Les virus adéno-associés (AAVs) font partie des vecteurs les plus efficaces pour le transfert de gène, en particulier dans la rétine. Ils sont utilisés aussi bien pour des études biologiques que pour la thérapie génique. Malgré cela, il reste encore des barrières qui limitent leur utilisation. Nous proposons ici d’utiliser une technique d’évolution dirigée pour surmonter ces barrières et améliorer l’efficacité des AAVs en tant que vecteurs de gènes. Dans un premier temps, nous avons créé trois librairies virales hautement diversifiées basées sur l’AAV2. Ces librairies étaient constituées de capsides modifiées aléatoirement pour leur donner de nouvelles propriétés. Nous avons ensuite réalisé deux types de sélections. D’une part, nous avons sélectionné nos librairies virales dans le système visuel de la souris pour obtenir une capside capable de transport axonal antérograde trans-synaptique afin de pouvoir étudier simultanément l’activité et la connectivité de réseaux neuronaux. Cette sélection a fortement convergée vers une capside nommée AAV2-7mD, dont la capacité de transport axonal antérograde trans-synaptique est plus efficace que les AAVs 1 et 2. D’autre part, nous avons sélectionné nos librairies virales directement sur des explants de maculas de rétine humaine afin découvrir une capside capable de traverser la membrane limitante interne de la macula humaine. Ceci a pour but d’avoir un vecteur efficace pour des traitements de thérapie génique par voie intra-vitréenne. Cette librairie a commencé à converger mais nous sommes toujours en attente du dernier cycle de sélection. Nous traitons donc dans cette thèse des résultats de deux évolutions dirigées sur l’AAV2 afin de créer des vecteurs de gènes plus performants dans le système visuel. / Adeno-associated viruses (AAVs) are among the most efficient vectors for gene transfer, particularly in the retina. They are used for asking biological questions as well as for gene therapy. Nonetheless, some barriers are still restraining their use. Here, we used a directed evolution method to overcome those barriers and improve the efficiency of AAVs for gene transfer. First, we created three highly diversified viral libraries based on AAV2. Those libraries were based on randomly modified capsids displaying new properties. Then, we did two types of selections. On one hand, we selected our libraries in the retinofugal pathway in order to obtain a capsid with enhanced axonal anterograde trans-synaptic transport capacities, so we could study simultaneously the activity and the connectivity of neuronal networks between the retina and the brain. This selection converged strongly toward a new capsid, named AAV2-7mD, with enhanced axonal anterograde trans-synaptic transport capacities compared to AAV1 and AAV2. On the other hand, we directly selected our viral libraries on human macular explants, to select capsids capable of crossing the human macular inner limiting membrane. Such a capsid would be very useful for retinal gene therapy via intravitreal injections. This library started to converge but we are still waiting to complete the last cycle of selection. In this thesis we discuss the results of these two directed evolution studies on AAV2 to create enhanced gene delivery vectors in the visual system.

