Estudo da remodelagem reversa miocárdica através da análise proteómica do miocárdio e do líquido pericárdico

Valve replacement remains as the standard therapeutic option for aortic stenosis patients, aiming at abolishing pressure overload and triggering myocardial reverse remodeling. However, despite the instant hemodynamic benefit, not all patients show complete regression of myocardial hypertrophy, being at higher risk for adverse outcomes, such as heart failure. The current comprehension of the biological mechanisms underlying an incomplete reverse remodeling is far from complete. Furthermore, definitive prognostic tools and ancillary therapies to improve the outcome of the patients undergoing valve replacement are missing. To help abridge these gaps, a combined myocardial (phospho)proteomics and pericardial fluid proteomics approach was followed, taking advantage of human biopsies and pericardial fluid collected during surgery and whose origin anticipated a wealth of molecular information contained therein. From over 1800 and 750 proteins identified, respectively, in the myocardium and in the pericardial fluid of aortic stenosis patients, a total of 90 dysregulated proteins were detected. Gene annotation and pathway enrichment analyses, together with discriminant analysis, are compatible with a scenario of increased pro-hypertrophic gene expression and protein synthesis, defective ubiquitinproteasome system activity, proclivity to cell death (potentially fed by complement activity and other extrinsic factors, such as death receptor activators), acute-phase response, immune system activation and fibrosis. Specific validation of some targets through immunoblot techniques and correlation with clinical data pointed to complement C3 β chain, Muscle Ring Finger protein 1 (MuRF1) and the dual-specificity Tyr-phosphorylation regulated kinase 1A (DYRK1A) as potential markers of an incomplete response. In addition, kinase prediction from phosphoproteome data suggests that the modulation of casein kinase 2, the family of IκB kinases, glycogen synthase kinase 3 and DYRK1A may help improve the outcome of patients undergoing valve replacement. Particularly, functional studies with DYRK1A+/- cardiomyocytes show that this kinase may be an important target to treat cardiac dysfunction, provided that mutant cells presented a different response to stretch and reduced ability to develop force (active tension). This study opens many avenues in post-aortic valve replacement reverse remodeling research. In the future, gain-of-function and/or loss-of-function studies with isolated cardiomyocytes or with animal models of aortic bandingdebanding will help disclose the efficacy of targeting the surrogate therapeutic targets. Besides, clinical studies in larger cohorts will bring definitive proof of complement C3, MuRF1 and DYRK1A prognostic value.A substituição da válvula aórtica continua a ser a opção terapêutica de referência para doentes com estenose aórtica e visa a eliminação da sobrecarga de pressão, desencadeando a remodelagem reversa miocárdica. Contudo, apesar do benefício hemodinâmico imediato, nem todos os pacientes apresentam regressão completa da hipertrofia do miocárdio, ficando com maior risco de eventos adversos, como a insuficiência cardíaca. Atualmente, os mecanismos biológicos subjacentes a uma remodelagem reversa incompleta ainda não são claros. Além disso, não dispomos de ferramentas de prognóstico definitivos nem de terapias auxiliares para melhorar a condição dos pacientes indicados para substituição da válvula. Para ajudar a resolver estas lacunas, uma abordagem combinada de (fosfo)proteómica e proteómica para a caracterização, respetivamente, do miocárdio e do líquido pericárdico foi seguida, tomando partido de biópsias e líquidos pericárdicos recolhidos em ambiente cirúrgico. Das mais de 1800 e 750 proteínas identificadas, respetivamente, no miocárdio e no líquido pericárdico dos pacientes com estenose aórtica, um total de 90 proteínas desreguladas foram detetadas. As análises de anotação de genes, de enriquecimento de vias celulares e discriminativa corroboram um cenário de aumento da expressão de genes pro-hipertróficos e de síntese proteica, um sistema ubiquitina-proteassoma ineficiente, uma tendência para morte celular (potencialmente acelerada pela atividade do complemento e por outros fatores extrínsecos que ativam death receptors), com ativação da resposta de fase aguda e do sistema imune, assim como da fibrose. A validação de alguns alvos específicos através de immunoblot e correlação com dados clínicos apontou para a cadeia β do complemento C3, a Muscle Ring Finger protein 1 (MuRF1) e a dual-specificity Tyr-phosphoylation regulated kinase 1A (DYRK1A) como potenciais marcadores de uma resposta incompleta. Por outro lado, a predição de cinases a partir do fosfoproteoma, sugere que a modulação da caseína cinase 2, a família de cinases do IκB, a glicogénio sintase cinase 3 e da DYRK1A pode ajudar a melhorar a condição dos pacientes indicados para intervenção. Em particular, a avaliação funcional de cardiomiócitos DYRK1A+/- mostraram que esta cinase pode ser um alvo importante para tratar a disfunção cardíaca, uma vez que os miócitos mutantes responderam de forma diferente ao estiramento e mostraram uma menor capacidade para desenvolver força (tensão ativa). Este estudo levanta várias hipóteses na investigação da remodelagem reversa. No futuro, estudos de ganho e/ou perda de função realizados em cardiomiócitos isolados ou em modelos animais de banding-debanding da aorta ajudarão a testar a eficácia de modular os potenciais alvos terapêuticos encontrados. Além disso, estudos clínicos em coortes de maior dimensão trarão conclusões definitivas quanto ao valor de prognóstico do complemento C3, MuRF1 e DYRK1A.Programa Doutoral em Biomedicin

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Influência da periodontite no peptidoma salivar e nas proteases

