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1
Mehat, M. S. "Investigation of stem cell-derived retinal pigmented epithelium transplantation." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/1540121/.
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Retinal pigment epithelium (RPE) cells perform a variety of roles that are principally directed at maintaining retinal function and homeostasis and their loss causes conditions such as age-related macular degeneration (AMD) and Stargardt disease (STGD). Regeneration of the loss or dystrophic RPE cells with stem cell-derived RPE may provide a potential therapeutic option. STGD is the commonest form of juvenile-onset, inherited macular degeneration. In order to determine the safety and efficacy of embryonic stem (ES) cell-derived RPE transplantation for the treatment of STGD, twelve subjects with STGD received a suspension of hES-derived RPE cells in escalating dose cohorts. Following the intervention, areas of sub retinal pigmentation were noted in all participants, suggestive of engraftment and survival. Transplanted hES-derived RPE cells were often observed overlying regions of atrophic Bruch's membrane (BrM). There was no evidence of tumorigenicity, immune adverse events or other serious safety concerns related to the transplanted cells. Furthermore, there was no significant change in visual function in the study eye of any participant. In order to improve the efficacy of ES-derived RPE transplantation, I developed an improved rodent model of retinal degeneration that features focal regions of atrophic BrM. I used a diode laser to selectively ablate RPE and observed focal regions of RPE atrophy with corresponding changes in choroidal vasculature that resemble those observed in retinal degenerative disease. Moreover, transplantation of human ES- and human induced pluripotent stem cell (iPS)-derived RPE resulted in re-population and restoration of RPE morphology post ablation. Although transplanted ES-derived RPE survived well on healthy BrM, attachment and survival of RPE is compromised on BrM that exhibits AMD or senescent changes. A potential strategy to promote survival and engraftment where there is damaged BrM is to deliver ES-derived RPE on a carrier substrate. I used a bespoke electrospun scaffold consisting of poly(e-caprolactone) and seeded this with either hESC or hiPSC-derived RPE. Transplantation of ES-derived RPE resulted in a functional monolayer of RPE with correct orientation and polarity on a biodegradable, porous and biocompatible membrane. These studies support further work in large animal models using ES-derived RPE scaffolds to restore the RPE in the presence of a compromised BrM.
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Weigel, Andrea Lynn. "Gene expression profiling of the retinal pigmented epithelium oxidative stress response in vitro and in vivo /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2002. http://uclibs.org/PID/11984.
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Wood, John P. M. "Induction of cell death within the retina and retinal pigmented epithelium : the role of protein kinase C." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363764.
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Más, Gómez Néstor [Verfasser], and Olaf [Akademischer Betreuer] Strauß. "Endogenously expressed bestrophin-1 modulates calcium signaling in the retinal pigmented epithelium / Néstor Más Gómez. Betreuer: Olaf Strauß." Regensburg : Universitätsbibliothek Regensburg, 2012. http://d-nb.info/1025386140/34.
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Raisler, Brian. "Adeno-associated virus type-2 mediated expression of pigmented epithelium derived factor or kringles 1-3 of angiostatin reduced neovascular retinopathies." [Gainesville, Fla.] : University of Florida, 2003. http://purl.fcla.edu/fcla/etd/UFE0002385.
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Sun, Jianan. "Protective Effects of Human iPS-Derived Retinal Pigmented Epithelial Cells in Comparison with Human Mesenchymal Stromal Cells and Human Neural Stem Cells on the Degenerating Retina in rd1 Mice." Kyoto University, 2016. http://hdl.handle.net/2433/215387.
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Tretiach, Marina Louise. "Bovine Models of Human Retinal Disease: Effect of Perivascular Cells on Retinal Endothelial Cell Permeability." Thesis, The University of Sydney, 2005. http://hdl.handle.net/2123/1153.
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Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true â barrier characteristicsâ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.
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Tretiach, Marina Louise. "Bovine Models of Human Retinal Disease: Effect of Perivascular Cells on Retinal Endothelial Cell Permeability." University of Sydney, 2005. http://hdl.handle.net/2123/1153.
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Doctor of Philosophy (Medicine)Background: Diabetic vascular complications affect both the macro- and microvasculature. Microvascular pathology in diabetes may be mediated by biochemical factors that precipitate cellular changes at both the gene and protein levels. In the diabetic retina, vascular pathology is found mainly in microvessels, including the retinal precapillary arterioles, capillaries and venules. Macular oedema secondary to breakdown of the inner blood-retinal barrier is the most common cause of vision impairment in diabetic retinopathy. Müller cells play a critical role in the trophic support of retinal neurons and blood vessels. In chronic diabetes, Müller cells are increasingly unable to maintain their supportive functions and may themselves undergo changes that exacerbate the retinal pathology. The consequences of early diabetic changes in retinal cells are primarily considered in this thesis. Aims: This thesis aims to investigate the effect of perivascular cells (Müller cells, RPE, pericytes) on retinal endothelial cell permeability using an established in vitro model. Methods: Immunohistochemistry, cell morphology and cell growth patterns were used to characterise primary bovine retinal cells (Müller cells, RPE, pericytes and endothelial cells). An in vitro model of the blood-retinal barrier was refined by coculturing retinal endothelial cells with perivascular cells (Müller cells or pericytes) on opposite sides of a permeable Transwell filter. The integrity of the barrier formed by endothelial cells was assessed by transendothelial electrical resistance (TEER) measurements. Functional characteristics of endothelial cells were compared with ultrastructural morphology to determine if different cell types have barrier-enhancing effects on endothelial cell cultures. Once the co-culture model was established, retinal endothelial cells and Müller cells were exposed to different environmental conditions (20% oxygen, normoxia; 1% oxygen, hypoxia) to examine the effect of perivascular cells on endothelial cell permeability under reduced oxygen conditions. Barrier integrity was assessed by TEER measurements and permeability was measured by passive diffusion of radiolabelled tracers from the luminal to the abluminal side of the endothelial cell barrier. A further study investigated the mechanism of laser therapy on re-establishment of retinal endothelial cell barrier integrity. Müller cells and RPE, that comprise the scar formed after laser photocoagulation, and control cells (Müller cells and pericytes, RPE cells and ECV304, an epithelial cell line) were grown in long-term culture and treated with blue-green argon laser. Lasered cells were placed underneath confluent retinal endothelial cells growing on a permeable filter, providing conditioned medium to the basal surface of endothelial cells. The effect of conditioned medium on endothelial cell permeability was determined, as above. Results: Co-cultures of retinal endothelial cells and Müller cells on opposite sides of a permeable filter showed that Müller cells can enhance the integrity of the endothelial cell barrier, most likely through soluble factors. Low basal resistances generated by endothelial cells from different retinal isolations may be the result of erratic growth characteristics (determined by ultrastructural studies) or the selection of vessel fragments without true âbarrier characteristicsâ in the isolation step. When Müller cells were co-cultured in close apposition to endothelial cells under normoxic conditions, the barrier integrity was enhanced and permeability was reduced. Under hypoxic conditions, Müller cells had a detrimental effect on the integrity of the endothelial cell barrier and permeability was increased in closely apposed cells. Conditioned medium from long-term cultured Müller cells and RPE that typically comprise the scar formed after lasering, enhanced TEER and reduced permeability of cultured endothelial cells. Conclusions: These studies confirm that bovine tissues can be used as a suitable model to investigate the role of perivascular cells on the permeability of retinal endothelial cells. The dual effect of Müller cells on the retinal endothelial cell barrier under different environmental conditions, underscores the critical role of Müller cells in regulating the blood-retinal barrier in health and disease. These studies also raise the possibility that soluble factor(s) secreted by Müller cells and RPE subsequent to laser treatment reduce the permeability of retinal vascular endothelium. Future studies to identify these factor(s) may have implications for the clinical treatment of macular oedema secondary to diseases including diabetic retinopathy.
