Magister Pharmaceuticae - MPharm / The aim of this study was to investigate the mechanism of fenfluramine-induced pulmonary phospholipidosis, by comparing the profile and levels of induced phospholipids in the rat and the mode of phospholipase inactivation, both relative to that produced by chlorphentermine. Wistar and BD9 rats were injected with fenfluramine (FF) and chlorphentermine (CP) intra-peritoneally daily over a six week period to induce phospholipidosis. The lungs isolated from such treated and untreated animals, were grouped into unlavaged lungs and lungs to be lavaged and from the latter group the alveolar macrophages were isolated. Small sections of the unlavaged lungs were microscopically examined to verify the induction of phospholipidosis. Further the levels of phosphatidyl choline (PC), spingomyelin (SPM), phosphatidyl ethanolamine (PE), phosphatidyl glycerol (PG), phosphatidyl inositol (PI), phosphatidyl serine (PS) and phosphatidic acid (PA) were determined in both groups of lungs using a TLC method. To assess whether the drug-mediated inactivation of the phospholipases (PL) occurred via direct inhibition of the enzymes or via the drug-phospholipid complex, the hydrolysis of the above phospholipids by PL-A or PL-C were monitored using colorimetric methods. The feasibility of the phospholipid-drug complex-mediated mechanism was further explored, by assessing the effect the two drugs had on the phase transition temperature of the phospholipids. Electron microscopy revealed the presence of hypertrophied and elevated counts of alveolar macrophages in the treated-Wistar and -BD9 rats. In the FF- and CP treated Wistar and BD9 rats there were, compared to the saline-treated rats, a 200 % and 235 % increase in macrophage counts, respectively, for the FF-treated rats and a 700 % and 965 % increase in macrophage counts, respectively, for the CP treated rats. The levels of all the phospholipids in the unlavaged lungs of both rat strains were elevated, except that for PG, PS and PA. In both rat strains following the treatment with both drugs the PG levels were not elevated and the PS levels were not elevated following CP treatment. Following the treatment for both drugs, the PA levels were also not elevated in the BD9 rats. Relative to the levels found in the unlavaged lungs of the control rats, the increases ranged from a minimum of 9 to a maximum of 216 %. In general, Wistar rats appeared to be more susceptible to both FF and CP treatment. In both rat strains, lavaging of the lungs considerably reduced the levels of phospholipids remaining in the lung and the differences between the treated and untreated animals became less striking. The addition of FF or CP, whether directly to the enzyme, or in the form of the drug phospholipid complex, resulted in significant decreases in the PL-A-mediated or PL-C-mediated hydrolysis of virtualy all the test phospholipids. The average decrease ranged from 0.811 to 4.04 ,.,.FFAbbb ,.,.1-1sample min-I, for the PL-A activity and 0.023 to 0.827 ,.,.gIp'CC100 ,.,.1-1 sample min-I, for the PL-C activity. In the case of FF, the inhibition of PL-A activity could not be ascribed exclusively to either direct inhibition of the enzyme or reduced susceptibility of the phospholipid substrate-drug complex. The PL-C activity appeared to be inhibited to a greater extent via the phospholipid substrate-drug complex rather than by direct inhibition. On the other hand, CP induced a small, but significantly greater degree of inhibition of PL-A activity, more via direct inhibition, rather than by the phospholipid substrate-drug complex. The PL-C activity appeared to be inhibited to a greater extent via phospholipid substrate-drug complexation than by direct inhibition. From the above data, considered collectively, it was not possible to declare either of the two possible mechanisms as the more likely one for FF or CP-induced inhibition of the phospholipases. The feasibility of the indirect mode was further explored, by determining the phase transition temperatures for the phospholipid-drug complexes of each drug. The addition of each drug caused a depression of the phase transition temperature of all the phospholipids with a .1T'dd ranging from 0.52 to 15.73 °C. This appears to support the notion that both drugs bind to the phospholipids and the differences in the extent of the phase transition temperature depression of the individual phospholipids may indicate differences in the binding capacities of these drugs. The following major conclusions may be drawn from the results of this investigation. Fenfluramine induces a phospholipidosis syndrome in the lungs of Wistar and BD9 rats that are histologically similar to that induced by CP. It induces the elevation of essentially the same phospholipids as CP, primarily in the alveolar spaces and macrophages, and by implication, most likely via similar mechanisms. For both FF and CP, both direct inhibition and phospholipid-drug complex-mediated inhibition of phospholipases were found to be a viable mechanism for this syndrome. The mechanism for FF-induced pulmonary phospholipidosis thus appears to be similar to that of CP; small quantitative differences in essentially similar mechanisms, may explain the differences in the levels of induced phospholipidosis found in this study.

