This dissertation presents an investigation into the structure of macromolecular protein assemblies by mass spectrometry. The experiments described within are designed to systematically assess the analytical utility of surface-induced dissociation (SID) tandem mass spectrometry in the characterization of multi-subunit protein complexes. This is accomplished by studying the effects of ion-surface collision on the fragmentation products of protein assemblies that vary by mass, number of subunits, and protein structural features. The dissociation energetics and mechanisms of protein complexes are considered by examining the influence of ion internal energy and sub-oligomeric protein structure on the dissociation process. Conditions are first established for the preservation of “native” protein quaternary structure and applied to previously characterized systems for proof-ofconcept. These conditions are subsequently extended to determine the molecular weight and subunit stoichiometry of several small heat shock proteins. Native mass spectrometry is then combined with limited proteolysis experiments to characterize the subunit interface of a unique small heat shock protein, Hsp18.5 from Arabidopsis thaliana, identifying regions of the protein essential for preservation of the native dimer. The dissociation of non-covalent protein assemblies is then explored on a quadrupole time-of-flight (Q-TOF) mass spectrometer, modified for the study of ion-surface collisions. This instrument allows ions to be dissociated through collisions with a surface or more conventional collisions with gas atoms. The dissociation of protein complexes is explored by both activation methods beginning with specific and non-specific dimers with masses less than 40 kDa. These studies are extended to larger assemblies with as many as 14 subunits weighing over 800 kDa, and are applied to both homo- and hetero-oligomeric protein complexes. Activation of a protein complex with “n” subunits through multiple collisions with inert gas atoms results in asymmetric dissociation into a highly charged monomer and complementary (n-1)-mer regardless of protein size or subunit architecture. This process is known to occur through an unfolding of the ejected subunit, and limits the amount of structural insight that can be gleaned from such studies. Collision at a surface however, results in more charge and mass symmetric fragmentation, and in some instances reflects the substructure of the protein assembly under investigation. The differences observed between the CID and SID of protein complexes is attributed to the rapid deposition of large amounts of internal energy deposited upon collision at a more massive target such as a surface. The ion activation time-frame and energy transfer efficiency are proposed to induce dissociation on a time-scale that precedes subunit unfolding providing access to dissociation pathways that are inaccessible by traditional means of activation. The systems studied here represent the largest ions fragmented via surface collisions within a mass spectrometer, and the fragmentation products observed by SID demonstrate its promise for expanding the role of mass spectrometry in the field of structural biology.

The electric charge distribution of molecules such as H₂ and D₂ is inversion-symmetric so that permanent dipole moments do not exist: such molecules are infrared-inactive. It is therefore interesting that gaseous, liquid, and solid hydrogen and its isotopes actually absorb infrared radiation, for example if gas densities are sufficiently high. The observed absorption arises from electric dipole moments induced by intermolecular interactions. It is of a supermolecular origin, due to binary (or higher-order) molecular complexes that may be transient (i.e., in a collisional encounter) or relatively stable (van-der-Waals molecule). Interaction-induced electric dipoles arise from the same mechanisms that generate the intermolecular forces: exchange forces, dispersion forces, and multipolar induction. Recently the induced dipole and potential energy surfaces of H₂ pairs have been obtained by advanced quantum-chemical calculations. Interaction-induced absorption, more commonly called collision-induced absorption (CIA), by H₂ pairs is an important opacity source in the atmospheres of various types of planets and cool stars, such as late stars, low-mass stars, brown dwarfs, certain white dwarfs, etc., and therefore of special astronomical interest. The emission spectra of cool white dwarf stars differ significantly from the expected blackbody spectra of their cores, mainly due to collision-induced absorption by collisional complexes of hydrogen and helium in the stellar atmospheres. Before proceeding to the frequencies and temperatures of interest it is good to check the new potential energy surface and induced dipole surface in all possible ways by comparison with existing isotopic laboratory measurements. Furthermore, the new potential energy surface is directly compared with previously available, well established intermolecular potential energy surfaces. The electric charge distributions of deuterium and hydrogen are very similar. The new potential energy and induced dipole surfaces were originally obtained to facilitate the computation of the collision-induced absorption of hydrogen. However, by replacing the rotovibrational wavefunctions of H₂ with those of D₂ the surfaces can also be used to calculate the collision-induced absorption of deuterium pairs, thereby probing them further. At the temperature of 298K existing measurements of the collision-induced absorption of D₂--D₂ gas are compared with our quantum scattering calculations in the D₂ fundamental band (approximately 2,500cm⁻¹ to 4,500cm⁻¹). Furthermore, measurements of the collision-induced absorption of deuterium (D₂) in the D₂ first overtone band (about 5,250cm⁻¹ to 7,250cm⁻¹) at 201K are reported. These measurements are compared with ab initio calculations of the absorption spectra. Close agreement of measured and calculated spectra is seen.

Thesis (M.S.)--University of Michigan, 1991.

Thesis (M.S.)--University of Michigan, 1995.

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 2001. / Typescript. Bibliography: leaves 82-94. Also available online.

