The proper organization and differentiation of the anterior segment is pivotal for normal eye development. Neural crest-derived POM cells are key contributors to correct anterior segment formation, differentiating to form the monolayered corneal endothelium. Mice with homozygous null mutations in the forkhead transcription factor gene, Foxc1, fail to develop a proper corneal endothelium stabilized by adherens junctions, with the endothelium adhering to the lens, preventing anterior chamber separation. The aim of this study was to evaluate the interaction between Foxc1 and the adherens junction protein, N-cadherin, as well as an associated gene, Msx1, during key stages in corneal endothelium development. Foxc1 was over-expressed in E12.5 and E13.5 POM cells and qPCR was carried out to determine the effect of Foxc1 on N-cadherin and Msx1 gene expression. Data showed over-expression of Foxc1 in wildtype E12.5 and E13.5 POM cells to cause significant fluctuations in N-cadherin and Msx1 expression (p < 0.05). POM cells were then transfected with a Foxc1 knock-down plasmid or the Foxc1 overexpression plasmid to evaluate the effect of Foxc1 on N-cadherin protein expression by Western blot analysis, however, these results were inconsistent with the gene expression analyses with no significant differences in N-cadherin expression detected. N-cadherin protein expression and localization was then further assessed by means of immunocytochemistry (ICC) and confocal microscopy in monolayer and hanging-drop POM cell cultures. Both qPCR and confocal microscopy data showed consistency, indicating increased amounts of N-cadherin in E12.5 cells relative to E13.5 cells, with membrane-bound N-cadherin showing a clear lattice-work pattern in hanging drop culture. Foxc1 over-expression/knock-down studies on E12.5 and E13.5 POM cells together suggest that N-cadherin is transcriptionally regulated by Foxc1 and that Foxc1 has a threshold level at which it is able to exert control over N-cadherin in POM cells. Foxc1 expression is therefore essential in establishing N-cadherin adhesion junctions in the corneal endothelium. Preliminary data also suggests that Msx1 may directly interact with Foxc1 in POM cells, however, further studies must be undertaken to verify and establish the effects of Foxc1/N-cadherin/ Msx1 interaction in the development of a cohesive, integrated corneal endothelium and functional anterior segment. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

Corneal endothelial development is an intricate process driven by finely tuned gene expression. Its formation is necessary for the continued normal development of the anterior segment of the eye. The presence of an inductive lens able to secrete factors such as TGFβ2 as well as the expression of Foxc1 and Pitx2 is essential to corneal endothelial development, as in the absence of any of these; the corneal endothelium fails to form. Corneal endothelial development begins as peri-ocular mesenchyme (POM) cells migrate into the space between the lens and surface ectoderm at E11.5. From E12.5, these cells begin to transition from a mesenchymal to an epithelial/endothelial (MET) phenotype, differentiating into a monolayered endothelium by E15 characterised by inter-cellular junctions. To study the initial process of development, immortalised POM cell lines from E12.5 and E13.5 embryos were used. Expression of the key genes, the transcription factors, Foxc1 and Pitx2 and two genes involved in EMT/MET, Slug and Tsc22, were analysed at these stages to establish the developmental norm. The effect of the lens on these expression levels was then determined. To establish whether TGFβ2 is the lens secreted signal responsible for gene expression changes, cells were subjected to TGFβ2 treatment. In all these experiments, the role of Foxc1 in regulating gene expression was determined by Foxc1 overexpression and knockdown. The effect of the lens on cellular proliferation and on the expression and cellular arrangement of N-cadherin, a junction protein was also determined. The results showed that, at E12.5, the lens downregulates Foxc1 and Pitx2 expression, is a potent inducer of Tsc22 expression and is required for maintaining Slug levels. TGFβ2 was shown to play a role in Foxc1 and Pitx2 downregulation. Analysis suggests that Tsc22 expression is responsive to lens signals, but that TGFβ2 is not the signal responsible for its downregulation between E12.5 and E13.5. The lens has no effect on Slug expression in the presence of Foxc1, but when Foxc1 is silenced, Slug is induced. Thus, Foxc1 plays a crucial regulatory role in Slug expression. At E13.5, as differentiation is initiated, Foxc1 expression remains responsive to the lens and to TGFβ2. Pitx2 expression is still induced by the lens but, at this stage, TGFβ2 does not play a part in Pitx2 regulation suggesting involvement of other unknown lens secreted signals. Other lens secreted signal/s were also shown to downregulate Tsc22 and Slug at this stage. The lens was implicated in MET as it was shown to have an effect on N-cadherin localisation in 3-dimensional culture. E12.5 Spheroids exposed to E6 lenses formed a distinct lattice arrangement of N-cadherin compared to the uniform distribution in control cells. Although the 13.5 control cell aggregates also showed a lattice framework, it was more pronounced in the lens treated cells. The transcriptional role of Foxc1 was determined by overexpression and knockdown experiments where Foxc1 overexpression and knockdown upregulated Tsc22 and downregulated Pitx2 and Slug at E12.5. At E13.5, Pitx2 was downregulated and Slug was upregulated in response to aberrant expression of Foxc1. This was illustrative of the sensitivity these genes have to Foxc1 expression during development. It is known that the presence of a functioning lens and Foxc1 are essential for proper development of the corneal endothelium, which in turn is necessary for normal eye development. The understanding of the precise molecular mechanisms required for corneal endothelial development and the processes requisite for cell proliferation and differentiation has important consequences for providing further insight into the pathophysiology of anterior segment dysgenesis and glaucoma. Previous studies suggest that stem-cell like qualities are conferred in cells undergoing EMT. Such an investigation may lead to application in regenerative medicine such as the bioengineering of corneal tissue. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

