The epithelial sodium channel (ENaC) made up of α, β and γ subunits is located at the apical membrane of polarised epithelia and mediates transport of sodium ions into the cells. Tight control of ENaC function is essential for maintaining sodium homeostasis, blood volume and blood pressure. Controlling the number of active channels present at the cell surface appears to be critically important in regulating ENaC activity. The neural precursor cell expressed developmentally down-regulated gene 4 (Nedd4) family of proteins (eg. Nedd4-2) ubiquitinate ENaC and decrease its cell surface expression. The activity of Nedd4-2 is modulated by serum and glucocorticoid-induced kinase (SGK), which phosphorylates Nedd4-2 and increases cell surface expression of ENaC. The c̲o̲pper m̲etabolism gene M̲URR1 d̲omain 1 (COMMD1) protein is a recently identified ENaC binding partner and negative regulator of channel activity. Studies by other groups suggest that COMMD1 is also involved in the processes of intracellular protein trafficking and ubiquitin-dependent protein degradation. The aims of this study were 1). To characterise the interactions between COMMD1 and ENaC. 2). To identify the mechanism(s) by which COMMD1 down-regulates ENaC activity. Here protein-protein interaction studies were used to show that a recently identified conserved C-terminal domain (the COMM domain) in COMMD1 is essential for its binding to ENaC. The binding site for COMMD1 in βENaC was found to be located in its N-terminal domain. COMMD1 was shown to down-regulate ENaC by increasing ubiquitin modification of ENaC and by decreasing the cell surface population. COMMD1 was found to interact with SGK and formed a complex with SGK and Nedd4-2. Ussing chamber studies of Na⁺ transport showed that COMMD1 attenuated the stimulation of ENaC by SGK and abolished insulin-stimulated ENaC current in epithelial cells. Conversely, knock-down of COMMD1 increased ENaC current in mammalian epithelial cells. These data suggest that COMMD1 plays a role in regulating ENaC activity in epithelial cells and its effect is likely mediated via SGK. In addition COMMD1 was found to bind to the adaptor protein subunit [mu]2. Mutations in COMMD1 that disrupt its interaction with [mu]2 impair its ability to decrease cell surface expression of ENaC in Cos-7 cells, therefore COMMD1 may also have a role in the endocytosis of ENaC by linking cell surface ENaC to the clathrin-dependent endocytosis machinery. In summary, this study investigated the interactions between COMMD1 and ENaC and identified that the SGK/Nedd4-2 pathway is involved in the COMMD1-mediated ubiquitination and down-regulation of ENaC activity.

The products of the Cdx genes, Cdx1, Cdx2 and Cdx4, are known to play essential roles in many developmental processes including neural tube closure, axial elongation and patterning the anterior-posterior axis of the developing embryo. Cdx1 and Cdx2 are both expressed in the endoderm of the embryo and persist throughout adulthood in the intestinal epithelium, but their functions and mechanisms of action in this lineage are poorly understood, in part due to the peri-implantation lethality of Cdx2-/- mice. To circumvent this limitation, a conditional loss of function strategy was used to inactivate Cdx2 in the intestinal epithelium. These conditional mutants were also crossed to Cdx1-/- mice, which are viable and fertile, to examine potential functional compensation between these family members. The major findings of this study are that Cdx2 regulates patterning and differentiation of the small intestinal epithelium, while Cdx1 does not appear to make a contribution to either process. Furthermore, Cdx operates upstream of Notch ligand Delta-like 1 (Dll1) in endoderm and mesoderm derivatives, demonstrating that Cdx function is similar in different lineages. Finally, Cdx2 cannot fulfill the requirement for Cdx1 in regulation of its own promoter in the intestine. This is the first in vivo evidence that these two family members have context-dependent functional specificity. Altogether, this study underscores critical roles and mechanisms of action for Cdx members in the developing intestine and mesoderm.

