A high frequency of retinitis pigmentosa (RP) is found in Northern Sweden. In an inventory of autosomal recessive RP patients in Västerbotten County, a great number of cases with a unique phenotype was noticed, denoted Bothnia Dystrophy (BD). The aim of the study was to describe the phenotype, to determine the chromosomal location, and to identify the gene. Patients typically show night blindness from early childhood. Symptoms of defect macular function with a decrease of visual acuity can appear in early adulthood. The retinal fundus shows irregular white spots in a central, and parafoveal pattern along the arcades. Centrally areolar maculopathy develops and round circular atrophies are observed in the periphery. The disease was shown to be associated with a missense mutation in the RLBP1 gene resulting in an amino acid substitution (R234W) in the cellular retinaldehyde-binding protein (CRALBP). The R234W mutation was found in a homozygous state in 61 patients affected with BD. Ten patients were heterozygous for the R234W mutation, and presented a similar phenotype. No additional mutations in the coding sequence or exon-intron junctions were found. CRALBP is localised in retinal pigment epithelium (RPE), and Müller cells of the retina. In the RPE, CRALBP functions as a carrier protein for endogenous retinoids. Dark adaptometry and electrophysiologic testing showed an initial loss of rod function followed by a progressive reduction of the cone responses in older ages. A compromised rod function, dysfunction of the Müller cells, and indications of a disturbed function of the inner retina were found. With prolonged dark adaptation, a gradual increase in retinal sensitivity to light and an improvement of the ERG components occurred. The findings indicate a prolonged synthesis of photopigments, retardation of the visual process in the retinal pigment epithelium and a loss of retinal cells probably starting at a relative early age in BD. To evaluate the subjective visual function in BD patients, a battery of objective tests of visual function and composite score of the 25-item NEI-Visual Function Questionnaire (VFQ-25) were analyzed. We found that weighted distance logMAR visual acuity (WVA), had the strongest association with subjective visual function, and that there was a considerable loss of subjective and objective visual function with increasing age in BD patients. The prevalence of BD is as high as 1:3600 in Västerbotten County. The possibility that recycling of retinoids localized in the RPE might be impaired in BD might give future therapeutic possibilities. Due to the large and clinically well-characterized set of patients with this disease, they constitute a suitable study group.

Les rétinites pigmentaires (RP) sont des pathologies rétiniennes cécitantes d'origine génétique caractérisées par une perte des photorécepteurs. Nous avons ciblé des formes de RP autosomique dominante consécutives à des mutations dans le gène du pigment visuel de la RHODOPSINE, du facteur d'épissage PRPF31 et du facteur de transcription impliqué dans le développement des photorécepteurs NR2E3. Les fibroblastes, issus de biopsies de peau de patients, ont été reprogrammés en cellules iPS par une technique dite non intégrative. Après stabilisation des cellules iPS, nous avons vérifié leur propriété de pluripotence et l'absence d'anomalies caryotypiques.Les cellules iPS porteuses d'une mutation sur le gène RHODOPSINE ont été différenciées dans le lignage des photorécepteurs. Nos résultats montrent que les photorécepteurs porteurs de la mutation P347L du le gène RHODOPSINE récapitulent la dégénérescence observée chez les patients.Nous montrons que les cellules de l'épithélium pigmentaire rétinien (EPR) dérivées de cellules iPS porteuses de la mutation Cys294X du gène PRPF31 présentent des problèmes d'adhésion cellulaire due à l'absence de lame basale. Leur activité de phagocytose est alors perturbée, suggérant qu'un dysfonctionnement de l'EPR pourrait être à la base de la RP causée par la mutation Cys294X du gène PRPF31. Les modèles développés nous ont permis de mieux comprendre les processus à la base de la pathogénèse de certaines RP. Ces modèles associés à des protocoles de criblage, pourraient permettre d'évaluer l'efficacité et la toxicité de nouvelles molécules pharmacologiques, mais également être utilisés pour valider des approches de thérapie génique. / Retinitis pigmentosa (RP) is an inherited retinal diseases characterized by a loss of photoreceptors. We focused specific forms of autosomal dominant RP with mutations in the rod visual pigment