Browsing by Author "Neves, Sofia"
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- Análise 2D no SAMESPORS: guia práticoPublication . Neves, SofiaObjective: Through an 2D analysis, the serum proteome of Cystic Fibrosis (CF) will be study by comparing the proteins expression level in healthy versus CF carriers and CF patients. The software “Progenesis SameSpots” will be use for the 2D image analysis.
- Diabetes mellitus tipo 2 (DMT2) associada a Sindrome de Apneia Obstrutiva do Sono (SAOS): um estudo proteómicoPublication . Vaz, Fátima; Valentim-Coelho, Cristina; Neves, Sofia; Penque, Deborah; Barbara, CristinaIntrodução: A prevalência da SAOS é elevada em doentes com DMT2. O não tratamento da SAOS pode levar ao agravamento ou desenvolvimento da DMT2. Temos vindo a demonstrar que a SAOS altera o proteoma do glóbulo vermelho (GV). A SAOS aumenta a overoxidação da peroxirredoxina 2 (PRDX2) (enzima antioxidante), o que pode levar à desregulação da homeostasia do GV e ao desenvolvimento de doenças metabólicas. Após tratamento com ventilação não invasiva (PAP), esta overoxidação diminuiu seguida de um aumento de PRDX2 decamérica overoxidada com funções chaperone na proteção celular (Feliciano et al. 2017). No presente estudo, fomos investigar o estado redox/oligomérico da PRDX2 em doentes DMT2 com SAOS, antes/após PAP, para melhor compreender a interligação entre estas patologias. Material e métodos: Amostras de GVs de controles (n=22 sendo 3 DMT2) e doentes SAOS antes/após 6 meses de tratamento com PAP (n=29 sendo 8 DMT2) foram analisadas por western-blot não reduzido, com anticorpo para a PRDX2 e PRDXSO2/3 (overoxidada). Os grupos foram comparados estatisticamente e correlacionados com dados clínicos e bioquímicos. Resultados: Nos GVs de doentes DMT2/SAOS, o nível de monómeros da PRDX2 mostrou-se aumentado e diminuía após PAP. Contudo, o nível destes monómeros PRDXSO2/3 estava diminuído e não se alterou com o tratamento. Após PAP, o nível de decâmeros PRDX2SO2/3 foi também menor nestes doentes. Os níveis de monómeros PRDX2 e PRDX2SO2/3 correlacionaram-se negativamente com os níveis de insulina/triglicéridos e HbA1C, respetivamente. Após PAP, os níveis de decâmeros PRDX2SO2/3 correlacionou-se positivamente com os níveis de adrenalina. Conclusões: O estado redox/oligomérico da PRDX2 do GV é diferencialmente modulado nos doentes DTM2/SAOS em comparação com doentes SAOS. Decâmeros PRDXSO2/3 induzidos pelo tratamento e associadas à função protetora “chaperone” estão diminuídos em doentes DMT2/SAOS. O impacto clínico destas descobertas, necessita de mais investigação e validação.
- Impacto do fumo do cigarro passivo no proteoma humano: em busca de biomarcadores precoces de risco para a saúdePublication . Neves, Sofia; Pacheco, Solange A.; Vaz, Fátima; Valentim-Coelho, Cristina; Saraiva, Joana; James, Peter; Simões, Tânia; Penque, DeborahOs não-fumadores expostos ao fumo do cigarro passivo ou, simplesmente fumo passivo (FP), apresentam um risco acrescido de desenvolver diversas doenças graves. No entanto, os mecanismos moleculares que explicam estes efeitos continuam pouco esclarecidos, o que reforça a necessidade de identificar biomarcadores capazes de avaliar o risco associado a esta exposição. Neste estudo, analisámos o proteoma do epitélio nasal e do plasma de indivíduos não-fumadores saudáveis expostos ao FP no local de trabalho, num contexto ainda enquadrado pela Lei n.º 37/2007, utilizando uma abordagem proteómica ‘shotgun’ por espectrometria de massa. No epitélio nasal, observámos um aumento de proteínas envolvidas em vias centrais do metabolismo energético, como a Gliceraldeído-3-fosfato desidrogenase (GAPDH) e a Triosefosfato isomerase (TPI1), sugerindo uma possível reprogramação metabólica induzida pela exposição. Identificámos também uma diminuição da tubulina beta-4B (TUBB4B), relacionada com a organização do citoesqueleto, e um aumento da proteína anti-apoptótica SERPINB3, apontando para alterações em processos de morte e sobrevivência celular. No plasma, destacaram-se o aumento da Butirilcolinesterase (BChE) e a diminuição da Proteína de ligação à vitamina D (GC), ambas associadas à resposta a xenobióticos e a processos de lesão tecidular. Foram ainda detetadas alterações em proteínas reguladoras da inflamação sistémica, como C1R, C1QC, HRG e PROS1. A expressão diferencial de APOA4 e SERPINF2 sugere, adicionalmente, a ativação de mecanismos relacionados com risco aterotrombótico. Em conjunto, estes resultados contribuem para aprofundar a compreensão das vias biológicas que ligam a exposição ao fumo passivo ao risco acrescido de cancro e de doenças cardiovasculares, e apresentam um conjunto promissor de potenciais biomarcadores para avaliação do risco associado à exposição ao FP.
