Search

Heart failure is one of the leading causes of death worldwide. Hypertension can initiate myocardial remodelling processes which, often via fibrotic triggers through the renin-angiotensin-aldosterone system, can lead to the development of heart failure. A main contributor of these pathways is angiotensin II, increased levels of which can induce volume and pressure overload in the cardiovascular system, making it an important factor in both hypertension and associated cardiovascular disease. A main process during cardiac remodelling is fibrosis which can be divided into two types: reactive and replacement fibrosis. The latter refers to the changes via scar formation at an injury site while the former (interstitial or perivascular fibrosis) can happen as a response to changes in the physical or chemical environment within the tissue such as hypertension or inflammation. Fibrillary collagen is an important extracellular matrix component and abundantly deposited during fibrosis. Collagen can have various subtypes based on its structure which can add different characteristics to the tissue. During collagen biosynthesis, cis- or trans-proline containing pro-α chains can be integrated into the protein, where chains containing cis isomer are associated with more distensible and abnormal collagen and those with trans isomer with more rigid triple helix collagen. Other factors can also influence the development of heart failure via the myocardial remodelling processes, such as inflammatory and angiogenic pathways. Heart failure can be diagnosed and assessed in the clinic via blood tests and imaging techniques such as ultrasound, magnetic resonance imaging (MRI), computerised tomography (CT), and single-photon emission computed tomography (SPECT) / positron emission tomography (PET). This thesis aimed to investigate the effect of increased angiotensin II and subsequent hypertension on the levels of myocardial collagen synthesis and to test PET radiotracers cis-4-18F-fluoro-L-proline and trans-4-18F-fluoro-L-proline for the detection of myocardial fibrosis and potential differentiation of the types of collagen fibers. The overarching hypothesis of the project was that myocardial fibrosis can be imaged non-invasively with PET in a rat pressure overload model with via persistent hypertension resulting in end-organ damage. A hypertensive rat model with myocardial remodelling was established via angiotensin II infusion using osmotic mini-pumps. Treatment length and dosage were tested and the optimal protocol was chosen to induce myocardial fibrosis. The model was assessed for myocardial collagen content as well as markers of inflammation and vasculature. On a separate set of experiments, the performance of PET radiotracers, cis-4-18F-fluoro-L-proline and trans-4-18F-fluoro-L-proline, was assessed in naïve rats to understand their in vivo metabolism and kinetics. Then, the optimised animal model of hypertensive heart failure and PET imaging protocols were used to investigate whether the new imaging probes could visualise areas if increased collagen synthesis and whether the uptake was related to the type of collagen involved. Using 500 ng/kg/min angiotensin II dose for 4 weeks duration was adequate to induce myocardial fibrosis and hypertension in the rat model. The fibrosis pattern was mainly perivascular in nature. Immunostaining also showed increased CD68 in the myocardium of rats on 250 ng/kg/min but not with the higher dose. The highest percentage of cells stained positive for all three of CD68, TSPO and isolectin B4 was found in the atria of animals on the higher angiotensin II dose, which area also showed the most fibrosis overall. Both radiotracers were successfully assessed in naïve rats, showing no metabolism and favourable kinetics in vivo, allowing for simplified quantification of radiotracer uptake. Myocardial radiotracer uptake in the angiotensin II treated cohort showed increased myocardial signal with the trans-4-18F-fluoro-L-proline radiotracer but no significant differences with cis-4-18F-fluoro-L-proline compared to vehicle treated animals. Animals undergoing the imaging experiment showed increased myocardial fibrosis similarly to rats in the model development experiments. Myocardial fibrosis develops during hypertension induced via angiotensin II treatment, and this change can be measured with PET imaging targeting collagen biosynthesis. Further investigation into the types of collagens involved and how they contribute to pathology needs to be carried out to characterise the underlying biological processes in more detail. Fluoroproline radiotracer PET imaging can become a valuable tool for the assessment of fibrosis pathology both in terms of early detection and disease progression.

Aim: To evaluate the performance of a state-of-the-art digital (dPET/CT) and long field-of-view (LAFOV) positron emission/computer tomography (PET/CT) system and to quantify how this translates into improved image quality, lesion detection, diagnostic certainty, lower dose, faster examinations and improved dynamic range. In doing so, some of the first evidence for these novel systems is provided. Methods: This thesis presents five studies which evaluate state-of-the-art dPET/CT systems by means of a matched-pair comparison of a digital with an analogue system, their performance and influence on diagnostic certainty and interrater reliability, the feasibility of reduced applied radiopharmaceutical activities in digital systems, the clinical performance of a novel LAFOV PET/CT system and the feasibility of using these for late acquisition of images for the diagnosis of recurrent prostate cancer. Results: dPET/CT systems revealed higher detection rate compared to an analogue PET/CT system as well as improved interrater reliability and diagnostic certainty. dPET/CT systems allow for 75% reduction in applied radiopharmaceutical activity or scan time. LAFOV PET/CT furnish orders of magnitude increases in count density, the possibility to reduce the total examination time to 30s or reductions in applied activity as well as the ability to examine at much later time points once multiple half-lives have elapsed without detriment to image quality. Conclusion: Digital and LAFOV PET/CT systems exhibit improved performance when compared to analogue and short-axial field-of-view systems, with demonstrated improvement in clinically relevant outcomes such as improved detection rate and diagnostic certainty.

Background: Calcification is one of the body's primary responses to injury and a key pathological feature of cardiovascular disease. Calcification activity can now be imaged using 18F-sodium-fluoride positron emission tomography and computed tomography which targets microcalcification (diameter <50 μm), providing different information to established macroscopic calcification (minimum diameter 200 to 500 μm) imaged with computed tomography. Indeed 18F-sodium fluoride PET has now been explored in a wide range of conditions including coronary atherosclerosis, aortic stenosis and bioprosthetic valve disease, offering a marker of vascular injury and disease activity in each condition studied. At the moment, we lack reliable and reproducible methods for predicting myocardial infarction in patients with established coronary artery disease. Moreover, there remain major uncertainties regarding disease activity within the retained native aortic valve as well as bioprosthetic valve durability following transcatheter aortic valve implantation. In this thesis, I will address the following questions: 1) What is the optimal method for quantifying microcalcification activity and how reproducible is it? 2) Can we further refine it? 3) Can 18F-sodium fluoride PET/CT predict myocardial infarction in patients with advanced atherosclerosis? 4) Can machine learning improve the ability of 18F-sodium fluoride PET/CT to predict events? 5) Can 18F-sodium fluoride PET/CT predict native aortic valve disease progression and bioprosthetic valve degeneration in patients with transcatheter aortic valve implantation? Methods and Results: Study one: 18F-sodium fluoride reproducibility We aimed to establish the observer repeatability and interscan reproducibility of coronary 18F-sodium fluoride positron emission tomography (PET) uptake using a novel semi-automated approach, coronary microcalcification activity (CMA). Nineteen patients with established coronary artery disease were included for analysis. CMA was assessed twice in 43 coronary vessels on two PET/CT scans performed 12±5 days apart. We demonstrated that CMA is a repeatable and reproducible global measure of coronary atherosclerotic activity. There was excellent intraclass correlation for intraobserver and interobserver repeatability as well as interscan reproducibility (all ≥0.991). There was 100% intraobserver, interobserver and interscan agreement for the presence (CMA > 0) or absence (CMA=0) of coronary18F-sodium fluoride uptake. Mean CMA was 3.12± 0.62 with coefficients of repeatability of 0.24 and 0.22 for intraobserver, 0.30 and 0.29 for interobserver and 0.33 and 0.32 for interscan analysis at a per-vessel and per-patient level respectively. Study two: Refining 18F-sodium fluoride uptake We aimed to evaluate the impact of respiratory-averaged computed tomography attenuation correction (RACTAC) compared to standard single-phase computed tomography attenuation correction (CTAC) map, on the quantitative measures of coronary atherosclerotic lesions of 18F-sodium fluoride in PET/CT. Twenty-three patients with calcified plaques in the coronary arteries were included. We evaluated 34 coronary lesions using CTAC and RACTAC reconstructions. We demonstrated that respiratory-averaged and standard single-phase attenuation correction maps provide similar and reproducible methods of quantifying coronary 18F-sodium fluoride uptake on PET/CT. Assessments of the individual vessel microcalcification burden (CMA) revealed no major differences between PET images reconstructed using CTAC and RACTAC scans (median [IQR] CMA: CTAC = 0.10 [0-1.0], RACTAC = 0.15 [0-1.03], p=0.19). Bland-Altman plots of the CMA values revealed a high degree of agreement when comparing the per vessel burden, with coefficient of reproducibility of 0.17. Study three: Coronary 18F-sodium fluoride PET/CT In a multicentre study, we investigated whether coronary 18F-sodium fluoride PET uptake predicts future myocardial infarction in patients with established coronary artery disease, and whether it can provide additional prognostic information over and above current methods of risk stratification including clinical risk scores, coronary calcium scoring and the severity of obstructive coronary artery disease. We studied 293 participants (65±9 years; 84% male) with established coronary artery disease. Patients underwent 18F-sodium fluoride PET/CT and were followed-up for fatal or non-fatal myocardial infarction over 42 [31-49] months. Total coronary 18F-sodium fluoride uptake was determined using coronary microcalcification activity (CMA). Fatal or non-fatal myocardial infarction occurred only in patients with increased coronary 18F-sodium fluoride activity (20/203 CMA > 0 versus 0/90 CMA=0; p<0.001). On receiver operator-curve analysis, fatal or non-fatal myocardial infarction prediction was highest for 18F-sodium fluoride CMA, outperforming coronary calcium scoring, modified Duke coronary artery disease index, cardiac risk scores (areas under curve: 0.76 versus 0.54, 0.62, 0.52 and 0.54; p<0.001 for all). Patients with CMA > 1.56 had >7-fold increase in fatal or non-fatal myocardial infarction (hazard ratio 7.1, 95% confidence interval 2.2 to 25.1; p=0.003) independent of age, gender, risk factors, segment involvement and coronary calcium scores, presence of coronary stents, coronary stenosis, cardiac risk scores, the Duke coronary artery disease index and recent myocardial infarction. Study four: Artificial intelligence in 18F-sodium fluoride PET/CT We undertook a post-hoc analysis of the population of patients from study three. Coronary 18F-sodium fluoride PET/CT and CT angiography-based quantitative plaque analysis have shown promise in refining risk stratification in patients with coronary artery disease. We combined both novel imaging approaches to develop an optimal machine-learning model for the future risk of myocardial infarction in patients with stable coronary disease. We demonstrated that our machine learning approach has overcome the challenges posed by co-linearity of these variables and, for the first time, demonstrated that this information is complementary and additive with the combination of both providing the most robust outcome prediction. On univariable receiver-operator-curve analysis, only 18F-sodium fluoride coronary uptake emerged as a predictor of myocardial infarction (c- statistic 0.76, 95% confidence interval [CI] 0.68-0.83). When incorporated into machine-learning models, clinical characteristics showed limited predictive performance (c-statistic 0.64, 95% CI 0.53-0.76;) and were outperformed by a quantitative plaque analysis-based machine-learning model (c-statistic 0.72, 95% CI 0.60-0.84). After inclusion of all available data (clinical, quantitative plaque and 18F-sodium fluoride PET), we achieved a substantial improvement (p=0.008 versus 18F-sodium fluoride PET alone) in the model performance (c-statistic 0.85, 95% CI 0.79-0.91). Study five: 18F-sodium fluoride in transcatheter aortic valves We undertook a multicentre cross-sectional observational cohort study to determine whether the retained native aortic valves in patients undergoing transcatheter aortic valve implantation (TAVI) demonstrate evidence of ongoing disease progression. Additionally, since long-term durability of transcatheter aortic valves is yet to be established, we aimed to establish whether bioprosthetic valve durability or degeneration was appreciably different from patients with surgical aortic valve replacement (SAVR). Patients with TAVI or bioprosthetic SAVR underwent baseline echocardiography, CT angiography and 18F-sodium fluoride PET/CT. Subsequently patients underwent serial echocardiography to assess for changes in valve hemodynamic performance (change in peak aortic velocity) and evidence of structural valve dysfunction. Comparisons were made to matched patients with bioprosthetic SAVR who had undergone the same imaging protocol. We enrolled 47 patients (81±6 years old, 79% male) with TAVI from 3 high volume centres. We demonstrated that 18F-sodium fluoride uptake within the native aortic valve is higher with longer duration of implantation suggesting disease activity continues despite immobilisation of the valve leaflet.