Purposes: To use optical coherence tomography (OCT) to image and quantify the effect of various corneal conditions, in terms of corneal, stromal and epithelial thickness, and light backscatter. To assess the changes caused by overnight orthokeratology (Corneal Refractive Therapy; CRT^TM) lens wear, keratoconus and laser in-situ keratomileusis (LASIK) refractive surgery, each of which may lead to topographical alterations in corneal thickness either by temporary moulding, degeneration, or permanent laser ablation, respectively. <br /><br /> Methods: Topographical thickness of the cornea was measured using OCT in all studies. The CRT^TM studies investigated myopic and hyperopic treatment, throughout the day. The myopic studies followed lens wear over a 4 week period, which was extended to 12 months, and investigated the thickness changes produced by two lenses of different oxygen transmissibility. CRT^TM for hyperopia (CRTH^TM) was evaluated after a single night of lens wear. <br /><br /> In the investigation of keratoconus, OCT corneal thickness values were compared to those obtained from Orbscan II (ORB) and ultrasound pachymetry (UP). A new fixation device was constructed to aid in the measurement of topographical corneal and epithelial thickness along 8 directions of gaze. Pachymetry maps were produced for the normal non-lens wearing cornea, and compared with the rigid gas permeable (RGP) lens wearing cornea and the keratoconic cornea. <br /><br /> Thickness changes prior to, and following LASIK were measured and monitored throughout six months. Myopic and hyperopic correction was investigated individually, as the laser ablation profiles differ for each type of procedure. The LASIK flap interface was also evaluated by using light backscatter data to monitor healing. <br /><br /> Results: Following immediate lens removal after myopic CRT^TM, the central cornea swelled less than the periphery, with corneal swelling recovering to baseline levels within 3 hours. The central epithelium decreased and mid-peripheral epithelium increased in thickness, with a more gradual recovery throughout the day. There also seemed to be an adaptation effect on the cornea and epithelium, showing a reduced amount of change by the end of the 4 week study period. The thickness changes did not alter dramatically during the 12 month extended study. In comparing the two lens materials used for myopic CRT^TM (Dk/t 91 vs. 47), there were differences in stromal swelling, but no differences in the central epithelial thinning caused by lens wear. There was a statistically insignificant asymmetry in mid-peripheral epithelial thickening between eyes, with the lens of lower Dk causing the greater amount of thickening. Hyperopic CRT^TM produced a greater increase in central stromal and central epithelial thickness than the mid-periphery. Once again, the stroma recovered faster than the epithelium, which remained significantly thicker centrally for at least six hours following lens removal. <br /><br /> Global pachymetry measurements of the normal cornea and epithelium found the periphery to be thicker than the centre. The superior cornea and epithelium was thicker than the inferior. In the measurement of the keratoconic cornea, OCT and ORB correlated well in corneal thickness values. UP measured greater values of corneal thickness. The keratoconic epithelium was thinner than normal, and more so over the apex of the cone than at the centre. The location of the cone was most commonly found in the inferior temporal region. Central epithelial thickness was thinner in keratoconics than in RGP lens wearers, which in turn was thinner than in non-lens wearers. <br /><br /> Following LASIK surgery for both myopia and hyperopia, the topographical OCT thickness profiles showed stromal thinning in the areas of ablation. The central myopic cornea showed slight regression at 6 months. During early recovery, epithelial thickness increased centrally in hyperopes and mid-peripherally in myopes. By the end of the 6 month study, mid-peripheral epithelial thickness was greater than the centre in both groups of subjects. The light backscatter profiles after LASIK showed a greater increase in backscatter on the anterior side of the flap interface (nearer the epithelium), than the posterior side (in the mid-stroma) during healing. The flap interface was difficult to locate in the OCT images at 6 months. <br /><br /> Conclusion: All the CRT^TM lenses used in this project produced more corneal swelling than that seen normally overnight without lens wear. In order for these lenses to be worn safely for long periods of time without affecting the health of the cornea, they need to be manufactured from the highest oxygen transmissible material available. The long-term effect of thinning on the epithelium's barrier properties needs to be monitored closely. <br /><br /> Global topographical thickness of the cornea and epithelium was measured using OCT in normal, RGP lens wearing and keratoconic eyes. Corneal and epithelial thickness was not symmetrical across meridians. The epithelium of RGP lens wearers was slightly thinner than normal, but not as thin as in keratoconics, suggesting that the epithelial change seen in keratoconus is mainly due to the condition. <br /><br /> Post-LASIK corneal and epithelial thickness profiles were not the same for myopic and hyperopic subjects, since the ablation patterns vary. Epithelial thickening in the mid-periphery had not recovered by six months in myopes or hyperopes, possibly indicating epithelial hyperplasia. Light backscatter profiles were used to monitor the recovery of the LASIK flap interface, showing the band of light backscatter around the flap interface to decrease as the cornea healed.

