Retinose Pigmentar (RP) é uma doença hereditária que conduz progressivamente à cegueira. Mais de 150 mutações da rodopsina associadas à RP foram descritas, e causam a alteração da sua conformação. Esta tese testou a hipótese de que pequenas moléculas auxiliam na formação da rodopsina e/ou reduzem a morte dos fotorreceptores. As mutações da RP, N15S e P23H, revelaram diferenças quanto às características e gravidade devido à má-formação das proteínas mutantes. Ligação de pequenas moléculas (retinóides, íons metálicos, clorofilas e antocianinas) à rodopsina foi demonstrada in vitro. O derivado da clorofila, Ce6, mostrou-se mais efetivo, conferindo maior estabilidade e foi então testado em ratos submetidos à degeneração por luz ou em modelos de RP (P23H e S334ter). Observou-se uma proteção contra a degeneração por luz e uma significante diminuição da degeneração no P23H. Em contraste, Ce6 causou um aumento na degeneração dos fotorreceptores do S334ter. Finalmente, resultados clínicos, bioquímicos e in vivo foram comparados e mostraram estar altamente relacionados. / Retinitis Pigmentosa (RP) is an inherited disease that progressively leads to blindness. More than 150 mutations associated with RP are known in rhodopsin, causing its misfolding. This thesis tested the hypothesis that small molecules can rescue folded rhodopsin and/or reduce photoreceptor cell death. RP mutations, N15S and P23H, revealed differences in characteristics and severity of misfolding of the mutant proteins. Binding of small molecule classes (retinals, metal ions, chlorophylls and anthocyanins) to rhodopsin was demonstrated in vitro. The chlorophyll derivative, Ce6, was most effective in conferring stability and therefore tested in rats subjected to light-damage and RP rat models, P23H and S334ter. Protection against the light-induced retinal degeneration and more importantly a significant slowing of the photoreceptor degeneration rate in the P23H rat were observed. In contrast, Ce6 increased photoreceptor degeneration in the S334ter rat. Finally, clinical, biochemical and in vivo rat data were compared and it was found to be highly correlated.

Estudos prévios do nosso laboratório têm mostrado a presença e importância das GTPases Rho, proteínas de baixo peso molecular que são ativadas por diversos sinais extracelulares, em neurônios da retina adulta, durante o desenvolvimento e na degeneração induzida por luz. O objetivo deste trabalho foi determinar por meio de imunocitoquímica a distribuição de alguns membros desta família (RhoA, Rac1 e Cdc42) nos fotorreceptores da retina de camundongos BalbC em situações normais e de adaptação ao claro e ao escuro. Os resultados mostraram que Rac1, Cdc42 e RhoA são expressos principalmente no segmento interno e nas possíveis terminações de cones e bastonetes na camada plexiforme externa. Em condições diferentes de estimulação não ocorreu translocação das GTPases, como ocorre com outras proteínas envolvidas na fototransdução. Os resultados sugerem que elas possam desempenhar papéis importantes na reorganização do citoesqueleto ou no processo de fototransdução, regulando outras proteínas e/ou protegendo contra danos induzidos pela luz. / Previous studies from our laboratory have shown the presence and importance of the Rho family of small GTPases, proteins that are activated by different extracellular signals, in neurons of the adult retina, during development and light-induce degeneration. The aim of the present work was to determine by immunocytochemistry the distribution of RhoA, Rac1 and Cdc42 in photoreceptors of the BalbC mice retina in different conditions of light/dark adaptation. Our results showed that Rac1, RhoA and Cdc42 are mainly expressed in the inner segments and presumptive axon terminals of cones and rods in the outer plexiform layer. In addition, there was no evidence of Rho GTPases translocation under different conditions of stimulation, as shown for some phototransduction proteins. The results suggest that Rho GTPases may play important roles in the reorganization of the cytoskeleton or in the phototransduction process, regulating other proteins and/ or protecting against light-induced damage.

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Plants absorb light for photosynthesis, but not all is used. Excess light energy may lead to photoinhibition of photosynthesis and irreversible photooxidative damage. Plants have evolved mechanisms for energy dissipation under high light stress. One such response may involve production of ELIP. It is of interest to know what signal(s) may be involved in ELIP expression. My hypothesis is that redox status of the chloroplast photosynthetic electron transport Chain (PETC) and/or chlororespiration may induce ELIP expression. Using the Arabidopsis thaliana immutans (im) chlororespiratory mutant, this hypothesis was tested. Etiolated seedlings of this variegated mutant were subjected to various light intensities over 0-24 hr period and ELIP mRNA levels were analyzed. These were compared with the wild type plants treated in the same manner. It was found that mature thylakoids may not be required for ELIP expression, and that both photoreceptor-dependent and independent components may be involved in ELIP expression.

