Browsing by Author "Charro, Nuno"
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- 2-DIGE of Red Blood Cells from Sickle-Cell Disease Patients with Severe Vaso-Occlusion.Publication . Vaz, Fátima; Charro, Nuno; Morais, Anabela; Lavinha, João; Penque, Deborah
- 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.
- Optimizing the discovery of predictors of vaso-occlusion in sickle-cell disease by proteomicsPublication . Lavinha, João; Charro, Nuno; Vaz, Fátima; Morais, Anabela; Penque, DeborahPainful crises are the major sickle-cell disease (SCD) clinical manifestation probably due to significant increase in dense red blood cells (RBC) and reduction of their ability to pass through capillaries. Using proteomic strategies (see figure below), we aimed to discover novel SCD prognosis biomarkers as early predictors of the transition from steady-state to vaso-occlusive crises thus, allowing a prompt and specific therapeutic intervention.
- Plasma and red blood cell proteome in sickle-cell diseasePublication . Charro, Nuno; Vaz, Fatima; Morais, Anabela; Lavinha, João; Penque, DeborahSickle-cell disease (SCD) is a clinically heterogeneous autosomal recessive monogenic chronic anaemia characterized by recurrent episodes of severe vaso-occlusion, haemolysis and infection. Painful crises are the major SCD clinical manifestation probably due to significant increase in dense red blood cells (RBC) and reduction of their ability to pass through capillaries. Using proteomic strategies, we aim to discover novel and better SCD prognosis biomarkers as early predictors of the transition from steady-state to crisis namely vaso-occlusive episodes, thus, allowing a prompt and specific therapeutic intervention
- Profiling the erythrocyte membrane proteome isolated from patients diagnosed with chronic obstructive pulmonary diseasePublication . Alexandre, Bruno; Charro, Nuno; Blonder, Yosip; Lopes, Carlos; Almeida, Antonio Bugalho; Veenstra, Timothy; Penque, Deborah; Azevedo, Pilar; Chan, K.C.; Prieto, D.A.; Issaq, H.Structural and metabolic alterations in erythrocytes play an important role in the pathophysiology of Chronic Obstructive Pulmonary Disease (COPD). Whether these dysfunctions are related to the modulation of erythrocyte membrane proteins in patients diagnosed with COPD remains to be determined. Herein, a comparative proteomic profiling of the erythrocyte membrane fraction isolated from peripheral blood of smokers diagnosed with COPD and smokers with no COPD was performed using differential 16O/18O stable isotope labeling. A total of 219 proteins were quantified as being significantly differentially expressed within the erythrocyte membrane proteomes of smokers with COPD and healthy smokers. Functional pathway analysis showed that the most enriched biofunctions were related to cell-to-cell signaling and interaction, hematological system development, immune response, oxidative stress and cytoskeleton. Chorein (VPS13A), a cytoskeleton related protein whose defects had been associated with the presence of cell membrane deformation of circulating erythrocytes was found to be down-regulated in the membrane fraction of erythrocytes obtained from COPD patients. Methemoglobin reductase (CYB5R3) was also found to be underexpressed in these cells, suggesting that COPD patients may be at higher risk for developing methemoglobinemia. This article is part of a Special Issue entitled: “Integrated omics— Functional applications to blood and blood therapeutics”.
- Proteomics in Detection and Monitoring Chronic Lung Diseases: The Human Nasal Epithelium as a Molecular ModelPublication . Simões, Tânia; Charro, Nuno; Alexandre, Bruno; Penque, DeborahAsthma and chronic obstructive pulmonary disease (COPD) are major causes of mortality and morbidity worldwide. The current state-of-art diagnosis and management schemes are suboptimal for both diseases as the incidence of asthma has risen by 250% over the last two decades and COPD is estimated to become the third leading cause of death worldwide within the next decade. Additionally, these diseases represent a very important threat to global economies in direct and indirect medical costs and lost working days [1,2]. Asthma is a chronic inflammatory disorder of the airways associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness and coughing. These episodes are usually associated with widespread, but variable, airflow obstruction within the lung [1]. Chronic airflow obstruction is also characteristic of COPD but, in contrast to asthma, is not fully reversible, even under the action of bronchodilators, and is usually progressive. A combination of small airway disease -obstructive bronchiolitis - and parenchymal destruction - emphysema, leads to COPD clinical manifestation [2]. A number of factors influence a person’s risk of developing these lung diseases, which include host factors, primarily genetic, and environmental factors, such as allergens and tobacco smoke in asthma and COPD, respectively
- 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.