RHODOPSIN, the ubiquitous splicing factor PRPF31 and the transcription factor involved in the development of photoreceptors NR2E3. Fibroblasts from skin biopsies of patients were reprogrammed into iPS cells by a non-integrative approach. After stabilization of iPS cell lines, we verified their pluripotency property and the absence of karyotype abnormalities. Based on the retinal differentiation protocol, iPS cells carrying a mutation in the RHODOPSIN gene have been differentiated in the photoreceptor lineage. Our results showed that the photoreceptors expressing the mutated form of RHODOPSIN summarizing the process of degeneration observed in RP patients. We show that retinal pigment epithelium (RPE) cells derived from iPS cells carrying a mutation in the PRPF31 gene lack basal membranes and have cell adhesion disorders. Consequently, their phagocytic activity is disturbed, suggesting that a malfunction of the RPE could be the primary step of the development of RP caused by mutation Cys294X in the PRPF31 gene. The models developed from specific-patient iPS cells have enabled us to better understand the processes underlying the pathogenesis of some RP. These models associated with screening protocols could be used to evaluate the efficacy and toxicity of new pharmacologic compounds but also used to validate new gene therapy approaches.

Introduction : Les rétinopathies pigmentaires (RP) sont caractérisées par une grande hétérogénéité clinique et génétique rendant difficile la fiabilité du pronostic évolutif. Les nouvelles techniques d’évaluation laissent envisager la découverte de nouveaux marqueurs prédictifs palliant à ce problème. Matériel et méthode : Les patients sont majeurs et atteints de RP type bâtonnets-cônes. Le projet de thèse comprend l’étude de la qualité de vie (QDV), l’identification de nouveaux marqueurs prédictifs et l’examination du traitement par optogénétique. Résultats : 1) Un diamètre du champ visuel inférieur à 20° et une acuité visuelle inférieure à 0.3 représentent un seuil fonctionnel de dégradation de la QDV et de l’état émotionnel du patient. 2) L’intérêt des examens ophtalmologiques varie selon le stade de progression de la maladie. L’OCT est un examen révélé important à chaque stade de la RP. Le fond d’œil autofluorescent n’est pertinent que lorsqu’un anneau hyper-autofluorescent (HAF) est présent. 3) L’Optique Adaptative (OA), un potentiel nouveau marqueur innovant mais non-standardisé, révèle une diminution de la densité en cônes dans toutes les zones de l’anneau HAF. 4) Le traitement par optogénétique ne peut être proposé qu’à une minorité de patients avec une RP avancée ayant encore des cônes résiduels centraux. Conclusion : Les examens de suivi anatomo-fonctionnels à préconiser varient selon l’évolution de la RP. L’OA fournit de riches espoirs pour le suivi des patients RP par la visualisation des structures fines de la rétine. Les données de QDV et d’état émotionnel, sont indispensables pour mieux appréhender la maladie et avérer de l’efficacité d’un traitement. / Introduction: Retinitis pigmentosa (RP) are characterized by a great clinical and genetic heterogeneity making difficult any reliable evolutionary prognosis. New assessment techniques presage discovering new predictive markers overcoming this problem. Materials and Methods: RP patients are over 18 years old and have been diagnosed with rod-cone dystrophy. This thesis studies the impacts on quality of life (QOL), new predictive markers and optogenetics treatments. Results: 1) A diameter of less than 20° visual fields combined with a visual acuity of less than 0.3 set a functional level of degradation of QOL. 2) The benefits of ophthalmic examinations at follow-up RPs vary with stages of the disease. However the OCT exam has been proven beneficial throughout all PR stages; the autofluorescent funduscopy is only relevant when a hyper-autofluorescent (HAF) ring is present in the retina. 3) Adaptive Optics (AO), a new innovative marker not yet standardized, reveals a decrease in the cones density in all areas of the HAF ring. 4) Optogenetics treatments, based on a neuromodulation method, can only be offered to a minority of patients with advanced RP still having central residual cones. Conclusion: The anatomical and functional follow-up examinations to advocate to RP patients vary according to the stages of RP. OA provides rich hopes for monitoring RP patients by viewing the fine structures of the retina. Besides, data on QOL and emotional states are essential to better understand the RP disease and prove the effectiveness of a treatment.

