<p>Lens development is an intricate process governed by growth factor signaling and a hierarchy of transcription factors that regulate important processes required for normal lens development.</p> <p>Midline hedgehog (Hh) signaling has been implicated in lens defects including cyclopia and lens degeneration in rodents and fish. A lens specific model of hedgehog signaling has not been examined, and it was unknown whether the lens is able to respond to Hh signals. To investigate this question, and to determine any consequences of abnormal Hh signaling on lens development, a mouse model of constitutively active smoothened in the surface ectoderm and derivatives, (including the lens), was created. These mutants exhibited ectopic expression of FoxE3 by E12.5, and ectopic Pax6 expression by E15.5, along with deregulation of the lens cell cycle and lens degeneration.</p> <p>Similar to the Hh signaling pathway, normal expression of the transcription factor Activating Protein-2 (AP-2, <em>tcfap2</em>), in the lens, was shown to be essential for the maintenance of an epithelial cell phenotype, and the regulation of the lens cell cycle. AP-2α has been shown to be important at the placode stage of development for correct separation of the lens vesicle away from the overlying surface ectoderm. Defects resulting from the loss of AP-2α at this stage do not manifest until E12.5, at time at which AP-2β expression is lost in the lens, suggesting possible redundancy between the two AP-2 family members in early lens development.</p> <p>To investigate this possible redundancy, <em>Tcfap2a </em>and <em>Tcfap2b</em> were conditionally deleted from the lens at E9.5 (AP-2α/β DKOs). These family members were shown to play redundant roles during early lens development, with the double mutants exhibiting more severe defects than those seen in the AP-2α single knockout model A more nasally positioned lens stalk and a rotated lens were observed. Severe corneal defects and deregulation of the lens cell cycle were also evident.</p> <p>Roles for AP-2α in later lens development were unknown. To examine whether or not this transcription factor continues to play a role in lens epithelial cell maintenance subsequent to lens vesicle separation, a mouse model with <em>Tcfap2a</em> conditionally deleted from the lens during these later stages of development was created (MRL10-AP-2α). These mutants displayed a disorganized and multilayered lens epithelial cell layer with elongated epithelial cells that abnormally expressed fiber cell specific β/γ crystallins. These mutants also exhibited defects in cell adhesion between the epithelium and fiber cells, as well as between the epithelium and capsule, and exhibited fiber cell defects including vacuoles.</p> <p>Together, the work presented in this thesis outline previously unknown roles for Hh and AP-2 signaling in lens development. Both Hh and AP-2 are required for the maintenance of a normal lens epithelial cell phenotype and regulation of the cell cycle. This thesis also illustrates the requirement (and redundant roles) for AP-2α and AP-2β at the lens placode stage of development.</p> / Doctor of Philosophy (Medical Science)

Our lab has shown that conditionally disrupting the tcfap2beta gene, responsible for the activating protein-2beta (AP-2beta) transcription factor, exclusively in the craniofacial neural crest cells, leads to anterior segment dysgenesis. Subsequent loss of the corneal endothelium results in the adherence of the iris to the corneal stroma, causing closure of the iridocorneal angle. The activating protein-2beta neural crest cell knockout (AP-2beta NCC KO) model involves a complete blockage of the both the conventional (through the trabecular meshwork) and non-conventional (uveoscleral) pathways for aqueous humor drainage, and therefore it could be used as a powerful experimental model for glaucoma. As shown by our previous work, elevated intraocular pressure (IOP) and a 35% decrease in the number of cells in the retinal ganglion cell (RGC) layer was observed in AP-2beta NCC KO mice by 2 months; 6 to 11 months sooner than other reported mouse models of glaucoma. These observations suggested that the AP-2beta NCC KO mouse could be a novel