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Al-Hosaini, Heba. "Age-related changes in retinal pigment epithelium." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444089/.
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The retinal pigment epithelium (RPE) is a monolayer of hexagonal organized cells located between the choriocapillaris and the neurosensory retina. As the RPE is implicated in a range of eye diseases, an understanding of its structure and ability for self renewal is critical for therapeutic strategies. Analysis of human RPE cells at the extreme periphery of the retina reveals a population larger in size than those in the centre, they are highly irregular and form an annulus of 4-5 mm. Although binucleation in humans is rare, 10% of these cells are binucleated. In the central region these large binucleated cells are only found adjacent to drusen, which are age-related lipid-rich deposits. Compared with humans, rat RPE is relatively homogeneous, however, the majority of its cells are binucleated, particularly in the central region. Human and rat RPE also shows different patterns of aging. In humans, the centre of the retina shows a significant reduction in RPE cell density with age, which was not observed in aged rats. The capacity of mature RPE cells to enter the cell cycle was investigated using a proliferative marker in rats. Here a subpopulation of mature peripheral RPE cells had the capacity to enter the cell cycle, and one-third of these cells completed cellular division. As RPE proliferation occur in response to retinal detachment, this was performed on rats and the patterns of gene expression in RPE examined. An increase was observed in nestin, PCNA and Ki67 expression, which was also confirmed at a protein level by immunohistochemistry. These results suggest that RPE cells have the capacity to proliferate and may possibly differentiate if subjected to appropriate stimuli in a normal retina.
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Donahue, Vicki S. "Phospholipase c activity in retinal pigment epithelium." Virtual Press, 1997. http://liblink.bsu.edu/uhtbin/catkey/1041916.
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The role of the retinal pigment epithelial cells on the viability and renewal of photoreceptors has been well demonstrated in the Royal College of Surgeons (RCS) strain of rat. These rats are characterized by an inherited time-dependent degeneration of their photoreceptors. This degeneration is apparently due to the inability of the retinal pigment epithelial cells to adequately ingest fragments of photoreceptor membrane that are shed during the course of photoreceptor membrane renewal. The buildup of photoreceptor material in the interphotoreceptor space ultimately leads to the degeneration of photoreceptors in these animals. With regard to the pigment epithelial cells, neither the mechanism mediating the ingestion process in normal rats nor the nature of the defect of this process in RCS rats is understood.It is the goal of this proposed research to assay for the presence of phospholipase C in retinal pigment epithelial (RPE) cells and to determine possible modulators of the enzyme in an attempt to associate this with the process of phagocytosis.Department of Biology
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Warner, Jacqueline. "Ionic transport across mammalian retinal pigment epithelium." Thesis, University of Westminster, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292857.
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Chen, F. K. "Retinal pigment epithelium transplantation in retinal diseases." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1318070/.
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Age-related macular degeneration (AMD) and inherited macular diseases (IMD) are retinal disorders that can cause blindness through atrophy of the retinal pigment epithelium (RPE) or choroidal neovascularisation (CNV). RPE transplantation in severe forms of neovascular AMD has been performed with promising short-term outcomes. However, this approach has not been evaluated in atrophic types of AMD or IMD. Furthermore, the long-term outcomes of photoreceptors cell function rescue by RPE reconstruction in neovascular AMD is unknown. Current surgical techniques are complex with associated high complication rates. Therefore, other treatment approaches to reconstruct the RPE are required. This thesis aims to examine whether long-term photoreceptor cell function rescue can be achieved through RPE reconstruction by investigating the outcomes of autologous RPE transplantation or full macular translocation in AMD and IMD. A further aim is to determine the feasibility of a new approach to reconstruct the RPE using human embryonic stem cell (hESC). A prospective study of autologous RPE-choroid grafts in 9 patients with atrophic macular disease secondary to AMD or IMD demonstrated that submacular RPE graft can support retinal function and fixation. However, there was a high surgical and post-operative complication rates and the overall visual acuity and reading ability declined. Long-term follow-up demonstrated that the graft can maintain retinal function for over 2 years in some patients. A retrospective review of long-term outcomes following autologous RPE-choroid grafts and full macular translocation in 12 and 40 patients with neovascular AMD, respectively, showed that rescue of retinal function beyond 2 years is possible. A visual acuity of 6/12 was achieved and maintained for over 2 years in 8% and 15% of patients who had patch graft and translocation, respectively. However, overall visual acuity outcomes were limited by delayed post-operative complications such as recurrent CNV and cystoid macular oedema. A prospective porcine experiment showed that subretinal implant of hESC derived-RPE was feasible and human donor cell can survive in vivo for up to 6 weeks. However, there was significant loss of the hESC-RPE which may have occurred intra-operatively or during the first 2 weeks post-operatively. Macrophages were noted at the site of the graft suggesting some inflammatory and immunological responses to the human cells, polyester substrate or surgical trauma. The work in this thesis has provided the proof of principle that reconstruction of the RPE can maintain retinal function in atrophic and neovascular macular diseases over the long-term. A novel approach using hESC-RPE on an artificial substrate may be a more feasible and safer alternative to current clinical techniques of RPE reconstruction.
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Chalour, Naïma. "La réponse des cellules gliales de Müller à l'amyloïde-β et au stress oxydant dans la dégénérescence rétinienne." Thesis, Paris 5, 2012. http://www.theses.fr/2012PA05T006/document.
