'une etude rapportait que le cd14, recepteur aux lipopolisaccharides bacteriens, exprime a la surface des monocytes etait sensible a l'action de serine proteinases, nous avons emis l'hypothese que l'hle et la cg pourraient etre candidates a cette elimination du cd14. La premiere etude se focalise sur les effets de l'elastase alors que la seconde etude traite du role de la cathepsine g sur ce recepteur. Ces deux serine proteinases purifiees ont la propriete de cliver le cd14 monocytaire. Meme si la consequence ultime est identique, ces deux enzymes ont des modes d'action assez differents sur le cd14. L'une et l'autre agissent via leur activite enzymatique, mais leurs sites de proteolyse ne sont pas similaires. La cg cliverait en un site (ou peut etre plusieurs tres proches) dans la partie carboxyterminale de la molecule alors que l'hle cliverait le cd14 en de nombreux peptides. Lors de conditions plus physiologiques pour lesquelles monocytes et polynucleaires neutrophiles sont co-incubes et actives, dans le but que les polynucleaires neutrophiles liberent le contenu de leurs granules azurophiles, une baisse de l'expression du cd14 monocytaire est aussi observee. La consequence physiologique est un blocage de l'activation cellulaire induite par le lps telle que l'atteste la diminution de synthese de tnf-, une cytokine pro-inflammatoire. Cette observation illustrerait un possible role anti-inflammatoire de ces enzymes.
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Wong, Chak-lui Carmen. "Regulations and functions of rho-kinases in hepatocellular carcinoma". Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B42182001.
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YONAMINE, CAMILA M. "Clonagem de serino proteases do veneno da cascavel Crotalus durissus terrificus e expressão da giroxina em célula de mamífero". reponame:Repositório Institucional do IPEN, 2007. http://repositorio.ipen.br:8080/xmlui/handle/123456789/11593.
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Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP
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Ma, Yan y 馬妍. "Role of the Ca2+ / calmodulin-dependent protein kinase II pathway in the cardioprotective effect of estrogen". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2008. http://hub.hku.hk/bib/B41290744.
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Wong, Chak-lui Carmen y 黃澤蕾. "Regulations and functions of rho-kinases in hepatocellular carcinoma". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B42182001.
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The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,2008-2009published_or_final_version
Pathology
Doctoral
Doctor of Philosophy
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Zu, Yi. "SIRT1 promotes cell proliferation and prevents cellular senescence through targeting LKB1 in primary porcine aortic endothelial cells". Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B43085805.
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Yonamine, Camila Miyagui. "Clonagem de serino proteases do veneno da cascavel Crotalus durissus terrificus e expressão da giroxina em célula de mamífero". Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/85/85131/tde-16052012-081641/.
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As serino proteases participam de diversos processos fisiológicos (tal como o de coagulação) e patológicos. Essas enzimas estão amplamente distribuídas entre as espécies, são também toxinas dos venenos de serpentes, sendo denominadas SVSPs (snake venom serine proteases). Essas SVSPs são multifuncionais e contêm uma tríade catalítica formada pelos aminoácidos HDS. Algumas SVSPs são comercialmente disponíveis, sendo indicadas para o tratamento de infarto do miocárdio, tromboses e embolia pulmonar. No veneno de Crotalus durissus terrificus estão descritas até o momento, apenas duas SVSPs sendo que a mais estudada é a giroxina que representa cerca de 2,5% do veneno total. No presente estudo foi reportado a clonagem de sete serino proteases amplificadas a partir de uma biblioteca