Mestrado em Bioquímica - Bioquímica ClínicaPeriodontitis is a complex immuno-inflammatory disease that results from a pre-established infection of gingiva, mainly due to Gram negative bacteria, which colonize deeper in gingival sulcus and latter in periodontal pocket. The progressive unresolved inflammation promote connective tissue loss and alveolar bone resorption, leading to several histopathological changes, namely destruction of periodontal ligament, deepening of periodontal pocket, bone loss and even tooth loss. Despite the efforts of the omics, until now there is no available biomarker for periodontitis, forcing diagnosis to continue based on certain clinical parameters such as clinical attachment level, probing depth, bleeding on probing and alveolar bone radiography. Peptidomic approaches seem promising to find surrogate markers for periodontitis. In that sense, saliva has been attracting researchers due to its diagnostic potential, ease and non-invasive nature of collection. The salivary peptidome is highly influenced by proteolytic events. In order to disclose the proteolytic events taking place in saliva, salivary peptidome was characterized and the salivary proteases were predicted applying, for the first time, filter-aided sample preparation (FASP) approach to saliva. Thus, saliva samples were incubated in spin filters for 18 or 115 hours, at 37 ºC, to promote saliva autolysis and the generation of novel peptides. In ex vivo conditions, proline-rich proteins (PRP), P-B peptide, histatin 1 and statherin came out as the most susceptible proteins to proteolysis. Peptide fragments were mainly attributed to cathepsins L1, K and MEP1A. The described endoProteoFASP approach avoids the use of synthetic peptides and exogenous proteases and could be very helpful in future studies targeting the characterization of salivary proteases and peptidome from pathophysiological conditions associated with remarkable proteolytic events. Following an endoProteoFASP approach and making also use of zymographic studies, the salivary peptidome and the proteolytic activity was studied in chronic periodontitis (CP). Overall, CP is associated with increased gelatinolytic and collagenolytic activity, which is mainly attributed to metalloproteases, remarkably MMP9. Proteomic and peptidomic data corroborated the inflammatory status, and demonstrated that intact histatin 1 may play an important role in the defense response against oral pathogens. The application of the endoProteoFASP approach to study the salivary peptidome of CP subjects resulted in the identification of 8 surrogate peptide markers, which may be used in multiplex to identify CP. These peptides belong to acidic PRP and to P-B peptide. Particularly, P-B peptide fragments exhibited domains with potential predicted antimicrobial activity, proposing a novel function to this protein. Therefore, the endoProteoFASP strategy looks promising for large-scale application to the study of the salivary degradome in CP.A periodontite consiste numa doença imuno-inflamatória complexa que resulta de uma infeção pré-estabelecida nos tecidos periodontais de suporte, sobretudo devido a bactérias Gram negativas que colonizam progressivamente os sulcos gengivais, originando a formação de bolsas periodontais. Progressivamente, a inflamação conduz à perda de tecido conjuntivo e do osso alveolar, conduzindo a várias alterações histopatológicas, nomeadamente a destruição do ligamento periodontal, o aprofundamento das bolsas periodontais, a perda de osso alveolar e até mesmo perda de dentição. Apesar do esforço das ómicas, ainda não existem biomarcadores para a periodontite, pelo que o diagnóstico é baseado em certos parâmetros clínicos como o nível de aderência, a profundidade de sondagem, a presença de hemorragia pós-sondagem e a radiografia ao osso alveolar. As abordagens da peptidómica parecem promissoras na pesquisa de marcadores para a periodontite. Nesse sentido, a saliva tem atraído os investigadores, devido ao potencial de diagnóstico, à facilidade e natureza não invasiva da sua recolha. O peptidoma salivar é altamente influenciado por eventos proteolíticos. De modo a compreender a proteólise que ocorre na saliva, o peptidoma salivar foi caracterizado e as proteases foram previstas aplicando, pela primeira vez, a metodologia FASP (filter-aided sample preparation) ao estudo da saliva. Para tal, as amostras de saliva foram incubadas em filtros spin durante 18 ou 115 horas, a 37ºC, para promover a autólise salivar e a produção de novos péptidos. Em condições ex vivo, as proteínas ricas em prolina (PRPs), o péptido P-B, a histatina 1 e a staterina, surgiram como as mais suscetíveis à proteólise. Os fragmentos peptídicos foram atribuídos sobretudo à atividade das catepsinas L1, K e à meprina A. A abordagem endoProteoFASP descrita evita o uso de péptidos sintéticos e de proteases exógenas e pode ser útil, futuramente, na caracterização do peptidoma e das proteases salivares em condições patofisiológicas, associadas a eventos proteolíticos. Seguindo a abordagem endoProteoFASP e recorrendo também a zimografias, o peptidoma salivar e a atividade proteolítica foram estudadas na periodontite crónica (CP). De uma forma geral, a CP está associada a um aumento das atividades gelatino- e colagenolítica, as quais foram atribuídas a metaloproteases, sobretudo à MMP9. Os dados da proteómica e peptidómica corroboram a presença de inflamação e demonstraram que a histatina 1 intacta pode ser importante na defesa contra os patogéneos orais. A aplicação da abordagem endoProteoFASP ao estudo do peptidoma salivar nos indivíduos com CP resultou na identificação de 8 potenciais marcadores peptídicos, que em conjunto podem identificar a CP. Estes péptidos pertencem às PRPs ácidas e ao péptido P-B. Particularmente, os fragmentos deste último apresentam domínios com potencial atividade antimicrobiana, sugerindo uma nova função para esta proteína. Desta forma, a estratégia endoProteoFASP parece promissora na aplicação, em larga escala, ao estudo do degradoma salivar na CP