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Biopeptides are synthesised as large pro-protein precursors that have to undergo proteolytic cleavage at positively charged amino acids (Lys and Arg) in order to become active. This cleavage is mediated by a family of subtilin/kexin related calcium dependent serine endoproteinases named prohormone convertases. The present thesis focuses on the endocrine member of the family named PC1/3. PC1/3 is expressed in the regulated secretory pathway of endocrine and neuroendocrine cells, where it was shown to activate various peptide hormones such as proopiomelanocortin (POMC), pro-insulin and pro-glucagon. PC1/3 is synthesized as a large precursor containing a signal peptide, a propeptide, a catalytic domain, a P domain and a C-terminal domain. The activation of the enzyme requires the sequential removal of the signal peptide, the propeptide and ultimately the C-terminal domain. / The structural characterisation of the enzyme is compromised by the difficulty in producing a sufficient amount of recombinant PC1/3. In this thesis it is clearly demonstrated that the production of PC1/3 using Baculovirus technology can be greatly improved by modifying the expression vector in insect cells (Spodoptera frugiperda). In addition, the intracoelemic injection of insect larvae (Tricoplusia ni) with the Baculovirus encoding the recombinant PC1/3 is shown to be a very efficient method for the production of a large amount of prohormone convertases. / It was previously demonstrated that the propeptide is essential for the folding of the enzyme and act as a tight binding inhibitor of the enzyme until the latter reaches the appropriate compartment for substrate cleavage. To assess the role of certain residues within the propeptide in the inhibition of the cognate enzyme, a mutational analysis by alanine scan was conducted. The results demonstrate that the substitution of a single amino acid can affect markedly the inhibition behavior, potency and selectivity of the propeptide towards the enzyme. Moreover, this mutational analysis allowed the first experimental mapping of the sequence involved in propeptide degradation once its function is achieved. / However, PC1/3 also possesses a C-terminal domain which must also be cleaved to allow the full activation of the enzyme. Previous studies showed that this domain is implicated in the sorting of the enzyme to secretory granules. In addition, over expression experiments showed that the C-terminal domain can inhibit the cleavage of certain substrates by PC1/3. The results, presented here, suggest that the CT-peptide acts as a non-essential activator of PC1/3, in vitro, which adds a supplementary level of complexity to the activation process of the enzyme. / Finally, based upon our results, it can be proposed that PC1/3 is a very complex enzyme capable of controlling its enzymatic activity through the coordinate action of its various domains. This exceptional mode of self-regulation is unique among all protease families.

Inducible LAK cell responses were studied in HIV-seropositive individuals lacking clinical symptoms, and overt AIDS patients. Inducible LAK cell responses have been operationally defined as, non-MHC-restricted and antigen-nonspecific cytotoxic activity observed following IL-2 stimulation. HIV-seropositive asymptomatic individuals exhibited an enhanced LAK cell response against HIV-infected targets while lysis of uninfected targets remained at control levels. LAK activity of AIDS patients however, was significantly diminished when compared to healthy controls. Immunomagnetic negative selection depletion experiments indicated that LAK cell activity is mediated primarily by CD56-expressing lymphocytes, both at the progenitor and effector cell level. Of interest, in HIV-seropositive asymptomatic individuals we observed the emergence of a second CD8-expressing cytotoxic population that mediates IL-2-induced non-MHC-restricted and antigen-nonspecific cytotoxicity. Overall we demonstrated that CD56-expressing LAK cells of HIV-seropositive patients exhibited a decreased ability to mediate cytotoxicity on a per cell basis against a panel of different targets. In vivo, this inhibition may be amplified by decreases in absolute numbers of CD56-expressing lymphocytes per ml of blood. HIV-infection therefore results in dramatic changes on the number, function and phenotype of the effector cells mediating IL-2 inducible LAK cell responses.

