Within the health sciences, action research is a methodology well suited to the goal of collaboratively improving practice. As the Royal College of Radiology recommends the use of published clinical trials as guides for achieving higher standards of accuracy, it is important for radiologists to reflect deeply on the results from diagnostic accuracy studies. When the results of the gold standard (or reference standard) are used to confirm a particular diagnosis or disease by comparing the diagnostic accuracy to a newer or index test, this is referred to as diagnostic accuracy research. In the reporting of all research, every effort must be made to reduce the incidence of bias. In 2003, the STARD (Standards for Reporting Diagnostic Accuracy) tool was developed for clinicians to enhance the quality of reporting diagnostic accuracy studies. Based on previous studies, experiential knowledge, and an extensive review of the literature, this research demonstrates that the STARD tool is not being fully optimized. The overall aim of this research was to conduct a work-based project within the department of radiology to develop a revised tool, based on the current STARD, which could then be used to more accurately report and interpret the results of radiology diagnostic accuracy trials. This study was conducted in accordance with participatory action research. Methods The development of this new reporting tool was conducted in collaboration with a group of physicians, and in two distinct phases. First, a needs assessment was sent to eight radiological experts who had agreed to participate in the study. Based on their responses, and feedback from my mentor and colleagues, the next phase of tool development was done using the Delphi technique, after two rounds of which consensus was met. Each phase and cycle iteration to complete the needs assessment and Delphi technique are synonymous with the cycles of action research. The new reporting tool was named the RadSTARD (Radiology Standards for the Reporting of Diagnostic Accuracy Studies), and an elaboration document was written to provide guidance to the end-user. Radiology residents and Fellows at The Ottawa Hospital were then asked to rate their level of confidence in interpreting a diagnostic accuracy article specific to radiology while referring to the RadSTARD. They were also provided a second diagnostic article, the STARD tool, and an elaboration document for comparison. Data was collected using questionnaires that allowed for additional comments. Findings The validation phase of the RadSTARD tool was completed via triangulation of data, as both a quantitative and qualitative analysis was completed. The results found no significant statistical difference between the two groups as per the Mann-Whitney and chi-square analysis. Likewise, both physician groups indicated that they found RadSTARD increased their level of confidence when interpreting the diagnostic accuracy article. Concomitantly, when combined, 96% of the two physician groups indicated they would use the tool again. Interpretation These results may be interpreted as generalizable, as there was no discrepancy or statistical difference found in the results between the radiology residents’ and Fellows’ scores, despite the differences in their level of training. Both groups found the RadSTARD tool and elaboration document to be beneficial to them when interpreting the literature. RadSTARD is thus a reliable tool that can be used to validate the results of diagnostic accuracy studies specific to radiology. It will aid radiologists in reporting and interpreting radiology diagnostic accuracy studies, impacting their practice for generations to come.

T cell "memory inflation" is the sustained induction of effector memory cells that home to peripheral tissues and retain their functionality. Defining the mechanisms that drive these non-classical memory responses may contribute towards the development of novel prophylactic or therapeutic vaccines. A model of memory inflation based on responses to β-galactosidase delivered by a non-replicating adenoviral vector provides a robust tool for investigating the underlying mechanisms. This work has shown that these responses are not dependent upon the human cytomegalovirus (HCMV) promoter within the model, this being the only part of the model that is CMV-derived. This model has been used to test the hypothesis that bypassing antigen processing would result in inflationary memory responses to CD8+ T cell epitopes that are not normally the targets of such responses. When the β-gal497-504 restricted epitope (ICPMYARV) was expressed as a minigene in a recombinant adenovirus vector, inflationary CD8+ T cell responses were induced, instead of the classical responses obtained with full-length β-galactosidase. Similar results were obtained with the M45985-993 (HGIRNASFI) epitope from the mouse cytomegalovirus M45 protein. These data demonstrate that the polypeptide context of a CD8+ T cell epitope may determine whether classical or inflating memory responses are induced. This could be relevant to the design of recombinant antigens in adenoviral vectors, which have emerging therapeutic and prophylactic applications.

