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The study of bacteria is important for understanding disease, the development of drugs and understanding bacterial ecosystems. Bacterial cells have a cell wall which is made of a biopolymer called peptidoglycan. In Gram positive species of bacteria like S. aureus and B. subtilis the peptidoglycan cell wall layer is ~25 nm thick. AFM is a powerful tool for understanding biological structures and processes. By immobilising the S. aureus cells in a silicon wafer etched with holes, AFM can be performed on living bacteria in a liquid environment. In this work small amplitude tapping mode AFM, combined with optimisation of imaging buffers has allowed for high resolution images of living S. aureus cells to be taken at different time points in the cell cycle revealing the architecture of the cell wall peptidoglycan with molecular resolution on living bacteria. Different structures have been seen on the cells that range from the freshly formed tightly spaced concentric rings to the mature loose mesh which are a result of the remodelling of the cell wall. Cells that are seen to be undergoing a division event have also been imaged revealing how the splitting process develops in S. aureus as two halves of the dividing cell separate into the two daughter cells. A set of experiments have been performed to study the architecture of the cell wall in mutants and under the influence of disruptive chemicals including detergent and lysostaphin. The experiments showed two distinct peptidoglycan architectures in S. aureus, the young rings and the mature mesh like structure. Comparing the organisation of the peptidoglycan in the mutants and the wild type cells showed that the features seen are made of peptidoglycan. A series of quantitative analyses have been performed on the high resolution images of ringed architecture including measuring the concentricity of the rings and comparing this concentricity in the dense and less dense ringed structures which shows that the rings at both densities have a common centre that they orbit. A bearing analysis technique has been developed for the analysis of the holes that are seen within the mature architecture that allows the depths, densities and areas of holes to be quantified at different depths in the cell wall layer. This analysis shows that some of the holes are as deep as the thickness of the whole cell wall layer and up to 50% of the cell wall material can lay in the deepest 10 nm of the cell wall thickness that was imaged. An analysis of the diameter of the peptidoglycan fibres seen in the mesh architecture was performed to measure the distribution of the fibre widths which was then used to calculate an estimate for the average number of glycan chains within a fibre. Imaging of another Gram positive bacterial species, B. subtilis, has been performed using the same small amplitude tapping technique. A method of physical immobilisation using etched silicon wafers was developed for the rod shaped cells then a protein immobilisation process (Cell-Tak) was tested. Using Cell-Tak allowed high resolution images to be taken of the cell wall structure of B. subtilis revealing a previously unreported mesh like disordered organisation, similar to the mature architecture in S. aureus. The same analysis techniques that had been developed for the mature architecture in S. aureus were performed on the images from B. subtilis revealing holes that measure over 20 nm deep into the cell wall thickness as had been measured in S. aureus. The first stages in developing a protocol for imaging outer membrane proteins in situ in Gram negative bacteria were made. BtuB is a protein that is responsible for the transport of vitamin B12 in E. coli. The BtuB proteins were overexpressed and reconstituted into lipid vesicles then imaged on a mica surface using small amplitude tapping mode. Partially deflated vesicles showed evidence of thousands of proteins on the surface with some proteins forming ringed structures. The work aims to develop understanding of the cell wall architecture at molecular scale and how it changes over the cell cycle which could aid in understanding how these antibiotics that target the cell wall synthesis work.

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.

A requirement for online monitoring has emerged owing to the susceptibility of fibre reinforced composite materials to sub-surface damage. Acoustic emission (AE) monitoring is understood to detect damage well before catastrophic failure; research in AE sensing therefore continues to attract significant attention. The research presented herein provides a review of a fibre-optic-based AE sensor design. Developmental work was performed to evaluate both sensor fabrication and packaging-related issues. The characteristics of the sensor were found to be influenced by: (i) the type of optical fibre used for fabrication; and (ii) preparation of optical fibres prior to sensor fabrication. The use of a small-diameter packaging substrate revealed improvements in sensor performance. The fibre-optic AE sensor was successfully embedded in a uni-directional composite laminate that was fabricated using autoclave processing. The embedded fibre-optic sensor was found to provide higher sensitivity to simulated AE compared with a surface-mounted sensor. Sensor characterisation trials were performed using simulated AE; a low directional sensitivity was observed. Modal analysis revealed a preferential sensitivity to the A0 wavemode; this sensor design may therefore be suited to the detection of delamination in FRCs. Finally, the sensor was shown to successfully detect interlaminar crack propagation under Mode-I loading.

Deeply Virtual Compton Scattering (DVCS) is the simplest interaction that allows access to Generalised Parton Distributions (GPDs), a theoretical framework describing nucleon structure. The strong interest in GPDs results from the fact that they can be used to determine the total angular momentum of quarks inside the nucleon and provide a 3-dimensional picture of nucleon structure. The measurement of the DVCS process is facilitated by the interference with a competing interaction known as the Bethe-Heitler process which has the same final state. DVCS information is obtained from the asymmetrical in distribution of the real photon around the azimuthal angle $\phi$ at HERMES. Beam charge and beam helicity asymmetries, extracted from DVCS events with an unpolarised hydrogen target recorded during the 2006-2007 and 1996-2007 data taking periods, are presented in this thesis. The asymmetry amplitudes are presented over the range of HERMES kinematic acceptance, with their dependence on kinematic variables t, xB and Q^2 also shown and compared to a phenomenological model.