The importance of arenavirus glycoprotein processing has only been understood within the past decade, with the majority of work focused on the Old World arenaviruses. Evidence has shown that SKI-1/S1P (subtilisin kexin isozyme-1/site 1 protease) is the cellular protease responsible for glycoprotein cleavage in Old and New World arenaviruses. Furthermore, glycoprotein cleavage is shown to be necessary for the production of infectious virus particles in Lassa and Junín viruses. In this thesis, evidence is provided that the recently emerged Chapare virus (New World) is also processed by SKI-1/S1P. Additionally, novel serpin-based SKI-1 inhibitors were shown to effectively prevent SKI-1 mediated cleavage. Using a wide panel of recombinant vesicular stomatitis viruses expressing New World arenavirus glycoproteins, these inhibitors were capable of significantly reducing viral titres. This provides strong evidence that SKI-1 inhibitors can be used as an effective treatment against the majority of New World Clade B arenaviruses and LASV in vivo.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone produced by the enteroendocrine L-cell that potently stimulates insulin secretion. Although signaling pathways promoting GLP-1 secretion are well characterized, the mechanism by which GLP-1 containing granules fuse to the L-cell membrane remain elusive. RT-PCR and protein analysis indicate that vesicle-associated membrane protein 2 (VAMP2) is expressed and localized to secretory granules in the murine GLUTag L-cell model. VAMP2, but not VAMP1, interacted with the core SNARE complex protein, Syntaxin 1a, in GLUTag cells. Tetanus toxin (TetX) cleavage of VAMP2 in GLUTag cells prevented glucose-dependent insulinotropic peptide (GIP)- and oleic acid (OA)-stimulated GLP-1 secretion, as well as K+-stimulated exocytosis from GLUTag cells. Although components of membrane rafts were detected in GLUTag cells, their role in GLP-1 secretion remains to be determined. Together, these findings indicate an essential role for VAMP2 in GLP-1 exocytosis from the GLUTag cell.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone produced by the enteroendocrine L-cell that potently stimulates insulin secretion. Although signaling pathways promoting GLP-1 secretion are well characterized, the mechanism by which GLP-1 containing granules fuse to the L-cell membrane remain elusive. RT-PCR and protein analysis indicate that vesicle-associated membrane protein 2 (VAMP2) is expressed and localized to secretory granules in the murine GLUTag L-cell model. VAMP2, but not VAMP1, interacted with the core SNARE complex protein, Syntaxin 1a, in GLUTag cells. Tetanus toxin (TetX) cleavage of VAMP2 in GLUTag cells prevented glucose-dependent insulinotropic peptide (GIP)- and oleic acid (OA)-stimulated GLP-1 secretion, as well as K+-stimulated exocytosis from GLUTag cells. Although components of membrane rafts were detected in GLUTag cells, their role in GLP-1 secretion remains to be determined. Together, these findings indicate an essential role for VAMP2 in GLP-1 exocytosis from the GLUTag cell.

Caveolin-1 is a marker protein for caveolae and can be a regulator of intracellular signaling pathways that contribute to the pathogenesis of human diseases. In the present study, the structural and functional changes of the lung in caveolin-1 null mice (Cav-1-/-) were assessed. Respiratory mechanics, measured using a small animal ventilator, revealed heightened central airway resistance (Rn), tissue resistance (G) and tissue elastance (H) in response to inhaled methacholine. The respiratory hyperreactivity is associated with increased collagen deposition around central and peripheral airways in Cav-1-/- mice; however, no difference was found in smooth muscle α-actin quantity between mouse strains. Similar to our in vivo findings, tracheal rings from Cav-1-/- mice mounted on an isometric wire myograph exhibited enhanced maximum active contractile force without a change in sensitivity (EC50) to methacholine. Rho kinase (ROCK1/2), protein kinase C (PKC) and extracellular signal regulated kinase 1/2 (ERK1/2) signaling were assessed as possible sources of the enhanced airway reactivity observed in Cav-1-/- mice. Inhibition of Rho kinase markedly blunted in vivo lung function responses (Rn) and (G) and ex vivo smooth muscle responses to methacholine. In fact, inhibition of Rho kinase completely eliminated any difference in response between mouse strains. Thus, our data indicate that Cav-1 may regulate mechanisms, such as Rho/Rho kinase signaling, that determine airway smooth muscle contraction and airway fibrosis; thus, it could be an important regulator of airway biology and physiology in health and disease.

Currently, the HIV pandemic remains a major global health challenge. In order to effectively control and cure HIV-1 infection, it is necessary to perform greater research on host-HIV interactions and develop novel preventive and therapeutic approaches. The human cytidine deaminase APOBEC3G (A3G) is the first identified host restriction factor, which can serve as an initial line of defense against HIV-1 by inducing lethal mutations on proviral DNA and disrupting viral reverse transcription and integration. In order to better understand the action of A3G on HIV-1 replication, my study was focused on characterizing the interplay between A3G and HIV-1 reverse transcriptase (RT). The results indicated that A3G directly bound to RT, which contributed to A3G-mediated inhibition of viral reverse transcription. Overexpression of the RT-binding polypeptide A3G65-132 was able to disrupt wild-type A3G and RT interaction, consequently attenuating the anti-HIV effect of A3G on HIV replication. While the potent antiviral activities of A3G make it an attractive candidate for gene therapy, the actions of A3G can be counteracted by HIV-1 Vif during wild-type HIV infection. In order to overcome Vif-mediated blockage and maximize the antiviral activity of A3G, this protein was fused with a virus-targeting polypeptide (R88) derived from HIV-1 Vpr, and various mutations were then introduced into R88-A3G fusion protein. Results showed that Vif binding mutants R88-A3GD128K and R88-A3GP129A exhibited very potent antiviral activity, and blocked HIV-1 replication in a CD4+ T lymphocyte cell line as well as human primary cells. In an attempt to further determine their potential against drug resistant viruses and viruses produced from latently infected cells, R88-A3GD128K was chosen and delivered by an inducible lentiviral vector system. Expression of R88-A3GD128K in actively and latently HIV-1 infected cells was shown to be able to inhibit the replication of both drug sensitive and resistant strains of HIV-1. In conclusion, this thesis has demonstrated one of the mechanisms that how A3G can disrupt HIV-1 reverse transcription. Meanwhile, an A3G-based anti-HIV-1 strategy has been developed, which provides a proof-of-principle for a new gene therapy approach against this deadly virus.