Orientadores: Jacqueline Mendonça Lopes de Faria, Jose Buttori Lopes de Faria / Dissertação (mestrado) - Universidade Estadual de Campinas Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T12:22:32Z (GMT). No. of bitstreams: 1 Rosales_MarianaAparecidaBrunini_M.pdf: 16380865 bytes, checksum: 2583696b31144f3e138bf4910979d918 (MD5) Previous issue date: 2010 / Resumo: A retinopatia diabética (RD) é a principal causa de cegueira adquirida em adultos em idade produtiva nos países desenvolvidos. A hipertensão (HA) é um importante fator de risco associado ao desenvolvimento da RD. O estresse oxidativo pode explicar como a hiperglicemia inicia os processos bioquímicos envolvidos na patogênese da RD. A retina possui vários mecanismos de defesa para minimizar o estresse oxidativo como "scavengers" de baixo peso molecular e sistemas enzimáticos. Sob condições do diabetes (DM), ocorre um aumento das espécies reativas, como o superóxido (O2·-) e óxido nítrico (NO·), conseqüentes da indução da reação da glicação. O produto desta reação, peroxinitrito (ONOO-), pode diretamente causar lesões em proteínas, lipídios e DNA. Embora muitos mecanismos patológicos da RD sejam focados nos danos vasculares, tem sido descrito que o DM também induza danos nas células de Müller da retina neural que pode anteceder as anormalidades vasculares. A neurodegeneração retiniana pode ser detectada pelo aumento da imunoreatividade da proteína ácidica fibrilar glial (GFAP) em astrócitos e em células de Muller. O acúmulo de matriz extracelular (ECM) é instrumental para a quebra de barreira hematoretiniana da retina presente precocemente na patogênese da RD. O objetivo deste estudo foi investigar a eficácia do antioxidante tempol, mimético da superóxido dismutase (SOD), na prevenção das alterações precoces na retina de um modelo de RD que combina hipertensão e diabetes. Foram utilizados neste estudo ratos espontaneamente hipertensos (SHR) e seu controle normotenso (WKY) com 4 semanas de idade. O diabetes foi induzido experimentalmente através de injeção intravenosa de estreptozotocina. Após a confirmação da indução do DM, os ratos SHR (DM-SHR) foram randomizados para receber ou não tratamento com injeção de tempol intraperitonealmente (DM-SHR-tempol). Após 20 dias, os ratos foram eutanaziados e suas retinas foram coletadas para extração de proteínas, ensaios colorimétricos e imunohistoquímica. Os animais diabéticos apresentaram os marcadores precoces de RD: houve aumento da expressão do GFAP no grupo SHR e DM-SHR comparado ao grupo WKY e aumento da FN no grupo DM-SHR comparado ao SHR e WKY. O balanço oxidativo, avaliado pela produção de superóxido e pelos produtos finais do óxido nítrico, assim como a defesa antioxidante, estimada pela expressão e atividade da Cu/Zn SOD, revelou um desequilíbrio acentuado no grupo DM-SHR em relação ao grupo SHR e WKY. Como resultado, o produto peroxinitrito detectada por imuno-histoquímica para nitrotirosina (NT) foi maior no grupo DM-SHR. A atividade da enzima poli (ADPribose) polimerase (PARP) detectada pela ribosilação, e a super regulação da expressão da óxido nítrico sintase induzida (iNOS) foram exacerbadas neste grupo. A administração do TEMPOL impediu o dano oxidativo, reduziu a ativação exacerbada da PARP e diminuiu os níveis de i-NOS nos ratos DM-SHR. Como conseqüência, os marcadores estruturais precoce da RD, GFAP e FN, foram prevenidos pelo TEMPOL. Estes resultados fornecem uma base racional para o desenvolvimento com fins farmacológicos do TEMPOL na prevenção e tratamento da RD. / Abstract: Diabetic retinopathy (DR) is the leading cause of blindness among working-age adults in developed countries. Hypertension (HA) is an important risk factor associated with the development of DR. Oxidative stress may explain how the hyperglycemia initiates the biochemical processes involved in the pathogenesis of DR. The retina has several defense mechanisms to minimize oxidative stress as scavengers of low molecular weight and enzyme systems. Under conditions of diabetes (DM), there is an increase of reactive species such as superoxide (O2o -) and nitric oxide (NO o), induced by glycation reaction. The product of this reaction, peroxynitrite (ONOO-), can directly damage proteins, lipids and DNA. Although many pathologic mechanisms of DR have been focused on vascular