Recent studies of hoplonenemertean planuliform larvae have clarified their development and provided insight into larval evolution within the phylum. However, an assessment of viviparous development using modern techniques is lacking. To help facilitate a comprehensive comparative evaluation of developmental diversity within hoplonemerteans, we have conducted a confocal laser scanning microscopy investigation of the development in Prosorhochmus americanus, one of the few viviparous hoplonemertean species. Phalloidin staining provides evidence of a modified transitory larval epidermis, and reveals that the foregut, midgut, proboscis, central nervous system, and body wall musculature form early in development, consistent with observations for planktonic and encapsulated hoplonemertean larvae. However, invaginations characteristic of these larvae were not observed. Acetylated tubulin labeling and light microscopy shows that embryos are uniformly ciliated, and some specimens possess a caudal ciliary cirrus and/or apical tuft which are characteristic of planktonic larvae. These are interpreted as vestigial structures in the non-swimming P. americanus embryos. The findings provide additional evidence that hoplonemerteans exhibit a form of metamorphosis in their life history and thus exhibit indirect development. However, a comparative assessment of larval features in P. americanus suggests an evolutionary trend towards direct development in this species.

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.

Aspergillus nidulans is a filamentous fungus whose cells have highly polarized growth. This requires many genes including hypA, which affects hyphal morphogenesis by promoting tip cell growth and suppressing growth in basal regions. The hypA locus was cloned previously by complementing the hypA1 phenotype. The hypA orthologues in Saccharomyces cerevisiae and Schizosaccharomyces pombe are essential, whereas hypA deletion strains in Aspergillus nidulans are viable but morphologically abnormal. The A. nidulans hypA locus has two temperature sensitive alleles, hypA1 and hypA6. These alleles were generated independently, but using the same mutagen, and were identified and characterized by different groups. Although the hypA1 strain has been shown to sporulate at restrictive temperature, the hypA6 strain was described as having a restrictive arrest phenotype, which suggested it was a lethal defect and was at odds with the report that hypA was dispensable for growth. To resolve this discrepancy, my study compared the hypA1 and hypA6 strains using morphometry, and compared their genetic lesions. I show that the hypA1 and hypA6 phenotypes are indistinguishable and mutation events in their coding regions are identical. Both hypA1 and hypA6 strains were able to sporulate at restrictive temperature as shown by scanning electron microscopy. Their cell morphologies were indistinguishable after growth under restrictive conditions, as were their repolarization kinetics when restrictive-grown cells were shifted to permissive temperature. Sequencing the hypA locus from a hypA6 strain, and comparing it to a hypA1 strain showed that they had identical lesions (G329R), which is consistent with the morphometric analysis. Unexpectedly, the hypA1 and hypA6 strains shared additional non-conservative amino acid substitutions, K885F and E932K, with their wildtype parent A28 compared to the strain used for complementing hypA1. The repair of these two amino acid changes can not rescue hypA1 or hypA6 phenotype at 42C. The S. cerevisiae homologue of hypA protein is TRS120p, which is involved in endomembrane trafficking. FM4-64 endomembrane staining of hypA1 and hypA6 strains showed they had aberrant endomembrane arrays at restrictive versus permissive temperature, which recovered the wildtype pattern after a return to permissive conditions. This is consistent with the similarities between the hypA and TRS120 sequences. The disparity between the published descriptions of the hypA1 and hypA6 restrictive phenotypes has been resolved, and the allele renamed hypA1/A6.

Aspergillus nidulans is a filamentous fungus whose cells have highly polarized growth. This requires many genes including hypA, which affects hyphal morphogenesis by promoting tip cell growth and suppressing growth in basal regions. The hypA locus was cloned previously by complementing the hypA1 phenotype. The hypA orthologues in Saccharomyces cerevisiae and Schizosaccharomyces pombe are essential, whereas hypA deletion strains in Aspergillus nidulans are viable but morphologically abnormal. The A. nidulans hypA locus has two temperature sensitive alleles, hypA1 and hypA6. These alleles were generated independently, but using the same mutagen, and were identified and characterized by different groups. Although the hypA1 strain has been shown to sporulate at restrictive temperature, the hypA6 strain was described as having a restrictive arrest phenotype, which suggested it was a lethal defect and was at odds with the report that hypA was dispensable for growth. To resolve this discrepancy, my study compared the hypA1 and hypA6 strains using morphometry, and compared their genetic lesions. I show that the hypA1 and hypA6 phenotypes are indistinguishable and mutation events in their coding regions are identical. Both hypA1 and hypA6 strains were able to sporulate at restrictive temperature as shown by scanning electron microscopy. Their cell morphologies were indistinguishable after growth under restrictive conditions, as were their repolarization kinetics when restrictive-grown cells were shifted to permissive temperature. Sequencing the hypA locus from a hypA6 strain, and comparing it to a hypA1 strain showed that they had identical lesions (G329R), which is consistent with the morphometric analysis. Unexpectedly, the hypA1 and hypA6 strains shared additional non-conservative amino acid substitutions, K885F and E932K, with their wildtype parent A28 compared to the strain used for complementing hypA1. The repair of these two amino acid changes can not rescue hypA1 or hypA6 phenotype at 42C. The S. cerevisiae homologue of hypA protein is TRS120p, which is involved in endomembrane trafficking. FM4-64 endomembrane staining of hypA1 and hypA6 strains showed they had aberrant endomembrane arrays at restrictive versus permissive temperature, which recovered the wildtype pattern after a return to permissive conditions. This is consistent with the similarities between the hypA and TRS120 sequences. The disparity between the published descriptions of the hypA1 and hypA6 restrictive phenotypes has been resolved, and the allele renamed hypA1/A6.

