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.

The unique gene, pleiotrophin (ptn) was identified in the expressed sequence taq (EST) derived from the developing tilapia brain in our lab. The cDNA full length of ptn was cloned. ptn play a role in the differentiation of nerve cell. In this study, bioinformatics were searched for EE723939.1 (ptn), which is a gene with 1026 bp of cDNA sequence, open reading frame(ORF) is 483bp, and deduced as 160 amino acids. The protein of PTN was expressed in the prokaryotic system, BL21(E.coli), and purified with Ni-NTA affinity chromatography. In the present studies, ptn, cloned from tilapia, Oreochromis mossambicus. The influence of ptn on the proliferation of Neuro-2a cell was also investigated.The ORF of ptn was cloned, and the pEGFP-ptn plasmid was constructed. The distribution of ptn in the pEGFP-ptn transfected Neuro-2a cell was identified by fluorescence and laser confocal microscopy.

In this thesis the methods of optical beam induced current (OBIC), multi-photon excitation, and confocal microscopy were employed to study InGaN LED¡¦s. Recently, important breakthrough and achievement have been made in the developments of InGaN based opto-electronic components. As a result, it is important to characterize the properties and the performance of InGaN based devices with various techniques. In this thesis, we have used 2-photon OBIC microscopy to observe various such LED¡¦s. We found that the LED¡¦s exhibit dotted pattern which can not be seen under 1-photon excitation. In addition, we have employed micro-spectroscopy to characterize the active layer of these LED¡¦s. These results will be discussed in this thesis in detail.

In this research we consider two different approaches for microfabricating physical templates to be used in template directed colloidal self-assembly experiments. Fabrication of templates, usable with confocal microscopy, forms an essential part of observation and analysis of template directed colloidal self-assembly studies. We use existing laboratory based microfabrication methods for patterning thin glass coverslips and polymeric films. These templates when used for directing colloidal self-assembly along with confocal microscopy analysis provide us with relevant information on the effect of confined geometries of the template on particle packing and order. The first method of template fabrication involves ultraviolet photolithography, thin film deposition, and glass micro machining. Various stages of the process were optimized while selecting reactive ion etch (RIE) and nickel etch mask with a suitable etch recipe for microfabrication of patterns on thin multi-component glass coverslips. Pattern dimensions were shown to be nearly commensurate with patterns on the microfiche, which was used as a field mask. In another approach, mechanical machining for fabricating polymeric templates was attempted on poly(methyl methacrylate) films spin coated on thin glass cover slips. The mechanical machining was implemented using computer numerical control (CNC) machines with the pattern dimensions in the range of 50 Mu m-150 Mu m. The glass and polymeric templates were used in template directed colloidal self-assembly experiments us ing polystyrene or silica particles. Confocal microscopy was used to obtain images of particle packing in template geometries. Imaging of the particles confined in the template geometries show increased particle concentration along pattern walls and corners. Inherent pattern irregularities and roughness possibly resulted in limited order in particle. Using a simple fortran program, image stack generated from confocal microscopy is used for obtaining images of particle packing in four different view planes which includes top, side, cross sectional and diagonal view of the image stack. The results from this research show the application of simple microfabrication processes for creating physical templates for template directed colloidal self-assembly. Confocal microscopy imaging combined with fortran image processing program can provide images of particle packing in different view planes. These images of the particles confined in various pattern geometries illustrate greater possibility of packing order in straight and regular pattern geometries or profiles.

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

Thesis (M.S.)--California Polytechnic State University, 2009. / Title from PDF title page; viewed on January 5, 2010. Major professor: Lily Laiho, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Engineering." "December 2009." Includes bibliographical references (p. 79-83). Also available on microfiche.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2003. / Vita. Includes bibliographical references.

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Chemotaxis is a process whereby single- and multi-cellular organisms migrate in response to external chemical stimuli. This directed cell movement is regulated by complex signaling pathways and is implicated in embryonic development, immune response, and the metastasis of cancer cells. Dictyostelium discoideum, social amoebae with the ability to migrate and aggregate in response to chemoattractants such as cAMP, have been used as a model system to study chemotaxis. Preliminary research suggests that protein kinase (PKA) is involved in some of the signaling pathways that regulate chemotaxis. The role of PKA in chemotaxis was investigated, first, by characterizing the phenotype of PKA null cells using established cell biological and biochemical assays. Furthermore, spatiotemporal regulation of critical cytoskeletal proteins was probed in wild-type and PKA null cells using confocal fluorescence microscopy, indicating misregulation of both F-actin and Myosin II in pkaC- and pkaR- cells. Finally, preliminary work was done to lay the groundwork for experiments exploring possible PKA targets mediating TORC2 function in chemotaxis.