La rétine est un tissu spécialisé dans le traitement de l'information visuelle par l'intermédiaire des photorécepteurs, cônes et bâtonnets, et des neurones de deuxième ordre, les cellules bipolaires et les cellules ganglionnaires dont les axones forment le nerf optique. Notre groupe s'intéresse à élucider les mécanismes génétiques impliqués dans les maladies rares stationnaires, comme dans la cécité nocturne congénitale stationnaire (CNCS), ou progressives comme dans la dystrophie de type bâtonnet-cône (DBC). Cette thèse apporte de nombreuses connaissances sur la physiologie rétinienne. D'une part, nous avons identifié GPR179, un nouveau gène impliqué dans la CNCS complète, étudié la localisation de la protéine et la physiopathologie des protéines mutantes. Nous avons également créé et caractérisé fonctionnellement un nouveau modèle souris invalidé pour GPR179 qui nous a permis de mieux approcher la première synapse rétinienne entre les photorécepteurs et les cellules bipolaires adjacentes. D'autre part, nous avons caractérisé le génotype et le phénotype de l'un des modèles les plus utilisés de la DBC, le rat P23H. Nous avons ensuite développé une approche d'édition génomique pour invalider les mutants RHO ayant un effet dominant négatif en testant in vitro, ex vivo et in vivo les meganucleases, TALEN (Transcription Activator-Like Effector Nuclease) puis le système CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9). / The first steps in vision occur in the retina when rod and cone photoreceptors transform light into a biochemical signal, which gets processed by bipolar cells, ganglion cells and finally by the brain. Our group investigates genetic causes and mechanisms involved in inherited stationary and progressive retinal diseases as congenital stationary night blindness (CSNB), and rod-cone dystrophy (RCD), also called retinitis pigmentosa. This thesis gives several insights on the retinal physiology. On one hand, we identified GPR179, a new gene mutated in complete CSNB, studied the localization and the physiopathology of missense and splice-site mutations. We also delivered a new knock-out mouse model which we functionally characterized, and studied GPR179 partners to provide a better understanding of the first visual synapse between photoreceptors and ON-bipolar cells. On the other hand, we genotypically and phenotypically characterized one of the most popular RCD model, the P23H rat model. There is currently no treatment for RCD and different therapeutic strategies are under investigation. We wanted to deliver the basis for a genome editing approach for RHO mutations, acting as a dominant negative effect, which cannot be addressed by current gene replacement strategies. We opened the field by performing in vitro, ex vivo and in vivo genome editing experiments using meganucleases, TALEN (Transcription Activator-Like Effector Nuclease) and finally CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) and revealed how challenging the setting of genome editing strategies was.

L’information visuelle est transmise de la rétine au cerveau par les cellules ganglionnaires. Il existe plusieurs types de cellules ganglionnaires, chaque type formant une mosaïque qui couvre l’intégralité de la scène visuelle. Comprendre la manière dont ces neurones encodent collectivement la scène visuelle est essentiel pour au moins deux raisons: - La manière dont une population de neurones encodent collectivement une information sensorielle reste jusqu’à aujourd’hui mystérieuse. La rétine est un système idéal pour étudier cette question: elle a en effet une structure en couches 2D qui se prête idéalement à l’enregistrement d’une population complète de neurones à grande échelle, et elle opère une transformation complexe de la scène visuelle. - Certaines maladies qui mènent à la cécité ne connaissent aujourd’hui pas de traitement. Plusieurs stratégies de restauration visuelle basées sur la stimulation directe de cellules ganglionnaires sont le sujet de recherches actives. Il pourrait être nécessaire de reproduire en imitant le code neural produit par la rétine pour optimiser les résultats de ces stratégies thérapeutiques. Au cours de ma thèse, j’ai travaillé sur deux questions complémentaires, qui sont liées à ces deux sujets respectivement. / Visual information is conveyed from the retina to the brain through ganglion cells. Ganglion cells are divided in different cell types, and each of them form a mosaic sampling the entire visual scene. Understanding how these neurons encode the visual scene is essential for at least two reasons: -It is still unclear how a population of neurons collectively code sensory information. The retina is an ideal system to study this issue: while it performs complex processing on the visual stimulus, its 2-D structure makes it suitable for large-scale recordings of complete populations of neurons. -Some diseases leading to blindness have currently no cure. Several visual restoration strategies based on the direct stimulation of ganglion cells are currently being investigated. Emulating the retinal code may be necessary to optimize the results of these therapeutic approaches. In my thesis I have worked on two complementary questions, that are related to these two topics.