Within neuroblastoma (NB), approximately 50% of patients are considered high-risk, presenting widespread distant metastasis and, frequently, MYCN amplification. NB is further characterised by the selective presence of the noradrenaline transporter (NET-1) and the GD2 disialoganglioside in a majority of cases. Targeted therapies against both NET-1 and GD2 have improved patient survival, however, many patients eventually relapse from disease. mIBG (meta-iodobenzylguanadine) is a noradrenaline analogue used for diagnosis and treatment of NB, yet despite 90% of patients presenting NET-1 expressing tumours and being characaterised as 'mIBG avid', less than 50% respond to targeted 131I-mIBG radiotherapy. This may be, in part, due to the limitations of spatial resolution and image quantification of single photo emission computed tomography (SPECT) acquisitions performed with 123I-mIBG. To address these deficiencies I have investigated the use of a fluorinated noradrenaline analogue, 18F-mFBG, for use with positron emission tomography (PET) imaging. Of note, a first-in-human study using 18F-mFBG has demonstrated clear benefits of using this tracer compared to 123I-mIBG, yet the tracer has not been fully translated to clinical practice due to a complicated radio-synthesis. The ability of pharmacological intervention to modulate NET-1 expression in NB has been highlighted with preclinical and clinical studies using the histone deacetylate (HDAC) inhibitor, vorinostat. I wanted to build on this work by investigating compounds that may increase NET-1 expression whilst simultaneously targeting other pathways critical in patients with high-risk disease, in particular the PI3k/Akt/mTOR pathway. I hypothesised that drugs targeting mTORC1/2 can increase NET-1 expression in NB models, to which I investigated the use of 18F-mFBG as a tool to quantify these changes in vitro and in vivo. GD2 expression is also thought to be ubiquitously expressed in NB and GD2-targeted immunotherapy (Dinutuximab/Dinutuximab-Beta) has provided promising improvements in the event free survival of patients compared to historical 'standard therapies'. However, NB tumours with positive, but low, GD2 expression are likely to relapse. Current methods to investigate tumour GD2 expression are invasive (tumour biopsies) and only provide a snapshot of the disease. In an attempt to better understand the efficacy and pitfalls of GD2 immunotherapy, I have developed an 89Zr-labelled GD2 antibody that could provide a means of measuring the tumour GD2 expression level in situ.

The use of cell-based therapies as a living drug have gained considerable attention for the treatment of a range of conditions such as genetic diseases, autoimmune disorders and cancer. The emergence of genetically engineered T cells expressing chimeric antigen receptors (CAR T-cells), together with modulations of immune checkpoints have brought the rapidly advancing field of immunotherapy into a new, revolutionary spotlight with a renewed interest in cell-based therapies. In vivo imaging and tracking of cells can be used to non-invasively improve the accuracy, efficacy and safety of novel immune-modulatory treatments and cell therapies. Positron Emission Tomography (PET) is a powerful non-invasive imaging technique that can be utilised for spatial and longitudinal tracking of different cell types. The long half-life PET isotope zirconium-89 (t1/2 = 78.4 h) has become increasingly available and [⁸⁹Zr]Zr-Oxine has recently emerged as a promising candidate for direct cell labelling and longitudinal cell tracking. However, the water insolubility and synthesis method of [⁸⁹Zr]Zr-Oxine may limit its implementation as a routine clinical imaging tool and little work has been performed looking at alternatives to this compound. The aim of this thesis was to investigate alternative approaches and methods for the direct labelling of cells using zirconium-89. This work also describes the first use of zirconium-89 in Cambridge and the set-up of the infrastructure and methods. First, the sensitivity of detecting 89Zr-labelled cells on clinical PET systems was investigated that can inform on the minimal cellular radioactivity needed for detection in prospective clinical studies. Secondly, a peptide-based approach was developed whereby cell penetrating peptides were tested for 89Zr-labelling, purification, quality control and their in vitro properties. ⁸⁹Zr-labelled peptides were subsequently employed for direct cell labelling in comparison to [⁸⁹Zr]Zr-Oxine in preparation for preclinical studies.

Frontotemporal dementia (FTD) is clinically and pathologically diverse, encompassing the behavioural variant FTD; non-fluent variant primary progressive aphasia; and semantic variant primary progressive aphasia. These are usually associated with either tau or TDP-43 pathology, with highly variable clinicopathological correlations. Neuroinflammation also contributes to the pathogenesis of FTD, but its relevance to the disease spectrum is incompletely understood. There is a critical need for better understanding of how drivers of pathophysiology, such as neuroinflammation and protein aggregation, relate to the heterogeneity of clinical disease in vivo. This knowledge gap currently forms a significant barrier to the development of effective treatments in FTD. I review the clinical, pathological and genetic features of FTD and the role of PET for measuring in vivo components of pathophysiology in this setting. I then describe a series of case studies and group analysis of FTD syndromes, using positron emission tomography (PET) radioligands to visualise and quantify different aspects of pathophysiology in vivo. [18F]AV-1451tau was introduced primarily to study tau pathology in Alzheimer’s disease using, which differs from FTD tauopathy in several respects. I examined the sensitivity and specificity of [18F]AV-1451 in FTD, in vivo, through (i) [18F]AV-1451 imaging of the FTLD-tau pathology in a case of FTD due to a MAPT 10+16 mutation in the microtubule associated protein tau, and a second pre-symptomatic case with the same mutation; (ii) [18F]AV-1451 imaging of a cohort of seven cases with Semantic Dementia and one case of FTD from a C9orf72 expansion, both strongly associated with TDP-43 pathology without tau; and (iii) the increase in [18F]AV-1451 binding, and changes in the distribution of binding, in thirty one patients spanning the three major FTD syndromes in comparison to matched controls. The literature on the role of neuroinflammation in FTD is more limited. I used the PET ligand [11C]PK-11195, as an established marker of activated microglia. I report the elevation in [11C]PK-11195 binding, and the change in its distribution, in a case of a pre-symptomatic MAPT 10+16 mutation carrier; and in twenty nine patients spanning the three major FTD syndromes in comparison to matched controls. In addition to reporting the correlations between PET ligand binding and disease severity, I describe the relationship across regions and across syndromes between [18F]AV-1451 and [11C]PK-11195 binding. In view of the marked variations in affinity of [18F]AV-1451 for different tau isoforms and TDP43-pathology, my analyses focus on multivariate distributions rather than absolute binding potential. The results show high correlations between [18F]AV-1451 and [11C]PK-11195 binding in each FTD syndrome. However, in the healthy MAPT 10+16 carrier, the distribution of elevated [11C]PK-11195 binding is much more extensive that the elevation of [18F]AV-1451, suggesting that inflammation might precede the aggregation of tau. I discuss the limitations of the PET ligands, and summarise the insights into FTD pathogenesis arising from my series of observational studies. The role of new PET ligands, and the integration of PET in future clinical trials are discussed.

Preclinical Positron Emission Tomography/Computed Tomography (PET/CT) is a well-established non-invasive imaging tool for studying disease development/progression, the development of novel radiotracers and pharmaceuticals for clinical applications. Over the last five years more than 8,200 preclinical studies using PET/CT were conducted. Despite this pivotal role, standardisation of preclinical PET/CT protocols, including CT absorbed dose guidelines, is essentially non-existent. Therefore, the first and second aims of this project were: (1) to quantitatively assess the variability of current preclinical PET and CT acquisition and reconstruction protocols in routine use across multiple centres and scanners; and (2) to propose optimized standardised acquisition and reconstruction PET/CT protocols for routine scanning procedures across all sites in a preclinical PET/CT laboratory. By assessing quantitative accuracy (known versus measured) and precision (reduced variability) of currently used routine protocols between five different sites/scanners (Bruker Albira, Mediso nanoPET/CT, Sedecal Super Argus, Siemens Inveon and Trifoil LabPET/CT), standard protocols were determined. Thereby, irrespective of scanner characteristics the least biased empirical quantitative and qualitative protocol results defined the standard protocol. In essence, neutralizing the manufacturers' difference, replacing scanner variability for scanner similarity to establish global standard protocols. The analysis of sites’ routine protocol results revealed significant quantitative differences between all five sites/scanners. Whereas the standard protocols put forth improved accuracy and precision across all sites. Additionally, the large disparity and measured amounts of CT absorbed ionising radiation amongst sites brought to light the lack of preclinical radiation guidelines and dose regulations. Unregulated CT radiation dose is of great concern. CT ionising radiation is known to have biological adverse effects. Thus, overexposure of radiation will potentially cause unnecessary animal suffering and confound research outcomes. Overall, the proposed standard CT protocol reduced radiation doses. The implementation of preclinical PET/CT standardised protocols, developed and tested in this project, will provide more robust, reliable and reproducible translational data sets for clinical applications. In accordance with the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) objectives, the refinement of PET/CT protocols and reduction of CT absorbed dose impacts animal welfare positively and potentially reduces the number of animals used. Reducing study variability in site and across sites through standardisation of protocols improves statistically significant results using less animals. For study specific imaging parameters in preclinical PET/CT rodents are commonly used to design the protocol. The third aim of this project strives to develop a tissue equivalent material (TEM) anthropomorphic rodent phantom for the replacement of animals when designing and optimizing varying in vivo rodent imaging protocols. Using a TEM phantom reduces potential biological experimental variability caused by the animals and increases reproducibility of findings. To address this aim, twenty-four commercially available 3D printing materials were X-rayed for the evaluation Hounsfield units (HU). A comparison of calculated 3D material attenuation coefficients and accepted tissue attenuation coefficient was also done. CT images were acquired using four CT protocols and the developed standard CT protocol. Based on measured material HUs compared to accepted tissue/organ HU values, four materials were chosen for testing and further evaluation in a 3D printed phantom prototype was undertaken. In order to obtain the anatomical features of the rodent a CT acquisition of a scheduled 1 rodent was acquired. The CT images were used for the 3D printing design. A 3D printed (TEM) anthropomorphic rodent phantoms was printed and tested. Measured HU analysis of the phantom TEM materials shows promise as a replacement strategy. This imaging protocol optimisation approach is also in line with the NC3Rs objective of replacing and/or avoiding the use animals.

Neuroinflammation and dysregulation of glutamate in schizophrenia A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health by Catherine J Gregory, June 2019 Background: Schizophrenia is a mental illness with a lifetime prevalence of 7 in 1000 people. In some, symptoms can persist despite treatment with antipsychotic medication, leading to a poor quality of life. There has been a drive to better understand the pathophysiology behind schizophrenia, with the hope of developing more effective treatments. There is good evidence for a role of hypofunction of the glutamatergic N-methyl-D-aspartate receptor (NMDAR) in schizophrenia, as well as for a role of neuroinflammation. However, how these processes may vary across the illness duration and with antipsychotic exposure is unclear. In particular, how these processes may be linked is uncertain. There is emerging evidence that they may be linked via neuroinflammation-mediated activation of the kynurenine pathway of tryptophan degradation and the production of neuroactive metabolites such as the endogenous NMDAR antagonist kynurenic acid. However, no study to date has measured neuroinflammation and NMDAR hypofunction in the same patients. Methods: Patients with recent onset (n=20) and chronic (n=21) schizophrenia were recruited along with age- and sex-matched healthy controls (n=19). Positron emission tomography (PET) and the 18kDa translocator protein (TSPO) radioligand [11C]-(R)-PK11195 measured TSPO availability as an indication of microglial activation, a putative measure of neuroinflammation. 1H magnetic resonance spectroscopy (MRS) measured concentrations of glutamate, glutamine and gamma-aminobutyric acid (GABA) as an indication of NMDAR function, as well as myo-inositol, a putative glial marker. Results: In the first study of its kind, we found a positive correlation between TSPO availability and glutamate in antipsychotic-free patients with recent onset illness, which was not present in antipsychotic-exposed or chronic patients. We also found a reduction in TSPO availability in antipsychotic-free recent onset schizophrenia compared to controls but no difference in antipsychotic-exposed or chronic patients. We found no difference in glutamate, glutamine or GABA between recent onset or chronic schizophrenia and controls. We found a weak positive correlation between myo-inositol and TSPO availability in schizophrenia but no difference in myo-inositol concentration between patients and controls. Conclusions: Our results provide evidence for a link between TSPO availability and glutamate concentration in antipsychotic-free recent onset schizophrenia. This association appears to be moderated by antipsychotic medication. The lack of group differences in TSPO availability or glutamate suggest that significant neuroinflammation leading to glutamatergic dysfunction may only be present in a subset of schizophrenia, such as those with treatment resistance. Further investigation is needed in this cohort of patients. We also found preliminary evidence in support of a role for myo-inositol as a potential MR marker of microglia, but further confirmatory evidence is needed.

