Background Hereditary transthyretin amyloid (ATTRm) amyloidosis is a systemic disease mainly affecting the peripheral nervous system and the heart. The disease is inherited in an autosomal dominant manner with a varying penetrance. It is caused by mutations in the transthyretin (TTR) gene. Today more than 100 disease causing mutations are known. The V30M mutation that is endemic in northern Sweden is the best studied and comprises the majority of the reported disease cases in the world. In ATTRm amyloidosis caused by the V30M mutation two distinct sub populations are seen, one with disease onset early in life and a mainly neuropathic disease and the other with late onset disease and both neuropathic disease and a progressive cardiomyopathy. These phenotypical findings have in Swedish patients been tied to differences in amyloid fibril composition. Generally, patients with early onset disease have amyloid fibrils containing only full length transthyretin (type B) whereas patients with late onset disease have amyloid containing both full length and fragmented transthyretin (type A). Until recently, the only available treatment for the disease has been liver transplantation. Patients with type A fibrils, especially males, have significantly worse survival after liver transplant due to progressive amyloid cardiomyopathy. Furthermore, it appears that type A fibrils may be the most common finding in other mutations. This thesis work aims to in depth investigate the impact amyloid fibril composition has on cardiac manifestations of the disease and on the outcome of available and novel modalities for cardiac amyloid imaging. Methods The four studies included in the thesis were done as part of the on going clinical research at the Swedish centre for transthyretin amyloidosis in Umeå.  Patients in whom amyloid fibril composition had been determined were included. Available echocardiographic data were analysed to find predictors for left ventricular hypertrophy and systolic function as measured by strain analysis in a large cohort of 105 patients (paper I). Serial 12-lead electrocardiograms from 98 patients were gathered and retrospectively interpreted and analysed to investigate the impact of amyloid fibril composition and disease progression on frequency and development of ECG abnormalities (paper IV).  DPD scintigraphy, cardiac biomarkers, clinical data and echocardiograms were analysed in a cohort of 53 consecutive patients. to assess the impact of amyloid fibril composition on the outcome of DPD scintigraphy and its relationship with cardiac hypertrophy. (paper II). To evaluate the usefulness of positron emission tomography (PET) using the amyloid specific tracer PIB, 10 patients, five with each fibril type, were selected and examined. The patients selected had a similar age of onset and similar echocardiographic findings (paper III). Results Paper I: Type A fibrils, male gender and age were independent factors associated with increased LV thickness. The distribution of amyloid fibril composition did not differ between the sexes, but in patients with type A fibrils, females had lower median cardiac wall thickness (p<0.01and better left ventricular septal strain (p=0.04).The gender differences were not apparent in patients with type B fibrils. Paper II: Ninety-seven per cent of patients with type A fibrils had pathological cardiac DPD uptake compared to none of the patients with type B fibrils. Among patients with normal septal thickness, none of 15 patients with type B fibrils had positive scintigraphy compared with 2 out of 2 with type A fibrils (P<0.01) Cardiac biomarkers, demographic data and cardiac biomarkers were significantly different, but could not differentiate between type A and type B fibrils in individual patients. Paper III: All patients had pathological cardiac PIB retention. In patients with type B fibrils the retention was significantly higher (p<0.01) than in patients with type A fibrils. Based on the selection criteria, no significant differences were seen in various echocardiographic measurements. Paper IV: All patients had a high prevalence of AV-blocks, LAH and anterior infarction pattern. Patients with type A fibrils had significantly more electrocardiographic abnormalities compared to those with type B fibrils, both at an early stage of diseases and at later follow up. Conclusion Type A fibrils are associated with more pronounced cardiac involvement, which appear to be more severe in males than in females. In study II we showed that DPD scintigraphy appears to be a very good tool for non-invasive determination of amyloid fibril composition. Papers III and IV show that patients with type B amyloid have cardiac involvement even without echocardiographic or DPD-scintigraphic evidence of amyloid cardiomyopathy and that ECG abnormalities are common irrespectively of amyloid fibril composition, and increase with time for both groups.