Abstract Glaucoma is a progressive neuropathy of the optic nerve. It causes degeneration of ganglion cell axons resulting in defects in the retinal nerve fibre layer (RNFL) and characteristic changes in the optic nerve head (ONH). The Heidelberg Retina Tomograph (HRT) is a confocal scanning laser imaging device, which measures the topography of the ONH and the adjacent RNFL. To quantify the measurements of the ONH topography, various stereometric parameters are calculated. The change in the stereometric parameters of the HRT was studied in 34 eyes with glaucomatous progression in RNFL photographs and 34 eyes without progression. The change in only one stereometric parameter, the cup shape measure, showed a statistically significant correlation with the progression of the RNFL defect. An optimised change in the best three-parameter combination had 77% sensitivity and 79% specificity for progression. The change in the stereometric parameters was compared in 51 eyes with glaucomatous progression in stereoscopic ONH photographs and 425 eyes without progression. The parameters having the best correlation with progression include cup:disc area ratio, vertical cup:disc ratio, cup volume and rim area. The parameter with the largest area under the receiver operating characteristics curve (0.726) was the vertical cup:disc ratio. A change of 0.007 in the vertical cup:disc ratio had a sensitivity of 80% and a specificity of 65% for progression. The factors having the most significant effect on the sensitivity and specificity of the stereometric parameters for progression were the reference height difference and the mean topography standard deviation, indicating image quality. The change in image quality and age also showed a consistent, but variably significant influence on all parameters tested. Exercise was associated with an increase in variance in 17 of the 18 stereometric parameters. In conclusion, the change in the stereometric parameters provides useful information on ONH topography, especially when image quality is excellent. However, the evaluation of glaucomatous progression should not rely solely on the stereometric parameters of the HRT.

Recent advances in retinal imaging have made it possible to take measurements of retinal oxygen saturation noninvasively in humans. This allows studying the supply of oxygen in healthy and diseased retinae, thereby advancing our understanding of both the normal functioning of the retina and of retinal pathologies. However, retinal oximetry is still a research tool only and requires further improvement before being used in a clinical setting. Here, a single-wavelength flickering light was used to increase retinal blood flow in healthy subjects. This increase is revealed by both vasodilation and an increase in retinal oxygen saturation. A flickering light stimulus provides the means to assess the sensitivity of any retinal oximetry system, as such systems should be able to pick up this increase in retinal blood flow. In addition, the flickering light allows for com- parison to be made within rather than between subjects and can be used to examine the activation of the eye. This reduces the influence of potential confounding factors between subjects including differences in fundus pigmentation and illumination. The most commonly used method to measure retinal oxygenation is the optical density ra- tio (ODR) approach. The standard approach is to compute the average ODR for each vessel segment by combining the hundreds of individual ODR readings and then to use the mean of these segment averages as a measure of oxygen saturation. Alternatively, it has been suggested that the peak location of Gaussian functions fitted to histograms of individual ODR readings can be used as an measure of retinal oxygenation. In response to a 10Hz flickering light, the venular diameter increased by 3.44% (SEM: ±0.53%) (n=16, p<0.05) and the arteriolar diameter by 1.87% (±0.72 %) (p<0.05). The optical density ratio, measured with the Gaussian fit, decreased in the venules from 0.713 (±0.015) to 0.694 (±0.015) (p<0.05). No changes in arteriolar optical density ratios were measured. The post-flicker measurement was computed as the average of up to four post-flicker datasets obtained at 10s, 20s, 30s and 40s after onset of flickering. These results suggest that the flickering light increased retinal blood flow. The mean absolute percentage error was lower in venules for the Gaussian fit method than for the gold standard method for datasets taken at 30s and 40s after onset of flickering. Thus, the Gaussian fit method was more robust. All measurements were taken with a custom-made retinal oximeter. The pixel intensity of the blood vessel and the intensity on either side of the vessel had to be extracted to compute the individual optical density ratios. This required the automatic extraction of the retinal vasculature. Two such algorithms were developed and applied to two databases of retinal fundus images: the DRIVE and the novel DR HAGIS database. One algorithm was purely based on the pixel intensities, while the other made use of oriented Gabor filters. These two algorithms segmented the images to a similar accuracy (DRIVE: 94.56% and 94.54%, DR HAGIS: 95.83% and 95.71% for the intensity and Gabor filter based algorithm, respectively) and performed as well as a human expert (DRIVE: 94.73%). These algorithms were of sufficient quality to extract individual segments for the oximetry study and to align fundus images.

