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Dielectrophoresis (DEP) is a term which describes the motion of polarisable particles induced by a non-uniform electric field. It has been the subject of research into a variety of fields including nanoassembly, particle filtration and biomedicine. The application of DEP to the latter has gained significant interest in recent years, driven by the development of microfluidic “Lab-on-a-chip” devices designed to perform sophisticated biochemical processes. It provides the ability to characterise and selectively manipulate cells based on their distinct dielectric properties in a manner which is non-invasive and label free, by using electrodes which can be readily integrated with microfluidic channels. Under appropriate conditions a biological cell will experience a DEP force directing it either towards or away from concentrations in the electric field. At a so-called “crossover frequency” the cell is effectively invisible to the field resulting in no DEP force, a response typically observed in the 1 kHz to 1 MHz range. Its value is a function of cell membrane dielectric properties and has been the subject of research directed at devices capable of using it to both characterise and sort cells. The aim of this work was to investigate the behaviour of a higher frequency crossover referred to as fxo2, predicted to occur in the 1 MHz to 1 GHz range. At these frequencies the electric field is expected to penetrate the cell membrane and behave as a function of intracellular dielectric properties. Standard lithography techniques have been used to fabricate electrodes carefully designed to operate at these frequencies. The existence of fxo2 was then confirmed in murine myeloma cells, in good agreement with dielectric models derived from impedance spectroscopy. A temperature dependent decrease in its value was observed with respect to the time that cells were suspended in a DEP solution. This decrease is consistent with previous studies which indicated an efflux of intracellular ions under similar conditions. An analytical derivation of fxo2 demonstrates its direct proportionality to intracellular conductivity. Direct control of the crossover was achieved by using osmotic stress to dilute the intracellular compartment and thereby alter its conductivity. By using a fluorophore which selectively binds to potassium, a strong relationship has been demonstrated between the value of fxo2 and the concentration of intracellular potassium. Measurements of fxo2 for an unfed culture demonstrated a correlation with viability and subtle shifts in its distribution were caused by the early stages of chemically induced apoptosis.

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Francisella tularensis, a Gram-negative coccobacillus, is an extremely virulent intracellular pathogen. Infection of humans with this pathogen results in tularemia, a life-threatening disease. An approximately 35 kb region found in the F. tularensis genome exhibits many features of a pathogenicity island. This region has a lower G+C content than the average G+C content of the F. tularensis genome, and is surrounded by transposable elements. Results from both a previous study and our present study demonstrated that at least four genes located on the Francisella pathogenicity island (FPI) are required for virulence. This represents the first description of a pathogenicity island in F. tularensis. The FPI-encoded proteins, however, have no significant similarities to any known bacterial proteins. Therefore, we believe that the FPI genes may encode a cluster of novel virulence factors, although the mechanism and their characteristics remain to be determined.

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Interleukin (IL)-1α and IL-1β are pivotal to the initiation and orchestration of inflammation. Unlike most cytokines, IL-1 does not have a signal peptide and therefore secretion requires 2 independent processes; an initial signal to induce the up-regulation of the inactive precursor (pro-IL-1) and a second signal to drive cleavage and subsequent secretion. Whereas many previous studies have focused on the mechanisms that drive IL-1 secretion, the aims of this thesis were to investigate the processes that regulate the intracellular precursors of IL-1 (pro-IL-1α and pro-IL-1β). The hypothesis here was that regulation of these precursors may serve to control the vigour of IL-1 secretion and, ultimately, may influence the potency of pro-inflammatory responses. Post-translational modifications were of particular interest in this thesis, as these modifications are becoming increasingly important to immune system function. Ubiquitination is an important post-translational modification whereby ubiquitin, an 8.5kDa protein, is covalently bound to lysine residues on substrate proteins. In chapter 2, evidence was provided to show that in murine DC, IL-1α and IL-1β are polyubiquitinated and that, in both DC and macrophages, this polyubiquitination drives the proteasomal degradation of IL-1. In addition, these data demonstrated that in the presence of a second signal, polyubiquitinated IL-1 is still available for secretion. Overall, these investigations highlight that the polyubiquitination and proteasomal degradation of IL-1 serves as an essential process in the regulation of IL-1 and, therefore, should be considered as an extra dimension to the current two-signal paradigm of IL-1 release. To support this work, an immortalized bone marrow derived murine macrophage cell line and a human monocyte cell line that both stably express fluorescent IL-1β were employed to measure the rate of IL-1β degradation. In these investigations, it was shown that fluorescence is a reliable readout for measuring IL-1β degradation in these cell lines. In addition, it was demonstrated that that TLR-stimulation leads to an inhibition in IL-1β ubiquitination and degradation. Together, the work presented herein highlights that ubiquitination actively regulates the vigour of IL-1β protein expression and thus may be an important regulator of inflammation. To complement this work, a broader approach was taken, whereby the interactome of pro-IL-1β was explored using a human protein microarray. In these investigations, a human proteome microarray containing 19,951 unique proteins was used to identify proteins that bind human recombinant pro-IL-1β. In these analyses, calmodulin was identified as a particularly strong hit, with a SNR of ~11. Using an ELISA-based protein-binding assay, the interaction of recombinant calmodulin with pro-IL-1β, but not mature IL-1β, was confirmed and shown to be calcium dependent. Finally, using small molecule inhibitors it was demonstrated that both calcium and calmodulin were required for nigericin induced IL-1β secretion in human monocytes. Collectively, the evidence presented in these investigations suggests that following calcium influx, pro-IL-1β interacts with intracellular calmodulin and that this interaction is central for IL-1β processing and release. In addition, a number of other potentially important pro-IL-1β-interacting proteins were also identified in this work, including IL22RA2 and PLCXD3. Overall, the work presented in this thesis serves to highlight that IL-1 is regulated by a broad range of potentially important intracellular processes. We postulate that these processes may be pivotal in the regulation of inflammation and thus the maintenance of homeostasis.