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La dégénérescence maculaire liée à l’âge ou DMLA est une pathologie oculaire qui touche près d’un million de personnes en France, et représente la première cause de cécité légale dans les pays industrialisés. C’est une affection multifactorielle (environnement, génétique), dans laquelle les stress inflammatoires, métaboliques et oxydants interviennent et aboutissent à la mort des photorécepteurs. L’apparition des drusen (dépôts de matériel extracellulaire contenant de l’amyloïde-β (Aβ)), entre les cellules de l’épithélium pigmentaire de la rétine (EPR) et la membrane de Brush, représente un facteur de risque de développement de la DMLA. De plus, le 4-hydroxynonenal (4-HNE) est un marqueur de stress oxydant dans la rétine de patients de différentes pathologies dégénératives comme la DLMA. L’identification des mécanismes moléculaires et cellulaires impliqués dans les dégénérescences rétiniennes la pathogenèse de la DMLA constitue un enjeu de santé publique, puisqu’elle permettrait de développer de nouvelles stratégies thérapeutiques anti-dégénératives.Le but de mon travail de thèse a été dans un premier temps de mieux comprendre le rôle de l’Aβ dans la dégénérescence rétinienne.Nous avons montré que l’Aβ induit une activation rapide des cellules microgliales, une gliose soutenue des cellules gliales de Müller (CGM), un œdème dans la rétine interne et une apoptose des photorécepteurs. La dégénérescence des photorécepteurs est en corrélation avec une activation soutenue de PERK, impliquée dans la voie pro-apoptotique de la réponse UPR. Par ailleurs la gliose des CGM est caractérisé par une délocalisation des canaux Kir4.1, une diminution de l’expression d’AQP4 et de la glutamine synthetase (GS), et une augmentation de l’expression des canaux Kir2.1 et du transporteur GLAST1, suggérant une dérégulation de l’homéostasie rétinienne contrôlée par ces protéines. Nous avons montré que l’inhibition de la réponse inflammatoire, par l’utilisation de l’indomethacine, un inhibiteur non stéroïdien de de la cyclooxygénase (COX) 2, réverse l’effet de l’Aβ sur l’expression des canaux Kir4.1 et sur GLAST1 mais pas celle de la GS et d’AQP4, suggérant un couplage partiel entre la gliose et la réponse inflammatoire dans notre modèle d’injection sous-rétinienne d’Aβ.Dans un deuxième temps, nous nous sommes intéressés au rôle du 4-HNE dans les CGM, un produit de peroxydation lipidique, qui est produit dans la rétine sous l’effet de l’Aβ. Nous avons observé qu’un stress oxydant unique et létal induit par le 4-HNE, entraîne la mort des CGM par apoptose dépendante de l’activation des caspases. L’utilisation d’antioxydants impliqués dans la régénération du glutathion (GSH), protège contre la mort des CGM. L’analyse du transcriptome des CGM soumises au 4-HNE a permis de mettre en évidence une réponse transcriptionnelle adaptative des CGM : une activation de la défense anti-oxydante, de la réponse UPR (unfolded protein response) au stress du réticulum endoplasmique, et un phénotype anti-inflammatoire. Par ailleurs, la surexpression de l’APP (amyloid protein precursor), dont l’expression du transcrit est augmentée sous l’effet du stress oxydant dans les CGM, protège ces cellules contre la mort induite par le 4-HNE. Cette protection est associée à une augmentation des capacités anti-oxydantes et à une activation de la voie de survie de la réponse UPR. L’ensemble de nos résultats montre un rôle de l’Aβ dans la dégénérescence des photorécepteurs et indique que le métabolisme de l’APP, ainsi que les voies de survie et pro-apoptotique de la réponse UPR pourraient constituer des cibles thérapeutiques contre la dégénérescence rétinienne induite par l’Aβ ou les stress oxydantsAge related macular degeneration (AMD) is a leading cause of blindness in western countries and affects one million people in France. Multiple risk factors (genetics, environment) are involved in the pathogenesis of AMD. In addition, the AMD pathogenesis is strongly associated with chronic oxidative stress and inflammation that ultimately lead to photoreceptor death. AMD is characterized by the formation of drusen, extracellular deposits, including amyloid-β (Aβ), between the retinal pigmented epithelium and Bruch’s membrane. Moreover, 4-hydroxynonenal (4-HNE) is an oxidative stress marker of different retinal diseases including AMD. The determination of molecular and cell mechanisms involved in retinal degeneration and the pathogenesis of AMD is required in order to develop new therapeutic anti-degenerative approaches. The aim of our study was first to investigate the role of Aβ in retinal degeneration. We demonstrated that subretinal injection of Aβ induces an early activation of microglial cells, a sustained retinal Müller glial (RMG) cells gliosis, an oedema in the internal part of retina and photoreceptors apoptosis. The photoreceptors apoptosis was correlated with a sustained activation of PERK, a kinase implicated in the pro-apoptotic pathway of UPR (unfolded protein response). In addition, RMG gliosis has been characterized by a Kir4.1 channel redistribution, a down-regulation of AQP4 and glutamine synthetase (GS) expression, and an up-regulation of Kir2.1 channel and GLAST1 transporter expression, suggesting a dysregulation of the retinal homeostasis which is controlled by these proteins. The inhibition of the inflammatory response using indomethacin, a non-steroidal and non-specific cyclooxygenase (COX) 2 inhibitor, reversed Aβ-induced Kir4.1 channel redistribution and GLAST1 up-regulation but not GS and AQP4 down-regulation, suggesting a partial coupling between gliosis and inflammatory response in retinal degeneration after subretinal injection of Aβ in mice. The second part of our study aimed to investigate the effects on RMG cells of 4-HNE, a lipid peroxidation product that is up-regulated in retina after Aβ injection. We have shown that a single lethal oxidative stress using 4-HNE induces RMG cells apoptosis associated with caspase 3 and caspase 9 activation. Pre-treatment of RMG cells with anti-oxidative molecules involved in glutathione regeneration restored cell viability. Transcriptome analysis of RMG cells treated with 4-HNE showed an adaptive transcriptional response consisting in an activation of anti-oxidative stress cell defense, activation of UPR in response to endoplasmic reticulum stress and anti-inflammatory phenotype. APP (amyloid protein precursor) overexpression, which the transcript is up-regulated in RMG cells under oxidative stress, protects from 4-HNE-induced cell death. This protection is associated with an up-regulation of anti-oxidative cell defense and an activation of the pro-survival pathway of UPR. Our study pinpoints the role of Aβ in photoreceptors degeneration and suggests that targeting APP metabolism, pro and anti-apoptotic pathways of the UPR response may hel develop selective methods against retinal degeneration implicating Aβ and oxidative stress
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Gagliardi, Giuliana. "Generation and selection of photoreceptor precursors from human-induced pluripotent stem cells for cell therapy Generation of storable retinal organoids and retinal pigmented epithelium from adherent human iPS cells in xeno-free and feeder-free conditions Characterization and transplantation of CD73-positive photoreceptors isolated from human iPSC-derived retinal organoids." Thesis, Sorbonne université, 2018. http://www.theses.fr/2018SORUS082.
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Les maladies dégénératives de la rétine représentent un défi majeur de santé publique pour lequel les techniques thérapeutiques restent aujourd’hui insuffisantes et limitées. L'une des approches les plus prometteuses est la thérapie cellulaire, c'est-à-dire le remplacement d’une typologie cellulaire spécifique. Dans ce contexte, les cellules souches pluripotentes pourraient être utilisées comme source illimitée de cellules rétiniennes. Le protocole développé par notre groupe pour la génération de photorécepteurs transplantables à partir de cellules souches induites à la pluripotence (iPSCs) a été adapté à des conditions compatibles avec les normes dites « Good Manufacturing Practice » (GMP) afin de passer rapidement à une application clinique. L'antigène de surface CD73 a été caractérisé comme un marqueur spécifique des photorécepteurs dérivés de iPSCs humaines. Nous avons développé une stratégie de tri basée sur le ciblage magnétique de CD73, qui permet d’obtenir une population homogène de photorécepteurs. Nous avons démontré l’absence du risque de développement de tumeur lié à la transplantation de ces cellules. Enfin, les cellules CD73-positives sont capables de survivre et acquérir un certain niveau de maturité après transplantation chez un modèle de dystrophie rétinienne. Bien que la compétence de cellules greffées à former de connections synaptiques fonctionnelles et à rétablir la fonction visuelle est encore à démontrer, ce travail apporte un nouvel outil pour l’utilisation de photorécepteurs dérivés de iPSCs humaines pour des applications thérapeutiques ainsi que pour l’étude des pathologies concernant les photorécepteursRetinal degenerative diseases represent a major public health challenge, for which there are currently no effective treatments. Cell therapy represent a promising therapeutic approach, consisting in specifically replacing degenerated cells with new cells. In this perspective, pluripotent stem cells could be used as an unlimited source of retinal cells. The work presented here aimed at contributing to the development of a cell therapy product for the treatment of photoreceptor degenerative diseases. We have demonstrated the possibility to generate and store retinal organoids from human induced pluripotent stem cells (iPSCs) using raw media complying with Good Manufacturing Practice (GMP). We have characterized the surface antigen CD73 as a marker of photoreceptors in human retinal organoids derived from human iPSCs. We have established a protocol based on the magnetic labelling of CD73-positive cells allowing for the separation of a homogenous population of photoreceptors. We could demonstrate their safety by showing the absence of tumor development upon transplantation of these cells in an immune-compromised host. Finally, CD73-positive cells had the ability to survive and to reach a certain degree of maturation in a dystrophic retinal environment. Although the ability of donor cells to establish functional synaptic connections and mediate a significant rescue of the visual function remains to be assessed, this work provides an advancement for the use of iPSC-derived photoreceptors in clinical applications and for the study of photoreceptor diseases
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Devine, Lesley. "Cellular interactions between lymphocytes and retinal pigment epithelium." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338652.