and cost-effective experimental model for glaucoma if the RGC loss occurred progressively rather than due to a congenital defect. The purpose of this research project was to investigate how the retinal ganglion cell layer and macroglial activity changes with respect to age in the AP-2beta NCC KO mutant through immunofluorescence. Specifically, it was investigated whether the loss of RGCs was progressive and due to the increased IOP caused by the blockage of the uveoscleral drainage pathway. A significant decrease in the number of RGCs was observed between P4 and P10 in the retinal periphery of both WT and AP-2beta NCC KO mice (p<0.05), which is indicative of the programmed cell death that occurs due to retinal pruning during development. No statistical difference between WT and AP-2beta NCC KO mice phenotypes was observed at postnatal day 4 (P4), suggesting that no developmental defect resulted in the significant loss of RGCs at 2 months. In all other time points investigated, while no statistical difference was found between WT and the AP-2 NCC KO mutant, a clear downward trend was present in the AP-2 NCC KO mutant retinal ganglion cell layer from P10 to P40. There was also an expression of glial fibrillary acidic protein (GFAP) by Müller cells, indicating the presence of neuroinflammation at P35 and P40. This substantiates the potential P42 starting point of neurodegeneration our lab previously observed. This was further corroborated with Müller cell-associated expression of GFAP at P35 and P40 exclusively in the AP-2beta NCC KO mouse. Overall, we have shown that the retinal damage observed in our AP-2beta NCC KO mouse is not due to a developmental defect, but rather occurs over time. Thus, this mouse model, which appears to block both the conventional and unconventional uveoscleral pathways, has a profound effect on aqueous humor drainage. As a result, the model requires relatively little time to observe an increase in IOP and subsequent RGC loss. Our findings suggest that the AP-2beta NCC KO mouse can be a novel, powerful, and extremely cost-effective experimental model for glaucoma. / Thesis / Master of Science (MSc)

Les travaux présentés dans cette thèse décrivent l'analyse angulaire complète dépendante du temps du canal vecteur-vecteur B_d^0 -> J/Psi (K_S^0 Pi^0)*0. Après un rappel consacré à la violation de CP dans le système des mésons B est traitée la phénoménologie des canaux vecteur-vecteur du type charmonium-K*(892). La méthode pour la mesure des amplitudes de transversité des canaux B -> J/Psi K*(892), basée sur l'utilisation d'un pseudo-maximum de vraisemblance, est ensuite exposée. Les résultats obtenus avec 81.9 fb-1 de données collectées par le détecteur BABAR au pic de la résonance Upsilon(4S) sont |A_0|^2 = 0.565 +- 0.011 +- 0.004, |A_para|^2 = 0.206 +- 0.016 +- 0.007, |A_perp|^2 = 0.228 +- 0.016 +- 0.007, delta_para = -2.766 +- 0.105 +- 0.040 et delta_perp = 2.935 +- 0.067 +- 0.040. A noter que (delta_para, delta_perp) -> (-delta_para, Pi - delta_perp) est aussi une solution. Les phases fortes delta_para et delta_perp sont à > 3 sigma de factorisation. L'analyse de l'asymétrie avant-arrière en fonction de la masse K Pi a révélé la présence d'une onde S cohérente qui interfère avec le K*(892). C'est la première mise en évidence de la présence de cette onde dans le système K Pi provenant de la désintégration d'un méson B. La particularité du canal B_d^0 -> J/Psi (K_S^0 Pi^0)*0 est d'avoir une distribution qui dépend de la différence des temps propres des 2 mésons B provenant de la désintégration Upsilon(4S) -> B_d^0 Bbar_d^0 mais aussi des trois angles de transversité, permettant de mesurer sin 2beta mais aussi cos 2beta. Les résultats obtenus avec un ajustement par maximum de vraisemblance sont sin 2beta = -0.10 et cos 2beta = 3.32 avec les amplitudes de transversité fixées aux valeurs précédentes. L'autre solution pour les phases fortes conduit à la même valeur de sin 2beta mais change le signe de cos 2beta. Des considérations théoriques basées sur la conservation de l'hélicité du quark favorisent le choix des phases fortes données ci-dessus conduisant au signe positif pour cos 2beta. Ce signe de cos 2beta est celui prédit par le Modèle Standard.