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 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.

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.

A novel colloidal model system is developed for the purpose of achieving simultaneous three-dimensional (3D) confocal imaging and optical tweezing of impurity probe particles embedded in a dense material of colloidal host particles. First, the colloidal host particles are developed from 3-trimethoxysilyl-propylmethacrylate (TPM), and the solvent mixture is tuned to match the refractive index and the density of the particles. A sedimentation-diffusion equilibrium profile of the TPM particles was imaged in 3D to establish suitability of the system for 3D confocal laser scanning microscopy, and to study its phase behaviour and particle dynamics. Then, core-shell particles, which consist of a high refractive index core and a TPM shell, are synthesised to be used as the impurities. The versatility of the two-step coating procedure with TPM has been demonstrated on several core materials, and the optical properties of the core-shell particle are established using digital holographic microscopy and their optical trapping strengths. Together with the TPM host particles, the core-shell particles are shown to be suitable impurity probes in dense colloidal materials, as they can be manipulated using optical tweezers in all three-dimensions, whilst the structure and dynamics of the surrounding host particles can be imaged simultaneously using fast confocal laser scanning microscopy. Subsequently, to demonstrate the capability of the TPM-based colloidal model system, the depletion potential of a pair of core-shell probe particles embedded in a sea of TPM host particles has been measured using optical tweezing. This is derived from comparing the direct pair potential between the core-shell particles in a TPM refractive index matching solvent, and the potential of mean force for the core-shell particles embedded in a dense fluid region of the TPM host particles. Direct visualisation of the liquid structures of the TPM host particles in the binary system around the probe particles has been linked to the form of the depletion potential, and its oscillatory nature as a function of the particle separation. Lastly, the formation of colloidal dumbbell particles is discussed. The dumbbell formation has been rationalised in terms of the total surface energy of droplet formation on spherical surfaces and the calculations are compared with experimental results from coating various seed particles with TPM. Using optically anisotropic dumbbell particles and tuning the refractive index of the dispersion medium, a preliminary two-trap experiment has been conducted which shows unusual trapping behaviour when a time-delayed feedback trapping trajectory has been applied.

No description available.

Zebrafish is a frequently used model organism with an array of transgenic lines that have been used indevelopmental and physiological studies. We aim to generate novel transgenic zebrafish reporter lines to study subpopulations of spinal neurons in vivo. The gene editing system called CRISPR/Cas9 system was used to knock in reporter genes such as green fluorescent protein (GFP) or Gal4 transcription factor, to generate transgenic fish lines. Zebrafish embryos were injected with gRNAs targeting gabrb1 or nr4a2a and GFP or Gal4 plasmid, respectively. F0 larvae were screened, positive fish were raised until sexual maturity, and founders characterized to verify germline insertion. Three founders were found for gabrb1 and the location and the direction of the insert verified. The GFP expression was studied during development and differential expression patterns were identified whereas all founders had expression in brain and spinal cord. In parallel, positive fish from the Gal4 injections were raised and will be screened. Immunohistochemistry was performed to check if nr4a2a is expressed in the same cells as known neuronal markers. However, no co-localization was detected. The three gabrb1 founders identified in this study highlight the challenges into creating stable transgenic lines recapitulating true expression of the gene of interest. Sequencing, in-situ hybridization and immunohistochemistry should be performed to verify the line. A possible reason for the varying expression may be that through the knock-in we may interfere with regions regulating gene. The nr4a2a-Gal4 line will be used to perform functional studies. Those experiments will be performed using reporter genes, such as opsins or GCaMP, controlled by Upstream Activation Sequence (UAS). These transgenic lines will provide important insights regarding neuronal subpopulations that express gabrb1 and nr4a2a to unravelhow the locomotor network is formed.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19972号 / 薬科博第63号 / 新制||薬科||7(附属図書館) / 33068 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 橋田 充, 教授 佐治 英郎, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM