Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2002. / Vita. Bibliography: 116-128.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 96-113).

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [100]-125).

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [115]-144).

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 192-203). Also available online.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Medical Microbiology and Immunology. Title from pdf file main screen (viewed on April 24, 2010). Includes bibliographical references.

La mucoviscidose est une maladie génétique caractérisée par une obstruction des voies respiratoires, des infections et une inflammation pulmonaire résultant du recrutement massif de neutrophiles qui sécrètent des protéases : l’élastase, la protéase 3 et la cathepsine G. Ces protéases peuvent être sécrétées selon deux voies, la dégranulation ou la sécrétion de NETs (Neutrophil Extracellular Traps), qui sont des fibres de chromatine auxquelles elles sont associées et décrites comme des structures antimicrobiennes. Dans le milieu extracellulaire, la dérégulation du contrôle de l’activité des protéases par leurs inhibiteurs conduit à la dégradation progressive du tissu pulmonaire. Nous avons montré que cette dérégulation était modulée par l’interaction des protéases avec l’ADN présent dans les sécrétions bronchiques des patients et que le ciblage de ces protéases par des inhibiteurs exogènes pouvait être amélioré in vitro par de la DNase ou de la polylysine qui compacte l’ADN. Ce polypeptide est également bactéricide vis-à-vis des pathogènes majeurs de la mucoviscidose, S. aureus et P. aeruginosa. Nos travaux montrent également que les NETs sont sécrétés dans les poumons des patients où ils constituent un réservoir de protéases actives potentiellement délétère et n’ont pas d’effet bactéricide vis-à-vis de S. aureus et P. aeruginosa. Nos travaux montrent que les voies de signalisation conduisant à la sécrétion des NETs varient selon le stimulus, générant des structures aux propriétés différentes. / Cystic fibrosis (CF) is a hereditary disease characterized by the obstruction of the airways, infections and a chronic lung inflammation due to a massive recruitment of neutrophils that secrete proteases: the elastase, the proteinase 3 and the cathepsin G. These proteases can be secreted by two mechanisms, namely degranulation and the secretion of NETs (Neutrophil Extracellular Traps), which are chromatin fibers to which they are bound and that have been described as antimicrobial structures. In the extracellular environment, the dysregulation of these proteases control by their inhibitors leads to progressive lung tissue degradation. We have shown that this dysregulation was influenced by the interaction of the proteases with the DNA found in the lung secretions of CF patients and that targeting these proteases with exogenous inhibitors could be improved in vitro by DNase or polylysine, which compacts DNA. This polypeptide also presents a bactericidal effect towards the major CF-associated pathogens, S. aureus and P. aeruginosa. Our work also shows that NETs are secreted in the lungs of CF patients, where they are a potentially deleterious reservoir of active proteases, and that they do not display any bactericidal effect towards S. aureus and P. aeruginosa. Our work shows that the signalization pathways leading to NETs secretion vary depending on the stimulus, generating structures that present different properties.

Made available in DSpace on 2015-07-22T20:49:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-24. Added 1 bitstream(s) on 2015-08-11T03:26:21Z : No. of bitstreams: 1 Publico-204.pdf: 1677799 bytes, checksum: 7f10174cddd62a0a9683b2af760805d8 (MD5) / O mosquito Ae. aegypti é o vetor clássico da febre amarela e do dengue. Em insetos hematófagos, a digestão do sangue é realizada por enzimas proteolíticas, que também podem estar envolvidas na relação parasita-vetor. O processo de alimentação com sangue requer que os artrópodes hematófagos sejam capazes de inibir as defesas hemostáticas do hospedeiro, como por exemplo, com a presença de inibidores das enzimas da cascata da coagulação. Com a finalidade de conhecer melhor sobre a biologia do vetor do dengue, o objetivo deste trabalho foi a expressão e a caracterização de um inibidor de serinoproteases do tipo Kazal presente em mosquito Ae. aegypti. Para isso, o inibidor recombinante (rAaTI) e sua forma truncada rAaTIΔ (sem a região C-terminal) foram expressos em sistema Pichia pastoris e purificados por cromatografia de afinidade. O rAaTI purificado foi capaz de inibir a atividade amidolítica da tripsina, plasmina e fracamente a trombina. O rAaTI foi também capaz de prolongar o tempo de coagulação, e há indícios que o inibidor se ligue em alguma região ocupada pela heparina ou pela anitrombina III. O análise do transcrito mostrou que o AaTI está presente na glândula salivar e em intestino de fêmeas 3 e 24 h após a alimentação com sague, e também em todos os estádios larvais, em pupa e em machos, o que sugere que este inibidor possa ter outras funções no desenvolvimento do mosquito. O rAaTI ainda foi capaz de inibir fortemente as tripsinas de larva de 4° instar, e tripsina de fêmeas 24 h após a alimentação, sugerindo que também possa estar envolvido com a modulação destas enzimas na fase larval e no final da digestão com sangue. / The Aedes aegypti mosquito is the classic vector of yellow fever and dengue fever. In haematophagous insects, blood digestion is carried out by proteolitic enzymes, which can also be envolved in parasite-vector relationship. Blood feeding requires that haematophagous insects are able to inhibit host hemostatic defenses, containing inhibitors of coagulation cascade enzymes, for example. In order to understand the vector bilogy, the aim of this work was expression and characterization of a Kazal-type serine protease inhibitor present in Aedes aegypti mosquito. Recombinant inhibitor (rAaTI) and its truncated form rAaTIΔ (without Cterminal region) were expressed in Pichia pastoris system and purified by affinity chromatography. Purified rAaTI was able to inhibit amidolytic activity of trypsin, plasmin and weakly inhibited thrombin. rAaTI was also able to prolong coagulation time, and there are evidences that inhibitor bind in some region occupied by heparin or antithrombin III. Transcription analyzes showed that AaTI is present in salivary gland and midgut of females 3 and 24 h after blood feeding, and in all larval instars, pupae and males, which suggest that inhibitor may have other functions in mosquito development. rAaTI was also able to strongly inhibit trypsin from 4th instar lavae , and female trypsin 3 and 34 h after blood feeding, suggesting that rAaTI may be involved in trypsin modulation in larval phase and in the end of blood difgestion. / TEDE / BV UNIFESP: Teses e dissertações

SmSP1 is a chimerical serine protease consisted of three domains (cub, LDLa and trypsin-like) and found in Schistosoma mansoni. Its characterization was performed by molecular techniques such as PCR screen, qRT-PCR and RNA interference (RNAi) to gain information about expression profile, level expression and susceptibility to RNAi. Further, protein expression was carried out to gain an antigen for immunization and recombinant for biochemical studies. Results of PCR screen and qRT-PCR suggested possible function of SmSP1 in egg and adult stages but SmSP1 gene was not found susceptible to RNAi in NTS. Recombinant from E. coli was successfully used for immunization. Active recombinant was likely expressed in Pichia pastoris but expression conditions are unstable and expression optimization is necessary.