Understanding the structural basis of specificity and promiscuity of paralogous enzymes is important for deciphering molecular mechanisms and is a necessary step towards designing enzyme-specific modulators. The main objective of this thesis is to provide structural insights that relate protein local sequences to their observed binding and activity profiles through the study of two human protein families – cytosolic sulfotransferases (SULTs) and sirtuins (SIRTs). This was achieved by comparing the family-wide ligand binding fingerprints of these two enzyme families with the structural details of their corresponding enzyme-ligand co-crystal structures. The hSULT enzyme family was profiled against a focused library through binding and activity assays. This suggested a number of novel compounds that bind to the less well-characterized SULT members (SULT1C3 and SULT4A1), and revealed additional broad-spectrum hSULT inhibitors. Based on the profiling data, three enzyme/co-factor/ligand complex structures were solved using X-ray crystallography. The structure of SULT1C2•PAP(3'-phosphoadenosine 5'-phosphate)•pentacholorphenol(PCP) provided a rationale for a novel SULTs inhibition mechanism that depends on substrate acidity. The SULT1B1•PAP•resveratrol structure suggested that the hydrogen-bonding coordination of the 5-OH group on resveratrol is the structural determinant for the observed substrate preference towards resveratrol. SULT2A1•PAP•lithocholic acid(LCA) ternary complex structure confirms that the specificity of SULT2A1 for lithocholic acid derives from its high hydrophobicity in the substrate binding pocket. The same approach was used to interrogate the interaction of the sirtuins with their peptide substrates. The binding and enzymatic assays for human sirtuins have suggested that SIRT1 and SIRT2 are generally less discriminate against substrates while class IV sirtuins - SIRT6 and SIRT7 might be highly specific enzymes. Three different biochemical and kinetic assays showed that SIRT6-dependent histone deacetylation is about 1,000 times slower than for other highly active sirtuins. To understand the molecular basis for the specificity and low activity of SIRT6, I determined the first set of crystal structures for SIRT6 in complex with ADPr (ADP ribose) and the non-hydrolyzable analog of OAADPr (2’-O-acetyl-ADP ribose) – NAADPr (2’-N-acetyl-ADP ribose). The structures revealed human SIRT6 has unique structural features including a splayed zinc-binding domain, lacks a helix bundle and the conserved, highly flexible, NAD(+)-binding loop, which contribute to its observed biochemical behavior.

Focused ultrasound therapy can be enhanced with microbubbles by thermal and cavitation effects. However, localization of treatment becomes difficult as bioeffects can occur outside of the target region. Spatial control of gas bubbles can be achieved with acoustic vaporization of perfluorocarbon droplets. This study was undertaken to determine the acoustic parameters for bubble production by droplet vaporization and how it depends on the acoustic conditions and droplet physical parameters. Droplets of varying sizes were sonicated in vitro with a focused ultrasound transducer and varying frequency and exposure. Simultaneous measurements of the vaporization and inertial cavitation thresholds were performed. The results show that droplets cannot be vaporized at low frequency without inertial cavitation occurring. However, the vaporization threshold decreased with increasing frequency, exposure and droplet size. In summary, we have demonstrated that droplet vaporization is feasible for clinically-relevant sized droplets and acoustic exposures.

The goal of this research is to improve the breast cancer screening process based on magnetic resonance imaging (MRI). In a typical MRI breast examination, a radiologist is responsible for visually examining the MR images acquired during the examination and identifying suspect tissues for biopsy. It is known that if multiple radiologists independently analyze the same examinations and we biopsy any lesion that any of our radiologists flagged as suspicious then the overall screening process becomes more sensitive but less specific. Unfortunately cost factors prohibit the use of multiple radiologists for the screening of every breast MR examination. It is thought that instead of having a second expert human radiologist to examine each set of images, that the act of second reading of the examination can be performed by a computer-aided detection and diagnosis system. The research presented in this thesis is focused on the development of a computer-aided detection and diagnosis system for breast cancer screening from dynamic contrast-enhanced magnetic resonance imaging examinations. This thesis presents new computational techniques in supervised learning, unsupervised learning and classifier visualization. The techniques have been applied to breast MR lesion data and have been shown to outperform existing methods yielding a computer aided detection and diagnosis system with a sensitivity of 89% and a specificity of 70%.

