Dynamical heterogeneity (DH) has been observed in many systems approaching the glass or jamming transition. Whether DH has a structural origin is under heated debate. To provide a deeper understanding, in this thesis I investigate the microscopic dynamics in weakly attractive colloidal systems by confocal fluorescence microscopy. The van Hove density-density correlation function is applied to our systems. Separable fast and slow populations emerge in the self part (svH), while the distinct part shows a strong signature of DH close to the gel transition. At intermediate time, svH shows a purely exponential tail, mainly arising from the fast population. I show that this broad tail is a direct consequence of the occurrence of rare large jumps that are statistically distributed. The slow population tends to form a space-spanning backbone, and its mean squared displacement close to the gel transition exhibits a plateau, whose height is consistent with the range of attraction, suggesting a bonding mechanism for the dynamical arrest. I further examine various quantities characterizing local structure and local dynamics and a strong correlation is identified between them. Subsequently, I develop order parameters for quantifying amorphous structure and apply them to our systems. I find that attractive colloidal systems exhibit higher order under higher attraction tension, while hard spheres become more ordered under higher compression. Finally, I investigate the effect of the range of attraction on the structure and dynamics of attractive colloidal systems. I observe that the system with shorter range of attraction forms a denser and more heterogeneous structure. Meanwhile, I observe an even stronger dynamical heterogeneity. These observations provide further evidence of a connection between structural heterogeneity and dynamical heterogeneity in these systems, providing guidance for a theoretical description of the dynamical arrest as well as the relaxation mechanisms upon gelation and its relation to solidification in glasses. / In order to do all of this, I first implemented full 3D subpixel resolution localization of particles and improved particle tracking algorithms tailored for the sorts of heterogenous dynamics these systems exhibit, that otherwise confounds existing methods such that the very relaxation mechanisms would be missed. This allows us to obtain unprecedented precision in positions of all of the particles and complete tracking, both of which are essential for correctly determining system properties that depend on measured particle dynamics.

In eukaryotes, the ubiquitin-proteasome system (UPS) degrades most cellular protein. Inhibition of the UPS has been associated with different disease states and can affect various intracellular processes including the activation of heat shock protein (hsp) gene expression. During cellular stress, HSPs act as molecular chaperones by inhibiting protein aggregation and assisting in their refolding once normal conditions are re-established. In the present study, Withaferin A (WA), a steroidal lactone with possible anti-inflammatory and antitumor properties, was found to inhibit proteasome activity and induce the expression of hsp genes in the amphibian model system, Xenopus laevis. Treatment of Xenopus kidney epithelial A6 cells with WA produced an increase in the accumulation of ubiquitinated protein and a significant decrease in chymotrypsin-like activity. Furthermore, immunoblot analysis revealed that WA induced HSP30 and HSP70 accumulation. For example, cells treated with 5 μM WA for 18 h resulted in the optimal accumulation of HSP30 and HSP70. Northern blot analysis revealed that exposure of cells to 5 μM WA induced hsp30 and hsp70 mRNA accumulation in a time-dependent manner up to 12 h. The activation of heat shock factor 1 (HSF1) DNA-binding may be involved in WA-induced hsp gene expression in A6 cells, since pretreatment with the HSF1 inhibitor, KNK437, reduced the accumulation of HSP30 and HSP70. Also, WA acted synergistically with mild heat shock to enhance HSP accumulation to a greater extent than the sum of both stressors individually. In cells recovering from WA, the relative levels of HSP30 and HSP70 accumulation remained elevated from 6 to 12 h after removal of WA. Immuocytochemical analysis and laser scanning confocal microscopy revealed that WA-induced HSP30 accumulation occurred primarily in the cytoplasm with some staining in the nucleus in a granular or punctate pattern. Prolonged exposure to WA resulted in some disorganization of the actin cytoskeleton as well as large cytoplasmic HSP30 staining structures in some cells. Prior exposure of cells to WA treatment conferred thermotolerance since it protected them against a subsequent thermal challenge at 37 °C. In conclusion, this study has shown that WA can induce an inhibition of proteasome activity and an increase hsp gene expression. Activating the heat shock response is a potential avenue for novel drug therapies, which can confer cytoprotection in disease states involving cytotoxic protein aggregation.

Monothiolate protected gold nanoclusters with near IR luminescence underwent a five-to-ten fold enhancement of quantum efficiency by heating in the presence of excess thiols. Two monothiolate nanoclusters, mercaptosuccinic acid and tiopronin, were shown to benefit from this procedure. Emission maximum around 700-900 nm is favorable for bioimaging applications due to reduction of background signal from autofluorescence. Dithiolate lipoic acid protected gold nanoclusters with higher near IR quantum efficiency present an interesting candidate for biological imaging due to the difference in hydrophobicity, resistance to quenching by divalent cations and cell growth media, and retained quantum efficiency when coupled to agents such as polyethylene glycol. Intracellular and nuclear internalization of mercaptosuccinic gold nanoclusters demonstrate a potential vector for delivery of nuclear targeting agents. The small size, chemical stability, high luminescence, and potential for targeting various intracellular domains make gold nanoclusters worthwhile for further studies as potential bioimaging probes.

