Introdução: a ataxia espinocerebelar tipo 7 (SCA7) é um distúrbio neurodegenerativo autossômico dominante causado por uma repetição CAG expandida (CAGexp) no gene ATXN7, resultando na inserção de uma poliglutamina (poliQ) alongada na proteína ataxina-7. Em consequência, pacientes com SCA7 desenvolvem ataxia, espasticidade e outros sintomas neurológicos. A SCA 7 se destaca de outras SCAs por se associar à distrofia retiniana, causando deficiências visuais que podem levar à cegueira. Sendo uma das mais raras SCAs, pequenas séries de casos têm aparecido na literatura. Poucas delas buscaram correlacionar os achados neurológicos com os oftalmológicos; e a fase pré-clínica jamais foi sistematicamente investigada. Objetivo: descrever os achados neurológicos e oftalmológicos de uma coorte de casos de SCA 7, comparando as manifestações encontradas em sujeitos sintomáticos com as encontradas em portadores assintomáticos e em parentes não portadores, em uma abordagem exploratória que buscou levantar potenciais biomarcadores de progressão da doença. Métodos: trata-se de um estudo transversal onde pacientes com diagnóstico molecular de SCA7 realizado na nossa instituição foram identificados em nossos arquivos protegidos. Tanto eles como seus parentes foram convidados a participar da presente investigação. Sujeitos em risco de 50% foram incluídos se tivessem mais de 18 anos. Após o consentimento, dados clínicos e demográficos foram coletados entre junho de 2016 e setembro de 2017. A seguir, todos os participantes realizaram uma bateria de escalas clínicas voltadas à medida da ataxia (SARA, CCFS, PATA e 8 MW) e das manifestações neurológicas (NESSCA e INAS); um questionário de qualidade de vida relacionada à visão (NEI-VFQ 25); avaliação da acuidade visual melhor corrigida (AVMC), desvio médio em campimetria computadorizada (MD) e espessuras da mácula e da camada de células ganglionares na tomografia de coerência óptica (OCT). A escala SARA e a AVMC foram escolhidas como as variáveis de referência para a gravidade dos quadros. A análise molecular do ATXN7 foi feita, mas participantes do estudo e avaliadores foram mantidos cegos para seus resultados; os indivíduos em risco interessados em receber seus resultados foram enviados para o programa de testes pré-sintomáticos. Como não houve critérios a priori para estimar tamanhos de efeito e como a SCA7 é uma condição rara, não houve como decidir um tamanho de amostra. O estudo foi exploratório e por isso não foram feitas correções para múltiplas testagens. Um p de 0,05 foi eleito para definir significância, e testes estatísticos foram aplicados de acordo com as características das variáveis em estudo. Resultados: 12 portadores sintomáticos (grupo 2) e 8 indivíduos em risco (3 portadores - grupo 1 - e 5 não-portadores - grupo 0) foram incluídos neste estudo. Todas as variáveis contínuas à exceção da CAGexp tiveram distribuição 4 normal. A AVMC estava reduzida em todos os participantes sintomáticos e claramente diferente entre estes e os outros dois grupos (p <0,0001, ANOVA), enquanto os portadores assintomáticos e os não portadores tiveram resultados semelhantes. A AVMC média foi 20/143, 20/18 e 20/20 nos grupos 2, 1 e 0, respectivamente. Não surpreendentemente, o NEI-VFQ 25 também demonstrou uma diferença estatisticamente significativa, mas o que foi inesperado foi a forma progressivamente diferente entre os 3 grupos (grupo 0 = 92,76 ± 6,7; grupo 1 = 74,9 ± 55,5; grupo 2 = 58,0 ± 21,3) (p= 0,012, ANOVA com Tukey) O MD mostrou um padrão linear estatisticamente significativo para piorar do grupo controle (-1,34 ± 1,15dB) para o assintomático (-2,81 ± 1,66dB) e do grupo assintomático para sintomático (-10,54 ± 6,95dB) (p = 0,027, ANOVA com Tukey). Além disso, o MD correlacionou-se com a AVMC (p = 0,020; r = 0,660) e apresentou tendência de correlação com a SARA (p= 0,073; r= -0,535). As medidas de espessura macular distinguem completamente os 3 grupos (grupo 0 = 243,6 ± 22,2 μ; grupo 1 = 204,5 ± 14,1 μ; grupo 2 = 137,95 ± 34,6 μ) (p = 0,0001, ANOVA) e também se correlacionou significativamente com os dois critérios planejados de gravidade, SARA (p = 0,050; r = -0,577) e AVMC (p = 0,007; r = 0,730). Discussão: alterações oftalmológicas estavam