Integral to the physiological and biochemical properties of the plant, the primary cell wall (PCW) is of great economical interest. Transglycanases are a class of cell-wall remodelling enzymes hypothesised to be involved – among other functions – in cellular elongation and strengthening of the PCW. At present only four transglycanases have been convincingly characterised but the potential existence of many more is likely. To detect potential novel transglycanase activity, broad spectrum fluorescent and radioactive assays were conducted using a variety of potential donor and acceptor substrates. Enzyme extracts were sourced from a range of plants that represented the majority of the plant kingdom. Beansprout extracts reproducibly displayed significant incorportation of radioactivity and fluorescence when incubated with an α-arabinan or β- galactan donor and labelled xyloglucan oligosaccharide (XGO) acceptor. However, further analysis indicated the presence of xyloglucan contamination in donor polysaccharides and thus the activity observed was xyloglucan endotransglucosylase (XET). It has been hypothesised in the literature that linkages formed between the hemicellulosic and pectic matrices may be due to the activity of a transglycanase. This study has found no evidence to support this. In addition, during identification of the gene responsible for mixed-linkage β- (1,3),(1,4)-glucan : xyloglucan endotransglycosylase (MXE) activity – observed in Equisetum – a heterologous Pichia pastoris expression system was developed allowing the synthesis of a novel recombinant hetero-transglycanase (HTG) conferring predominant MXE activity and of five previously unstudied recombinant XET-active xyloglucan endotransglycosylase/ hydrolases (XTHs).

By serial subcultivation on media containing egg-white lysozyme a highly resistant variant of M. lysodeikticus bacteriophage was selected. The parent strain was sensitive to 0.1 pg per ml. lysozyme while the variant was resistant to 4000 pg per ml. The two strains were examined in detail and it was found that the resistant strain differed genotypicelly from the sensitive strain in its ability to acetylate certain cell wall hydroxyl groups. This was the sole reason for lysozyme resistance. It was found that deacetylation of the resistant strain by ample chemical treatment restored its sensitivity to lysozyme while acetylation of the sensitive strain rendered it resistant.

Severe stenosis may cause critical flow conditions related to artery collapse, plaque cap rupture which leads directly to stroke and heart attack. In this paper, a nonlinear viscoelastic model and a numerical method are introduced to study dynamic behaviors of the tube wall and viscous flow through a viscoelastic tube with a stenosis simulating blood flow in human carotid arteries. The Mooney-Rivlin material model is used to derive a nonlinear viscoelastic thin-wall model for the stenotic viscoelastic tube wall. The mechanical parameters in the Mooney-Rivlin model are calculated from experimental measurements. Incompressible Navier-Stokes equations in the Arbitrary Lagrangian-Eulerian formulation are used as the governing equation for the fluid flow. Interactions between fluid flow and the viscoelastic axisymmetric tube wall are handled by an incremental boundary iteration method. A Generalized Finite Differences Method (GFD) is used to solve the fluid model. The Fourth-Order Runge-Kutta method is used to deal with the viscoelastic wall model where the viscoelastic parameter is adjusted to match experimental measurements. Our result shows that viscoelasticity of tube wall causes considerable phase lag between the tube radius and input pressure. Severe stenosis causes cyclic pressure changes at the throat of the stenosis, cyclic tube compression and expansions, and shear stress change directions in the region just distal to stenosis under unsteady conditions. Results from our nonlinear viscoelastic wall model are compared with results from previous elastic wall model and experimental data. Clear improvements of our viscoelastic model over previous elastic model were found in simulating the phase lag between the pressure and wall motion as observed in experiments. Numerical solutions are compared with both stationary and dynamic experimental results. Mooney-Rivlin model with proper parameters fits the non-linear experimental stress-strain relationship of wall very well. The phase lags of tube wall motion, flow rate variations with respect to the imposed pulsating pressure are simulated well by choosing the viscoelastic parameter properly. Agreement between numerical results and experimental results is improved over the previous elastic model.