damage, it has been reported that DM also induces damage in nonvascular retinal neurons of Müller cells occurred prior to vascular abnormalities. The retinal neurodegeneration can be detected by increased immunoreactivity of glial fibrillary acidic protein (GFAP) in astrocytes and Muller cells. The extracellular matrix accumulation (ECM)) is instrumental to blood retinal barrier breakdown present in early pathogenesis of DR. The objective of this study was to investigate the efficacy of the antioxidant TEMPOL, mimetic of superoxide dismutase (SOD) in the prevention of early changes in the retina in a model that combines hypertension and diabetes. Four-week-old spontaneously hypertensive rats (SHR) and their normotensive control (WKY) were used in this research. Diabetes was induced experimentally by intravenous injection of streptozotocin. SHR diabetic (DM-SHR) rats were andomized to receive or not treatment with intraperitoneal injection of tempol (DM-SHR-tempol). fter 20 days, the rats were euthanized and their retinas were collected for protein extraction, colorimetric assays and immunohistochemistry. The presence of exacerbated early markers of DR was detected by increased GFAP expression in SHR and SHR-DM group compared to WKY and FN in group DM-SHR compared to SHR and WKY. The oxidative balance, evaluated by superoxide production and nitric oxide end-product levels and the counterpart antioxidant defense, estimated by the expression and activity of Cu / Zn SOD showed a marked unbalance in DM-SHR group compared to SHR and WKY group. As a result, the product peroxynitrite detected by immunohistochemistry for nitrotyrosine (NT) was higher in the DM-SHR. The activity of the enzyme poly (ADP-ribose) polymerase (PARP) detected by ribosylation, and the upregulation of inducible nitric oxide synthase (iNOS) expression were exacerbated in this group. The administration of TEMPOL prevented the oxidative damage, reduced the exacerbated activation of PARP and decreased iNOS levels in DM-SHR rats. As a result, the structural markers of early RD, GFAP and FN, were prevented by tempol. These findings provide a rationale for the development with pharmacological purposes of tempol in the prevention and treatment of DR. / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Diabete Melito (DM) é uma endocrinopatia frequentemente diagnosticada na clínica de pequenos animais e desenvolve-se pela deficiência relativa ou absoluta de insulina, sendo caracterizada pela hiperglicemia crônica. Complicações associadas ao DM são frequentes, dentre elas podemos citar catarata e retinopatia. A catarata, que é a oftalmopatia mais frequente nos cães, impossibilita à fundoscopia e nestes casos a avaliação da função retiniana pode ser feita com o auxílio do eletrorretinograma de campo total. O objetivo deste estudo foi avaliar a progressão da retinopatia diabética por meio de eletrorretinograma (ERG) de campo total pré e pós-operatória (180 dias) em cães portadores de catarata madura e hipermadura submetidos à facoemulsificação. Vinte e quatro cães (Grupo Diabético (DM) = 12 cães; Grupo Não Diabético (NDM) = 12 cães) de raças diversas foram avaliados. Previamente ao ERG, todos os cães foram submetidos ao exame oftalmológico completo e ultrassom ocular. Todos os cães foram sedados seguindo o mesmo protocolo. ERG de campo total foi realizado com sistema eletrodiagnóstico Veris 2000 e cúpula de estimulação (Ganzfeld), segundo o protocolo da ISCEV contendo 5 respostas. Para obtenção dos registros utilizou-se eletrodo bipolar Burian Allen. Avaliou-se a amplitude pico a pico das 5 respostas e o tempo de culminação da onda-b na resposta de bastonetes, máxima resposta, cones e flicker para cada registro em condições escotópicas e fotópicas. As respostas obtidas em cada olho no momento basal e após 6 meses foram comparadas por meio do teste de Wilcoxon. O teste de Mann-Whitney foi utilizado para comparação de respostas dos olhos operados (OP) com os olhos contralaterais (OC), assim como para comparação entre os grupos DM e NDM. O efeito da cirurgia e do tempo de DM foi ainda avaliado por meio do cálculo das diferenças entre as respostas no momento pré e pós 6 meses de cada olho (delta OP e delta OC). Observou-se diferenças entre OP e OC, no momento pré, nas respostas dos bastonetes e da máxima