presentes já nas fases pré-clínicas da doença, quando os escores obtidos das escalas neurológicas ainda não distinguem portadores assintomáticos de não portadores: a espessura macular medida por OCT e o MD medido pela campimetria computadorizada. Esses achados demonstram que o processo neurodegenerativo já se encontra em curso e é detectável por essas medidas anatômicas e funcionais da retina. Além disso, ambas as alterações detectadas em fases pré-clínicas, ao serem estudadas no grupo total de portadores sintomáticos e assintomáticos, se correlacionaram com os nossos padrões-ouro da gravidade da doença, SARA e AVMC. Os dois achados - início em fase pré-clínica e correlação com a progressão da doença medida por escores independentes - sugerem que a espessura macular medida por OCT e o MD medido pela campimetria computadorizada são potenciais candidatos a biomarcadores de estado (de progressão da doença) desde fases pré-manifestas na SCA7. / Background: spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder caused by an expanded CAG repeat (CAGexp) at ATXN7 gene, resulting in the insertion of an elongated polyglutamine (polyQ) into the ataxin-7 protein. As a consequence, patients with SCA7 develop ataxia, spasticity and other neurological symptoms. SCA7 stands out from other SCAs by associating it with retinal dystrophy, causing visual deficiencies that can lead to blindness. Being one of the rarest SCAs, small series of cases appear in the literature. Few of them sought to correlate neurological findings with ophthalmologic findings; and the preclinical stage has never been systematically investigated. Objective: to describe the neurological and ophthalmological findings of a cohort of cases of SCA7, comparing the manifestations found in symptomatic subjects with those found in asymptomatic carriers and in non-carrier relatives in an approach exploratory study that sought to raise potential biomarkers of disease progression. Methods: patients with a molecular diagnosis of SCA7 performed at our institution were identified in our protected files. Both they and their relatives were invited to participate in the present investigation. Subjects at risk of 50% were included if they were older than 18 years. After consent, clinical and demographic data were collected between June 2016 and September 2017. All participants then performed a battery of clinical scales aimed at the measurement of ataxia (SARA, CCFS, PATA and 8 MW) and neurological manifestations (NESSCA and INAS); a visual function questionnaire (NEI-VFQ 25); assessment of better corrected visual acuity (AVMC), mean deviation in computerized campimetry (MD), and thickness of the macula and ganglion cell layer on OCT. The SARA and AVMC scale were chosen as the reference variables for the severity of the frames. Molecular analysis of ATXN7 was done, but study participants and evaluators were kept blind to their results; the individuals at risk interested in receiving their results were sent to the presymptomatic testing program. As there were no a priori criteria for estimating effect sizes and because SCA7 is a rare condition, there was no way to decide on a sample size. The study was exploratory and therefore no corrections were made for multiple tests. A p of 0.05 was chosen to define significance, and statistical tests were applied according to the characteristics of the variables under study. Results: 12 symptomatic carriers (group 2) and 8 individuals at risk (5 carriers - group 1 - and 3 non-carriers - group 0) were included in this study between June 2016 and September 2017. All continuous variables with the exception of CAGexp had normal distribution. AVMC was reduced in all symptomatic participants and clearly different between these and the other two groups (p <0.0001, ANOVA), while asymptomatic and non-carriers had similar results. The mean BCVA was 20/143, 20/18 and 20/20 in groups 2,1 and 6 0, respectively. Not surprisingly, NEI-VFQ 25 also showed a statistically significant difference, but what was unexpected was the progressively different form between the 3 groups (group 0 = 92.76 ± 6.7, group 1 = 74.9 ± 55, 5, group 2 = 58.0 ± 21.3) (p = 0.012, ANOVA with Tukey). The MD showed a statistically significant