No description available.

Forkhead (Fox) proteins are transcription factors that function in many processes including development, metabolism and cell cycle regulation. This gene family is divided into subfamilies that appear to originate from a common ancestor. I have identified the evolutionary selection pressures acting on individual amino acid positions in the FoxA, FoxC, FoxD, FoxI, FoxO and FoxP subfamilies. The patterns of selection observed allowed for the prediction of residue function and identification of residues that differentiate orthologs and paralogs. The subfamily structure and negative selection found within the subfamilies indicates that after gene duplication, differentiation of subfamilies through amino acid changes and subsequent negative selection on these changes has occurred. Meanwhile, the observed neutral changes and positive selection allow for further protein differentiation. Within the FoxC subfamily, positive selection was identified at one amino acid site in the inhibitory domain. Mutation of this site in FOXC1 alters transactivation activity and the effects of mutants on transactivation activity are different on different reporters. The mutant effects were consistent with those of known disease causing mutations, supporting the predicted positive selection. The inhibitory domain is known to function in reducing FOXC1 transactivation activity and influences protein stability. Here I additionally show that loss of the inhibitory domain and mutation of the positively selected site can reduce FOXC1 DNA binding. Co-transfection of FOXC1 and TLE4, a repressor protein that can potentially bind to the inhibitory domain, was shown to increase FOXC1 transactivation activity. The effects of a novel disease causing FOXC1 inhibitory domain mutation on FOXC1 function were also assessed. The mutation reduced FOXC1 transactivation activity and increased protein half-life both of which may lead to disease. Regulation of FOXC1 activity is critical for normal function and this work has furthered our knowledge of how the inhibitory domain influences FOXC1 activity. I have provided biological evidence for the theory that positive selection acts at the amino acid level to optimize protein function. I have also shown that both changes in transcription factor proteins and the cis-regulatory region of target genes have the potential to contribute to evolutionary adaptation.

D.Phil. (Optometry) / An important issue in the quantitative analysis of optical systems is, for example, the question of how to calculate an average of a set of eyes. An average that also has an optical character as a whole and is representative or central to the optical characters of the eyes within that set of eyes. In the case of refraction, an average power is readily calculated as the arithmetic average of several dioptric power matrices. The question then is: How does one determine an average that represents the average optical character of a set of eyes, completely to first order? The exponential-mean-log transference has been proposed by Harris as the most promising solution to the question of the average eye. For such an average to be useful, it is necessary that the exponential-mean-log-transference satisfies conditions of existence, uniqueness and symplecticity, The first-order optical nature of a centred optical system (or eye) is completely characterized by the 4x4 ray transference. The augmented ray transference can be represented as a 5x5 matrix and is usually partitioned into 2x2 and 2x 1 submatrices. They are the dilation A, disjugacy B, divergence C, divarication D, transverse translation e and deflection 1t. These are the six fundamental first-orders optical properties of the system. Other optical properties, called derived properties, of the system can be obtained from them. Excluding decentred or tilted elements, everything that can happen to a ray is described by a 4x4 system matrix. The transference, then, defines the four A, B, C and D fundamental optical properties of the system…

<b>Purpose:</b> Assess the relationship between tear film drying and sensation between blinks. <b>Methods:</b> MATLAB sampled a slitlamp video camera, a potentiometer and a microphone while subjects kept one eye open for as long as possible. 23 subjects rated the intensity of the ocular sensation while video and voice data were collected simultaneously. The tear drying on the cornea was measured. <b>Results:</b> The sensation was triphasic. Two linear functions described the latter 2 parts of the data (r &#8805; 0. 95). The correlation between TBUT and the elbow in the time-discomfort function was 0. 72. Extent of tear film drying was linearly correlated to time (median correlation = 0. 88). The correlation between the discomfort elbow and image elbow was 0. 93 with single data pair for each subject. Analysis of sensation characteristics showed significant differences between itching and burning for both intensity and time (p = 0. 03 and p = 0. 02 respectively). <b>Conclusions:</b> Simultaneous recording of ocular surface appearance, discomfort intensity and attributes of sensation provide novel information about the development of discomfort during ocular surface drying. The rapid increase in discomfort proceeding blinking has been quantified and the relationship between the time course of drying and discomfort is elucidated.

