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This thesis introduces Pulse Project (2011-2016), a creative research series exploring an ecology of complex relations between art, humanities, medicine, and technology. In this series, I embody transdisciplinary research practice through my performing as an instrument or medium between others and myself, and between cultural traditions for understanding and mediating the body. Drawing upon my expertise as a clinical acupuncturist with training in biomedicine, I use Chinese medicine and music theories to inform my algorithmic soundscape compositions. These soundscapes are not sonifications of modern Euro-American principles of circulation or embodiment but offer another perspective to conceive of/listen to the interior spaces of the body-in-being. In this transdisciplinary study, practice-based research is used to give form to the transdisciplinary research process by producing performances, personalised sound works, graphic notations and interpersonal correspondences that interweave artistic, medical and technological ways knowing together into new material configurations. This project also questions the distinctions between premodern and modern, East and West and self and other. To investigate these distinctions, Pulse Project interrogates the aesthetic and philosophical axioms underpinning modern and contemporary art, medicine and technology through using premodern Chinese medicine and music theories in tandem with cutting edge technology. Thus, this research travels laterally between cultures, practices and epochs and calls for a radical change in conceiving of the body in ‘oriental’ and ‘occidental’ terms in order to both reduce ethnocentrism and also to travel beyond the tired bifurcations between mind and body, self and others and Western and othered cultures. In combining art, technology and diverse medicines together with contemporary digital culture, this project opens transverse lines of inquiry that create new interconnections between disciplinary practices, whilst at the same time, it generates new forms of cultural engagement through performance and sound works.

Carbamazepine (CBZ) is an effective antiepileptic drug but has been associated with hypersensitivity reactions in up to 10% of patients. These reactions range from mild maculopapular exanthema to life-threatening conditions such as Stevens-Johnson syndrome and toxic epidermal necrolysis. The identification of CBZ-specific T cells and strong associations with specific human leukocyte antigen alleles provide evidence for immunological involvement. CBZ is extensively metabolised and forms several reactive metabolites. The aim of this thesis was to investigate the complex relationships between CBZ, its metabolism, the immune system, and genomics. Direct and microsomal incubations demonstrated that carbamazepine 10,11-epoxide (CBZE), the major metabolite of CBZ, formed a protein conjugate with human serum albumin (HSA) at His146. The same CBZE-modified HSA was also detected in patients tolerant of CBZ therapy. A second His146 CBZ-modified HSA adduct was identified in microsomal incubations, formed as a product of arene oxide providing the first chemical evidence that reactive metabolites of CBZ can modify soluble proteins. Healthy volunteers (n=8) and patients prescribed CBZ therapy (n=72) were recruited to investigate the influence of genetic variation on CBZ metabolism. Patient demographics and a mixture of rich and sparse pharmacokinetic (PK) samples were collected. Plasma levels of CBZ and four major metabolites were measured using a novel high performance liquid chromatography tandem mass spectrometric assay. There was significant variation in observed plasma concentrations of CBZ (14-fold) and its metabolites (approximately 30-fold). A population PK model was developed with nonlinear mixed effects modelling using the PK and clinical data collected from patients. Completion of autoinduction, total daily dosage and concomitant therapy with phenytoin were significant covariates that influenced the CBZ PK. Analysis of variance demonstrated that two single nucleotide polymorphisms (SNPs) in the gene ABCB1 and a single SNP in EPHX1 were significantly associated with altered plasma concentrations of CBZE. T cell clones (TCCs) were generated to CBZ and CBZE from two patients with a history of hypersensitivity to CBZ. All TCCs were CD4+ and secreted the cytokines IFN-γ, IL-13, granzyme B and perforin. TCC activation was MHC class II restricted and all TCCs were stimulated when CBZ was freshly added into the incubation mixture indicative of direct activation. A single TCC was activated when antigen presenting cells (APCs) were pulsed with CBZ indicative of a hapten mechanism. Transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) from patients with a known history of CBZ hypersensitivity identified 9 CBZ/CBZE-specific mRNA and 39 CBZ/CBZE- miRNA transcripts. Pathway analysis mRNA and miRNA changes showed that antiviral response, psoriasis and inflammation were the most significant functions associated with exposure of cells from cases to CBZ and CBZE. In conclusion, these studies show that CBZ is transformed to stable and reactive metabolites, and these metabolites together with the parent drug, lead in susceptible individuals to an orchestrated response which involves transcriptional and immunological activation.

