Regulação da fibrose pulmonar pela expressão de c-Met em células imunes

Dissertação de mestrado em Ciências da SaúdeIdiopathic pulmonary fibrosis (IPF) is a rapid debilitating lung disease, the most common and lethal form of interstitial lung diseases, which causes remain unknown. Currently, there is no medical therapy capable of reverse the disease progression. This disease comprises a chronic inflammation, where neutrophil-derived proteases have been described as key mediators of tissue damage and consequently decline of lung function. Recently, it was demonstrated that hepatocyte growth factor receptor (c-Met) expression is required for neutrophils’ extravasation to inflamed tumors, through inflammatory stimuli. Accordingly, we hypothesized that inflammatory cells recruitment, as neutrophils, is attenuated during pulmonary fibrosis in mice with c-Met-deleted immune cells, with subsequent decline of fibrosis progression and lung function preservation. In this way, the purpose of this thesis was to understand the role of c-Met expression in immune cells in the pathogenesis of pulmonary fibrosis and associated-inflammation. To answer this, bleomycin (BLM)-induced lung fibrosis model was used in c-Met fl/fl/Tie2:Cre-positive transgenic (TG) mice, in which c-Met-floxed gene is excised from hematopoietic and endothelial cells. Survival analysis was performed until day 21 after BLM administration, in which TG mice exhibited increased survival and less weight loss compared to wild-type (WT). Next, lung fibrosis extent was determined 14 days after administration on Masson’s trichrome stained lung sections. TG mice presented significantly reduced fibrotic score, characterized by reduced alveolar destruction and extracellular matrix deposition. Moreover, using hydroxyproline assay, collagen levels were diminished in TG mice lungs, in accordance with the significant decrease of collagen (Col1a1 and Col1a2), as well as a fibroblast marker (S100a4) and a profibrotic factor (Igf-1) transcript’ levels. At day 7 after administration, tissue cytotoxicity, assessed by lactate dehydrogenase assay, was reduced in TG mice. This comes in agreement with reduction of inflammatory cells recruitment, specifically neutrophils and inflammatory macrophages, evaluated by flow cytometry. Moreover, TG mice presented an increase in transcript’ levels of proinflammatory cytokines (Tnf-α, IL-1β, IL-6 and iNos). Taken together, our findings propose that c-Met deletion in immune cells attenuates pulmonary fibrosis progression, probably due to reduction of inflammatory cells, specifically neutrophils and inflammatory macrophages, in injured lung and consequent decrease in the production of profibrotic factors, as Igf-1.A fibrose pulmonar idiopática (IPF) é uma doença pulmonar debilitante e rápida, sendo a forma mais comum e letal de doenças pulmonares intersticiais, cujas causas permanecem desconhecidas. Atualmente, não há terapia médica capaz de reverter a progressão da doença. Esta doença compreende uma inflamação crónica, na qual as proteases derivadas de neutrófilos foram descritas como mediadores chave do dano tecidular e, consequentemente, declínio da função pulmonar. Recentemente, foi demonstrado que a expressão do recetor do fator de crescimento de hepatócitos (c-Met) é necessária para o extravasamento de neutrófilos para tumores inflamados, aquando dos estímulos inflamatórios. Nesse sentido, hipotetizamos que o recrutamento de célulasinflamatórias, como neutrófilos, está atenuado durante a fibrose pulmonar em ratinhos com deleção de c-Met em células hematopoiéticas e endoteliais, com subsequente diminuição do processo fibrótico e função pulmonar preservada. Desta forma, o objetivo desta tese foi compreender o papel da expressão de c-Met em células imunes na patogénese da fibrose pulmonar e inflamação associada. Para responder a isso, o modelo de fibrose pulmonar induzido por bleomicina (BLM) foi usado em ratinhos transgénicos (TG) c-Met fl/fl/Tie2:Cre-positiva, nos quais o gene c-Met é deletado em células hematopoiéticas e endoteliais. A análise de sobrevivência foi realizada até ao dia 21 após a administração de BLM, os ratinhos TG exibiram maior sobrevivência e menor perda de peso em comparação com o tipo selvagem (WT). Em seguida, a extensão da fibrose pulmonar foi determinada 14 dias após a administração, em secções de pulmão coradas com tricrómio de Masson. Os ratinhos TG apresentaram classificação fibrótica pulmonar significativamente reduzida, caracterizados pela redução da destruição alveolar e deposição de matriz extracelular. Além disso, utilizando o ensaio de hidroxiprolina, os níveis de colagénio estavam reduzidos em ratinhos TG, o que vem de acordo com a diminuição significativa dos níveis de transcritos de colágeno (Col1a1 e Col1a2), bem como de um marcador de fibroblastos (S100a4) e um fator pró-fibrótico (Igf-1). No dia 7 após a administração, a citotoxicidade do tecido, avaliada pelo ensaio da lactato desidrogenase, estava reduzida nos ratinhos TG. Isto está de acordo com a redução do recrutamento de células inflamatórias, especificamente neutrófilos e macrófagos inflamatórios, avaliados por citometria de fluxo. Além disso, os ratinhos TG apresentaram um aumento nos níveis de transcritos de citocinas pró-inflamatórias (Tnf-α, IL-1β, IL-6, iNos). Em conjunto, os nossos resultados sugerem que a deleção de c-Met em células imunes atenua a progressão da fibrose pulmonar, provavelmente devido à redução de células inflamatórias, especificamente neutrófilos e macrófagos inflamatórios, no pulmão lesionado e consequente diminuição da produção de fatores pró-fibróticos, como o Igf-1.O financiamento para a realização deste projeto provém dos fundos do Programa Operacional Regional do Norte (NORTE 2020), sobre o Acordo de Parceria PORTUGAL 2020, através do Fundo Europeu de Desenvolvimento Regional (FEDER). O financiamento da FEDER advém também do Programa Operacional Fatores de Competitividade – COMPETE, e dos fundos nacionais através da Fundação para a Ciência e Tecnologia sobre POCI-01-0145-FEDER-007038; e sobre o projeto NORTE-01-0145-FEDER-000013