linear pattern to worsen from the control group (-1.34 ± 1.15dB) to the asymptomatic (-2.81 ± 1.66dB) and from the asymptomatic to the symptomatic group (-10.54 ± 6, 95dB) (p = 0.027, ANOVA with Tukey). In addition, MD correlated with AVMC (p = 0.020; r = 0.660) and showed a correlation tendency with ARDS (p = 0.073; r = -0.535). The macular thickness scores completely distinguish the 3 groups (group 0 = 243.6 ± 22.2 μ, group 1 = 204.5 ± 14.1 μ, group 2 = 137.95 ± 34.6 μ) (p = 0.0001, ANOVA ...) and also correlated significantly with the two planned criteria of severity, SARA (p = 0.050, r = -0.577) and AVMC (p = 0.007, r = 0.730). Conclusion: ophthalmologic changes were present already in the preclinical stages of the disease, when the scores obtained from the neurological scales did not yet distinguish asymptomatic non-carrier patients: macular thickness measured by OCT and MD measured by computerized campimetry. These findings demonstrate that the neurodegenerative process is already underway and is detectable by these anatomical and functional measures of the retina. In addition, both changes detected in preclinical stages, when studied in the total group of symptomatic and asymptomatic carriers, correlated with our gold standard of disease severity, SARA and AVMC. The two findings - pre-clinical onset and correlation with disease progression measured by independent scores - suggest that the macular thickness measured by OCT and MD as measured by computerized campimetry are potential candidates for disease biomarkers (disease progression) from pre-manifest stages in SCA7.

Les yeux forment des avant-postes visuels importants du cerveau. Comme les autres organes, la rétine sensorielle des yeux est vulnérable aux effets nocifs des facteurs environnementaux, tels que la lumière et l'oxygène. Dans ce travail, nous nous sommes concentrés sur l’impact de l’exposition à une lumière constante et l’hyperoxie prolongée sur l'architecture et la fonction rétinienne. Dans la première partie de notre étude, nous avons montré qu’ une exposition de sept jours à une lumière constante perturbe la phagocytose des bâtonnets et cônes et régule négativement leur renouvellement dans la « rétine riche en cônes " d’Arvicanthis ansorgei. Notre étude donne un aperçu sur la physiopathologie des cônes, ce qui représente la principale source de handicap visuel dans une variété de pathologies rétiniennes, y compris la rétinite pigmentaire (RP) et la dégénérescence maculaire liée à l'âge (DMLA). Dans la deuxième partie de notre étude, nous avons montré qu’ une exposition de cinq jours à l’hyperoxie entraîne chez les souris néonatales une perte significative de cellules ganglionnaires dans les régions périphériques de la rétine, et de cellules à mélanopsine (ipRGC). L’exposition prolongée à l’hyperoxie perturbe également la capacité de photoentrainment des animaux probablement due à la perte des ipRGC et la perte de la rhodopsine dans les segments externes des bâtonnets chez les animaux traités. / Eyes form important visual outposts of the brain. Just like other organs, sensory retina in the eyes is also vulnerable to the injurious effects of environmental factors; such as light and oxygen. In this work, we have focused on the impacts of constant prolonged light and hyperoxia on the retinal architecture and function. In the first part of our study, we show that seven days of constant light disrupts rod and cone phagocytosis and downregulates their turnover in the “cone rich retina” of Arvicanthis ansorgei. The study gives an insight on the cone pathophysiology, which represents the major source of visual handicap in a variety of retinal pathologies, including retinitis pigmentosa (RP) and age-related macular degeneration (AMD). In the second part of our study, we show that five days of hyperoxia treatment in the neonatal mice results in the significant loss of retinal ganglion cells in the peripheral regions; the loss of melanopsin expressing retinal ganglion cells (ipRGC) was found to be significant. Hyperoxia also affects the photoentrainment capability of the animals probably because of the loss of ipRGC and the loss of rhodopsin in the outer segments of the photoreceptors in the treated animals.