An experimental investigation into the feasibility of imaging multiple medical radioisotopes with the ProSPECTus Compton camera is presented. The aim of the project is to develop a Compton imaging system configured with segmented solid state detectors, for the purpose of alleviating the limitations of conventional Single Photon Emission Computed Tomography (SPECT) systems. The ProSPECTus system consists of a Si(Li) and HPGe planar detector housed in a magnetic field compatible custom made cryostat. The investigation presented in this thesis has been undertaken with a prototype ProSPECTus system, which consists of Si(Li) and HPGe planar detectors housed in separate cryostats. Analysis of the results attained from investigations into the optimum coincidence window, noise thresholds and image reconstruction techniques as part of this thesis are to be used to inform future developments and measurements. The work presented focuses on an investigation of simultaneous data acquisition with the medical radioisotopes 99mTc and 123I, which emit gammarays with energies of 141 keV and 159 keV, respectively. The experimental methodology of defining parameters for timing and noise thresholds is described along with system performance in terms of image quality and efficiency. A custom designed phantom is used to build source complexity and to provide qualitative and quantitative assessment of the imaging capabilities of ProSPECTus using analytical and iterative reconstruction algorithms. It has been determined that a spatial resolution of 3 mm is achievable, using iterative reconstruction of data acquired from a 139Ce point source of energy 166 keV. Quantitative analysis has revealed that an Edge Response is a good measure of determining the resolvability of extended source distributions. It has been proved that the spectroscopic response enables ProSPECTus to image 99mTc and 123I simultaneously. This would otherwise require complex peak deconvolution techniques in clinical SPECT systems. An improvement in efficiency of up to a factor of 2.5 over conventional SPECT systems is demonstrated, with suggestions for further improvements, indicating a factor of 23 is achievable with the next generation system. This would potentially reduce patient dose or increase patient throughput. Suggestions are presented for the iterative reconstruction algorithm to facilitate enhanced imaging with the next generation ProSPECTus system.

Optical coherence tomography (OCT) is a versatile and powerful imaging technique widely used in biomedical applications. It employs non-destructive radiation and performs non-contact micrometre-scale cross-sectional imaging of the sample structure. However, classic OCT systems generally apply a single-point detection scheme, which creates inefficiencies in terms of the experimental alignment of system, the point-by-point signal acquisition process and the measurement speed. One of its variants, full-field optical coherence tomography (FF-OCT) employs parallel illumination and directly acquires en-face images with a complementary camera, hence omitting the need of electromechanical lateral scans as in classic OCT systems. Current FF-OCT systems could offer more efficient measurement procedures as well as superior imaging performance, however, they are neither economically viable nor universally applicable to different applications. There is a need for a simplified low cost system to make such a powerful technology readily available for a wide range of applications. In this thesis, the development of a low cost simple FF-OCT system is described from its system setup, experimental procedures, data analysis, and system performance. The system consists of only essential components including probing lens and a beam-splitter, together with a low cost infrared LED source and CMOS camera. During the measurement, the system only requires to control the axial movement of the sample arm and the image acquisition by the camera. For the imaging of a sample with a depth of 100 um, the FF-OCT measurement only takes less than two minutes. The time-efficient measurement with the simple system offers great advantage over the developed phase-shifting FF-OCT system, which requires lengthy measurement and excessive operations, despite the decoupling of signal strength and instantaneous phase with penetration depth. Therefore, compared to state-of-the-art systems, it has the advantage of being low-cost, fast image acquisition speed and simple experimental operations. For the data analysis of tomographic imaging, the axial position of a structural feature is determined by that of the envelope, which is obtained by processing raw FF-OCT signal with Hilbert transform. The imaging performance of the simple system is measured to have a spatial resolution of 3.6 x 10.3 um2 (axial x lateral) and a system sensitivity of 74 dB. The characterisation of small-size pharmaceutical pellet coatings, bovine corneal layers and paint films is to demonstrate the potential of the simple FF-OCT system for the tomographic imaging. The layered structures and internal morphology features can be revealed by analysing the measured FF-OCT B-scan images and A-scan signals. First of all, the simple FF-OCT system is capable of performing accurate and quick measurements of pellet coatings, which are validated by the XuCT technique. FF-OCT imaging can provide a spatial characterisation of coating layers, an accurate determination of coating thickness, and an estimation of coating uniformity and porosity, making the simple system a powerful tool for the coating evaluation of similar pharmaceutical pellets. Secondly, the simple system can detect corneal surfaces and the two anterior layers of bovine cornea. This could permit the prediction of the corneal oedematous state and epithelial erosions by the analysis of the FF-OCT results of the corneal structure. Thirdly, the simple system is capable of revealing the surface and subsurface of basecoat and clearcoat films. The measurement of their paint thicknesses is also verified by the reference profilometry results. FF-OCT imaging can provide further spatial evaluation of a paint film and the areal thickness map could be obtained. The study of these paint samples with the simple system might provide an indication for the FF-OCT measurement of industrial automotive paint. For the data analysis of the surface topography, the axial position of the surface is obtained by applying interpolation and a minimum search algorithm to the raw FF-OCT signal. This allows sub-micrometre depth precision to be obtained with the simple system. In the validation of the measurement of the surface topography, a less than 10 nm deviation of the FF-OCT measurement is found compared to the AFM measurement of a nanostructured step-like surface. The capability of the simple system for the surface topography is further illustrated by the determination of the electrode thickness of semiconductor microelectronics. By analysing the phase change upon reflections and the optical path lengths during the measurement, the step-like structure and the sandwich configuration can be revealed from the measured FF-OCT surface maps. The usefulness of the simple system is presented in the surface topography of PMMA models. It is demonstrated that the areal refractive power can be obtained by analysing the 2-D curvature of the FF-OCT measured surface map, which is useful in the identification of surface irregularity.