Introdução: A Retinose Pigmentar (RP) é uma doença hereditária da retina, caracterizada por perda da função visual, principalmente devido à degeneração dos fotorreceptores (bastonetes e cones). Objetivo: Avaliar os efeitos de uma única injeção intravítrea de fração mononuclear de células da medula óssea (FMMO) CD34+ em pacientes portadores de RP. Métodos: Ensaio clínico aberto, não randomizado, prospectivo, observador mascarado, no qual 20 pacientes, portadores de RP, com boa fixação ao exame de campo visual, foram incluídos. Única injeção intravítrea (IIV) de FMMO foi aplicada em apenas um dos olhos de cada paciente, enquanto que os olhos contralaterais serviram como controle e foram submetidos à injeção simulada. As avaliações incluíram: melhor acuidade visual corrigida (MAVC); campo visual estático - estratégia 30-2 (Octopus 900); microperimetria (MAIA - Center Vue) para avaliar estabilidade de fixação e sensibilidade macular; eletrorretinografia de campo total (ERG) e multifocal (mfERG) - padrão da ISCEV usando aparelho Espion E2 (Diagnosys LLC) e tomografia de coerência óptica (OCT). Os exames foram realizados antes da injeção e 4, 16, 32 e 48 semanas após. Resultados: Não houve diferença significativa na MAVC durante o seguimento. A diferença entre MAVC medida após 48 semanas e a basal foi de -0,04 ? 0,02 logMAR nos olhos tratados frente a -0,03 ? 0,01 logMAR nos controles (p=0,3898). A melhora da sensibilidade macular foi discretamente maior nos olhos com FMMO: 1,0 ? 0,5 dB do que nos olhos contralaterais: 0,2 ? 0,5 dB, mas sem significância estatística (p=0,0569). Não se observou mudança na estabilidade de fixação. A perda de desvio médio (MD) do campo visual dos olhos tratados (0,33 ? 0,70 dB) foi discretamente menor do que nos olhos controle (1,12 ? 0,58 dB) (p=0,0761). Nenhuma diferença significativa foi observada nas amplitudes e latências das respostas eletrorretinográficas durante o período avaliado. Não se verificou nenhuma complicação e nem efeito colateral após a injeção. Conclusão: A aplicação intravítrea de FMMO contendo células CD34+ mostrou-se segura em pacientes com RP. Observou-se, ainda, discreta melhora na sensibilidade macular, mas esta não foi significativa estatisticamente. Estudos futuros são necessários para esclarecer o potencial uso dessas células em distrofias retinianas. / Introduction: Retinitis pigmentosa (RP) is a hereditary disease of the retina, characterized by loss of visual function, mainly due to degeneration of the photoreceptors (rods and cones). Objective: To evaluate the effects of a single intravitreal injection of bone marrow mononuclear fraction (BMMF) containing CD34+ cells in patients with RP. Methods: Open trial, non-randomized, prospective, masked observer, in which 20 patients with RP with good fixation in visual field examination were included. Single intravitreal injection of BMMF was performed in only one eye of each patient, while the contralateral eyes served as control and underwent shaw injection. Evaluations included: best corrected visual acuity (BCVA); static visual field - strategy 30-2 (Octopus 900); microperimetry (MAIA - Center Vue) to evaluate fixation stability and macular sensitivity; full-field (ERG) and multifocal (mfERG) electroretinograms according to the ISCEV using Espion E2 (Diagnosys LLC) and optical coherence tomography (OCT). The exams were performed before the injection and 4, 16, 32 and 48 weeks after. Results: There was no significant difference in BCVA during follow-up. The difference measured in BCVA between 48 weeks and baseline was 0.04 ? 0.02 logMAR in treated eyes versus -0.03 ? 0.01 logMAR in controls (p=0.3898). The improvement in macular sensitivity was slightly higher in BMMF eyes: 1.0 ? 0.5 dB than in contralateral eyes: 0.2 ? 0.5 dB, but without statistical significance (p=0.0569). No change in fixation stability was observed. The mean deviation loss (MD) of the visual field in treated eyes (0.33 ? 0.70 dB) was slightly lower than in the control eyes (1.12 ? 0.58 dB) (p=0.0761). No significant difference was observed evaluating amplitudes and latencies of ERG and mfERG responses during the follow-up. No complications or side effects were observed after the injection. Conclusion: The intravitreal injection of BMMF containing CD34 + cells was shown to be safe in patients with RP. There was still a slight improvement in macular sensitivity, but this was not statistically significant. Future studies are needed to clarify the potential use of these cells in retinal dystrophies.