- Investigating the impact of COVID-19 vaccines on the red blood cell immune function by omics-based approachesPublication . Saraiva, Joana; Coelho, Cristina Valentim; Vaz, Fátima; Antunes, Marilia; Neves, Sofia; Ricardo, Peliano; Andrade, Odília; Miranda, Armandina; Melo, Aryse; Roque, Carla; Guiomar, Raquel; Mohammad, Hamza; Soares, Nelson; Penque, DeborahThe role of red blood cells (RBC) in the immune system is increasingly recognized. However, RBC-derived molecules with an immunomodulatory role in health and disease, as well as in vaccine immunogenicity are still poorly investigated. Taking as a model the emergent COVID-19 vaccines, we aimed to investigate whether vaccines induce proteome and/or metabolome changes in RBCs able to affect T-cell immune activity, as a mechanistic test for vaccine immunization regulated by RBCs. Our ultimate goal is to identify RBC immunomodulators as potential co-adjuvants in the formulation of next-generation vaccines with bolstered efficacy and duration. A biobank of blood samples collected longitudinally under ‘omics’ quality control from subjects (n=39) that underwent vaccination for COVID-19 between April and September 2021 was created. This biobank is associated with extensive clinical data, including demographic data, COVID-19 PCR diagnosis, hematological and vaccine effectivity data. Linear Mixed Models, were used to evaluate the association between biometrical characteristics, health related habits, vaccine technology and vaccine effectivity and hematological parameters, along the different time-points (t0-t4) under study, i.e, before and after (24-72h or 30 days) of the first and second dose of vaccine. Statistical analyses were performed using R software version 4.1.2. Results showed significant differences (p<0.05) before/after vaccination in a set of hematological variables (e.g., hemoglobin, lymphocytes and monocytes values), as well in terms of vaccine effectivity and vaccine technology (mRNA or adenovirus – based vaccines). Preliminary data from proteomics and metabolomics analysis of RBCs along the different time-points (t0-4) of immunization response will be also presented and discussed. The knowledge gained with this project can generate important evidence-based recommendations intended to optimize vaccine immunization, by recognizing the impact of blood cells such RBCs in the immune system regulation.
- Módulo de Proteómica: Análise 2DPublication . Neves, Sofia; Velentim-Coelho, CristinaAnálise de géis 2DE pelo "software" "SameSpot"(Progenesis) com identificação de biomarcadores candidatos.
- Obstructive sleep apnea associated with Diabetes mellitus Type 2: a proteomic studyPublication . Vaz, Fátima; Valentim-Coelho, Cristina; Neves, Sofia; Feliciano, Amelia; Antunes, Marília; Pinto, Paula; Barbara, Cristina; Penque, DeborahBackground: We previously showed that Obstructive sleep apnea (OSA), a common public health concern causing deleterious cardiometabolic dysfunction, induced proteomic alterations in red blood cells (RBC) such as changes in the redox-oligomeric state of peroxiredoxin 2 (PRDX2)1-2. Herein, we aimed to investigate whether OSA patients with Type 2 Diabetes Mellitus before and after positive airway pressure (PAP) treatment present similar changes in the RBC antioxidant protein PRDX2 to better understand the molecular basic mechanisms associated with OSA and OSA outcomes. Methods: RBC samples from control snorers (n=22 being 3 diabetics) and OSA patients before and after six month of PAP-treatment (n=29 being 8 diabetics) were analysed by non-reducing western blot using antibody against PRDX2 or PRDXSO2/3 to measure the total and overoxidized levels of monomeric/dimeric/multimeric forms of PRDX2. Results: We confirmed previously data by showing that in OSA RBC the overoxidation on the monomeric forms of PRDX2 was higher compared to controls. After PAP treatment, this overoxidation decreased followed by an increase of multimeric-overoxidized forms of PRDX2 described to be associated with chaperone protective function. In contrast, the level of PRDX2 monomers in RBC diabetic OSA, although higher abundant its overoxidation level was much lower than those observed in OSA without comorbidity and did not significant change after treatment. Moreover, the level of PAP-induced PRDX2-overoxidized-multimers was also lower in these diabetic OSA patients. The level of overoxidized monomeric/dimeric forms of PRDX2 correlated negatively with levels of insulin / triglycerides and HbA1C, respectively. After PAP, the level of (overoxidized) PRDX2SO2/3 multimers correlated positively with adrenaline levels. Conclusions: The redox/oligomeric state of RBC PRDX2 that is regulated by overoxidation of the active cysteines was differentially modulated in diabetic OSA patients compared to OSA without this comorbidity. PAP-induced overoxidized oligo forms of PRDX2 that is associated with chaperone protective function showed decreased in OSA patients with diabetes. The clinical impact of these findings needs further investigation and validation.
