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In the quest to master ocular surface regeneration, one must isolate the stem cells at the limbus and understand them. The stem cell niche is a concept that-was first described in 1978 and subsequently gained interest and became widely accepted. The work presented in Chapter 2 sought to characterize the stem cell niche at the ocular surface, and in doing so led to further understanding of stem cells at the limbus. In Chapter 3 the sampling of infant tissue provided further insight into the niche at that age group. In Chapter 4, Desmoglein 3 was hypothesized to be a negative stem cell marker. Finally in Chapter 5, the Amniotic Membrane was investigated as a possible surrogate stem cell niche. The stem cells at the limbus have not been isolated yet, but hopefully we are one step closer to mastering ocular surface reconstruction.

The lens is an ideal tissue to study growth and ageing since it continually grows throughout life accumulating cells with no protein turnover. The lenticular structural proteins, the crystallins, are distributed in layers that follow a chronological pattern. In this thesis, crystallin distribution patterns were investigated in concentric fractions of eye lenses from nine species. Clear lenses were used in order to examine changes occurring with development and ageing without the confounding factor of cataract. For the majority of species studied this was the first study to investigate and identify lens crystallin distributions. Lens proteins were separated using a fractionation technique following the lenticular growth mode. Individual crystallin subunits were further isolated and identified by size-exclusion chromatography, electrophoresis and mass spectrometry. Proportions of HMW, MMW and LMW proteins across each lens were determined by Bradford Assay. a, ~ and y-crystallin subunits were identified in each lens studied with comparable distribution patterns in phylogenetically similar species. a-crystallin proportions decreased from lens periphery to centre in all species and was not detected in the centre of amphibian and fish lenses. y-crystallins represented the most prominent soluble proteins in the lens centre with largest increases from the lens periphery seen in amphibian and fish, corresponding to their high refractive indices. Insoluble protein proportions increased towards the lens centre in all species. Taxon-specific crystallins were not identified in any species studied. Protein distribution patterns affect the optics of the lens; this structure/function relationship is important in understanding lens transparency. Protein distribution patterns in transparent lenses from in this thesis will assist understanding of changes in lenticular biochemistry taking place when transparency is compromised by cataract formation. Results from this thesis will also aid determination of phylogenetic relationships among species studied.

La formation et l’intégrité de la barrière hémato-rétinienne (BHR) sont nécessaires au maintien d’une bonne vision et la violation de cette barrière contribue à l’apparition d’un grand nombre de pathologies rétiniennes tel que la rétinopathie diabétique (RD) ou l’occlusion de la veine centrale de la rétine (OVCR). La dystrophine Dp71 est une protéine du cytosquelette associée à la membrane qui s’exprime majoritairement dans les cellules gliales de Müller. Son absence a été associée à une augmentation de la perméabilité vasculaire liée à la délocalisation et à la diminution de l’expression des canaux AQP4 et Kir4.1. La souris Dp71-null est donc un excellent modèle d’étude des pathologies rétiniennes présentant une rupture de la BHR. L’ensemble de nos résultats démontrent que chez la souris déficiente en Dp71 ayant une rupture de la BHR, il est possible de restaurer une perméabilité et une homéostasie rétinienne normale grâce à la surexpression de la Dp71 via les virus adéno-associés. Cette étude est à la base du développement de nouvelles stratégies thérapeutiques dans le traitement de pathologies associées à une rupture de la BHR et à un œdème maculaire, comme la RD ou l’OVCR. / Formation and maintenance of the blood-retinal barrier (BRB) is required for proper vision and breaching of this barrier contributes to the pathology in a wide variety of retinal conditions such as diabetic retinopathy (DR) or Central retinal vein occlusion (CRVO). Dystrophin Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller glial cells, its absence has been related to BRB permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels. Dp71-null mouse is thus an excellent model to approach the study of retinal pathologies showing blood-retinal barrier permeability. Our results collectively demonstrated that in Dp71 deficient mouse with compromised barriers, normal BRB permeability and retinal homeostasis can be restored through over-expression of Dp71 via adeno-associated virus. This study is the basis for development of new therapeutic strategies in dealing with diseases with BRB breakdown and macular edema such as DR or CRVO.