Superhydrophobic surfaces are able to entrap gas pockets in-between surface roughness elements when submerged in water. These entrapped gas pockets give these surfaces the potential to reduce drag due to the overlying flow being able to locally slip over the gas pockets, resulting in a mean slip at the surface. This thesis investigates the different effects that slip and the texturing of the surface have on turbulence over superhydrophobic surfaces. It is shown that, after filtering out the texture-induced flow, the background, overlying turbulence experiences the surface as a homogeneous slip boundary condition. For texture sizes, expressed in wall units, up to $L^+ \lesssim 20$ the only effect of the surface texture on the overlying flow is through this surface slip. The direct effect of slip does not modify the dynamics of the overlying turbulence, which remains canonical and smooth-wall-like. In these cases the flow is governed by the difference between two virtual origins, the virtual origin of the mean flow and the virtual origin experienced by the overlying turbulence. Streamwise slip deepens the virtual origin of the mean flow, while spanwise slip acts to deepen the virtual origin perceived by the overlying turbulence. The drag reduction is then proportional to the difference between the two virtual origins, reminiscent of drag reduction using riblets. The validity of slip-length models to represent textured superhydrophobic surfaces can resultantly be extended up to $L^+ \lesssim 20$. However, for $L^+ \gtrsim 25$ a non-linear interaction with the texture-coherent flow alters the dynamics of the background turbulence, with a reduction in coherence of large streamwise lengthscales. This non-linear interaction causes an increase in Reynolds stress up to $y^+ \lesssim 25$, and decreases the obtained drag reduction compared to that predicted from homogeneous slip-length models.

The dematiaceous (melanised) fungus Lomentospora (Scedosporium) prolificans is a life-threatening opportunistic pathogen of immunocompromised humans, resistant to anti-fungal drugs. Melanin has been shown to protect human pathogenic fungi against antifungal drugs, oxidative killing and environmental stresses. To determine the protective role of melanin in L. prolificans to oxidative killing (H2O2), UV radiation and the polyene anti-fungal drug amphotericin B, targeted gene disruption was used to generate mutants of the pathogen lacking the dihydroxynaphthalene (DHN)-melanin biosynthetic enzymes polyketide synthase (PKS1), tetrahydroxynapthalene reductase (4HNR) and scytalone dehydratase (SCD1). Infectious propagules (spores) of the wild-type strain 3.1 were black/brown, whereas spores of the PKS-deficient mutant ΔLppks1::hph were white. Complementation of the albino mutant ΔLppks1::hph restored the black-brown spore pigmentation, while the 4HNR-deficient mutant ΔLp4hnr::hph and SCD-deficient mutant ΔLpscd1::hph both produced orange-yellow spores. The mutants ΔLppks1::hph and ΔLp4hnr::hph showed significant reductions in spore survival following H2O2 treatment, while spores of ΔLpscd1::hph and the ΔLppks1::hph complemented strain ΔLppks1::hph:PKS showed spore survivals similar to strain 3.1. Spores of the mutants ΔLp4hnr::hph and ΔLpscd1::hph and complemented strain ΔLppks1::hph:PKS showed spore survivals similar to 3.1 following exposure to UV radiation, but survival of ΔLppks1::hph spores was significantly reduced compared to the wild-type strain. Strain 3.1 and mutants ΔLp4hnr::hph and ΔLppks1::hph:PKS were resistant to amphotericin B while, paradoxically, the PKS1- and SCD1-deficient mutants showed significant increases in growth in the presence of the antifungal drug. Melanin was shown to play no role in the protection of the pathogen from immune cell recognition and killing by alveolar macrophages, with similar degrees of engulfment, and spore viabilities, of mutant and wild-type strains after phagocytosis. Contrary to expectations, the albino PKS-deficient mutant was significantly more virulent than the melanised wild-type strain during pathogenicity studies in the invertebrate infection model Galleria mellonella, with levels of virulence restored to near wild-type levels in the complemented strain ΔLppks1::hph:PKS. Taken together, the results presented in this thesis show that melanin protects L. prolificans from UV radiation and from oxidative killing by H2O2, consistent with its survival in extreme environmental habitats. However, melanin was 3 not found to play a role in the resistance of the pathogen to the antifungal drug amphotericin B, to protect the fungus from immune cell recognition or killing by alveolar macrophages, or to its pathogenicity.

