Desarrollo de una combinación de biomarcadores relacionados con el estrés oxidativo y la inflamación en el glaucoma: Aplicaciones de combinación de biomarcadores para el diagnóstico del glaucoma

• Autores: Azza Dammak
• Directores de la Tesis: Fernando Huete Toral (dir. tes.), Gonzalo Carracedo Rodríguez (dir. tes.)
• Lectura: En la Universidad Complutense de Madrid ( España ) en 2024
• Idioma: español
• Número de páginas: 278
• Títulos paralelos:
  • Development of a combination of biomarkers related to oxidative stress and inflammation in Glaucoma: Applications of biomarkers combination for glaucoma diagnostic
• Tribunal Calificador de la Tesis: Jesús Carballo Álvarez (presid.), María Serramito Blanco (secret.), Margarita Calonge Cano (voc.), Marianna Bacellar Galdino (voc.), David P. Piñero Llorens (voc.)
• Programa de doctorado: Programa de Doctorado en Óptica, Optometría y Visión por la Universidad Complutense de Madrid
• Materias:
  • Química
    • Bioquímica
      • Biología molecular
      • Farmacología molecular
• Enlaces
  • Tesis en acceso abierto en: Docta Complutense
• Resumen
  • español

    Chapter I: General Introduction The pathophysiology of glaucoma is characterized by the progressive degeneration of retinal ganglion cells, causing irreversible blindness. It is manifested by the high resistance of aqueous humour drainage, causing intraocular pressure, inflammation, and the production of reactive oxygen substances. The imbalance between prooxidative and antioxidant capacities leads to the release of several biomarkers.

    Chapter II: justification, hypothesis, and objective The importance of biomarkers in diagnosing glaucoma has a critical role in disease evolution. In-vivo and incorporating in-vitro models is imperative for exploring glaucoma-related biomarkers. The hypothesis examines the release of a combination of biomarkers, using in-vivo models, in-vitro models, and human ocular samples. The objective is to advance our understanding of glaucoma by identifying a combination of biomarkers associated with oxidative stress and inflammation.

    Chapter III: Development of biomarkers combination In-Vitro This chapter aimed to quantify the selected biomarkers. The research revealed that human TM cells secreted baseline levels of OPN. Exposure to TGF-beta 2 significantly increased OPN levels in TM cells, and this effect was exacerbated by concurrent mechanical strain. Mechanical strain exacerbates the fibrotic effects of recombinant TGF-beta 2, likely due to the upregulation of TGF-beta1 and TGF-beta 2. In arising retinal pigment epithelia cells, the combination of mechanical strain and relevant stressors (t-BHP + glucose) led to a significant decrease in OPN gene expression. Additionally, strain significantly increased OPN and MMP-9 gene expression. Our results highlight the complex signalling crosstalk in ocular tissues and underscore the importance of identifying tissue-specific biomarkers.

    Chapter IV: Development of new glaucoma model and testing the biomarker combination In-Vivo The purpose of the study was to develop a new glaucoma model in rabbits induced by intracameral injection of gold nanoparticles, while using chondroitin sulphate as a reference high intraocular pressure model. The model was characterized by measuring the concentration of selected biomarkers. By comparing the concentration of biomarkers in tears, AH, and retina among the model group induced by injection of GNP in PBS, the positive control group induced by injection of CS in PBS and control group that received injection of PBS, we were able to characterize the model group and justify the change in the levels of biomarkers. Injection of 25nm GNP twice weekly for five consecutive weeks results in the increase of IOP from 12,7 ± 1.8 mmHg at the baseline to 21.24 ± 5 mmHg, and the injection of CS weekly for four weeks results in the increase of IOP from 12,5 ± 2 mmHg to 22,37± 4 mmHg after six weeks. The concentration of biomarkers was quantified in tears, AH, and retina.

    Chapter V: Development of a new biomarker combination in human samples This chapter focuses on quantifying the concentration of selected biomarkers in AH samples from glaucoma patients compared to healthy patients who underwent cataract surgery. Two different studies were conducted using distinct techniques and samples. In the first step, biomarkers in AH samples were quantified using ELISA kit. The selected biomarkers, including OPN, MMP-9, TNF-alpha, and IL-10, are significantly increased in the AH of glaucoma patients compared to the control group, confirming their involvement in glaucoma. In the second step, the biomarkers in AH were detected and quantified using Multiplex AYOXAA technology. Significant changes were observed in IL-8, IL10, IL-6, and TNF-alpha levels between the two groups.

  • English

    This doctoral thesis is achieved as part of the European Union's Horizon 2020 research and innovation program under the Marie -Curie grant agreement N° 813440 ORBITAL Ocular Research by Integrated Training and Learning. The thesis has been carried out within a context of collaboration between the spin-off company Ocupharm Diagnostics and the Loyola University Chicago, Ophthalmology and molecular pharmacology and Neuroscience department (Maywood, Chicago) to develop a new cocktail of biomarkers related to oxidative stress and inflammation in glaucoma based on the research of biomarkers that have been identified in the literature and tested in cells, in animal models, and human samples...