Passive acoustic mapping (PAM) is a versatile technique for monitoring of therapeutic ultrasound, in particular the generation of cavitation and subsequent bubble dynamics. The objective of this thesis was to apply PAM to investigate the activity of different types of cavitation agent and any correlation to therapeutic effects. The work focuses particularly on a new type of agent in the form of microbubbles that can be magnetically targeted, which have shown great potential for localised drug delivery. In this thesis PAM was used to study the behaviour of magnetic microbubbles (MMB) in tissue phantoms, in vitro cell experiments and in vivo mouse models. In tissue phantoms magnetic localisation of microbubble-induced cavitation activity was demonstrated and resolved using PAM. Under clinically relevant flow conditions an increase in the energy of cavitation on the order of 2-5 times was observed using PAM, which was similar to doubling the injected microbubble dose. To facilitate cell experiments a novel chamber system was used with improved variants of MMB as well as condensed magnetic nanodroplets which were shown to enhance uptake of fluorescent small interfering RNA (siRNA), transfection of knockdown siRNA and paclitaxel-induced cell kill. Magnetic targeting was associated with increased cavitation power in PAM as well as increased treatment efficacy measured by biological methods including fluorescence microscopy and flow cytometry. Finally, improved MMB were tested for the first time in vivo under real-time B-mode imaging and PAM, followed by fluorescence and MR imaging to assess distribution. MMB cavitation activity was of similar magnitude to the commercial contrast agent SonoVue®. Cavitation induced by the microbubbles and magnetic targeting were both associated with increases in fluorescence and MRI contrast in tumours. The therapeutic potential of MMB and monitoring power of PAM were thus demonstrated.

The development of an effective human immunodeficiency virus type 1 (HIV-1) vaccine is the best solution to the global HIV-1 pandemic. This has proven difficult due to the high mutation rate and genetic diversity of the virus. One way to overcome this is for a T-cell vaccine to target highly functional conserved regions of HIV-1 across all clades, mutations would typically incur a cost on viral fitness. Our first conserved vaccine, HIVconsv, is a clade alternating consensus immunogen comprised of 14 conserved regions across the HIV-1 proteome. HIVconsv showed very promising results in mice and human clinical trials, inducing HIV-1 specific CD8+ T cells of high magnitude and breadth. In my work, four conserved mosaic immunogens were designed in-silico, to maximize the coverage of potential 9-mer T cell epitopes. The aim was to develop a new conserved mosaic vaccine to enhance the breadth and depth of the vaccine elicited responses. The first, tHIVcmo, designed to complement tHIVconsv and tConsv1, tConsv2 and tConsv3 were designed to complement each other in a trivalent vaccine. The immunogens were expressed from a plasmid DNA (D), ChAdV63 (C) and a MVA (M) vector, used in heterologous prime boost regimens DDDCM and CM. HHD transgenic mice, expressing a chimeric human HLA-A*02:01 were used to asses the breadth and depth of vaccine elicited T cells. I assessed whether or not combining tHIVcmo to tHIVconsv had any benefits, and compared this bivalent combination to the trivalent conserved mosaic vaccines. Over 70 known human HLA-A*02:01 epitopes of HIV-1 across major clades where used for analysis. The trivalent mosaic vaccines elicited T cell responses with greater breadth, depth, magnitude and killing efficacy compared to that of the bivalent or HIVconsv elicited responses. These are the first data on mosaic vaccines with regards to known human epitopes, using a model with a human HLA molecule and the first conserved mosaic vaccines tested. In a separate series of experiments, the effects of the tPA leader sequence were examined, comparing HIVconsv with and without tPA. Experiments in BALB/c mice have revealed that the addition of a tPA leader sequence had no added benefit on our T cell vaccine. Specificity, magnitude, breadth, depth and killing efficacy were all not affected by the addition of tPA to the vaccine.