Advancement in Image Acquisition Equipment and progress in Image Processing Methods have brought the mathematicians and computer scientists into areas which are of huge importance for physicians and biologists. Early diagnosis of diseases like blindness, cancer and digestive problems have been areas of interest in medicine. Development of Laser Photon Microscopy and other advanced equipment already provides a good idea of very interesting characteristics of the object being viewed. Still certain images are not suitable to extract sufficient information out of that image. Image Processing methods have been providing good support to provide useful information about the objects of interest in these biological images. Fast computational methods allow complete analysis, in a very short time, of a series of images, providing a reasonably good idea about the desired characteristics. The thesis covers application of these methods in 3 series of images intended for 3 different types of diagnosis or inference. Firstly, Images of RP-mutated retina were treated for detection of rods, where there were no cones present. The software was able to detect and count the number of cones in each frame. Secondly, a gastrulation process in drosophila was studied to observe any mitosis and results were consistent with recent research. Finally, another series of images were treated where biological cells were observed to undergo mitosis. The source was a video from a photon laser microscope. In this video, objects of interest were biological cells. The idea was to track the cells if they undergo mitosis. Cell position, spacing and sometimes contour of the cell membrane are broadly the factors limiting the accuracy in this video. Appropriate method of image enhancement and segmentation were chosen to develop a computational method to observe this mitosis. Cases where human intervention may be required have been proposed to eliminate any false inference.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 68 pages. Includes Vita. Includes bibliographical references.

Les processus de plasticité cérébrale entrainés par la perte visuelle restent un domaine méconnu dans le champ des neurosciences. La vision centrale et périphérique, le meilleur compromis évolutionniste entre une bonne résolution spatiale et un volume d’espace échantillonné maximal, sont traitées par des régions différentes du cerveau. Par conséquent, étudier et comprendre l’impact de la perte visuelle centrale ou périphérique dans ces régions constitue une étape cruciale dans l’étude du cerveau visuel. Afin d’étudier ces processus, nous avons utilisé deux modèles de perte visuelle sélective de la vision centrale (la dystrophie maculaire de Stargardt) et périphérique (la rétinopathie pigmentaire), et nous avons évalué l’impact à terme de ces deux types de désafférentation sur la structure et la connectivité fonctionnelle du cerveau. 1. Plasticité structurelle induite par la perte sélective de la vision centrale ou périphérique. Nous avons étudié l’épaisseur corticale (EpCo) et de l’entropie corticale (EnCo, marquer de complexité synaptique) du lobe occipital, région pour laquelle nous disposons d’une cartographie complète des régions cytoarchitectoniques. Nous avons constaté que la perte de la vision centrale induit un amincissement des régions appartenant au flux dorsal, tandis que la perte de la vision périphérique occasionne un amincissement du cortex visuel primaire (CVP), ainsi que des régions du flux ventral et dorsal. Ces effets étaient inattendus si on se rapporte au modèle canonique qui associe la vision centrale au flux ventral et la vision périphérique au flux dorsal. La normalité de l’EnCo dans ces régions, suggère que la complexité synaptique est préservée dans les réseaux neuronaux résiduels. Nous avons identifié des modifications de l’EnCo seulement en cas de perte de la vison centrale, où l’augmentation de l’EnCo dans des régions impliquées dans la reconnaissance des objets pourrait traduire une réponse adaptative à la perte de la haute résolution spatiale de cette partie du champ visuel. Cette augmentation de la complexité synaptique pourrait compenser une éventuelle perte neuronale et être responsable de la normalité de l’EpCo dans ces régions. 