PROTEIN-TRIGGERED SUSTAINED DRUG RELEASE CONTROLLED BY APTAMERS BOUND TO OLIGOMER-CROSSLINKED ALGINATE

Sustained drug release systems provide many advantages over traditional delivery methods such as extending the time in which the drug is found to be within an effective concentration within the therapeutic window, which decreases the frequency of administration of the drug, and increases patient compliance. Research using polyacrylamide crosslinked by oligomers containing an aptamer sequence, has demonstrated a pulsatile release over 50 minutes triggered by a 2 mM target adenosine concentration. This thesis aims to build off this concept by designing a system that delivers in a sustained manner when triggered by micromolar target concentrations reflective of disease in vivo, using macromolecular targets. For example, the disease wet age related macular degeneration (wet AMD) is associated with increased concentrations of the protein vascular endothelial growth factor (VEGF-A) – a macromolecule. Patients with wet AMD would benefit from the implantation of devices or microspheres that release drugs in a sustained manner in response to local VEGF concentrations. In this thesis, we hypothesize that the protein lysozyme, used to demonstrate proof-of-concept, could trigger the increased release of drugs from oligomer-crosslinked alginate. The objectives are to (i) demonstrate sustained release from alginate, (ii) design oligomer crosslinked alginate that degrades in response to lysozyme, and then (iii) use these systems to control the release of FITC-dextran with and without lysozyme. A series of control experiments and analyses were used to optimize the crosslinking of alginate by annealed oligomers. The cumulative release of FITC-dextran (MW 20,000) from oligomer crosslinked alginate increased by 3.4 μg when lysozyme (3 μM) was introduced at 48 hours, as opposed to controls which released only 0.2 μg. FITC-loaded alginate microspheres coated by oligomer-crosslinked alginate released 15% more FITC-dextran over 120 hours when placed into 3 μM of lysozyme than without lysozyme. Controls of alginate crosslinked with PEG or control oligomers (without a lysozyme aptamer sequence) had no changes in release with lysozyme. The incorporation of a lysozyme aptamer onto oligomers used to crosslink alginate disks or alginate coatings on microspheres resulted in different diffusion and release of FITC-dextran into PBS with or without lysozyme. This approach could be adapted for the delivery of drugs to diseases with specific protein profiles such as wet AMD.

Development of an activity disease score in patients with uveitis (UVEDAI)

To develop a disease activity index for patients with uveitis (UVEDAI) encompassing the relevant domains of disease activity considered important among experts in this field. The steps for designing UVEDAI were: (a) Defining the construct and establishing the domains through a formal judgment of experts, (b) A two-round Delphi study with a panel of 15 experts to determine the relevant items, (c) Selection of items: A logistic regression model was developed that set ocular inflammatory activity as the dependent variable. The construct “uveitis inflammatory activity” was defined as any intraocular inflammation that included external structures (cornea) in addition to uvea. Seven domains and 15 items were identified: best-corrected visual acuity, inflammation of the anterior chamber (anterior chamber cells, hypopyon, the presence of fibrin, active posterior keratic precipitates and iris nodules), intraocular pressure, inflammation of the vitreous cavity (vitreous haze, snowballs and snowbanks), central macular edema, inflammation of the posterior pole (the presence and number of choroidal/retinal lesions, vascular inflammation and papillitis), and global assessment from both (patient and physician). From all the variables studied in the multivariate model, anterior chamber cell grade, vitreous haze, central macular edema, inflammatory vessel sheathing, papillitis, choroidal/retinal lesions and patient evaluation were included in UVEDAI. UVEDAI is an index designed to assess the global ocular inflammatory activity in patients with uveitis. It might prove worthwhile to motorize the activity of this extraarticular manifestation of some rheumatic diseases.

MEF2C: expression, regulation and interaction with target genes in health and diseases

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Alterações histopatológicas em duas espécies de teleósteos expostos ao inseticida endosulfan.

Embora pesticidas produzam resultados positivos no controle de pragas, seus efeitos danosos em organismos "não alvo" não devem ser excluídos. Entre os vários pesticidas cuja toxicidade a peixes tem sido estudada, o endosulfan é um dos mais tóxicos. Entretanto, dados sobre as alterações histopatológicas provocadas em peixes por ação do endosulfan são escassos. Foram identificados os danos histopatológicos em branquias e figado dos peixes Brachydanio rerio e Hyphessobrycon bifasciatus após exposição a concentrações letais e subletais do pesticida. Os peixes foram expostos a concentração de 0 a 6,5 ppb de Thiodan técnico na água por 24 h, sendo que durante este período, amostras de peixes foram retiradas para analise histopatológica. Nas exposições subagudas os peixes foram submetidos a concentração media de 0,3 ppb do pesticida durante 21 dias. Foram realizados testes nos quais os peixes da especie H. bifasciatus alimentaram-se, durante 181 dias, com dieta contendo 8,9 ppm do inseticida. A microscopia óptica dos tecidos revelou predominância dos efeitos agudos sobre as branquias, observando-se necrose e destacamento do epitélio das lamelas branquiais. O figado demonstrou alterações histopatológicas características de efeitos subagudos, observando-se degeneração gordurosa e alterações nucleares dos hepatócitos. Os dados obtidos indicam necessidade de estudos adicionais sobre a toxicidade do endosulfan a longo prazo, visto que alguns dos danos histopatológicos identificados poderiam evoluir para alterações malignas.

Retinopatia na hipertensão arterial sistêmica

Este vídeo integra o curso Oftalmologia na Atenção Básica à Saúde (2016) que tem o objetivo de ampliar a capacidade de resolução clínico-oftalmológica na Atenção Básica à saúde. O vídeo tem enfoque na apresentação dos problemas causados pela hipertensão arterial sistêmica, como a retinopatia.