Asthma is a common chronic inflammatory condition which affects over 300 million people worldwide. Thickening of the subepithelial layer is a key feature of asthmatic airways and the extent of thickening has been correlated with severity of asthma and increased exacerbations. Recent epidemiological studies have shown a link between fungal sensitisation primarily with Aspergillus fumigatus (A. fumigatus) and exacerbations of asthma leading to increased morbidity and mortality. The airway epithelium acts as an initial defence barrier to inhaled allergens such as A. fumigatus and emerging evidence suggests that as well as orchestrating an allergic immune response, it initiates aspects of airway wall remodelling including subepithelial thickening. However, induction of a profibrogenic response by the airway epithelium following exposure to inhaled fungi associated with severe asthma has not been well documented. The epithelial expression and production of the profibrotic growth factors, TGF-β1, TGF-β2, IL-6, endothelin-1 and periostin, selected as implicated in the aetiology of asthma and their profibrotic activity, were investigated in response to both A. fumigatus spores and culture filtrate in vitro. Furthermore, in vivo chronic inhalation models using either live spores or culture filtrate from two different strains of A. fumigatus (AF293 and CEA10) were used to determine the ability of the fungi to induce murine airway wall remodelling. In vitro, spores from both strains were able to induce the expression and production of IL-6 and endothelin-1 from human bronchial epithelial cells but none of the other profibrotic growth factors. In vivo, despite spores from both strains inducing expression and production of IL-6 and endothelin-1, only CEA10 spores caused significant subepithelial collagen deposition however, both strains induced α-SMA, a myofibroblast and smooth muscle marker around the airways. As a secreted factor was suspected of driving airway wall remodelling, subsequent studies used culture filtrate produced by the two strains, AF293, a low and CEA10, a high protease producer in basal medium. Only AF293 culture filtrate induced IL-6 and endothelin-1 from human bronchial epithelial cells in vitro. However, in vivo, culture filtrate from both strains was able to induce IL-6 and endothelin-1 expression, with AF293 causing a more profound subepithelial collagen deposition and significantly increased α-SMA abundance. It was hypothesised that epithelial-derived endothelin-1 drives airway wall remodelling and hence Endothelin receptor A was inhibited in the in vivo culture filtrate inhalation model. A significant reduction in subepithelial collagen deposition and α-SMA localisation around the airways was demonstrated in mice receiving an Endothelin receptor A antagonist compared with culture filtrate alone. This thesis indicates that A. fumigatus exposure can drive features of airway wall remodelling such as subepithelial fibrosis possibly through the epithelial production of profibrotic growth factor, endothelin-1.

No description available.

Lipoarabinomannan (LAM) and the mycolyl-arabinogalactan (mAG) complex are two major entities found in the cell wall of Mycobacterium tuberculosis, the bacterium that causes tuberculosis in humans. Given their important roles in the viability and virulence of the pathogen, enzymes involved in these pathways represent a rich source of potential therapeutic drug targets. As fundamental understanding of substrateenzyme interactions is often essential in the drug discovery process, the purpose of this study was to investigate the substrate specificities of an -(16)-mannosyltransferase (ManT) and a -(15,6)-galactofuranosyltransferase (GlfT2), two key enzymes involved in the biosynthesis of LAM and mAG, respectively. Although the ManT activity had been detected using an established radioactive assay, its substrate specificity remained poorly defined. The current study focused on the design, synthesis and evaluation of acceptor substrate analogs of ManT. Among those analogs prepared were those containing methoxy-, hydrogen-, and amino-substituted carbohydrate residues as well as epimeric derivatives. A homologous series of oxygen- and sulfur-linked mannosides were also prepared. Evaluation of these analogs revealed the steric requirements and hydrogen bonding interactions of the enzyme, and the effect of acceptor length on mannosyltransferase activity. Also, these results provided additional insight into the role of ManTs and allowed the current proposed pathway of LAM to be further revised. Another objective of the current study was to understand how GlfT2 catalyzes the alternating -(15) and -(16)-galactofuranosyl transfers in a single active site. A panel of mono- and dideoxy trisaccharide derivatives was synthesized, in which hydroxyl groups at either or both C-5 and C-6 positions on the sugar residues at the reducing ends were selectively removed. Biological evaluation of these analogs using a spectrophotometric assay, and structural analysis of some of the enzymatic products, showed that the removal of the hydroxyl group(s) in the acceptors appeared to have no dramatic effect on either GlfT2 activity or the regioselectivity of its galactosylation. These results suggest that groups other than the C-5 and C-6 hydroxyl groups of the acceptors are more critical for the enzyme catalysis. The identification of these key elements would be the further objective of this project. The results from these fundamental studies provide important information about how these enzymes interact with their substrates at the molecular level. More importantly, this work will serve as the basis for the further design of potential inhibitors, which are potential lead compounds for novel therapeutic agents that are active against tuberculosis.