L’impact laser obtenu avec les systèmes actuels de photocoagulation laser de la rétine n’est pas maitrisé. L’enjeu est d'obtenir un confinement 3D de l'impact laser, c’est-à-dire de contrôler son positionnement et son extension, pour éviter de porter atteinte aux couches saines de la rétine. Un tel confinement pourrait être réalisé si l’on disposait d’un système laser plus ouvert et d’un dispositif permettant de corriger en temps réel les aberrations de l'œil. L’optique adaptative permet une telle correction ; cette technique est utilisée depuis une vingtaine d’années dans le domaine du diagnostic (imagerie). Cependant, sa mise en œuvre pour des applications thérapeutiques, telles que la photocoagulation laser, nécessite encore d’en améliorer la robustesse : alors qu’en imagerie il suffit de reprendre une image si la précédente est mauvaise, le confinement de l’impact laser doit être assuré en permanence durant toute la procédure chirurgicale. Le but de la thèse est de guider le développement ultérieur d’un système d’optique adaptative médical visant à assister un système de photocoagulation laser de la rétine. Le dimensionnement d’un tel système doit s’appuyer sur une solide connaissance des aberrations oculaires. Ainsi, une étude hautement résolue temporellement et spatialement des aberrations dynamiques de l’œil de grande ampleur a été réalisée et de cette étude, des conclusions sur le dimensionnement d’un système d’optique adaptative pour l’œil ont été tirées. Par ailleurs, un banc de test d’optique adaptative haute cadence a été conçu et intégré pour se confronter aux problèmes pratiques que pose la mise en place d’un système d’optique adaptative pour l’œil. / The laser lesion delivered by current retinal photocoagulation laser systems is not well controlled. The issue is to get a better confinement of the laser lesion that is to control the size of the laser lesion as well as the laser lesion positioning in the retina, in order to prevent any retinal damage. Such a confinement could be reached if the laser system featured a higher numerical aperture and was associated with a real-time correction of the aberrations of the eye. Adaptive optics gives access to such a correction; this technique has been used for the past twenty years for diagnosis (retinal imaging). However, further work is still to be achieved to improve the robustness of current adaptive optics systems before implementing adaptive optics in therapeutic systems, in particular in retinal laser photocoagulation systems. Indeed, unlike imaging where the acquisition can be repeated as many times as necessary, the confinement of the laser lesion must be maintained over time during the whole laser treatment. In this thesis, we provide guidance for the future design of an adaptive optics system for retinal photocoagulation. Such a design has to rely on a thorough knowledge of the ocular aberrations to correct. Thus, a highly temporally as well as spatially resolved aberrometry study on a large population was performed and conclusions on the design of an adaptive optics system for the eye were drawn from this study. Besides, a test bench was designed and set up to face the practical problems coming with the implementation of an adaptive optics system for the eye.