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.

Purpose: Hyper-reflective superficial cells were an unexpected finding while examining the corneal epithelium using confocal microscopy (CM), during an MSc thesis conducted in 2006 at the University of Waterloo, Canada. The author1 suggested that the appearance of these hyper-reflective cells could be associated with solution induced corneal staining (SICS) that was also observed in those participants who had manifested these hyper-reflective cells. However, this hypothesis has not been reported in the literature. This thesis aimed to investigate variables that could possibly predict the appearance of hyper-reflective superficial cells. These investigated variables were the effect of: contact lenses, contact lens solutions, lens/solution combinations, long-term use of certain contact lenses and solutions, age, dry eye symptom, topical anaesthetics and sodium fluorescein. In addition to this, the normal superficial epithelium of controls was defined. Methods: CM images of the superficial epithelium were obtained during the various experiments from: 32 non-contact lens wearing participants, 18 post-menopausal participants symptomatic of dry eye and 18 post-menopausal age-matched asymptomatic women and 147 adapted soft contact lens wearers. For one experiment CM was performed with the contact lens in situ, making the use of a topical anaesthetic unnecessary. Superficial cellular appearance of CM images was graded using a custom grading scale. Hyper-reflective cells were counted. Corneal staining was assessed using sodium fluorescein. Results: Results obtained during the various experiments revealed that hyper-reflective cells predominately appeared with the use of a specific lens/solution combination. Also, the number of hyper-reflective cells peaked after two hours of lens wear. It was also shown that when hyper-reflective cells occurred during an experiment, not every participant who was exposed to that specific lens/solution combination manifested hyper-reflective cells. Also, a great deal of inter-subject variability in observed numbers of hyper-reflective cells was noted. Conclusion: In conclusion, this thesis established that the hyper-reflective cells that were observed by Harvey were reproducible and may co-occur with corneal staining induced by a specific lens/solution interaction

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.

Purpose: Hyper-reflective superficial cells were an unexpected finding while examining the corneal epithelium using confocal microscopy (CM), during an MSc thesis conducted in 2006 at the University of Waterloo, Canada. The author1 suggested that the appearance of these hyper-reflective cells could be associated with solution induced corneal staining (SICS) that was also observed in those participants who had manifested these hyper-reflective cells. However, this hypothesis has not been reported in the literature. This thesis aimed to investigate variables that could possibly predict the appearance of hyper-reflective superficial cells. These investigated variables were the effect of: contact lenses, contact lens solutions, lens/solution combinations, long-term use of certain contact lenses and solutions, age, dry eye symptom, topical anaesthetics and sodium fluorescein. In addition to this, the normal superficial epithelium of controls was defined. Methods: CM images of the superficial epithelium were obtained during the various experiments from: 32 non-contact lens wearing participants, 18 post-menopausal participants symptomatic of dry eye and 18 post-menopausal age-matched asymptomatic women and 147 adapted soft contact lens wearers. For one experiment CM was performed with the contact lens in situ, making the use of a topical anaesthetic unnecessary. Superficial cellular appearance of CM images was graded using a custom grading scale. Hyper-reflective cells were counted. Corneal staining was assessed using sodium fluorescein. Results: Results obtained during the various experiments revealed that hyper-reflective cells predominately appeared with the use of a specific lens/solution combination. Also, the number of hyper-reflective cells peaked after two hours of lens wear. It was also shown that when hyper-reflective cells occurred during an experiment, not every participant who was exposed to that specific lens/solution combination manifested hyper-reflective cells. Also, a great deal of inter-subject variability in observed numbers of hyper-reflective cells was noted. Conclusion: In conclusion, this thesis established that the hyper-reflective cells that were observed by Harvey were reproducible and may co-occur with corneal staining induced by a specific lens/solution interaction