The work presented in this thesis investigates the use of new luminescent lanthanide complexes, both free and bound to the surface of gold nanoparticles, for protein labelling. Lanthanide complexes were shown to maintain their luminescence properties when conjugated to proteins and one complex also demonstrated participation in Förster resonance energy transfer when conjugated to a protein in an appropriate system. Furthermore, it was found that bovine serum albumin can act as a vehicle to transport luminescent lanthanide complexes into two human cell lines. Lanthanide complexes were then used to coat 13 nm gold nanoparticles for protein labelling within cells. The aim was to find a peptide sequence to preferentially bind to gold nanoparticles which could be expressed as part of a protein of interest, acting as a binding site within the cell. The interaction of peptides with gold nanoparticles was examined using several methods and, of the sequences tested, CCPGCC was found to have the highest affinity for the nanoparticles. This peptide was expressed in HeLa cells as part of green fluorescent protein. Co-localisation of the nanoparticles with the protein in cells could not be established through fluorescence microscopy, however, cell lysis revealed green fluorescence protein associated with nanoparticle aggregate.

The objectives of this thesis are to explore, design, fabricate and implement the use of advanced micro-engineered platforms to be exploited as versatile, novel device technologies. An increasing number of technologies require the fabrication of conductive structures on a broad range of scales and large areas. Here, we introduce advanced yet simple electrohydrodynamic lithography for patterning conductive polymers directly on a substrate with high-fidelity. We illustrate the generality of this robust, low-cost method by structuring thin films via electric-field-induced instabilities, yielding well-defined conductive structures with a broad range of feature sizes. We show the feasibility of the polypyrrole-based structures for field-effect transistors, which might herald a route towards submicron device applications. We also demonstrate a miniaturised platform technology for timely, sensitive and rapid point-of-care diagnostics of disease-indicative biomarkers. Our micro-engineered device technology (MEDTech) is based on reproducible electrohydrodynamically fabricated platforms for surface enhanced Raman scattering enabling tuneable, high-throughput nanostructures yielding high-signal enhancements. These, integrated within a microfluidic-chip provide cost-effective, portable devices for detection of miniscule biomarker concentrations from biofluids, offering clinical tests that are simple, rapid and minimally invasive. Using MEDTech to analyse clinical blood-plasma, we deliver a prognostic tool for long-term outcomes, in the hospital or at the point-of-care.

A commercial Ion Mobility Spectrometer that is designed to detect Chemical Warfare Agents (CWAs), is modified by the addition of a second ion gate, and connected to a commercial Ion Trap Mass Spectrometer (ITMS). The addition of the second gate allows selection of individual ion mobility peaks for m/z analysis in the ITMS. This was demonstrated with the organophosphorus ester ions (CWA nerve gas simulants). The ITMS was used to perform isolation and fragmentation of the CWA simulants ions produced in the IMS. For the organophosphates dimethyl methylphosphonate, diethyl methylphosphonate and diisopropyl methylphosphonate, two ion mobility peaks were observed, which are shown to be the ammoniated monomer and ammoniated dimer ions. Using an ElectroSpray Ionisation (ESI) - ITMS, the fragmentation pathway of dimethyl ethylphosphonate (DMEP) is investigated. The isotopomers of DMEP have unusual fragmentations, and density functional theory calculations are used to aid in the interpretation of the mechanisms involved in these fragmentations. Of note, it is shown that entropy must be taken into consideration, and hence the free energy of the final transition involved in the mechanism, so that the true rate-limiting steps can be determined. Preliminary fragmentations using ESI-ITMS of eighteen other organophosphorus esters have been undertaken. These give an insight as to which fragmentations will require further investigations involving Density Functional Theory (DFT) calculations and deuterated isotopomers to fully understand the mechanisms involved.

This thesis seeks to augment the understanding of gelled micro-particulate suspensions known as sheared/fluid gels, by investigating the use of dairy proteins (whey, WPI) as the gelling material. The research used a microstructural approach to probe the underlying design principles governing the formation, and subsequent material properties of WPI microgel systems. The work initially focused on preparing suspensions through both thermal and cold-set approaches. By controlling two key processing parameters: shear and gelling rate, it was shown that a range of suspension properties could be produced. In both cases, it was demonstrated that structural characteristics could be controlled, for tailored rheologies. The shear technology was then applied to a more complex system of oil and whey protein, resulting in the formation of microcapsules; as the WPI gelled around the oil droplets in a core-shell model. Again, controllable structural properties were obtained, however, the lipophilic core provided a reservoir for potential delivery. This multi-functional formulation was then investigated under gastro-intestinal conditions, highlighting controllable release as a function of the type of oil used in production. Therefore, the potential use of WPI/WPI-oil micro-particles have been presented as a multi-functional composite for both structure and delivery within food ingredients.

This work presents an evaluation of the applicability of gene reporter technology to monitor Escherichia coli stress in industrial conditions with special interest in recombinant protein production. Different reporter plasmids containing promoter sequences of genes of the heatshock response were utilized to monitor chaperone expression upon different sources of stress such as exposure to chemicals, temperature and anaerobic growth. Activation of the heat shock response was monitored by \(\beta\)-galactosidase activity from the reporter plasmid pQF50groE. Cultures responded to heat-shock, anaerobiosis and \(\beta\)-mercaptoethanol by increasing the expression of \(\sigma\)\(^{32}\)-related genes. The performance of fluorescence reporters containing varieties of GFP was measured by fluorimetry and flow cytometry. Low copy number plasmids were demonstrated to be better suited than medium-high copy plasmids to report stress in industrial conditions. Reporter plasmids containing the promoters of the chaperones DnaK and GroES were utilized to measure E. coli stress in reducing environments and during recombinant protein production. It was demonstrated that the production strategy caused an impact in the host physiology which determined the outcome of the process. Flow cytometry showed excellent potential to obtain reliable measurements providing data of reporter activity cell death and cell morphology.