La dégénérescence maculaire liée à l’âge ou DMLA représente la première cause de cécité légale dans les pays industrialisés. C’est une affection multifactorielle (facteurs environnementaux et génétiques), caractérisée par la dégénérescence des photorécepteurs et une dysfonction de l’épithélium pigmentaire de la rétine (EPR). La présence de dépôts sous-rétiniens, les drusen, qui sont un facteur de risque de développer la DMLA contiennent de l’amyloïde-b (Ab), le peptide neurotoxique impliqué dans la maladie d’Alzheimer (MA) et le facteur H du complément (CFH), qui est un des inhibiteurs solubles majeurs de l’activation de la voie du complément. L’Aβ est capable de lier le CFH et il existe une corrélation inverse entre les taux de CFH et d’Aβ dans le cerveau et la rétine de modèles murins de la MA, de plus, le polymorphisme H402Y dans le gène codant pour le CFH est associé à la moitié des cas de DMLA et est aussi un facteur de risque pour la MA. Au début de ma thèse, rien n’était connu sur les fonctions de la protéine précurseur de l’amyloïde β (APP), et du CFH dans la rétine. Ainsi, le but de ma thèse a été d’étudier d’une part le rôle physiologique de l’APP au cours de la différenciation de la rétine et à l’âge adulte chez la souris, et d’autre part le CFH dans la rétine normale chez la souris normale adulte et chez la souris Rd10, un modèle de dégénérescence des photorécepteurs. Nous avons montré par l’utilisation d’une souris dont le gène de l’APP est invalidé (APPko), une diminution stable de 35% de nombre de cellules amacrines glycinergiques et de 36% de celui des cellules bipolaires, associé à une désorganisation importante des laminations des synapses au stade adulte, et une augmentation transitoire à 50% du nombre cellules horizontales au cours de la phase précoce de la différenciation rétinienne. Nous avons identifié Ptf1a, facteur de transcription indispensable à la différenciation des cellules amacrines, comme la cible de l’APP. De plus, non montrons que SorLA, le récepteur de l’APP, est indispensable à la fonction de différenciation rétinienne de l’APP. Dans la deuxième partie de mon étude, j’ai montré que le CFH protége in vitro les cellules de l’EPR contre les effets létaux du stress oxydant. Après avoir caractérisé les processus associés à la dégénérescence des photorécepteurs chez la souris Rd10 (activation gliale, microgliale, activation de l’inflammation et de la voie du complément), j’ai montré que l’injection intrapéritonéale répétée de CFH humain durant la dégénérescence protège partiellement les photorécepteurs de la mort. L’ensemble de mon étude montre pour la première fois un rôle important de l’APP dans la différenciation de la rétine et un nouveau rôle, comme antioxydant, pour le CFH, qui pourrait être un agent thérapeutique ciblant des photorécepteurs et l’EPR dans les pathologies dégénératives rétiniennes. / Age-related macular degeneration (AMD) is the first cause of central vision loss, due to photoreceptor degeneration and retinal pigmented epithelium (RPE) dysfunction. It is a multifactorial disease (genetic and environmental factors) and subretinal deposits of materials called drusen, is a major risk to develop AMD. Drusen contain amyloïd β (Aβ), the major neurotoxic peptide involved in Alzheimer disease (AD), and complement factor H (CFH), one of the major inhibitor of the complement pathway. Aβ and CFH interact and there is an inverse correlation between CFH and Aβ in the brain and retina of mouse models of AD. Polymorphism H402Y of CFH may be a cause of AMD in as many as 50% of cases. At the beginning of my thesis, nothing was known about the role of the amyloid precursor protein (APP) in the retina, as well as CFH. Therefore, the aim of my work was to investigate the retinal functions