Growing evidence from animal and clinical studies suggests that cardiac cell therapy can restore perfusion and improve function in the ischemic/infarcted myocardium. However, cell therapy is hindered by insufficient cell numbers, inefficient cell homing and engraftment, and inadequate cellular interactions. Furthermore, the biological mechanisms and local effects of transplanted cells have not been well-elucidated. The research presented herein attempts to address some of these issues. In manuscript #1, a new subpopulation of circulating progenitor cells (CPCs), termed derived CD133+ cells, was generated from the CD133- fraction of human peripheral blood. The derived CD133+ progenitors appeared to have superior vasculogenic potential in vitro, which may prove to be beneficial in inducing vasculogenesis in ischemic tissues. Positron emission tomography (PET) with direct cell labeling and reporter gene techniques were employed to assess the fate of transplanted human CPCs in vivo at different subjects of investigation, and different stages of cell transplantation. In manuscript #2, PET imaging with 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) direct cell labeling was used to demonstrate that collagen-based matrices improve the early homing and retention of delivered CPCs in a rat ischemic hindlimb model. This mechanism conferred by the matrix may have implications on cell therapy at the early stages after transplantation. In manuscript #3, a more efficient, stable and accurate labeling method, hexadecyl-4-[18F]fluorobenzoate (18F-HFB) direct cell labeling, was developed to quantify cell distribution of transplanted CPCs in a rat myocardial infarction model. PET imaging of 18F-HFB-CPCs revealed significant cell washout from the myocardium immediately after intramyocardial injection, with only a small proportion of transplanted CPCs remaining in the target area in the first 4 hours after delivery. In manuscript #4, human CPCs transduced with lentiviral vectors showed stable expression of PET reporter genes. This reporter gene based-cell labeling technique can be developed for noninvasive tracking cells within a bioengineered matrix by PET, while preserving cell phenotype, viability and function. These studies contribute important insights into the biology and physiology of transplanted stem cells and the ability of delivery matrices to improve transplanted cell engraftment, survival, and function. I believe with further refinement, cell expansion, tissue engineering and PET imaging could facilitate the clinical applications of cell therapies in years to come.

Cardiac hypertrophy is a compensatory response to increased work load or stress on the heart, but over time can lead to heart failure and death. The molecular mechanisms underlying this disease are still not completely understood, however the Rho/Rho kinase pathway has been shown to play a role. N-[11C]-methyl-hydroxyfasudil, a PET radiotracer, binds to active Rho kinase and could be a possible tracer for hypertrophy. Hypertrophy was induced in vitro using the β-adrenergic receptor agonist isoproterenol to evaluate optimal Rho kinase activity. Rho kinase activity data was correlated to N-[11C]-methyl-hydroxyfasudil binding. Cardiac hypertrophy was verified with an increase in nuclear size (1.74 fold) and cell size (~2 fold), activation of hypertrophic signalling pathways, and increased Rho kinase activity (1.64 fold). This correlated to a 10.3% increase in N-[11C]-methyl-hydroxyfasudil binding. This data suggests that N-[11C]-methyl-hydroxyfasudil may be useful as a radiotracer for detecting cardiac hypertrophy and merits further in vivo investigation.

Diagnosis of diabetes mellitus, presence of hyperglycemia, and/or insulin resistance confer cardiovascular risk, particularly for diastolic dysfunction. Diabetes is associated with elevated myocardial norepinephrine (NE) content, enhanced sympathetic nervous system (SNS) activity, altered resting heart rate, and depressed heart rate variability. Positron emission tomography (PET) using the NE analogue [11C]meta-hydroxyephedrine ([11C]HED) provides an index of myocardial sympathetic neuronal integrity at the NE reuptake transporter (NET). The hypothesis of this project is that (i) hyperglycemia imparts heightened sympathetic tone and NE release, leading to abnormal sympathetic neuronal function in the hearts of diabetic rats, and (ii) these abnormalities may be reversed or prevented by treatments to normalize glycemia. Sprague Dawley rats were rendered insulin resistant by high fat feeding and diabetic by a single dose of streptozotocin (STZ). Diabetic rats were treated for 8 weeks with insulin, metformin or rosiglitazone, starting from either 1 week (prevention) or 8 weeks (reversal) after STZ administration. Sympathetic neuronal integrity was evaluated longitudinally by [11C]HED PET. Echocardiography measures of systolic and diastolic function were completed at serial timepoints. Plasma NE levels were evaluated serially and expression of NET and β-adrenoceptors were tested at the terminal endpoints. Diabetic rats exhibited a 52-57% reduction of [11C]HED standardized uptake value (SUV) at 8 weeks after STZ, with a parallel 2.5-fold elevation of plasma NE and a 17-20% reduction in cardiac NET expression. These findings were confirmed by ex vivo biodistribution studies. Transmitral pulse wave Doppler echocardiography established an extension of mitral valve deceleration time and elevated early to atrial velocity ratio, suggesting diastolic dysfunction. Subsequent treatment with insulin but not metformin restored glycemia, reduced plasma NE by 50%, normalized NET expression, and recovered [11C]HED SUV towards non-diabetic age-matched control. Diastolic dysfunction in these rats persisted. By contrast, early treatment with insulin, metformin, or rosiglitazone delayed the progression of diastolic dysfunction, but had no effect on elevated NE and reduced [11C]HED SUV in diabetic rats, potentially owing to a latent decrease in blood glucose. In conclusion, diabetes is associated with heightened circulating and tissue NE levels which can be effectively reversed by lowering glycemia with insulin. Noninvasive interrogation of sympathetic neuronal integrity using [11C]HED PET may have added value in the stratification of cardiovascular risk among diabetic patients and in determining the myocardial effects of glycemic therapy.