Hypersensitivity denotes a form of immune-mediated adverse reaction associated with a high degree of morbidity and mortality. Due to a lack of animal models, research has focussed on patient lymphocytes ex vivo. However, such studies bypass investigation of naïve T-cell activation. Subsequently, in vitro assays to assess the priming of healthy donor naïve T-cells have been developed to facilitate the discrete analysis of primary and secondary T-cell responses. While these assays have been used to assess the association of specific HLA alleles with hypersensitivity, for most drugs, the majority of individuals who are positive for an HLA risk allele do not develop a reaction. Thus, predisposition to hypersensitivity is likely mediated by other parameters. As polymorphisms in co-inhibitory pathways are associated with dysregulated immune responses, we first investigated the role of these pathways during the drug antigen (SMX-NO)-specific activation of T-cells. Antibody-mediated blockade of PD-L1 enhanced the activation of SMX-NO-primed naive, but not memory, T-cells. In comparison, inclusion of PD-L2 block had no effect, but in combination with PD-L1 block effectively dampened the enhanced T-cell activation seen with PD-L1-block alone. In comparison, inclusion of CTLA4 block enhanced the proliferative response of antigen-stimulated naive, but also memory T-cells, suggesting a greater regulatory role for CTLA4 than PD-1 during secondary T-cell responses. Blockade of TIM-3 had no effect on T-cell activation. Further investigation focussed on the kinetics of receptor expression. While all receptors were upregulated on naive and memory T-cells during the 3 week culture after antigen exposure, PD-1 was upregulated at earlier time points than CTLA4 and TIM-3 indicating a differential role for these receptors during early and late stage T-cell activation. Moreover, CTLA4 expression was induced in response to antigen far less on CD4+ T-cells suggesting that CTLA4 has a greater regulatory role during the drug antigen-specific activation of CD8+ T-cells. High expression of individual co-inhibitory receptors including PD-1 has previously been associated with exhausted T-cells, while other studies indicate that these cells are highly functional. To address this, we compared receptor expression with T-cell clone function. To assess function, we investigated the role of the newly identified Th17 and Th22 subsets in these responses. A range of Th1/Th2 cytokines were secreted in response to SMX-NO including IFN-γ, IL-13, and IL-5. While no IL-17 was detected, 50% of clones secreted IL-22. Further analysis uncovered two distinct antigen-responsive T-cell subsets that secrete Fas-L/IL-22 or granzyme B, the presence of which was confirmed utilising cells from SMX-hypersensitive patients. This is the first data to show production of IL-22 alongside IFN-γ by antigen-specific T-cells from drug-hypersensitive patients. Analysis of the level of individual co-inhibitory receptor expression found no correlation with T-cell proliferative capacity or secretion of cytokines/cytolytic molecules. Both SMX and SMX-NO, stimulate T-cells from hypersensitive patients. 9/10 healthy donors respond to SMX-NO, while only 30% respond to SMX in vitro. These observations were made using a whole lymphocyte population and thus we utilised an in vitro T-cell priming assay, whereby naïve or memory T-cells from healthy donors are stimulated with drug-antigen using mature dendritic cells, to assess the T-cell origins of antigen-responsive cells. Both naïve and memory T-cells were activated by SMX-NO in all donors. Although SMX failed to induce proliferative responses, 2/3 donors displayed SMX-induced IL-13 secretion. SMX and SMX-NO-responsive clones were subsequently generated from these donors from naïve and memory T-cell cultures indicating that the priming of naïve T-cells and the re-activation of memory T-cells has a role in the onset of SMX-induced hypersensitivity. As these responses were detected using cells from drug-naïve donors, the memory T-cell responses are likely a result of cross-reactivity with T-cells primed to an undetermined peptide antigen. Our data are the first to show that both hapten and parent drug can stimulate pre-existing memory T-cells from drug-naïve donors. PPD, a compound found in dyes used for hair colouring, is associated with ACD. Both PPD and a downstream oxidation product, BB, activate T-cells in allergic patients. Thus we used an in vitro T-cell priming assay to assess the propensity of PPD and BB to activate naïve and memory T-cells from healthy donors, and compared responses to those characterised from allergic patients. 