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These studies have characterized the peptide transport systems of Candida albicans, with a view to the rational design of peptide antifungal agents exploiting the 'smugglin' concept. In initial studies, a series of polyoxin complexes (peptide-nucleoside antibiotics) and individual components, were isolated from a batch of agricultural fungicide (Polyoxin Z). Isolated fractions were toxic to a particulate chitin synthetase preparation from Candida albicans. Different strains of Candida albicans exhibited varied sensitivities to a series of peptide analogues. From a sensitive strain, B2630, spontaneous mutants were selected for resistance to each analogue; certain mutants showed cross-resistance to other analogues and associated defects in peptide transport. A bacilysin-resistant mutant was cross-resistant to the other analogues and to m- fluorophenylalanylalanylalanine a but retained sensitivity to m- fluorophenylalanylalanylalanine. This mutant showed defective dipeptide transport but normal oligopeptide transport, and was unable to utilize Ala-Ala as a sole nitrogen source. Thus, Candida albicans has distinguishable mechanisms for dipeptide and oligopeptide transport which can be exploited for uptake of peptide-drug adducts. Peptide transport was shown to be stimulated by the presence of peptides (peptone) in the growth medium. On transferring cells from minimal to peptone medium, this stimulatory effect was shown to be rapid, independent of protein synthesis and to override ammonia regulation of peptide transport. The reduction of transport activity on transferring cells from peptone to minimal medium was also rapid. It was speculated that regulation of peptide transport is achieved by a rapid, reversible activation of preformed transport components, or a mechanism of exocytotic insertion and endocytic retrieval of preformed transporters. The effect of protein-modification reagents on transport activity was also examined. Dipeptide transport was specifically inhibited by N-ethyl-5-phenylisoxazolium-3'-sulphonate (Woodwards Reagent K), offerring potential for the specific labelling of the component(s) of this system. Peptide transport was shown not to be sensitive to osmotic shock though a series of uncharacterized polypeptides was released by the shock treatment.

We consider a novel extension of the recently proposed mimetic gravity. The latter is a scalar-tensor theory which is able to describe dark matter on cosmological scales. Moreover, this theory can be considered as a low energy limit of the projectable Horava-Lifshitz gravity. The proposed novel extension directly couples gradients of the mimetic scalar field to the curvature tensor. These couplings introduce into the energy momentum tensor an anisotropic stress which is non-vanishing even at the first order in perturbations around a cosmological background. Further we show that such terms modify the formula for the speed of sound of scalar perturbations and even more importantly change the speed of propagation for the gravitational waves. The appearance of the anisotropic stress and the consequent nontrivial speed of propagation of the gravity waves are new phenomena which were not present in the previously studied mimetic models. Furthermore, we demonstrate that the effective Newton's gravitational constant in the background Friedmann equations is shifted in the presence of the novel couplings of the mimetic scalar field. We calculate the quadratic action for scalar and tensor perturbations and briefly discuss possible instabilities. Finally we consider the current observational bounds on the model....

La présente recherche porte sur les applications possibles de la théorie mimétique de René Girard à des oeuvres cinématographiques américaines et européennes de trois périodes : l'entre-deux-guerres,les années 1970 et les années 2000.Cette étude se situe dans le champ d'une sociologie de l'imaginaire dont l'anthropologie du religieux constitue l'axe épistémologique principal.A partir des notions-clés de mimesis, de sacré, de sacrifice et de violence comme termes permettant de penser la sociogenèse, nous tentons de renouveler la lecture d'oeuvres classiques et récentes du cinéma d'auteur américain et européen. Le lien originel de la violence et du sacré constituerait,selon nous, un axe interprétatif essentiel pour la compréhension du lien du social à une violence fondatrice. Dans notre étude, nous analysons les oeuvres cinématographiques comme autant de mythes et de rituels affaiblis, héritiers du sacré et susceptibles à ce titre de transfigurer la violence.Quel sens donner à cette transfiguration esthétique aujourd’hui ? C’est en nous focalisant sur le passage historique du tiers au double, dont les films que nous analysons sont autant de jalons, que nous avons voulu reconstituer ce « parcours de la méconnaissance » qui semble se confondre avec la violence et le même. / The present study is concerned with the possible applications of René Girard’s mimetic theory toAmerican and European films from three different periods : the interwar period, the 1970s and the2000s.This work belongs to the field of the ‘sociology of the imaginary’, of which religious anthropologyconstitutes the main epistemological axis.Based on key notions such as mimesis, the sacred, sacrifice and violence, which facilitate thereflection on sociogenesis, this study represents an attempt to renew the interpretation of classic, aswell as recent, american or european art-house cinema. The original link between violence and thesacred may offer an essential interpretative axis for a proper understanding of the relationshipbetween social matters and a founding violence. In this work, we approach cinematic works asweakened forms of myth and ritual, heirs of the sacred which are, for this very reason, liable totransfigure violence. Which meaning can be given to this aesthetic transfiguration nowadays ? Byfocusing on the historical transition from the third to the double, of which the movies analyzed hereare milestones, I have attempted to reconstruct