The aim of this study was to investigate in vitro transdermal diffusion of a small peptide namely arginine vasopressin (AVP) with the aid of the novel PheroidTM drug delivery system. Generally, peptides seem unfit for transdermal permeation, but it was thought prudent to explore the suitability of this lipid-based system after success was achieved with entrapment of tuberculostatics, bacteria and viruses. Bestatin (a selective aminopeptidase inhibitor) was employed to circumvent any skin-related degradation of the active. Therefore, the effect of bestatin on the preservation of AVP during diffusion was investigated. Vertical Franz cell diffusion studies were conducted with female abdominal skin, with AVP at a concentration of 150 pglml in the donor phase and Hepes buffer as the receptor phase over a twelve-hour period. To prove entrapment of AVP within the lipid structures of the PheroidsTM, fluorescentlylabelled samples were monitored by means of confocal laser scanning microscopy (CLSM), which revealed definite entrapment. In vitro permeation profiles for AVP exhibited a biphasic character, with the majority of permeation occurring during the first two hours. The PheroidTM delivery system proved to be advantageous when applied as delivery medium. The inclusion of bestatin has an enhancing effect on permeation probably due to its protection of AVP. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

The object of this thesis is to present two novel applications of Spatiotemporal Image Correlation Spectroscopy (STICS) to biological systems. STICS is a technique which uses the correlations in pixel intensity fluctuations of an image time series, captured under fluorescence microscopy, to measure the speed and direction of a flowing population of fluorescently labeled molecules. The method was first applied to measure the dynamics of transport vesicles inside growing pollen tubes of lily flowers. The measured vector maps allowed to confirm the presence of actin filaments along the periphery of the tubes, as well as the presence of a reverse-fountain pattern in the apical region. In a second set of experiments, STICS was used to measure the retrograde flow of filamentous actin in migrating chick DRG neuronal growth cones. These results serve as proof of principle that STICS can be used to probe the response of the growth cone cytoskeleton to external chemical cues.

During inflammatory responses, macrophages make and secrete cytokines, including the proinflammatory cytokine TNF-alpha (TNF). TNF is a highly potent activator of immune responses with pleiomorphic effects throughout the body. TNF is a key causative agent of chronic inflammatory diseases and is of an intense clinical interest as a therapeutic target. At the outset of this thesis, little was known about how macrophages secrete TNF. Notably, the pathways, carriers and molecules that regulate TNF secretion had not been characterised. A main goal of this work was to identify compartment and molecules involved in the intracellular trafficking of TNF. Live cell imaging of GFP-TNF was established and this provided novel and important new insights into trafficking. Both endogenous and GFP-tagged TNF were followed in macrophages using fluorescence microscopy. The trafficking of other molecules in macrophages was also studied. The major findings of this work include the identification of a new two-step secretory pathway for TNF and other proteins from the trans-Golgi Network (TGN) to the cell surface. This pathway goes via the recycling endosome as an intermediate station. Pleiotrophic tubular-vesicular carriers containing TNF bud off the TGN for the post-Golgi trafficking of TNF and their characterization both in live cell imaging and in biochemical analysis of isolated vesicles constituted the main parts of this work. Functional studies, including endosome inactivation and overexpression of Rab11 mutants (proteins functioning at the level of the recycling endosome) revealed that recycling endosomes have indeed an essential role in the exocytic trafficking of TNF in macrophages. This thesis also provides further insight into recycling endosomes as a possible intermediate step in the exocytic trafficking of several other proteins including the adhesion protein E-Cadherin, that function at the cell surface. Finally, the last chapter of this thesis examines endocytic pathways in activated macrophages. Assays for fluid phase endocytosis and receptor-mediated endocytosis were established and the regulation of both pathways was compared. The results show that LPS has opposite effects on fluid phase and receptor mediated endocytosis, decreasing and increasing their activity respectively. Recycling of transferrin through the recycling endosome was also measured, providing a link with studies on TNF exocytosis. Overall, the work in this thesis has made a major contribution to our understanding of TNF trafficking in macrophages, of macrophage pathways more generally and of trafficking at a fundamental level. The findings herein set the stage for more in depth analysis at a single molecular level to explore TNF regulation in normal and disease cells.

Thesis (Ph.D.)--University of Western Australia, 2004.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 203-215). Available also in a digital version from Dissertation Abstracts.

Thesis (M.S.)--Missouri University of Science and Technology, 2009. / Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed August 14, 2009) Includes bibliographical references (p. 16-17).

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 99-102). Also available on the Internet.