The daf-2 gene is a key regulator of growth, metabolism and longevity in the nematode Caenorhabditis elegans. The DAF-2 receptor functions in a pathway that is analogous to mammalian insulin/insulin-like growth factor signalling and determines whether animals proceed with full reproductive development or arrest in a long-lived diapausal state known as the dauer larva. Temperature-sensitive hypomorphic mutants of daf-2 constitutively arrest as dauers when raised at non-permissive temperatures. At permissive temperatures the animals develop into adults that are long-lived compared to wild-type adults. These alleles of daf-2 can be separated into two distinct classes (1 and 2) based on their pleiotropic phenotypes. In addition to their phenotypic differences, the two classes also differ in their epistatic interactions with other genes involved in dauer formation, suggesting that the DAF-2 receptor has multiple signalling outputs. In this thesis I have investigated the nature of the daf-2 allele class difference using a range of methods, including sequence analysis and homology modelling of mutant receptors. This generated the prediction that signalling flux through the receptor is a determinant of the class difference, with class 2 alleles having an asymmetrical alteration in signal transduction through DAF-2. Experimental testing of these predictions suggest that some phenotypes such Eat and Unc may be associated with asymmetrical signalling, while others such as early larval arrest may be correlated with a reduction in receptor level at the plasma membrane. A comparative analysis of the DAF-2 receptor with its homologues from Caenorhabditis briggsae, Caenorhabditis remanei and the parasitic nematode Brugia malayi suggests the large C-terminal extension in the Caenorhabditis species, which shorter in Brugia and not present in vertebrates, is an adaptive trait that has evolved by exon duplication for rapid growth and development and may contribute to the shortevity of these species. In addition, I have also performed an analysis of ins-7 and ins-35, two putative ligands of DAF-2 that are differentially regulated by the transcription factor DAF-16, using RNAi. Knockdown of both these genes lead to slight lifespan extension (10-20%) at 20 °C in all genetic backgrounds and slight suppression (-10%) at 25 °C in long-lived daf-2 mutant backgrounds compared to controls. This suggests that INS peptides may function as agonists a 20 °C and antagonists at 25 °C, and that this behaviour may be independent of their transcriptional regulation.

The recent developments in experimental methods for gene data analysis, called microarrays, provide the possibility of interrogating changes in the expression of a vast number of genes in cell or tissue cultures and thus in depth exploration of disease conditions. As part of an ongoing program of research in Guy A. Rutter (G.A.R.) laboratory, Department of Biochemistry, University of Bristol, UK, with support from the Welcome Trust, we study the impact of established and of potentially new methods to the statistical analysis of gene expression data.

Studies on G-quadruplex DNA have grown rapidly due to the potential important roles this type of DNA may play in biology. Stabilisation of telomeric G-quadruplex DNA is thought to inhibit telomerase, an enzyme overexpressed in 85-90 % of cancerous cells. There are also evidences that formation of quadruplex DNA in the promoter region of certain oncogenes (e.g. c-MYC) can suppress their transcription. Therefore, targeting G-quadruplex DNA with small molecules could have interesting applications in cancer therapy. Although most of the quadruplex stabilising molecules reported to date are purely organic G-quadruplex binders, more recently, the ability of metal complexes to stabilise quadruplex DNA has gained more attention due to their unique structural and functional features. The research presented in this thesis aimed at expanding the metal salphen family of complexes to improve affinity and selectivity. The work in this thesis described the design and synthesis of a second generation of nickel(II), copper(II), zinc(II) and platinum (II) salphen complexes to explore the effect of different substituents of the ligand core on quadruplex DNA stabilisation and their selectivity towards quadruplex over duplex DNA. In addition to the above, heteroleptic cycloplatinated complexes have also been prepared in this research to investigate their potential as G-quadruplex DNA stabilisers. Several studies were carried out to elucidate the detailed binding mode between this family of complexes and quadruplex DNA. The interaction between the synthesised metal complexes with G-quadruplex DNA has been examined by means of fluorescent intercalator displacement (FID) assays, UV/Vis DNA titration, circular dichroism (CD) and fluorescence resonance energy transfer (FRET) melting assays. Selected complexes which have shown high quadruplex stabilisation, were co-crystallised with quadruplex DNA. Two X-ray crystal structures have been obtained showing that the metal complexes interact with human telomeric DNA via π-π stacking at the end of G-tetrad.

Porphyrin moieties were rigidly attached to DNA to generate an accurate molecular ruler. Molecular ruler analysis was conducted using steady-state fluorescence, circular dichroism and small angle X-ray scattering spectroscopic techniques, in an attempt to analyse the FRET, exciton coupling and scattering intensity between different porphyrin-porphyrin labelled DNA combinations. A 21-mer test sequence was labelled with a porphyrin in one position on one strand, and seven different positions on seven complementary strands, to overall give seven porphyrin-porphyrin inter-strand combinations. Steady-state fluorescence and circular dichroism spectroscopic analysis of the Soret band revealed individual Watson-Crick bases pair molecular ruler sensitivity. Small angle X-ray scattering attempts between metallated-porphyrin entities did not reveal sufficient scattering at low concentrations, in contrast, an iodinated analogue of the porphyrin system did displayed scattering correlating to different iodine iodine distances. After calibration of the porphyrin system, the moieties were applied to study protein-DNA interactions between Tus, a 36 KDa DNA binding protein, and Ter, a specific 21-mer DNA sequence. Molecular ruler nalysis of the complex required an extended version of the Ter DNA sequence to which modifications were attached. Established FRET pairs FAM and TAMRA were applied to investigate protein-DNA complexation. Native PAGE analysis revealed Tus binds to the extended DNA via a sliding mechanism. Fluorescence analysis of the established FRET pairs identified changes in fluorescence not correlating to changes in FRET, and instead was attributed to emission quenching upon protein binding. Applying the zinc and free base porphyrin version displayed subtle changes in the Soret band circular dichrosim upon complexation, indicating small DNA helical change upon complexation. A 45-mer DNA sequence was designed to form multiple hairpin-duplex conformations with the addition of an appropriate complementary strand. Attaching FRET pairs to the extremes of the DNA sequence enabled multiple DNA conformations, and hence FRET distances to be obtained from one doubly modified DNA sequence. The combinations were characterised by UV-Vis, fluorescence and circular dichroism spectroscopy. Finally, terpyridine labelled DNA sequences selectively formed DNA nanotubes through orthogonal hydrogen bonding and metal complexation interactions. Short DNA strands were designed to self-assemble into long duplexes through a sticky-end approach. Addition of weakly binding metals such as zinc induced the formation of tubular arrays consisting of DNA bundles 50-200 nm wide and 2-50 nm high. TEM displayed additional long distance ordering of the terpyridine-DNA complexes into fibers.