Proton-Exchange Membrane Fuel Cells are an alternative source of electricity generation for automobiles and stationary power plants. With increasing concerns on environmental issues, recent research has focused on maximizing the efficiency and durability as well as minimizing the costs of fuel cells. One of the main areas of research is optimizing the structure of the cathode catalyst layer. The main driving force of this thesis was the effective visualization of nanostructure of the ionomer, which is responsible for proton conduction in the cathode catalyst layer. However, challenges regarding sample preparation and radiation damage still need to be well understood. Different sample preparation techniques of catalyst inks and catalyst coated membranes were used for Scanning and Transmission Electron Microscopy, such as freeze fracturing, ultramicrotomy and Focused Ion Beam. Comparisons of the microstructure and chemical differences of all components, especially the ionomer, prepared by ultramicrotomy and Focused Ion Beam, was done with Transmission Electron Microscopy and Scanning Transmission X-ray Microscopy applied to the same catalyst coated membrane sample. Detailed spectroscopic information regarding components in both specimens was compared with C 1s and F 1s near edge X-ray absorption spectra recorded in a Scanning Transmission X-ray Microscope. Focused Ion Beam causes extensive damage to the carbon support and ionomer but prepares thinner sections than ultramicrotomy. This work makes it possible to understand the limitations of each sample preparation and compositional analysis technique in order to later apply one of them to image the ionomer in the catalyst layer at the nanoscale, hopefully using tomography techniques. / Thesis / Master of Materials Science and Engineering (MMatSE)

Advances in micro/nano-fabrication, thin electron transparent materials, holder designs, and acquisition methods have made it possible to perform meaningful experiments using liquid electron microscopy (liquid EM). Liquid EM provides researchers with micro-to-nano scale tools to explore biomaterials in liquid environments capable of capturing dynamic in situ reactions, providing characterization means in mimetic conditions to the human body. However, these emerging techniques remain in their infancy; limited work presents best practice strategies, and several challenges remain for their effective implementation, particularly for beam-sensitive, soft biological materials. This thesis seeks to address these shortcomings by exploring strategies for liquid EM of biomaterials and real-time dynamic processes using two key methods: room temperature ionic liquid (RTIL) treatment for scanning EM (SEM) and liquid cell transmission EM (TEM). With these techniques, the research explores the characterization of hard-tissue systems relevant to bone and seeks to provide new methods of exploring structurally biological culprits behind diseases like COVID-19. Research in this thesis is presented by increasing complexity, touching on three themes: (i) exploring liquid EM for the first time using RTILs for SEM of biological samples notably bone (static, micro-scale), (ii) developing new methods for high-resolution liquid biological TEM of viruses (static, nano-scale), and (iii) applying novel liquid TEM to dynamic biomineralization systems (dynamic, nano-scale). After review articles serve as introductory material in Chapter 2, in Chapter 3, healthy and pathological bone was explored in hydrated conditions with liquid SEM using a new workflow involving RTIL treatment, demonstrated to be highly efficient for biological SEM. Moving to the nanoscale, Chapter 4 presents a commercial liquid TEM option and a new liquid TEM clipped enclosure developed for imaging biological specimens, specifically virus assemblies such as Rotavirus and SARS-CoV-2. Combined with automated acquisition tools and low-dose direct electron detection, enclosures resolved high-resolution structural features in the range of ~3.5 Å – 10 Å and were correlatively used for cryo TEM. Chapter 5 applies these liquid TEM methods to study collagen mineralization, revealing in high-resolution the presence of precursor calcium phosphate mineral phases, important transitional phases to mineral platelets found in mineralized tissues. But – dynamic reactions were not captured, attributed to confinement effects, lack of heating functionality, and cumulative beam damage experienced. Chapter 6 overcomes these challenges by optimizing collagen-liquid encapsulation within a commercial liquid TEM holder mimicking physiological conditions at 37°C. Dynamic nanoscale interactions were highlighted, where evidence of the coexistence of amorphous precursor phases involving polymer-induced liquid as well as particle attachment was presented within this model. Several liquid TEM challenges remain particularly beam sensitivity and distribution for biomaterials, providing many exciting avenues in future to explore. Taken together, this thesis is advancing characterization through the development and applied use of new liquid EM strategies for studying biomaterials and dynamic reactions. Insights on these reactions and structures anticipate leading to a better understanding of diseases and treatment pathways, the key to moving Canada’s health care system forward. / Thesis / Doctor of Philosophy (PhD) / In the electron microscopy (EM) community, there is a need for improved methodologies for high-resolution liquid imaging of biological materials and dynamic processes. Imaging biological structures and reactions in hydrated biomimetic environments improves our understanding of their true nature, thus providing better insight into how they behave in the human body. While liquid EM methods have surged in publications recently, the field is still in its infancy; limited works present best practice strategies, and several challenges remain for their effective implementation. To address these shortcomings, this thesis aims to strategically explore the improvement of liquid EM of biomaterials and real-time dynamic processes through two key methods: room temperature ionic liquid treatment for scanning EM and liquid cell transmission EM. Using these novel techniques, the research explores the characterization of hard-tissue systems relevant to bone and seeks to provide new means of exploring structurally biological culprits behind diseases like COVID-19.