resposta (amplitude e tempo de culminação onda-b). No momento pós, foram observados diferenças entre OP e OC no tempo de culminação dos bastonetes e flicker, e nas amplitudes do potencial oscilatório, cones e flicker. No grupo NDM, não foram observados diferenças entre os momentos pré e pós assim como entre OP e OC. Quando comparadas as respostas dos grupos DM e NDM, observou-se valores significativamente maiores no grupo DM, no tempo de culminação da onda-b na máxima resposta do OP no momento pós, no tempo de culminação da onda-b na máxima resposta OC no momento pós e no tempo de culminação da onda-b da resposta de cones no momento pré cirurgia. Não foram observados diferenças entre delta OP e delta OC dentro de cada grupo e entre os grupos. Todos os pacientes que participaram do estudo recuperaram a visão após a cirurgia, sugerindo que a inflamação pós cirúrgica foi controlada com a medicação prescrita nos 2 grupos estudados. Conclui-se que a facoemulsificação representa mínimo impacto na evolução da retinopatia diabética em cães. A remoção cirúrgica da catarata não deve ser contraindicada nesta espécie, independentemente se o animal é ou não portador de DM, por resultar em melhoria da visão. / Diabetes mellitus (DM) is a common endocrinopathy in the small animals practice and it develops as a relative or absolute insulin deficiency characterized by a chronic hyperglycemia. There are frequent complications, such as cataracts, retinopathy, resulting in visual impairment. Cataracts are dogs most frequent ophthalmic affection and, since it prevents fundoscopic examination, full-field electroretinography is used for retinal functional evaluation. Therefore, to provide a better understanding of diabetic retinopathy (DR) progression in dogs with mature and hypermature cataracts undergoing phacoemulsification surgery, this study aims to analyze the full-field electroretinogram prior and 180 days post cataracts removal. Twenty four dogs (diabetic group (DM) = 12 dogs and non-diabetic group (NDM) = 12 dogs), from different breeds were evaluated. Before the ERG, all animals were submitted to a complete ophthalmic examination and ocular ultrasonography. All dogs were sedated following the same anesthetic protocol. The full-field ERG was recorded using 2000 VERIS system and a dome stimulator (ganzfeld) according to ISCEV five response standard. The recordings were obtained using bipolar Burian Allen electrodes. Peak to peak amplitude five response and b-wave culmination time in rod responses, maximum response, cone and flicker were evaluated in scotopic and photopic conditions. The responses obtained in each eye at baseline and after 6 months were compared using the Wilcoxon test. The Mann-Whitney test was used to compare the responses of the operated eyes (OP) and the contralateral eyes (OC) as well as for comparison between groups DM and NDMAs. The effect of surgery and duration of DM was assessed by calculating the differences between the responses prior and six months after surgery, of each eye (delta delta OP and OC). There were differences between OP and OC, in the baseline values, in rod responses and maximum response (amplitude values and b-wave implicit time). After phacoemulsification, differences were observed between OP and OC in b-wave implicit time of rods and flicker, and the amplitudes of the oscillatory potentials, cone and flicker. In the NDM group, no differences were observed between pre and post evaluations between OP and OC. Comparing the responses of DM and NDM groups, there were significantly higher values in the DM group, in b-wave implicit time and in maximum response of OP after caratact removal, and also, of the b-wave implicit time in the maximum response in OC after surgery and, finally, in the b-wave implicit time of cone response prior to the surgery. No differences were observed between delta OP and delta OC within each group and between groups. All patients recovered vision after surgery, suggesting that post-surgery inflammation was controlled by the prescribed medication in both groups. The phacoemulsification surgery did not appear to have a great impact on diabetic retinopathy evolution in dogs. Cataract surgery should not be contraindicated in this species, regardless of the presence of DM, as it can result in sight improvement.