Models of sensory processing have historically abstracted underlying biological circuits, due to unknown connectivity and/or complexity. In contrast, the use of tractable and anatomically well-characterized model organisms such as the fruit fly Drosophila melanogaster allows us to utilize biological constraints in models of sensory processing to understand underlying circuit mechanisms and make more accurate predictions. This approach has been used to dissect motion vision circuits, but investigations into color vision - a salient visual feature for many animals - have been limited. Here, we investigate the circuit mechanisms of the early color circuit of the fruit fly and assess its information processing capabilities. Using in vivo two-photon calcium imaging and genetic manipulations, we measure the chromatic tuning properties of photoreceptor axons and their primary targets in the medulla neuropil. At the level of photoreceptor axons, we show that opponent processes are the result of a dual mechanism - a direct pathway specific to insect physiology and an indirect pathway found across the animal kingdom. Both pathways are necessary to decorrelate incoming signals and efficiently represent chromatic information. We built an anatomically constrained model that is able to quantitatively reproduce these color opponent responses without fitting synaptic weights. Instead, we used electron-microscopy-derived synaptic count, an anatomically defined measure, as a proxy for synaptic weight, thereby linking structure to function. Downstream of photoreceptors, we find that neurons shift their tuning and become highly selective for particular directions in color space - similar to “hue-selective” neurons in primate cortex. To achieve this selectivity, these neurons require input from all types of photoreceptors and an interneuron that determines the neuron's preferred chromatic direction. We extended our anatomically constrained model to incorporate these downstream neurons and are able to predict their responses, qualitatively and quantitatively.In summary, the detailed reconstruction of the fly circuit anatomy predicts the mechanisms of multiple stages of color information processing and allows us to infer functional roles for each part of the circuit. The circuit motifs, we uncover, are shared across species and hint at convergent mechanisms that underlie the transformation from an opponent neural code to a hue selective code.

Light regulates many biological processes through light-sensitive proteins called opsins. Opsins are involved in vision, but they are also expressed in extraretinal tissue, where their roles are far less clear. Fish have large opsin repertoires, derived from a long history of gene duplication and divergence, making them useful models to study opsin diversity and function. I introduce the deep-sea sablefish (Anoplopoma fimbria) as a model for opsin research for three main reasons: i) the availability of a draft genome and transcriptome, simplifying the characterization of this species’ opsin repertoire, ii) the proximity of the only sablefish aquaculture facility in the world, providing exclusive access to a large number of individuals at all developmental stages, iii) the observation that sablefish occupy very different light environments during the course of development, ranging from well-lit shallow waters to the aphotic zone, which provides a light environment context for opsin gene expression data. My survey of the genome showed that sablefish have 36 distinct opsin genes (7 visual and 29 non-visual), even though they spend most of their lives in the dark. The sablefish opsin sequences and repertoire are similar to those of other teleost fish. To test the hypothesis that the sablefish opsin repertoire is being expressed/transcribed during the comparatively brief period of time when this species is exposed to light (the free-swimming larval stage through to the juvenile stage), I quantified the expression of five paralogous genes from a well-studied non-visual opsin family (OPN4’s) in the brain across life stages. Data show statistically stable expression of Opn4m1 and Opn4m3 among life stages, a rough association of Opn4x1 and Opn4m2 expression with age and light environment, and little-to-no expression of Opn4x2. I localized proteins encoded by the most highly expressed class of OPN4 genes in the brain, the Opn4m genes, to the surface of the optic tectum just below a cranial ‘window’; a zone that has been shown to express dozens of opsins in zebrafish (a distant relative, with their ancestor diverging more than 230 million years ago). Thus, in some cases, expression appears to be correlated with light exposure not only temporally, but also spatially. By studying non-visual opsins in sablefish, I have challenged and broadened the current understanding of opsin evolution and function in fish and provided the foundation for future studies to test brain regions for light-sensitivity, perform opsin gene knock-outs, and explore potential light-independent processes. / Graduate / 2023-09-06

A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.

Little is known about the visual capabilities of marine turtles. The ability to discriminate between colors has not been adequately demonstrated on the basis of behavioral criteria. I used a three-part methodology to determine if color discrimination occurred. FIrst, I exposed naèive, light-adapted hatchlings to either a blue, green or yellow light. I manipulated light intensity to obtain a behavioral phototaxis threshold to each color, which provided a range of intensities we knew turtles could detect. Second, I used food to train older turtles to swim toward one light color, and then to discriminate between the rewarded light and another light color ; lights were presented at intensities equally above the phototaxis threshold. Lastly, I varied light intensity so that brightness could not be used as a discrimination cue. Six turtles completed this task and showed a clear ability to select a rewarded over a non-rewarded color, regardless of stimulus intensity. Turtles most rapidly learned to associate shorter wavelengths (blue) with food. My results clearly show loggerheads have color vision. Further investigation is required to determine how marine turtles exploit this capability. / by Morgan Young. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.