La rétine est constituée de l’association d’un tissu neurosensoriel et de l’épithélium pigmentaire rétinien (EPR). Malgré la présence d’une barrière hémato-rétinienne, la rétine est contrainte aux changements de son milieu environnant, incluant les modifications du régime alimentaire et les stress environnementaux. Au cours du vieillissement, les échanges de nutriments et l’élimination des déchets métaboliques et cellulaires au travers de l’EPR diminuent. La dégénérescence maculaire liée à l’âge (DMLA) et la rétinopathie diabétique (RD) sont les pathologies rétiniennes les plus prévalentes dans les populations occidentales avant et après l’âge de 50 ans, respectivement. L’atrophie géographique et la DMLA néovasculaire constituent les stades avancés des maculopathies liées à l’âge. La RD affecte 60% des patients atteints de diabète de type 2 (T2D) dans les 15 années suivant l’apparition de la pathologie. Par ailleurs, les facteurs alimentaires interfèrent avec le développement de la DMLA et du T2D. Considérant l’épidémie mondiale de T2D d’une part, et l’allongement de l’espérance de vie d’autre part, les dépenses médico-sociales relatives à la prise en charge des patients atteints de T2D ou de DMLA sont à même de devenir un enjeu socioéconomique majeur. Le syndrome métabolique (SMet) est un des principaux facteurs de risque du T2D. Il peut ainsi être suggéré que limiter le développement du SMet pourrait potentiellement limiter l’incidence du T2D et de ses complications. Dans ce contexte, la consommation d’acides gras polyinsaturés à longue chaîne (AGPI-LC) de type oméga 3 fait partie des recommandations nutritionnelles pour la population afin de prévenir l’apparition du SMet et du T2D. Par ailleurs, une alimentation riche en AGPI-LC omega-3 est également associée à la réduction du risque de DMLA. En revanche, l’association entre T2D, RD et DMLA reste controversée, bien que quelques études basées sur des grandes cohortes rapportent une prévalence de DMLA augmentée chez les patients atteints de diabète ou de RD. Nos objectifs ont été d’évaluer premièrement si un SMet constituait un environnement favorable au développement des complications de type néovasculaires dans la rétine, et de tester secondairement l’efficacité d’AGPI-LC omega-3 dans la réduction des conséquences de ce SMet sur la rétine. Pour atteindre cet objectif, des rats ont été nourris avec un régime diabétogène enrichi en fructose afin de provoquer un SMet. Une néovascularisation choroïdienne a ensuite été induite en utilisant un modèle d’impacts laser au fond d’œil. Nous nous sommes intéressés tout d’abord à l’effet de ce régime à court terme et avons mis en évidence une diminution de la sensibilité des photorécepteurs de type cônes après 8 jours de régime, ainsi qu’une modification de l’expression des gènes et en particulier des sous-familles de cristallines. Ensuite, nos études se sont portées sur les effets de régime enrichi en fructose à long terme jusqu’à 6 mois. Le développement d’un SMet a été illustré par une augmentation de la masse grasse, une hyperinsulinémie, une hyperleptinémie et l’installation d’une stéatose hépatique. Ces rats ont développé une néovascularisation choroïdienne exacerbée après 1 et 3 mois de régime associée à une surexpression de facteurs pro-angiogéniques tels que VEGF et leptine .Une infiltration de macrophages dans la rétine et/ou l’activation des cellules microgliales résidentes a également été détectée. Les données électrorétinographiques ont suggéré une diminution de la sensibilité des photorécepteurs de type bâtonnets ainsi qu’une altération des fonctionnalités des cellules de la rétine interne à 6 mois. Dans un second temps, l’efficacité des AGPI-LC omega-3 (EPA+DHA) dans le but de réduire les conséquences du SMet dans la rétine a été testée. Nos données mettent en évidence qu’une forte dose d’EPA+DHA n’a cependant pas amélioré le SMet chez le rat. / The retina is the association of the neurosensory tissue and the retinal pigment epithelium (RPE). Despite the presence of the blood retinal barrier, the retina is submitted to changes of the external milieu, including dietary modulation and environmental stresses. With advanced age, the exchanges of nutrients and elimination of cellular and metabolic wastes via the RPE become limited. Age-related Macular Degeneration (AMD) and Diabetic Retinopathy (DR) are the most prevalent retinal pathologies in Western adult populations before and after the age of 50 years, respectively. Geographic atrophy and neovascular AMD are advanced stages of age-related maculopathies. DR afflicts 60% of type 2 diabetic (T2D) patients in the first 15 years of the disease. Dietary factors interfere in the development of both AMD and T2D. Accounting the worldwide epidemics of