This thesis describes the development of new magnetic resonance imaging (MRI) methods to characterize cardiac tissue with myocardial infarction (MI). Wall motion imaging (for visualizing myocardial contraction) and viability imaging (to identify MI) are two components of cardiac tissue characterization used for prognosis and treatment planning. MRI-based wall motion and viability methods are considered the gold standard in imaging, and characterization of MRI viability images has been correlated with inducibility for ventricular tachycardia (VT). However, viability imaging with MRI has limitations such as difficulty visualizing the blood-infarct border. Wall motion and viability images are acquired separately, each requiring cardiac gating and breath holds, leading to long scan times. A novel multi-contrast delayed enhancement (MCDE) sequence was developed that simultaneously acquires wall motion and viability images. In a patient study, the MCDE sequence was demonstrated to provide improved visualization of MI compared to the conventional inversion-recovery gradient echo (IR-GRE) sequence, particularly for small infarcts adjacent to the blood pool. MCDE images also provided accurate wall motion images that could be used to calculate the left ventricular ejection fraction. An image processing algorithm was developed to analyze MCDE images to segment and classify the infarct gray zone, which is hypothesized to represent heterogeneous infarct responsible for causing VT. In a study of 15 patients with MI, the MCDE-derived gray zone was shown to be less sensitive to image noise than the IR-GRE-derived gray zone, and did not require manual contours of the blood pool which contributes to additional variability in the IR-GRE gray zone analysis. Finally, a real-time delayed enhancement (RT-DE) method was developed to provide black-blood viability images without requiring cardiac gating or breath holds. RT-DE imaging was shown to have a high sensitivity for detecting MI in a study of 23 patients. The methods described in this thesis help expand the patient population that can undergo a cardiac viability exam and help improve the visualization of myocardial infarct. Further modifications in the pulse sequences to improve the temporal and spatial resolutions are proposed with the goal of predicting and guiding treatment of ventricular tachycardia resulting from myocardial infarct.

The spatiotemporal dynamics of ongoing beta band (15-30 Hz) cortical oscillations and the modulation of this neural activity by tactile input and movement provide insight into how the brain achieves proper sensorimotor processing. Earlier studies have shown that the synchrony of the cortical beta rhythms within and between central and peripheral neuronal populations is modulated during and following somatosensation or movement, and correlated with effective motor control. In addition, abnormal levels of beta oscillations in the basal ganglia are correlated with motor dysfunction in Parkinson’s disease. Numerous functional roles for the beta rhythm have been proposed – ranging from inhibition to the facilitation of long-range communication. However, the neural network that generates the sensorimotor beta rhythm and the functional significance of this activity have not been fully specified. Thus, I used magnetoencephalography to complete three studies of the beta rhythm in healthy right-handed adults. In the first study, I hypothesized that finger vibration at beta frequencies would generate stimulus-coherent neuronal firing in the neural network that generates the beta rhythm – thus revealing the nodes of this network. Data were analyzed for nineteen subjects (10 females). The coherent activity was revealed using a novel analysis technique that generated whole-brain maps of inter-trial synchrony during passive repetitive finger vibration at 23 Hz. These maps identified contralateral primary somatosensory cortex (SI), posterior parietal cortex, supplementary motor area and primary motor cortex (MI), and ipsilateral brainstem as nodes in the network. In the second study, I correlated changes in focused attention with modulations in beta band cortical responses to specify the functional significance of this activity. Data were analyzed for twelve subjects (7 females). With increased focused attention to the stimulus, I hypothesized that the beta band responses to finger vibration would be enhanced in areas involved in somatosensory processing. A transient increase in the magnitude of beta oscillations in MI (event-related synchronization) following vibration offset was significantly enhanced by attention, as compared to passive stimulation. In addition, attention caused the suppression of beta oscillations (event-related desynchronization, ERD) in ipsilateral SI beginning 1 second prior to vibration offset. Strong attention-modulation of the beta rhythm outside of contralateral SI implies that these changes are indicative of higher-order processing of afferent information. In the third study, I tested the hypothesis that synchrony between beta rhythms in contralateral MI and the relevant muscle supports effective neuronal communication. I correlated changes in task performance with corticomuscular coherence (CMC) during the sustained application of force to match a visually-presented target. Data were analyzed for eighteen subjects (9 females). As predicted, CMC in MI was significantly increased during improved performance in this task. This suggests that central-peripheral synchrony plays an important functional role in sustaining isometric muscle control. Concurrent beta ERD in bilateral SI and primary visual cortices during the contraction indicates the importance of afferent feedback in this task. Gender-related effects were not investigated in these studies. Beta band neuromagnetic responses to movement and somatosensation identify a pervasive neural network that is involved in processing the relevant properties of somatic input and regulating sustained motor output.