of both APP during retinal development and in adult mouse and CFH in normal mouse and in the Rd10 mouse model of photoreceptor degeneration. By using a mouse knock-out for app, we showed that APP regulates differentiation of retinal interneurons: it positively controls differentiation of glycinergic amacrine cells and bipolar cells, whereas it negatively controls differentiation of horizontal cells. Moreover, we identified the transcription factor, Ptf1-1, as a main transcriptional target of APP. Finally, we demonstrated that SorLA, an APP receptor, participates to the differentiation role of APP in the retina. In the second part of the study, I demonstrated that CFH protects RPE cells from lethal oxidative stress in cultures. Moreover, I showed that expression of CFH is very low in neural retina in comparison to the RPE/choroid/sclera complex in normal mice, and that CFH expression increases in both the neural retina and the RPE/choroid/sclera complex during photoreceptor degeneration in the Rd10 mice, suggesting an adaptive response of these tissues to degeneration. Intrapéritonéale injections of CFH partially protect photoreceptors from degeneration, suggesting a protective effect of CFH against retinal degeneration. Altogether, my study showed for the first time a physiological function of APP in the retina, and a new role for CFH in the protection of photoreceptors against degeneration, suggesting that it may be a therapeutic target against retinal degeneration.

In Canada alone, there were an estimated 800,000 visually impaired people in 2007, costing the federal government an annual amount of $15.8 billion in services, treatments and lost revenue. These costs are estimated to double by the year 2032, as the population ages. The leading causes of visual impairment and blindness is retinal degeneration, characterized by the progressive death of retinal cells. The research presented in this PhD thesis aimed to prevent retinal degeneration by over-expressing the X-linked Inhibitor of Apoptosis (XIAP) in retinal cells using plasmid and adeno-associated viral vectors. The work is divided into four sequential chapters targeted at developing an anti-apoptotic gene therapy strategy to prevent retinal cell death. The first chapter examines XIAP gene therapy in the treatment of Leber’s Hereditary Optic Neuropathy (LHON). In vitro studies using the 661W cone-photoreceptor cell line showed that XIAP over-expression significantly lowers cell death when 661W cells are exposed to a number of apoptotic stimuli. In a mouse model of Leber’s Hereditary Optic Neuropathy (LHON), XIAP expression in retinal ganglion cells (RGCs) protected the ultrastructure of the RGC axons within the optic nerve, in addition to providing evidence of functional protection. The second and third chapters further examine the potential for XIAP gene therapy in the treatment of retinal disease by developing an in vivo model of retinal detachment in cats, followed by evaluating the efficacy of XIAP gene therapy intervention. When XIAP was over-expressed in the photoreceptor cells, there was significant structural protection and trends in preservation of function in this model of degeneration. Finally, the fourth chapter explores an alternate method to viral gene therapy by evaluating the efficacy and toxicity of chitosan microparticles as a protein delivery system to the retina. Results show that chitosan microparticles are mucosal-adhesive and are non-toxic at low concentrations in vitro in 661W cells and in vivo in rats. This thesis work provides strong evidence that XIAP gene therapy is an effective method for preventing retinal degeneration, and works as a broad spectrum gene therapy strategy that can be applied to different forms of retinal degeneration.