- Occupational exposure to environmental tobacco smoke in hospitality venues: are genetic- or proteomics-based biomarkers predictive of respiratory diseases?Publication . Silva, M.J.; Vital, Nádia; Pacheco, Solange; Antunes, Susana; Neves, Sofia; Louro, Henriqueta; Torre, Vukosava M.; Vaz, Fátima; James, Peter; Lopes, Carlos; Marçal, Nelson; Bugalho de Almeida, António; Simões, Tânia; Penque, DeborahEnvironmental tobacco smoke (ETS) is recognized as an occupational hazard in the hospitality industry. Although Portuguese legislation banned smoking in most indoor public spaces, it is still allowed in some restaurants/bars, representing a potential risk to the workers’ health, particularly for chronic respiratory diseases. The aims of this work were to characterize biomarkers of early genetic effects and to disclose proteomic signatures associated to occupational exposure to ETS and with potential to predict respiratory diseases development. A detailed lifestyle survey and clinical evaluation (including spirometry) were performed in 81 workers from Lisbon restaurants. ETS exposure was assessed through the level of PM 2.5 in indoor air and the urinary level of cotinine. The plasma samples were immunodepleted and analysed by 2D-SDSPAGE followed by in-gel digestion and LC-MS/MS. DNA lesions and chromosome damage were analysed innlymphocytes and in exfoliated buccal cells from 19 cigarette smokers, 29 involuntary smokers, and 33 non-smokers not exposed to tobacco smoke. Also, the DNA repair capacity was evaluated using an ex vivo challenge comet assay with an alkylating agent (EMS). All workers were considered healthy and recorded normal lung function. Interestingly, following 2D-DIGE-MS (MALDI-TOF/TOF), 61 plasma proteins were found differentially expressed in ETS-exposed subjects, including 38 involved in metabolism, acute-phase respiratory inflammation, and immune or vascular functions. On the other hand, the involuntary smokers showed neither an increased level of DNA/chromosome damage on lymphocytes nor an increased number of micronuclei in buccal cells, when compared to non-exposed non-smokers. Noteworthy, lymphocytes challenge with EMS resulted in a significantly lower level of DNA breaks in ETS-exposed as compared to non-exposed workers (P<0.0001) suggestive of an adaptive response elicited by the previous exposure to low levels of ETS. Overall, changes in proteome may be promising early biomarkers of exposure to ETS. Likewise, alterations of the DNA repair competence observed upon ETS exposure deserves to be further understood. Work supported by Fundação Calouste Gulbenkian, ACSS and FCT/Polyannual Funding Program.
- Occupational second-hand smoke exposure: A comparative shotgun proteomics study on nasal epithelia from healthy restaurant workersPublication . Neves, Sofia; Pacheco, Solange; Vaz, Fátima; James, Peter; Simões, Tânia; Penque, Deborah; NevesNon-smokers exposed to second-hand smoke (SHS) present risk of developing tobacco smoke-associated pathologies. To investigate the airway molecular response to SHS exposure that could be used in health risk assessment, comparative shotgun proteomics was performed on nasal epithelium from a group of healthy restaurant workers, non-smokers (never and former) exposed and not exposed to SHS in the workplace. HIF1α-glycolytic targets (GAPDH, TPI) and proteins related to xenobiotic metabolism, cell proliferation and differentiation leading to cancer (ADH1C, TUBB4B, EEF2) showed significant modulation in non-smokers exposed. In never smokers exposed, enrichment of glutathione metabolism pathway and EEF2-regulating protein synthesis in genotoxic response were increased, while in former smokers exposed, proteins (LYZ, ATP1A1, SERPINB3) associated with tissue damage/regeneration, apoptosis inhibition and inflammation that may lead to asthma, COPD or cancer, were upregulated. The identified proteins are potential response and susceptibility/risk biomarkers for SHS exposure.