105 PPD- and 122 BB-responsive T-cell clones were generated from five allergic patients. More than 90% of patient BB-clones were CD4+, and all lacked cross-reactivity. In contrast, certain PPD-clones cross-reacted with BB, and 37.5% expressed CD8 promoting PPD as the central driving force behind the allergic reaction. However, upon utilising cells from healthy donors, neither naïve nor memory T-cells were activated by PPD suggesting the lack of important susceptibility factors. In comparison, both naïve and memory T-cells were activated by BB, which similarly to patients displayed a lack of cross-reactivity and secreted a similar panel of cytokines including IFN-γ, IL-13, and IL-22. In conclusion, the similar findings between drug and chemical antigen-responsive T-cells generated using an in vitro T-cell priming assay and those from patients highlight the promising use of this assay in the investigation of these responses. Indeed, the future definition and inclusion of (a) susceptibility factors and (b) the peptide-antigens responsible for naive T-cell priming will form a promising platform for the safe screening of drugs.

Vaccines have already made a significant impact on global healthcare with the eradication or reduction in disease states; however, further work is required to develop ‘safer’ vaccines by the use of sub-unit antigens. When considering immunisation, whilst the oral route is the most convenient route for drug and vaccine administration, most vaccines are still delivered via an injection. To be able to be given orally, vaccine antigens need to be associated with carrier systems to both protect the antigen from degradation within the harsh gut environment and to improve uptake by appropriate target sites, namely M cells located in the Peyer’s patches, which are responsible for secretory IgA and other mucosal responses. Therefore, niosomes have been considered within this thesis to enhance the protection and delivery of sub-unit vaccines via the oral route and intramuscularly. Initial work included using Design of Experiments to optimise niosome/ bilosome vesicles prepared from of Monopalmitoyl glycerol (MPG), Synthecol (Chol), Dicetyl Phosphate (DCP) and bile salt in terms of their vesicle size, surface charge, suspension pH and antigen loading. Optimisation studies, demonstrated that the ideal composition was a 5:4:1 ratio of MPG:Chol:DCP respectively. Langmuir balance and differential scanning calorimetry studies also demonstrated that, cholesterol inserts between the lipids within the bilayer hence, preventing crystallisation of the hydrophobic tails of the other surfactants. Using the optimised niosome formulation, oral biodistribution of these niosomes (3H) and the H3N2 (125I) antigen showed that of the dose administered (t=1h), significantly (p< 0.05) higher antigen (59.8%) was recovered within all organs of the gastrointestinal tract (GIT) when formulated with niosome vesicles compared to the free antigen (38 %). Uptake at the target site of the Peyer’s patches revealed that on average 1.4 % antigen was present within the Peyer’s patches when associated with the niosome vesicles compared to the free antigen dose which demonstrated only 0.4 % antigen was acquired within the Peyer’s patches. Furthermore, uptake within the mesenteric lymph tissues demonstrated 0.7 % antigen recovery when associated with niosomes compared to 0.2 % recovery of the free antigen dose. Hence, uptake studies demonstrated that niosome associated antigen show a 2-fold improved uptake and retention at the target sites compared to administering the free antigen alone. Reduction in vesicle size of the niosomal systems to 2 μm made no significant difference in recovery of either the vesicles or associated antigen in the mesenteric lymph tissue. However, when comparing the Peyer’s patches, whilst there was no significant difference in localisation of antigen, there was a greater recovery of vesicles within the Peyer's patches of the larger 6 μm vesicles (p< 0.05) in comparison to the 2 μm vesicle formulation, which is an indicator of achieving increased mucosal immunity. Within ferrets, the optimised niosomes containing bile salts were shown to lower median temperature differential change and show inflammatory cell counts in nasal washes to be comparable to the commercial vaccine administered intramuscularly after challenge with a clinical H1N1 isolate. In terms of intramuscular administration, niosomes incorporating H1N1 and H3N2 antigen provided thermostability when stored at elevated temperatures for 3 months at 40 °C confirmed by HAI and ELISA studies when compared to the commercial vaccine. In addition, the association of antigen confirmed the dose sparing ability of niosome preparations to elicit immune responses comparable to the control vaccine doses. In conclusion, this thesis demonstrates that niosomes can be used as delivery systems for peptides and protein sub-unit antigens by providing antigen protection thereby, enhancing vaccine efficacy when administered orally and intramuscularly.