2. Plasticité de la connectivité fonctionnelle des régions du cortex visuel primaire recevant les projections de la partie centrale » et périphérique champ visuel Dans cette étude, nous avons exploré et comparé la connectivité fonctionnelle des régions afférentées et desafférentées du CVP de sujets souffrant de dystrophie maculaire de Stargardt et retinitis pigmentosa, avec les régions afféréntées correspondantes du CVP de sujets avec une vision normale. Cette étude a révélé une réorganisation fonctionnelle distincte du CVP afférenté et désafférenté. Ainsi, le CVP qui reçoit les afférences visuelles résiduelles présente une connectivité fonctionnelle accrue avec des régions voisines, probablement afin de favoriser le traitement de l’information visuelle, tandis que le CVP désafférenté augmente sa connectivité fonctionnelle avec des régions plus éloignées, vraisemblablement pour contribuer aux fonctions supérieures et à des processus de type top-down. L’analyse comparative des données morphologiques et fonctionnelles suggère une correspondance des régions amincies du cortex visuel associatif avec des régions qui montrent une diminution de la connectivité fonctionnelle avec le CVP périphérique, et des régions présentant une augmentation de la complexité synaptique avec des régions qui montrent une connectivité fonctionnelle accrue avec le CVP périphérique. Ces données suggèrent que la désafferentation sensorielle du CVP périphérique est plus propice au développement d’une réorganisation cérébrale. Synoptique : Ces travaux révèlent un aspect inattendu de la plasticité cérébrale induite une perte isolée de la vision centrale ou périphérique. La réorganisation s’avère plus complexe que le laisser présager le modèle canonique actuel, vraisemblablement trop simple. / Cerebral plasticity induced by visual loss represents a poorly understood field of neuroscience, with numerous questions that don’t yet have an answer. Central and peripheral vision, the evolutionary compromise between spatial resolution and the sampled space volume, are processed in distinct areas of the brain. Understanding the impact of vision loss in theses regions, is of utmost interest for the study of visual brain. Herein, in two models of retinal disorders affecting central and peripheral vision (namely Stargardt macular dystrophy and retinitis pigmentosa), we specifically investigated the effects of the central and peripheral visual loss on brain morphology and its functional connectivity. 1. Morphological plastic changes induced by central and peripheral visual loss. We explored the effects of visual loss on cortical thickness (CoTks) and cortical entropy (CoEn, marker of synaptic complexity) in the cytoarchytectonic regions of the occipital lobe. Central visual loss associated thinning in dorsal stream regions, while peripheral visual loss in early visual cortex (EVC) and regions belonging both to dorsal and ventral stream. Theses effects were unpredicted by the canonical view “central vision – ventral stream”, “peripheral vision – dorsal stream”. Normal CoEn in theses areas suggests that synaptic complexity is preserved in the remaining networks. Only central visual field loss presented CoEn alterations, namely an increase in areas involved in object recognition, that likely reflects a synaptic complexity enhancement in response to the loss of the high spatial resolution of central vision. The gain in synaptic complexity could mask neuronal loss due to deafferentation and may account for the CoTks normality. 2. Plastic changes in the functional connectivity of central and peripheral EVC. We explored and compared to normally afferented EVC, the functional connectivity of afferented and deafferented parts of EVC and found that central and peripheral visual loss induce different patterns of reorganization. Residually afferented early visual cortex reinforce local connections presumably to enhance the processing of altered visual input, while deafferented EVC strengthen long-range connections presumably to assist high-order functions. Combined structural and functional data indicate that areas with reduced CoTks superpose with several areas presenting reduced functional connectivity with the peripheral EVC and that areas with increased CoEn superpose with several areas presenting increased functional connectivity with afferented peripheral EVC. These data point that alterations of the sensory input to the peripheral field are more prone to induce plastic changes. Overview : Data in the current work provide an interesting perspective about the plasticity following central or peripheral visual field loss and show that it is more complex than the canonical model would have let to presume.