Efficient separation of target protein from impurities is crucial in bioseparation for large scale production and purity of the target protein. Two separation process approaches were considered in this study. The first approach focused on identifying major impurity and optimization of solution properties for target protein purification. The second approach consisted of designing an adsorbent that interacted specifically with the target molecule. The first study included modification of protein solution properties (pH, ionic strength, buffer ions) in order to maximize lysozyme purification by a strong cation exchange resin. The interaction of phytic acid, a major impurity, present in transgenic rice extracts, that contributes to decreased lysozyme adsorption capacity on SP Sepharose was evaluated. The target protein was lysozyme, which is used in a purified form as a baby formula additive to reduce gastrointestinal tract infections. At constant ionic strength, lysozyme in pH 4.5 acetate buffer had a higher binding capacity and stronger binding strength than at pH 6.0. Lysozyme in sodium phosphate buffer of pH 6.0 exhibited lower adsorption capacity than in pH 6 Tris buffer. Binding capacity and strength were significantly affected by phytic acid in all studies buffers. The second study consisted of surface modification of microfiltration membranes for protein purification and separation and reduces fouling. This study describes adsorption and fouling of chemically modified microfiltration membranes with bovine serum albumin (BSA) and immunoglobulin G (IgG). Least fouling resulted with polyethylene glycol (PEG) membranes when BSA protein was used. Amine-functionalized membranes showed specific interaction with BSA. There was multi-layer deposition of IgG on amine-functionalized membrane. G3 membrane synthesized to selectively bind IgG seemed a noble option to separate IgG from a protein mixture.

Collagen I, a natural protein found in animal tissues, can self-assemble into fibrous matrices that support cell and tissue growth. Peptide mimics of collagen are able to recapitulate this selfassembly process towards the development of biomaterials for tissue engineering. In recent years, the metal mediated self-assembly of collagen mimetic peptides (CMPs) has allowed access to various particle morphologies. Herein, two studies are presented. In the first, NCOH-FOGER, a cell adhesive CMP capable of metal-triggered self-assembly, was utilized to develop a model system to mimic natural collagen’s interactions with endothelial cells. Notably, a cobalt(III)- NCoH-FOGER assembly was able to induce endothelial cells to form network-like structures. In the second, a CMP was modified to include an unnatural amino acid, L-4-trans-fluoroproline, which increased the thermostability of its folded state. The effect of this substitution on the morphology of self-assembled particles was evaluated.

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No description available.