L’objectif de cette thèse est la validation fonctionnelle d’implants rétiniens pour la restauration fonctionnelle de la vision chez des patients non voyants suite à la perte de leurs photorécepteurs. Ce travail a été réalisé sur modèle animal et a évalué expérimentalement de nouveaux protocoles de stimulation. Tout d’abord nous avons utilisé la technique de spectroscopie d’impédance pour simuler mathématiquement l’interface tissu-implantafin de caractériser la présence d’un espace entre le tissu et l’implant. La seconde partie compare par imagerie optique (IO) les caractéristiques de la réponse corticale évoquée par stimulation visuelle ou électrique de la rétine par prothèse sous rétinienne. Nous avons retrouvé que la taille de l’activation par l’implant rétinien est beaucoup plus grande que son correspondant visuel. Dans une troisième partie, est réalisée une évaluation in vitro de la performance des stimulations sur rétine isolée pour définir comment les cellules ganglionnaires réagissent à différents modes de stimulations. Ce travail a permis d’établir la courbe des réponses en fonction de l’intensité des stimulations électriques. Enfin, la thèse décrit un modèle animal de dégénérescence rétinienne qui présente des désorganisations de la rétine externe. Une analyse en IO a été réalisée sur ce modèle afin d’évaluer la réponse corticale aux stimuli visuels et électriques. Ce travail de thèse, par des approches physiques et physiologiques complémentaires, apporte un certain nombre de réponses qui devraient permettre d’améliorer l’utilisation de futures prothèses rétiniennes par une adaptation physique des matrices d’électrodes ou des patrons de stimulations utilisées / The aim of this thesis is the functional validation of retinal implants used for vision restoration in blind patients due to the loss of photoreceptors. This work was designed to develop an animal model to experimentally validate prototypes of new implants and new stimulation protocols pattern. Firstly we used the technique of impedance spectroscopy to simulate mathematically the tissue/implant interface. These data confirm the importance of reducing the space between the stimulating electrodes and retinal tissue, as well as the importance of physical characteristics of the electrical stimulus used. In a second approach, we have compared responses of visual cortical neuronal population using optical imaging (OI), evoked either by visual or electric retinal stimulation through subretinal prosthesis. This approach has demonstrated that the stimulation of an electrode induces cortical activation that the size of the cortical response to the retinal implant stimulation is much larger than its corresponding visual stimulus. In the third part, I performed in vitro experiment to measure the performance of stimulation at the level of ganglion cells of isolated retina. We have quantified the response curve as a function of the intensity of the electrical stimulation. Finally, the thesis describes a new animal model of outter retinal degeneration. OI was also performed on this model to assess the response to the visual and retinal prosthesis stimulations. This thesis, through complementary physical and physiological approaches, provides a number of responses that can potentially improve the use of retinal prostheses through specification of their design or patterns of stimulation.

Le système endocannabinoïde (eCB) est une cible thérapeutique intéressante pour traiter diverses conditions variées, allant de la modulation du système immunitaire à la prise en charge de la douleur neuropathique. De plus, le système eCB est impliqué dans les processus développementaux comme l’indique une exposition aux cannabinoïdes au cours du développement qui provoque des troubles neurofonctionnels. En raison de leur nature lipophile, les eCBs ne sont pas emmagasinés, mais sont plutôt synthétisés et dégradés sur demande par des enzymes. Ainsi, l’étude du patron d’expression de ces enzymes permettrait de mieux comprendre l’expression et ainsi le rôle joué par les eCBs pendant la formation du système nerveux central. Le récepteur CB1 est grandement distribué dans le système nerveux, alors que le récepteur CB2 est traditionnellement associé au système immunitaire. La découverte récente de l’expression et de l’impact fonctionnel du récepteur CB2 dans certains neurones, notamment au niveau rétinien, modifie la vision traditionnelle des rôles des eCBs. Notamment, une étude de notre laboratoire a montré que la délétion du récepteur CB2 chez des souris transgéniques (cnr2-/-) provoque une augmentation de l’amplitude de l’onde a en électrorétinographie, celle-ci reflétant l’activité des photorécepteurs rétiniens. Cette étude a mis en évidence l’importance du récepteur CB2 dans la vision, du moins au niveau rétinien. Jusqu’à ce jour, aucune étude ne s’est intéressée à l’impact des cannabinoïdes sur l’acuité visuelle. Nous avons caractérisé la distribution