The body of work presented here is a compendium of the multiple steps required for an investigator initiated trial of an existing medication (Valproic Acid- VPA) for a new indication (Retinitis Pigmentosa – RP). The chapters are listed in logical and chronological order of the process. In order to access patient records an expedited Institutional Review Board (IRB) application for retrospective chart review was submitted (Chapter 1). These records enabled the statistical analysis which not only laid the framework for the trial design, but also became the basis for two manuscripts (Chapter 2). Protocol development informed by the preliminary human studies (Chapter 3) was an instrumental part of the Investigational New Drug (IND) application (Chapter 3.5). This protocol along with the extensive case report forms that detail the intended data to be collected are included in the IND application. Because the Phase II clinical trial proposed attempting to identify the specific RP mutations of the subjects utilizing a National Eye Institute (NEI) study that enabled free genotyping services, two IRB applications were submitted (Chapter 3.6). The first was for approval of the NEI genotyping protocol, the second involved the VPA intervention. Two very different sources of funding for this trial were attempted (Chapter 4) – the NIH via the Challenge Grant mechanism and a private eye disease foundation (Foundation Fighting Blindness). In Chapter 5 I detail the alternate study designs that were considered and developed for this trial (and ultimately abandoned). Finally, in Chapter 6, I formally detail my suggestions to aid in the development of a comprehensive investigator initiated core facility at UMMMC. The goal of this project was two-fold. The first was to learn the entire process of trial and protocol design both from a Umass Institutional perspective as well as from the perspective of the FDA. The second goal was the very real prospect of helping patients with a blinding disease. This work was successful on both counts. IRB approval was received for all the submitted applications. The complexity and uniqueness of many aspects of these submissions culminated in a comprehensive learning experience. The process of working with the Umass Research Pharmacy as well as developing the industry contacts and know-how to develop a workable and financially feasible placebo were both particularly important learning experiences. FDA approval of the IND submission was also received, and the process of pre-communication and delving into the considerable and ever-changing rules and regulations resulted in an extensive and valuable knowledge base. While the practicality of funding has limited the ability of this trial to move forward at this point, given the extensive framework laid by this body of work, we are actively pursuing other opportunities. The third outcome of this work, while not as intentional, was the considerable process of determining the specific competencies and infrastructure that exist at UMMMC to enable investigator initiated drug intervention studies. While this institution is clearly moving rapidly in the direction of translational research, the many needs of these studies are often only clearly understood when the process is specifically undertaken. In completing the approval of this Phase II clinical trial, I was not only able to better understand and define the existing capabilities of UMMMC for this kind of research, I was able to add to that infrastructure when the existing knowledge or skill set was not available. In this manner, I was able to inform and guide many of the support personnel who guided me and have become a part of the strategic direction of UMMMC towards clinical translational research.

