Previous studies have shown that fish release alarming substances into the water to alert their kin to escape from danger. In our laboratory, we found that zebrafish produce trypsin and release it from their gills into the environment when they are under stress. By placing the zebrafish larvae in the middle of a small tank and then placing trypsin at one end of the tank, we observed that the larvae moved away from the trypsin zone and almost to the opposite end of the tank. This escape response was significant and did not occur in response to the control substances, bovine serum albumin (BSA), Russell's viper venom (RVV), and collagen. Also, previously, we had shown that the trypsin could act via a protease-activated receptor-2 (PAR2) on the surface of the cells. Therefore, we hypothesized that trypsin would induce a change in neuronal activity in the brain via PAR2-mediated signaling in cells on the surface of the fish body. To investigate whether the trypsin-responsive cells were surface cells, we generated a primary cell culture of zebrafish keratinocytes, confirmed these cells' identity by specific marker expression, and then incubated these cells with the calcium indicator Fluo-4 and exposed them to trypsin. By using calcium flux assay in a flow-cytometer, we found that trypsin-treated keratinocytes showed an increase in intracellular calcium release. To test whether PAR2 mediates the escape response to trypsin, we treated larvae with a PAR2 antagonist and showed that the trypsin-initiated escape response was abrogated. Furthermore, par2a mutants with knockdown of par2a by the piggyback knockdown method failed to respond to trypsin. Trypsin treatment of adult fish led to an approximately 2-fold increase in brain c-fos mRNA levels 45 mins after trypsin treatment, suggesting that trypsin signals may have reached the brain, probably via a spinothalamic pathway. Taken together, our results reveal a novel trypsin-initiated escape response in fish. These studies should enhance our understanding of fish communication in general and alarm behavior in particular. Furthermore, since pain receptors in other animals are also PAR2, our finding may be useful in exploring pathways of pain reception.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: This study describes: a) Characterization of traditional methodologies and testing methods used to purify and quantify trypsin and α-chymotrypsin b) Re-engineering / development of a new method for purifying trypsin and α-chymotrypsin that delivered higher product yields and improved control exercised over the process by investigating: i. Extraction methods ii. Centrifugation iii. Ultrafiltration iv. Chymotrypsinogen and trypsin crystallization v. Column chromatography vi. Investigation into different raw material sources for pancreatic enzyme production c) Development of kinetic and ELISA testing methodologies for in-process QC analysis. / AFRIKAANSE OPSOMMING: Hierdie Studie beskryf: a) Karakterisering van die ou prosessering metodes en toets metodes wat gebruik word om Tripsien en Alpha-chimotripsien te suiwer en te kwantifiseer. b) Herontwerp / ontwikkeling van 'n nuwe metode vir die suiwering Tripsien en Chimotripsien wat „n hoër opbrengs lewer en meer kontrole oor die proses uit oefen deur ondersoek in te stel na: i. Ekstraksie- metodes ii. Sentrifugering iii. Ultrafiltrasie iv. Chymotripsienogeen - en tripsien kristallisasie v. Kolom chromatografie vi. Ondersoek na verskillende rou materiaal bronne vir die produksie van pankreas ensieme. c) Die ontwikkeling van kinetiese- en ELISA toets metodes vir die in-proses kwaliteitkontrole.

Leishmania development in their vectors is closely connected with bloodmeal digestion. This thesis focuses on factors affecting bloodmeal digestion, egg development and Leishmania infection within the sand fly gut. First, we compared the effect of mammalian (rabbit) and avian (chicken) blood on digestion and eggs development in Phlebotomus duboscqi. Sand flies fed on chickens had twice lower protein concentrations in the midgut and significantly lower trypsin activity compared to those fed on rabbits. The highest differences in the trypsin activity were observed during first 24 hours post bloodmeal. In addition, females fed on chickens had slower eggs development and their eggs were 10 % smaller compared to those fed on rabbits. In the second part of the thesis we tested the effect of mosquito hormone TMOF on the trypsin activity and eggs development of Lutzomyia longipalpis. Rabbit blood with TMOF (28 mg/ml) was presented to the females via a membrane feeding system. Sand flies fed on blood with TMOF had 15 - 35 % less trypsin activity than control females fed on only rabbit blood. In addition, females fed on blood with TMOF had developed 30 % less eggs and their eggs were 12 - 24 % smaller compared to control group. However the effect of TMOF we observed was lower than that described previously...