rétinienne des enzymes diacylglycérol lipase alpha (DAGLα) et monoacylglycérol lipase (MAGL), responsables respectivement de la synthèse et de la dégradation du ligand eCB 2-arachidonoyl glycérol (2-AG), pendant le développement postnatal. L’enzyme DAGLα est présente dès la naissance et est grandement distribuée dans la rétine, notamment dans les photorécepteurs, les cellules horizontales, amacrines et ganglionnaires. L’enzyme MAGL apparait plus tardivement et est limitée aux cellules amacrines et de Müller. Nos résultats fonctionnels indiquent que l’acuité visuelle des animaux cnr2-/- est plus élevée autant chez les adultes que pendant le développement postnatal. L’administration répétée d’un agoniste inverse du récepteur CB2 produit une augmentation de l’acuité visuelle similaire à la délétion du récepteur CB2 par génie génétique et inversement, l’administration d’un agoniste du récepteur CB2 diminue l’acuité visuelle. Enfin, l’administration d’un inhibiteur de l’enzyme MAGL, responsable de la dégradation du 2-AG, induit une diminution de l’acuité visuelle similaire à celle obtenue par un agoniste du récepteur CB2 tandis que l’administration d’un inhibiteur de l’enzyme DAGL, responsable de la synthèse du 2-AG, provoque une augmentation de l’acuité visuelle. Ces résultats suggèrent que le 2-AG est fortement présent tôt lors du développement rétinien et qu’il pourrait être impliqué dans la maturation structurelle et fonctionnelle de la rétine. De plus, les expériences fonctionnelles ont démontré que les cannabinoïdes affectent non seulement la réponse rétinienne, mais aussi l’acuité visuelle de manière significative. En outre, ces résultats confirment que les cannabinoïdes induisent leurs effets sur la vision exclusivement par le récepteur CB2. Enfin, les résultats de cette thèse accroissent les connaissances actuelles dans un contexte de légalisation grandissante du cannabis à des fins récréatives, puisqu’ils mettent en évidence l’importance des impacts sur l’acuité visuelle. / The endocannabinoid (eCB) system is a great therapeutic target for the treatment of many diseases, ranging from immune system modulation to pain management. This system is implicated in developmental processes as indicated by neurofunctional afflictions following developmental exposition to cannabinoids. Since eCBs are lipophilic, they are not stored in vesicles but rather synthesized and degraded on demand by specific enzymes. Thus, the expression pattern of these enzymes could help to better understand the expression of eCBs, and their role during central nervous system maturation. The CB1 receptor is strongly distributed in the nervous system, while the CB2 receptor is traditionally associated with the immune system. The recent finding of the CB2 receptor expression and function in some neurons, especially in the retina, changes the dogma associated with cannabinoids. A study from our laboratory found that deletion of the CB2 receptor in transgenic mice (cnr2-/-) enhances the a-wave amplitude in electroretinography, this wave reflecting photoreceptor activity. This report highlighted the importance of the CB2 receptor in vision, at least in the retina. Until now, no study aimed at the impact of cannabinoids on visual acuity. We characterized the retinal distribution of diacylglycerol lipase alpha (DAGLα) and monoacylglycerol lipase (MAGL) enzymes, responsible for the synthesis and degradation of the eCB ligand 2-arachidonoyl glycerol (2-AG) during postnatal development. The enzyme DAGLα is expressed since birth and is greatly distributed across the retina such as in photoreceptors, horizontal, amacrine and ganglion cells. The enzyme MAGL is expressed later during development and is present only in amacrine and Müller cells. Our functional results show that the visual acuity of cnr2-/- mice is enhanced in adults and during postnatal development. The repeated administration of a CB2 receptor antagonist yielded a better visual acuity, and inversely a CB2 receptor agonist decreased the visual acuity. Furthermore, the administration of a MAGL inhibitor, the enzyme in charge of 2-AG degradation, induced a strong decrease in visual acuity, similar to that obtained with a CB2 receptor agonist. Inversely, a DAGL inhibitor, the enzyme responsible for 2-AG synthesis, caused an increase in visual acuity. These results suggest that 2-AG is strongly expressed early during retinal development and could be implicated in structural and functional maturation of the retina. Furthermore, we demonstrated that cannabinoids do not only affect retinal function, but also visual acuity. These results confirm that cannabinoids modulate their visual effects exclusively via the CB2 receptor. Finally, in recent years, many countries legalized cannabis for recreational and therapeutic use. The findings from this thesis increase the understanding of cannabinoids since they highlight the great impact of cannabinoids on the visual acuity.

