[en] This thesis is presented in the form of a compendium of published articles in accordance with the doctorate commission of the Universitat Autónoma de Barcelona. The main body of this thesis comprises original research articles published in indexed journals. The main subject of the research is the evaluation of Phosphorylation and Nitrosylation of interleuquines in the vitreous of patients with diabetic retinopathy. Samples from diabetic patients were analysed and compared with a control group of not diabetic patients. Article 1 Tyrosine Phosphorylation of Vitreous Inflammatory and Angiogenic Peptides and Proteins in Diabetic Retinopathy Invest Ophthalmol .Vis Sci. 2009;50:1378–1382) DOI:10.1167/iovs.08-2736 Impact factor 3,431 .To evaluate the degree of phosphorylation of vitreous proteins in patients with type 2 diabetes mellitus and diabetic retinopathy compared with a group of control subjects without diabetes and of similar age and sex. METHODS. In samples obtained after vitrectomy for diabetic retinopathy in patients and for macular hole in control subjects, immunoblot techniques were applied to a mini-array system for quantification of a wide range of chemokines and vasoactive peptides and proteins. Antiphosphotyrosine antibody was used for tyrosine phosphorylation evaluation and results were expressed as the percentage of variation compared with that in control subjects. RESULTS. Samples from eight patients with type 2 diabetes and from eight control subjects were analyzed. The total quantity of proteins analyzed was similar in both patients and control subjects. Tyrosine phosphorylation was very significantly decreased (_20%, P _ 0.05) in diabetic patients with respect to the control group in growth-related oncogene, human cytokine I-309, interleukin-13, monocyte colony-stimulating factor, macrophage-derived chemokine, stem cell factor, transforming growth factor-_1, angiogenin, and oncostatin M. A significant decrease in phosphorylation (between 20% and 40%, P _ 0.05) was observed in epithelial neutrophil-activating peptide 78; granulocyte colony-stimulating factor; granulocyte-monocyte– stimulating colony factor; IL-5, -6, -7, -8, -10, and -12p40p70; monokine induced by interferon-_; macrophage inflammatory protein 1-; and normal T expressed and secreted cytokine (RANTES) in comparison with that in the control subjects. The greatest decrease in phosphorylation status was found in IL-1-_ and -1. CONCLUSIONS. Diabetic retinopathy is associated with a decrease in tyrosine phosphorylation of many vitreous proteins which may indicate an alteration in protein functionality or action even before significant quantitative variations. Article 2 Diabetic Retinopathy Is Associated with Decreased Tyrosine Nitrosylation of Vitreous Interleukins IL-1 , IL-1 , and IL-7 Ophthalmic Res 2011;46:169–174 DOI: 10.1159/000323812 Impact factor 1.29 Objective: To simultaneously evaluate tyrosine nitrosylation and phosphorylation levels of vitreous interleukins of patients with diabetic retinopathy, in which abnormal tyrosine phosphorylation has been previously described. ResearchDesign and Methods: Specific immunoprecipitation of interleukins IL-1 _ , IL-1 _ , IL-2 and IL-7 was carried out in samples obtained during vitrectomy performed for proliferative diabetic retinopathy in patients (n = 12) and for macular hole in controls (n = 12). Tyrosine nitrosylation and phosphorylation levels of the immunoprecipitated interleukins were analysed by Western blot with the respective specific antibodies and correlated. The results were also correlated with the total amount of immunoprecipitated interleukin protein. The mean phosphorylation/nitrosylation ratios of these proteins in vitreous humour of both the control group and diabetic patients were determined Results: Diabetes was associated with decreased tyrosine nitrosylation of IL-1, IL-1 and IL-7and an increased tyrosine phosphorylation/nitrosylation ratio with respect to controls in IL-1 (1.58 8 0.22 vs. 2.74 80.39, respectively; p 0.05) and IL-7 (2.15 8 0.01 vs. 3.26 80.57, respectively; p 0.05). No significant changes were observedin nitrotyrosine or in the tyrosine phosphorylation/ nitrosylation ratio of IL-2. Conclusions: Proliferative diabetic retinopathy is associated with concomitant and simultaneous changes in both tyrosine phosphorylation and tyrosine nitrosylation status of specific pro-inflammatory interleukins present in the vitreous fluid such as IL-1 , IL-1 and IL-7. These changes could be related to the increase in pro-inflammatory activity detected in diabetes-induced retinopathy. Main conclusions Diabetic retinopathy is associated with alterations in the tyrosine phosphorylation status of protein inflammatory interleukins group, which could be the pathophysiological basis of retinal involvement. Tyrosine nitrosylation is not the result of changes in phosphorylation. Alterations in interleukin-nitrosylation may have an implication in the pathogenesis of diabetic retinopathy. The phospho-dephosphorylation mechanisms and nitro-denitrosilatión could be therapeutic targets in the future. These findings open an future research, which could be based on experimental models in which they assessed the effects that changes in the mechanisms of phosphorylation / tyrosine nitrosylation on the occurrence or progression of diabetic retinopathy.

