Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 98-102).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 90-103).

The new field of protein-based nano-technology takes advantage of the complex interactions between proteins to form unique structures with properties that cannot be achieved with traditional components. Microtubules (MTs), self assembled proteinaceous hollow filaments, offer promise in the development of MT-based nano-systems. The compelling need for the controlled assembly of 3D MT arrays is the fundamental motivation for the first part of this research. We report on the morphology of MTs grown in a crowded environment in the form of high viscosity fluids containing agarose and a novel process that enables the assembly of MTs supported by gel-based 3D scaffolds. Our research on MTs and their interaction with other molecules lead us to discover extraordinary spherulitic structures that changed the course of the project. The novel subject situate us into a complicated dilemma that question the nature of MT asters reported in experiments carried out in cells. The second part of this research is focused in the crystallization ofTaxol, a MT stabilizing molecule used as anti-cancer drug. It was confirmed via fluorescent and differential interference contrast microscopy that Taxol crystals can be decorated with fluorescent proteins and fluorochromes without perturbing their morphology. We used theoretical calculations to further investigate Taxol-fluorescent agent interactions. Furthermore, the crystallization of Taxol was studied in pure water, aqueous solutions containing tubulin proteins and tubulin-containing agarose gels. We demonstrated that tubulin is able to heterogeneously nucleate Taxol spherulites. To explain the formation of tubulin-Taxol nuclei a new, secondary Taxol-binding site within the tubulin heterodimer is suggested. Results presented in this work are important for in vivo and in vitro microtubule studies due to the possibility of mistaking these Taxol spherulites for microtubule asters. Thus, we are confirming the need for careful interpretation of fluorescence microscopy observations of MT structures when large concentrations of Taxol are used as stabilizing agent in cells.

Growth anisotropy, in which cells grow predominantly in one direction, is common in plant cells, and an essential event for plant form and function. The direction and degree of growth anisotropy are governed by the mechanical properties of the primary cell wall. When aligned in a parallel manner, cellulose microfibrils accommodate great resistance in the direction of their alignment to expansion driven by isotropic turgor pressure. Using the Arabidopsis thaliana inflorescence stem as a model system, field emission scanning electron microscopy (FESEM) analysis demonstrated that the establishment of parallel arrangement of microfibrils is closely correlated with anisotropic cell expansion. In the novel anisotropy 1 (any1) mutant allele of the primary cellulose synthase CesA1, growth defects were correlated with random cellulose microfibril patterns in some inflorescence stem tissues. Microtubules have been considered to be the most likely candidates for controlling the orientation of cellulose microfibrils. Recent studies have indeed demonstrated a close association of the plasma membrane-localized cellulose-synthase-complexes (CSCs) that produce cellulose and cortical microtubules. Despite this close association, microtubule disruption did not cause cellulose microfibrils to lose parallel alignment in the radial and inner periclinal walls of cells in the inflorescence stem, suggesting that microtubules influence mechanical properties of cellulose microfibrils other than orientation. X-ray diffraction analysis demonstrated that cellulose crystallinity in wild-type plants declines at the growth-promoting temperature of 29°C, whereas crystallinity fails to adapt and remains high in mor1-1, the temperature-sensitive mutant whose microtubule arrays become disorganized at its restrictive temperature (29°C). This finding suggests that organized microtubules are involved in reducing cellulose crystallinity that normally accompanies increased cell expansion. Live-cell imaging of CSCs by tracking a yellow fluorescent protein (YFP)-tagged CesA6 subunit in hypocotyl cells demonstrated that dynamic and well-organized microtubules affect the velocity, the direction of movement, and the density of CSCs, suggesting that there is a close relationship between microtubules and CSCs. Together with the finding that microtubules also control the distribution of COBRA, a GPI-anchored wall protein that is essential for growth anisotropy, I discuss the variety of roles microtubules play in anisotropic growth.