K pořízení snímků sítnice, která představuje nejdůležitější část lidského oka, je potřeba speciálního vybavení, kterým je fundus kamera. Z tohoto důvodu je cílem této práce navrhnout a implementovat systém, který bude schopný generovat takovéto snímky bez použítí této kamery. Navržený systém využívá mapování vstupního černobílého snímku krevního řečiště sítnice na barevný výstupní snímek celé sítnice. Systém se skládá ze dvou neuronových sítí: generátoru, který generuje snímky sítnic, a diskriminátoru, který klasifikuje dané snímky jako reálné či syntetické. Tento systém byl natrénován na 141 snímcích z veřejně dostupných databází. Následně byla vytvořena nová databáze obsahující více než 2,800 snímků zdravých sítnic v rozlišení 1024x1024. Tato databáze může být použita jako učební pomůcka pro oční lékaře nebo může poskytovat základ pro vývoj různých aplikací pracujících se sítnicemi.

To use the image of the retina for biometric or medical purposes, it is necessary that the image is of the highest quality and ideally covers the largest possible area. It is therefore necessary to move the sensing device to the most suitable position while ensuring its stability. A laboratory handling platform is used for this purpose. The platform is moved by stepper motors. A control board equipped with a microcontroller has been developed for their control, which allows very fine movements. The theoretical accuracy of the manipulator is up to 20 nm. Since it is necessary for sensing, to have proper retina illumination, the control system also includes adjustable illumination elements in the infrared and visible spectrum. The resulting system allows scanning of the retina of the eye from multiple angles and thereby effectively increase the area on which it is possible to further look for diseases or biometric features.

This doctoral thesis called “Photobiological safety of luminaires and light sources” has focused on an optical radiation and its relation to human body. The thesis describes possible consequences on living tissue during excessive exposure to optical radiation. Among others the work deals with the light source, lamp and optical devices evaluation in dependence of photobiological safety. There is created a method of light source evaluation procedure for photobiological safety in accordance with ČSN EN 62471. The meaning of this procedure is based on its hazard calculation from measured values of irradiation to different photometric distances. Validity of procedure is verified by light source measuring, in which visual angle and photometric distance have been changed. There we can also find an application of this procedure in the UV source evaluation, which helps to determine a safe distance away from UV sources not to create any danger. The evaluated UV sources include a UVA luminaire with a dominant wavelength of 365 nm or an arc of an electric welder. The thesis also describes a method of evaluating light sources for blue light hazard for an aphakic and pseudophakic eye. This method can also help to determine the hazard for the eye without lens or with some implanted lens. Furthermore, there was developed the method of the blue light hazard assessment depending on the age of an exposed person. In terms of the work there was also created a computer programme evaluating photobiological safety resulting from the spectral data having been stored in the library programme or from spectroradiometric measurement data. The work also marginally deals with the topic of circadian rhythms, which are closely related to photobiological manifestation in an human body. Above all the work compares light sources for which equivalent illuminances are calculated, consequently light sources have the same effect on suppressing the melatonin hormone production.

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