<b>Purpose:</b> Assess the relationship between tear film drying and sensation between blinks. <b>Methods:</b> MATLAB sampled a slitlamp video camera, a potentiometer and a microphone while subjects kept one eye open for as long as possible. 23 subjects rated the intensity of the ocular sensation while video and voice data were collected simultaneously. The tear drying on the cornea was measured. <b>Results:</b> The sensation was triphasic. Two linear functions described the latter 2 parts of the data (r &#8805; 0. 95). The correlation between TBUT and the elbow in the time-discomfort function was 0. 72. Extent of tear film drying was linearly correlated to time (median correlation = 0. 88). The correlation between the discomfort elbow and image elbow was 0. 93 with single data pair for each subject. Analysis of sensation characteristics showed significant differences between itching and burning for both intensity and time (p = 0. 03 and p = 0. 02 respectively). <b>Conclusions:</b> Simultaneous recording of ocular surface appearance, discomfort intensity and attributes of sensation provide novel information about the development of discomfort during ocular surface drying. The rapid increase in discomfort proceeding blinking has been quantified and the relationship between the time course of drying and discomfort is elucidated.

<p>Spectral&#8208;domain optical&#8208;coherence tomography (SDOCT) has recently emerged as a powerful new tool for noninvasive human retinal imaging. I have developed a low&#8208;cost, high resolution real&#8208;time Spectral Domain Optical Coherence Tomography (SDOCT) system optimized for rapid 3D imaging of the human retina in vivo. Then functional retinal OCT imaging such as polarization sensitive OCT (PSOCT) and Doppler OCT were also developed based on phase technique. Unique phase unwrapping method in retina is described to extract the total reflectivity, accumulative retardance and fast axis orientation of the retinal nerve fiber layer (RNFL). The polarization scrambling layer of the retinal pigment epithelium was segmented by employing single camera sequential scan bsed PSOCT. As an extension, synthetic wavelength method will be also introduced for phase unwrapping in cell imaging. Finally I present an algorithm for 3D refraction correction based on a vector representation which accounts for refraction of CT light in the cornea. Following 3D refraction correction of volumetric corneal datasets, we can estimate the corneal optical power, thickness and the individual wavefront aberrations of the epithelial and the refraction&#8208;corrected endothelial surfaces by using Zernike spectrum analysis.</p> / Dissertation

Aim: To assess the ability of critical flicker frequency (CFF) and optimal reading speed (ORS) to predict the potential vision in patients with cataract with and without ocular comorbidity. Methods: The two novel tests were compared with two well established potential vision tests (PVTs), the potential acuity meter (PAM) and the laser interferometer (LI). Measurements were made preoperatively in 1 eye of 88 subjects using the battery of 4 PVTs. Postoperative measurements were made with the CFF and the ORS. The subjects studied were consecutive cases over a 12-month period who fulfilled the inclusion and exclusion criteria, and agreed to participate in this study. Results: CFF was the PVT most resistant to the presence of cataract. Both CFF and ORS give a similar predictive precision in the presence of cataract and ocular comorbidity, although CFF seems more precise when the cataract is dense. Conclusions: The PAM and the LI showed a limited clinical capability in predicting postoperative visual acuity, particularly with dense opacities. The CFF shows the most promise as a PVT, particularly with dense cataract. Further evaluation is required for both CFF and ORS.

The changes in the human crystalline lens shape and its internal structure during accommodation and with aging are a fundamental component of the dynamic mechanism of accommodation and presbyopia, the loss of near vision with age. A better understanding of the crystalline lens changes during accommodation will help in developing new treatments to correct for presbyopia. The goal of this dissertation is to design and develop an imaging system to study the dynamic changes in lens shape during accommodative response. An imaging system based on spectral domain optical coherence tomography (SD-OCT) was developed with long axial range, high axial and lateral resolution and high speed for in vivo imaging the anterior segment along its entire length at video-rate. A slit-lamp mounted optical delivery scanning device for the extended depth SD-OCT system was developed. The delivery system was combined with a custom made unit that provides accommodation and disaccommodation step stimuli. A method to correct for the distortions of the OCT images was also developed that provides corrected two dimensional biometric data at different accommodative states.