Background-Pulmonary artery hypertension (PAH) is characterized by progressive vascular changes causing increased pulmonary resistance and eventual right heart failure (HF). It has been suggested that altered myocardial substrate utilization may be associated with right HF, however these changes have not yet been well characterized. The aim of this study was to evaluate in vivo right ventricular (RV) function and RV glucose and fatty acid metabolism in an experimental model of PAH using non-invasive positron emission tomography (PET) imaging and to investigate the effect of a novel endothelin receptor antagonist, Macitentan, on the development of PAH and RV energetics. Methods and Results-Severe PAH was induced in a total of 11 male Sprague-Dawley rats using a single injection of Sugen5416 followed by chronic hypoxia. The rats were then randomized to treatment or no treatment with Macitentan (30 mg/kg daily) Five and eight weeks post injection, substrate utilization was serially assessed with 2-[18F]fluoro-2-deoxyglucose (FDG) and 4-[18F]fluoro-6-thia-heptadecanoate (FTHA) PET scans for glucose and fatty acid metabolism respectively, and reported as a standardized uptake value (SUV). This data was correlated with in vivo functional measurements with echocardiography and multi gated acquisition scans. The Sugen-hypoxia (SuHx) model resulted in an increase in RV FDG uptake over 8 weeks (SUV control: 1.56 ± 0.38, week 5 SuHx: 4.06 ± 1.90, week 8 SuHx: 4.00 ± 1.60, p<0.005 between control and week 5 SuHx). RV FTHA data showed a trend towards increased uptake with onset of PAH at week 5 SuHx (SUV control: 1.50 ± 0.40, week 5 SuHx: 3.06 ± 1.10, p>0.05). Macitentan significantly decreased RV FDG uptake (SUV week 8 SuHx: 4.00 ± 1.60, week 8 SuHx +ERA: 2.54 ± 0.90, p<0.05). This was associated with improved RV ejection fraction (PAH week 8 untreated: 53.15 ± 9.9% vs PAH week 8 treated: 73.22 ± 4.8%, p<0.01) and improved pulmonary artery pressures measured by pulmonary artery acceleration time (PAH week 8 untreated: 17.32 ± 2.30 ms vs. PAH week 8 treated: 24.38 ± 3.90 ms, p<0.001). There was a strong correlation between increased pulmonary artery pressures and increased RV FDG uptake (r=0.87, p=0.001) as well as a significant inverse relationship between improved RV ejection fraction and decreased RV FDG uptake (r=-0.72, p=0.01). Conclusion-PAH is associated with metabolic changes in the RV, characterized by increased glucose uptake and a trend towards increased RV fatty acid uptake with onset of PAH. Macitentan attenuated RV FDG uptake and significantly increased RV function as well as hemodynamics compared to untreated group.

Injectable biomaterials have emerged as a treatment for myocardial infarction (MI). They can be applied either as an enhancement for cell therapy or as a stand-alone treatment for MI. The main focus of this study was to apply circulating angiogenic cells (CACs) with or without an injectable collagen matrix for MI treatment in a mouse model. Furthermore, a collagen-chitosan matrix was tested for modulating the myocardial maladaptive remodeling post-MI. First, the in vivo thermo-gelling and retention properties of the collagen matrix were validated using positron emission tomography (PET) tracer and quantum dot (Qdot) labelled matrix in MI mouse hearts. The therapeutic potential of the matrix ± CACs was then tested in a mouse MI model. The results showed that CACs-only and matrix-only treatments were associated with cardiac function preservation. However, in combination, CAC + matrix therapy had a synergistic effect and significantly improved cardiac function (echocardiography), perfusion and viability (PET scan), increased cell engraftment and arteriole density, and reduced the infarct size. CAC-matrix interaction through the integrin alpha2 receptor was essential for the observed therapeutic effect. In a third study, the addition of chitosan (a polysaccharide) to the collagen matrix was shown to reduce maladaptive remodeling post-MI by limiting cardiac fibroblast-to-myofibroblast differentiation and scar formation. In conclusion, these collagen-based hydrogels hold promise to enhance cardiac repair as a delivery scaffold for therapeutic cells, and/or as a stand-alone treatment, which can actively modulate the environment including the fibrotic process after MI.