de cDNA de glândula de veneno de um único espécime adulto de Crotalus durissus terrificus. Estes clones foram analisados com relação à organização do cDNA, estrutura e prováveis funções. A construção do modelo tridimensional da giroxina permitiu verificar as similaridades com tripsina, trombina e outras SVSPs. A glicosilação e a presença de muitas pontes dissulfetos dificultam a obtenção das SVSP recombinantes na forma solúvel e com atividade, por expressão em E.coli. Assim, neste trabalho foi abordada a expressão em células de mamífero (que realiza as modificações pós-traducionais) com resultados promissores. Para tanto, o peptídeo sinal de Igk, a seqüência madura e a região 3 UTR da giroxina foram clonados no vetor pED, originando um novo vetor (pED-Giro). Este vetor carrega o peptídeo sinal de Igk, o que possibilitou a secreção da giroxina para o meio de cultura. O vetor pED-Giro foi transfectado em células CHO DXB11 dhfr e COS-7. A giroxina foi detectada no extrato total das células COS-7 por western blot e, em seguida, purificada do meio de cultura com coluna de afinidade (Benzamidina Sepharose) e demonstrado sua integridade pelo ensaio de atividade esterásica.The serine proteases affect several physiological processes (such as the coagulation cascade) and pathological ones. These enzymes are widely distributed beyond the species; they are also toxins from snake venoms and are called SVSPs (snake venom serine proteases). These SVSPs are multifunctional and have a catalytic triad formed by HDS amino acids. Some of them are commercially available for use in clinical treatment for heart attack, tromboses and pulmonary embolism. So far, in Crotalus durissus terrificus venom only two SVSPs are described and gyroxin is considered the most studied SVSP which represents about 2,5% of the total venom. In the present study was reported the cloning of seven serine proteases amplified from a cDNA library of a venomous gland of a single adult specimen from Crotalus durissus terrificus venom. These clones have been analyzed in relation to the cDNA organization, structure and probable functions. The three-dimensional model of the gyroxin made possible the analysis of similarities with trypsin, thrombin and other SVSPs. The glycosylation and many disulfide bonds of the SVSPs make difficult the expression in E.coli to obtain the soluble recombinant toxin with activity. The expression in mammalian cells is very promising, because it is possible to make pos translation modification and to obtain the recombinant toxin secreted to the culture medium. The IgK signal peptide, the mature sequence and 3\'UTR region of gyroxin were cloned in the pED expression vector resulting in a new vector (pED-Giro). This vector carries the Igk signal peptide, which allows the secretion of the protein to the culture medium. The pED-Giro vector was transfected in CHO DXB11 dhfr and COS-7 cells. The gyroxin was detected in COS-7 total extract by western blot and after, purified from the medium culture and its integrity was confirmed by esterase activity assay.
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Devlin, Glyn L. "The mechanisms of serpin misfolding and its inhibition". Monash University, Dept. of Biochemistry and Molecular Biology, 2003. http://arrow.monash.edu.au/hdl/1959.1/9469.
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Zu, Yi y 祖毅. "SIRT1 promotes cell proliferation and prevents cellular senescence through targeting LKB1 in primary porcine aortic endothelial cells". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B43085805.
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Zhou, Zhenzhen. "Study of light dependent Arabidopsis phytochrome A signal transduction through FHY1 and its downstream gene expression regulation". Diss., Online access via UMI:, 2009.
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Oliveira, Ana Karina de. "Caracterização proteômica comparativa da agregação plaquetária induzida pela trombina e pela PA-BJ, uma serinoproteinase do veneno da Bothrops jararaca". Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-20072015-111254/.
