The production of retroviral vectors for gene therapy applications faces a number of challenges. Of primary concern is the low titre of vector stocks produced by packaging cells in culture and the inherent instability of retroviral vector activity. A systematic investigation of culture parameters that can effect vector titre was conducted. Physical and chemical factors including temperature, pH, medium composition, dissolved oxygen and serum concentration were all assessed. In addition, a number of studies were undertaken to assess the effects of packaging cell growth rate on vector production. The use of a packed bed, as a novel system for large-scale vector production, was also investigated. Prolonged production of retroviral vector stocks was demonstrated in the packed bed system with immobilised packaging cells. Determination of the critical culture parameters allowed optimisation of culture conditions, which can be continuously controlled in the packed bed system, thereby ensuring optimal vector production throughout the production period. Furthermore, vector decay rates can be reduced by the immediate collection of vectors into a recovery vessel. The studies within this thesis will aid the development of appropriate procedures for the large-scale production and handling of retroviral vector stocks for human gene therapy applications.

The work described in this thesis concerns the design, synthesis, DNA binding and biological activity of palladium(II) supramolecular cylinders that might be capable of recognizing a DNA four-way junction. An introduction to DNA structure will be presented, as well as the different binding modes of natural and synthetic agents that can recognise and bind to DNA. Since this work is focused on the design of large metallo-structures, the general principles of supramolecular chemistry will be summarised with particular emphasis on metallo-supramolecular structures. Palladium(II) supramolecular cylinders have already been reported and these show promising cytotoxicity against different cancer cell lines. However, these complexes present poor solubility in aqueous solutions. It is therefore the aim of this thesis to improve the water solubility of palladium(II) supramolecular cylinders without significantly changing their structure or quenching their cytotoxic activity. The DNA binding properties of the newly synthesised palladium(II) complexes will be presented. Several spectroscopic techniques, such as circular and linear dichroism and ethidium bromide displacement assays, as well as electrophoresis experiments were carried out and these will be discussed. Initial DNA four-way junction experiments and cytotoxicity studies will also be discussed.

In order to enhance the sensing capabilities of DNA, an array of tags and modified nucleic acids have been synthesised. This thesis studies both the sensing capabilities and the stability of electrochemical DNA reporters based on the redox active molecule ferrocene. The topics covered are as follows: 1. Two synthetic ferrocene nucleic acid (FcNA) DNA mimics and a redox-active tag based on two covalently connected ferrocene molecules were incorporated into the backbone of a DNA strand in a central position and the 5’ end of a DNA strand, respectively. These probes were then used to form mixed monolayers and their stability was assessed. DNA target detection was possible with square wave voltammetry due to the unusual reliance of this technique on the ‘critical frequency’ of the redox active molecule. 2. DNA probes containing thymine-modified FcNA reporter groups were investigated further due the ability of mercuric ions (Hg2+) to bind covalently to two opposite thymine groups. Detection of these ions was possible through both cyclic voltammetry and square wave voltammetry. 3. The FcNA-conjugated DNA probes are assessed for their potential as therapeutic antisense agents by exposing the single stranded and duplexed probes to a DNase and two types of exonuclease enzyme.

Biocatalysis processes use biologically-derived enzymes to perform fine-chemical synthesis. Whole-cell biocatalysis, using live microorganisms, offers protection against buffer conditions and denaturation, and allows turnover of effective enzymes. However, cells may still be damaged by reaction conditions. In nature, cell populations protect themselves by attaching to surfaces and producing a multi-component protective extracellular matrix. This multicellular mode of growth is termed a biofilm. Biofilms offer many advantages over individual free-floating cells which may be beneficial in whole-cell biocatalysis. The primary aim of this work was to develop a biofilm platform using non-pathogenic Escherichia coli strains as a generic host for various biocatalysis enzymes. To this end, a simple, inexpensive and reliable biofilm generation method was developed and optimised using quantitative assays and confocal laser scanning microscopy. Reporter gene technology was used to provide insight into the expression of the matrix component curli. Flow cytometry was employed to reveal curli expression heterogeneity in biofilm-forming populations. Biofilm-modulating plasmids were used to determine whether improvements could be made to the biofilm-forming strains and their relevant effects were observed. Lastly, three biocatalysis processes were tested in the biofilm biocatalyst with observation of effects on biofilm formation, curli expression and biocatalytic potential.