Entomopathogenic fungi, including Beauveria bassiana are being developed as an alternative to chemical insecticides. Their effectiveness can be enhanced through understanding of the mechanisms of response to environmental stresses and conditions. An aspect of repair of ultraviolet radiation induced DNA damage and response to high temperature were studied here. A region of the photolyase gene (phr-1), encoding cyclobutane pyrimidine dimer photolyase (CPD-PHR), an enzyme pivotal to DNA repair, was cloned, sequenced and identified for species of the genera Beauveria, Isaria, Lecanicillium, Metarhizium and Tolypocladium. The DNA and deduced amino acid sequences were analysed using several in silico methods and annotated for functionality. The data suggested that the DNA encoded a protein with conserved residues known in CPD-PHR function, which had structural homology with other CPD-PHRs and molecular phylogeny that was generally consistent amongst this group of fungi. These results are the first for a phr-1 from the genera Isaria, Lecanicillium and Tolypocladium. In bacteria and yeasts, tolerance to environmental stress was shown to be aided through an inducible phenomenon that involves extracellular sensory component (ESC) proteins in Escherichia coli, which have yet to be purified or sequenced. The presence of an ESC-like factor (ELF) was examined in cell-free filtrate (CFF) from B. bassiana cultures. It was revealed that the tolerance of conidiospores and blastospores (BS) to ultraviolet radiation or heat could be increased by preheated CFF, respectively, but not after pretreating the CFF with trypsin. Several novel polyacrylamide-based in situ and binding bioassays were developed to screen for and characterize ELF candidate (EC) proteins. Two were detected (EC1 and EC2) and EC1 was found to interact with BS, while bioassays with purified ECs showed that EC1 could increase heat tolerance in BS. De novo peptide sequencing revealed that the ECs were the same protein, but differed by EC1 being glycosylated. An expressed sequence tag from B. bassiana, encoding six peptides that were also found in the ECs, was identified in the public data base. This sequence information was exploited to amplify the remaining coding regions of the suspected ELF gene (elf) using polymerase chain reactions. Through this a 741 nucleotide open reading frame was cloned and sequenced. Structure-function analyses of the amino acid sequence encoded by the open reading frame revealed features that were consistent with the ECs, such as eight shared peptides, its nascent derived size (26 kDa), potential glycosylation sites and secretion signal peptide. In addition, other features such as the high proportion of cysteine residues and internal amino acid repeats will be discussed. The elf gene was inserted into an expression vector and introduced into the methylotrophic yeast Pichia pastoris for its controlled over-expression. Heterologously expressed ELF conferred elevated tolerance to heat in BS to similar levels produced by ELF synthesized by B. bassiana. Several functional and molecular features of the ELF system have certain commonalities with many agonist-receptor systems involved in signal transduction, but remain to be detailed. This is the first report of the cloning and functional analyses of elf and ELF, respectively, from any organism.

The chondroitin sulfate proteoglycan (CSPG) aggrecan forms link protein-stabilised complexes with hyaluronan (HA), via its N-terminal G1-domain, that provide cartilage with its load bearing properties. Similar aggregates (potentially containing new members of the link protein family), in which other CSPGs (i.e., versican, brevican and neurocan) substitute for aggrecan, may contribute to the structural integrity of many other tissues including skin and brain. In this thesis, cartilage link protein (cLP) and the G1-domains of aggrecan (AG1) and versican (VG1) were expressed in Drosophila S2 cells, purified to homogeneity and functionally characterised. The recombinant human proteins were found to have properties similar to those described for the native molecules. For example cLP formed dimers, and HA decasaccharides (HA 10-mers) were the minimum size that could compete effectively for their binding to polymeric HA. In addition, gel filtration and protein cross-linking/MALDI-TOF peptide fingerprinting showed that cLP and AG1 interact in the absence or presence of HA. Conversely, cLP and VG1 did not bind directly to each other hi solution yet formed ternary complexes with HA24. N-linked glycosylation of VG1 and AG1 was demonstrated to be unnecessary for either HA binding or the formation of ternary complexes. Additionally, the length of HA required to accommodate two G1-domains was found to be significantly larger for aggrecan than versican, which may reflect differences hi the conformation of HA stabilised on binding these proteins. To further investigate protein-HA interactions, fluorescent HA oligosaccharides were prepared and characterised. HA oligosaccharides labelled with the fluorophore 2-aminobenzoic acid (2AA) from four to 40 residues hi length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterisation of HA oligosaccharides was facilitated by 2AA derivitisation since it enhanced signals in MALDI-TOF mass spectrometry and improves fluorophore-assisted carbohydrate electrophoresis (FACE) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC) labelled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatised oligosaccharides, as demonstrated by their ability to compete for polymeric hyaluronan binding to VG1, AG1 and cLP. An electrophoretic mobility shift assay was used to study VG1 binding to 2AA-labelled HA 8-, 10-, 20-, 30- and 40-mers and although no stable VG1 binding was observed to labelled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA 10-mers was estimated from densitometry analysis of the free oligosaccharide. Interactions involving 2AA labelled HA 20-, 30-, and 40-mers with VG1 also displayed positive cooperativity. Therefore, oligosaccharides labelled with 2-aminobenzoic acid are biologically active and show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Dentistry / Master / Master of Orthodontics

published_or_final_version / abstract / Mechanical Engineering / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Promising strategies for treating diseases and conditions like cancer, tissue necrosis from injury, congenital abnormalities, etc., involve replacing pathologic tissue with healthy tissue. Strategies devoted to the development of tissue to restore, maintain, or improve function is called tissue engineering. Engineering tissue requires three components, cells that can proliferate to form tissue, a microenvironment that nourishes the cells, and a tissue scaffold that provides mechanical stability, controls tissue architecture, and aids in mimicking the cell’s natural extracellular matrix (ECM). Currently, there is much focus on designing scaffolds that recapitulate the topology of cells’ ECM, in vivo, which undoubtedly wields structures with nanoscale dimensions. Although it is widely thought that sub-microscale features in the ECM have the greatest vii impact on cell behavior relative to larger structures, interactions between cells and nanostructures surfaces is not well understood. There have been few comprehensive studies elucidating the effects of both feature dimension and geometry on the initial formation and growth of the axons of individual neurons. Reconnecting the axons of neurons in damaged nerves is vital in restoring function. Understanding how neurons react with nanopatterned surfaces will advance development of optimal biomaterials used for reconnecting neural networks Here, we investigated the effects of micro- and nanostructures of various sizes and shape on neurons at the single cell level. Compulsory to studying interactions between cells and sub-cellular structures is having nanofabrication technologies that enable biomaterials to be patterned at the nanoscale. We also present a novel nanofabrication process, coined Flash Imprint Lithography using a Mask Aligner (FILM), used to pattern nanofeatures in UV-curable biomaterials for tissue engineering applications. Using FILM, we were able to pattern 50 nm lines in polyethylene glycol (PEG). We later used FILM to pattern nanowells in PEG to study the effect of the nanowells on the behavior preadipocytes (PAs). Results of our cell experiments with neurons and PAs suggested that incorporating micro- and nanoscale topography on biomaterial surfaces may enhance biomaterials’ ability to constrain cell development. Moreover, we found the FILM process to be a useful fabrication tool for tissue engineering applications. / text

We are coming to understand that the ecological importance of diatoms is not limited to primary productivity, as many diatoms produce extracellular polymeric substances (EPS), which are vital components in algal and bacterial “biofilms.” While great effort has been made to chemically identify the types of molecules and polymers used to create and modify diatom EPS there is still much about the process we do not know. Rather than studying this process chemically, we have elected to search for the genes involved in EPS production and secretion. We assembled transcriptomes from three EPS-producing diatoms (Cyclophora tenuis, Lucanicum concatenatum, Thalassionema frauenfeldii) and two diatoms which do not (Astrosyne radiata, Thalassionema sp. ‘BlueH20’). In an attempt to limit the differences to EPS-related transcripts, the taxa were selected in a phylogenetic framework (which is also discussed in this dissertation), where EPS-producing taxa were closely-related to taxa which did not produce EPS (A. radiata, C. tenuis, L. concatenatum as one set, T. frauenfeldii and T. sp. ‘BlueH20’ as the other). The resulting pool of transcripts sorted for contigs which appeared in the EPS-producing taxa but not their closely-related non EPS-producing counterparts, and those contigs were then compared to two annotated diatom genomes and sorted by function, looking specifically for genes related to secretion, polysaccharide assembly or modification and carbohydrate metabolism. In the Thalassionema clade, 41 contigs with the aforementioned annotations were found, while 22 such contigs were found in the Cyclophora/Lucanicum/Astrosyne clade. These putative EPS-related markers are identified in this dissertation for further study on their function and evolution across diatoms. / text

No description available.