Cell death is an important target for imaging the early response of tumours to treatment. In this study a phosphatidylserine-binding protein (C2Am) has been derivatised with a fluorine-18 containing maleimide for imaging tumour cell death in a xenograft murine advanced colorectal cancer. A one-pot, two-step automated synthesis of N-(5-[18F]fluoropentyl)maleimide using a GE TRACERlab FXFN automated module (within 58±5.8 min (n = 12), >98% radiochemical purity and 12±3% decay corrected yield) has been developed. This was used to label the single cysteine present on C2Am within 30 min in PBS (Am=212000±30000 MBq/μmol (n = 3)). Using xenograft models of breast and colorectal cancer, and a TRAIL-R2 agonist for inducing cell death, the binding of [18F]FPenM-C2Am was tested in vitro and in vivo using biodistribution and dynamic PET imaging studies. Cell death detection was validated by cell death histology assays CC3 and TUNEL. For colorectal cancer, there was a positive correlation between [18F]FPenM-C2Am signal in tumours post treatment and the corresponding histologic markers of cell death CC3 (Pearson r = 0.82) and TUNEL (Pearson r = 0.95). [18F]FPenM-C2Am showed a favourable biodistribution profile, with predominantly renal clearance and minimal retention in spleen (0.79±0.05 %ID/g), liver (1.18±0.13 %ID/g), small intestine (0.97±0.25 %ID/g) and kidney (6.90±0.56 %ID/g) at 2 h after probe administration. In a xenograft model of colorectal cancer treated with a TRAIL-R2 agonist, for 24h, at 0.2-0.4 mg/kg, i.v. [18F]FPenM-C2Am generated tumour-to-muscle and tumour-to-blood ratios following treatment of 6.7±0.8-fold and 1.89±0.23-fold, respectively, at 2 h after administration. A statistically significant pairwise difference was obtained between the tumour-to-muscle contrast prior to and following therapy (P=0.0137, unpaired two-tailed t-test). Given the favourable biodistribution profile of [18F]FPenM-C2Am, and its ability to produce rapid and cell death-specific image contrast, this agent has potential for clinical translation. We have initiated cGMP manufacture and toxicology studies required for a Phase 1 trial. Aberrant cell surface glycosylation has been described as one of the key hallmarks of cancer. Monitoring glycosylation could provide an insight into tumour progression, proliferation and ultimately could potentially be used for monitoring treatment response. Aberrant glycosylation can be observed by harnessing the cell’s metabolism to incorporate into its glycome unnatural sugars bearing bioorthogonal chemical reporters. These reporters are targeted subsequently by fluorescent, magnetic or optoacoustic probes that allow imaging. As part of this work, we have demonstrated in vitro that a peracetylated cyclopropene mannosamine (Ac4ManNCCp)-modified sugar can be used as a tool for imaging hypersialylation in an advanced colorectal cancer model. Further optimisation of the probe to improve solubility is required to facilitate transitioning from in vitro to in vivo imaging. Nevertheless, overcoming this solubility issue would allow for facile labelling of surface glycans using PET radionuclides.

The ability to assess the distribution and extent of tau pathology and neuroinflammation in Progressive Supranuclear Palsy (PSP) and Alzheimer's disease (AD) 'in vivo' and to validate it using post mortem data is critical to develop reliable biomarkers for these disorders, and for tracking the effects of clinical trials using disease-modifying therapies. The main aim of my PhD was therefore to assess the utility of the PET radiotracers [18F]AV1451 and [11C](R)-PK11195 to compare the distribution and intensity of tau pathology and neuroinflammation respectively, in PSP and AD. I use both in vivo scanning and post mortem tissue from archival cases in the Cambridge Brain Bank. For the in vivo studies, I investigated whether tau accumulation and neuroinflammation relate to clinical measures of disease severity and cognitive impairment. My PhD was undertaken as part of the Neuroimaging of Inflammation in MemoRy and Other Disorders (NIMROD). Nineteen people with PSP-Richardson's syndrome, 15 patients with amnestic Alzheimer's disease and biomarker-positive mild cognitive impairment (MCI+) and 13 age- and sex-matched controls participated. Post mortem brain tissue from three subjects (one PSP case, one AD patient, and one control with similar age) were included. Autoradiographic and immunohistochemical analyses were conducted in different cases from those included in the [18F]AV1451 and [11C](R)-PK11195 PET in vivo studies. [18F]AV1451 PET imaging revealed distinct patterns of tau binding in AD and its prodromal state of MCI+, in comparison to PSP. The clinical syndromes of AD and MCI+ were associated with increased [18F]AV1451 BPND in widely distributed subcortical and cortical areas that have been consistently implicated in the pathogenesis and progression of AD (e.g. hippocampus, amygdala as well as frontal, parietal, temporal, and occipital cortices). Conversely, PSP was associated with a pattern of increased [18F]AV1451 uptake in the basal ganglia, midbrain, and dentate nucleus of the cerebellum, consistent with the pathophysiology of the disease. Despite this potential of [18F]AV1451 as a tau biomarker, caution in the interpretation of its binding targets is indicated by the neuropathological and autoradiographic data. In particular, while [18F]AV1451 strongly bound to AD-related tau pathology, non-specific binding of the same tracer can be found in PSP patients and controls. Furthermore, [18F]AV1451 uptake was not correlated with disease severity in the clinical groups. [11C](R)-PK11195 PET imaging revealed distinct patterns of inflammation in AD and PSP, and mirrored the pathological distribution for each disease seen by [11C](R)-PK11195 post mortem phosphor screen autoradiography and immunohistochemistry. In AD, microglial activation was observed in prefrontal cortex, and medial temporal lobe. In PSP microglial activation was observed in midbrain and basal ganglia. In both clinical cohorts, disease severity correlated with neuroinflammation in the regions most closely associated with principal neuropathological markers including tau aggregates.

Background: Aortic stenosis is not only characterized by progressive valve narrowing but also by the hypertrophic response of the left ventricle that ensues. In this most common valvular condition novel imaging approaches (cardiovascular magnetic resonance [CMR] and positron emission tomography [PET]) have shown promise in the assessment of disease progression and risk stratification. The central aim of this thesis was to investigate the potential of CMR imaging to refine risk prediction and to improve the imaging protocol of 18F-sodium fluoride PET for aortic stenosis. Methods and Results: Asymmetric wall thickening in aortic stenosis In a prospective observational cohort study, 166 patients with aortic stenosis (age 69, 69% males, mean aortic valve area 1.0±0.4cm2) and 37 age and sex-matched healthy volunteers underwent phenotypic characterisation with comprehensive clinical, imaging and biomarker evaluation. Asymmetric wall thickening on both echocardiography and cardiovascular magnetic resonance was defined as regional wall thickening ≥13 mm and >1.5-fold the thickness of the opposing myocardial segment. Asymmetric wall thickening was observed in 26% (n=43) of patients with aortic stenosis using magnetic resonance and 17% (n=29) using echocardiography. Despite similar demographics, co-morbidities, valve narrowing, myocardial hypertrophy and fibrosis, patients with asymmetric wall thickening had increased cardiac troponin I and brain natriuretic peptide concentrations (both p < 0.001). Over 28 [22, 33] months of follow-up, asymmetric wall thickening was an independent predictor of aortic valve replacement or death whether detected by magnetic resonance (HR=2.15; 95 CI 1.29 to 3.59; p=0.003) or echocardiography (HR=1.79; 95 CI 1.08 to 3.69; p=0.021). Animal model of pressure overload We performed serial Cardiac Magnetic Resonance (CMR) imaging every 2-week in 31 mice subjected to pressure overload (continuous angiotensin II infusion) for 6 weeks and investigated reverse remodelling by repeating CMR 1 month following normalization of afterload (n=9). Cine CMR was used to measure left ventricular volumes, mass, and systolic function whilst myocardial fibrosis was assessed using indexed ECV (iECV) calculated from T1-relaxation times acquired with a small animal modified look-locker inversion recovery sequence. During the initial phase of increased pressure afterload indices of left ventricular hypertrophy (0.091 [0.083, 0.105] vs 0.123 [0.111, 0.138] g) and myocardial fibrosis (iECV: 0.022 [0.019, 0.024] vs 0.022 [0.019, 0.024] mL) increased in line with blood pressure measurements (65.1±12.0 vs 84.7±9.2 mmHg) whilst left ventricular ejection fraction (LVEF, 59.3 [57.6, 59.9] vs 46.9 [38.5, 49.6] %) deteriorated significantly (all p≤0.01 compared to baseline). During the reverse remodelling phase blood pressure normalized (68.8±5.4 vs 65.1±12.0 mmHg, p=0.42 compared to baseline). Whilst LV mass (0.108 [0.098, 0.116] vs 0.091 [0.083, 0.105] g) and iECV (0.034 [0.032, 0.036] vs 0.022 [0.019, 0.024] mL) improved both remained elevated compared to baseline (p < 0.05). Similarly, the LVEF remained impaired 51.1 [42.9, 52.8] vs 59.3 [57.6, 59.9] %, p=0.03. There was a strong association between LVEF and iECV values during pressure overload (r=-0.88, p < 0.001). Gender differences in aortic stenosis Two hundred forty-nine patients (66±13 years, 30% women) with at least mild AS were recruited from two prospective observational cohort studies and underwent comprehensive Doppler echocardiography and CMR exams. On CMR, T1 mapping was used to quantify extracellular volume (ECV) fraction as a marker of diffuse fibrosis, and late gadolinium enhancement (LGE) was used to assess focal fibrosis. There was no difference in age between women and men (66±15 vs 66±12 years, p=0.78). However, women presented a better cardiovascular risk profile than men with less hypertension, dyslipidemia, diabetes, and coronary artery disease (all p≤0.10). As expected, LV mass index measured by CMR was smaller in women than in men (p < 0.0001). Despite fewer comorbidities, women presented larger ECV fraction [29.0 (27.4-30.6) vs. 26.8 (25.1-28.7) %, p < 0.0001] and similar LGE [4.5 (2.3- 7.0) vs. 2.8 (0.6-6.8) %, p=0.20] than men. In multivariable analysis, female sex remained an independent determinant of higher ECV fraction and LGE (both p≤0.05). Prior CT angiography for PET Forty-five patients (age 67.1±6.9 years, 76% males) underwent CTA (CTA1) and combined 18F-NaF PET/CTA (CTA2) imaging within 14 [10,21] days. We fused CTA1 from visit one with 18F-NaF PET from the second visit (PET) and compared visual pattern of activity, maximal standard uptake values (SUVmax) and target to background (TBR) measurements on (PET/CTA1) fused versus hybrid (PET/CTA2) data. On PET/CTA2, 226 coronary plaques were identified. Fifty-eight coronary segments from 28 (62%) patients had high 18F-NaF uptake (TBR>1.25), whilst 168 segments had lesions with 18F-NaF TBR ≤1.25. Uptake in all lesions was categorized identically on co-registered PET/CTA1. There was no significant difference in 18F-NaF uptake values between PET/CTA1 and PET/CTA2 (SUVmax: 1.16±0.40 vs. 1.15±0.39, p=0.53; TBR:1.10±0.45 vs. 1.09±0.46, p=0.55). The intraclass correlation coefficient for SUVmax and TBR was 0.987 (95%CI 0.983 to 0.991) and 0.986 (95%CI 0.981 to 0.992). There was no fixed or proportional bias between PET/CTA1 and PET/CTA2 for SUVmax and TBR. Cardiac motion correction of PET scans improved reproducibility with tighter 95% limits of agreement (±0.14 for SUVmax and ±0.15 for TBR vs. ±0.20 and ±0.20 on diastolic imaging; p < 0.001). Delayed PET imaging Twenty patients (67±7years old, 55% male) with stable coronary artery disease underwent coronary CT angiography and PET/CT both 1 h and 3 h after the injection of 266.2±13.3 MBq of 18F-NaF. We compared the visual pattern of coronary uptake, maximal background (blood pool) activity, noise, standard uptake values (SUVmax), corrected SUV (cSUVmax) and target to background (TBR) measurements in lesions defined by CTA on 1h vs 3h post injection 18F-NaF PET. On 1h PET 26 CTA lesions with 18F-NaF PET uptake were identified in 12 (60%) patients. On 3h PET we detected 18F-NaF PET uptake in 7 lesions which were not identified on the 1h PET. The median cSUVmax and TBR values of these lesions were 0.48 [interquartile range (IQR) 0.44-0.51] and 1.45 [IQR, 1.39-1.52] compared to -0.01 [IQR, -0.03-0.001] and 0.95 [IQR, 0.90-0.98] on 1h PET, both p < 0.001. Across the entire cohort 3h PET SUVmax values were similar to 1h PET measurements 1.63 [IQR, 1.37-1.98] vs. 1.55 [IQR, 1.43-1.89], p=0.30 and the background activity was lower 0.71 [IQR, 0.65-0.81] vs. 1.24 [IQR, 1.05-1.31], p < 0.001. On 3h PET, the TBR values, cSUVmax and the noise were significantly higher (2.30 [IQR, 1.70-2.68] vs 1.28 [IQR, 0.98-1.56], p < 0.001; 0.38 [IQR, 0.27-0.70] vs 0.90 [IQR, 0.64-1.17], p < 0.001 and 0.10 [IQR, 0.09-0.12] vs. 0.07 [IQR, 0.06-0.09], p=0.02). The median cSUVmax and TBR values increased by 92% (range: 33-225%) and 80% (range: 20-177%). Conclusions: In aortic stenosis, asymmetric wall thickening is associated with adverse prognosis, in this condition there are significant differences in the fibrosis burden between male and female patients and the adverse remodeling of the ventricle can be reproduced in a simple animal model of pressure overload. For 18F-NaF PET utilizing a CT angiography acquired before the PET acquisition enables adequate uptake quantification and delayed emission scanning facilitates image analysis.