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Plaquetas são fragmentos celulares anucleados, derivados de megacariócitos, que estão envolvidos em diversos processos fisiológicos e patológicos, como coagulação, inflamação, trombose, aterosclerose, e metástase e angiogênese tumorais. Para executar estas funções, plaquetas ativadas secretam uma fração solúvel de moléculas presentes em seus conteúdos granulares, que passam a interagir com outras moléculas e células adjacentes ao local da injúria, e com os próprios receptores plaquetários. No entanto, os mecanismos que regem a secreção em plaquetas ainda são pouco conhecidos. Neste sentido, o objetivo deste estudo foi analisar comparativamente a agregação de plaquetas ativadas por dois diferentes agonistas enzimáticos: a trombina, um dos mais importantes agonistas plaquetários, e a PA-BJ, uma serinoproteinase do veneno da Bothrops jararaca, que assim como a trombina, ativa plaquetas através dos receptores PAR-1 e PAR-4. Neste estudo foram utilizadas abordagens de espectrometria de massas e de bioinformática para caracterizar alterações nos proteomas do sedimento de plaquetas não ativadas e ativadas, e também, para em paralelo analisar as frações proteicas e peptídicas presentes no sobrenadante (secretoma). Nas análises do sedimento de plaquetas ativadas tanto por PA-BJ quanto por trombina, foi verificada a menor abundância das proteínas PBP, PF4, proteína S, fibronectina, fator V e alfa-1 antitripsina, entre outras, e que também foram identificadas no sobrenadante (secretadas), e o aumento de abundância das proteínas ADAM-10, tromboxano A2 sintase, integrina αlIb, miosina-9 e fosforilase b, que estão diretamente envolvidas na ativação/agregação. Por outro lado, verificamos que na secreção plaquetária induzida por trombina ocorreu o aumento de abundância de proteínas envolvidas na regulação da formação do coágulo, como a proteína S, PAI1 e antitrombina III, sugerindo que nos eventos disparados pela trombina, exista uma regulação rigorosa de sua ação no local da injúria vascular. Já na secreção induzida por PA-BJ, verificamos o aumento significativo das proteínas amiloide beta A4 e do fibrinogênio, envolvidas na ativação/agregação plaquetária, além da liberação e ativação de MMP1, indicando que esta metaloproteinase atue sinergicamente com a PA-BJ para a formação e estabilização do agregado plaquetário. Nas análises do secretoma de plaquetas não ativadas, identificamos pela primeira vez, a presença das proteínas catalase, anidrase carbônica, inibidor de elastase leucocitária e a glicoproteína rica em histidina, que estão envolvidas na inibição e regulação da ativação plaquetária. A análise da fração peptídica do sobrenadante plaquetário permitiu avaliar pela primeira vez o degradoma gerado no processo de agregação por PA-BJ e trombina. O conjunto de peptídeos resultante da ativação plaquetária pela PA-BJ é maior e mais complexo do que aquele gerado pela ação da trombina, sugerindo que as vias ativadas por ambas sejam diferenciais e sujeitas a diferentes controles de regulação da proteólise. Além disso, a degradação seletiva de algumas proteínas, e o conjunto de peptídeos gerados, poderiam ter um papel no controle da ativação e agregação plaquetárias. Em conjunto, nossos resultados demostram que, embora a PA-BJ e a trombina induzam a agregação plaquetária mediada pelos receptores PAR-1 e PAR-4, estas enzimas induzem vias diferentes, alterando a secreção plaquetária para levar à agregação.Platelets are anucleated cell fragments derived from megakaryocytes which are involved in many physiological and pathological processes, such as coagulation, inflammation, thrombosis, atherosclerosis, and tumor angiogenesis and metastasis. To perform these functions, activated platelets secrete a soluble fraction of molecules present in their granules, which then interact with other molecules and cells adjacent to the site of injury, and with platelet receptors. However, the mechanisms governing secretion in platelets are still poorly understood. Therefore, the objective of this study was to comparatively analyze the aggregation of platelets activated by two different enzyme agonists: thrombin, one of the most important platelet agonists, and PA-BJ, a serine proteinase from Bothrops jararaca venom, which, like thrombin, causes platelet aggregation mediated by the receptors PAR-1 and PAR-4. For this purpose, approaches of mass spectrometry and bioinformatics were used to characterize changes in the proteome of non-activated and activated platelets, and also to analyze proteins and peptides present in the supernatant of aggregated platelets (secretome). In the analysis of the sediment of platelets activated by PA-BJ and thrombin, various proteins, such as PBP, PF4, protein S, fibronectin, factor V, and alpha-1 antitrypsin, were detected in lower abundance while they were also identified as secreted, in the supernatant; likewise, proteins that are directly involved in the activation/aggregation, such as ADAM-10, thromboxane A2 synthase, integrin αIIb, myosin-9 and phosphorylase b were