Thesis (M.S.)--University of Michigan, 1995.

Thesis (M.S.)--University of Michigan, 1991.

Two murine models of acute inflammation, namely thioglycollate-induced peritonitis and carrageenan-induced paw edema, were optimized using non-steroidal anti-inflammatory drug indomethacin and corticosteroid dexamethasone. During the optimization phase, the presence of neutrophils, monocytes, macrophages, eosinophils, B cells and T cells in the peritoneal cavity at various time points after injection of thioglycollate medium was assessed via multicolor flow cytometry. Moreover, two different thioglycollate media (suppliers BD and Sigma-Aldrich) were compared for their ability to induce an inflammatory response. The optimization of thioglycollate-induced peritonitis and carrageenan-induced paw edema was followed by the evaluation of the anti-inflammatory activity of Ixodes ricinus cystatins G1 and G9 in both mouse models.

Humans are known as homeothermic endotherms. To ensure thermic balance at rest when exposed to cold, they dispose of two main thermogenic processes: shivering thermogenesis (ST) and non-shivering thermogenesis (NST). ST consists of involuntary muscle contractions and NST represents the component of Hprod that is not ST. While ST is difficult to tolerate, it is not yet known which nutrients and how much are required to stimulate NST and lower ST in the cold. Whether or not food caloric intake has an impact on the relative contribution of ST and NST to total Hprod remains to be determined. Therefore, the purpose of this thesis was 1) to quantify the effects of ingesting two quantities (1507 vs 3015 kJ) of same relative compositions on cold-induced whole-body Hprod and 2) to establish the effects of these two quantities of food on the relative contribution of ST and NST to total Hprod. Five healthy male participants were exposed to a 3h mild cold, using a liquid conditioned suit with water flowing at 15°C (COLD) or 33 °C (CON) for a total of 4 trials. Thermal, metabolic and shivering responses were measured at baseline, before and after shake ingestion. Results demonstrated that Hprod and ST intensity increased in the cold, while no significant differences were found between the ingested shakes at two different caloric equivalents. In addition, ST intensity did not change, which confirmed that NST remained the same between the two conditions. Thus, knowing that the caloric intake will not maximize the thermogenic effects in the cold (i.e. improve the comfort of the individual), is it more advantageous to bring food or additional clothing, for any activity? Clearly, more research on the exact pathways of each processes in the cold with food consumption needs to be made. To that extent, the investigation of the effect of food quality on changes in the thermogenic processes during cold exposure strikes us as a fascinating area for future research.

Noise pollution threatens marine ecosystems, where animals rely heavily on sound for navigation and communication. The largest source of underwater noise from human activity is shipping, and propeller-induced cavitation is the dominant source of noise from ships. Mitigation strategies require accurate methods for predicting cavitation-induced noise, which remains challenging. The present thesis explores prediction and modelling strategies for cavitation-induced noise from marine propellers, and provides insight into models that can be used both during propeller design and to generate intelligent vessel control strategies. I examined three distinct approaches to predicting cavitation-induced noise, each of which is discussed in one of the three main chapters of this thesis: a high-fidelity computational fluid dynamics scheme, a parametric mapping procedure, and the use of field measurements. Each of these three chapters presents different insight into the acoustic behaviour of cavitating marine propellers, as well both real and potential strategies for mitigating this critical environmental emission. A combined experimental and numerical study of noise from a cavitating propeller, focused on both the fundamental importance of experimental findings and the effectiveness of the numerical modelling strategy used, is detailed in the first main chapter of this thesis. The experimental results highlighted that loud cavitation noise is not necessarily associated with high-power or high-speed propeller operation, affirming the need for intelligent vessel operation strategies to mitigate underwater noise pollution. Comparison of the experimental measurements and simulations revealed that the simulation strategy resulted in an over-prediction of sound levels from cavitation. Analysis of the numerical results and experiments strongly suggested that the cavitation model implemented in the simulations, a model commonly used for marine propeller simulations, was responsible for the over-prediction of sound levels. Ships are powered primarily by combustion engines, for which it is possible to generate "maps" relating the emission of pollutants to the engine’s speed and torque; the second main chapter of this thesis presents the methodology I developed for generating similar "maps" relating the level of cavitation-induced noise to the speed and torque of a ship's propeller. A proof-of-concept of the method that used the model propeller from the first main chapter is presented. To generate the maps, I used a low-order simulation technique to predict the cavitation induced by the propeller at a range of different speed and torque combinations. A pair of semi-empirical models found in the literature were combined to provide the framework for predicting noise based on cavitation patterns. The proof-of-concept map shows a clear optimal operating regime for the propeller. The final main chapter of this thesis presents an analysis of field noise measurements of coastal ferries in commercial operation, the data for which were provided by an industrial partner. The key finding was the identification of cavitation regime changes with variation in vessel speed by their acoustic signatures. The results provide a basis for remotely determining which vessels produce less noise pollution when subject to speed limits, which have been implement in critical marine habitats, and which vessels produce less noise at a specific optimum speed. / Graduate