A new laser ablation mechanism of liquid water based on recent insights into its hydrogen bond dynamics has been studied and several applications of the ablation demonstrated. The mechanism, termed as Impulsive Heat Deposition through Vibrational Excitations (IHDVE), is based on the ability of the hydrogen bond network of water to rapidly thermalize vibrational O-H stretch excitations on a time scale of several picoseconds even for excitation intensities that are large enough to bring excited volumes far into the supercritical region. In this way, by using vibrationally resonant picosecond infrared laser pulses with sufficient energy, it is possible to drive ultrafast phase transitions in the excited water volume leading to a rapid and efficient ablation process of water and water rich targets with minimum perturbation of solute molecules of interest. The physics behind the IHDVE ablation process is outlined and the benefits of the IHDVE ablation are demonstrated for two important applications of tissue cutting and mass spectrometry of biomolecules. Finally, the development of two high power infrared laser systems suitable for the practical implementation of IHDVE is presented.

Prostate cancer is extremely prevalent, with shifting patient demographics leading to an increasing number of men balancing treatment efficacy with associated side-effects. Non-invasive characterization of disease – useful for guiding biopsy, to monitor disease progression during active surveillance, or for treatment planning of focal therapies – could have a significant impact on patient management. Through its excellent anatomic imaging capabilities and its ability to characterize physiologic properties, magnetic resonance imaging (MRI) has the potential to fulfill clinical goals; however, further improvements are necessary to maximize accuracy and impact. Thus, this thesis presents: 1) the development of a multi-parametric model to combine parameters derived from measurement of T2 relaxation, diffusion weighted imaging, and dynamic contrast-enhanced MRI to improve the discrimination between normal and malignant peripheral zone tissue; 2) determination of the impact that the presence of normal tissue within regions of tumour has on the measurement of apparent diffusion coefficient (ADC) and T2 relaxation in the peripheral zone; and 3) relationships between MRI measurement and underlying prostate tissue composition. A common patient cohort was used for all studies, with prostate cancer patients having in vivo MRI prior to prostatectomy followed by whole-mount histologic sectioning of the surgical specimens, facilitating the use of pathology as a gold-standard for all analyses. In the first study, the optimal multi-parametric model combines ADC, T2, and volume transfer constant (Ktrans) to yield the probability of malignancy for each voxel. Performance of the model is better than each single parameter, but not significantly so compared to ADC. The second study demonstrates that there is no difference in ADC and T2 between tumours containing significant portions of normal tissue and the surrounding normal tissue itself, indicating that full characterization of prostate cancer with MRI may be limited. Finally, by determining relationships between MRI parameters and tissue characteristics, the third study suggests mechanisms driving MR image appearance in the prostate, including the visualization of cancer. Taken together, this thesis presents potential improvements to prostate cancer imaging, and provides further insight into the interplay between the underlying histology and MRI.