Regulatory T cells (Tregs) are crucial controllers of inflammation. Patients with a genetic defect in the lineage defining transcription factor Foxp3 suffer from a broad spectrum of autoimmune diseases, including early manifestation of various types of skin inflammation. However, our knowledge about the properties of skin resident regulatory T cells under homeostatic and pathologic conditions and in particular in inflammatory diseases of the skin such as psoriasis is limited. In order to address this open question, phenotypic characterization and transcriptomic analyses of murine skin Tregs were performed. The majority of CD4+ T cells in homeostatic skin express the transcription factor Foxp3. These Tregs show a unique profile, are mainly thymic derived nTregs and are enriched in markers associated with a highly suppressive phenotype such as CD25, CD103, IL-10, ST2, and GATA-3. To delineate the role of Tregs in psoriasis-like inflammation, the imiquimod model of psoriasiform inflammation was used. Tregs are significantly increased during skin inflammation and retain their suppressive phenotype and trancriptome. Treg depletion before and during imiquimod treatment led to exacerbated inflammation and dense leukocytic infiltrate in the dermis and epidermis. Furthermore, the exacerbated inflammation was characterized by a significant increase in IL-12 and infiltration of IFN-γ and TNF-a producing, T-bet+ CD8+ and CD4+ T cells. IL-23 and IL-17 secreting dermal γδ T cells are pathogenic in the imiquimod model, but their cell numbers decreased in the absence of Tregs. Functional experiments demonstrated that myeloid-derived type I Interferon drove the exacerbated inflammation, which was dependent on CD4+ and CD8+ T cells. In conclusion, skin Tregs have a protective role in psoriasis-like inflammation and depletion of Tregs causes disease exacerbation and triggers an altered cytokine profile and cellular infiltrate. These findings assist in our understanding of tissue resident Tregs and how they maintain homeostasis and prevent inflammation in the skin.

Mast cells play a central role in many allergic and in ammatory conditions. These cells are activated following an intracellular rise in calcium, such as that which occurs after the activation of cell-surface receptors. One such important receptor is cysteinyl leukotriene (CysLT) receptor type 1 (CysLT1), which is activated by lipid mediators such as CysLTs LTC4, LTD4, and LTE4. CysLT1 stimulation leads to the hydrolysis of membrane phospholipids such as phosphatidylinositol 4 5-bisphosphate (PIP2) via phospholipase C-β , which results in the generation of diacylglycerol and inositol trisphosphate. Inositol trisphosphate transiently increases cytosolic calcium levels by releasing calcium from its internal stores. This transient phase is followed by an influx of external calcium caused by the opening of store-operated calcium release-activated calcium (CRAC) channels in the plasma membrane. To understand how CRAC channels are involved in receptor- driven calcium responses, I investigated whether the opening of CRAC channels regulates the production of cellular phosphoinositide. Using cytoplasmic calcium ion (Ca2+) imaging in the mast cell line RBL-2H3, I found that LTC4 induced repetitive calcium oscillations that ran down in the absence of external calcium and were sustained by calcium entry through CRAC channels. The molecular characterisation of CRAC channel components in RBL-2H3 cells revealed that LTC4 -mediated calcium oscillations were maintained through calcium entry via Orai1 and that the calcium signal could not be maintained by Orai3 or other calcium- permeable channels. Furthermore, STIM1 (but not STIM2) was the only homologue that supported calcium oscillations in RBL-2H3 cells. The inhibition of the cellular phosphoinositide pool by lithium chloride (LiCl) reduces calcium oscillations. Adding the substrate inositol rescued these oscillations, but only when external calcium was present. Pharmacologically blocking CRAC channels with a low concentration of CRAC channel blockers prevented the recovery of oscillations in LiCl-treated cells, even when inositol was present. To further understand how calcium entry contributes to the production of PIP2, I investigated whether PI4P- or PI5P-specific pools support the oscillatory calcium signal induced by LTC4. Accordingly, by using pharmacological blockers, concluded that PIP2 used in LTC4 -mediated calcium signalling is produced via the conversion of PI4P into PIP2 by PI5K1 kinases and that the cellular PI5P pool does not contribute to the calcium signal. Moreover, the conversion of PI4P into PIP2 was possible only when there was calcium entry via CRAC channels. Characterisation of the expressed PI5K1 kinases in RBL-2H3 cells revealed expression of only PIP5K1a and PIP5K1? and that both kinases are needed to maintain the oscillatory calcium signal induced by LTC4 and to provide an overlapping function. To further expand current understanding of how calcium regulates PI5K1 kinases, I specifically investigated how calcium entry regulates PIP5K1?. This was accomplished by looking into PIP5K1?-regulating proteins, of which talin is a focal adhesion protein shown to activate PIP5K1?. In this thesis, I show that the cleavage and activation of talin depend on calcium entry via CRAC channels, thereby elucidating a possible mechanism in how CRAC channels mediating calcium entry are involved in phosphoinositide production. This thesis identifies a new role for CRAC channels in mast cell activation. The opening of CRAC channels and calcium entry are required for PIP2 production and thus the maintenance of agonist-mediated calcium signalling.