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

Purposes: To use optical coherence tomography (OCT) to image and quantify the effect of various corneal conditions, in terms of corneal, stromal and epithelial thickness, and light backscatter. To assess the changes caused by overnight orthokeratology (Corneal Refractive Therapy; CRT^TM) lens wear, keratoconus and laser in-situ keratomileusis (LASIK) refractive surgery, each of which may lead to topographical alterations in corneal thickness either by temporary moulding, degeneration, or permanent laser ablation, respectively. <br /><br /> Methods: Topographical thickness of the cornea was measured using OCT in all studies. The CRT^TM studies investigated myopic and hyperopic treatment, throughout the day. The myopic studies followed lens wear over a 4 week period, which was extended to 12 months, and investigated the thickness changes produced by two lenses of different oxygen transmissibility. CRT^TM for hyperopia (CRTH^TM) was evaluated after a single night of lens wear. <br /><br /> In the investigation of keratoconus, OCT corneal thickness values were compared to those obtained from Orbscan II (ORB) and ultrasound pachymetry (UP). A new fixation device was constructed to aid in the measurement of topographical corneal and epithelial thickness along 8 directions of gaze. Pachymetry maps were produced for the normal non-lens wearing cornea, and compared with the rigid gas permeable (RGP) lens wearing cornea and the keratoconic cornea. <br /><br /> Thickness changes prior to, and following LASIK were measured and monitored throughout six months. Myopic and hyperopic correction was investigated individually, as the laser ablation profiles differ for each type of procedure. The LASIK flap interface was also evaluated by using light backscatter data to monitor healing. <br /><br /> Results: Following immediate lens removal after myopic CRT^TM, the central cornea swelled less than the periphery, with corneal swelling recovering to baseline levels within 3 hours. The central epithelium decreased and mid-peripheral epithelium increased in thickness, with a more gradual recovery throughout the day. There also seemed to be an adaptation effect on the cornea and epithelium, showing a reduced amount of change by the end of the 4 week study period. The thickness changes did not alter dramatically during the 12 month extended study. In comparing the two lens materials used for myopic CRT^TM (Dk/t 91 vs. 47), there were differences in stromal swelling, but no differences in the central epithelial thinning caused by lens wear. There was a statistically insignificant asymmetry in mid-peripheral epithelial thickening between eyes, with the lens of lower Dk causing the greater amount of thickening. Hyperopic CRT^TM produced a greater increase in central stromal and central epithelial thickness than the mid-periphery. Once again, the stroma recovered faster than the epithelium, which remained significantly thicker centrally for at least six hours following lens removal. <br /><br /> Global pachymetry measurements of the normal cornea and epithelium found the periphery to be thicker than the centre. The superior cornea and epithelium was thicker than the inferior. In the measurement of the keratoconic cornea, OCT and ORB correlated well in corneal thickness values. UP measured greater values of corneal thickness. The keratoconic epithelium was thinner than normal, and more so over the apex of the cone than at the centre. The location of the cone was most commonly found in the inferior temporal region. Central epithelial thickness was thinner in keratoconics than in RGP lens wearers, which in turn was thinner than in non-lens wearers. <br /><br /> Following LASIK surgery for both myopia and hyperopia, the topographical OCT thickness profiles showed stromal thinning in the areas of ablation. The central myopic cornea showed slight regression at 6 months. During early recovery, epithelial thickness increased centrally in hyperopes and mid-peripherally in myopes. By the end of the 6 month study, mid-peripheral epithelial thickness was greater than the centre in both groups of subjects. The light backscatter profiles after LASIK showed a greater increase in backscatter on the anterior side of the flap interface (nearer the epithelium), than the posterior side (in the mid-stroma) during healing. The flap interface was difficult to locate in the OCT images at 6 months. <br /><br /> Conclusion: All the CRT^TM lenses used in this project produced more corneal swelling than that seen normally overnight without lens wear. In order for these lenses to be worn safely for long periods of time without affecting the health of the cornea, they need to be manufactured from the highest oxygen transmissible material available. The long-term effect of thinning on the epithelium's barrier properties needs to be monitored closely. <br /><br /> Global topographical thickness of the cornea and epithelium was measured using OCT in normal, RGP lens wearing and keratoconic eyes. Corneal and epithelial thickness was not symmetrical across meridians. The epithelium of RGP lens wearers was slightly thinner than normal, but not as thin as in keratoconics, suggesting that the epithelial change seen in keratoconus is mainly due to the condition. <br /><br /> Post-LASIK corneal and epithelial thickness profiles were not the same for myopic and hyperopic subjects, since the ablation patterns vary. Epithelial thickening in the mid-periphery had not recovered by six months in myopes or hyperopes, possibly indicating epithelial hyperplasia. Light backscatter profiles were used to monitor the recovery of the LASIK flap interface, showing the band of light backscatter around the flap interface to decrease as the cornea healed.

