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>Magister Scientiae - MSc / Life is an emergent property of a complex network of interacting cellular-machines. Three-dimensional (3D), cellular structure captured at supra-atomic resolution has the potential to revolutionise our understanding of the interactions, dynamics and structure of these machines: proteins, organelles and other cellular constituents, in their normal functional states. Techniques, capable of acquiring 3D cellular structure at sufficient resolution to enable identification and interpretation of individual macromolecules in the cellular milieu, have the potential to provide this data. Advances in cryo-preservation, preparation and metal-coating techniques allow images of the surfaces of in situ macromolecules to be obtained in a life-like state by field emission scanning – and transmission electron microscopy (FE/SEM, FE/TEM) at a resolution of 2-4 nm. A large body of macromolecular structural information has been obtained using these techniques, but while the images produced provide a qualitative impression of three-dimensionality, computational methods are required to extract quantitative 3D structure. In order to test the feasibility of applying various photogrammetric and tomographic algorithms to micrographs of well-preserved metal-coated biological surfaces, several algorithms were attempted on a variety of FE/SEM and TEM micrographs. A stereoscopic algorithm was implemented and applied to FESEM stereo images of the nuclear pore basket, resulting in a high quality digital elevation map. A SEM rotation series of an object of complicated topology (ant) was reconstructed volumetrically by silhouette-intersection. Finally, the iterative helical real-space reconstruction technique as applied to cryo-TEM micrographs of unidirectionally heavy-metal shadowed. These preliminary results confirm that 3D information obtained from multiple TEM or SEM surface images could be applied to the problem of 3D macromolecular imaging in the cellular context. However, each of the various methods described here comes with peculiar topological, resolution and geometrical limitations, some of which are inherent shortcomings of the methodologies described; others might be overcome with improved algorithms. Combined with carefully designed surface experiments, some of the methods investigated here could provide novel insights and extend current surface-imaging studies. Docking of atomic resolution structures into low-resolution maps derived from surface imaging experiments is a particularly exciting prospect.

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Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Thesis (M.S.)--University of Missouri-Columbia, 2004. / Typescript. Includes bibliographical references (leaves 99-102). Also available on the Internet.

Thesis (M.S.)--University of Missouri-Columbia, 2004. / Typescript. Includes bibliographical references (leaves 99-102). Also available on the Internet.

Epidemiological studies highlight a direct association between the decline in respiratory health of the human population and increased environmental ultrafine particulate (UFP) exposure. This evidence, coupled with research identifying shared characteristics and toxicity between UFP and engineered nanomaterial (ENM), suggests that increased levels of ENM associated with the nanotechnology revolution could have a detrimental effect on human health. Although the link between respiratory disease and air pollution is well-established, toxicological data for ENM is limited. Current methods for the assessment of particle toxicity utilise a combination of both in vitro assays and in vivo animal testing. In some cases, these conventional assays provide unreliable results on account of nanoparticle interference. In this thesis, assays were undertaken to more fully understand the impact of a panel of ENMs on alveolar epithelial cell function and survival, as well as to assess the potential value of an alternative method for nanotoxicological screening. Eicosanoid profiling was used to assess both toxicity and inflammatory markers associated with a panel of ENMs, this technique is novel for the use in testing of ENM and the results show it has potential to be introduced/applied as an effective tool to predict a broad spectrum of detrimental effects of ENM in lung function. Submerged A549 cells, were used as a model of lung epithelial cells throughout. The secondary cell line is commonly used in in vitro research to examine the effect of toxins on respiratory health, specifically the alveolar region. A panel of ENM (SiO2, TiO2, NiO, ZnO and CuO) were selected to span from the benign to the highly toxic. ENM prepared in suspension were applied to the cells at 100cm2/mL for 24 h. This doctoral thesis focused on addressing the following aims: 1. To assess whether metallic ENM of differing chemical composition damage the cell membrane and/or mitochondria. 2. To determine whether ENM induce mitochondrial dysfunction through delivery or over-production of harmful reactive oxygen species (ROS) and, if so, to determine whether mitochondrial dysfunction results in activation of apoptosis. 3. To ascertain whether ENM alter the release of lipid inflammatory mediators using eicosanoid profiling. Mitochondrial function and membrane integrity assays revealed that CuO and ZnO induced mitochondrial dysfunction (~ 100% reduction in mitochondrial function), and promoted cell death (85 ± 7.5% cell lysis, ***P<0.001), respectively, when compared to control. In addition, superoxide production was increased by TiO2 alone (~ 100% increase, 0.0394 ± 0.0081 AU, **P<0.01), creating a discrepancy between assays. Analysis also revealed that metallic ENMs, specifically ZnO and CuO, significantly increased the production of prostaglandin E2 (~ 50%, 828 ± 119pg/sample, **P<0.01) and ~ 100%, 1439 ± 248pg/sample, ***P< 0.001), a pro-inflammatory eicosanoid, and elevated generation of a range of hydroxy-eicosatetraenoic acids (HETEs), suggesting induction of lipid peroxidation by these oxide derived ENMs. In conclusion, through the use of in vitro assays and eicosanoid analysis it was determined that ZnO and CuO ENM induce cell damage and death. However, although traditional in vitro assays are able to identify highly toxic ENM from the rest, they lack the ability to identify more subtle changes and, in some cases, are unreliable. By contrast, eicosanoid profiling has the ability to provide more detailed information regarding generation of both pro- and anti-inflammatory mediators, as well as oxidative stress, whilst avoiding the issues that are encountered through the use of current in vitro tests.

Call number: LD2668 .T4 1978 M63 / Master of Science