Cardiovascular diseases, such as stroke and heart attack, are one of the largest causes of death and morbidity in Canada. Atherosclerosis, or the thickening of the arterial wall, has been identified as the primary culprit lesion behind the end organ damage associated with cardiovascular diseases. Magnetic resonance imaging has taken a primary role in characterising the constituents of these atherosclerotic plaques. Of these components, MR detected intraplaque haemorrhage (IPH), or bleeding inside the vessel wall, appears to predispose a patient to future clinical events. This leads us to the conclusion that IPH is a secondary manifestation of plaque progression and complication, or somehow contributes to the complication of these atherosclerotic plaques. This thesis explores this second possibility, in line with the “iron hypothesis” of atherosclerosis which suggests that iron plays a significant, primary role in atherogenesis. In chapter two, the signal hyperintensity associated with IPH is correlated with the lipid oxidising potential of blood products in various oxidation states. It is found that the ferric paramagnetic species has a significantly greater ability to generate lipid oxide components and oxidise lipid surrogates. This finding suggests that it may be possible to alter the course of plaque progression by inhibiting the active ferric iron state. Chapter three explores an endogenous molecule, haptoglobin, whose purpose is to bind and inactivate free haemoglobin. It is shown that haptoglobin has the ability to modulate the MR signal intensity from IPH. Chapter four explores a mechanism to detect this imaging biomarker outside the MR environment using the electron paramagnetic resonance of the ferric haemoglobin. Results are shown in a custom made bench top system detecting ferric haemoglobin in an in vitro sample. This thesis provides more evidence for the iron hypothesis of atherosclerosis and explores methods of inhibiting and detecting this biomarker of disease.

Biochemical and structural studies of membrane protein usually require their stabilization in solution with detergents. However, purified membrane proteins often show reduced activity and stability in traditional detergents. Lipopeptide detergents (LPDs) are a new class of engineered amphiphiles that from small micelles and mimic the lipid bilayer more closely than traditional detergents. An LPD molecule consists of an α-helical peptide with fatty acyl chains covalently attached to both ends. These molecules self-assemble into cylindrical micelles with a hydrophilic exterior and a hydrophobic interior made up of the fatty acyl chains. Here we present the biophysical and biochemical properties of a model bacterial ABC transporter, MsbA, in different LPD variations. Four types of LPD molecules have been synthesized and have been categorized as original LPD, LPD5Q, LPD2, and LPD4 based on the peptide sequence. Dynamic light scattering, thermal aggregation, and ATPase activity were used to measure the biophysical properties of MsbA in LPDs and in a traditional detergent, n-decyl-β-D-maltoside (DM). The results show that MsbA-LPD particles are monodisperse with small hydrodynamic radii. When compared to DM, MsbA is thermodynamically more stable and has higher catalytic activity in LPDs. Membrane proteins have favorable biophysical properties in LPDs, suggesting that these detergents resemble the native lipid bilayer environment more closely. We also present crystal structures of three LPDs: LPD-12, LPD5Q-14 and LPD- 14. These structures reveal that LPD micelles are highly ordered with varying oligomeric states. The octomeric structure of the LPD-12 micelle is composed of four sets of antiparallel coiled-coil dimers, while the LPD5Q-14 micelle assembles as a nonamer of three trimers each with an “up-up-down” topology. The LPD-14 micelle, on the other hand, is a dodecamer of three tetramers with all helices assuming an antiparallel orientation. Overall, the structures of LPDs show highly ordered detergent micelles that are made up of repeated building blocks. Based on these results, we propose that LPDs can sample multiple conformational states, but the number of accessible conformations is significantly reduced relative to traditional detergents. Our results show that LPDs are an alternative platform for in vitro studies of membrane proteins. Future studies will focus on the crystallization of membrane proteins in LPDs and the further characterization of these complexes.

Biochemical and structural studies of membrane protein usually require their stabilization in solution with detergents. However, purified membrane proteins often show reduced activity and stability in traditional detergents. Lipopeptide detergents (LPDs) are a new class of engineered amphiphiles that from small micelles and mimic the lipid bilayer more closely than traditional detergents. An LPD molecule consists of an α-helical peptide with fatty acyl chains covalently attached to both ends. These molecules self-assemble into cylindrical micelles with a hydrophilic exterior and a hydrophobic interior made up of the fatty acyl chains. Here we present the biophysical and biochemical properties of a model bacterial ABC transporter, MsbA, in different LPD variations. Four types of LPD molecules have been synthesized and have been categorized as original LPD, LPD5Q, LPD2, and LPD4 based on the peptide sequence. Dynamic light scattering, thermal aggregation, and ATPase activity were used to measure the biophysical properties of MsbA in LPDs and in a traditional detergent, n-decyl-β-D-maltoside (DM). The results show that MsbA-LPD particles are monodisperse with small hydrodynamic radii. When compared to DM, MsbA is thermodynamically more stable and has higher catalytic activity in LPDs. Membrane proteins have favorable biophysical properties in LPDs, suggesting that these detergents resemble the native lipid bilayer environment more closely. We also present crystal structures of three LPDs: LPD-12, LPD5Q-14 and LPD- 14. These structures reveal that LPD micelles are highly ordered with varying oligomeric states. The octomeric structure of the LPD-12 micelle is composed of four sets of antiparallel coiled-coil dimers, while the LPD5Q-14 micelle assembles as a nonamer of three trimers each with an “up-up-down” topology. The LPD-14 micelle, on the other hand, is a dodecamer of three tetramers with all helices assuming an antiparallel orientation. Overall, the structures of LPDs show highly ordered detergent micelles that are made up of repeated building blocks. Based on these results, we propose that LPDs can sample multiple conformational states, but the number of accessible conformations is significantly reduced relative to traditional detergents. Our results show that LPDs are an alternative platform for in vitro studies of membrane proteins. Future studies will focus on the crystallization of membrane proteins in LPDs and the further characterization of these complexes.