- Supramolecular organizations in the aerobic respiratory chain of Escherichia coliPublication . Sousa, Pedro M.F; Silva, Sara T.N.; Hood, Brian L.; Charro, Nuno; Carita, João N.; Vaz, Fátima; Penque, Deborah; Conrads, Thomas P.; Melo, Ana M.P.The organization of respiratory chain complexes in supercomplexes has been shown in the mitochondria of several eukaryotes and in the cell membranes of some bacteria. These supercomplexes are suggested to be important for oxidative phosphorylation efficiency and to prevent the formation of reactive oxygen species. Here we describe, for the first time, the identification of supramolecular organizations in the aerobic respiratory chain of Escherichia coli, including a trimer of succinate dehydrogenase. Furthermore, two heterooligomerizations have been shown: one resulting from the association of the NADH:quinone oxidoreductases NDH-1 and NDH-2, and another composed by the cytochrome bo3 quinol:oxygen reductase, cytochrome bd quinol:oxygen reductase and formate dehydrogenase (fdo). These results are supported by blue native-electrophoresis, mass spectrometry and kinetic data of wild type and mutant E . coli strains.
- Tobacco Smoke Occupational Exposure: Biomarkers of Biological DamagePublication . Simões, Tânia; Milic, Vukosava D.; Pacheco, S.A.; Aguiar, Fátima; Gomes, Filomena; Louro, Henriqueta; Vital, Nádia; Antunes, Susana; Charro, Nuno; Bruno, Alexandre; Vaz, Fátima; Lopes, C.; Marçal, N.; Fragoso, E.; Proença, C.; Sekera, M.; Hagenfeld, Manuela; Silva, Maria João; Almeida, A.B.; Penque, Deborah; Ruivo, P.High concentration of toxic substances emanated from tobacco smoke in entertainment places such as restaurants, bars and nightclubs may compromise indoor air quality (IAQ) generating environments of likelihood health risk. Their employees, particularly those exposed to second-hand smoke, are at increased risk for developing chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Objectives In this work, we aimed at evaluating relationships between occupational ETS exposure, biological damage (DNA or proteome alterations) and putative respiratory dysfunctions. Materials and Methods A group of restaurants located in Lisbon has been studied, in which fine particles (smaller than 2.5µm; PM2.5), indicative of environmental tobacco smoke (ETS) contamination, were measured. After informed consent, workers were evaluated for acute exposure to ETS based on cotinine levels in urine and clinically evaluated for their respiratory health by spirometry measurements and chestpiece auscultation. Effects of ETS exposure on genotoxic lesions were evaluated by measuring DNA/chromosomes breaks in peripheral blood lymphocytes and buccal mucosa cells. Effects of ETS exposure on plasma proteins is being studied using a 2D-DIGE-MALDI-TOF/TOF approach. To achieve that goal, global proteome characterization is being carried based on the same individual plasma samples collected for genotoxic studies and were pooled according to previous criteria. Results Results have confirmed higher respirable particle levels in smoking-designated areas of those entertainment places, indicating an ETS contamination. Leukocytes from ETS-exposed-workers presented lower levels of genotoxic-induced damage in comparison with non-exposed workers, suggesting an ETS-induced stress adaption response in exposed-workers. By proteomics, we are now to investigating those workers for putative alterations on their plasma proteome to provide additional insights on the adaptative response mechanisms that might be activated by ETS exposition. Conclusion Altogether, this study provides information on indoor air quality of Lisbon smoking entertainment places, in particular ETS contamination, and may provide biomarker candidates for occupational ETS-exposure which might precede respiratory diseases on their employees.
- Tobacco smoke occupational exposure: biomarkers of biological damagePublication . Simões, Tânia; Torres, Vukosava; Pacheco, Solange; Louro, Henriqueta; Silva, Maria Joao; Charro, Nuno; Alexandre, Bruno; Vaz, Fatima; Penque, DeborahHigh concentration of toxic substances emanated from tobacco smoke in entertainment places such as restaurants, bars and nightclubs may compromise indoor air quality (IAQ) generating environments of likelihood health risk. Their employees, particularly those exposed to second-hand smoke, are at increased risk for developing chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Objectives In this work, we aimed at evaluating relationships between occupational ETS exposure, biological damage (DNA or proteome alterations) and putative respiratory dysfunctions.