This dissertation investigates the origins of tunable and efficient photochemistry in three different photoactive proteins, bacteriorhodopsin (BR), rhodopsin (RHO) and green fluorescent protein (GFP). In all cases, significant differences exist between the photoreactivity of model chromophores in solution and in the protein environment, in terms of excited state lifetime and efficiency of the primary photochemical process (opsin proteins) or the type of reaction (excited state proton transfer versus C=C double bond photoisomerisation for GFP). The work presented here investigates for each case to what extent the protein environment is necessary to alter the photochemistry of model chromophores in solution. For GFP and BR steric and electrostatic interactions between the protein pocket and the chromophore are shown to be likely responsible for shaping the excited state surface along which the photoreactions take place. For RHO it is suggested, contrary to current belief, that selection of a reactive ground state conformer might be the main effect generating the observed differences between solution and protein environment. The solution photochemistry of structurally modified retinal protonated Schiff bases, taken as model chromophores for the opsin proteins, is studied with continuous wave irradiation experiments and ultrafast transient spectroscopies. Surprisingly large differences are observed for the isomerisation reaction depending on the starting configuration (trans or cis) of the photoactive double bond. The current model for BR based on the tuning of the excited state barrier encountered along the isomerisation coordinate is expanded to include the changes in selectivity, speed and efficiency observed for a series of all-trans derivatives. For 11-cis, the photoisomerisation in solution is proposed to take place along a barrierless isomerisation coordinate, in contrast with the models currently available in literature. It is suggested that the protein might be discriminating between ground state conformers rather than significantly changing the topography of the reaction coordinate. For GFP, excited state Raman spectra are recorded for the wild-type protein, two mutants and a model chromophore in solution. It is suggested that the high frequency vibrational modes observed in the excited state spectra of the proteins but not of the model chromophore in DMSO are a sign of a tighter chromophore environment that inhibits the photoisomerisation reaction occurring in solution.

In patients with age-related macular degeneration, a monolayer of cells in the eyes called retinal pigment epithelium differ from healthy ones in morphology. It is therefore important to quantify the morphological changes, which will help us better understand the physiology, disease progression and classification. Classification of the RPE morphometry has been accomplished with whole tissue data. In this work, we focused on the spatial aspect of RPE morphometric analysis. We used the second-order spatial analysis to reveal the distinct patterns of cell clustering between normal and diseased eyes for both simulated and experimental human RPE data. We classified the mouse genotype and age by the k-Nearest Neighbors algorithm. Radially aligned regions showed different classification power for several cell shape variables. Our proposed methods provide a useful addition to classification and prognosis of eye disease noninvasively.

In the developing visual system, the axon of a pre-synaptic cell must be guided to a post-synaptic partner. Retinal ganglion cells (RGCs) in the eye are an excellent model to study this process. Multiple classes exist that respond to specific types of light input, and these project to different destinations in the brain that process distinct types of information. The RGC axons that navigate to the lateral geniculate nucleus (LGN) do so in a class-specific manner. Axons from RGCs that mediate non-image forming functions innervate the ventral LGN (vLGN) and the intergeniculate leaflet (IGL). Axons from RGCs that process image-forming information bypass these regions to innervate the dorsal LGN (dLGN). The extracellular protein reelin was identified as a potential factor in RGC axonal targeting of the vLGN and IGL, and the reeler mutant mouse used to study the effects of its functional absence. Anterograde labeling of RGCs and their axons with Cholera toxin B (CTB) revealed reduced patterns of retinal innervation to the vLGN and IGL in mutant mice. Moreover, the absence of functional reelin resulted in axons incorrectly growing into inappropriate regions of the thalamus. We identified these misrouted axons as those of the intrinsically photosensitive RGCs (ipRGCS), a class of RGCs known to project to the affected subnuclei. In contrast to defects in ipRGC targeting, no deficits were seen in retinogeniculate or corticothalamic projections in classes of axons that normally target the dLGN. Immunohistochemistry did not reveal any effects of the absence of the functional reelin on the LGN cytoarchitecture, which is unlike many other brain regions altered in the reeler. In summary, results suggest that intact reelin is required for class-specific retinogeniculate targeting to the vLGN and IGL. The defects are likely to be in targeting and not in neuronal positioning.