The strong association of the human leukocyte antigen HLA-B27 (B27), with spondyloarthropathies (SpA), was discovered more than four decades ago, yet the role B27 plays in disease pathogenesis remains unclear. It has been demonstrated that HLA-B27 can form non-classical FHC forms, including B27 FHC homodimers (B272) and misfolded FHCs. Two leading theories of SpA pathogenesis proposed that both cell surface NC-B27 (the B27 homodimer theory) and intracellular (the B27 misfolding and UPR theory) can drive immune responses in SpA. Several studies demonstrated that cell surface NC-B27 binds to immune receptors and suggested that these interactions can activate Th17 immunity. My DPhil project focuses on the investigation of the cell surface expression of NC-B27 molecules in cells and tissues obtained from AS patients and B27 TG2 rats. I used both existing reagents and a novel HD6 antibody, generated against B272, to examine the expression of HLA-B27 by flow cytometry, confocal microscopy and immunoprecipitation. This thesis provides evidence that the expression of HD6- and HC10-reactive molecules in AS patients is rather restricted to specific cell types (e.g. osteoclasts) and possibly particular environmental factors and tissue types (e.g. synovial fluid and joint tissue). My data using B27 TG2 rats demonstrated that resident joint cells express HD6- and HC10-reactive molecules before the onset of clinical manifestations. Furthermore, I showed increased expression of NC-B27 on antigen presenting cells (APCs), and CD161+ and CD8+ cell populations in blood and secondary lymphoid organs. Based on these findings I hypothesized that cell surface expression of NC-B27 may not only be crucial for activation of Th17-driven immune responses, but may also shift the balance between immune tolerance and activation. Importantly, the expression of potentially pathogenic HD6-reactive molecules is restricted to specific cell and tissue types hence HD6 antibody may be a potential future therapeutic agent in SpA.

Spin-half nuclei in a magnetic field possessing a population difference in excess of that expected at thermal equilibrium are said to be hyperpolarised. Dynamic Nuclear Polarisation (DNP) is a method to generate hyperpolarised systems, pumping nuclear spins into a given state at low temperature. Hyperpolarised samples can be rapidly dissolved in a hot solvent, and maintain a highly polarised spin population that relaxes to the new thermal equilibrium over several minutes. It is possible to inject hyperpolarised molecules into an organism, sample the population difference through NMR spectroscopy, and obtain spectra with a large ( > 104) increase in their signal-to-noise ratio compared to thermal equilibrium. The use of hyperpolarised [1-13C]pyruvate allows for the quantification of the rate of [1-13C]lactate production, increases of which are a defining characteristic of cancer; or the rate of 13C-bicarbonate production, which is altered in heart disease. This thesis describes the development of magnetisation-efficient sequences of spectrally-and-spatially selective radiofrequency pulses and rapid imaging readouts that are able to image the metabolism of hyperpolarised [1-13C]pyruvate in three dimensional space, with a reconstructed resolution of 1 x 1 x 2 mm3. The technique's sensitivity to metabolism is demonstrated, and attempts are made to image cancer that has metastasised to the brain prior to the point of conventional detection through its metabolic phenotype. It is also used to image metabolism in the post ischaemic myocardium, where it reports on regions of non-viable tissue. Additionally, hyperpolarised 13C-urea is used as a 'metabolically inert' probe of perfusion in the myocardium. Flow-sensitisation gradients and a spiral readout allow 13C-urea to be resolved in the myocardium. The ability to 'co-polarise' [1-13C]pyruvate and 13C-urea is demonstrated. This technique could simultaneously resolve perfusion deficits and metabolic changes in the acutely damaged heart, with several potential advantages over current clinical methods. Finally, several methods for analysing data generated by hyperpolarised experiments are proposed: weighted averaging for combining spectroscopic data of varying quality; novel Bayesian approaches to quantifying metabolic rate constants of interest following the injection of hyperpolarised [1-13C]pyruvate; and the Fast Padé Transformation as an improved method for the reconstruction of truncated hyperpolarised spectral data. DNP is a technique that, following its development for particle physics experiments in the 1970s, is transitioning to clinical practice as a safe, novel molecular imaging modality. It is hoped that the techniques proposed in this text ultimately find wide utility in the assessment of cancer and heart disease, which combined are responsible for approximately 50% of deaths occurring in the developed world.

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