Positron emission tomography (PET) is a non-invasive medical imaging technique that allows visualisation and quantification of biochemical, physiological and pharmacological processes in living subjects. This is achieved through application of radiotracers – compounds labelled with positron emitting radionuclides. Neurodegeneration is the progressive loss of neurons resulting in impairment of brain function leading to cognitive decline and can affect movement. The underlying pathology of many neurodegenerative diseases is misfolding of proteins such as α-synuclein, the key pathological hallmark of Parkinson’s disease. Also implicated in the processes of neurodegeneration is neuroinflammation, which is observed by the activation of microglia – the immune cells of the brain. Activation of microglia is associated with the upregulation of the 18 kDa mitochondrial translocator protein (TSPO). This work has involved the synthesis and characterisation of novel compounds that have the potential for being applied as radiotracers for imaging α-synuclein fibrils (project 1), or TSPO (project 2) via PET. Over the course of project 1 a library of compounds was synthesised based upon structural modifications of a lead structure identified from the literature. These compounds then underwent screening via biophysical methodologies in order to determine their affinity to α-synuclein fibrils. This stage of the work involved the development of a novel biophysical technique – microscale thermophoresis (MST). A general automated radiosynthetic method to afford the [18F]fluoro-derivatives of these compounds has also been developed, and preliminary in vitro autoradiography studies and an in vivo microPET scan has been performed. For project 2, an automated radiosynthetic method was developed to produce [18F]GE387, a lead compound identified through collaboration with GE Healthcare. This radiotracer has then been applied to preliminary in vitro autoradiography and an in vivo microPET study using rats with induced neuroinflammation alongside control rats.

A characteristic feature of Chronic Obstructive Pulmonary Disease (COPD) is an abnormal inflammatory response in the lungs to inhaled particles or gases. The ability to assess and monitor this response in the lungs of COPD patients is important for understanding the pathogenic mechanisms, but also provides a measure of the activity of the disease. Disease activity is more likely to relate to lung inflammation rather than the degree of airflow limitation as measured by the FEV1. Preliminary studies have shown the 18F fluorodeoxyglucose positron emission tomography (18F FDG-PET) signal, as a measure of lung inflammation, is quantifiable in the lungs and is increased in COPD patients compared to controls. However, the methodology requires standardisation and any further enhancement of the methodology would improve its application to assess inflammation in the lungs. I investigated various methods of assessing FDG uptake in the lungs and assessed the reproducibility of these methods, and particularly evaluated whether the data was reproducible or not in the COPD patients (smokers and ex-smokers). This data was then compared with a group of healthy controls to assess the role of dynamic 18F FDG-PET scanning as a surrogate marker of lung inflammation. My data showed a good reproducibility of all methods of assessing FDG lung uptake. However, using conventional Patlak analysis, the uptake was not statistically different between COPD and the control group. Encouraging results in favour of COPD patients were nonetheless shown using compartmental methods of assessing the FDG lung uptake, suggesting the need to correct for the effect of air and blood (tissue fraction effect) when assessing this in a highly vascular organ like the lungs. A prospective study analysis involving a bigger cohort of COPD patients would be desirable to investigate this further.

BACKGROUND: Cardiovascular disease, principally manifest as myocardial infarction or stroke, is the dominant cause of death worldwide and despite therapeutic advances, the global burden of these conditions continues to increase. In order to address this ongoing disease burden, there is a clear need to more effectively target the use of existing and novel diagnostic investigations and medical therapies. Emerging cardiovascular biomarkers include the biochemical, such as high-sensitivity cardiac troponin, and the radiological, such as computed tomography coronary angiography (CTCA) and 18Ffluoride positron emission tomography (PET). Cardiac troponins can now be reliably quantified in clinically stable or asymptomatic populations and provide information about myocardial pathophysiology, whilst CTCA can non-invasively quantify atherosclerotic burden and 18F-fluoride PET imaging offers insight into plaque vulnerability. Improved targeting of diagnostic investigations requires more reliable estimation of pre-test probability of coronary disease whilst optimizing the use of pharmacological or interventional treatments requires more accurate prognostic stratification. Achieving both objectives in an equitable manner across all population groups will depend upon updated clinical guidelines containing improved risk models and enhanced management pathways. The objective of this thesis was to investigate the potential clinical benefit of novel approaches to the diagnostic and prognostic assessment of coronary heart disease. EVALUATION OF THE 2016 NATIONAL INSTITUTE FOR HEALTH AND CARE EXCELLENCE (NICE) GUIDANCE ON THE ASSESSMENT OF SUSPECTED STABLE ANGINA. A post-hoc analysis was undertaken of the Scottish COmputed Tomography of the HEART (SCOT-HEART) trial of 4,146 participants with suspected angina randomised to assessment with computed tomography coronary angiography or standard care. Patients were dichotomised according to guideline definitions into groups representing possible angina and non-anginal presentations. The primary (diagnostic) endpoint was diagnostic certainty of angina at 6 weeks and the prognostic endpoint comprised fatal and non-fatal myocardial infarction. In 3,770 eligible participants, CTCA increased diagnostic certainty more in those with possible angina (relative risk [RR] 2.22 (95% CI 1.91-2.60), p < 0.001) than those with non-anginal symptoms (RR 1.30 (1.11-1.53), p=0.002; pinteraction < 0.001). In the possible angina cohort, CTCA did not change rates of invasive angiography (p=0.481) but markedly reduced rates of normal coronary angiography (hazard ratio [HR] 0.32 (0.19-0.52), p < 0.001). In the non-anginal cohort, rates of invasive angiography increased (HR 1.82 (1.13-2.92), p=0.014) without reducing rates of normal coronary angiography (HR 0.78 (0.30-2.05), p=0.622). At 3.2 years of follow-up, fatal or nonfatal MI was reduced in patients with possible angina (3.2% to 1.9%; HR 0.58 (0.34- 0.99), p=0.045) but not in those with non-anginal symptoms (HR 0.65 (0.25-1.69), p=0.379). Overall the updated NICE guidance on patient assessment maximises the benefits of CTCA with respect to diagnostic certainty, the use of invasive coronary angiography, and reductions in fatal and non-fatal myocardial infarction. Patients with non-anginal chest pain derive minimal benefit from CTCA, which instead increases rates of invasive investigation. EXTERNAL VALIDATION OF THE PROSPECTIVE MULTICENTER IMAGING STUDY FOR EVALUATION OF CHEST PAIN (PROMISE) TOOL FOR DETERMINING MINIMAL-RISK OF CORONARY ARTERY DISEASE. The PROspective Multicenter Imaging Study for Evaluation of chest pain (PROMISE) minimal-risk tool was recently developed to identify patients with suspected stable angina at very low risk of coronary artery disease and clinical events. The external validity of this tool was investigated within the context of the Scottish Computed Tomography of the HEART multicenter randomised controlled trial of patients with suspected stable angina due to coronary artery disease. Model discrimination and calibration was determined amongst 1,764 patients in whom complete CCTA data were available and compared with the European Society of Cardiology guideline-endorsed Coronary Artery Disease Consortium (CADC) risk score. The PROMISE minimal-risk tool improved discrimination compared with the CADC model (c-statistic 0.785 vs 0.730, p < 0.001) and was improved further following re-estimation of covariate coefficients (c-statistic 0.805, p < 0.001). Model calibration was initially poor (c2 197.6, Hosmer-Lemeshow [HL] p < 0.001), with significant overestimation of probability of minimal risk, but improved significantly following revision of the PROMISE minimal-risk intercept and covariate coefficients (c2 5.6, HL p=0.692). HIGH-SENSITIVITY CARDIAC TROPONIN I IN THE DIAGNOSIS OF STABLE CORONARY ARTERY DISEASE In a pre-specified sub-study of the Scottish COmputed Tomography of the Heart trial, plasma cardiac troponin was measured using a high-sensitivity single molecule counting assay in 943 adults with suspected stable angina who had undergone coronary computed tomography angiography. Rates of obstructive coronary artery disease were compared with the pre-test probability determined by the European Society of Cardiology Coronary Artery Disease Consortium risk model with and without cardiac troponin concentrations. External validation was undertaken in an independent study population from Denmark comprising 487 patients with suspected stable angina. Higher cardiac troponin concentrations were associated with obstructive coronary artery disease with a 5-fold increase across quintiles (9 to 48%, p < 0.001) independent of known cardiovascular risk factors (odds ratio [OR] 1.35 [95% confidence interval (CI) 1.25-1.46] per doubling of troponin). Cardiac troponin concentrations improved the discrimination of the ESC model for identifying obstructive coronary artery disease (c-statistic 0.785 to 0.800, p=0.003) and improved classification into ESCrecommended categories of clinical risk (net reclassification improvement 0.143 [95% CI, 0.093-0.193]). The revised model achieved similar improvements in discrimination and net reclassification when applied in the external validation cohort. HIGH-SENSITIVITY CARDIAC TROPONIN I IN CARDIOVASCULAR RISK STRATIFICATION OF PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE AND HEIGHTENED CARDIOVASCULAR RISK. The association between plasma high-sensitivity cardiac troponin I concentration and cardiovascular events in patients with chronic obstructive pulmonary disease and heightened cardiovascular risk was examined within the context of a double-blind randomised controlled trial of inhaled corticosteroids and bronchodilators (1 placebo arm and 3 different treatment arms). Plasma cardiac troponin I concentrations were measured with a high-sensitivity assay in a subgroup of 1,599 patients. The cardiovascular endpoint was a composite of cardiovascular death, myocardial infarction, stroke, unstable angina and transient ischaemic attack during follow-up of 1.5 years. Baseline plasma cardiac troponin I concentrations were above the lower limit of detection (1.0 ng/L) in 1,559 (97.5%) patients and were unaffected by inhaled therapies at 3 months (p > 0.05 for all). Compared with the lowest tertile (cardiac troponin I ≤3.0 ng/L), patients in the highest tertile (≥ 5.5 ng/L) were at greater risk of cardiovascular events (hazard ratio 3.0, 95% confidence interval 1.5 to 6.2, p=0.002) and cardiovascular death (hazard ratio 9.6, 95% confidence interval 2.6 to 35.6, p < 0.001) after adjustment for cardiovascular risk factors. There were no differences in COPD exacerbations between tertiles even after adjustment (p > 0.05)., REPRODUCIBILITY OF CORONARY 18F-FLUORIDE PET-CT IMAGING The inter-observer and scan-rescan reproducibility of coronary 18F-fluoride PET-CT imaging was investigated in 20 patients with clinically stable but high risk multi-vessel coronary artery disease who underwent repeated 18F-fluoride PET-CT scans 11.5±4.5 days apart. Scan analysis using the currently accepted approach of normalisation to a referent coronary segment (TBRREFERENT) identified 10 (50%) patients with evidence of focal coronary 18F-fluoride uptake and demonstrated moderate agreement across observers on a per-patient level (k = 0.56). This was similar to the level of agreement achieved with visual assessment alone (k = 0.64). Reproducibility was improved by semi-quantitative reporting combining visual assessment with a threshold uptake value for determining the presence of tracer uptake (k = 0.84). Using the optimised approach achieved excellent agreement on overall segmental uptake counts (intra-class correlation = 0.97). CONCLUSION: Cardiovascular diagnostic and prognostic assessments represent a complex endeavour and established tools for risk prediction can demonstrate suboptimal predictive accuracy when evaluated in patient cohorts that are independent of the population used for model derivation.

