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Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Mark F. Hamilton. Vita. Includes bibliographical references.

The osteoclast ruffled border is a highly convoluted, complex membrane that is necessary for bone resorption. It is thought to form following mass lysosomal fusion with the boneapposing plasma membrane and vesicular trafficking is vital for its formation and function. The aim of this PhD was to better understand the ultrastructure, formation and function of the ruffled border using TEM and advanced imaging techniques. Ruffled border reformation following calcitonin treatment was visualised and the stages of ruffled border formation were described. Ruffled borders in healthy and osteopetrotic osteoclasts were also imaged by TEM and characterised using a morphological grading system. The key findings of this thesis are as follows: (1) vacuoles, not lysosomes, are the primary contributors of membrane to the ruffled border and the membrane projections of the ruffled border form passively as a consequence of channel formation, not actively by membrane folding, (2) extracellular vesicles are located, and appear to be released, at the ruffled border. Various functional aspects of the ruffled border were also investigated. Vesicles near the ruffled border were identified and characterised by immunoelectron microscopy based on their content and morphology. We found no morphological defects in ruffled borders in mice deficient in Plekhm1. In osteoclasts derived from patients with a SNX10 mutation, we found that while the cells retained the capacity to form well-developed ruffled borders, they did so less often than healthy control osteoclasts. Importantly, we observed that even in a population of healthy osteoclasts, ruffled border morphology is highly heterogeneous because they are at different stages in the resorption cycle. In conclusion, the data in this thesis provide novel findings, previously unseen details regarding how resorbing osteoclasts interact with the bone surface, and have revealed unique insights into ruffled border morphology, formation and the vesicles with which it interacts.

Coded-aperture systems are indirect imaging systems that have been used to image x-ray and (gamma)-ray sources. Coded aperture systems are also capable of recording tomographic information and because they involve no detector motion they are natural candidates for use in dynamic studies in nuclear medicine. Computer simulations suggest that an orthogonal-view coded-aperture system, which circumvents the problem of limited angular view, is capable of restoring clinically useful tomographic information. The restoration is performed with the aid of the iterative back-projection algorithm which is shown to yield the Moore-Penrose generalized inverse in the limit of many iterations. The convergence behavior of this algorithm is also examined. In order to improve reconstructions, the problems of optimizing coded aperture design is addressed. The concept of "alignment" is introduced in which the aperture parameters are adjusted until the system is tuned to measure well the object class of interest. A mean-square error figure of merit is derived that indicates the degree of alignment of a system. Aperture design may then be seen as a multidimensional optimization problem in which system parameters are adjusted in order to find a global minimum value for the figure of merit. The figure of merit presumes the use of an optimum restoration filter in the reconstruction process. Various restoration algorithms are suggested which fulfill this requirement. Finally, simple proof-of-principle simulations are given that demonstrate a degree of plausibility to the alignment approach.

Lanthanide-doped upconversion nanoparticles (UCNPs) are perceived as promising novel near-infrared (NIR) bioimaging agents characterised by high contrast and high penetration depth. However, the interactions between charged UCNPs and mammalian cells have not been thoroughly studied and the corresponding intracellular uptake pathways remain unclear. Herein, my research work involved the use of hydrothermal method and ligand exchange approach to prepare UCNP-PVP, UCNP-PEI, and UCNP-PAA. These polymer-coated UCNPs demonstrated good water dispersibility, the similar size distribution as well as similar upconversion luminescence efficiency. However, the positively charged UCNP-PEI evinced greatly enhanced cellular uptake in comparison with its neutral or negative counterparts, as revealed by cellular uptake studies. Meanwhile, it was discovered that cationic UCNP-PEI could be effectively internalized mainly through the clathrin endocytic machanism. This study is the first report on the endocytic mechanism of positively charged lanthanide-doped UCNPs. Furthermore, it allows us to control the UCNP-cell interactions by tuning surface properties. Glioblastoma multiforme (GBM) is the most common and malignant form of primary brain tumors in humans. Small molecule MRI contrast agents are used for GBM diagnosis and preoperative tumor margin delineation. However, the conventional gadolinium-based contrast agents have several disadvantages, such as a relatively low T1 relaxivity, short circulation half lives and the absence of tumor targeting efficiency. Multimodality imaging probes provide a better solution to clearly delineate the localization of glioblastoma. My research work also involved the development of multimodal nanoprobes for targeted glioblastoma imaging. Two targeted paramagnetic/fluorescence nanoprobes were designed and synthesized, UCNP-Gd-RGD and AuNP-Dy680-Gd-RGD. UCNP-Gd-RGD was prepared through PEGylation, Gd3+DOTA conjugation and RGD labeling of PEI-coated UCNP-based nanoprobe core (UCNP-NH2). It adopted the cubic NaYF4 phase, had an average size of 36 nm by TEM, and possessed a relatively intense upconversion luminescence of Er3+ and Tm3+. It also exhibited improved colloidal stability and reduced cytotoxicity compared with UCNP-NH2, and a higher T1 relaxivity than Gd3+DOTA. AuNP-Dy680-Gd-RGD was synthesized through bioconjugation of amine-modified AuNP-based nanoprobe core (AuNPPEG- NH2) by a NIR dye (Dy680), Gd3+DOTA and RGD peptide. It demonstrated a size of 3–6 nm by TEM, relatively strong NIR fluorescence centered at 708 nm, longterm physiological stability, and an enhanced T1 relaxivity compared with Gd3+DOTA. Targeting abilities of both UCNP-Gd-RGD and AuNP-Dy680-Gd-RGD towards overexpressed integrin αvβ3 receptors on U87MG cell surface was confirmed by their enhanced cellular uptake visualized by confocal microscopy imaging and quantified by ICP-MS, where their corresponding control nanoprobes were used for comparison. Furthermore, targeted imaging capabilities of UCNP-Gd-RGD and AuNP-Dy680-Gd- RGD towards subcutaneous U87MG tumors were verified by in vivo and ex vivo upconversion fluorescence imaging studies and by in vivo and ex vivo NIR fluorescence imaging and in vivo MR imaging studies, respectively. These two synthesized targeted nanoprobes, with surface-bounded cyclic RGD peptide and numerous T1 contrast enhancing molecules, are applicable in targeted MR imaging glioblastoma and delineating the tumor boundary. In addition, UCNP-Gd-RGD favors the upconversion luminescence with NIR-to-visible nature, while AuNPDy680- Gd-RGD possesses NIR-to-NIR fluorescence, and both lead to their potential applications in fluorescence-guided surgical resection of gliomas. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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Thesis (Ph. D.)--Dept. of Dentistry, University of Adelaide, 1990. / Typescript (Photocopy). A stereo-viewer is provided ... to facilitate fusing of the three dimensional CT reconstructions and the stereo wire frame models--Pref. v. 2. Includes bibliographical references (leaves [244]-263).

Thesis (M.S.)--University of Nevada, Las Vegas, 1998. / Typescript (photocopy). Vita. Abstract. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 53-54).