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

Cancer morbidity and mortality is greatly reduced when the disease is diagnosed and treated early in its development. Tissue biopsies are the gold standard for cancer diagnosis, and an accurate diagnosis requires a biopsy from the malignant portion of an organ. Light, guided through a fiber optic probe, could be used to inspect regions of interest and provide real-time feedback to determine the optimal tissue site for biopsy. This approach could increase the diagnostic accuracy of current biopsy procedures. The studies in this thesis have characterized changes in tissue optical signals with carcinogenesis, increasing our understanding of the sensitivity of optical techniques for cancer detection. All in vivo studies were conducted on the dimethylbenz[alpha]anthracene treated hamster cheek pouch model of epithelial carcinogenesis. Multiphoton microscopy studies in the near infrared wavelength region quantified changes in tissue morphology and fluorescence with carcinogenesis in vivo. Statistically significant morphological changes with precancer included increased epithelial thickness, loss of stratification in the epithelium, and increased nuclear diameter. Fluorescence changes included a statistically significant decrease in the epithelial fluorescence intensity per voxel at 780 nm excitation, a decrease in the fluorescence lifetime of protein-bound nicotinamide adenine dinucleotide (NADH, an electron donor in oxidative phosphorylation), and an increase in the fluorescence lifetime of protein-bound flavin adenine dinucleotide (FAD, an electron acceptor in oxidative phosphorylation) with precancer. The redox ratio (fluorescence intensity of FAD/NADH, a measure of the cellular oxidation-reduction state) did not significantly change with precancer. Cell culture experiments (MCF10A cells) indicated that the decrease in protein-bound NADH with precancer could be due to increased levels of glycolysis. Point measurements of diffuse reflectance and fluorescence spectra in the ultraviolet to visible wavelength range indicated that the most diagnostic optical signals originate from sub-surface tissue layers. Optical properties extracted from these spectroscopy measurements showed a significant decrease in the hemoglobin saturation, absorption coefficient, reduced scattering coefficient and fluorescence intensity (at 400 nm excitation) in neoplastic compared to normal tissues. The results from these studies indicate that multiphoton microscopy and optical spectroscopy can non-invasively provide information on tissue structure and function in vivo that is related to tissue pathology. / Dissertation

This study was conducted to determine if electroporation could deliver membrane-impermeant molecules intracellularly to intact, physiologically competent monolayers that mimic the intestinal epithelium. The long-term effects of electroporation on these monolayers were studied to determine the kinetics with which monolayers recover barrier function. The ability of electroporation to introduce biologically active molecules, e.g., plasmid DNA and siRNA, into these monolayers, to either express a protein of interest or modify cellular function, was also studied. Results showed that intracellular uptake of calcein, a small tracer molecule, and bovine serum albumin, a globular protein, occurred uniformly throughout the monolayers and increased as a function of voltage, pulse length, and pulse number. There was no significant difference in uptake resulting from single and multiple pulses of the same total exposure time. Barrier function recovery depended on the electroporation conditions applied, with some monolayers recovering normal physiologic function within a day. Electroporation also increased the permeability of the monolayers to calcein and BSA, possibly through a combination of increased paracellular and transmonolayer transport. When compared to cationic lipid transfection (lipofection), transfection of intestinal epithelial monolayers with reporter plasmids by electroporation was more efficient in situations where high concentrations of DNA, and as a result, higher levels of expression were needed. Although uptake of DNA was high after electroporation and increased with increasing amounts of DNA, overall expreseion efficiency was still low (~3%). Electroporation-mediated transfection of intestinal epithelial monolayers with a plasmid that expressed inflammation inhibitor protein, IκВα was not always successful, probably because of low levels of protein expression. Introduction of the much smaller siRNA molecules into the monolayers by electroporation, on the other hand, was very successful at inhibiting the production of the nuclear envelope proteins lamin A and lamin C. The results of these experiments demonstrated that electroporation can introduce a wide variety of molecules intracellularly into model intestinal epithelia. These results should be useful to identify optimal electroporation conditions for transporting drugs, proteins, and genes into intestinal and, possibly, other epithelia for local drug and gene therapy, as well as for development of improved models of intestinal epithelium.

Summary Clmatis Armandi has been used frequently in traditional Chinese medicine for the treatment of diuretic symptoms. The mechanism of its action is unclear. Possible action of this substance may involve alternation of electrolyte transport through the epithelia membranes. In this study¡Atransepithelial conductance of frog skin was measured in vitro in voltage-clamped Ussing chambers. Adding Clematis Armandi extracts to apical surface induced a conductance increment of 1.21 £gS and an apical to serosal Isc of 28.78 £gA/cm2. The Isc can not be completely blocked by apical application of amiloride. Nifedipine and TEA had no effect on Clematis Armandi induced Isc decrease. These data indicate that frog skin is highly responsive to the concentrated Clematis Armandi extracts. The increase in Isc reflects changes in transepithelial transport of Na+ ions modulated at apical membrane. The enormous increase in transepithelial conductance suggests that in additional to enhancement of amiloride-sensitive Na+ channels, Clematis Armandi may also modulate other pathways, such as Cl- ion channel modulation, which needs further investigation.

Thesis (M. Sc.)--York University, 2000. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 111-119). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ59179.