T2D in the one hand, and the improvement of life expectancy in the other hand, medical care to the patients is expected to worsen the socioeconomic burden of both T2D and AMD. Metabolic syndrome (MetS) is one of the major risk factor for T2D. Lowering the development of MetS would potentially lessen the incidence of T2D, and its complications. The daily intake of omega 3 long chain polyunsaturated fatty acids (LC-PUFA) is now recommended by health agencies for the prevention of MetS. Meanwhile dietary omega 3 LC-PUFA are associated with reduced risk of AMD. The association between T2D, DR, and AMD remains controversial, although large-scale population-based studies have reported increased prevalence of AMD in patients with diabetes or DR. Our objectives were first to evaluate whether MetS would represent a favorable environment for the development of neovascular complications in the retina, and second to test the efficacy of omega 3 LC-PUFA to reduce the consequences of MetS in the retina. For that purpose, a pro-diabetogenic high fructose diet was fed to rats to induce MetS and choroidal neovascularization was triggered by laser impacts in the eye fundus. We focused first on short term diet periods, and showed impairment on cone photoreceptor sensitivity after 8 days, as well as changes in gene expression in relation to crystallin sub-families. A long term - up to 6 months - fructose diet period triggered MetS as illustrated by body fat increase, hyperinsulinemia, hyperleptinemia and liver steatosis. Rats exhibited exacerbated laser-induced choroidal neovascularization after 1 and 3 months of feeding, that was associated with up-regulation of genes coding pro-angiogenic factors such as VEGF and leptin, as well as infiltration of macrophages and/or activation of retinal microglia. Electroretinographic data showed decreased sensitivity of rod photoreceptors and inner retinal cell functionality at 6 months of feeding. In a second time, the efficacy of dietary omega 3 LC-PUFA (EPA plus DHA) to reduce the consequences of MetS in the retina was tested. Our data showed that a high dose of EPA+DHA in rats did not improve MetS. Furthermore, side effects were generated as illustrated by localized atrophy in the retina submitted to the combination of laser-impacts, and normal light exposure. These works allowed us to suggest that MetS generated a favorable environment in the retina for the development of neovascular complications. Meanwhile, the sensitivity of cones and rods was impaired by MetS. Accounting the deleterious long term effects of omega 3 LC-PUFA in the retina, caution may be taken while recommending massive supplementation with omega 3 LC-PUFA in the context of MetS.

Cette thèse s'inscrit dans le contexte de mieux comprendre l'effet de la température ainsi que celui de l'acide docosahexaénoïque (DHA) alimentaire dans les performances de vision chez les juvéniles de Dicentrarchus labrax. La rétine est contrainte aux changements de son milieu environnant, incluant les modifications du régime alimentaire et les stress environnementaux comme la température. Par ailleurs, le loup de Méditerranée est une espèce ectotherme, carnivore et pélagique. Son métabolisme, sa physiologie et son comportement dépendent de la température extérieure mais aussi de la composition alimentaire en acides gras et plus spécialement en DHA. Cette étude a rapporté l'impact de l'augmentation (28°C) et de l'abaissement (18°C) de la température du milieu mais aussi l'influence des différentes concentrations de DHA alimentaire sur la composition lipidique ainsi que l'abondance d'ARNm des enzymes clés du métabolisme lipidiques (FADS 2, ELOVL 5, SREBP-1, PLA 2 et la triglycéride lipase) et l'expression des gènes liés à la fonction visuelle (rhodopsine) et à la production de la mélatonine. / This thesis is in the context of more understanding the effect of temperature and different dietary levels of docosahexaenoic acid (DHA) in vision performance of juveniles Dicentrarchus labrax. The retina is constrained by environmental change, including changes in diet and environmental stresses such as temperature. In addition, sea bass is an ectothermic, carnivorous and pelagic species. Their metabolism, physiology and behavior rely on the external temperature but also on the dietary composition of fatty acids and especially DHA. This study reported that the impact of the increase (28°C) and decrease (18°C) of the temperature of the water but also the influence of different dietary DHA concentrations on the lipid composition, abundance of ARNm of key lipid metabolism enzymes (FADS 2, ELOVL 5, SREBP-1, PLA 2 and triglyceride lipase) and the expression of the genes related to the visual function (rhodopsin) and the production of melatonin.