The cytoskeleton plays an important role in trafficking proteins and other macromolecular moieties throughout the cell. Viruses have been thought to depend heavily on the cytoskeleton for their replication cycles. However, studies, including one in our lab, found that some viruses are not inhibited by anti-microtubule drugs. This study was undertaken to evaluate the replication of viruses from several families in the presence of cytoskeleton-inhibiting drugs and to examine the intracellular localization of the proteins of one of these viruses, Sindbis virus, to test the hypothesis that alternate pathways are used if the cytoskeleton is inhibited. We found that Sindbis virus (Togaviridae, positive-strand RNA), vesicular stomatitis virus (Rhabdoviridae, negative-strand RNA), and Herpes simplex virus 1 (Herpesviridae, DNA virus) were not inhibited by these drugs, contrary to expectation. Differences in the localization of the Sindbis virus were observed, suggesting the existence of alternate pathways for intracellular transport.

Bacteria are implicated in endodontic infections. They not only infect the root canal lumen but also invade the dentinal tubules where they may remain untouched by contemporary chemomechanical preparation during root canal therapy. The contentious issue is whether the bacteria within these tubules contribute to secondary infections. Many studies have shown that clinicians fail to completely eradicate them during root canal therapy. At present there are no techniques available to detect the effectiveness of the current chemomechanical treatment regime within dentinal tubules. It is difficult to detect bacteria within the dentinal tubules. Culturing techniques have been used routinely as they are versatile and easy to use. However, they are unable to show the distribution of the bacteria within the dentinal tubules. Scanning electron microscopy, on the other hand, shows detailed surface structure in association with bacteria. Histological examination of root dentine specimens under the light microscope also shows the distribution of bacteria within the specimen but not viability. The dilemma posed by these existing techniques is that the results offer limited information; either demonstrating bacterial viability or bacterial distribution within specimens. No techniques able to show both the viability and the distribution of bacteria within the dentinal tubules have been reported to date. Fluorescent stains, in particular SYTO�9 and propidium iodide (LIVE/DEAD� Baclight[TM] viability kit, Molecular Probes Inc., Eugene, Oregon), have made it possible not only to stain bacteria but to differentiate live and dead bacteria. The combination of these two stains has yet to be applied to dental hard tissue in situ and they provide the basis for this investigation. The aim of this study was to evaluate the potential of the LIVE/DEAD� Baclight[TM] stains in conjuction with confocal laser scanning microscopy in the development of a technique to evaluate the viability and distribution of bacteria within dentinal tubules. This was extended to demonstrate the application of this technique by examining three different means of root canal disinfection both qualitatively and quantitatively. An important aspect of this study was to maintain bacterial viability, as well as to get maximum bacterial invasion into dentinal tubules. Results indicated that when the root canals were instrumented with Protaper� files and then irrigated with sodium hypochlorite (NaOCl) and ethylene diaminetetraacetic acid with cetrimide (EDTAC), there was more bacterial invasion into the dentinal tubules than when the root canals were only irrigated with NaOCl and EDTAC. Daily replenishments of nutrients resulted in deeper bacterial invasion into the dentinal tubules. Bacteria colonized the dentinal tubules up to a distance of 594 � 133 [mu]m from the canal. In the untreated tubules, 96 � 4 % of bacteria remained viable (green-fluorescent), whereas the Amoxicillin-treated tubules contained 94 � 6 % dead (red-fluorescent) bacteria. The calcium hydroxide-treated tubules resulted in 92 � 7 % bacterial death while the laser-treated tubules contained 81 � 12 % dead cells, frequently displaying an inner zone of dead cells surrounded by an outer zone of viable cells. The application of the fluorescent stains combined with confocal microscopy offers a new method for assessing the in vitro efficacy of root canal disinfection regimens.

Thesis (Ph.D.)--Murdoch University, 2002. / Thesis submitted to the Division of Veterinary and Biomedical Sciences. Bibliography: leaves 237-283.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2008. / Includes bibliographical references (leaves 107-123). Also available in electronic version.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

Thesis (Ph. D.)--Lehigh University, 1996. / Includes vita. Includes bibliographical references (leaves 78-90).