Immunolocalization of proteins within the cell is a significant and powerful tool that improves understanding of cellular functions and processes, such as molecule secretion during immune responses. Immunogold electron microscopy (IEM) is an immunohistochemistry technique that uses gold-conjugated antibodies and electron microscopy (EM) to identify and localize antigens at the ultrastructural level. Here, we are trying to develop and optimize an IEM staining protocol that targets glomerular proteins of interest in Minimal Change Disease (MCD), and eliminates background staining, and preserves tissue morphology. Using this optimized protocol, we hope to learn more about the relationship between IgG and Nephrin in MCD. Kidney biopsies diagnosed with MCD, Membranous Nephropathy (MN), and Thin Basement Membrane Disease (TBMD) and previously embedded in paraffin blocks were retrieved from the tissue archive of the Renal Pathology Laboratory at Boston Medical Center. MN and TBMD were selected as positive controls for IgG and Nephrin staining protocols, respectively. Co-staining of IgG and Nephrin was performed after the protocols for each target were optimized. During protocol development, it was observed that section quality is significantly affected by the angle and sharpness of the knife, and the thickness of the section. Moreover, section quality highly impacted gold particle localization. Ultimately, co-staining of IgG and Nephrin was successful in MCD cases. However, further improvements are needed to optimize IgG and Nephrin staining, and in turn, our understanding of MCD.

The objective of the current study was to determine the efficacy of 3% acetic acid in reducing Salmonella in pork trimming and the effects of such treatment on meat quality. For 15-s dipping and 5-log CFU/pork cube inoculation, only 0.2- to 0.3-log reduction was observed (P ≤ 0.026). Acetic acid worked best at 75 s and 50°C, providing 1.4-log reduction (P < 0.001), damaging Salmonella cell membranes. When an inoculated pork cube was placed at the geometrical center of 2.3-kg pork trimming, dipping at 50°C for 75 s only reduced Salmonella by 0.2 log (P = 0.040). Although dipping slightly increased lightness (P < 0.001) and decreased redness (P ≤ 0.008) on the meat surface, no inside color change was detected (P = 0.120). Neither lipid oxidation (TBARS, P = 0.644), protein solubility (P = 0.187), nor water-holding capacity (P = 0.076) were affected by treatments.

Following the discovery of graphene, 2D nanostructures have been noted for their potential in a range of high-impact applications, such as sensing, catalysis, and composite reinforcement. Liquid-phase exfoliation and chemical vapour deposition have been demonstrated and indicate the feasibility of mass-scale production. With the advent of mass-produced 2D nanostructures a key focus of research is to characterise these materials. This thesis is concerned with imaging and structural properties of the 2D nanomaterials, hexagonal boron nitride (h-BN), molybdenum disulfide (MoS₂), tungsten disulfide (WS₂), titanium disulfide (TiS₂) and hexabenzocoronene (HBC), produced via liquid phase exfoliation. HBC strictly speaking is not 2D nanomaterial, however, it can be viewed as transition molecule from benzene to graphene. The data used for characterisation is based primarily on electron diffraction and, in particular, aberration corrected annular dark field (ADF) scanning transmission electron microscopy (STEM). The incoherent nature of ADF STEM provides direct atomic imaging without the contrast reversals upon focus changes seen in conventional high-resolution transmission electron microscopy (HRTEM). The main structural feature investigated in this thesis was the stacking sequences in few-layers h-BN, MoS₂, WS₂ and TiS₂. Simple stacking (AAA) can be distinguished from Bernal (ABA) and rhombohedral (ABC) on the basis of intensity ratio, I_{10̅10}/I_{11̅20} , in diffraction patterns and indirectly in HRTEM images. Nonetheless acquisition of the diffraction patterns suitable for analysis can be challenging due to the sample issues. Non-bulk stacking sequences were reliably confirmed for all above 2D nanomaterials on the basis of atomically resolved ADF STEM. 20 h-BN, 28 MoS₂, 5 WS₂ and 6 TiS₂ nanoflakes were imaged and analysed. Amongst them 2 h-BN, 9 MoS2, 4 WS2 and 1 TiS2 nanoflakes displayed non-bulk stacking. Hence, it appears that 2D WS2 has the greatest affinity for non-bulk stacking. Finally, an interesting structural transformation was observed in HBC molecules. Under the influence of electron beam HBC agglomerates were transformed into crystalline phase with 90^o symmetry.

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

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