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Sheridan, Carl Michael. "The retinal pigment epithelium and its extracellular matrix." Thesis, University of Liverpool, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366682.
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Shahabi, G. "Dynamic features of the mature retinal pigment epithelium." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1352450/.
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The retinal pigment epithelium (RPE) is a monolayer of cells that are vital for visual function and play a key role in the maintenance of the photoreceptors. Within RPE cells are melanin granules which absorb stray light and minimise scatter within the eye, therefore, protecting the RPE from damage. Albinos lack this protective capacity of melanin as a result of mutations of the tyrosinase gene. The first half of this thesis investigates heterogeneity within the RPE in both pigmentation phenotypes. Furthermore, the effect of ageing on the RPE is examined. Immunohistochemistry highlighted the molecular heterogeneity of the RPE and how this varies with pigmentation phenotype. In aged animals, SEM revealed abnormalities occurring within the photoreceptor outer segments of albinos, while ERGs and QRT-PCR illustrated that albinos show the signs of an age-related decline in visual function much sooner than pigmented animals. These studies revealed that in the outer retina, albinism is a progressive disease and not simply a congenital abnormality. The second half of this thesis investigates migration and proliferation of RPE cells in healthy pigmented animals. Using BrdU and DiI it was established that individual RPE cells have the ability to migrate. In addition, the effect of inducing lesions in different retinal locations was studied to determine whether this affects the response of RPE cells to the damaged area. The results conveyed that a single unilateral lesion in the RPE caused an upregulation of proliferating cells not only in the lasered eye, but also the contralateral unlasered eye in a quadrant specific manner. An additional experiment investigated the effect of treating rats with Glatiramer Acetate and found that it elevates levels of cell proliferation in the RPE of treated animals. Together, the data presented in this thesis demonstrate that the mature RPE is a dynamic heterogeneous epithelial cell layer.
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Ulbrich, Stefan, Jens Friedrichs, Monika Valtink, Simo Murovski, Clemens M. Franz, Daniel J. Müller, Richard H. W. Funk, and Katrin Engelmann. "Retinal Pigment Epithelium Cell Alignment on Nanostructured Collagen Matrices." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-136023.
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We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α2 were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α2 was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α2 expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α2-mediated matrix binding was verified by preincubation with an α2-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriersDieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Cahyadi, S. "Zinc in the retinal pigment epithelium and choriocapillaris interface." Thesis, University College London (University of London), 2012. http://discovery.ucl.ac.uk/1344183/.
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The highest concentration of zinc in human tissues is found in the retinal pigment epithelium (RPE)-choroid complex. Despite the association of zinc deficiency with age-related macular degeneration (AMD) and the widespread use of zinc supplementation to slow the progression of AMD, very little is known about how zinc affects the RPE and the choroid. Molecular and cell biology techniques were used to uncover how changes in zinc levels could play a role in regulating the RPE-choroid complex. First, QRT-PCR was used to assess the expressions of all 24 known zinc transporters in cadaveric human RPE, cultured RPE cells and cells isolated from other parts of the retina, ZIP12 was identified as a potentially important transporter to regulate zinc levels at the RPE-choroid interface. As there is very little published about ZIP12, bioinformatics and data mining were used to understand how this protein might function. Confirmation of these predictions was achieved through the cloning and expression of V5-tagged ZIP12 protein in different cell lines. Based on these experiments, we concluded that ZIP12 is a plasma membrane transporter that mediates zinc influx. In parallel, we tested the hypothesis that extracellular zinc levels in Bruch’s membrane might be involved in regulating both the RPE as well as the fenestrated choroidal capillaries using cultures of ARPE19 and bEND5 cells respectively. The presence of extracellular zinc in the growth media affected the characteristics of ARPE19 cells as well as fenestrae formation in bEND5 cells. In summary, the range of zinc transporter at the RPE-choroid interface was defined and properties of one particular transporter, ZIP12 which may have a specific role at this site, were elucidated. Using cellular systems some of the effects of zinc on the RPE-choroid complex were investigated. Future studies are required to elucidate the role of zinc in the AMD pathogenesis.
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Ulbrich, Stefan, Jens Friedrichs, Monika Valtink, Simo Murovski, Clemens M. Franz, Daniel J. Müller, Richard H. W. Funk, and Katrin Engelmann. "Retinal Pigment Epithelium Cell Alignment on Nanostructured Collagen Matrices." Karger, 2011. https://tud.qucosa.de/id/qucosa%3A26646.
Full textAbstract:
We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α2 were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α2 was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α2 expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α2-mediated matrix binding was verified by preincubation with an α2-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriers.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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Lane, Carol. "Transplantation of retinal pigment epithelial cells." Thesis, University of Southampton, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316114.
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Zhang, Yadan. "Mechanism(s) by which pigment epithelium-derived factor regulate angiogenesis." Thesis, Cardiff University, 2007. http://orca.cf.ac.uk/54652/.
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Pigment Epithelium-derived Factor (PEDF), a natural protein possessing both neuroprotective and anti-angiogenic properties, is a very unique and attractive candidate as a therapeutic agent in the management of pathological neovascular diseases, such as tumours, age-related macular degeneration (AMD) and diabetic retinopathy. While it is well-known that PEDF can exert powerful effects on various tissues and cells, the underlying mechanism of PEDF's action is not well understood. This study investigated the relationship between vascular endothelial growth factor (VEGF)/PEDF and VEGFR-1 /VEGFR-2 by exploring Presenilin-l (PS-l) dependent regulated intramembrane proteolysis (RIP). Work on this non-classical pathway was initiated by Cai et al., (2006) using in vitro models of bovine retinal microvascular endothelial cells (BRMECs). Current study used BRMECs and human retinal pigment epithelial (HRPE) cells. In this study, BRMECs and HRPE cells were isolated and cultured. BRMECs were used as an angiogenic cell type while HRPE cells were used as an angiogenic regulator cell type. The characteristics of endothelial and epithelial cells and the localisation of VEGFR-1, VEGFR-2 and PS in BRMECs and HRPE cells were determined using immunocytochemistry techniques. The effects of VEGF and PEDF on VEGFR-1, VEGFR-2 and PS were assessed using immunocytochemistry and Western blotting, y-secretase activity in BRMECs and HRPE cells treated with various growth factors were analysed using a y-secretase activity kit. The role of VEGF on the production of PEDF and the expression of VEGFR-1, VEGFR-2 and PS in HRPE cells was investigated at both the transcriptional and translational levels. The techniques, VEGF-small interfering ribonucleic acid (VEGF-siRNA), reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and Enzyme-linked immunosorbent assay (ELISA) were used for the investigation. Results obtained from the project showed that PEDF had a regulatory role in the counterbalance of VEGFR-1 and VEGFR-2 expression in cultured BRMECs. PEDF upregulated y-secretase activity and PS-1 expression in BRMECs while VEGF acted as an antagonist of the effect of PEDF. In contrast, in HRPE cells, VEGF upregulated y-secretase activity and PEDF acted as an antagonist of the effect of VEGF. VEGF-siRNA induced a reduction of PEDF at both transcriptional and protein levels and a reduction of VEGFR-1 at the protein level. The effects of VEGF and PEDF on VEGFR-1 and VEGFR-2 may be cell type dependent. This study strengthens the view that PEDF can exert different regulatory effects on the same molecule (s) in different cell types. PEDF acts either antagonistically to VEGF or synergistically dependent upon the target molecule. Deciphering the cellular and molecular mechanisms underlying these interactions will not only contribute to our understanding of PEDF's action but also provide the foundation to maximise the therapeutic potential of this protein.