The precise structure of the adult Drosophila eye results from a coordinated process of cell sorting, differentiation and selective cell death in the retinal epithelium. Mutations in the gene echinus cause supernumerary pigment cells due to insufficient cell death. This study reports the identification of echinus and the characterization of its role in Drosophila retinal development. Using a combination of deletion mapping, gene expression analysis and genomic sequencing, echinus was cloned and several alleles were sequenced. echinus encodes a ~180kDa protein containing an ubiquitin hydrolase domain at its N-terminus and a polyglutamine tract at its C-terminus. echinus is expressed in the retina during pupal development and mutants of echinus have decreased levels of apoptosis during several stages of retinal development. Defects in the cell sorting process that precedes cell death are also observed in echinus loss-of-function mutants and echinus overexpression can cause defects in ommatidial rotation and the morphology of cone cells. echinus is a positive regulator of DE-cadherin and Enabled accumulation in adherens junctions of retinal epithelial cells. Genetic interactions were observed between echinus and the genes wingless, enabled and expanded. An immunofluorescence assay in Drosophila S2 cell cultured demonstrated that Echinus localizes to intracellular vesicles that do not appear to be endocytic in nature, and the C-terminal region of Echinus was shown to be necessary for this association. A protein interaction screen using an immunoprecipitation and mass spectrometry approach identified interactions between Echinus and the vesicle coat protein Clathrin, the scaffolding protein RACK1 and the casein kinase I epsilon (Dco). Co-immunoprecipitation additionally identified an interaction between Echinus and Enabled. This work has revealed echinus to be an important regulator of cell sorting and adherens junction formation in the developing retina and has identified multiple interactions between echinus and enabled, a regulator of the actin cytoskeleton. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Electroretinagram (ERG) is the recording of electrical activity of retinal cells elicited by light stimulation, which has been widely used to help diagnose different types of retinal dysfunctions. The ERG response signal is a short non-stationary signal that contains overlapping components. Different Digital Signal Processing (DSP) techniques are investigated using MATLAB to study the time-frequency responses of the ERG signal such as Short Time Fourier Transform (STFT), Continuous Wavelet Transform (CWT) and Discrete Wavelet Transform (DWT). The Photopic ERG signal was processed and analyzed in this thesis and the results of each technique have been investigated in detail. The Photopic ERG components have been extracted using DWT and ERG Models.

Studies on the survival of central nervous system of hypoxia-tolerant species under challenges of reduced energy availability have characterised adaptive mechanisms of brain at the cell and tissue level that lead to reduced excitability and protection. However, evidence of hypoxic suppression of retinal activity in these species has not been followed up with mechanistic studies. Microspectrofluorometric monitoring of intracellular free Ca2+ concentration ([Ca2+]i) is useful for identifying cellular mechanisms that may lead to adaptive strategies, as unregulated increases in [Ca2+]i cause toxicity. Horizontal cells (HCs) are second order retinal neurons that receive tonic excitatory input from photoreceptors, and possess voltage-gated Ca2+ conductances and other channels that can facilitate toxic increases in [Ca2+]i under conditions of reduced energy availability (modeled as oxygen-glucose deprivation, OGD). It was demonstrated that isolated HCs of the hypoxia-tolerant goldfish display spontaneous, transient [Ca2+]i activity (SA) which decreased in amplitude and area under the curve following OGD or glucose removal (20 min) without recovery. SA was shown to be dependent on extracellular Ca2+ influx through voltage-gated Ca2+ channels, though mechanisms of SA generation and regulation has yet to be determined. Additionally, glutamate-elicited peak increases in [Ca2+]i were reduced after 20 min of OGD. The removal of O2 during OGD insult seemed to be protective as an increase in baseline [Ca2+]i was seen during and following glucose removal under normoxic conditions. The mechanisms mediating these decreases in spontaneous and elicited [Ca2+]i activity are currently unknown, though candidate pathways are discussed. This thesis contributes a hint towards how HCs may tolerate conditions of low energy availability, which may also inform investigations on their role in situ during these insults.