La rétinopathie diabétique (RD) se compose d’une part d’une ischémie rétinienne périphérique et d’autre part d’une exsudation rétinienne responsable d’un œdème maculaire diabétique, première cause de cécité chez les moins 55 ans. Les traitements utilisés actuellement sont non spécifiques et traitent les complications tardives de la RD. Les phases précoces de la RD ne sont donc pas ciblées. L’hyperglycémie chronique entraine un stress oxydant et une activation des PKC qui participent à l’altération des BHR. L’objectif de ce travail a été 1°) d’étudier l’implication de la PKCζ et de la voie Rho/ROCK/Myosine II sur la physiopathogénie de la RD et 2°) de montrer l’effet bénéfique de leurs inhibiteur sur les BHR et sur la reperfusion des capillaires rétiniens. Nous avons confirmé l’hyperactivation de la PKCζ et de la voie Rho/ROCK/Myosine II chez les rats diabétiques et leur participation à la rupture de la BHR externe. Le traitement par leurs inhibiteurs respectifs normalise l’activation des deux enzymes et restaure l’intégrité anatomique et fonctionnelle de la BHR externe. De plus l'hyperactivation de ROCK altère la perfusion rétinienne par 1) constriction focale artériolaire, 2) protrusions membranaires endoluminales des cellules endothéliales (blebbing) et 3) vasoconstriction capillaire diffuse. Nous avons montré que l'ensemble de ces phénomènes étaient réversibles par traitement intravitréen de son inhibiteur le Fasudil. De manière importante le traitement par Fasudil induit également une diminution du VEGF rétinien responsable de la perméabilité des barrières et témoin indirect de l’ischémie rétinienne. Ces travaux éclairent la physiopathogénie de la RD et ouvre des perspectives thérapeutiques permettant de cibler les événements précoces de la RD. / Diabetic retinopathy (DR) mainly results from peripheral retinal ischemia and exudation leading to sight threatening complications such as retinal neovascularization or macular edema. This latter represents the main cause of visual loss among working age individuals. Current treatments address late complications of DR and are non-specific. Therefore, early events are currently not addressed. Chronic hyperglycemia increases oxidative stress and activates PKC leading to blood retinal barrier (BRB) breakdown. The aims of the present work were two fold. First, to assess the implication of PKCζ and the Rho/ROCK/Myosin II pathway on the pathogenesis of DR and second, to assess whether their specific inhibitors have the potential to restore the phenotype. Herein we have demonstrated the pathogenic role of PCKζ and ROCK hyperactivation on the development of diabetes induced external BRB breakdown. Furthermore their inhibitors restored the morphologic and functional aspect of the external BRB. We also found that ROCK hyperactivation was responsible for altered retinal perfusion through several mechanism namely 1) focal constriction of retinal arterioles; 2) endoluminal protrusions of the endothelial cell membrane (blebs) and 3) capillary diffuse vasoconstriction. We were able to demonstrate that all this aspects were reversible by Fasudil, a ROCK inhibitor, administrated into the vitreous. Of importance this treatment decreased also retinal VEGF that is a well-known factor responsible for barrier breakdown and a surrogate marker for retinal ischemia. To conclude the present findings not only shed light on the mechanisms of DR but also open new therapeutic avenues addressing the early events of DR a current unmet medical need.

Diabetic retinopathy is an ischemic retinal neovascular disease causing vision loss among adults. The studies presented involve the design and testing of a gene therapy vector to inhibit retinal revascularization, similar to that found in diabetic retinopathy. Gene therapy has proven to be an effective method to introduce therapeutic proteins to treat retinal diseases. Targeting a specific cell type and expression of therapeutic proteins according to the tissue microenvironment should have an advantage over traditional gene therapy by avoiding unwanted transgene expression. Hypoxia plays a significant role in the pathophysiology of many retinal ischemic diseases. Retinal Mèuller cells provide structural and functional support to retinal neurons, as well as playing a significant role in retinal neovascularization. Targeting Mèuller cells may be an effective strategy to prevent retinal neovascularization under pathological conditions. ... The hypoxia regulated, glial specific vector successfully reduced the abnormal neovascularization in the periphery by 93% and reduced the central vasobliterated area by 90%. A substantial amount of exogenous endostatin was produced in the retinas of P17 OIR mice. A significant increase in human endostatin protein and reduced vascular endothelial growth factor (VEGF) were identified by Western blot and ELISA, respectively. These findings suggest hypoxia-regulated, glial cell-specific scAAV mediated gene expression may be useful to prevent blindness found in devastating retinal diseases involving neovascularization. / by Manas Ranjan Biswal. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Su Huan Xing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 79-89). / Abstracts in English and Chinese. / Abstract --- p.i / 中文摘要 --- p.iii / Acknowledgements --- p.iv / Abbreviations Frequently Used --- p.v / Table of contents --- p.vi / Chapter Chapter1 --- General Introduction --- p.1 / Chapter Chapter2 --- Effects of an intravitreal optic nerve graft on the sprouting and regeneration of axotomized retinal ganglion cells --- p.17 / Chapter Chapter3 --- Effects of an intravitreal pre-injured optic nerve graft on the sprouting and regeneration of axotomized retinal ganglion cells --- p.44 / Chapter Chapter4 --- Effects of co-transplantation of an optic nerve graft and a peripheral nerve graft into the vitreous body on the sprouting and regeneration of axotomized retinal ganglion cells --- p.60 / Chapter Chapter5 --- General discussion --- p.74 / References --- p.79 / Tables --- p.90