BACKGROUND Aortic stenosis is a common and potentially fatal condition in which fibro-calcific changes within the valve leaflets lead to the obstruction of blood flow. Severe symptomatic stenosis is an indication for aortic valve replacement and timely referral is essential to prevent adverse clinical events. Calcification is believed to represent the central process driving disease progression. 18F-Fluoride positron emission tomography computed tomography (PET-CT) and CT aortic valve calcium scoring (CT-AVC) quantify calcification activity and burden respectively. The overarching aim of this thesis was to evaluate the applications of these techniques to the study and management of aortic stenosis. METHODS AND RESULTS REPRODUCIBILITY The scan-rescan reproducibility of 18F-fluoride PET-CT and CT-AVC were investigated in 15 patients with mild, moderate and severe aortic stenosis who underwent repeated 18F-fluoride PET-CT scans 3.9±3.3 weeks apart. Modified techniques enhanced image quality and facilitated clear localization of calcification activity. Percentage error was reduced from ±63% to ±10% (tissue-to-background ratio most-diseased segment (MDS) mean of 1.55, bias -0.05, limits of agreement - 0·20 to +0·11). Excellent scan-rescan reproducibility was also observed for CT-AVC scoring (mean of differences 2% [limits of agreement, 16 to -12%]). AORTIC VALVE CALCIUM SCORE: SINGLE CENTRE STUDY Sex-specific CT-AVC thresholds (2065 in men and 1271 in women) have been proposed as a flow-independent technique for diagnosing severe aortic stenosis. In a prospective cohort study, the impact of CT-AVC scores upon echocardiographic measures of severity, disease progression and aortic valve replacement (AVR)/death were examined. Volunteers (20 controls, 20 with aortic sclerosis, 25 with mild, 33 with moderate and 23 with severe aortic stenosis) underwent CT-AVC and echocardiography at baseline and again at either 1 or 2-year time-points. Women required less calcification than men for the same degree of stenosis (p < 0.001). Baseline CT-AVC measurements appeared to provide the best prediction of subsequent disease progression. After adjustment for age, sex, peak aortic jet velocity (Vmax) ≥ 4m/s and aortic valve area (AVA) < 1 cm2, the published CT-AVC thresholds were the only independent predictor of AVR/death (hazard ratio = 6.39, 95% confidence intervals, 2.90-14.05, p < 0.001). AORTIC VALVE CALCIUM SCORE: MULTICENTRE STUDY CT-AVC thresholds were next examined in an international multicenter registry incorporating a wide range of patient populations, scanner vendors and analysis platforms. Eight centres contributed data from 918 patients (age 77±10, 60% male, Vmax 3.88±0.90 m/s) who had undergone ECG-gated CT within 3 months of echocardiography. Of these 708 (77%) had concordant echocardiographic assessments, in whom our own optimum sex-specific CT-AVC thresholds (women 1377, men 2062 AU) were nearly identical to those previously published. These thresholds provided excellent discrimination for severe stenosis (c-statistic: women 0.92, men 0.88) and independently predicted AVR and death after adjustment for age, sex, Vmax ≥4 m/s and AVA < 1 cm2 (hazards ratio, 3.02 [95% confidence intervals, 1.83-4.99], p < 0.001). In patients with discordant echocardiographic assessments (n=210), CT-AVC thresholds predicted an adverse prognosis. BICUSPID AORTIC VALVES Within the multicentre study, higher continuity-derived estimates of aortic valve area were observed in patients with bicuspid valves (n=68, 1.07±0.35 cm) compared to those with tri-leaflet valves (0.89±0.36 cm p < 0.001,). This was despite no differences in measurements of Vmax (p=0.152), or CT-AVC scores (p=0.313). The accuracy of AVA measurments in bicuspid valves was therefore tested against alternative markers of disease severity. AVA measurements in bicuspid valves demonstrated extremely weak associations with CT-AVC scores (r2=0.08, p=0.02) and failed to correlate with downstream markers of disease severity in the valve and myocardium and against clinical outcomes. AVA measurements in bicuspid patients also failed to independently predict AVR/death after adjustment for Vmax ≥4 m/s, age and gender. In this population CT-AVC thresholds (women 1377, men 2062 AU) again provided excellent discrimination for severe stenosis. CONCLUSIONS Optimised 18F-fluoride PET-CT scans quantify and localise calcification activity, consolidating its potential as a biomarker or end-point in clinical trials of novel therapies. CT calcium scoring of aortic valves is a reproducible technique, which provides diagnostic clarity in addition to powerful prediction of disease progression and adverse clinical events.

Background Although abdominal aortic aneurysm (AAA) growth is non-linear, serial measurements of aneurysm diameter are the mainstay of aneurysm surveillance and contribute to decisions on timing of intervention. Aneurysm biology plays a key part in disease evolution but is not currently routinely assessed in clinical practice. Magnetic Resonance Imaging (MRI) and Positron Emission Tomography-Computed Tomography (PET-CT) provide insight into disease processes on a cellular or molecular level, and represent exciting new imaging biomarkers of disease activity. Macrophage-mediated inflammation may be assessed using ultrasmall superparamagnetic particles of iron oxide (USPIO) MRI and the PET radiotracer 18FSodium Fluoride (18F-NaF) identifies microcalcification which is a response to underlying necrotic inflammation. The central aim of this thesis was to investigate these imaging modalities in patients with AAA. Methods and Results USPIO MRI: MULTI-CENTRE STUDY In a prospective multi-centre observational cohort study, 342 patients (85.4% male, mean age 73.1±7.2 years, mean AAA diameter 49.6±7.7mm) with asymptomatic AAA ≥4 cm anteroposterior diameter underwent MRI before and 24-36 hours after intravenous administration of USPIO. Colour maps (depicting the change in T2* caused by USPIO) were used to classify aneurysms on the basis of the presence of USPIO uptake in the aneurysm wall, representing mural inflammation. Intra- and inter-observer agreement were found to be very good, with proportional agreement of 0.91 (kappa 0.82) and 0.83 (kappa 0.66), respectively. At 1 year, there was 29.3% discordant classification of aneurysms on repeated USPIO MRI and at 2 years, discordance was 65%, suggesting that inflammation evolves over time. In the observational study, after a mean of 1005±280 days of follow up, there were 126 (36.8%) aneurysm repairs and 17 (5.0%) ruptures. Participants with USPIO enhancement (42.7%) had increased aneurysm expansion rates (3·1±2·5 versus 2·5±2·4 mm/year; difference 0·6 [95% confidence intervals (CI), 0·02 to 1·2] mm/year, p=0·0424) and had higher rates of aneurysm rupture or repair (69/146=47·3% versus 68/191=35·6%; difference 11·7%, 95% CI 1·1 to 22·2%, p=0·0308). USPIO MRI was therefore shown to predict AAA expansion and the composite of rupture or repair, however this was not independent of aneurysm diameter (c-statistic, 0·7924 to 0·7926; unconditional net reclassification -13·5%, 95% confidence intervals -36·4% to 9·3%). 18F-NaF PET-CT: SINGLE-CENTRE STUDY A sub-group of 76 patients also underwent 18F-NaF PET-CT, which was evaluated using the maximum tissue-to-background ratio (TBRmax) in the most diseased segment (MDS), a technique that showed very good intra- (ICC 0.70-0.89) and inter-observer (ICC 0.637-0.856) agreement. Aneurysm tracer uptake was compared firstly in a case-control study, with 20 patients matched to 20 control patients for age, sex and smoking status. 18F-NaF uptake was higher in aneurysm when compared to control aorta (log2TBRmax 1.712±0.560 vs. 1.314±0.489; difference 0.398 (95% CI 0.057, 0.739), p=0.023), or to non-aneurysmal aorta in patients with AAA (log2TBRmax 1.647±0.537 vs. 1.332±0.497; difference 0.314 (95% CI 0.0685, 0.560), p=0.004). An ex vivo study was performed on aneurysm and control tissue, which demonstrated that 18F-NaF uptake on microPET-CT was higher in the aneurysm hotspots and higher in aneurysm tissue compared to control tissue. Histological analysis suggested that 18F-NaF was highest in areas of focal calcification and necrosis. In an observational cohort study, aneurysms were stratified by tertiles of TBRmax in the MDS and followed up for 510±196 days, with 6 monthly serial ultrasound measurements of diameter. Those in the highest tertile of tracer uptake expanded more than 2.5 times more rapidly than those in the lowest tertile (3.10 [3.58] mm/year vs. 1.24 [2.41] mm/year, p=0.008) and were also more likely to experience repair or rupture (15.3% vs. 5.6%, log-rank p=0.043). In multivariable analyses, 18F-NaF uptake on PET-CT emerged as an independent predictor of AAA expansion (p=0.042) and rupture or repair (HR 2.49, 95% CI1.07, 5.78; p=0.034), even when adjusted for age, sex, body mass index, systolic blood pressure, current smoking and, crucially, aneurysm diameter. Conclusion These are the largest USPIO MRI and PET-CT studies in AAA disease to date and the first to investigate 18F-NaF. Both USPIO MRI and 18F-NaF PET-CT are able to predict AAA expansion and the composite of rupture and repair, with 18F-NaF PETCT emerging as the first imaging biomarker that independently predicts expansion and AAA events, even after adjustment for aneurysm diameter. This represents an exciting new predictor of disease progression that adds incremental value to standard clinical assessments. Feasibility and randomised clinical trials are now required to assess the potential of this technique to change the management and outcome of patients with AAA.