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2016-03-09T13:28:58Z No. of bitstreams: 1 PDF - Géssica Laize Berto Gomes.pdf: 960450 bytes, checksum: 01f35541007decb425986338cf77d27a (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-07-21T21:00:52Z (GMT) No. of bitstreams: 1 PDF - Géssica Laize Berto Gomes.pdf: 960450 bytes, checksum: 01f35541007decb425986338cf77d27a (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-07-21T21:01:00Z (GMT) No. of bitstreams: 1 PDF - Géssica Laize Berto Gomes.pdf: 960450 bytes, checksum: 01f35541007decb425986338cf77d27a (MD5) / Made available in DSpace on 2016-07-21T21:01:00Z (GMT). No. of bitstreams: 1 PDF - Géssica Laize Berto Gomes.pdf: 960450 bytes, checksum: 01f35541007decb425986338cf77d27a (MD5) Previous issue date: 2014-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Brazilian production of grains has grown every year, transforming Brazil in one of the main cereals and oilseeds producers. However, the post-harvest losses are huge, causing production loss by the inadequacy of storage, especially in order of pest occurrence. In the case of sesame, there has been a marked occurrence of P. interpuctella, which attack stored grains accompanied by other pests causing economic damage to the crop. With this, the Breeding Program Sesame Embrapa Cotton seeks alternatives to control these pests by genotypes that express high levels of protein inhibitors (PIs) with ability to inhibit proteolytic digestive enzymes of insects. The objective of this study was to detect trypsin inhibitors in genotypes of sesame (S. indicum L.) with potential use in biotechnological applications and programs to improve the species, aiming selection of genotypes resistant to stored grain pests, so we proceeded to study PIs in 10 sesame genotypes belonging to the Active Germplasm Bank at Embrapa Cotton. The total protein extracts of accessions were extracted and tested in vitro with bovine pancreatic trypsin (BPT) and intestinal homogenate of larvae of P. interpuctella. After selection of the most promising genotypes, we proceeded to a protein fractionation with ammonium sulfate. The protein fractions obtained were analyzed for inhibitory activity with BPT and intestinal homogenate of larvae of P. interpuctella, and yet, as the thermal stability and hydrogen ion and bioassays with P. interpuctella. Data were analyzed by Tukey test at 5% of probability. The results showed the presence of trypsin inhibitors in the total protein extracts of 10 sesame genotypes, ranging from 51% to 90% inhibition, among which, the BRS-SEDA, CNPA G3 and CNPA G4 stood out with 87%, 88% and 90% inhibition, respectively. These genotypes inhibitory activity have been demonstrated for the intestinal homogenate of larvae of P. interpuctella, with inhibition between 60% and 76%. The fractions F2 of the three genotypes were relatively stable to changes in temperature and pH. In the bioassay, the larvae of P. interpuctella. were susceptible to protein extracts of the three genotypes of sesame, with a maximum of 83% mortality of larvae in different strengths with total protein extracts and 58% in fractions F2, however, did not observe statistically a significant difference between genotypes. In this regard, BRS-SEDA, CNPA G3 and CNPA G4 have protein inhibitors that can prevent the development of P. interpuctella, suggesting candidates with potent insecticidal properties to control stored grain pests. / A produção brasileira de grãos tem crescido a cada ano levando o Brasil a um dos principais produtores de cereais e oleaginosas. No entanto, as perdas na pós-colheita são enormes provocando uma redução da produção em função do controle inadequado no armazenamento, especialmente em função da ocorrência de pragas. No caso do gergelim, tem havido uma ocorrência acentuada de P. interpuctella, que ataca os grãos armazenados acompanhado de outras pragas, causando danos econômicos à cultura. Com isto, o Programa de Melhoramento Genético de Gergelim da Embrapa Algodão busca alternativas para controlar essas pragas, por meio de genótipos que expressem altos níveis de inibidores proteicos (IPs) com capacidade de inibir enzimas proteolíticas digestivas de insetos. Objetivou-se com este trabalho detectar inibidores de tripsina em genótipos de gergelim (Sesamum indicum L.) com potencial uso na aplicação biotecnológica e no programa de melhoramento da espécie, visando seleção de genótipos resistentes a pragas de grãos armazenados. Assim, procedeu-se o estudo de IPs em 10 genótipos de gergelim, pertencentes ao Banco Ativo de Germoplasma da Embrapa Algodão. Os extratos proteicos totais dos acessos foram extraídos de sementes de gergelim e testados in vitro com tripsina pancreática bovina (TPB) e homogenato intestinal de larvas de P. interpuctella. Após seleção dos genótipos mais promissores, procedeu-se um fracionamento proteico com sulfato de amônia. As frações proteicas obtidas foram analisadas quanto a atividade inibitória com TPB e homogenato intestinal de larvas de P. interpuctella, e ainda, quanto a estabilidade térmica e hidrogeniônica e em bioensaios com P. interpuctella. Os dados foram analisados pelo teste de Tukey a 5% de probalibilidade. Os resultados obtidos mostraram a presença de inibidores de tripsina nos extratos proteicos totais dos 10 genótipos de gergelim, com variação de 51% a 90% de inibição, dentre os quais, o BRS-SEDA, CNPA G3 e CNPA G4 se destacaram com 87%, 88% e 90% de inibição, respectivamente. Nesses genótipos foram evidenciadas atividade inibitória para o homogenato intestinal de larvas de P. interpuctella, com inibição entre 60% e 76%. As frações F2 dos três genótipos foram relativamente estáveis às variações de temperatura e pH. No ensaio biológico, as larvas de P interpuctella mostraram-se suscetíveis aos extratos proteicos dos três genótipos de gergelim, com mortalidade máxima de 83% das larvas em diferentes dosagens com extratos proteicos totais e 58% nas frações F2, contudo, estatisticamente não observou-se diferença significativa entre os genótipos. Neste aspecto, os genótipos BRS-SEDA, CNPA G3 e CNPA G4 possuem inibidores proteicos capazes de impedir o desenvolvimento P. interpuctella, o que sugere potentes candidatos com propriedades inseticidas no controle de pragas de grãos armazenados.