O presente estudo avaliou diferentes aspectos funcionais da visão de diabéticos do tipo 2 com e sem retinopatia diabética. A visão desses pacientes foi avaliada em diferentes níveis do sistema visual por meio da aplicação de uma bateria de testes psicofísicos e eletrofisiológicos de grande sensibilidade. O objetivo foi caracterizar as alterações visuais provocadas pela doença antes do aparecimento de retinopatia, e avaliar o efeito da presença da retinopatia sobre as alterações funcionais precoces. Materiais e métodos: 31 pacientes diabéticos com retinopatia não proliferativa leve (15 , 16 ; idade = 59 ± 09; tempo de diabetes = 10 ± 06 anos); 36 pacientes diabéticos sem retinopatia (16 , 20 idade = 56 ± 11; tempo de diabetes = 06 ± 04 anos) e 30 sujeitos controle (13 , 17 ; idade = 44 ± 10 anos) foram submetidos a exames de (1) eletrorretinograma multifocal - mfERG; (2) sensibilidade ao contraste acromático segregada em componentes magno (MC) e parvocelulares (PC) Teste do pedestal; (3) perimetria visual computadorizada branco/branco e azul/amarelo e (4) teste quantitativo de visão de cores Teste de cores de Cambridge. A comparação dos dados de pacientes e controles foi feita com ANOVA e a comparação da capacidade de detecção do dano funcional entre os testes foi feita por meio da análise de curvas ROC. Resultados: ambos os grupos de pacientes manifestaram perdas significantes no mfERG, sensibilidade ao contraste e visão de cores. Em ambos os protocolos de perimetria apenas os pacientes com retinopatia tiveram perdas significativas. As respostas de pacientes com e sem retinopatia não foram significantemente diferentes na maior parte das medidas. Diferenças significativas foram encontradas entre os dois grupos em parte das latências do mfERG e na visão de cores, nos limiares do eixo tritan,. Na análise entre testes, o mfERG teve os maiores índices de sensibilidade e especificidade, seguido por visão de cores, sensibilidade ao contraste e perimetria, nesta ordem. Na comparação das áreas sob as curvas ROC não houve diferença significativa apenas entre os testes 1, 2 e 4. Conclusões: foram encontradas perdas funcionais estatisticamente significativas na avaliação psicofísica e eletrofisiológica da visão de pacientes diabéticos do tipo 2 sem e com retinopatia diabética. O presente trabalho confirma e amplia o crescente corpo de evidencias de perdas funcionais observadas precocemente no curso da diabetes, na ausência de alterações morfológicas (vasculares) detectáveis na retina. A interpretação dessas perdas precoces é de que tenham origem neural. Nossos achados indicam que a presença de retinopatia leve não teve efeito significativo sobre a maior parte dos aspectos funcionais analisados, corroborando a noção de origem neural das perdas. O efeito da diabetes sobre a função neural não parece ser específico a nenhuma das vias de processamento visual, como indicado pelos testes de sensibilidade ao contraste e de visão de cores. Os achados deste trabalho confirmam a hipótese de que, para o sistema visual, a diabetes seja uma doença neurodegenerativa da retina que pode estabelecer- se mesmo na ausência de retinopatia. Assim, a avaliação dos aspectos funcionais da visão deve ser melhor que a avaliação morfológica da retina para a identificação de pacientes diabéticos sob risco de perda visual. / The present study evaluated different aspects of visual function in type 2 diabetics both with and without retinopathy. The approach was to evaluate vision at several levels of the visual system by means of application of a battery of modern, sensitive psychophysical and electrophysiological tests. The goals were to characterize the changes in visual function underlying the disease prior to the onset of retinopathy, and to verify how the early losses are affected once retinopathy has occurred. Materials and methods: 31 diabetic patients with non-proliferative retinopathy (15 , 16 ; age = 59 ± 09 years; duration of diabetes = 10 ± 06 years); 36 diabetic patients without retinopathy (16 , 20 ; age = 56 ± 11 years; duration of diabetes = 06 ± 04 years) and 30 controls (13 , 17 ; age = 44 ± 10 years) were evaluated with: (1) multifocal electroretinogram - mfERG; (2) achromatic contrast sensitivity segregated into magno- (MC) and parvocellular (PC) pathways Pedestal test; (3) white-onwhite and blue-on-yellow computerized visual perimetry and (4) quantitative computerized color vision test Cambridge Colour Test (CCT). An ANOVA was performed for the statistical comparison among groups and ROC curve analysis was used to compare the diagnostic power of the different tests. Results: both diabetic patient groups manifested significant functional losses compared to controls in the mfERG, the Pedestal test and the CCT. In the mfERG, both patient groups had significantly smaller amplitudes and longer latencies than controls in one or more of the signature mfERG waveform components. For the Pedestal Test, both patient groups manifested losses in both the M-targeting and P-targeting paradigms. The CCT chromatic discrimination test found significant losses along all three color confusion axes (protan, deutan, tritan) in both patient groups. In the visual fields, only the patients with retinopathy exhibited significant losses compared to controls. In the comparison between patients with and without retinopathy, no statistical differences between results were found, except for some of the mfERG latencies and the color discrimination in the tritan axis. The ROC analysis showed that the mfERG had the higher combined sensitivity and specificity indexes, followed by the CCT, pedestal test and perimetry with decreasing indexes in this order. Conclusions: statistically significant losses were found in psychophysical and electrophysiological assessment of visual function of type 2 diabetic patients with and without diabetic retinopathy. The present work strongly confirms the growing body of evidence that functional losses with neural etiology occur in type 2 diabetes before any vascular changes are clinically detectable in the retina. In the majority of the measures, the presence of retinopathy increased the functional losses, but did not determine significant differences in relation to the losses observed in the diabetics with normal fundus. According to our results from the Pedestal Test and the Cambridge Colour Test, the neural damage is not selective to either of the visual processing pathways. The results are consistent with the hypothesis that diabetes is a neurodegenerative disease of the retina whose establishment may occur even in the presence of retinopathy. Thus, the assessment of functional status rather than morphological examination seems a better approach to identify diabetic patients under vision threat.