Introduction. Fused Positron Emission Tomography and Computed Tomography (PET/CT) is an emerging investigative tool in cardiovascular disease that provides an imaging-based quantification of pathophysiological processes of interest. The purpose of this thesis was to study the application of PET to identify fundamental pathophysiological processes driving 3 forms of cardiovascular disease: aortic stenosis, myocardial infarction, and atherosclerosis. Methods. Aortic Stenosis. Patients with a spectrum of calcific aortic valve disease (n=121) who underwent PET-CT imaging for the identification of valvular calcification (18Ffluoride) and inflammation (18F-fluorodeoxyglucose, 18F-FDG) underwent serial imaging and clinical follow-up over 2 years. Baseline imaging findings were compared with echocardiographic and CT markers of disease progression and clinical outcome. Myocardial Infarction. Patients underwent PET-CT imaging with 18F-fluciclatide (a novel αvβ3-selective radiotracer highlighting active angiogenesis, inflammation and fibrosis) after ST-segment elevation MI (n=21), alongside stable patients with chronic total occlusion (CTO) of a major coronary vessel (n=7), and healthy volunteers (n=9). Myocardial radiotracer uptake was compared with clinical and cardiac magnetic resonance imaging (CMR) markers of infarction and remodeling. Atherosclerosis. Patients with a spectrum of atherosclerotic disease categorized as stable or unstable (recent MI) underwent PET/CT imaging with 18F-fluciclatide (n=46). Thoracic aortic 18F-fluciclatide uptake was compared with aortic atherosclerotic burden quantified by CT plaque thickness, plaque volume and calcium scoring. Histological validation. Tissue from the aortic valve, myocardium and carotid arteries of study subjects was acquired and examined ex vivo using histology and autoradiography. Results. Aortic Stenosis. Baseline valvular 18F-fluoride uptake correlated strongly with the rate of progression in AVC (r=0.80, p < 0.001) and with haemodynamic progression (mean aortic valve gradient r=0.32, p=0.001). It emerged as independently associated with clinical outcome after age and sex-adjustment (HR 1.55 [1.33-1.81], p < 0.001). 18F-FDG demonstrated moderate correlations with disease progression as assessed by CT (r=0.43, p=0.001) and echocardiography (18F-FDG r=0.30, p=0.001), and was associated with clinical outcomes independent of age and sex (HR 1.35 [1.16-1.58], p < 0.001). Valvular 18F-fluoride uptake correlated with immunohistochemical markers of calcification activity. There was no correlation between 18F-FDG uptake and inflammation. Myocardial Infarction. 18F-Fluciclatide binding was demonstrated in ex vivo peri-infarct myocardium and uptake was increased in vivo at sites of acute infarction compared to remote myocardium (tissue-to-background ratio (TBRmean) 1.34±0.22 vs 0.85±0.17 respectively, p < 0.001) and myocardium of healthy volunteers (TBRmean 1.34±0.22 vs 0.70±0.03; p < 0.001). There was no 18F-fluciclatide uptake at sites of established prior infarction in patients with CTO, with myocardial activity similar to healthy volunteers (TBRmean 0.71±0.06 vs. 0.70±0.03,p=0.83). 18F-Fluciclatide uptake occurred at sites of regional wall hypokinesia (wall motion index ≥1 vs 0; TBRmean 0.93±0.31 vs 0.80±0.26 respectively, p < 0.001), was increased in segments displaying functional recovery (TBRmean 0.95±0.33 vs 0.81±0.27, p=0.002) and associated with increase in probability of regional recovery. Atherosclerosis. 18F-Fluciclatide vascular binding ex vivo co-localised with regions of increased αvβ3 integrin expression, and markers of inflammation and angiogenesis. 18F-Fluciclatide uptake in vivo correlated with measures of aortic atherosclerotic burden: plaque thickness (r=0.57, p=0.001), total plaque volume (r=0.56, p=0.001) and the CT aortic calcium score (r=0.37, p=0.01). Patients with recent MI had greater aortic 18F-fluciclatide uptake than those with stable disease (TBRmax 1.33 vs 1.21, p=0.01). Conclusions. In a range of cardiovascular diseases, PET-CT can provide insights into key pathophysiological processes, guide patient risk stratification and prognosis, and identify important biomarkers of disease activity that can be used for the development of future therapeutic interventions.

Introduction: In Positron Emission Tomography (PET), the use of resolution modelling (RM) in iterative image reconstruction enables the modelling of aspects of detection which result in mispositioning of measured data and the subsequent blurring of reconstructed images. RM reconstruction can result in significant improvements in spatial resolution, voxel variance and count rate bias and could be a software alternative to detection hardware that is able to achieve higher resolution. Such hardware typically consists of small scintillation crystals, small bore diameters and depth of interaction discrimination, such as for the High Resolution Research Tomograph (HRRT, Siemens), which used a double crystal layer phoswich detector system. However, RM implementation comes with penalties such as slower rates of convergence, potentially higher region of interest variance and Gibbs artefacts. Methods: Assessment of the benefits and drawbacks of RM was done in the first part of this thesis together with the measurement and modelling of spatially varying resolution kernels for different scanner configurations and PET isotopes for the HRRT. It is also unclear as to whether high resolution scanning offers significant advantages over clinical PET-CT scanners for applications in the head. Through direct comparison to our HRRT, we explore whether there are significant advantages of high resolution scanning for an application in the head over clinical PET-CT. For this comparison our Biograph TruePoint TrueV (Siemens) optimised for whole body imaging was used and a novel clinical study using both scanners was set where we scanned Neurofibromatosis 2 (NF2) patients with vestibular schwannomas (VS) using [18F]fluorodeoxyglucose (FDG) and [18F]fluorothymidine (FLT). The clinical objective was to assess if uptake within VS of FLT and FDG could be measured and whether this uptake was predictive of tumour growth. Finally an assessment of the feasibility and impact of reducing the original injected activities in our clinical study was performed using bootstrapping resampling. Conclusions: RM provides greater but additive improvements in image resolution compared to DOI on the HRRT. Isotope specific image based RM could be estimated from published positron range distributions and measurements using fluorine-18. With the clinical project, uptake of FDG and FLT within the VS lesions was observed, these uptake values were correlated to each other, and high uptake was predictive of tumour growth with little differences in predictive power between FLT and FDG. Although there were benefits of the HRRT for imaging small lesions, in our clinical application there was little difference between the two scanners to discriminate lesion growth. Using the PET-CT scanner data and knowledge of lesion location, doses could be reduced to 5-10% without any significant loss of ability to discriminate lesion growth.

Systemic inflammatory networks and local signaling cascades trigger culprit pathogenic mechanisms relating clinical cardiovascular disease (CVD) risk factors to atherosclerotic plaque progression and rupture. Imaging vascular inflammation affords a valuable marker of atherosclerotic disease activity to reveal important mechanistic insights for CVD research, to quantify early anti-inflammatory effects of new atherosclerosis drugs, and, ultimately, to help improve CVD risk prediction. While carotid, aortic, and peripheral arterial inflammation can be measured by 18F-fluorodeoxyglucose (FDG) PET-computed tomography (CT), as a glucose analog, high 18F-FDG signal spillover owing to physiological myocardial muscle metabolism prevents reliable coronary interpretation. Lack of cell specificity, and the influence of hypoxia on 18F-FDG uptake within macrophages and other plaque cells, are further limitations that drive the search for an alternative PET tracer for imaging inflammation in atherosclerosis. Up-regulation of the G-protein coupled receptor somatostatin receptor subtype-2 (SST2) occurs on the cell surface of activated macrophages. The central hypothesis tested in this thesis is that vascular SST2 PET imaging using 68Ga-DOTATATE might offer a more accurate marker of macrophage inflammation than 18F-FDG, with superior coronary imaging and therefore better power to discriminate high-risk vs. low-risk atherosclerotic lesions. Comprehensive molecular, histological and clinical evaluation of this experimental CVD imaging biomarker was undertaken, including a prospective clinical study with head-to-head comparison to 18F-FDG in coronary, carotid, and aortic vasculature. In vitro data showed that (i) target SSTR2 gene expression occurred in “pro-inflammatory” M1 macrophages, (ii) specific 68Ga-DOTATATE ligand binding to SST2 receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and (iii) and carotid SSTR2 mRNA was highly correlated with both the pan-macrophage marker CD68 and in vivo 68Ga-DOTATATE PET signals. In clinical imaging, increased 68Ga-DOTATATE inflammatory signals correctly identified culprit vs. non-culprit arteries in patients with acute coronary syndrome and transient ischemic attack/stroke. 68Ga-DOTATATE also demonstrated good diagnostic accuracy for high-risk coronary CT features, and strong correlations with clinical CVD risk factors and 18F-FDG-defined vascular inflammation. While 18F-FDG also differentiated culprit vs. non-culprit carotid and high-risk coronary arteries, myocardial 18F-FDG overspill rendered coronary scans uninterpretable in most patients. In contrast, 68Ga-DOTATATE allowed unimpeded coronary interpretation in all patients. Findings of this thesis provide compelling evidence, from gene, to cell, to plaque, to patient, that SST2 PET imaging using 68Ga-DOTATATE provides a quantifiable marker of macrophage-related atherosclerotic inflammation and disease activity. Further studies are needed to establish whether 68Ga-DOTATATE PET can improve CVD risk prediction when added to current clinical methods, or offer a novel imaging platform to rapidly test the anti-inflammatory capacity of emerging atherosclerosis drugs. Broader translational applications of 68Ga-DOTATATE PET include possible use in diagnosis and therapeutic monitoring of vasculitis, endocarditis, myocarditis, and other manifestations of cardiovascular inflammation.

Previous research has established a bidirectional interaction between the brain and the skin, the so-called 'brain-skin axis'; novel aspects of which were explored in human subjects in this thesis. Study 1 used positron emission tomography (PET) with the radioligand [11C]-(R)-PK11195 co-registered with magnetic resonance imaging (MRI) to investigate the relationship between systemic inflammation and neuroinflammation of the brain in psoriasis. Patients with chronic plaque psoriasis (n=12) and Healthy controls (n=12) underwent brain scanning and measurement of their peripheral inflammatory markers. Inflammatory markers were increased (significantly for interleukin-8) in psoriasis patients when compared with controls but brain neuroinflammation was not. There was heterogeneity in the degree of neuroinflammation in both groups which did not correlate with any measured variables. These findings suggest that in this population, the systemic inflammation associated with chronic plaque psoriasis does not induce brain neuroinflammation. Study 2 investigated the impact of psychosocial stress on cutaneous mast cell (MC) function. In sub-study 2.1, Healthy subjects were exposed to either the modified Trier paradigm (an acute, experimental psychosocial stressor, n=12) or sat quietly for an equivalent period of time (controls, n=12). The stressor did not affect MC density but significantly reduced MC degranulation, suggesting that acute psychosocial stress confers a degree of resilience to MC degranulation in heathy human skin. Sub-study 2.2 employed corticotropin-releasing hormone (CRH) as a surrogate 'chemical stressor' in an ex vivo full-thickness human skin organ culture model (n=5). Significant MC degranulation was induced by CRH, an effect replicated by Etanercept (tumour necrosis factor (TNF)-alpha inhibitor) supplementation of the culture medium. It is plausible that Etanercept triggers MC degranulation and TNF-alpha release which is subsequently neutralised by Etanercept binding; although this remains speculative. Both MC and Langerhans' cells (LC) are important in the cutaneous stress response. Study 3 investigated the effect of histamine, the prototypical granule-associated MC mediator on LC migration. Healthy human subjects (n=20) underwent skin prick testing (SPT) with histamine and normal saline (control). Two and four hours post-SPT biopsies were taken and analysed for the presence of LC. Histamine SPT had no impact on LC migration when compared to normal saline SPT. These findings suggest that histamine at this concentration does not directly influence LC migration. This thesis enhances our knowledge of brain-skin interactions which I hope will ultimately benefit patients with skin disease.

Computed tomography imaging has revolutionised modern medicine and we can now study the body in greater detail than ever before. Cardiac computed tomography has the potential to provide information not just on coronary anatomy, but also on myocardial function, perfusion and viability. This thesis addresses the optimisation and validation of computed tomography imaging of the heart using a wide volume 320-multidetector scanner. Computed tomography coronary angiography now has diagnostic accuracy comparable to invasive coronary angiography. However, radiation dose remains an important concern. It is therefore important to minimise computed tomography radiation dose while maintaining image quality. I was able to demonstrate that iterative reconstruction and patient tailored imaging techniques led to a 39% reduction in radiation dose in computed tomography coronary angiography, while maintaining subjective and objective assessments of image quality. In addition, I demonstrated that diagnostic images can be obtained in 99% of unselected patients presenting with suspected coronary artery disease when using single heart-beat 320- multidetector computed tomography coronary angiography. Computed tomography myocardial perfusion imaging can provide additional and complementary information as compared to computed tomography coronary angiography that can aid diagnosis and management. I established both quantitative and qualitative assessment of computed tomography myocardial perfusion imaging and validated it against both a clinical “gold-standard”, fractional flow reserve during invasive coronary angiography, and a physiological “gold-standard”, positron emission tomography with oxygen-15 labelled water. Finally, I was able to show that techniques to reduce radiation dose can also be applied to computed tomography myocardial perfusion imaging, leading to a 60% reduction in radiation dose, while maintaining image quality. In my thesis, I have established that comprehensive cardiac angiographic and perfusion imaging can be performed with wide volume computed tomography in a broad generalizable population of patients with relatively low radiation exposure. These techniques provide both structural and functional assessments from a single imaging modality that are valid and readily applicable to the clinic in the assessment and management of patients with suspected coronary artery disease.