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Heller, Janosch Peter Dave. "Transplantation of retinal pigment epithelium in age-related macular degeneration." Thesis, University of Cambridge, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708198.
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Penishkevich, Ya I. "Laboratory testing of retinal pigment epithelium dysfuction in diabetic retinopathy." Thesis, БДМУ, 2022. http://dspace.bsmu.edu.ua:8080/xmlui/handle/123456789/19663.
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Lu, Tianlin. "Studies on the Mechanism behind Retinal Pigment Epithelium (RPE) Reprogramming." Miami University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=miami1575301308695243.
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Shahhossein-Dastjerdi, Saeed. "Human Retinal Pigment Epithelium In Physiological Ageing And Ocular Pathology." Thesis, The University of Sydney, 2020. https://hdl.handle.net/2123/26382.
Full textAbstract:
There is significant interest in human induced pluripotent stem cell (hiPSC) therapy and RPE derived from hiPSCs as a therapeutic approach for age-related macular degeneration (AMD). Limited knowledge of the physiology of human RPE makes it difficult to relate the properties of RPE-hiPSCs to normal RPE cells. No published reports have shown RPE-hiPSCs to be capable of autophagy and exocytosis of waste products. So, transplantation of RPE cells lacking these key functions could be responsible for failure to restore long term vision. Twenty eyes, with no ophthalmic history, and 2 with AMD, were quantified by transmission electron microscopy. Also, a physiologically relevant model was developed for measuring heat shock protein (HSP) responses to oxidative stress in human primary RPE (hpRPE) cultures via aging. The RPE changes that occur during aging and AMD are summarised in Chapter 1. Methods & materials are explained in Chapter 2. The ultrastructure of RPE during aging is described in Chapter 3. The main finding is that, melanosomes migrate apically while mitochondria migrate to the perinuclear region adjacent to the basolateral extracellular space (BES) at 6 year (Y). Autophagy, lipofuscin deposition at the BES are evident at 6Y. Drusen formation from 25Y and thickening of Bruch’s membrane are evident via aging. Chapter 4 shows measurement of markers of mitochondrial responses to oxidative stress in hpRPE cultures. This Chapter shows how RPE cells keep their functions during aging. Two cases of AMD are evaluated in Chapter 5. In Chapter 6, possible mechanisms are discussed in relation to RPE detachment and AMD. RPE cells are incapable to keep initial responses to oxidative stress because of cytoplasmic lipofuscin coalescence and alteration of HSP expression. Finally, drusen accumulation and dysfunctional BES predispose to pigment epithelial detachment and choroidal neovascularisation.
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Turner, Lesley-Anne. "The development of large area patterning techniques for the characterisation of nerve and retinal cell responses to nano and micro scale topographies." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/the-development-of-large-area-patterning-techniques-for-the-characterisation-of-nerve-and-retinal-cell-responses-to-nano-and-micro-scale-topographies(ef4f67a5-8581-49a9-a1ae-d6cb25e65756).html.
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Cells respond to chemical, mechanical and topographical cues both in vivo and in vitro. Much research has been carried out into the effects of chemical signals and to a lesser degree, mechanical. However, less is known about cell responses to topographical cues, particularly to topographies with nanoscale dimensions. Understanding how cells respond to topography is of particular interest to the field of tissue engineering, where it is crucial to characterise the effects that biomaterial surfaces have on the cells that they come into contact with. Observations of the impact that topographic signalling has on cells, within two tissue engineering systems, are discussed in this thesis. These systems are: polymer conduits for peripheral nerve regeneration and thin films for the replacement of the retinal pigment epithelium. Understanding the effects that micro and nano scaled topographies have on nerve and retinal cell regeneration is important for successful development and implementation of appropriate tissue engineered devices. In order to fabricate topographical patterns on biomaterial surfaces, a number of fabrication techniques were investigated. The fundamental requirement of these techniques was for reliable production of uniform nano and micro scale topographical patterns over large lateral areas (millimeter scale). Initially, the suitability of electrohydrodynamic lithography (EHDL) was assessed. EHDL is a relatively new technique, first published in 2000, which employs electrostatic forces to pattern thin polymer films. Subsequently, techniques traditionally associated with the computing industry, such as e-beam lithography and reactive ion etching, were evaluated. Following successful pattern fabrication, NG108-15 and ARPE-19 cells were cultured on grooved topographies. Against a baseline parameter of elapsed time, the cell morphologies and their propensity for alignment with the grooves was rigorously assessed and compared. ARPE-19 and NG108-15 cell responses differed from one another, and were sensitive to varying groove dimensions. Ultimately, the developing morphologies (for both cell types) proved to be clearly dependent on groove dimensions and elapsed time.
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Tuglu, Mehmet Ibrahim. "Analysis of the behaviour of retinal pigment epithelial (RPE) cells in relation to their maintenance in the epithelium during development of the chick eye." Thesis, University of Liverpool, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.480920.
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Franz, Tamara Anne. "Induction of the retinal pigment epithelium of the chicken embryonic eye." Master's thesis, University of Cape Town, 1997. http://hdl.handle.net/11427/26993.
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During development of the eye, invagination of the optic cup gives rise to a double layered neuroepithelium, part of which differentiates into the retinal pigment epithelium (RPE). The molecular mechanisms which control differentiation of the RPE are not known. The present study was undertaken to determine 1) when induction of the RPE has occurred in chicken embryos and 2) to investigate whether contact with the presumptive neural retina (NR) is required for RPE differentiation. In order to investigate when RPE induction has occurred, early expression of two genes involved in pigmentation were investigated. Digoxigenin-labeled tyrosinase and tyrosinaserelated protein-2 (TRP-2) riboprobes were synthesised and used in ISH reactions on embryonic eye tissue. Tyrosinase transcripts were first detected at stage 19.5 (70-71 hours) and TRP-2 transcripts were detected a few hours earlier at stage 18.5 (67-69 hours) of embryonic development. These results indicate that induction has occurred by stage 18.5, approximately ten hours before distinct granules are visible in the RPE. The tyrosinase and TRP-2 transcripts were always localised first in the optical axis of the eye in the region where pigment granules are first present. This indicates that differentiation of the RPE proceeds from the optical axis of the eye cup outwards towards the periphery and that induction of the RPE may also proceed in this direction. To determine whether the presumptive NR is required for RPE induction, synthetic barriers were inserted into the uninvaginated optic vesicle of chicken embryos at stage 11 (40-45 hours) of development. The embryos were cultured in vitro until the optic vesicle had invaginated and sectioned to locate the barrier. Results suggest that contact with the presumptive NR may not be necessary for RPE induction.
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Wolk, Alyson M. "The Role of the Retinal Pigment Epithelium in Sorsby Fundus Dystrophy." Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1606842751125309.