Aberrations in Epidermal Growth Factor Receptor (EGFR/ErbB1) signalling are the most common oncogenic stimuli in human glioblastoma multiforme (GBM). Interactions between mutant and wildtype ErbB family members in GBMs are of biological and potential therapeutic importance. In this thesis, we describe our work developing and optimizing a novel in situ proximity ligation assay (PLA) for dimerization and activation analysis of EGFR mutants prevalent in GBMs. Utilizing this novel in situ platform for EGFR dimerization analysis, we seek to systematically interrogate the dimerization capacity and activation status amongst EGFR and EGFR mutants. Our in vitro analysis using this platform demonstrates the aberrant homo-/hetero-dimeric properties of EGFRvIII and EGFRc958 mutants, the two most common mutants associated with EGFR amplification in GBMs. In addition, dimer phospho-activation status can be detected using in situ PLA with ≥ 16-fold sensitivity and ≥ 17-fold signal-to-noise than phospho-EGFR measurements currently undertaken with IHC or IF. These aberrant features are not overexpression dependent but appear independent of cellular expression levels, suggesting inherent properties of the mutant receptors. This EGFR dimerization/activation detection platform may also be useful for evaluating novel anti-EGFR therapeutics. Our data suggests that various EGFR monoclonal antibody therapies have unique dimerization blocking abilities and that certain mutant EGFR dimer configurations can evade blockage by anti-EGFR treatments. Furthermore, we report for the first time the detection of wt- and EGFRvIII dimerization in GBM specimens, in keeping with our prior cell line data, and a potential feature of prognostic or diagnostic value in GBMs harbouring them. Additionally, we demonstrate the utility of this platform for measuring pEGFR and total EGFR expression on tissue samples, which has not been efficacious to date with conventional antibody-mediated techniques. Results from this thesis may therefore provide novel insights into the interaction and activation characteristics of EGFR mutants prevalent in GBMs, as well as the efficacy of current anti-EGFR therapies to target these mutants. In summary, these findings demonstrate the successful application of a novel in situ EGFR molecular detection platform which may have clinical utility in diagnostic evaluation or stratification of GBM patient subgroups for prognosis and treatment. Furthermore, since PLA allows specimen assessment of not only expression and activation, but also dimerization, which is not evaluated by current IHC techniques, it will likely serve as a way to evaluate promising anti-EGFR strategies directed at preventing EGFR dimerization and activation.

The aim of this study was to test the application of an in situ–formed synthetic polyethylene glycol (PEG) as a biodegradable membrane with a variety of graft materials and hyperbaric oxygen (HBO) for enhanced bone regeneration. Critical-sized rabbit calvarial defects were created in bilateral parietal bones. Group 1 served as a control with unfilled defects, Group 2 had defects filled with morcelized autogenous bone, and Group 3 had defects filled with biphasic calcium phosphate. One defect was protected PEG membrane and half the animals were subjected to HBOT treatment. The unsupported membrane didn’t produce the desired bone regeneration in the unfilled and bone grafted groups. HBO didn't ameliorate the bone grafted or ceramic filled defects over the 6-week time period. Caution is recommended with the membrane over unsupported defects. Future assessments with HBO should be completed at the 12-week time point.

Activation of the unfolded protein response follows induction of endoplasmic reticulum (ER) stress, resulting in widespread inhibition of protein expression. FLIP protein is particularly sensitive to stresses that perturb protein translation; as such, a reduction in FLIP is likely an early outcome of ER stress. Due to the anti-apoptotic role of FLIP, it is anticipated that potential decreases in FLIP would bring about an increase in sensitivity to death receptor stimuli and anoikis, a form of anchorage-dependent cell death. It was hypothesized that induction of ER stress results in downregulation of FLIP expression, resulting in sensitization of resistant tumour cells to death receptor stimuli and anoikis. From this hypothesis, it was determined that induction of ER stress through treatment of cells with brefeldin sensitized tumour cells to Fas-mediated cell death and anoikis. Moreover, over-expression of FLIP appeared to protect against ER stress-induced sensitization to cell death.

