Percorrer por autor "Carvalho, Ana Sofia"
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- Estratégia Nacional para a Medicina Genómica - PT_MedGen: desafios e prioridadesPublication . Almeida, Fernando de; Vicente, Astrid Moura; Calado, Patrícia; Santos, Manuel; Carvalho, Ana Sofia; Águas, Cíntia; Pinto, Cátia Sousa; Silva, Mário Jorge Gaspar da; Melo, Ana Portugal; Oliveira, Mónica Duarte Correia de; Feijó, Joana; Vilarinho, Laura; Oliveira, CarlaO presente documento visa propor o conceito e as linhas de ação prioritárias da Estratégia Nacional para a Medicina Genómica (PT_MedGen). O documento baseia-se na auscultação de alguns dos principais stakeholders nacionais, representados na Comissão nomeada pelo Despacho n.o 5135/2021 coordenada pelo INSA, e ainda na consulta de outras entidades e peritos de relevância. A estratégia PT_MedGen tem a meta global de criar infraestruturas e processos que permitam a adoção de abordagens de medicina personalizada na prática clínica, a par com a contribuição para a iniciativa 1+MG. Esta estratégia promoverá ainda a investigação, a inovação, a competitividade e a internacionalização, permitindo a criação de conhecimento e valor significativos na área da saúde.
- Overview of proteomics studies in obstructive sleep apneaPublication . Feliciano, Amélia; Torres, Vukosava M.; Vaz, Fatima; Carvalho, Ana Sofia; Matthiesen, Rune; Pinto, Paula; Malhotra, Atul; Bárbara, Cristina; Penque, DeborahObstructive sleep apnea (OSA) is an underdiagnosed common public health concern causing deleterious effects on metabolic and cardiovascular health. Although much has been learned regarding the pathophysiology and consequences of OSA in the past decades, the molecular mechanisms associated with such processes remain poorly defined. The advanced high-throughput proteomics-based technologies have become a fundamental approach for identifying novel disease mediators as potential diagnostic and therapeutic targets for many diseases, including OSA. Here, we briefly review OSA pathophysiology and the technological advances in proteomics and the first results of its application to address critical issues in the OSA field.
- Quantitative proteome analysis of an antibiotic resistant Escherichia coli exposed to tetracycline reveals multiple affected metabolic and peptidoglycan processesPublication . Jones-Dias, Daniela; Carvalho, Ana Sofia; Moura, Inês Barata; Manageiro, Vera; Igrejas, Gilberto; Caniça, Manuela; Matthiesen, RuneTetracyclines are among the most commonly used antibiotics administrated to farm animals for disease treatment and prevention, contributing to the worldwide increase in antibiotic resistance in animal and human pathogens. Although tetracycline mechanisms of resistance are well known, the role of metabolism in bacterial reaction to antibiotic stress is still an important assignment and could contribute to the understanding of tetracycline related stress response. In this study, spectral counts-based label free quantitative proteomics has been applied to study the response to tetracycline of the environmental-borne Escherichia coli EcAmb278 isolate soluble proteome. A total of 1484 proteins were identified by high resolution mass spectrometry at a false discovery rate threshold of 1%, of which 108 were uniquely identified under absence of tetracycline whereas 126 were uniquely identified in presence of tetracycline. These proteins revealed interesting difference in e.g. proteins involved in peptidoglycan-based cell wall proteins and energy metabolism. Upon treatment, 12 proteins were differentially regulated showing more than 2-fold change and p<0.05 (p value corrected for multiple testing). This integrated study using high resolution mass spectrometry based label-free quantitative proteomics to study tetracycline antibiotic response in the soluble proteome of resistant E. coli provides novel insight into tetracycline related stress.
- Quantitative proteome analysis of an Escherichia coli exposed to tetracycline reveals multiple affected cytoplasmic metabolic processesPublication . Jones-Dias, Daniela; Carvalho, Ana Sofia; Moura, Inês Barata; Dill, Brian; Molina, Henrik; Manageiro, Vera; Caniça, Manuela; Matthiesen, RuneBackground: Many studies have already focused on the evaluation of variations in protein expression caused by antibiotic exposure in tetracycline resistant microorganisms. However, little is known about the metabolic response of genetically resistant bacterial populations to antibiotic exposure. In this study we used a liquid chromatography-mass spectrometry-based (LC-MS/MS) proteomics to evaluate the global cytoplasmic metabolic changes of a resistant Escherichia coli isolate, when challenged with tetracycline. Material/methods: IsolateEcAamb278 was recovered from a soil sample, used for the intensive farming of tomato plants and showed nonsusceptibility to all categories of β-lactams except carbapanems and cefoxitin, and tetracycline. Previous genomic characterization revealed the presence of TEM-1, CTX-M-1, Sul2,TetA and TetB resistance determinants. Single colonies of E. coliEcAamb278 were used to extract crude cytoplasmic protein fraction using non detergent cell lysis buffer. The proteomic profile of E. coli EcAmb278 was determined by label-free LC-MS/MS upon tetracycline stress in a comparative study, using as referencethe proteome of the same strain nonexposed to antibiotics. The samples were analyzed by nano LC-MS/MS, using a Q-Exactive mass spectrometer. All data was searched with VEMS. Proteins were quantified by spectral counting and mziXIC, followed by iBAQ estimation. Results: The complete proteome yielded about 1484 proteins, using a 1% FDR as cut off. The comparison of the proteome profiles of the two E. coli EcAamb278 samples pointed to several proteins with altered expression under tetracycline stress conditions. The twelve most significant (FDRcorrected p<0.05) proteins differentially regulated by more than two-fold were involved in DNA metabolism, transcription, virulence, intracellular trafficking and secretion, biosynthesis of vitamins, other processes, and unknown functions. For instance, (1) acyl-CoA thioester hydrolase, involved in biosynthesis of coenzyme A; (2) bacterial NAD+-dependent DNA ligase, plays a critical role in DNA replication, recombination and repair in all living organisms; and (3) adhesion protein FimH, which confirmed the pathogenicity of this environmental-borne E. coli. Overall, several other detected proteins were associated with the process of antibiotic resistance, virulence and acquisition and transfer of foreign DNA (AcrA, TolC, MdtE, Omps, TnsE, Colicin peptides, among others). Conclusions: These results indicate that E. coli responses to tetracycline are related to protein translation as well as metabolic regulation. We observed differentially regulation of several metabolic proteins though not belonging to the canonical antibiotic resistance pathway, suggesting an integrated metabolic bacteria response to antibiotic exposure, which can potentially be explored for drug development.