Retinal hypoxia is associated with many retinal diseases, such as diabetic retinopathy. Current retinal research suggests that retinal hypoxia appears prior to the onset of diabetic retinopathy. The preliminary association of retinal hypoxia to the early stages of diabetic retinopathy is stimulating the development of new technologies to measure the oxygen content of retinal tissue. Frequency domain phosphoresence lifetime imaging (PLI) is a promising technology that enables the mapping of the oxygen content across the entire retina in the form of two-dimensional images. The two-dimensional images generated from the PLI process are a spatial mapping of the retinal tissue's oxygen tension. Currently, the phosphorescent based oxygen tension PLI measurements contain contaminating auto-fluorescent signals in addition to the desired phosphorescent signals. These auto-fluorescent signals artificially inflate the oxygen tension readings due to the nature of fluorescent signals in phosphorescent imaging. Additionally, the maps generated through PLI appear to contain oxygen tension information from both the retinal vasculature and the choroidal vasculature. The choroidal vasculature is situated directly behind the retina and can have a different oxygen tension value than the retinal vasculature. This research enhanced the PLI system by mathematically eliminating the contaminating auto-fluorescent signals and investigated the methods aimed at separating the PO2s of the retinal and choroidal vasculature beds. In addition, the application of the enhanced PLI technology to the investigation of retinal oxygen changes in a rat model of type I diabetes yielded results that suggest a hyperoxic to hypoxic trend prior to the onset of diabetic retinopathy.

Ocular fundus image can provide information on pathological changes caused by local ocular diseases and early signs of certain systemic diseases. Automated analysis and interpretation of fundus images has become a necessary and important diagnostic procedure in ophthalmology. Among the features in ocular fundus image are the optic disc, fovea (central vision area), lesions, and retinal vessels. These features are useful in revealing the states of diseases in the form of measurable abnormalities such as length of diameter, change in color, and degree of tortuosity in the vessels. In addition, retinal vessels can also serve as landmarks for image-guided laser treatment of choroidal neovascularization. Thus, reliable methods for blood vessel detection that preserve various vessel measurements are needed. In this paper, we will examine the pathological issues in the analysis of retinal vessels in digital fundus images and give a survey of current image processing methods for extracting vessels in retinal images with a view to categorize them and highlight their differences and similarities. We have also implemented two major approaches using matched filter and mathematical morphology respectively and compared their performances. Some prospective research directions are identified. / Singapore-MIT Alliance (SMA)

Experimental and clinical data have documented the adverse effects of perinatal ethanol intoxication on peripheral organs and the central nervous system. There is little known, however, about potential damaging effects of perinatal ethanol on the developing visual system. The purpose of this study was to examine the effects of neonatal ethanol intoxication on RGC morphology, estimate the total number of neurons in RGC layer and dorsolateral geniculate nucleus (dLGN), and on the eye-specific fiber segregation in the dLGN), in YFP and C57BL/6 mice pups. Ethanol (3 g/kg/day) was administered by intragastric intubation throughout postnatal days (PD) 3-20 or 3-10. Intubation control (IC) and untreated control (C) groups were included. Blood alcohol concentration (BAC) was measured in separate groups of pups on PD3, PD10, and PD20 at 4 different time points, 1, 1.5, 2 and 3 h after the second intubation. Numbers neurons in the RGCs and dLGN were quantified on PD10, PD20 using unbiased stereological procedures. The RGC images were taken using a confocal microscope and images were traced using Neurolucida software. On PD9, intraocular injections of cholera toxin-

The extent to which cortical maps may reorganize in adult humans is a significant and topical debate in visual neuroscience. Though there are conflicting findings, evidence from humans and animals indicates that the topography of the visual cortex may change after retinal deafferentation. Remarkably, this reorganization seems to be possible in adults, whose brains are less amenable to plastic change. If adult visual reorganization is legitimate, an understanding of its causes and consequences could be profound considering the millions suffering from age-related visual disorders. This dissertation explores whether visual training may yield a reorganization of sensory maps in the adult visual cortex. It describes research in which patients, diagnosed with age-related macular degeneration (AMD), underwent visual rehabilitation therapy. Functional brain scans and behavioral tests were conducted pre and post training. These interventions generated valuable knowledge regarding whether "reorganized" activity is a true rewiring of feed forward cortical processes or an artifact of attentional feedback. The rehabilitation training produced demonstrable differences in activation patterns along the primary visual cortex (V1), but sparse improvement in the behavioral tests. In contrast, there was significant improvement in fixation tests which assessed oculomotor control. These results suggest that the nature of reorganized activity has more to do with attentional mechanisms than feed forward reorganization. Future investigations could benefit from examining the brain sites that govern visual attention in the frontal and parietal cortices. These areas may have more to do with visual adaptation in AMD patients than V1.