A nonlinear finite element model was developed to study the behaviour of unstiffened steel plate shear walls. The model was validated using the results from quasi-static and dynamic experimental programs. With the validated finite element model, the performance of 4-storey and 8-storey Type D (ductile) and Type LD (limited-ductility) steel plate shear walls with moment-resisting beam-to-column connections was studied under spectrum-compatible seismic records. A design procedure that aims to achieve optimal seismic behaviour for steel plate shear walls was proposed. The proposed method uses the concepts of indirect capacity design principles of CAN/CSA-S16-01 to identify the infill plates that are likely to yield in the design earthquake. The proposed method was used for the design of two 4-storey and one 8-storey shear walls. Design axial forces and moments in the boundary columns for the shear walls were shown to be in good agreement with nonlinear seismic analysis results. Results also showed that some of the other capacity design methods available generally underestimate the maximum design forces in the columns, while others can be overly conservative. The effect of loading rate on the dynamic behaviour of steel plate shear walls was also investigated, as was the P-Delta effect in terms of its influence on seismic demand in shear and flexure. A shear strength model of the infill plate with circular openings at any location was developed based on a strip model where all the strips with perforations were partially discounted. A design method for steel plate shear walls with perforations was introduced. The method was applied for the design of boundary columns of a 4-storey steel plate shear wall with perforations. The predicted design forces in the columns for the 4-storey perforated shear wall agreed well with the forces obtained from nonlinear seismic analysis. Finally, an improved simple formula for estimating the fundamental period of steel plate shear walls was developed by regression analysis of the period data obtained from frequency analysis of series of steel plate shear walls. In addition, the effectiveness of a shearflexure cantilever formulation for determining fundamental periods and P-Delta effects of steel plate shear walls was studied. / Structural Engineering

Abstract In silico analysis showed that Arabidopsis thaliana gene AT5G09530 encodes a uniquely repetitive, proline-enriched protein that is conserved across species, and is likely secreted to the cell wall. Based on its most common amino acid repeat motif, I named the gene PELPK1 and its putative paralog PELPK2 (AT5G09520). Reporter (GUS) expression showed that the PELPK1 upstream genomic region is sufficient for expression in the aleurone layer during seed germination, and is induced throughout the plant by biotic factors (especially Pseudomonas syringae infection), defense chemicals (MeJa, salicylic acid), and mechanical wounding, consistent with the presence of conserved regulatory elements. Sub-cellular localization of a translational fusion of PELPK1 with GFP showed that the protein was secreted into seed-coat aleurone cells and to the cell walls of other tissues. Based on these results, it was concluded that the PELPK1 is a cell wall-associated protein and is most actively transcribed during radicle penetration of the seed coat and during pathogen and wounding responses. A proteomic survey of aleurone proteins failed to identify PELPK1, although several proteins not previously associated with this tissue were identified. Mutational analysis demonstrated that RNAi silencing significantly down-regulated the transcript abundance of PELPK1. Phenotypic analysis showed that RNAi plants exhibited significantly slower germination and root growth when the medium was supplemented with sucrose (100mM). Conversely, constitutive overexpression (OX) of PELPK1 enhanced seed germination and root elongation as compared to wild-type (WT). Analysis of soil-grown plants showed slower emergence and slower vegetative growth for RNAi lines, while OX plants exhibited faster emergence and enhanced vegetative growth and flowering as compared to WT. However, PELPK1 RNAi and OX lines did not differ from WT in response to treatment with pathogens. These results show that the abundance of PELPK1 is positively correlated with plant growth rate under some conditions. PELPK1 may influence growth through CW modification or other independent pathways. / Plant Biology