- Sequence variation at KLK and WFDC clusters and its association to semen hyperviscosity and other male infertility phenotypesPublication . Marques, Patrícia Isabel; Fonseca, Filipa; Carvalho, Ana Sofia; Puente, Diana A.; Damião, Isabel; Almeida, Vasco; Barros, Nuno; Barros, Alberto; Carvalho, Filipa; Azkargorta, Mikel; Elortza, Felix; Osório, Hugo; Matthiesen, Rune; Quesada, Victor; Seixas, SusanaSTUDY QUESTION: Are kallikreins (KLKs), the whey-acidic-protein four-disulfide core domain (WFDCs) and their neighbors, semenogelins (SEMGs), known to play a role in the cascade of semen coagulation and liquefaction, associated with male infertility? SUMMARY ANSWER: Several KLK and SEMG variants are overrepresented among hyperviscosity, asthenozoospermia and oligozoospermia, supporting an effect of abnormal semen liquefaction on the loss of semen quality and in lowering male reproductive fitness. WHAT IS KNOWN ALREADY: In the cascade of semen coagulation and liquefaction the spermatozoa coated by EPPIN (a protease inhibitor of the WFDC family) are entrapped in a cross-linked matrix established by SEMGs. After ejaculation, the SEMG matrix is hydrolyzed by KLK3/2 in a fine-tuned process regulated by other KLKs that allows the spermatozoa to increase motility. STUDY DESIGN SIZE, DURATION: This study includes a cohort of 238 infertility-related cases and 91 controls with normal spermiogram analysis. The remaining 126 controls are healthy males with unknown semen parameters. Sample collection was carried out from June 2011 to January 2015 and variant screening from May 2013 to August 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: We performed a screening by massive parallel sequencing in a pooled sample (N = 222) covering approximately 93 kb of KLK (19q13.3-13.4) and WFDC (20q13) clusters, followed by the genotyping of most promising variants in the full cohort. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 160 common and 296 low-frequency variants passed the quality control filtering. Statistical tests disclosed an association with hyperviscosity of a KLK7 regulatory variant (P = 0.0035), and unveiled a higher burden of deleterious mutations in KLKs than expected by chance (P = 0.0106). KLK variants found to be overrepresented in cases included two substitutions likely affecting the substrate binding pocket, two nonsynonymous variants overlapping in the three-dimensional structure and two mutations mapping in consecutive N-terminal residues. Other variants identified in SEMGs possibly contributing to hyperviscosity and asthenozoospermia consisted of three replacements predicted to modify targets of proteolysis (P = 0.0442 for SEMG1 p.Gly400Asp) and a copy number variation associated with a reduced risk of oligozoospermia (P = 0.0293).
- WNK1 phosphorylation sites in TBC1D1 and TBC1D4 modulate cell surface expression of GLUT1Publication . Henriques, Andreia F.A.; Matos, Paulo; Carvalho, Ana Sofia; Azkargorta, Mikel; Elortza, Felix; Matthiesen, Rune; Jordan, PeterGlucose uptake by mammalian cells is a key mechanism to maintain cell and tissue homeostasis and relies mostly on plasma membrane-localized glucose transporter proteins (GLUTs). Two main cellular mechanisms regulate GLUT proteins in the cell: first, expression of GLUT genes is under dynamic transcriptional control and is used by cancer cells to increase glucose availability. Second, GLUT proteins are regulated by membrane traffic from storage vesicles to the plasma membrane (PM). This latter process is triggered by signaling mechanisms and well-studied in the case of insulin-responsive cells, which activate protein kinase AKT to phosphorylate TBC1D4, a RAB-GTPase activating protein involved in membrane traffic regulation. Previously, we identified protein kinase WNK1 as another kinase able to phosphorylate TBC1D4 and regulate the surface expression of the constitutive glucose transporter GLUT1. Here we describe that downregulation of WNK1 through RNA interference in HEK293 cells led to a 2-fold decrease in PM GLUT1 expression, concomitant with a 60% decrease in glucose uptake. By mass spectrometry, we identified serine (S) 704 in TBC1D4 as a WNK1-regulated phosphorylation site, and also S565 in the paralogue TBC1D1. Transfection of the respective phosphomimetic or unphosphorylatable TBC1D mutants into cells revealed that both affected the cell surface abundance of GLUT1. The results reinforce a regulatory role for WNK1 in cell metabolism and have potential impact for the understanding of cancer cell metabolism and therapeutic options in type 2 diabetes.