The anterior cingulate cortex (ACC) can influence emotional and motivational states in primates by its dense connections with many neocortical and subcortical regions. Pyramidal neurons serve as the basic building blocks of these neocortical circuits, which have been extensively studied in other brain regions, but their morphological and electrophysiological properties in the primate ACC are not well understood. In this study, we used whole-cell patch clamp and high-resolution laser scanning confocal microscopy to reveal the general electrophysiological properties and detailed morphological features of layer 2 and 3 pyramidal neurons in ACC (area 24/32) of the rhesus monkey. Neurons from both layers had similar passive membrane properties and action potential properties. Morphologically, dendrites of layer 3 ACC neurons were more complex than those of layer 2 neurons, by having dendrites with longer total dendritic lengths, more branch points and dendritic segments, spanning larger convex hull volumes. This difference in total dendritic morphology was mainly due to the apical dendrites. In contrast, the basal dendrites displayed mostly similar features between the two groups of neurons. However, while apical dendrites extend to the same layer (layer 1), the basal dendrites of layer 3 extended into deeper layers than layer 2 because of the difference in soma-pia distance. Thus, basal dendrites of the two groups of neurons receive different laminar inputs. Analysis of spines showed that more spines were found in neurons of layer 3 apical dendritic arbors than layer 2 neurons. However, the apical spine densities were similar between neurons in the two layers. Thus, while higher spine number suggests that layer 3 neurons receive more excitatory input than layer 2 neurons, the similar spine density suggests similar spatial and temporal summation of these inputs. The combined effects of increased number of excitatory input and higher dendritic complexity in layer 3 than in layer 2 ACC neurons suggest the additional information received by layer 3 neurons, especially in the apical dendrites, might undergo more complex integration.

Basic information about nail behaviour, under passive and especially iontophoretic condition, lacks in the literature. Thus, this thesis aims to fill gaps in the nail understanding by studying the potential and feasibility of the application of iontophoresis to human nail. The iontophoretic and passive delivery of Sodium Fluorescein (SF) and Nile Blue Chloride (NBC) were studied, in vitro, in order to determine their pathways as well as their depth and uniformity of penetration into the nail. The permselective properties of the nail were investigated by characterizing the contribution of electroosmosis, using mannitol as a marker, and by studying the flux of two inorganic cations, sodium and lithium, during in vitro experiments. Finally, the feasibility of transungual iontophoresis and the extraction of sodium and chloride ions from the body through the nail plate were performed on a group of human volunteers. Iontophoresis led the fluorescent markers slightly deeper into the nail plate than passive diffusion. The delivery of the bianion and of the cation was not different. Both compounds mainly penetrated the nail via the transcellular pathway. Electroosmosis resulted only in a slight enhancement of the mannitol fluxes compared to passive diffusion and the fluxes presented high variability, especially at pH 7.4 and when the current was applied in the anode-to-cathode direction. The delivery of the two inorganic cations was significantly higher at pH 7.0 than at pH 4.0 and supported that nails hold a negative charge at physiological pH. Ions were easily extractable through the nail plate during in vivo iontophoresis and all volunteers' feedbacks supported iontophoresis as an acceptable technique. This thesis demonstrated the feasibility and potential of in vivo transungual iontophoresis.

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2007.

Abstract Both in the closed and open eye state the superior limbus is covered by the upper lid. This region is of physiological interest and clinical importance because in chronic hypoxia, neovascularization of the cornea commonly occurs here. The limbal region in general is additionally of importance as the stem cells which are the source of the new corneal cells are located in the epithelium of the limbus and these are vital for normal functioning and are affected under certain adverse conditions. Purpose: In this experiment I examined corneal morphology in the limbal area and in particular under the upper lid in order to primarily examine the variation in the corneal limbal epithelial and total thickness as well as epithelial and endothelial cell density. Methods: I measured 30 eyes OD/OS (chosen randomly) of thirty healthy subjects aged from 18 to 55 years in the first study and twelve participants in the second study, with refractive error ≤ ±4 D and astigmatism ≤ 2 D. The thickness and cell density of five positions: superior, inferior, temporal, nasal limbal and central cornea was determined with optical coherence tomography (OCT) and confocal microscopy. At least three scans of each position were taken in both studies with OCT. At least 40 of 100 adjacent sagittal scans of each image were measured using OCT software program. In the confocal study, image J software was used to determine cell densities. Results: The epithelial and corneal limbal thickness were significantly thicker than the epithelial and central corneal thickness (p<0.05). The limbal, inferior cornea is thinner than the three other positions and the temporal region of the cornea is the thickest both in epithelial and total cornea. Epithelial cell density was significantly lower in the superior cornea than the four other positions. There was no significant difference in the endothelial cell density. Conclusions: Using OCT with high resolution and cross-sectional imaging capability and confocal microscope with high magnification, I found that the limbal cornea is significantly thicker than the central cornea both in total and in epithelial thickness. In the limbus, one might expect the superior cornea (under the lid) to be thickest (because of the expected hypoxia) whereas I found the temporal cornea was thickest. The epithelial cell density was lower in the superior cornea but there was no significant difference in cell densities in the endothelium. Further morphological investigation is of interest.

Abstract Both in the closed and open eye state the superior limbus is covered by the upper lid. This region is of physiological interest and clinical importance because in chronic hypoxia, neovascularization of the cornea commonly occurs here. The limbal region in general is additionally of importance as the stem cells which are the source of the new corneal cells are located in the epithelium of the limbus and these are vital for normal functioning and are affected under certain adverse conditions. Purpose: In this experiment I examined corneal morphology in the limbal area and in particular under the upper lid in order to primarily examine the variation in the corneal limbal epithelial and total thickness as well as epithelial and endothelial cell density. Methods: I measured 30 eyes OD/OS (chosen randomly) of thirty healthy subjects aged from 18 to 55 years in the first study and twelve participants in the second study, with refractive error ≤ ±4 D and astigmatism ≤ 2 D. The thickness and cell density of five positions: superior, inferior, temporal, nasal limbal and central cornea was determined with optical coherence tomography (OCT) and confocal microscopy. At least three scans of each position were taken in both studies with OCT. At least 40 of 100 adjacent sagittal scans of each image were measured using OCT software program. In the confocal study, image J software was used to determine cell densities. Results: The epithelial and corneal limbal thickness were significantly thicker than the epithelial and central corneal thickness (p<0.05). The limbal, inferior cornea is thinner than the three other positions and the temporal region of the cornea is the thickest both in epithelial and total cornea. Epithelial cell density was significantly lower in the superior cornea than the four other positions. There was no significant difference in the endothelial cell density. Conclusions: Using OCT with high resolution and cross-sectional imaging capability and confocal microscope with high magnification, I found that the limbal cornea is significantly thicker than the central cornea both in total and in epithelial thickness. In the limbus, one might expect the superior cornea (under the lid) to be thickest (because of the expected hypoxia) whereas I found the temporal cornea was thickest. The epithelial cell density was lower in the superior cornea but there was no significant difference in cell densities in the endothelium. Further morphological investigation is of interest.

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.