Retroviral vectors are a promising tool for gene therapy. However, there are two major problems to overcome if a viable commercial production process is to be established. These are the instability of virus particles and the low virus titres. The characteristics of the producer cells were determined in batch culture, semicontinuous culture and semi-continuous culture at 32°C. Additionally, cell attachment, growth and virus production on various macroporous microcarriers was assessed under static and stirred conditions. Alternative strategies for the cultivation of cells were also investigated. These included spinner basket, packed bed and spinner flask cultures with semi-continuous feeding and packed bed, fixed bed, fluidised bed and stirred tank cultures with continuous perfusion of culture medium. Of these the fixed bed bioreactor had the highest cell specific productivity and was capable of running for 28 days. The fluidised bed bioreactor had the highest reactor productivity, due to the higher cell number. Optimisation of culture medium was performed with regard to serum concentration. The greatest production was observed at an initial serum concentration of 2.5% (v/v). The findings in this thesis will assist the development of an efficient method for the production of clinical grade retro viral vectors for gene therapy applications.

The purpose of this research was to contribute to the development of H\(_2\) production technologies from biomass. The study integrated thermochemical processes to achieve biomass hydrolysis with biological methods to then obtain H2 by the fermentation of these hydrolysates using E. coli. Different strains of E. coli were tested under controlled conditions in 3 L scale fermentations with the aim to find the most useful strain for the fermentation process in terms of H\(_2\) produced and the subsequent hydrogen production potential of the organic acid co-products in a downstream photofermentation. Among the strains tested FTD89, FTD67 and RL009 gave the best results, however ethanol was successfully abolished by strain RL009 making this strain more suitable for long term fermentations. Model polysaccharide compounds such as starch and cellulose, and representative food and lignocellulosic wastes were hydrolysed in hot compressed water in the presence of CO\(_2\) under pressure and various temperatures to produce hydrolysates with high sugar content suitable for fermentation for H\(_2\) production. Optimum hydrolysis conditions for maximum sugar yields for each compound were determined. Fermentation of the obtained hydrolysates yielded acceptable amounts of H\(_2\) after their ‘detoxification’ with activated carbon (AC), comparable to H\(_2\) yielded by the glucose controls in all cases. The maximum yield of glucose after HCW treatment was obtained from starch at 200 °C yielding 548 g C.kg C initial starch\(^{-1}\); maximum glucose yield from cellulose was 225 g C.Kg C initial cellulose\(^{-1}\) obtained from cellulose hydrolysis at 250 °C, and the glucose yield from food waste was 45.5 g.g food waste\(^{-1}\). The main degradation product (DP) from these hydrolysates was 5 Hydroxymethylfurfural (5-HMF), whereas the main DP obtained from the lignocellulosic wastes was Furfural. Both were successfully removed by AC treatment. The best hydrolysate obtained from wastes was evaluated for H\(_2\) production at 3 L scale. Despite obtaining low H\(_2\) yields improvements would be possible and are discussed. Fermentations for H\(_2\) production at pilot plant scale were also trialled, indicating key areas for future development for successful scale up.

Thesis (Ph. D.)--Lehigh University, 2000. / Includes vita. Includes bibliographical references (leaves 181-185).

Thesis (PhD--Mechanical Engineering)--University of Auckland, 2009. / " A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Engineering." "Centre for Advanced Composite Materials." Includes bibliographical references.

Cost effective processing of wheat straw using solid state fermentation (SSF) would provide a source for value added chemicals from agricultural waste biomass. Fungi natural breakdown lignocellulosic biomass hence could have received a lot of attention. In this study the ability of S. lacrymans to convert straw waste was compared with other Basidiomycetes (Postia placenta, Phanerochaete chrysosporiom, and Schizophyllum commune). S. lacrymans out performed the other Basidiomycetes both in its growth (as measured by ergosterol and fatty acid production (linoleic acid);18:2n6c) , and in the comounds released which included; total soluble phenols, total reducing sugars, and low molecular organic chemicals (MW<400). Non-enzymatic breakdown requiring the presence of Fe2+ was also demonstrtated and influenced by the production of quinone and low molecular organic acid. The amount of the fungal extract used and the concentration of chelator/reducing agents also affected the production of Fe2+. Changes in the lignocellulose structure was also detected as key functional group, such as the pyranose ring and aromatic skeletal vibration were significantly reduced following culture with S. lacrymans and a significant reduction in mass was measured. Iron reductase genes IR1 and IR2 suspected to be involved in the lignocellulose degradation were cloned. It seems that these genes are more closely related to the cellulose binding module (CBM) family instead of cellobiose dehydrogenase (CDH) genes as first suspected. IR1 has an open reading frame of 774 bp which encoding 258 amino acid (55 kDa), whilst IR2 642 bp encoding 214 amino acid ( 49 kDa). The IR1 gene contains a CBM1 domain which is lacking in IR2. Gene expression analysis using qRT-PCR showed that in the early stage of fungal growth, the level of IR2 genes expression was higher than IR1 while IR1 became more dominant in the latter stages of culture. The time at which these genes are highly expressed correlated with the release of soluble and aromatic phenolic compounds. The functionality of the recombinant IR1 and IR2 on the decomposition of lignocellulose was shown using several assays including; iron reductases, nitrated lignin and the reduction of electron acceptor (DCPIP). In addition, using both synthetic and nature sources of cellulose or lignocellulose (Avicel and wheat straw powder) the recombinant IR proteins were shown to break down cellulose. This suggested that these enzymes represent a significant addition to those currently used within biomass based biorefineries.