published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

No description available.

In vertebrates the cornea must maintain its transparency throughout adult life to ensure sight, and understanding the mechanisms underpinning corneal homeostasis are fundamental to developing new treatments to cure or prevent blindness. This study investigated the role the planar cell polarity pathway plays in the directed migration of adult corneal epithelial cells, in maintaining the homeostatic environment of the eye and during wound healing. RT-PCR confirmed, for the first time, the expression of multiple core PCP genes within human corneal epithelial (HCE) cells. Components of the PCP pathway were pharmacologically and genetically manipulated during wound healing of corneal epithelial cells and the importance of the downstream target JNK, and core PCP gene Vangl2, during wound healing was demonstrated. Manipulation of core PCP components was found also to directly affect the ability of HCE cells to realign and migrate in response to physical topographical cues in vitro. This study therefore indicated that PCP may regulate the directed migration of corneal epithelial cells as they travel over the basement membrane. Using conditional knockout mice the loss of Vangl2, a core PCP gene, and its effect on both planar and the apical-basal polarity of the corneal epithelium was investigated. Severe morphological defects were observed in Vangl2-null mice indicative of underlying problems in apical-basal polarity of the epithelial cells. The basement membrane of Vangl2-null cells was largely absent in vivo, which suggested that at least some of the planar defects were secondary to an unexpected failure of apical-basal polarity. This study has shown for the first time that PCP plays a crucial role in the maintenance of an adult vertebrate tissue, particularly during wound healing and maintenance of the corneal epithelium. It has also indicated a role for the core PCP gene, Vangl2, in setting up apical-basal polarity of these adult cells.

Corneal degenerative diseases and opacity are leading causes of corneal impairment and blindness worldwide. Like many epithelial tissues, the constant renewal of transparent corneal epithelial cells is essential for a lifelong healthy cornea and optimal vision. The limbus (the boundary between the cornea and the conjunctiva) is believed to be the site that harbours adult stem cells responsible for corneal maintenance and repair after injury, referred to as limbal epithelial stem cells (LESCs). In the basal limbal epithelium, an active LESC subset divides to yield progenitor cells that migrate centripetally into the corneal epithelium for cell renewal. This asymmetric division however, is assumed to be regulated by a balance between cell renewal and loss of cells from the corneal surface. The search for specific LESC molecular markers has been difficult and to date there are few if any candidate markers that unambiguously identify LESCs but not their immediate progeny. Consequently, LESC clonality, activity and proliferative dynamics have remained poorly understood. In addition, the nature of the regulatory molecular pathways involved during LESC activity is still an open key question. In this research project, we identified stem cells on the ocular surface of the eye, assayed their activity and demonstrated quantitively for the first time how the cornea responds to damage. The retention of DNA labelling reagents in control and wounded corneas was combined with clonal analyses of cell division and migration using mice mosaic for reporter LacZ expression. Corneal transplant in LacZ reporter transgenic mice showed migration of LacZ-positive cells into the donor corneal button, with long-term disruption of patterns of migration. Corneal epithelial scrape wounds at the periphery also showed long– term disruption. Label retention suggested a small but statistically significant proliferation response of LESCs to injury, but this was attenuated or absent in aging mice and Pax6 mutants. The Hippo signalling pathway has been shown to have promising results in regulating stem cell activity and proliferation in many organs, however, its effect on LESC proliferation is unknown. Here, we investigated the regulatory role of the Hippo−YAP signalling pathway during cell proliferation, and determined whether the label retention assay in a uniform population of dividing cells is a real indicator of slow-cycling cells in vivo. Cell-cycling kinetics, Abstract v proliferation rate, and label retention were determined in a spontaneous human corneal epithelial (HCE-S) cell line, using double-labelling IdU and EdU thymidine analogues. During homeostasis, HCE-S cells underwent approximately one cell cycle per day, however, cells in which YAP-dependent signalling was activated by 17-Allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of heat shock protein 90 (Hsp90), showed slower proliferation rate and longer cell-cycle time. In vitro label-retention assay in confluent cultures estimated number of ~3-4 cell cycles needed to dilute out the label from slow-cycling cells in vivo. The data are suggestive that the Hippo pathway has a potential regulatory role in proliferative corneal epithelium, and that label-retention assay is a real indicator of slow-cycling cells. Furthermore, this research observed the proliferative dynamics of conjunctival stem cells. Clonal analysis of patterns of cell growth in the conjunctiva were analysed following tamoxifen-induction of LacZ transgene activity. The long−term presence of coherent patches of LacZ-positive cells suggested the presence of long-lived conjunctival stem cells but that the turnover time for the bulbar conjunctival epithelium may be more than 16 weeks. The key results of this research are that the limbus is the niche for stem cells, that there is a unidirectional migration of cells from the limbus to the corneal epithelium during homeostasis, but this is disrupted, perhaps permanently, by wounding. We find only a mild response of limbal epithelial stem cells to acute corneal injury, which is reduced to no response with age and is dependent on genetic background.

Abstract not available

Thesis (Ph. D.)--University of Rhode Island, 2005. / Typescript. Includes bibliographical references (leaves 114-122).

The primary function of the lens capsule of the eye unfolds during the process of accommodation; wherein, tension imposed onto its equator is released, allowing the elastic capsule to mold the underlying lens nucleus and cortex into a more quasispherical morphology to change focus from distant to near objects. Given its highly mechanical nature, it is prudent to study the native lens capsule from the perspective of biomechanics for such applications as understanding the mechanism of accommodation. Further, cataract surgery introduces alterations to the geometry of the lens capsule that lead to changes in resident cell behavior from quiescent to contractile and synthetic. Though resultant changes in capsule histology are well documented little has been done to quantify the corresponding altered mechanics, which is important for elucidating related post-surgical pathologies and improving prosthetic lens design. In this study we present the first data on the in situ multiaxial mechanical behavior of the native and hyperglycemic anterior lens capsule in both the porcine and human models. From these data, native stresses in the lens capsule are calculated using a finite element analysis, and alterations in the corresponding strain field are calculated after the introduction of a continuous circular capsulorhexis, which is imposed during cataract surgery. Finally, we quantify both the altered mechanical behavior and contractile loads imposed onto the lens capsule after cataract surgery.