Positron Emission Tomography (PET) has great value in research and clinical applications from oncology to neurodegenerative disorders. However, there is a barrier in translating biological knowledge into new PET applications due in part to the lack of efficient, widely applicable methods for labelling compounds with PET radioisotopes. Herein, a generic approach to radiolabelling is presented which is direct, broadly applicable and potentially adaptable to either of the two most commonly used PET radioisotopes, 11C and 18F. This approach employs the advantages of solid phase synthesis to achieve selective release of only the desired radiolabelled product from a solid support in a single step, simplifying purification and hence improving synthetic efficiency. Polystyrene resin was functionalised with a 1,2-diol group; this allowed the covalent attachment of compounds bearing boronic acid groups via formation of a boronate ester linkage. A Suzuki-Miyaura reaction with methyl iodide was used to cleave a model compound from the resin in 61% conversion after five minutes. This reaction was adapted to develop a fully automated radiosynthesis with [11C]- methyl iodide which generated a radiolabelled model compound in 2 – 7% non-decay-corrected radiochemical yield. This provided proof of concept for the simultaneous cleavage of compounds from the resin and radiolabelling with 11C. A boronic acid precursor of the known radiotracer [11C]-M-MTEB was attached to the resin and successfully radiolabelled with 11C in 2.4% non-decay-corrected radiochemical yield and 96 – 100% radiochemical purity under the same conditions. Furthermore, the potential adaptability of this solid phase approach to 18F radiolabelling was demonstrated by treatment of the resin-bound small molecules and peptides with potassium bifluoride, which released the compounds rapidly as trifluoroborate salts.

Background: Mounting evidence suggests that inflammation is involved in the pathophysiology of both Major Depressive Disorder (MDD) and schizophrenia. The presence of inflammation in the brain, however, is less clear. Microglial activation, a measure of neuroinflammation, can be quantified using PET ligands that bind to the Translocator Protein (TSPO) which is overexpressed by activated microglia. Previous PET studies using TSPO radioligands have shown some evidence for neuroinflammation in both MDD and schizophrenia. However some of these studies have been confounded by antidepressant/antipsychotic medication, low numbers and mild severity. We aimed to address some of these issues and investigate the relationship between neuroinflammation and peripheral inflammation, medication status, symptom severity and cognitive function. Method: Fourteen patients in a Major Depressive Episode (MDE) of at least moderate severity, sixteen patients with a diagnosis of schizophrenia of at least moderate severity and a total of eighteen age and gender-matched healthy volunteers underwent a 60 minute dynamic PET scan with the TSPO radioligand [11C](R)-PK11195 on the High Resolution Research Tomograph (HRRT). Parametric maps of binding potential (BPND) were generated using the simplified reference tissue model and a grey matter cerebellum input function. All of the MDD patients were antidepressant-free for at least eight months prior to scanning. Of the sixteen schizophrenia patients, eight were antipsychotic-free (for at least twelve months) and eight were on a long-acting injection of risperidone or paliperidone. All patients and healthy volunteers were medically healthy and had drug or alcohol abuse within the previous year. Results: We found a 26% mean increase in BPND values, indicative of microglial activation, in MDD patients compared to healthy volunteers. Exploratory analysis revealed significantly higher [11C](R)-PK11195 binding in the anterior cingulate cortex (ACC). We found no significant correlations between [11C](R)-PK11195 binding and peripheral markers of inflammation or with symptom severity. We also found a mean 27% increase in BPND values in the schizophrenia patients compared to healthy volunteers. There were significant correlations between [11C](R)-PK11195 and negative symptoms across multiple brain regions. When breaking the cohort down according to medication status, there was no difference between antipsychotic-free patients and healthy volunteers. However, mean BPND values were 30% higher in the ACC. The medicated patients exhibited higher BPND values than healthy volunteers, with a mean increase of 48%. Exploratory t-tests revealed significant increases in dorsolateral prefrontal cortex and ACC.Conclusions: Our findings are largely consistent with previous PET findings of increased microglial activation in a sample of antidepressant-free patients in a moderate-to-severe MDE, suggesting that neuroinflammation is present in MDD. We also investigated neuroinflammation in antipsychotic-free patients for the first time and found no evidence of microglial activation. However it is likely that the subgroup sample was underpowered. The medicated patients exhibited a 48% increase in [11C](R)-PK11195 binding compared to controls, suggesting that either medication or duration of illness might potentiate microglial activation. Our findings also point to an association between neuroinflammation and the negative symptoms of schizophrenia. The PET findings from both cohorts are largely overlapping, suggesting that neuroinflammation is not specific to either disorder but rather a common mechanism. This could reflect a common aetiology and/or an overlap in symptoms. Our findings suggest that inflammation could be used as a potential biomarker as well as a target for novel treatment strategies in both MDD and schizophrenia.

Alzheimer’s disease (AD) is a progressive neurodegenerative disease resulting in alterations in memory, language, executive function and emotional behaviour. Although it can be characterised by symptoms, by the time they arise significant pathological alterations have already emerged in the central nervous system, namely increased amyloid plaques, neurofibrillary tangles and neuronal loss. Despite known pathological hallmarks the exact aetiology of AD is poorly understood and no current treatments are available. However, there is growing interest in the role of neuroinflammation in AD, with increases observed in the early stages of disease and with disease progression. Moreover, it has been suggested that peripheral inflammation can influence neuroinflammation and worsen neurodegeneration. Using Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRS) we can non-invasively measure biomarkers of neuroinflammation and degeneration allowing multi-modal investigation of its role in normal aging and AD. Considering this the objectives of this study were to (i) Use PET and MRS to investigate neuroinflammatory and metabolite alterations in transgenic (TG) models of AD and their wildtype (WT) animals. (ii) Assess rates of cognitive decline in these models using memory based tests. (iii) Investigate relatively new TgF344AD rat as an AD model by characterising younger time-points than previously reported. (iv) Investigate the contribution of peripheral inflammation on AD progression. PET and MRS imaging was carried out longitudinally in the APPswe×PS1de9 mouse. Neuroinflammation was confirmed ex vivo and cognitive ability was assessed by behavioural tests. Results revealed significantly increase hippocampal and thalamic neuoinflammation in old TG mice as assessed by [18F]DPA-714 PET and supported by immunohistochemistry. Reduced neuronal marker N-acetlyaspartate was seen with age and was exacerbated in the TG mice. Accelerated cognitive decline was also seen in TG mice. PET and MRS imaging was carried out at 6 and 12 months in the TgF344AD model, which expresses amyloid and tau pathology as well as neuronal loss. No cognitive decline was observed in TG rats; however increased anxiety behaviour was seen. Increased [18F]DPA-714 PET was observed as an effect of gene in the thalamus at 6 months and the hypothalamus at 12 months. Increases in glutamate were seen with age in the TG rats but not the WTs. Increased inflammation and metabolite alterations were seen with aging. The effect of peripheral urinary tract infection (UTI) on cognition and imaging out was assessed. Imaging was carried out prior to and after re-current UTI. Infection induced cognitive decline in infected TG but not WT rats. Infection had an increasing effect on hypothalamic neuroinflammation in WT rats but a decreasing effect on TG rats, which masked the original gene differences. This thesis is set out in the alternative format with each experimental study represented as a chapter. Results in this thesis implicate neuroinflammation in AD development and progression. In addition, we report systemic infection-CNS interactions accelerating cognitive decline in AD and highlight the importance of understanding the effects of comorbidities in disease.

The expansion of [18F]-radiolabelling methodologies is vital for the advancement of Positron Emission Tomography (PET) as a medical imaging tool. Novel protocols will simplify access to current PET tracers and allow development of original tracers, potentially enhancing the imaging quality or permit imaging of a wider range of clinical problems. A general introduction is presented in Chapter 1, covering PET, application of fluorine-19 in the pharmaceutical industry, the production of fluorine-18 and the current state-of -art for [18F]-radiolabelling. The results chapters are divided in to two parts; Part 1 (Chapters 2 - 4) concern the synthesis of [18F]fluoroarenes and Part II (Chapters 5 and 6) discuss diversity oriented radiofluorination techniques, targeting novel [18F]motifs. In Part I, Chapter 2 describes the use of hypervalent iodine reagents to meditate an umpolung approach for fluorination and radiofluorination of N-arylsulfonamides and anilides. Chapter 3 again utilises hypervalent iodine reagents, but as spirocyclic iodonium(III) ylide precursors, being applied to semi-automated and automated microfluidic conditions. Chapter 4 discusses the development of conditions for the production and isolation of radiopharmaceuticals employing Cu(II)-mediated radiofluorination of aryl boronic pinacol esters. In Part II, Chapter 5 discusses the applicability of halogen exchange reactions to reach polyfluorinated motfis and Chapter 6 focuses on the application of a single diazo precursor to access three polyfluorinated motifs with single-step reactions. Finally, Chapter 7 provides experimental data for compounds discussed in this thesis.

In translational medicine, complementary functional and morphological imaging techniques are used extensively to observe physiological processes in vivo and to assess structural changes as a result of disease progression. The combination of magnetic resonance imaging (MRI) and positron emission tomography (PET) provides excellent soft tissue contrast from MRI with exceptional sensitivity and specificity from PET. This thesis explores the use of sequentially acquired PET and MR images to improve the quantification of small animal PET data. The primary focus was to improve image-based estimates of the arterial input function (AIF), which defines the amount of PET tracer within blood plasma over time. The AIF is required to produce physiological parameters quantifying key processes such as metabolism or perfusion from dynamic PET images. The gold standard for AIF measurement, however, requires serial blood sampling over the course of a PET scan, which is invasive in rat studies but prohibitive in mice due to small total blood volumes. To address this issue, the geometric transfer matrix (GTM) and recovery coefficient (RC) techniques were applied using anatomical MR images to enable the extraction of partial volume corrected image based AIFs from mouse PET images. A non-invasive AIF extraction method was also developed for rats, beginning with the optimization of an automated voxel selection algorithm to assist in extracting MR contrast agent signal time courses from dynamic susceptibility contrast (DSC) MRI data. This procedure was then combined with dynamic contrast enhanced (DCE) MRI to track a combined injection of Gadolinium-based contrast agent and PET tracer through the rat brain. By comparison with gold standard tracer blood sample data, it was found that normalized MRI-based AIFs could be successfully converted into PET tracer AIFs in the first pass phase when fitted with gamma variate functions. Finally, a MR image segmentation method used to provide PET attenuation correction in mice was validated using the Cambridge split magnet PET/MR scanner?s transmission scanning capabilities. This work recommends that contributions from MR hardware in the PET field of view must be accounted forto gain accurate estimates of tracer uptake and standard uptake values (SUVs). This thesis concludes that small animal MR data taken in the same imaging session can provide non-invasive methods to improve PET image quantification, giving added value to combined PET/MR studies over those conducted using PET alone.

The fluorinase enzyme, originally isolated from Streptomyces cattleya, has the unique ability to generate a C–F bond from aqueous fluoride ion and S-adenosylmethionine, making the fluorinase an attractive biochemical tool for radiolabelling biomolecules with fluorine-18 for application to positron emission tomography (PET). The inherent substrate specificity of the enzyme is, however, limiting, as only small modifications to the natural nucleoside substrate were known to be tolerated. This thesis describes an exploration and expansion of the substrate scope of the fluorinase enzyme, and its application to radiolabelling biomolecules for PET. The design and synthesis of a novel acetylene bearing substrate for the fluorinase, 5'-chloro-5'-deoxy-2-ethynyladenosine (ClDEA) is described. ClDEA proved an excellent substrate for the fluorinase, and the kinetics of the transformation and binding affinities of the new substrate and product were investigated. The fluorinated acetylenic product was demonstrated to undergo a copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction with an azide bearing RGD peptide, and this methodology was investigated for the synthesis of a novel fluorine-18-bearing prosthetic group for the synthesis of a radiolabelled RGD peptide, which was assessed in vivo in a rat. After the demonstration that the fluorinase can be used for “last step” radiolabelling of bioactive peptides, the synthesis of dimeric and tetrameric RGD-bearing substrates for the fluorinase was investigated. These large constructs underwent efficient enzymatic fluorination, and the fluorinated products showed increased binding affinity to their targets, compared to monomeric analogues. The challenges encountered during radiolabelling of these multimers with fluorine-18 using the fluorinase are discussed. A difluoromethyl-bearing nucleoside substrate (F₂DA) was synthesised as a potential substrate in the reverse direction for the fluorinase, to further probe the substrate specificity if the fluorinase. Upon incubation with the enzyme, F₂DA did not appear to undergo reaction, despite the demonstration that F₂DA binds to the enzyme. Finally, the optimisation of a fluorinase-based protocol for the synthesis of the PET radiotracer [¹⁸F]fluoroacetate is described. The enzymatic method proved unsuitable for a small animal study due to contamination of the final product, and a chemical method was investigated and optimised as an alternative approach. [¹⁸F]Fluoroacetate synthesised using the developed chemical method was employed in an in vivo evaluation of acetyl CoA synthetase (ACSS2) activity in healthy and tumour-bearing mouse models, in an study to assess the activity of ACSS2 in breast and colon cancer models in mice.