La qualité du sommeil se modifie avec l’âge, et les troubles du sommeil seraient associés au déclin cognitif et à un risque accru de développer une maladie d’Alzheimer (MA). Cependant, les mécanismes cérébraux sous-tendant cette association restent mal compris. L’objectif de cette thèse était de contribuer à une meilleure compréhension des corrélats cérébraux structuraux, fonctionnels et moléculaires des principales modifications objectives du sommeil dans le vieillissement, et d’explorer les liens avec les performances cognitives. Nos résultats montrent que les altérations des premiers cycles de sommeil et de l’activité à ondes lentes sont associées à un hypométabolisme, une hypoperfusion et/ou une diminution du volume de substance grise au niveau des aires fronto-cingulaires et hippocampiques. De plus, la présence d’un syndrome d’apnées obstructive du sommeil et l’altération de la microstructure du sommeil paradoxal étaient significativement associés à une augmentation de la charge amyloïde, respectivement au niveau du cortex cingulaire postérieur et du précunéus, ou de manière plus diffuse. En revanche, les liens avec la cognition restaient subtils voire absents, certaines modifications cérébrales étant asymptomatiques. Ainsi, le sommeil pourrait être un facteur de résilience face aux premières altérations neuropathologiques de la MA. Ces résultats supportent la nécessité de dépister et traiter les pathologies du sommeil dans le vieillissement, avant l’apparition des premiers déficits cognitifs, dans l’espoir de ralentir le déclin cognitif. / Sleep changes are a major feature of the ageing process, and sleep disturbances are increasingly recognized as a risk factor for cognitive decline and Alzheimer’s disease (AD). However, the brain mechanisms underlying this association are still unclear. The objective of this thesis was to deepen our understanding about brain structural, functional and molecular correlates of the main objective sleep changes in ageing, and to assess the potential links with cognitive performance. Our results demonstrate that the fragmentation of the first sleep cycles and the alteration of slow wave activity, are associated with reduced gray matter metabolism, perfusion and/or volume in fronto-cingulate and hippocampal areas. Moreover, sleep-disordered breathing and rapid eye movement sleep microstructure alterations were related to increased amyloid burden respectively in the posterior cingulate cortex and precuneus, or more widespread neocortical areas. However, associations with cognitive performance remained subtle or inexistent, suggesting early and asymptomatic associations between sleep and brain changes. Therefore, sleep may contribute to resilience processes and may help to cope with early neuropathological changes in AD. These results support the need to screen and treat sleep disturbances in older adults, before the onset of the first cognitive signs, in order to slow cognitive decline.

[18F]Fluorodeoxyglucose ([18F]FDG) Positron Emission Tomography/Computed Tomography (PET/CT) imaging is a powerful tool in the diagnosis of cancer and subsequent treatment planning. New state-of-the-art PET/CT scanners have the capacity to generate images of superb quality. The new scanners feature detectors with increased sensitivity and a new generation of reconstruction algorithms that produce higher quality images than the scanners they are replacing. In addition to the scanner, the scan duration, amount of administered [18F]FDG activity, and the anatomy of the patients themselves are also determining factors of image quality. There is evidence suggesting that [18F]FDG PET image quality is significantly reduced for larger patients, jeopardizing lesion detection. Two possible solutions to this problem are to (i) increase injected activity or (ii) increase scan duration. Increasing scan duration is preferable but not always possible in a busy clinic. Increasing injected activity is necessary but a proper scaling regimen with patient size must be determined in order to achieve consistent image quality. The aim of the work presented in this thesis was to achieve higher quantification accuracy and consistent image quality for all patients scanned with [18F]FDG PET. Because quantitative PET/CT images require corrections for image degrading effects, for which attenuation correction is the main contributor and is performed based on CT images, the first step in this project was to develop software tools to automate the analysis of phantom images for CT quality assurance. The next step was to optimize the reconstruction parameters for whole body [18F]FDG PET based on a phantom experiment. Finally, a retrospective study was conducted using patient [18F]FDG PET images to characterize the relationship between patient anatomical characteristics and image quality. This work concludes by suggesting optimized reconstruction parameters and a scaling regimen for injected [18F]FDG activity. With the implementation of these recommendations it can be possible to obtain images with increased quantitative value while delivering less dose to patients and maintaining a uniform level of image quality between different patients. / Graduate