Background: Respiratory syncytial virus (RSV) is one of the leading causes of acute lower respiratory tract infection and childhood hospitalization worldwide. However, there are currently no vaccines or antivirals available to prevent or treat RSV infections. Of the 11 proteins encoded by RSV’s negative-sense single-stranded RNA genome, the nucleoprotein, phosphoprotein, and large polymerase interact through well characterized domains to form the RNA-dependent RNA polymerase complex. This polymerase complex is essential for viral replication and virulence, which makes it an excellent antiviral target. Previous studies have shown that the nucleoprotein-phosphoprotein interaction of the polymerase complex can be disrupted by synthetic peptides of the last 21 C-terminal (P220-241) or the first 29 N-terminal (P1-29) amino acids of the phosphoprotein. Objective: The Mahony lab has also previously demonstrated that P220-241 conjugated to a maltose binding protein (MBP) and HIV-1 Tat cell penetrating peptide (CPP) could inhibit up to 90% of RSV A replication in vitro. However, the bacterial derived MBP is immunogenic. This study builds on these findings by developing and evaluating the efficacy of a P220-241 peptide mimetic conjugated to human thioredoxin (hTrx) carrier protein and a P1-29 peptide mimetic conjugated to MBP. Methods and Results: Inverse PCR and In-Fusion® cloning was used to clone a hTrx-P220-241 plasmid, which was then expressed as a recombinant protein and purified by affinity chromatography for functional analysis. HTrx-P220-241 was shown to specifically interact with RSV nucleoprotein in a glutathione S-transferase (GST) pull down assays and it could successfully enter into LLC-MK2 cells. However, upon challenge with RSV A, LLC-MK2 cells that were incubated with increasing concentrations of hTrx-P220-241 did not inhibit RSV A replication when assessed by indirect immunofluorescence microscopy. The MBP-P1-29 construct did not exhibit any significant cytotoxicity in LLC-MK2 cells nor BEAS-2B cells. Upon challenge with RSV A, LLC-MK2 cells and BEAS-2B cells pre-treated with MBP-P1-29 demonstrated a dose-dependent inhibition of RSV replication in vitro, with a percent inhibition of infection of 80% and 60% respectively. Furthermore, MBP-P1-29 also reduced the release of infectious progeny virion by up to 74% in LLC-MK2 cells and 34% in BEAS-2B cells. Conclusion: Phosphoprotein peptide mimetics targeting essential nucleoprotein-phosphoprotein interaction are a promising approach in the development of therapeutic treatments for RSV. In this study, a P220-241 peptide mimetic conjugated to a human thioredoxin scaffold protein was not able to inhibit RSV A replication while a P1-29 peptide attached to a maltose binding protein was effective in reducing RSV replication in vitro. Thus, further studies are required to evaluate a P1-29 peptide mimetic against different RSV A and B strains and to find an appropriate human carrier protein to attach it to. / Thesis / Master of Science (MSc) / Respiratory syncytial virus is a respiratory illness that is one of the leading causes of childhood hospitalization worldwide. RSV infects almost all infants at least once by the age of two. It can also repeatedly infect individuals throughout their lives, which puts the elderly and individuals with weak immune, cardiac or pulmonary systems at risk. There are also no approved vaccines or antiviral treatments available to prevent or combat a RSV infection, which highlights the pressing need for the development of new antiviral drugs. This thesis focuses on developing and evaluating the efficacy of two different antiviral peptides, which both target and disrupt the formation of the viral machinery required for the replication of the RSV genome.