O objetivo deste estudo foi descrever a distribuição dos neurofilamentos (NFs) e da proteína associada ao microtúbulo do tipo 2 (MAP-2) em retinas humanas e murinas. Para isso, usamos camundongos C57BL/6, submetidos à cirurgia estereotáxica para realização de lesão eletrolítica no colículo superior direito provocando degeneração retrógrada de células ganglionares da retina. Utilizamos ensaios de imunohistoquímica e PCR em tempo real (qPCR) para a caracterização dessas proteínas nas duas espécies. Na retina humana, observou-se que NFs e MAP-2 estão presentes nas células ganglionares do tipo M. No modelo animal, houve diminuição dos NFs e aumento de MAP-2, na análise de imuno-histoquímica. Já o ensaio com qPCR mostrou um aumento e diminuição da expressão dos NFs e MAP-2, respectivamente. Assim, concluímos que houve alterações na expressão do RNAm e na marcação dos NFs e do MAP-2 nas retinas murinas, e esses resultados podem ser extrapolados para os seres humanos, uma vez que essas proteínas estão presentes nas células M que são inicialmente afetadas no glaucoma. / This study aimed to describe the distribution of NFs and type 2 protein associated with microtubule (MAP-2) in human retinas of these proteins and evaluate a model of retrograde retinal ganglion cell degeneration in murine retinas. To achieve this, we submitted C57bl/6 to a stereotaxic surgery for superior colliculus electrolytic lesion in the right side. The characterization of these proteins was obtained through immunohistochemical essays and real-time PCR (qPCR). The results revealed that both proteins are present in the ganglion cell M in the human retina. In the experimental animal model the immunohistochemical essays demonstrated decrease of NFs and increased MAP-2. However, the qPCR analysis demonstrated increased NFs and decreased MAP-2 expression. We can conclude that there was variation of mRNA expression and structural protein levels in the experimental retina. And, the results related to NFs and MAP-2 in this animal model can be extrapolated to humans, as these proteins are also present in the human ganglion cell that are affected early in glaucoma.

O objetivo deste estudo foi descrever a distribuição dos neurofilamentos (NFs) e da proteína associada ao microtúbulo do tipo 2 (MAP-2) em retinas humanas e murinas. Para isso, usamos camundongos C57BL/6, submetidos à cirurgia estereotáxica para realização de lesão eletrolítica no colículo superior direito provocando degeneração retrógrada de células ganglionares da retina. Utilizamos ensaios de imunohistoquímica e PCR em tempo real (qPCR) para a caracterização dessas proteínas nas duas espécies. Na retina humana, observou-se que NFs e MAP-2 estão presentes nas células ganglionares do tipo M. No modelo animal, houve diminuição dos NFs e aumento de MAP-2, na análise de imuno-histoquímica. Já o ensaio com qPCR mostrou um aumento e diminuição da expressão dos NFs e MAP-2, respectivamente. Assim, concluímos que houve alterações na expressão do RNAm e na marcação dos NFs e do MAP-2 nas retinas murinas, e esses resultados podem ser extrapolados para os seres humanos, uma vez que essas proteínas estão presentes nas células M que são inicialmente afetadas no glaucoma. / This study aimed to describe the distribution of NFs and type 2 protein associated with microtubule (MAP-2) in human retinas of these proteins and evaluate a model of retrograde retinal ganglion cell degeneration in murine retinas. To achieve this, we submitted C57bl/6 to a stereotaxic surgery for superior colliculus electrolytic lesion in the right side. The characterization of these proteins was obtained through immunohistochemical essays and real-time PCR (qPCR). The results revealed that both proteins are present in the ganglion cell M in the human retina. In the experimental animal model the immunohistochemical essays demonstrated decrease of NFs and increased MAP-2. However, the qPCR analysis demonstrated increased NFs and decreased MAP-2 expression. We can conclude that there was variation of mRNA expression and structural protein levels in the experimental retina. And, the results related to NFs and MAP-2 in this animal model can be extrapolated to humans, as these proteins are also present in the human ganglion cell that are affected early in glaucoma.

Diabetic retinopathy and age-related macular degeneration are two of the most common retinal diseases in these days, which can lead to partial or full loss of sight. Due to it, it is necessary to create new approaches enabling to detect these diseases and inform the patient about his condition in advance. The main objective of this work is to design and to implement an algorithm for retinal diseases classification based on images of the patient's retina of previously mentioned diseases. In the first part of this work, there is described in detail each stage of each disease and its the most frequent symptoms. In this thesis, there is also a chapter about fundus camera, which is a tool for image creation of human eye retina. In the second part of this thesis, there is proposed an approach for classification of diabetic retinopathy and age-related macular degeneration. There is also a chapter about algorithmic methods which can be used for image processing and object detection in image. The last part of this thesis contains the test results and their evaluation. Assessment of success of proposed and implemented methods is also part of this chapter.