Bibliography: leaves 161-213. / xviii, 219, [48] leaves of plates : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Analyses of six rare alpha 1-antitrypsin alleles were undertaken to determine their molecular basis, assess the biosynthesis directed by these alleles and, examine their clinical correlation. / Thesis (M.D.)--University of Adelaide, Dept. of Medicine, 1992

The field of proteomics encompasses the study of identities, interactions, and dynamics of all proteins expressed by a living system. Research in this dissertation blends biochemical and quantitative proteomics techniques to increase the latitude of biological applications for the bottom-up mass spectrometry proteomics approach. Together, isolation of selected protein “targets,” such as multiprotein complexes, and quantitative characterization yields information essential for more detailed understanding of microbial cell function. Often, a challenging aspect of characterizing a variety of biochemically enriched samples is limited protein yield. This dissertation describes an enzymatic proteolysis protocol employing an organic/aqueous solvent that alleviates excessive handling steps to reduce losses during sample preparation for small quantities of protein samples. Presence of artifactual, non-specific proteins in enriched protein complex isolates complicates biological interpretation of specific protein interactions. Heterologous expression of affinity-tagged bait proteins may also cause unintended collateral effects. A series of local and global protein isotope ratio measurements were performed to differentiate authentic interactions from artifactual interactions among affinity-isolated complexes and assess collateral effects, respectively. Protein localization provides clues regarding protein function. To infer protein localization, quantitative proteomics techniques were used to estimate protein enrichment of cold osmotic shock periplasmic isolates. Protein isotope ratios indicating enrichment, combined with identification of amino-terminal signal peptide cleavages, increase confidence of periplasmic localization. Collectively, this dissertation provides a framework for tailoring biochemical and quantitative techniques for targeted characterization of microbial protein isolates.

No description available.

Detection trace compound from human serum by matrix-assited laser desorption ionization mass spectrometry and second dimensional gel ectrophoresis

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 176-182). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 176-182). Also available on the Internet.