Right ventricular function (RV) is one of the most important predictors of prognosis in many cardiovascular disease states. Despite the significance of RV function to survival, there are no therapies that directly nor selectively improve RV function. As well, the basis for RV failure is poorly understood. This is particularly relevant for patients with pulmonary arterial hypertension (PAH), where RV failure in the setting of pressure overload is the leading cause of death. PAH will be introduced in the 2nd chapter of this thesis by comparing and refining contemporary mortality risk assessment strategies. I will then explore 1) RV neurohormonal function and, 2) RV energetics, two molecular pathways thought to be involved in the pathogenesis and progression of maladaptive RV failure. I employed small animal molecular imaging using positron emission tomography (PET) to non-invasively investigate these pathways. The PET imaging techniques employed in this thesis have the unique potential for translation to human studies, to further explore disease mechanisms.

Proton therapy, a relatively young modality in radiation therapy, has proven useful in cases where a sharp dose gradient or low secondary irradiation is required. In Canada proton therapy it was performed at the TRIUMF Proton Therapy Facility in the treatment of large or difficultly positioned ocular melanomas. This rare primary malignant cancer of the eye has a poor prognosis if untreated. Patient vision sparing is critical for quality of life and is strongly affected by the accuracy of the chosen treatment. Reduction in irradiation of critical structures is a proven strength of proton therapy due to the high dose-gradient and finite range in tissue. But, with the advantage of steep dose gradients, comes the requirement of precision target positioning and planning. Monte Carlo particle transport software is a valuable tool for understanding treat- ment doses in cases where measurement is time consuming or difficult. Accurate simulation of primary proton dose to water aids in the evaluation of beam charac- teristics and allows for study into improving dose application for patient treatment. In this work, a full Monte Carlo model of the TRIUMF proton therapy facility was developed. Measurements were taken in water to validate simulated results within 2% over the treatment depth for a wide range of beam modulations. The second advantage of proton therapy lies in its reduced dose bath to healthy tissue. This is especially important in pediatric cases where extraneous dose comes with a high risk of secondary carcinogenesis. Whereas multi-angle photon treatments necessarily irradiate large volumes of healthy tissue to produce a flat target dose, proton treatments may irradiate a target with a single beam. With this advantage comes a trade-off - protons produce a large number of neutrons as they are prepared for patient treatment. These neutrons are the largest contributor to secondary dose in proton therapy and must be well modeled and shielded to ensure patient safety. The second part of this work involves the measurement of secondary neutron doses in the TRIUMF treatment room. Measurements were validated within 20% of simulated values with uncertainties dominated by calibration of the detector. Neutron doses to an anatomic human model showed that calibrated secondary doses were in line with similar treatment facilities reporting globally. Simulations indicated that the source of neutrons was primarily in the unshieldable region of the beamline opening. Thus the total treatment time was the determining factor in secondary dose to the patient. With primary proton dose well modeled, it became possible to study the pre- cision of treatment and possible avenues for improvement. The beam modulation wheels and optimization scheme was developed in the late 90‘s when computational and manufacturing technologies were less developed. Updated optimization methods indicated that moving to a smooth scheme of energy modulation, as opposed to a stepped modulation wheel, could improve distal dose sharpness. This was contrary to the long-held belief that there was an optimal number of steps for modulation. The third portion of this work explored the use of 3D printers to enable the fabri- cation of smoothly transitioning modulator wheels. Materials and printer methods were studied, indicating a strong candidate in the PolyJet TM method for beam mod- ulation. Both stepped and newly-optimized smooth modulator wheels were printed and validated. Total turnaround time for modulator production was under 24 hours - proving the feasibility of patient-specific beam modulation. The last portion of this work explored the use of positron emitting isotopes for dose validation. Protons traversing tissue or plastic generate β + emitting isotopes via nuclear interactions. The resulting back-to-back annihilation photons can be re- constructed into the isotope distribution produced by the beam. This can potentially provide information about beam position in the target and hence position of a phan- tom or patient. An anatomic 3D printed eye phantom was designed and irradiated to test the feasibility of this method. While a strong isotope signal was reconstructed, the test did not yield a viable technique due to the low resolution of the phantom scan. The phantom position was poorly reconstructed using the transmission scan. Despite this, it could be possible to improve this method by using other methods for phantom position registration. / Graduate