[es] Esta tesis se presenta en forma de compendio de publicaciones según la normativa aprobada por la Comisión de Doctorado de la Universitat Autònoma de Barcelona. El núcleo principal se basa en artículos originales publicados en revistas indexadas. Los trabajos evalúan los niveles de fosforilación y nitrosilación de tirosinas de las interleuquinas del vítreo de pacientes afectos de retinopatía diabética. Para ello se han analizado muestras de vítreo de los mismos y se han comparado con pacientes no diabéticos Artículo 1 Tyrosine Phosphorylation of Vitreous Inflammatory and Angiogenic Peptides and Proteins in Diabetic Retinopathy Investigative Ophthalmology and Visual Science 2009; 50: 1378–1382 DOI:10.1167/iovs.08-2736 FACTOR IMPACTO ………………3,431 Objetivo Estudiar el grado de fosforilación de tirosinas en un amplio conjunto de péptidos y proteínas relacionados con procesos de angiogénesis y de inflamación en muestras de humor vítreo de pacientes diabéticos tipo 2 con Retinopatía diabética proliferativa. Comparar los resultados obtenidos con las muestras de vítreo de pacientes sin retinopatía diabética, de sexo y edades similares. Material y Método Estudio comparativo entre muestras obtenidas durante la VPP realizada por complicaciones derivadas de la RDP con las obtenidas en las VPP por agujero macular idiopático en los sujetos control. Estudio comparativo entre muestras obtenidas durante la VPP realizada por complicaciones derivadas de la RDP con las obtenidas en las VPP por agujero macular idiopático en los sujetos control. Obtención de las muestras de humor vítreo: Vitrectomía Pars Plana con intercambio de aire para la obtención de muestra no diluida , se realiza una centrifugación inmediata y se congela a – 80° C Se aplicaron técnicas de inmunoblot en un sistema de mini-array para la cuantificación de una amplia gama de quimioquinas, péptidos vasoactivos y proteínas con cuantificación de bandas software multi Gauge v 3.0 Los resultados se expresaron como el porcentaje de variación en comparación con sujetos control. Muestra: 8 pacientes afectos de Retinopatía diabética proliferativa Control: 8 pacientes con Agujero macular idiopático La cantidad total de proteínas analizadas fue similar en los pacientes y sujetos control (48). Fosfosforilación de tirosinas sin cambios significativos (p< 0,05) en pacientes diabéticos respecto al grupo de control: Crecimiento regular del oncogén α (GRO α ) IL-2 , IL-3, IL-4 Interferón (FN- ϒ) Proteína quimiotáctica de monocitos (MCP-1-2-3) Factor de necrosis tumoral (TNF α , β) Factor de crecimiento epidérmico (EGF) Factor de crecimiento similar a la insulina (IGF) Trombopoyetina Factor de crecimiento vascular endotelial (VEGF) Factor de crecimiento derivado de las plaquetas (PDGF 88) Fosfosforilación de tirosinas con disminución moderada 15%-20% (p< 0,05) en pacientes diabéticos respecto al grupo de control: Crecimiento regular del oncogén (GRO) Citoquina humana I-309 Interleuquina (IL-13) Factor estimulante de las colonias de monocitos Quimioquina derivada de los macrófagos Factor de las células madre