- Occupational secondhand smoke exposure - A proteomic analysisPublication . Neves, Sofia; Pacheco, Solange; Vaz, Fátima; Simões, Tania; James, Peter; Simões, Tânia; Penque, DeborahBackground: WHO have stated that near 900 000 deaths per year result from exposure to Second- Hand Smoke (SHS). SHS exposure has been linked to cancer, respiratory and cardiovascular diseases and diabetes. However, the associated underlying molecular mechanisms remain to be elucidated. The objective of this proteomics study is to uncover putative key molecules involved in these mechanisms that can be used to predict and monitor diseases risks associated with occupational SHS exposure. Methods: In total, 25 Lisbon restaurants agreed to participate. Nasal epithelium and urine samples were collected from their employees (n=52) for proteomics analysis and cotinine evaluation of SHS exposure, respectively. The subjects were classified as never smoker (N), former smoker (F) and smoker (S); exposed (NE=11; FE=10; SE=4) or non-exposed (N=11; F=8; S=8) to SHS. All subjects were healthy and showed no significant differences in parameters like age, time in the workplace, tobacco smoking habits and spirometry evaluation of pulmonary function. Urine cotinine levels showed significantly elevated in the exposed subjects compared to non-exposed, confirming SHS exposure. Nasal epithelium samples were analyzed by shotgun proteomics using an ESI-LTQOrbitrap mass spectrometer. The “MS raw data” was submitted to “PatternLab for Proteomics” software, with “Comet” search machine algorithm, from where the identified proteins were submitted to a “ClueGO” functional annotation & enrichment analyses in “Cytoscape” software, with the propose to shed some light about the molecular biology involved in the cellular response to the SHS exposition. Results: In NE subjects the SHS is associated with the biologic terms of “Lactate dehydrogenase complex” and “Pentose-Phosphatase Shunt”, also with “Glutathione peroxidase activity” and “Tcell apoptotic process”. At the other end the FE individuals present a specific proteome enriched in biologic information with terms as the “L-Lactate dehydrogenase complex” and the “Peroxisome” as was expected by the results above for the NE cohort; but there were also other different terms as: “Peripheral T cell lymphoma”, “Central carbon metabolism in cancer”, “Myelodysplastic syndrome”, “Monocyte & Granulocyte & Macrophage & Leukocyte Chemotaxis”, Nucleossome, variant H3.1-H2A2-H2B.1&Others” and finally “DNA replication-dependent chromatin assembly”. Conclusions: Proteome of nasal epithelium seems to be modulated by SHS exposure and this is a different and perhaps cumulative process between NE and FE individuals.
- Occupational secondhand smoke exposure may modify the proteoma expression of human nasal epitheliumPublication . Neves, Sofia; Pacheco, Solange; Vaz, Fátima; Martins, Inês; James, Peter; Simões, Tânia; Penque, DeborahThe tobacco is one of the biggest public health threats, smoking kills more than 7 million people/year worldwide and more than 890,000 are deaths resulting from exposure to Second Hand Smoke (SHS). In adults, SHS is associated to cardiovascular and respiratory diseases, including coronary heart disease and lung cancer, through pathological and molecular mechanisms not yet understood. In this study, we aimed to investigate the SHS effects on airway proteome in exposed workers. Nasal epithelium was collected from hospitality workers (non-smokers=40; smokers=12) long-term exposed and non-exposed to SHS at the workplace. Samples were analyzed by shotgun proteomics using an ESI-LQT Orbitrap XL mass spectrometer. The generated MS raw data was submitted to ‘PatternLab for Proteomics 4.0’ for peptide identification and relative quantification by label-free - extracted ion chromatograms (XIC). Golden rules were applied to obtain reliable data such as the inferred proteins must have at least one unique peptide identified to be considered and be detected in at least 80% of the cohort. Two proteins were found to be differentially expressed in the no-smokers exposed to SHS compared with the control: BPI fold-containing family A member 1 (BPIFA1) and Heat shock Protein Beta-1 (HSPB1). The first protein plays a role in the airway inflammatory response after exposure to irritants substances and the second is associated as a regulator of actin filament dynamics. Our findings support the indication that in non-smokers the prolonged exposure to SHS can lead to airway proteome modulation. When validated, the uncovered proteins can be promising candidates to “susceptibility/risk” and/or “response” biomarkers for SHS exposure.
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