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Morales, Shawn A. "Examining the role of the retinal pigment epithelium in proliferative vitreoretinopathy." Diss., Restricted to subscribing institutions, 2008. http://proquest.umi.com/pqdweb?did=1621832581&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Höhne, Katja [Verfasser]. "Pigment Epithelium-derived Factor in Augen von Aderhautmelanom-Patienten / Katja Höhne." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2009. http://d-nb.info/1027498183/34.
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Carnagarin, Revathy. "Pigment Epithelium-derived Factor and Insulin Crosstalk in Skeletal Muscle Biology." Thesis, Curtin University, 2017. http://hdl.handle.net/20.500.11937/59666.
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PEDF is an important regulator of skeletal muscle metabolism, in particular the insulin–dependent metabolic processes. The attenuation of PEDF signalling is an effective approach to achieve better control over the insulin resistance of metabolic disorders and the PEDF-insulin antagonistic cross talk involved in the modulation of skeletal muscle biology could be an effective strategy to achieve controlled bone formation counteracting the PEDF-induced heterotrophic ossification.
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Rosales, Mariana Aparecida Brunini 1983. "O estresse nitrosativo na patogênese da retinopatia diabética = implicações na barreira hemato-retiniana externa e possíveis alvos terapêuticos = Nitrosative stress in the pathogenesis of diabetic retinopathy: implications in the outer blood retinal barrier and possible therapeutics targets." [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/309781.
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Orientadores: Jacqueline Mendonça Lópes de Faria, José Butori Lopes de FariaTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: A patogênese da retinopatia diabética (RD) está associada ao estresse nitrosativo. Alterações na barreira hemato-retiniana (BHR) externa, formada pelas células do epitélio pigmentar da retina (EPR), estão associadas às fases precoces da RD e podem acarretar no desequilíbrio da manutenção dos fotorreceptores e consequentemente promoverem mudanças nas células neuronais da retina. O estresse nitrosativo como conseqüência do aumento da produção de óxido nítrico (NO¿) produzido pela super expressão da óxido nítrico sintetase induzida (iNOS) esteve presente em todas as camadas da retina, inclusive no EPR em condições de RD experimental in vivo precoce ou na linhagem celular humana do EPR (ARPE-19) expostas à alta concentração de glicose. O tratamento com agentes químicos como a S-nitrosoglutationa (GSNO), ou naturais (cacau enriquecido com polifenol) atuaram em diferentes vias de inibição da iNOS, prevenindo o estresse nitrosativo. Para o estudo in vivo com o colírio de GSNO (artigo I) foram utilizados animais espontaneamente hipertensos (SHR) com 4 semanas de idade. O diabetes (DM) foi induzido por STZ. Após a confirmação do DM (48 horas), os animais foram divididos em 6 grupos: controles (CTs) veículo; GSNO 900nm e GSNO 10?m ou DMs veículo; GSNO 900nm e GSNO 10?m. O efeito do tratamento com colírio de GSNO foi dependente da presença ou ausência da condição do DM. Nos animais CT, o GSNO atuou como um agente nitrosativo e nos animais DM preveniu o aumento da expressão da iNOS, preservando a retina funcional. Os estudos in vitro, demonstraram que o efeito do GSNO foi deletério ou protetor dependente da concentração de glicose. Nas células ARPE-19 expostas a condições normais de glicose, o tratamento promoveu um aumento na produção de NO¿ sem aumentar a expressão de iNOS e nas células sob alta glicose induziu uma modificação pós-translacional de proteína, a S-glutationilação da iNOS prevenindo o estresse nitrosativo. No estudo do cacau (artigo II), foi avaliado in vitro (ARPE-19 exposta a alta concentração de glicose) o seu efeito protetor dependente da concentração de polifenóis. Para isso foram testadas duas formulações de cacau que diferiram somente na concentração de polifenol: 0,5% para o cacau com baixo teor de polifenol e 60,5% para o cacau com alto teor de polifenol. A epicatequina (EC), encontrada na concentração de 12% no cacau com alto teor de polifenol foi tão eficaz quanto o próprio e esteve envolvida no controle da expressão da iNOS através da estimulação do receptor ?-opióide (DOR) diminuindo os níveis de TNF-?. A modulação da iNOS, preveniu a S-nitrosilação da caveolina-1 (CAV-1) e diminuição da expressão das junções intercelulares claudina-1 e ocludina através da prevenção da interação CAV-1?junções. Em ambos os estudos, o alvo terapêutico foi a iNOS em duas diferentes modalidades: modificação pós-translacional de proteína e modulação do TNF-? via DOR no EPR em modelos experimentais de RD. Os tratamentos apresentados neste trabalho demonstraram a iNOS como alvo terapêutico e mostraram-se eficaz em conter danos funcionais e morfológicos promovidas pela situação de mimetismo do DM no EPR demonstrando o importante papel da iNOS no desenvolvimento da RD
Abstract: The pathogenesis of diabetic retinopathy (DR) is associated with nitrosative stress. Changes in outer blood-retinal barrier (BRB), formed by retinal pigment epithelium cells (RPE) are associated in the early stages of DR and can cause imbalance in the maintenance of photoreceptors and thereby cause changes on retinal neuronal cells. The nitrosative stress as a result of increased production of nitric oxide (NO) produced by overexpression of nitric oxide synthase (iNOS) was present in all layers of the retina and mainly in RPE cells in early in vivo experimental DR or in human RPE cell line (ARPE-19) exposed to high glucose condition. Therapy with chemical agents such as S-Nitrosoglutathione (GSNO) or natural compounds (enriched cocoa polyphenol) acted in different pathways of iNOS inhibition, preventing nitrosative stress. For the in vivo study with GSNO eye drops (article I), it were used spontaneously hypertensive rats (SHR) rats with 4 week old. Diabetes (DM) was induced by streptozotocin (STZ). After DM confirmation (48 hours), the animals were divided into 6 groups: controls (CTs) vehicle; GSNO 900nm and GSNO 10?m or DMs vehicle; GSNO 900nm e GSNO 10?m. The effects of treatments were dependent on glucose concentration. In CT animals, GSNO acted as a nitrosative agent and in DM rats prevented iNOS overexpression, preserving the retina function. In vitro study showed that GSNO protective or deleterious effects were dependent on the glucose concentration. In ARPE-19 cells exposed to normal glucose, the treatment promoted an increase of NO¿ production without increase iNOS expression and in cells under high glucose (HG) condition induced post-translational protein modification, S-glutationylation of iNOS, preventing nitrosative stress. In the study with cocoa (article II), it was evaluated its protective effect dependent on concentration of polyphenols in ARPE-19 cells under HG condition. For this study, the composition of cocoa was the same in both preparations with the only difference in the amounts of polyphenol, 0.5% for low polyphenol cocoa (LPC) and 60.5% for high polyphenol cocoa (HPC). Epicatechin (EC), found in 12% of HPC was similarly protective compare to HPC and it was involved in controlling iNOS expression by stimulation of the delta opioid receptor decreasing TNF- ? levels. The modulation of iNOS prevented S-nitrosylation of caveolin-1 (CAV-1) and decreased expression of claudin-1 and occluding tight junctions by preventing CAV-1/junctions interactions. The treatments presented here showed iNOS as a therapeutic target containing functional and morphological changes promoted by DM milieu in RPE showing the important role of iNOS in the development of DR
Doutorado
Clinica Medica
Doutora em Clínica Médica
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Lightfoot, Ruth M. "Retinal pigment epithelial dystrophy (RPED) in the dog." Thesis, Royal Veterinary College (University of London), 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.339266.
