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High molecular weight mouse nerve growth factor(H M W-NGF), in addition to its effects on certain neural elements, is also chemotactic for human polymorphonuclear leukocytes. One of the subunits of H M W-NGF is a protease of the serine family and its active site contains a serine residue and a closely-neighboring histidine residue that are both essential for proteolysis. Elimination of enzyme activity by irreversibly blocking the single serine has no effect on leukotaxis, but blocking the histidine abolishes leukotaxis. These results suggest the possibility that part of the proteolytic active site of this enzyme may have evolved to perform more than one, completely different, biologic function - proteolysis as well as nonproteolytically mediated chemotaxis.

Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) are critical for the protection of organisms against pathogens and cancer. The process by which these cells eliminate infected or transformed cells are through two basic mechanisms, receptor-mediated interactions, or delivery of contents from intracellular cytotoxic granules. Granules are comprised of perforin and a family of serine proteases, called granzymes. Upon entry into target cells, these proteins work together to initiate cellular death pathways. Previous and extensive biochemical studies had already established that granzyme B (GrB) was a powerful inducer of apoptosis, but sensitive assays to confirm its release from cytotoxic cells were lacking. We hypothesized that GrB release, measured by ELISPOT, directly assessed the lytic potential of antigen-specific cytotoxic cells. Indeed, data provided in this thesis established a strong correlation between GrB release and target cell lysis. Our results imply that GrB could be a promising tool to assess cell-mediated immunity during vaccine development. However, several other independent studies in grB-/- mice demonstrated that additional granzymes were capable of clearing viruses and tumorigenic cells. Granzyme H (GrH) is highly and constitutively expressed in human NK cells, and therefore, we hypothesized that it was also an effective cytotoxic molecule. Our experiments established that GrH-induced cell death by a mechanism distinct from those of GrB and Fas. We identified a GrH substrate, DFF45/ICAD, and showed that GrH induced mitochondrial damage through a Bid-independent mechanism. Furthermore, cell death was dependent on Bax and/or Bak, but independent of caspase activation. Hence, we have elucidated an alternative cytotoxic pathway that could be employed to eliminate target cells with immune evasion strategies targeted to GrB or Fas. Finally, control of serine proteases by endogenous inhibitors is important to numerous biological processes, including apoptosis. We hypothesized that as GrH displayed chymase activity, the serine protease inhibitor anti-chymotrypsin (ACT) would impair GrH function. Our data established that ACT effectively attenuated GrH cytotoxicity and prevented proteolysis of a GrH substrate. Collectively, this thesis describes a novel GrH inhibitor, provides a new tool to evaluate cell-mediated immunity, and provides evidence of an alternative mechanism of cytotoxicity.

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Lors de la réponse immunitaire de la drosophile, la voie Toll est activée lors d'un challenge immunitaire par des bactéries à Gram positif ou des champignons. Ce mécanisme est initié soit par la reconnaissance de motifs moléculaires associés aux pathogènes (PAMPs) qui activent la voie de reconnaissance, soit par des facteurs de virulence et des protéases produits par les agents pathogènes qui activent la voie des signaux de danger. Le travail que j'ai effectué a pour but de caractériser les différentes molécules impliquées dans ces cascades protéolytiques en amont de Toll. Cela permettra de reconstituer ces cascades in vitro et de comprendre comment elles sont organisées, comment et où des complexes peuvent être formés. La première partie concerne les approches génétiques utilisées pour générer des mutants des gènes pouvant être impliqués dans l'activation de la voie Toll par la voie des PAMPs. La deuxième partie se concentre sur un homologue inactif de protéase à sérine appelé spheroide et sur son implication dans la voie de reconnaissance des signaux de danger. Pour la première fois, nous avons pu démontrer qu'une protéase inactive est requise dans la cascade protéolytique, et plus particulièrement dans la détection des signaux de danger après un challenge immunitaire par des bactéries pathogènes à Gram positif. / The Toll pathway in Drosophila immune response is activated upon immune challenge with Gram positive bacteria and fungi. This can be achieved either through recognition of Pathogen Associated Molecular Patterns (PAMPs), which triggers the recognition cascade; or by virulence factors and proteases produced by the pathogens, which triggers the danger signal cascade. The work I have done aimed to characterize the various molecules involved in proteolytic cascade supstream of Toll. This will help to reconstitute these cascades in vitro and understand how they are organized, how and where complexes could be formed. The first part focuses on genetic approaches used to generate mutants for genes suggested to be involved in the activation of Toll pathway via the recognition cascade. The second part focuses on an inactive serine protease, aserine protease homolog spheroide and its involvement in the danger signal cascade. For the first time, we could demonstrate that an inactive protease is required in the proteolytic cascade,involved in the sensing of danger signais upon immune challenge with pathogenic Gram-positive bacteria.

The anticancer activity of several platinum compounds is due to the formation of complexes with DNA. We hypothesize that the size and shape of the platinum compounds would impact interaction with proteins, and these interactions may be partly responsible for the anticancer activity. Chymotrypsin and subtilisin are serine proteases that have a histidine residue in the active site. We are investigating the inhibition of the digestive enzymes chymotrypsin and subtilisin by analogs of the anticancer drug cisplatin and trying to discern trends in the inhibition as the active site residues vary. In our research, we found that the enzyme subtilisin did not show any significant inhibition with different platinum compounds we used, while chymotrypsin showed inhibition only with the potassium tetrachloroplatinate and this inhibition is concentration dependent

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