Avancement dans l'acquisition d'image et le progrès dans les méthodes de traitement d'image ont apporté les mathématiciens et les informaticiens dans les domaines qui sont d'une importance énorme pour les médecins et les biologistes. Le diagnostic précoce de maladies (comme la cécité, le cancer et les problèmes digestifs) ont été des domaines d'intérêt en médecine. Développement des équipements comme microscope bi-photonique à balayage laser et microscope de fluorescence par réflexion totale interne fournit déjà une bonne idée des caractéristiques très intéressantes sur l'objet observé. Cependant, certaines images ne sont pas appropriés pour extraire suffisamment d'informations sur de cette image. Les méthodes de traitement d'image ont été fournit un bon soutien à extraire des informations utiles sur les objets d'intérêt dans ces images biologiques. Rapide méthodes de calcul permettent l'analyse complète, dans un temps très court, d'une série d'images, offrant une assez bonne idée sur les caractéristiques souhaitées. La thèse porte sur l'application de ces méthodes dans trois séries d'images destinées à trois différents types de diagnostic ou d'inférence. Tout d'abord, Images de RP-muté rétine ont été traités pour la détection des cônes, où il n'y avait pas de bâtonnets présents. Le logiciel a été capable de détecter et de compter le nombre de cônes dans chaque image. Deuxièmement, un processus de gastrulation chez la drosophile a été étudié pour observer toute la mitose et les résultats étaient cohérents avec les recherches récentes. Enfin, une autre série d'images ont été traités où la source était une vidéo à partir d'un microscopie photonique à balayage laser. Dans cette vidéo, des objets d'intérêt sont des cellules biologiques. L'idée était de suivre les cellules si elles subissent une mitose. La position de la cellule, la dispersion spatiale et parfois le contour de la membrane cellulaire sont globalement les facteurs limitant la précision dans cette vidéo. Des méthodes appropriées d'amélioration de l'image et de segmentation ont été choisies pour développer une méthode de calcul pour observer cette mitose. L'intervention humaine peut être requise pour éliminer toute inférence fausse. / Advancement in Image Acquisition Equipment and progress in Image Processing Methods have brought the mathematicians and computer scientists into areas which are of huge importance for physicians and biologists. Early diagnosis of diseases like blindness, cancer and digestive problems have been areas of interest in medicine. Development of Laser Photon Microscopy and other advanced equipment already provides a good idea of very interesting characteristics of the object being viewed. Still certain images are not suitable to extract sufficient information out of that image. Image Processing methods have been providing good support to provide useful information about the objects of interest in these biological images. Fast computational methods allow complete analysis, in a very short time, of a series of images, providing a reasonably good idea about the desired characteristics. The thesis covers application of these methods in 3 series of images intended for 3 different types of diagnosis or inference. Firstly, Images of RP-mutated retina were treated for detection of rods, where there were no cones present. The software was able to detect and count the number of cones in each frame. Secondly, a gastrulation process in drosophila was studied to observe any mitosis and results were consistent with recent research. Finally, another series of images were treated where biological cells were observed to undergo mitosis. The source was a video from a photon laser microscope. In this video, objects of interest were biological cells. The idea was to track the cells if they undergo mitosis. Cell position, spacing and sometimes contour of the cell membrane are broadly the factors limiting the accuracy in this video. Appropriate method of image enhancement and segmentation were chosen to develop a computational method to observe this mitosis. Cases where human intervention may be required have been proposed to eliminate any false inference.

Le syndrome de Bardet-Biedl (BBS) est considéré comme l’une des causes les plus fréquentes de rétinopathie pigmentaire dite syndromique. Il a été démontré une connexion entre les protéines BBS et les structures du cil primaire. Le cil primaire est un organelle formé par une fine évagination de la membrane plasmique soutenu par une ossature de microtubules. Dans la rétine, le photorécepteur (PR) est une cellule ciliaire composée d’un segment interne et d’un segment externe reliés par un cil primaire modifié. Au cours de ce travail, nous avons mis en évidence que le stress du réticulum endoplasmique est à l’origine du processus apoptotique car un défaut ciliaire dans le PR entraine l’accumulation de protéines dans le segment interne et déclenche une réponse au stress cellulaire appelé unfolded protein response. Nous avons développé un traitement pharmacologique modulant ce stress cellulaire afin de ralentir l’apoptose des PR dans un modèle murin BBS. Cette approche pharmacologique a montré son efficacité dans le maintien et la fonctionnalité des PR. Elle pourrait potentiellement être applicable à d’autres ciliopathies rétiniennes. / Bardet-Biedl syndrome (BBS) is one of the most frequent cause of syndromic retinitis pigmentosa. BBS proteins are related to primary cilium structure and function. The primary cilium is microtubule-based antenna-like structure at the surface of the cell. In the retina, the photoreceptor (PR) is a ciliated cell composed of an inner and an outer segment linked by a modified primary cilium. In this study, we demonstrated that endoplasmic reticulum stress induces unfolded protein response due to protein accumulation in the inner segment in case of ciliary defect in the PR leading to apoptosis. We designed a pharmacological treatment to alleviate PR apoptosis in a BBS mouse model. This pharmacological approach was efficient to protect PR from apoptosis and maintain their functionality. This treatment could be applicable to others retinal ciliopathies.