In photodynamic therapy (PDT), the combination of light, photosensitizer and molecular oxygen generates reactive oxygen species, including singlet oxygen (1O2), which is regarded as the primary cytotoxin and effector with most clinical photosensitizers. PDT has gained some acceptance for the treatment of cancer and other conditions. However, its clinical utility and effectiveness has been limited by variability in treatment response and failure to integrate adequate treatment planning and dosimetry. Direct PDT dosimetry through the detection of ultra-weak near-infrared 1O2 luminescence emission at 1270 nm (SOL) collapses the complexity of PDT into a single parameter, the 1O2 concentration. Prior to the present studies, it was shown that SOL was well correlated with PDT response in vitro and in vivo under controlled experimental conditions. However, SOL detection is technically challenging because of the very low radiative probability of 1O2 (~ 10-8 in biological environments), dynamic background signals and limited sensitivity of suitable photodetectors in this wavelength region. A technologically simpler and less costly PDT dosimetry approach is to use photosensitizer photobleaching to estimate the 1O2 dose. The first objective in this thesis was to characterize the dynamics of SOL measurements, in particular the influence of oxygen depletion, in order to improve the quantification of SOL and its use as an accurate PDT dose metric. Subsequently, direct comparison of SOL and photobleaching-based dosimetry during in vitro PDT treatment with meso-tetra(hydroxyphenyl)chlorin (mTHPC) showed that SOL dosimetry is robust but that photobleaching-based dosimetry can fail under hypoxic conditions. However, the latter can be salvaged through the identification of a previously unreported 613 nm emission from mTHPC that indicates hypoxia. These studies were carried forward into an in vivo dorsal skin-fold window chamber tumor model, which showed promising initial correlation between 1O2 dose and tumor response. This work also identified SOL detection limitations and opportunities for further development. Additionally, SOL measurements were used as a ‘gold standard’ to evaluate novel activatable PDT beacons and a novel “PDT biodosimeter” based on STAT3 cross-linking. Future work includes further tumor dose-response studies, characterization of novel photosensitizing agents, improvement on signal detection and processing, and studies in normal human skin.

In photodynamic therapy (PDT), the combination of light, photosensitizer and molecular oxygen generates reactive oxygen species, including singlet oxygen (1O2), which is regarded as the primary cytotoxin and effector with most clinical photosensitizers. PDT has gained some acceptance for the treatment of cancer and other conditions. However, its clinical utility and effectiveness has been limited by variability in treatment response and failure to integrate adequate treatment planning and dosimetry. Direct PDT dosimetry through the detection of ultra-weak near-infrared 1O2 luminescence emission at 1270 nm (SOL) collapses the complexity of PDT into a single parameter, the 1O2 concentration. Prior to the present studies, it was shown that SOL was well correlated with PDT response in vitro and in vivo under controlled experimental conditions. However, SOL detection is technically challenging because of the very low radiative probability of 1O2 (~ 10-8 in biological environments), dynamic background signals and limited sensitivity of suitable photodetectors in this wavelength region. A technologically simpler and less costly PDT dosimetry approach is to use photosensitizer photobleaching to estimate the 1O2 dose. The first objective in this thesis was to characterize the dynamics of SOL measurements, in particular the influence of oxygen depletion, in order to improve the quantification of SOL and its use as an accurate PDT dose metric. Subsequently, direct comparison of SOL and photobleaching-based dosimetry during in vitro PDT treatment with meso-tetra(hydroxyphenyl)chlorin (mTHPC) showed that SOL dosimetry is robust but that photobleaching-based dosimetry can fail under hypoxic conditions. However, the latter can be salvaged through the identification of a previously unreported 613 nm emission from mTHPC that indicates hypoxia. These studies were carried forward into an in vivo dorsal skin-fold window chamber tumor model, which showed promising initial correlation between 1O2 dose and tumor response. This work also identified SOL detection limitations and opportunities for further development. Additionally, SOL measurements were used as a ‘gold standard’ to evaluate novel activatable PDT beacons and a novel “PDT biodosimeter” based on STAT3 cross-linking. Future work includes further tumor dose-response studies, characterization of novel photosensitizing agents, improvement on signal detection and processing, and studies in normal human skin.

While extirpative surgery is curative for localized clear cell renal cell carcinoma (ccRCC), many patients develop recurrences or present with metastatic disease. Several aspects of ccRCC biology have been investigated, but these have been done in cell lines, which are known to poorly represent the tumour. Since cell lines are amenable to a wide array of experimental testing, the studies presented here demonstrate a novel method to generate ccRCC cell lines from primary tumours, which increases the rate of primary tumour cell line generation four fold. Additionally, ccRCC cells do not grow in serum-free media, which has been shown to be beneficial in other cancers. Therefore, we interrogated the effect of exogenous growth factors to optimize our serum-free media growth conditions, among which TGFb1 appeared to elicit the largest mitogenic effect. Once optimized, these findings will provide a valuable tool for understanding ccRCC tumour cell biology and identifying therapeutic targets.