Thesis (MSc (Chemistry and Polymer Science))--University of Stellenbosch, 2009. / Thesis submitted in partial fulfilment for the degree of Master of Science (polymer science) at Stellenbosch University / ENGLISH ABSTRACT: The goal of this study was to synthesize hybrid hydrogels via a chemical crosslinking mechanism through use of chain end functional poly(N-vinyl pyrrolidone)(PVP) with various topologies. The crosslinking chemistries should be benign in nature i.e. at physiological pH ranges and at 37 °C. The degradation products should be biologically tolerable and renal clearance should be possible (< 30 000 g/mol PVP0. PVP of various topologies, controlled molar mass and quantitative chain end functionality was obtained via Reversible Addition Fragmentation chain Transfer (RAFT) mediated polymerization (PDI = 1.1- 1.4). The synthesized polymers were chain end functionalized to introduce thiol or aldehyde moieties. Thiol chain ends were obtained through post polymerization modification of xanthate functional PVP with either aminolysis or reduction. The aldehyde moiety was obtained by post polymerization modification of xanthate end functional PVP with sequential hydrolysis and thermolysis. Thiol functional four arm star PVP was reacted with acrylate difunctional poly(ethylene glycol) (DIAC PEG) crosslinker under standard Michael addition conditions. In order to obtain thioether crosslinked hydrogels from tetra functional star PVP molecules it was found that a minimum thiol functionalization of 30% and a molar ratio of acrylate:thiol of 1:1.1 is required. The Schiff base reaction was used to synthesize imine or secondary amine (after reduction) crosslinks with the lysine residues on either lysozyme or bovine serum albumin (BSA) or the primary amines of bis-(2-amino ethyl)amine). Hydrogels were obtained from aldehyde functionalized PVP molecules with a fraction of functional aldehyde chain ends of 0.88 for difunctional molecules and 0.50 for tetra functional star PVP molecules with lysozyme or BSA crosslinkers. The reaction rate was favoured by lowered pH (<6.0) and an optimum molar ratio of amine : aldehyde of 1:0.8. Hydrogels were analyzed by equilibrium swelling calculations to determine the molar mass between crosslinks and the estimated pore size. In both crosslinking systems the properties of the formed hydrogels were seen to be affected by molar ratio used and by the topology of the crosslinking agent. PVP BSA and PVP PEG hydrogels were tested for 24 h and 48 h cell viability by using H9C2 myoblast cells. A concentration range of 0.25 x 10(2) to 0.01 g/mL was studied. Cell mortality was tested by Trypan blue staining and results were verified with MTT assay. A very low cell death precentage (<37%)was observed. Cells even appeared to experience a stimulatory effect after 48 h of exposure at low concentrations of PVP PEG hydrogel treatments. The properties of the formed hydrogel could be tuned by the molar mass ratios of PVP and crosslinker. The functionality of the crosslinker directly affects the molar mass between crosslinks and thus indirectly the degradation profile. It was concluded that PVP molecules with various topologies, well-defined molar masses and chain end functionality could be obtained via RAFT mediated polymerization. Obtained polymers were successfully modified and crosslinked to obtain hydrogels with stoichiometrically tuneable properties i.e. initial swelling ratio, degradation time, molar mass between crosslinks. The hydrogels had very positive cell viability results that would definitely justify further research into these materials as “tissue-mimetic” materials. / AFRIKAANSE OPSOMMING: Die doel van die studie is om poli(N-viniel pirollidoon) (PVP) gebaseerde hibried hidrogelle te sintetiseer deur middel van kovalente kruisbindings met toepaslike kruisverbinder molekules. Die chemiese reaksies betrokke in die vorming van hierdie kovalente kruisbindings moet gematig van aard wees, by fisiologiese pH en by 37 °C plaasvind. Die degradasieprodukte van die hibried-hidrogel moet biologies verdraagsaam en ook uitskeibaar deur die endokrinologiese sisteem wees. PVP van verskillende topologieë, beheerde molêre massa en kwantitatiewe kettingendfunksionaliteit is berei deur ‘n omkeerbare addisie-fragmentasiekettingoordrag (OAFO)-beheerde polimerisasieproses (PDI = 1.1-1.4). Xantaat-kettingend-PVP is aangepas na thiol of aldehied kettingendfunksies. Thiolendfunksies is verkry deur middel van ‘n aminolisasie-reaksie. Xantaat kettingend-PVP is stapsgewys gemodifiseer deur hidroliese en verhittingstappe om die aldehied ketting-endfunksionaliteit te bekom. Thiol ketting-endfunksionele vier-armige ster-PVP is kovalent gebind aan difunksionele poly(etileen glikol) deur middel van die Michael-addisiereaksie. PVP PEG hidrogelle het slegs gevorm met vier-armige ster-PVP molekules wat oor ‘n minimum van 30 % thiol-funksionaliteit beskik het en ‘n optimale molêre massa verhouding van 1:1.1 vir ankrilaat to thiol. Die Schiff-basisreaksie is gebruik om hidrogel te sintetiseer wat met imiene of amiene (na redusering) kovalente bindings gekruisbind is. In hierdie sisteme het hidrogel slegs gevorm as die aldehied-PVP molekules oor ‘n fraksie funksionele kettingend-waarde van 0.88 vir dialdehied-PVP molekules en 0.5 vir vier armige ster-PVP molekules beskik het. Die reaksie snelheid van die Schiff-basis kovalente bindings is bevoordeel deur die pH te verlaag (≤ 6.0) en ‘n gunstige molêre massa verhouding van 1:0.8 vir die nukleofiel teen oor die akseptor molekule is waargeneem. Ewewigswel berekeninge is gebruik om die molêre massa tussen kruisbindings en die gemiddelde benaderde porieë binne die drie-dimensionele interne struktuur van die hydrogel te bepaal. Die seltoksisiteit van PVP-BSA en PVP-PEG hidrogelle is oor 24 h en 48 h in die teenwoordigheid van H9c2 mioblast-selle getoets. Die hydrogel behandelings is uitgevoer in ‘n konsentrasie reeks van 0.25 x 10(2) tot 0.01 g/mL. Selmortaliteit is getoets deur ‘n Trypan-blou verkleuringstudie. Hierdie resultate is ondersteun deur MTT sel-lewensvatbaarheidstoetse. ‘n Lae selmortaliteit (≤ 37 %) is waargeneem en, opspraakwekkend, het van die selle na 48 h verhoogde vitaliteit getoon in die teenwoordigheid van lae konsentrasies PVP-PEG hidrogelle. Dit is bevind dat hidrogel eienskappe deur stoichiometriese molêre massa verhoudings asook die keuse in die topologie van kruisverbinder beïnvloed word. Hierdie eienskappe het ‘n direkte effek op die degradasieprofiel van die gevormde hidrogel. Samevattend dus is PVP molekules met ‘n variasie van topologieë, spesifieke molêre massas en kettingfunksionalitete deur middel van OAFO-gemedieerde polimerisasies gesintetiseer. Xantaatkettingendfunksionele PVP-molekules kon suksesvol omgeskakel word na die kettingendfunksionaliteit van ons keuse om ‘n hibriedhidrogel met stoichiometries-manupileerbare eienskappe te sintetiseer. Die positiewe sel-lewensvatbaarbheidstudie resultate staaf verdere ondersoeke in hierdie PVPgebaseerde hibried hidrogelmateriaal as ‘n weefsel nabootsingsmateriaal.