Dopamine is an important neurotransmitter that is involved in several human functions such as reward, cognition, emotions and movement. Abnormalities of the neurotransmitter itself, or the dopamine receptors through which it exerts its actions, contribute to a wide range of psychiatric and neurological disorders such as Parkinson’s disease and schizophrenia. Thus far, despite the great interest and extensive research, the exact role of dopamine and the causalities of dopamine related disorders are not fully understood. Here we have developed multimodal imaging methods, to investigate the release of dopamine and the distribution of the dopamine D2-like receptor family in-vivo in healthy humans. We use the [11C]PHNO PET ligand, which enables exploration of dopamine-related parameters in striatal regions, and for the first time in extrastriatal regions, that are known to be associated with distinctive functions and disorders. Our methods involve robust approaches for the manual and automated delineation of these brain regions, in terms of structural and functional organisation, using information from structural and diffusion MRI images. These data have been combined with [11C]PHNO PET data for quantitative dopamine imaging. Our investigation has revealed the distribution and the relative density of the D3R and D2R sites of the dopamine D2-like receptor family, in healthy humans. In addition, we have demonstrated that the release of dopamine has a functional rather than a structural specificity and that the relative densities of the D3R and D2R sites do not drive this specificity. We have also shown that the dopamine D3R receptor is primarily distributed in regions that have a central role in reward and addiction. A finding that supports theories that assigns a primarily limbic role to the D3R.

Background: Gliomas are the most frequent primary brain tumours in adults with two main histological subtypes: astrocytoma and oligodendroglioma. Translocator protein (TSPO) is a pro-inflammatory molecule over-expressed predominantly in activated microglia under pathological conditions. In astrocytoma samples, TSPO has also been found to be up-regulated and correlated with the malignancy of the tumours. [11C]-(R)PK11195 is a selective radioligand for the TSPO widely applied in clinical PET studies. We used [11C]-(R)PK11195 PET to investigate in vivo cerebral TSPO expression and microglial activation in patients with gliomas of different histological subtypes and grades. Methods: 24 glioma patients and 10 healthy volunteers underwent volumetric MRI and dynamic [11C]-(R)PK11195 PET scans. Tissue time-activity curves (TACs) were extracted from tumour regions and normal grey and white matter of the brains. Parametric maps of binding potential (BPND) were generated with the simplified reference tissue model. Co-registered MRI/PET was used to guide tumour biopsies. Tumour tissue was quantitatively assessed for TSPO expression and microglial infiltration by immunohistochemistry. Results: Three types of tumour TAC were observed in gliomas (grey matter-like kinetics, white matter-like kinetics and mixed kinetics), which differed between low-grade astrocytomas and low-grade oligodendrogliomas but were independent of the tumour grade. [11C]-(R)PK11195 BPND also differed between the two subtypes of low-grade gliomas, and low-grade gliomas demonstrated lower BPND than high-grade gliomas. 4 cases of high-grade glioma with minor or no contrast enhancement on MRI showed pronounced [11C]-(R)PK11195 binding. Immunohistochemistry confirmed that expression of TSPO correlated with [11C]-(R)PK11195 BPND of the tumour. It was related mainly to expression by neoplastic cells while the contribution from tumour-infiltrating microglia was minimal. When compared with control subjects, increased [11C]-(R)PK11195 BPND was found in patients’ normal appearing cerebral structures, being more prominent in the tumour-bearing than the tumour-free hemisphere. This extra-tumoral [11C]-(R)PK11195 binding was correlated with the duration of epileptic seizures, the symptom shared by the majority of our patients. Conclusions: Gliomas show differences in [11C]-(R)PK11195 kinetics and binding that are related to histological subtype and grade. Neoplastic cells rather than activated microglia are the main cellular sources expressing TSPO and determine the [11C]-(R)PK11195 binding within the tumours. [11C]-(R)PK11195 PET has the potential to detect malignant transformation of non-enhancing gliomas and facilitate the targeting of more aggressive areas in tumour biopsy. The high extra-tumoral [11C]-(R)PK11195 binding indicates widespread microglial activation in otherwise normal appearing cerebral structures of glioma patients. It is associated with epilepsy and could open up novel therapeutic perspectives for seizure control in this patient population.

Introduction. Aortic stenosis is characterised both by progressive narrowing of the valve and the hypertrophic response of the left ventricle. The purpose of this thesis was to study the contribution of inflammation and calcification to valve narrowing using Positron Emission and Computed Tomography (PET/CT) and to investigate the hypertrophic response using cardiovascular magnetic resonance (CMR). Methods. PET/CT studies. Patients with aortic sclerosis and mild, moderate and severe stenosis were prospectively compared to matched control subjects. Aortic valve severity was determined by echocardiography. Calcification and inflammation in the aortic valve and coronary arteries were assessed by sodium 18-­‐fluoride (18F-­‐NaF) and 18-­‐fluorodeoxyglucose (18F-­‐FDG) uptake using PET. CMR studies. Consecutive patients with moderate or severe aortic stenosis undergoing CMR were enrolled into a registry. Patients who received gadolinium contrast were categorised into absent, mid-­‐ wall or infarct patterns of late gadolinium enhancement (LGE) by blinded independent observers. Patients follow-­‐up was completed using patient questionnaires, source record data and the National Strategic Tracing Scheme. After excluding those patients with concomitant triggers to LV remodeling, the extent and patterns of hypertrophy were investigated based upon measurements of indexed LV mass, indexed LV volume and the relative wall mass. Results. PET/CT studies. 121 subjects (20 controls; 20 aortic sclerosis; 25 mild, 33 moderate and 23 severe aortic stenosis) were studied. Quantification of tracer uptake within the valve demonstrated excellent inter-­‐observer reproducibility with no biases and limits of agreement of ±0.21 (18F-­‐NaF) and ±0.13 (18F-­‐FDG) for maximum tissue-­‐to-­‐background ratios (TBR). Activity of both tracers was higher in patients with aortic stenosis than control subjects (18F-­‐NaF: 2.87±0.82 vs 1.55±0.17; 18F-­‐ FDG: 1.58±0.21 vs 1.30±0.13; both P<0.001). 18F-­‐NaF uptake displayed a progressive rise with valve severity (r2=0.540, P<0.001) with a more modest increase observed for 18F-­‐FDG (r2=0.218; P<0.001). Amongst patients with aortic stenosis, 91% had increased 18F-­‐NaF (>1.97) and 35% increased 18F-­‐ FDG (>1.63) uptake. Increased 18F-­‐NaF uptake was also observed in the coronary arteries in a subset of patients with atherosclerosis. These patients (n=40) had higher rates of prior cardiovascular events (p=0.016) and angina (p=0.023), and higher Framingham risk scores (p=0.011). CMR studies. 143 patients (aged 68±14 years; 97 male) were followed up for 2.0±1.4 years and 27 died. Compared to those with no LGE (n=49), univariate analysis revealed that patients with mid-­‐wall fibrosis (n=54) had an eight-­‐fold increase in all-­‐cause mortality despite similar aortic stenosis severity and coronary artery disease burden. Patients with an infarct pattern (n=40) had a six-­‐fold increase. Mid-­‐wall fibrosis (HR 5.35 [95% CI 1.16-­‐24.56]; P=0.03) emerged as an independent predictor of all cause mortality by multivariate analysis. The pattern of LV remodelling was studied in 91 patients (61±21 years; 57 male) and displayed wide variation comprising normal ventricular geometry (n=11), concentric remodelling (n=11), asymmetric remodelling (n=11), concentric hypertrophy (n=34), asymmetric hypertrophy (n=14) and LV decompensation (n=10). The magnitude of the hypertrophic response was unrelated to the severity of aortic valve narrowing. Conclusions. Modern imaging techniques have provided important insights in to the pathology underlying aortic stenosis and suggest that valvular calcification and myocardial fibrosis have a key role. Both represent important potential targets for future therapeutic interventions.

Since its inception around 1975, Positron Emission Tomography (PET) has proved to be an important tool in medical research as it allows imaging of the brain function in vivo with high sensitivity. It has been widely used in clinical dementia research with [18F]2-Fluoro-2-Deoxy-D-Glucose (FDG) and amyloid tracers as imaging biomarkers in Alzheimer's Disease (AD). The high resolution offered by modern scanner technology has the potential to provide new insight into the interaction of structural and functional changes in AD. However, the high resolution of PET is currently limited by movement and resolution (even for high resolution dedicated brain PET scanner) which results in partial volume effects, the undersampling of activity within small structures. A modified frame-by-frame (FBF) realignment algorithm has been developed that uses estimates of the centroid of activity within the brain to detect movement and subsequently reframe data to correct for intra-frame movement. The ability of the centroid to detect motion was assessed and the added benefit of reframing data for real clinical scans with patient motion was evaluated through comparison with existing FBF algorithms. Visual qualitative analysis on 6 FDG PET scans from 4 blinded observers demonstrated notable improvements (ANOVA with Tukey test, p < 0.001) and time-activity curves were found to deliver biologically more plausible activity concentrations. A new method for Partial Volume Correction (PVC) is also proposed, PARtially-Segmented Lucy-Richardson (PARSLR),that combines the strength of image based deconvolution approach of the Lucy-Richardson (LR) Iterative Deconvolution Algorithm with a partial segmentation of homogenous regions. Such an approach is of value where reliable segmentation is possible for part but not all of the image volume or sub-volume. Its superior performance with respect to region-based methods like Rousset or voxel-based methods like LR was successfully demonstrated via simulations and measured phantom data. The approach is of particular importance for studies with pathological abnormalities where complete and accurate segmentation across or with a sub-volume of the image volume is challenging and for regions of the brain containing heterogeneous structures which cannot be accurately segmented from co-registered images. The developed methods have been shown to recover radioactivity concentrations from small structures in the presence of motion and limited resolution with higher accuracy when compared to existing methods. It is expected that they will contribute significantly to future PET studies where accurate quantitation in small or atrophic brain structures is essential.

Positron emission tomography (PET) employs short half-life positron emitting isotopes, typically 18F, for in vivo measurement of physiological processes. Easy access to structurally diverse radiolabelled probes would accelerate the rapid progress of PET imaging but, to date, radiochemistry is still limited by cost and efficiency. Nucleophilic fluorination with 18F-fluoride is the preferred “non-carrier-added” methodology in the synthesis of 18F-labelled pharmaceuticals because it leads to radiotracers with a high specific activity, a key feature allowing for investigations to be performed in sub-toxic doses. Chapter 1 serves as an introduction on radiochemistry, especially focussing on current radiosynthetic methods for the synthesis of 18F-labelled aromatics. Aromatic compounds without electron-withdrawing groups are notoriously difficult to label with 18F-fluoride. In this thesis, we present two novel methodologies to deliver 18F-labelled aromatic compounds from nucleophilic 18F-fluoride. Chapter 2 details the experimental efforts towards “Convergent Radiosynthesis” (Scheme 1). We proposed a convergent synthetic tactic that allows for simultaneous reaction between three or more substrates, including an 18F-labelled building block. This chemistry has been validated by the radiosynthesis of various structural scaffolds which are not responsive to direct nucleophilic fluorination. Chapter 3 presents our research into “Oxidative Nucleophilic 18F-Fluorination” (Scheme 2). We proposed that electron-rich aromatics, such as phenols, which are not responsive to nucleophilic fluorination may undergo umpolung reactivity under oxidative conditions. This “umpolung strategy” allows for the direct transformation from 18F-fluoride to 4-[18F]fluorophenol. Potentially, this established oxidative fluorination strategy could be adapted to the radiosynthesis of radiotracers containing a 4-fluorophenol sub-motif, such as 6-fluoro-meta-tyrosine. An appropriate precursor has been validated for the prospective radiosynthesis of 6-[18F]fluoro-meta-tyrosine.