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Tovell, Victoria E. "Purinoceptor signalling in human retinal pigment epithelial cells." Thesis, University of East Anglia, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435973.
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Chang, Yongen. "Important role of tetraspanin CD81 in integrin-dependent retinal phagocytosis /." Access full-text from WCMC :, 2007. http://proquest.umi.com/pqdweb?did=1467888711&sid=14&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Boutzen, Jocelyn. "Contribution à la modélisation d’interface biologique par spectroscopie d’impédance : application au suivi de l’épithélium pigmenté de la rétine durant sa croissance et face à diverses perturbations." Thesis, Paris Est, 2019. http://www.theses.fr/2019PESC2044.
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Cette thèse porte sur l’étude de l’interface entre électrodes et cellules épithéliales de la rétine : l’épithélium pigmenté. Les cellules RPE (Retinal Pigment Epithelium) qui le constitue forment une monocouche qui à confluence est constituée de cellules de forme polygonale. Elles sont juxtaposées et en contact intime les unes avec les autres par la présence de jonctions serrées. Un épithélium pigmenté endommagé est souvent associé à des pathologies de la vision. La spectroscopie d’impédance est une méthode de mesure qui permet d’étudier de manière non destructive un milieu composé d’éléments diélectriques et conducteurs. Cette mesure s’applique particulièrement bien aux cellules épithéliales. On applique ceci à l’étude du tapis cellulaires. Les membranes cellulaires remplissent le rôle de milieu diélectrique alors que les milieux ioniques intra et extracellulaires peuvent être considérés conducteurs. On peut en première approche analyser le module de l’impédance mesurée à une fréquence donnée afin de suivre le développement des tissus. Par exemple dans le domaine des implants à électrodes les fréquences autour de 1 KHz sont couramment citées. On peut par la suite mesurer l’impédance dans une gamme de fréquence plus importante et appliquer un modèle composé de dipôles électriques aux mesures. L’analyse des paramètres extraits peut donner une interprétation plus fine de l’état du tapis cellulaire. Deux notions seront principalement abordées dans cette thèse. Tout d’abord l’étude de l’utilisation de l’élément à constante de phase (CPE) dans la représentation du tapis cellulaire. Ensuite dans le cadre de ce modèle on va étudier le tapis cellulaire face à différentes perturbationsThis manuscript focuses on studying the interface between an electrode and epithelial cells of the retina: the Retinal Pigment Epithelium (RPE). The cells that are part of this epithelium develops until they form a monolayer of juxtaposed cells with close lateral contact involving the presence of tight junctions. A damaged epithelium is often associated with sight alterations.Impedance spectroscopy is a measurement method that allows to study materials containing both conducting and dielectric elements in a non–destructive way. We apply this technique to the RPE cells layer. Cells membranes are the dielectric part while the intra and extracellular mediums are the conductive parts of this material. In a first stage one can measure the impedance at a fixed frequency as a way to follow tissues development. As an example, the 1 KHz frequency is often considered in characterizing electrodes from implanted devices. One can also measure the impedance over a wider bandwidth and apply an electric model circuit to the data. The extracted parameters can give a better interpretation of the state of the cell layer. In this work, two part will be mainly investigated. First we will evaluate the use of a constant phase element in part of the electrical model describing the cell layer. Second, and considering the same model, we will observe the reaction of the model when the cells are subject to various perturbations
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Armstrong, Ian. "A study of the transport characteristics of mammalian retinal pigment epithelium (RPE)." Thesis, Cardiff University, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.276870.
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Pitsillides, Costas M. 1973. "Monitoring intracellular cavitation during selective laser targeting of the retinal pigment epithelium." Thesis, Massachusetts Institute of Technology, 2002. http://hdl.handle.net/1721.1/89901.
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Porpino, Meschede I. "Interactions between the endocytic and phagocytic pathways in the retinal pigment epithelium." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1391573/.
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The retinal pigment epithelium (RPE) is a monolayer of highly polarised cells that lies between the photoreceptors and choriocapillaris and performs a crucial role in the maintenance of visual function. The RPE phagocytoses and digests shed photoreceptor outer segments, transports nutrients, ions and water and secretes various essential growth factors that support surrounding cells. Despite the importance of membrane traffic pathways in the RPE little is known about endocytosis in these cells. Furthermore, although the early stages of phagocytosis of photoreceptor outer segments have been well characterised, the processing of the phagosome after engulfment is poorly understood. The first aim of this PhD project was to characterise the organisation of the endocytic pathway in the RPE and identify potential endocytic cargos that could be used to monitor endocytosis in RPE cells in culture. The second aim was to identify methods of characterising sequential stages of phagosome maturation in the RPE in situ and determine whether the same stages could be reproduced in cultured RPE cells. The final aim was to identify mechanisms underlying phagosome maturation, focusing in particular on the role of interactions with the endocytic pathway. Endocytic compartment markers and potential endocytic cargos were localised in situ on retinal sections and in vitro using primary porcine RPE cells. In situ and in vitro studies showed that Rab11a, a marker for apical recycling endosomes, is found distributed throughout the cell and not restricted to an apical compartment as seen in other epithelial cells. Transferrin receptors are expressed on both apical and basal plasma membranes in the RPE and both fluid phase probes and transferrin endocytosed from apical and basal surfaces meet in a common endocytic compartment. Phagosome maturation was investigated by immuno-electron microscopy using antibodies to two different rhodopsin epitopes. Loss of a C-terminal cytoplasmically exposed epitope was an early step in phagosome maturation, occurring before phagosome:lysosome fusion, which allowed the distinction of early and late phagosomes. Here it is demonstrated that the same stages of phagosome maturation occurred, albeit more slowly, in cultured porcine RPE challenged with isolated porcine outer segments. Treatment with the protease inhibitor, leupeptin, inhibited loss of the C-terminal rhodopsin epitope, suggesting that limited proteolysis can occur within the maturing phagosome. Finally, by loading the endocytic pathway from the basal surface and the phagocytic pathway from the apical surface it was possible to demonstrate acquisition of endocytic content by the maturing phagosome, the timing of which suggests that interaction with the endocytic pathway is likely to be a factor in the limited proteolysis of rhodopsin that occurs in the maturing phagosome. This work opens the possibility to investigate defects in endocytic and phagocytic pathways in retinal disease and determine how these defects could lead to phenotypes associated with aging and retinal degeneration.
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Thomas, Sara E. "Mechanisms of Xanthophyll Uptake in Retinal Pigment Epithelial Cells." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1478183410555123.
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Folarinde, Micheal Shola. "A Comparative Study Between Genotypes and Ages of Eyes Using Morphometric Measures of Retinal Pigment Epithelial Cells." Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/math_theses/117.
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Aged-related macular degeneration (AMD) is a common eye condition among people older than 65 years and is a leading cause of vision loss. It gradually destroys the macula, the part of the eye that provides sharp, central vision needed for seeing objects clearly. This study aims to test the hypothesis that the morphology of retina pigment epithelium, a key site of AMD pathology, can reflect the various stresses aging and AMD progression impose. We first identify and separate the young and old age group for mouse eyes. Then we classify, the mouse eyes using two genotypes (C57BL/6L, RD10), and two age group (young, old).We show that without dimensional reduction, the cell area and shape measures do not provide good classification of the mouse eyes.
But with the dimension reduction at the eye level, the cell area and shape measures provide excellent classification for mouse genotype and age.
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Heussen, Florian Moritz Antonius. "Autologous translocation of choroid and retinal pigment epithelium in age-related macular degeneration /." Köln, 2008. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000252843.