identified in higher abundance in platelets activated by PA-BJ and thrombin. Moreover, we found that in the thrombin-induced platelet secretion there was increased abundance of proteins involved in the regulation of blood clot formation, such as protein S, and antithrombin III PAI1, suggesting that in the events triggered by thrombin, there is strict regulation of its action at the site of vascular injury. In the analysis of the secretion induced by PA-BJ, we found a significant increase in amyloid beta A4 protein and fibrinogen, which are involved in the platelet activation/aggregation, in addition to the release and activation of MMP-1, indicating that this metalloproteinase acts synergistically with PA-BJ in the formation and stabilization of the platelet thrombus. In the analysis of the non-activated platelet secretome, we identified for the first time the presence of catalase, carbonic anhydrase, leukocyte elastase inhibitor and histidine-rich glycoprotein, which are involved in the inhibition and regulation of platelet activation. The analysis of the peptide fraction of the supernatant of activated platelets enabled the characterization, for the first time, of the degradome generated in the process of aggregation by thrombin and PA-BJ. The resulting set of peptides generated upon platelet activation by PA-BJ is larger and more complex than that generated by the action of thrombin, suggesting that the pathways activated by both are differential and are subject to different controls of proteolysis. Furthermore, the selective degradation of some proteins, and the set of generated peptides could play a role in the control of platelet activation and aggregation. Taken together, our findings demonstrate that although both PA-BJ and thrombin induce platelet aggregation via PAR-1 and PAR-4, these enzymes activate different pathways to cause platelet secretion and aggregation.
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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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Lauro, Andrea Marie. "The design and synthesis of novel serine proteinase inhibitors". Diss., Georgia Institute of Technology, 1989. http://hdl.handle.net/1853/30032.
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Sigle, Leah T. "Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi". Thesis, Kansas State University, 2011. http://hdl.handle.net/2097/13140.
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Master of ScienceDepartment of Entomology
Marcelo Ramalho-Ortigao
Sand flies (Diptera:Psychodidae) are vectors of parasites of the genus Leishmania transmitted to suitable vertebrate host during blood feeding. For blood feeding arthropods, including sand flies, blood meal digestion requires the secretion of inhibitory molecules, such as Kazal-type serine proteinase inhibitors that are involved in preventing the blood from coagulating within the mouthparts and the midgut. Previous studies have identified such molecules in mosquitoes, ticks, and triatomine bugs. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). We are interested in the role of these proteins as inhibitors of coagulation cascades, in addition to their potential effects on blood digestion in P. papatasi. Ppkzl1 is similar to thrombin and trypsin inhibitors in triatomines and mosquitoes and Ppkzl2 is similar to Kazal-type inhibitors in mosquitoes with unknown function. Analyses of expression profiles indicated that although both transcripts are expressed prior to blood feeding in the midgut of P. papatasi they are tightly regulated by the blood meal. Reverse genetics studies using RNAi-targeted knockdown of PpKzl1 and PpKzl2 by dsRNA injection did not result in a detectable effect on mRNA expression levels. Thus, we expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess activity against various serine proteinases. Recombinant PpKzl2 inhibited chymotrypsin at nanomolar levels and also inhibited thrombin and trypsin at micromolar levels, suggesting that native PpKzl2 is an active serine proteinase inhibitor and may regulate digestive enzymes and thrombin in the midgut. Leishmania development within the sand fly midgut is faced with several barriers that can severely impact the parasites. For transmission to occur, parasites must be able to overcome these barriers including digestive proteinases, escape from the peritrophic matrix, and midgut attachment. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sand fly midgut. Thus, targeting serine proteinase inhibitors may provide a new strategy to prevent transmission of Leishmania.
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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/.
Texto completoResumen
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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Van, Gent Diana. "Evolution of serine proteinase inhibitors and gene expression of α-1-antitrypsin". Thesis, University of Nottingham, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408668.
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