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.

Artifacts caused by head motion are a substantial source of error in fMRI that limits its use in neuroscience research and clinical settings. Real-time scan-plane correction by optical tracking has been shown to correct slice misalignment and non-linear spin-history artifacts, however residual artifacts due to dynamic magnetic field non-uniformity may remain in the data. A recently developed correction technique, PLACE, can correct for absolute geometric distortion using the complex image data from two EPI images, with slightly shifted k-space trajectories. We present a correction approach that integrates PLACE into a real-time scan-plane update system by optical tracking, applied to a tissue-equivalent phantom undergoing complex motion and an fMRI finger tapping experiment with overt head motion to induce dynamic field non-uniformity. Experiments suggest that including volume by volume geometric distortion correction by PLACE can suppress dynamic geometric distortion artifacts in a phantom and in vivo and provide more robust activation maps.

Multiple-mouse MRI (MMMRI) accelerates biomedical research by imaging multiple mice simultaneously. To date, MMMRI has been explored in three ways: shielded transmit-receive coils, shielded transmits coil with separate unshielded receive coils; and finally shielded transmit-receive coils with independent gradient coils. However alternative transmit coil configurations and possible benefits of eliminating shielding have not yet been explored. The goal of this thesis is to test possible radiofrequency configurations with and without shielding for the purpose of improving image quality for MMMRI. Results demonstrate that using an unshielded transmit-receive coil array provided a 20% improvement over an identical shielded coil. A new unshielded 7-coil MMMRI array is presented, minimizing the ghosting between image overlap using mutual inductance minimization and a sensitivity encoding (SENSE) reconstruction. The final array provided high resolution images (90µm) of up to seven live mice simultaneously with appropriate signal-to-noise for automated analysis.

Artifacts caused by head motion are a substantial source of error in fMRI that limits its use in neuroscience research and clinical settings. Real-time scan-plane correction by optical tracking has been shown to correct slice misalignment and non-linear spin-history artifacts, however residual artifacts due to dynamic magnetic field non-uniformity may remain in the data. A recently developed correction technique, PLACE, can correct for absolute geometric distortion using the complex image data from two EPI images, with slightly shifted k-space trajectories. We present a correction approach that integrates PLACE into a real-time scan-plane update system by optical tracking, applied to a tissue-equivalent phantom undergoing complex motion and an fMRI finger tapping experiment with overt head motion to induce dynamic field non-uniformity. Experiments suggest that including volume by volume geometric distortion correction by PLACE can suppress dynamic geometric distortion artifacts in a phantom and in vivo and provide more robust activation maps.

Multiple-mouse MRI (MMMRI) accelerates biomedical research by imaging multiple mice simultaneously. To date, MMMRI has been explored in three ways: shielded transmit-receive coils, shielded transmits coil with separate unshielded receive coils; and finally shielded transmit-receive coils with independent gradient coils. However alternative transmit coil configurations and possible benefits of eliminating shielding have not yet been explored. The goal of this thesis is to test possible radiofrequency configurations with and without shielding for the purpose of improving image quality for MMMRI. Results demonstrate that using an unshielded transmit-receive coil array provided a 20% improvement over an identical shielded coil. A new unshielded 7-coil MMMRI array is presented, minimizing the ghosting between image overlap using mutual inductance minimization and a sensitivity encoding (SENSE) reconstruction. The final array provided high resolution images (90µm) of up to seven live mice simultaneously with appropriate signal-to-noise for automated analysis.

The role of antioxidants in cancer has been controversial for a long time. Although the public’s belief is that antioxidants prevent and/or inhibit cancer, there is increasing evidence to suggest the opposite: that cancer cells require antioxidants to survive. We wanted to interrogate the role of antioxidants in cancer by investigating both upstream regulators and downstream effectors of antioxidant signaling. We have identified protein tyrosine phosphatase non-receptor type 12 (PTPN12) as a novel regulator of antioxidant signaling in cancer. PTPN12 reduces reactive oxygen species (ROS) levels by promoting activity of the forkhead box O (FOXO) family of antioxidant transcription factors. We have also elucidated the impact of glutathione (GSH), the most abundant antioxidant in the cell, on tumorigenesis. We have found that GSH is required for cancer initiation, yet dispensable once transformation has occurred due to compensation provided by the thioredoxin (TXN) antioxidant pathway. Together, these studies expand our knowledge of the role of antioxidants in cancer and provide numerous avenues of research for the future.

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.