Search

Artificial olfaction has the potential to revolutionise medical diagnosis, speed up threat detection, and provide long-term data collection on atmospheric pollution. Current technology provides a means to detect some compounds, but detection speed is slow, and accuracy is often balanced against cost and reusability of devices. Insects have long been the inspiration for artificial olfaction and are able to detect low concentrations of compounds over vast areas and navigate towards these targets. A deeper understanding of how this is achieved could inform the design of better artificial olfactory devices. What features of the insect olfactory system allow the rapid detection of miniscule concentrations of a wide variety of compounds? How does an insect discriminate odours using a limited number of olfactory receptor types?Using UAS/GAL4 technology, Drosophila melanogaster larvae expressing only a single functional olfactory sensory neuron (OSN), of their full repertoire of 21, were used to explore peripheral olfactory responses. Three different fly lines, which each expressed different olfactory receptor (OR) types, were used to record the electrophysiological responses of the peripheral OSNs to a panel of biologically significant odours, at differing concentrations. These responses were compared to those from a leading electronic nose (metal oxide sensors) for the same odour conditions, and the features of the responses were characterised. A novel odour delivery system was also developed to accurately deliver these odours and concentrations repeatably, and was validated using a commercially available photoionisation detector. The ability to correctly select the odour from which a response was recorded, out of a choice of five odours, at two concentrations each, was scrutinised using a classifier algorithm. The three OSN types achieved 53%, 62% and 75% accuracy, respectively. This is the first instance that Drosophila larvae have been conclusively shown to be able to discriminate odour concentrations using only a single peripheral OSN. The two metal oxide sensors achieved 92% and 95% accuracy under the same conditions. Whilst the features of the responses used to discriminate odours differed between the biological and electronic systems, the time frame required for correct classification of sensor data was only, in some cases, three seconds longer than in OSNs (~0.5 seconds), indicating that metal oxide sensors may have a useful role to play in biologically inspired artificial olfaction.

The Ras superfamily of small G-proteins cycle between an active GTP bound form and an inactive GDP bound form. This cycle is controlled by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Sos, a GEF for Ras proteins, is a ubiquitously expressed 150 kDa protein that is recruited to the cell membrane by Grb2 on phosphorylation of a receptor, where it promotes GDP release from Ras, enabling formation of the active Ras.GTP complex. Study of the interaction of the catalytic domain of mSos1 (CmSos1) with N-Ras in vitro was hampered by low expression levels of CmSos1 in E.coli. The gene for CmSos1, coding for residues 577-1076, was reconstructed following codon optimisation and expressed in E.coli. Yields for a 4 Litre culture were 120 mg of pure CmSos1 protein, an ~100 fold enhancement in expression levels. This enabled the interaction of CmSos1 with N-Ras to be studied, using the fluorescent analogue of GDP, 2-deoxy-3-O-N-methlyanthraniloylGDP (mdGDP) for transient kinetic and equilibrium measurements. The mechanism of nucleotide exchange has been proposed to be a substituted mechanism, exchange factors stabilising a nucleotide free binary complex of GEF.G- protein allowing rebinding of nucleotide via a transient ternary complex. The formation of a weak ternary N-Ras.mdGDP.CmSos1 complex has been studied using fluorescence anisotropy measurements. Addition of excess GDP to N- Ras.mdGDP.CmSos1 enabled the release of mdGDP from the ternary complex to be analysed. These processes have been followed over millisecond time scales using stopped-flow apparatus. The nucleotide free N-Ras.CmSos1 complex has been purified and characterised. The reverse reaction, analysing the association of nucleotide to the N-Ras.CmSos1 complex, has been studied. Computer simulation of this data has led to rate and equilibrium constants for the steps of the reaction being proposed. The interaction of different nucleotides and nucleotide bound states has also been observed. The interaction of the proposed catalytic domain of aPIX (a Dbl homology exchange factor) with Rac1 has been studied. Low exchange activity has been observed, leading to the proposal that other factors are required for the observed in vivo activity of PIX on Rho family proteins.

The phenomenon of electrogenerated chemiluminescence has been examined as a potential analytical technique for the determination of trace amounts of aromatic polycyclic hydrocarbons and related compounds. An apparatus has been constructed to establish the optimum excitation conditions for the observation of ecl emission from such compounds, and to obtain analytical data at high sensitivity. Long-wavelength eel emission peaks arising from decomposition products and excited dimer formation have been examined, and related to parameters of the applied signal. An attempt has been made to utilise ecl for the analysis of aromatic polycyclic hydrocarbons separated from tobacco residues by preparative scale gas chromatography.