Browsing by Author "Faria, Daniel"
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- Molecular profiling of the human nasal epithelium: a proteomics approachPublication . Simoes, Tania; Charro, Nuno; Blonder, Josip; Faria, Daniel; Couto, Francisco; Chan, King C.; Waybright, Timothy; Isaaq, Haleem J.; Veenstra, Timothy D.; Penque, DeborahA comprehensive proteomic profiling of nasal epithelium (NE) is described. This study relies on simple subcellular fractionation used to obtain soluble- and membrane-enriched fractions followed by 2-dimensional liquid chromatography (2D-LC) separation and tandem mass spectrometry (MS/MS). The cells were collected using a brushing technique applied on NE of clinically evaluated volunteers. Subsequently, the soluble- and the membrane-protein enriched fractions were prepared and analyzed in parallel using 2D-LC-MS/MS. In a set of 1482 identified proteins, 947 (63.9%) proteins were found to be associated to membrane fraction. Grand average hydropathy value index (GRAVY) analysis, the transmembrane protein mapping and annotations of primary location deposited in the Human Protein Reference Database (HPRD) confirmed an enrichment of hydrophobic proteins on this dataset. Ingenuity Pathway Analysis (IPA) of soluble fraction revealed an enrichment of molecular and cellular functions associated with cell death, protein folding and drug metabolism while in membrane fraction showed an enrichment of functions associated with molecular transport, protein trafficking and cell-to-cell signaling and interaction. The IPA showed similar enrichment of functions associated with cellular growth and proliferation in both soluble and membrane subproteomes. This finding was in agreement with protein content analysis using exponentially modified protein abundance index (emPAI). A comparison of our data with previously published studies focusing on respiratory tract epithelium revealed similarities related to identification of proteins associated with physical barrier function and immunological defence. In summary, we extended the NE molecular profile by identifying and characterizing proteins associated to pivotal functions of a respiratory epithelium, including the control of fluid volume and ionic composition at the airways' surface, physical barrier maintenance, detoxification and immunological defence. The extent of similarities supports the applicability of a less invasive analysis of NE to assess prognosis and treatment response of lung diseases such as asthma, cystic fibrosis and chronic obstructive pulmonary disease.
- Serum proteomics signature of Cystic Fibrosis patients: A complementary 2-DE and LC–MS/MS approachPublication . Charro, Nuno; Hood, Brian L.; Faria, Daniel; Pacheco, Paula; Azevedo, Pilar; Lopes, Carlos; Bugalho de Almeida, António; Couto, Francisco M.; Conrads, Thomas P.; Penque, DeborahComplementary 2D-PAGE and ‘shotgun’ LC–MS/MS approaches were combined to identify medium and low-abundant proteins in sera of Cystic Fibrosis (CF) patients (mild or severe pulmonary disease) in comparison with healthy CF-carrier and non-CF carrier individuals aiming to gain deeper insights into the pathogenesis of this multifactorial genetic disease. 78 differentially expressed spots were identified from 2D-PAGE proteome profiling yielding 28 identifications and postulating the existence of post-translation modifications (PTM). The ‘shotgun’ approach highlighted altered levels of proteins actively involved in CF: abnormal tissue/airway remodeling, protease/antiprotease imbalance, innate immune dysfunction, chronic inflammation, nutritional imbalance and Pseudomonas aeruginosa colonization. Members of the apolipoproteins family (VDBP, ApoA-I, and ApoB) presented gradually lower expression from non-CF to CF-carrier individuals and from those to CF patients, results validated by an independent assay. The multifunctional enzyme NDKB was identified only in the CF group and independently validated by WB. Its functions account for ion sensor in epithelial cells, pancreatic secretion, neutrophil-mediated inflammation and energy production, highlighting its physiological significance in the context of CF. Complementary proteomics-based approaches are reliable tools to reveal pathways and circulating proteins actively involved in a heterogeneous disease such as CF.
- Utilidade da Proteómica na Compreensão da Patogenia Molecular Proximal da Doença Cerebral AlcoólicaPublication . Peneda, Jorge; Charro, Nuno; L. Hood, Brian; Fonseca, Aidil; Hagenfeldt, Manuela; Miranda, Armandina; Zerimech, Farid; Gomes, Filomena; Neto, Domingos; P. Conrads, Thomas; Faria, Daniel; M Couto, Francisco; Penque, DeborahO avanço técnico instrumental no estudo da Proteómica permite aprofundar a interpretação da patogenia molecular de múltiplas doenças. Na doença degenerativa cerebral o mecanismo patogénico inflamatório crónico e imunológico tem sido consensual como será o caso deste órgão alvo preferencial estrutural e funcional em circuitos neuronais susceptíveis na doença alcoólica crónica. A perda da quiescência da impermeabilidade da barreira hemato-encefálica na doença alcoólica, torna acessível o estudo de marcadores moleculares proteicos no sangue periférico, com o apoio da bioinformática e cuidado escrutínio abrangente bibliográfico de proteínas selectivas individuais explícitas e, implícitas intervenientes directas/indirectas na disfunção bioquímica da nevróglia (glicose) e da transmissão neural. Objectivo: Valorização interpretativa no comportamento do complexo perfil proteómico electivo na patogenia molecular da doença cerebral sob efeito pró imunoinflamatório sistémico do metabolismo do etanol e endotoxémia na doença cerebral alcoólica (DCA). Material e métodos: Estudo longitudinal de 27 doentes com alcoolismo crónico activo (T0) e valorização comparativa com abstinência controlada (T1) terapêutica, psicologia de grupo e dieta standardizada em dois subgrupos: (A n=17) com elevados níveis relativos de indicadores de stresse oxidativo/nítrico versus (B n=10) com baixos níveis destes indicadores. Métodos laboratoriais – proteómica e bioquímica descrita pelos autores em 4th EuPA Scientific Meeting, A Proteomics Odyssey Towards Next Decades, Estoril, Portugal, October 23-27 2010:110 111. Resultados Sintéticos: Frequência de rácios A/B: 54.2% de 354 proteínas totais descriminadas têm influência directa/indirecta no SNC. Do total, 51.8% aumentadas e 53.1% diminuídas. Evolução de rácios sob abstinência em cada grupo: AT0/AT1 de 146 proteínas 40.4% diminuídas e 59.6% aumentadas; BT0/BT1 de 130 proteínas 50.8% diminuídas e 49.2% aumentadas; e ainda valorização funcional descriminada de proteínas individuais do total de 340. Conclusões: O stresse oxidativo/nítrico altera o perfil proteómico quantitativo e qualitativo sob consumo activo com indiciadores moleculares múltiplos para um estado global pró-inflamatório e pró-apoptoico (glicose e degenerescência neural). A reversibilidade clínica parcial é acompanhada por variações evolutivas quantitativas e qualitativas do perfil proteómico o que permite valorizar a importância do stresse oxidativo/nítrico como indução patogénica molecular proximal na degenerescência da (DCA).