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Kobayashi, Kaori. "Expression of 17 β-hydroxysteroid dehydrogenase type IV in chick retinal pigment epithelium." Kyoto University, 1997. http://hdl.handle.net/2433/202174.
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Ozaki, Shiro. "Influence of the sensory retina on healing of the rabbit retinal pigment epithelium." Kyoto University, 1997. http://hdl.handle.net/2433/202179.
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Chen, Rui. "Effects of bioflavonoids on cultured human retinal pigment epithelial cells." Doctoral thesis, Universitätsbibliothek Leipzig, 2016. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-206176.
Full textAbstract:
The thesis describes the effects of various plant flavonoids (curcumin, epigallocatechin-3-gallate [EGCG], luteolin, apigenin, myricetin, quercetin, and cyanidin) on the physiological properties and viability of cultured human retinal pigment epithelial (RPE) cells. It is described that, with the exception of EGCG, all flavonoids tested decrease dose-dependently the RPE cell proliferation, migration, and secretion of VEGF. Luteolin, apigenin, myricetin, and quercetin decreased the viability of RPE cells at higher concentrations, by triggering cellular necrosis. Curcumin decreased the viability of RPE cells via induction of early necrosis and delayed apoptosis. The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, and increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein. Myricetin caused caspase-3 independent RPE cell necrosis mediated by free radical generation and activation of calpain and phospholipase A2. The myricetin- and quercetin-induced RPE cell necrosis was partially inhibited by necrostatin-1, a blocker of programmed necrosis. The author concludes that the intake of curcumin, luteolin, apigenin, myricetin, and quercetin as supplemental cancer therapy or in the treatment of retinal diseases should be accompanied by careful monitoring of the retinal function. Possible beneficial effects of EGCG and cyanidin in the treatment of retinal diseases should be examined in further investigations.
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Hamel, Christian. "Caractérisation, clonage moléculaire et expression de RPE65 protéine de l'épithélium pigmenté de la rétine." Montpellier 1, 1994. http://www.theses.fr/1994MON1T032.
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Laird, Dale W. "Characterization of the specific ligand-receptor interactions between rod outer segments and retinal pigment epithelial cells." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/28850.
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An in vitro phagocytosis assay system was developed and characterized for studying the specific receptor-mediated phagocytosis of bovine ROS by bovine RPE cells. The phagocytosis of ROS was detected qualitatively by electron microscopy and quantitatively by treating RPE cells with radioiodinated ROS or by probing ROS-treated RPE cells with a radiolabeled antirhodopsin monoclonal antibody.
The binding sites for various antirhodopsin monoclonal antibodies were localized as an essential step in their application as immunochemical probes for analysis of the structure and function of rhodopsin. Five monoclonal antibodies raised against rhodopsin have been shown to be directed against the N-terminal regions on the basis of their reactivity to an immunoaffinity purified 2-39 glycopeptide, a 2-16 tryptic glycopeptide and a 1-16 synthetic peptide as measured by radioimmune competition assays. Limited proteolysis, immunogold-dextran labeling and competitive inhibition studies identified two antirhodopsin monoclonal antibodies which bound to internal cytoplasmic loop regions of rhodopsin. Finally, the binding sites for these and other C-terminal specific antirhodopsin monoclonal antibodies were used to elucidate the proposed transmembrane helical model of rhodopsin.
An antirhodopsin monoclonal antibody (rho 4D2), which bound to rhodopsin
in glutaraldehyde-fixed ROS plasma membranes, was employed as an
immunocytochemical probe in studying the possible role of rhodopsin in the
binding and phagocytosis of rod outer segments. An immunoaffinity purified
2-39 N-terminal rhodopsin glycopeptide, a synthetic 1-16 peptide analogue of
rhodopsin and phospholipid vesicles reconstituted with rhodopsin were all
found to be ineffective in inhibiting the phagocytosis of ¹²⁵I-labeled ROS by RPE cells. In essence, these results provided compelling evidence that rhodopsin in the ROS plasma membrane does not function as the ligand for recognition by RPE cells.
The molecular properties of the ROS cell surface ligand(s), which are involved in recognition by bovine RPE cells, were studied by limited-proteolytic digestion in conjunction with quantitative phagocytosis assays. Mildly trypsin-treated ROS were found to be less effectively phagocytized than untreated ROS by bovine RPE cells. Moreover, the glycopolypeptides (34kD and 24kD) released from the ROS cell surface by trypsin were capable of inhibiting ROS phagocytosis. The ROS plasma membrane specific, ricin-binding, 230kD glycoprotein was observed by SDS-gel electrophoresis and western blotting to be highly trypsin sensitive under these conditions. Hence, ricin affinity chromatography and immunoaffinity chromatography were employed in an attempt to purify this 230kD glycoprotein from ROS membranes. Enriched preparations of the 230kD glycoprotein were reconstituted into phospholipid vesicles and effectively used to inhibit the phagocytosis of ROS by RPE cells. In summary, a ROS plasma membrane specific, 230kD glycoprotein has been identified and isolated; this protein may act as a ligand in specific ligand-receptor interactions between ROS and RPE cells.Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
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Tschernutter, Marion. "Gene therapy for retinal degeneration due to a defect in the retinal pigment epithelium." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445131/.
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The Royal College of Surgeons (RCS) rat is a well characterised model of autosomal recessive retinitis pigmentosa (RP) due to a defect in the retinal pigment epithelium (RPE). It is homozygous for a null mutation in the gene encoding Mertk, a receptor tyrosine kinase found in RPE cells, which is required for phagocytosis of shed photoreceptor outer segments. The absence of Mertk results in accumulation of outer segment debris. This subsequently leads to progressive loss of photoreceptor cells. Recently, MERTK has been established as a human retinal dystrophy gene. Retinal dystrophies are the most common cause of visual impairment in the Western World, for which no effective treatment exists. In order to evaluate the efficacy of virus mediated gene replacement therapy in the RCS rat, we produced recombinant adeno-associated viruses (AAV) and lentiviruses containing murine Mertk cDNA. Vectors were subretinally injected into the right eye of 10 day old RCS rats the left eye was left untreated as an internal control. Animals were examined at various time points by light and electron microscopy, electroretinography, and ophthalmoscopy. A detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits is presented in this thesis. AAV-2-mediated gene therapy resulted in preservation of retinal function for more than 9 weeks, when there is no activity in untreated eyes. Photoreceptors were still present at this time point and debris layer thickness was reduced. After subretinal delivery of human immunodeficiency virus type 1 (HIV-1) based lentiviral vectors carrying a functional copy of Mertk to the RCS rat eye, correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function was observed for up to 7 months, the latest time point evaluated. Whilst this was an improvement of the rescue compared to that achieved with AAV-2, lentiviral vectors raise more safety concerns regarding clinical application. Due to these biosafety issues, gene therapy vectors based on non-human lentiviruses and integration-deficient vectors have been developed. As part of this project, the potential of equine infectious anemia virus (EIAV) and non-integrating HIV-1-based vectors for the management of retinal degenerative disorders has been evaluated. The results presented in this thesis support the use of viral vectors for the treatment of retinal dystrophies. However, the development of an efficient therapy depends on the identification of patients and characterisation of pathological changes. Therefore, a panel of DNA samples from patients with autosomal recessive and sporadic forms of RP was screened for mutations in the MERTK gene. A new homozygous frame- shifting deletion was identified in four affected members of a family with RP. Clinical examination of these patients showed distinctive clinical signs that may improve the chances of identifying further patients and families in the future.