Cette thèse a pour objet l’étude de nouvelles méthodes de traitement d’image appliquées à l’analyse d’images numériques du fond d'œil de patients diabétiques. En particulier, nous nous sommes concentrés sur le développement algorithmique supportant un système de dépistage automatique de la rétinopathie diabétique. Les techniques présentées dans ce document peuvent être classées en trois catégories: (1) l’évaluation et l’amélioration de la qualité d’image, (2) la segmentation des lésions, et (3) le diagnostic. Pour la première catégorie, nous présentons un algorithme rapide permettant l’estimation numérique de la qualité d’une seule image à partir de caractéristiques extraites de la vascularisation et de la couleur du fond d'œil. De plus, nous démontrons qu’il est possible d’augmenter la qualité des images et de supprimer les artefacts de réflexion en fusionnant les informations extraites de plusieurs images d’un même fond d'œil (images capturées en changeant le point d’attention regardé par le patient). Pour la deuxième catégorie, deux familles de lésion sont ciblées: les exsudats et les microanévrysmes. Deux nouveaux algorithmes pour l’analyse des images du fond d'œil sont proposés et comparés avec les techniques existantes afin de démontrer leur efficacité. Dans le cas des microanévrismes, une nouvelle méthode basée sur la transformée de Radon a été développée. Dans la dernière catégorie, nous présentons un algorithme permettant de diagnostiquer la rétinopathie diabétique et les œdèmes maculaires en analysant les lésions détectées par segmentation d’image; à partir d’une seule image, notre algorithme permet de diagnostiquer une rétinopathie diabétique et/ou un œdème maculaire en ~ 22 secondes sur une machine à 1,6 GHz avec 4 Go de RAM; de plus, nous montrons les premiers résultats de notre algorithme de détection d'œdème maculaire basé sur des images du fond d'œil multiples, qui peut éventuellement permettre d’identifier le gonflement de la macula même si aucune lésion n’est visible. / In this Ph.D. thesis, we study new methods to analyse digital fundus images of diabetic patients. In particular, we concentrate on the development of the algorithmic components of an automatic screening system for diabetic retinopathy. The techniques developed can be categorized in: quality assessment and improvement, lesion segmentation and diagnosis. For the first category, we present a fast algorithm to numerically estimate the quality of a single image by employing vasculature and colour-based features; additionally, we show how it is possible to increase the image quality and remove reflection artefacts by merging information gathered in multiple fundus images (which are captured by changing the stare point of the patient). For the second category, two families of lesion are targeted: exudate and microaneurysms; two new algorithms which work on single fundus images are proposed and compared with existing techniques in order to prove their efficacy; in the microaneurysms case, a new Radon transform-based operator was developed. In the last diagnosis category, we have developed an algorithm that diagnoses diabetic retinopathy and diabetic macular edema based on the lesions segmented; starting from a single unseen image, our algorithm can generate a diabetic retinopathy and ma cular edema diagnosis in _22 seconds on a 1.6 GHz machine with 4 GB of RAM; additionally, we show the first results of a macular edema detection algorithm based on multiple fundus images, which can potentially identify the swelling of the macula even when no lesions are visible.