CK2 is a ubiquitous serine/threonine protein kinase implicated in numerous cellular processes as well as in tumorigenesis. CK2 is composed of two catalytic (αα, αα’, α’α’) subunits and two regulatory (ββ) subunits that assemble to form the active CK2 holoenzyme. CK2 has been shown to phosphorylate, interact with, and regulate other proteins, including other protein kinases. CK2 substrates can be initially bound by the CK2β regulatory subunit, which acts as a docking site to facilitate phosphorylation and mediate CK2 substrate specificity. In a screen to identify novel CK2β interacting proteins, I identified three novel CK2β interactors, including the mitogen- and stress-activated kinase 2 (MSK2), which I pursued for further characterization. MSK2, and the closely related isoform MSK1, are nuclear kinases that are activated following mitogen stimulation or cellular stress, including UV radiation, by the ERK1/2 and p38-MAPK signaling cascades, respectively. However, factors that differentially regulate MSK1 and MSK2 have not been well characterized. In my thesis, I demonstrate that CK2, which contributes to NF-κB activation following UV radiation in a p38-dependent manner, physically interacts with MSK2 but not MSK1 and that CK2 inhibition specifically impairs UV-induced MSK2 kinase activation. A putative site of CK2 phosphorylation was mapped to MSK2 residue serine-324 and when substituted to alanine (S324A) also compromised MSK2 activity. RNA interference-mediated depletion of MSK2 in human MDA-MB-231 cells, but not MSK1 depletion, resulted in impaired UV-induced phosphorylation of NF-κB p65 at serine-276 in vivo, which was restored by the ectopic expression of MSK2 but not by MSK2-S324A. Furthermore, UV-induced p65 transactivation capacity was dependent on MSK2, MSK2 residue S324, and p65-S276. These results suggest that MSK1 and MSK2 are differentially regulated by CK2 during the UV response and that MSK2 is the major protein kinase responsible for the UV-induced phosphorylation of p65 at S276 that positively regulates NF-κB activity in MDA-MB-231 cells.

The motivation of this study lies in the necessity for a microfocal therapy to specifically target diseased areas in vascular pathologies such as age-related macular degeneration (AMD). AMD is the most common cause of legal blindness among people over the age of 60 in developed countries. This degenerative condition affects the macula, the central region of the retina, severely impairing detailed vision and hindering everyday activities. Worldwide, 25-30 million people live with some form of AMD. Among them, ~10% suffer from the more advanced and damaging form, wet-AMD, which causes rapid and severe loss of central vision. To date, there is no cure or long-term alternative for this degenerative disease despite intensive research efforts. With recent developments in biophysical tools and experimental procedures, in this study, we demonstrate a highly-localized therapeutic option: two-photon (2-photon) photodynamic therapy (PDT) that could be advantageous for the cure of wet-AMD, either alone or in combination with recently discovered anti-angiogenic therapies. This new approach offers selective targeting of the diseased area, thus minimizing damage to the surrounding sensitive healthy eye tissues, which is a major concern with the clinically-used, standard wide-beam, one-photon (1-photon) PDT. The objective of the research was to test the feasibility of microfocal 1-photon and the inherently localized 2-photon PDT, their optimization and also to evaluate the efficacy of existing 1-photon and novel 2-photon photosensitizers. In this thesis, I illustrated the in vitro (endothelial cell monolayer) and in vivo (window chamber mouse (WCM)) models that can be used to quantitatively compare the 2-photon efficiency of photosensitizers. Using the in vitro model, I compared the 2-photon efficacy of clinically used 1-photon PDT drugs Photofrin and Visudyne, and showed that the Visudyne is an order of magnitude better 2-photon photosensitizer than Photofrin. With the WCM model, I demonstrated a novel designer 2-photon photosensitizer is 20 times more efficient than Visudyne for single vessel occlusion. I also generated the drug and light dose reciprocity curve for localized single-vessel microfocal PDT. This is a necessary step towards applying the method to the relevant ocular models of AMD, which is the next phase for this research.

Intensity modulated radiotherapy (IMRT) can potentially increase the dose delivered to liver tumours while sparing normal tissues from dose. More complex IMRT, with more modulation of the radiation beam is more susceptible to geometric and dosimetric uncertainties than simpler radiotherapy plans. Simple breath-hold liver IMRT using few radiation beam segments (<30) was investigated in 27 patients to determine the quality of treatment in terms of tumour dose coverage and normal tissue sparing as compared to index IMRT using >30 segments. In all 27 plans number of segments was reduced to <30 without compromising tumour coverage or normal tissue dose constraints, at the expense of dose conformity. Delivered tumour and normal tissue dose did not differ statistically between IMRT plans when accounting for treatment residual geometric error. This research supports considering the use of simple IMRT for treatment of liver cancer, except when loss of dose conformation is undesirable (i.e. very high doses).

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.