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Investigating the Molecular Mechanisms of Crosstalk Between NF-κB and Nrf2 Signalling Pathways In order to maintain tissue homeostasis, cells must respond swiftly to inflammatory and oxidative challenges, ensuring appropriate processes are sequentially activated and repressed when stress conditions are normalised. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and Nuclear Factor-κB (NF-κB) are the key regulators of cellular antioxidant and inflammatory responses respectively. Imbalance between these processes is a contributing factor in many human diseases, including neurodegeneration, autoimmune disorders and cancers. Pharmacological and genetic studies suggest that there is functional crosstalk between these two pathways, however mechanistic details of conditional dominance between them remain unclear. To address this, we have generated multiple, low expression, fluorescent molecular tools that allow monitoring of the distribution and activity of Nrf2 and NF-κB proteins in primary living neurons and astrocytes. This allowed us to characterise the cell-specific antioxidant and inflammatory signalling, as well as to understand the patterns in basal activity. We also investigated patterns of crosstalk in a cancer-related context utilising a previously generated SK-N-AS neuroblastoma cell line, engineered to stably express Nrf2-Venus, from a bacterial artificial chromosome (BAC). This model cell-line was first extensively characterised in order to define conditional Nrf2 responses to antioxidant compounds in real-time. We then proceeded to investigate the effect of acute inflammation on the cellular antioxidant activity, in order to define the extent and nature of functional crosstalk between NF-κB and Nrf2. Data from these studies provide definitive proof of a self limiting reciprocal mechanism of interplay in neuroblastoma cells, in which NF-κB-mediated inflammatory signalling promotes an increase in Nrf2 transcription which then in turn supresses further NF-κB signalling. In addition, results from single cell imaging and population level studies show that Nrf2 responses are fine tuned, by either Keap1-mediated mechanism of repression. To define the intricate mechanisms of basal Nrf2 activity, we utilised a photoswitchable fluorescent protein fusion, which provided the first direct measurements of Nrf2 nuclear import and export dynamics in live-cells. Finally, we used the sophisticated live cell imaging approaches to define the mechanism of action of the Nrf2 inhibiting drug brusatol. Significantly, these results contradict published data and reveal a more general explanation for the potential therapeutic utility of brusatol.

Background: Non-contrast CT-based surrogates of regional ventilation derived from pulmonary images acquired at multiple inflation levels have been proposed as alternatives to established modalities. However, their physiological accuracy has yet to be validated prior to clinical translation. Purpose: To address the hypothesis that these surrogates can provide information comparable to a direct measure of ventilation from hyperpolarised gas MRI ventilation via: i. development of a methodology for registering CT and gas MRI. ii. comparison of these surrogates with gas MRI at the lobar level. iii. evaluation of the impact of inflation levels when comparing gas MRI and ventilation CT. iv. development of an image acquisition and analysis framework to facilitate spatial correlations of both techniques. v. assessment of the effect of using different gases on the correlation. Methods: i. A method to indirectly register gas MRI to CT via same-breath 1H-structural MR images was developed and its accuracy was assessed. ii. A ventilation model based on expansion of lobar CT segmentations was compared with gas MRI lobar ventilation measurements. iii. The spatial overlap of ventilation CT was compared to gas MRI acquired at two different inflation levels. iv. An image acquisition protocol was designed to minimise differences in acquisition settings between scans such as posture and breathing manoeuvre and analysis methods were developed to enable direct regional and voxel level correlations. v. The effect of using two different noble gases, namely, 3He and 129Xe, on correlation with ventilation CT was assessed. Results: i. The indirect method of registration was more accurate than direct registration. ii. Despite subtle differences, lobar ventilation measurements derived from CT and hyperpolarised gas MRI were comparable. iii. Comparison of ventilation CT and gas MRI varied with inflation state. iv. The spatial correlation between ventilation CT and gas MRI increased at coarser levels. v. A marked improvement in correlation was observed for 3He and 129Xe MRI in contrast to when ventilation CT was compared with either 3He and 129Xe MRI. Conclusion: Although CT-based surrogates of ventilation show promise for replacing established ventilation modalities such as hyperpolarised gas MRI, particularly at coarser levels, they cannot be assumed to be equivalent to the techniques they purport to replace.