In recent years, concern about a new epidemic of retinopathy of prematurity (ROP) has focused attention on the increasing incidence of the disease and the factors responsible for its most severe consequences. Two studies designed to address these issues were done using data from three sources: the B.C. Health Surveillance Registry (Registry), Physicians's Notices of Livebirth (PNOB), and the Vancouver General Hospital (VGH). In the first study, Registry and PNOB records were used to determine crude annual birth weight-specific incidence rates for ROP in infants liveborn in the Province of British Columbia (B.C.) in the period 1952-1983. These rates showed that, in B.C., the original epidemic of the disease ended in 1954. Linear regression lines fitted for each of four birth weight categories showed that, in the 29 year period after 1954, there was a significant increase in the incidence of ROP-induced blindness in infants weighing less than 1000 grams at birth. To refine this observation, the data were sub-divided: the 29 year period, to two smaller periods, 1955-1964 and 1965-1983; the less than 1000 gram birth weight category to two sub-categories, 500-749 and 750-999 grams. Since the inter-period incidence should have been similar if the birth weight-specific incidence had not changed since the end of the original epidemic, the crude weight-specific rates for ROP-induced blindness in the early period were used to calculate the expected number of cases in the later period. When weight-standardized incidence ratios (SIR's) and 95% confidence limits were calculated, the results showed that, in the 750-999 gram sub-category, the SIR was significantly increased. Infants born in the period 1965-1983 were 3.07 times more likely to be ROP: blind than their equal weight counterparts in the earlier period. In infants weighing 500-749 and 1000 grams or more, there was no evidence to suggest an increase in incidence after 1954. The second study was done to determine the cofactors that differentiate infants who are blinded by ROP from those who are not. Infants were included if (i) they were born in B.C. between 1955 and 1983, (ii) they were known to the Registry as being ROP: blind (cases) or not blind (controls), and (iii) they were born in or admitted to the VGH within 28 days of birth. When the data from all three data sources were dichotomized and analyzed using univariate techniques, two variables, respiratory distress syndrome (RDS) and neonatal weight loss, showed a significantly protective effect. The effect of RDS disappeared when the data were stratified by birth interval indicating that the observed association was confounded by time. When the variables were reanalyzed in continuous form, none were significantly associated with visual outcome. However, since the power of the cofactor study was extremely low, none of the variables that were included can be eliminated as potential cofactors for the induction of blindness in infants with ROP. / Medicine, Faculty of / Population and Public Health (SPPH), School of / Graduate

Orientador: Carlos Eduardo Steiner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T13:53:01Z (GMT). No. of bitstreams: 1 Assis_NilmaAlmeidade_M.pdf: 1405023 bytes, checksum: 12e3861c31dae0e759ab571d2d029476 (MD5) Previous issue date: 2006 / Resumo: A retinopatia diabética é uma complicação freqüente no diabetes melito tipo 1, acometendo quase a totalidade de pacientes, em graus variados, após 20 anos de doença. A interferência de fatores ambientais como a manutenção de um estado hiperglicêmico na sua fisiopatologia já foi comprovada, mas ainda não foi esclarecido porque alguns pacientes desenvolvem essa complicação de maneira grave e precoce. Nos últimos anos, diversos estudos têm sugerido a participação de fatores genéticos nesse processo. O fator de crescimento endotelial vascular (VEGF), potente indutor da angiogênese, foi associado à retinopatia diabética por alguns autores, pelo aumento da sua expressão, em virtude de mutações em sua região promotora. Neste trabalho foi realizada uma avaliação clínico-laboratorial, a análise do SNP rs833061 (- 460) e a pequisa da deleção de 18 pares de bases em -2549, ambas na região promotora do gene VEGF em 114 pacientes com diabetes melito tipo 1, de três centros de referência em diabetes no Brasil ¿ Hospital das Clínicas da Unicamp, Instituto Estadual de Diabetes e Endocrinologia Luiz Capriglione (RJ) e Santa Casa da Misericórdia de São Paulo (SP). Houve associação entre retinopatia diabética proliferativa e nefropatia, porém nenhum paciente apresentou a deleção em -2549, nem o alelo -460 C em homozigose. Tais resultados sugerem que esses polimorfismos na região promotora do gene VEGF não interferem na predisposição à retinopatia diabética na população estudada e que outros agentes ambientais e/ou genéticos devem ser significativos / Abstract: Diabetic retinopathy is a frequent complication of diabetes mellitus type 1 and almost all patients develop it after twenty years of disease. The causes of these complications are not clear, but several environmental factors such as chronic hyperglicaemia may act in this predisposition, however, it is not clearly understood why some individuals develop it in a severe and precocious way. The participation of genetic factors as the vascular endothelial growth factor (VEGF), a potent angiogenic mediator, was already confirmed for some authors. In this study we investigate whether polymorphisms in VEGF gene are associated with proliferative diabetic retinopathy. One hundred-fourteen patients with diabetes mellitus type 1 underwent a clinical and laboratorial study, as well as the analysis of two polymorphisms: rs833061 and the deletion of 18 bp at -2549 both on the promoter region of the VEGF gene. There was an association between nephropathy and retinopathy in our patients but none of the individuals presented the deletion at -2549 or the allele C in rs833061 in homozygous state. These results suggest that such polymorphisms in the promoter region of the VEGF gene do not interfere in the predisposition to diabetic retinopathy in our population and that other environmental and/or genetic factor may be more relevant / Mestrado / Genetica Medica / Mestre em Ciências Médicas

Orientadores: Jacqueline Mendonça Lópes de Faria, José Butori Lopes de Faria / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T10:41:17Z (GMT). No. of bitstreams: 1 Rosales_MarianaAparecidaBrunini_D.pdf: 28953374 bytes, checksum: e9a2824bad639c7cbd3628c24c4308ad (MD5) Previous issue date: 2014 / 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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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22320号 / 医博第4561号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 大森 孝一, 教授 森田 智視 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

L’angiogenèse physiologique est un processus clé du développement embryonnaire et chez l’adulte. Une anomalie de la formation des vaisseaux sanguins est à l’origine de nombreuses pathologies. Une meilleure compréhension des mécanismes de l’angiogenèse est un pré-requis essentiel à la mise au point de nouvelles stratégies thérapeutiques ayant pour objectif d’inhiber ou stimuler cette angiogenèse pour mieux traiter l’ensemble de ces pathologies. Au cours de ces dernières années, les voies de signalisation Wnt/Fzd sont apparues comme jouant un rôle fondamental dans le développement vasculaire. Au début de cette thèse, un premier projet nous a permis de montrer le rôle important de Fzd7 dans le contrôle de la perméabilité vasculaire, in vitro et in vivo, via la voie canonique et la régulation des complexes jonctionnels dépendantes de la VE-cadhérine. La deuxième partie de ce travail s’est focalisé sur le rôle de Fzd7 dans la formation des vaisseaux. Nous avons mis en évidence que Fzd7 contrôle la vascularisation post-natale de la rétine chez la souris. La voie de signalisation Fzd7/DVL/β-caténine régule le sprouting et la prolifération des cellules endothéliale (CE) via l’activation de la voie Notch, tandis que la voie de signalisation de Fzd7 contrôle la migration des CE via la régulation de MMP2/9 indépendamment de la voie Notch. Enfin, la troisième partie de cette thèse a eu pour objectif d’étudier l’implication de Fzd7 sur l’angiogenèse pathologique. Nos résultats préliminaires indiquent que Fzd7 participe aux phases de vaso-oblitération et de néovascularisation dans un modèle de rétinopathie induite par l’oxygène chez la souris, suggérant que Fzd7 pourrait être une nouvelle cible dans le traitement des rétinopathies. / Physiological angiogenesis is a key process in embryonic development but also in adult. Abnormal formation of blood vessels is the cause of many diseases. A better understanding of the mechanisms of angiogenesis is an essential prerequisite for the development of new therapeutic strategies aimed to inhibit or stimulate angiogenesis to better address these pathologies. In recent years, the Wnt/Fzd signaling pathways appeared to play a key role in vascular development. At the beginning of this study, a first project allowed us to show the important role of Fzd7 in controlling vascular permeability in vitro and in vivo, through the canonical pathway and the regulation of VE-cadherin junctional complexes. The second and main part of this work focused on the role of Fzd7 in the formation of blood vessels. We have demonstrated that Fzd7 controls postnatal vascularization of mice retina. The signaling pathway Fzd7/DVL/β-catenin regulates the sprouting and proliferation of endothelial cells (EC) through activation of Notch signaling, but also controls EC migration through MMP2/9 but independently of the Notch pathway. Finally, the third part of this work aimed to study the involvement of Fzd7 on pathological angiogenesis. Our preliminary data indicate that Fzd7 regulates vaso-obliteration and neovascularization in a mice model of oxygen-induced retinopathy suggesting that Fzd7 could be a new target for the treatment of retinopathy.