Project Fact for minors: Fostering alternative care for troubled minors (JUST/2015/RCHI/AG/PROF/9578)

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Project Fact for minors: Fostering alternative care for troubled minors (JUST/2015/RCHI/AG/PROF/9578)

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Monitorização da qualidade da água em aproveitamentos hidroagrícolas

Mestrado em Engenharia do AmbienteO presente trabalho identifica os principais impactes provocados pela agricultura de regadio, sobretudo nos recursos onde esta mais intervém (água e solo). Um uso excessivo da água, fertilizantes e pesticidas, uma inadequada mobilização dos solos, práticas de drenagem e irrigações incorrectas, induzem à perda quantitativa e qualitativa da água, do solo e da biodiversidade. Medidas preventivas devem de ser tomadas, como é o caso da monitorização da qualidade da água em aproveitamentos hidroagrícolas, sendo este um instrumento de grande importância na gestão ambiental. O caso de estudo apresentado neste trabalho é o do Aproveitamento Hidroagrícola do Baixo Mondego, cujo plano de monitorização da qualidade da água é elaborado desde de 2000 até aos dias de hoje. Este plano inclui análises às águas superficiais de rega (Canal Condutor Geral), de drenagem (quatro zonas agrícolas: Vale Central, Vale do Pranto, Vale do Foja e Vale do Arunca) e subsuperficiais do Vale Central. Os vários parâmetros de qualidade da água são analisados fazendo-se referência aos limites aceitáveis por lei. No caso da água superficial de rega é boa para o seu uso, quanto às águas de drenagem muitas vezes ultrapassam os valores estipulados por lei, o que se deve a possíveis contaminações fora da área de estudo. No caso mais problemático dos nitratos são registados valores elevados às entradas das áreas agrícolas, diminuindo ao longo do vale, registando-se valores inferiores à saída destes. À saída do vale do Foja e Pranto foi possível arranjar uma boa correlação (r2 perto de 1) para os valores de condutividade com outros parâmetros. Estes valores apresentam-se elevados, principalmente nos meses de Outubro onde ocorre as primeiras chuvas de Outono (após um período de estiagem), arrastando os sais existentes no solo. A difícil estimativa dos caudais, efectuada no plano de monitorização, é um entrave para determinar a carga de nutrientes que passam em cada secção. São propostos contributos para a melhoria do plano de monitorização, com o intuito de efectuar as observações que permitam depois realizar o balanço de nutrientes, de modo a avaliar os fluxos de nutrientes provenientes das áreas agrícolas. Uma das observações que se torna necessária é a medição do caudal em determinadas secções de linhas de água, assim como apresentar alternativas às despesas relativas às análises da qualidade da água suportadas pela ABOFHBM. Relativamente a esta última alternativa, a ABOFHBM poderá adquirir um medidor de bancada multi-parâmetros e um medidor portátil de pH, condutividade, sólidos suspensos totais e temperatura, com interesse a nível económico.This work identifies the major impacts caused by irrigated agriculture, particularly in the resources where it intervene more (water and soil). An excessive use of water, fertilizers and pesticides, inadequate mobilization of the soil, drainage practices and improper irrigation, induce qualitative and quantitative loss of water, soil and biodiversity. Preventive measures must be taken, such as monitoring of water quality in irrigation projects, which is a very important tool in environmental management. The case study presented in this work is the “Aproveitamento Hidroagrícola do Baixo Mondego”, whose plan for monitoring the quality of water is drawn from 2000 until the present day. This plan includes analysis of surface water irrigation (Channel Driver General), drainage (four agricultural zones: Central Valley, the Valley of Pranto, Valley of Foja and Valley of Arunca). The various parameters of water quality are analyzed by making reference to the limits acceptable by law. In the case of surface water irrigation this is good for that use, for the drainage water often exceed the limits set by law, which is due to possible contamination outside the study area. In the most problematic case of nitrates are recorded high values to the inputs of agricultural land, decreasing over the valley, registering below the output of these. At the outside of valley of Foja and valley of Pranto was possible to get a good correlation (r2 close to 1) for the conductivity values with other parameters. These levels were high, especially during October which is the first rains of autumn (after a period of drought) dragging the salts in the soil. The rough estimate of the flow carried in the monitoring plan, makes it difficult to determine the load of nutrients that are in each section. Contributions are proposed to improve the monitoring plan in order to make the observations possible after take stock of nutrients in order to assess the flow of nutrients from agricultural lands. One point that is required is the measurement of flow in certain sections of water lines, and present alternatives to the cost of the analysis of water quality backed by ABOFHBM. For that alternative, ABOFHBM can purchase a meter multi-parameter bench and a portable pH meter, conductivity, total suspended solids and temperature, with an economic interest

Interstitial macrophages lead early stages of bleomycin-induced lung fibrosis and induce fibroblasts activation

A progressive fibrosing phenotype is critical in several lung diseases. It is irreversible and associated with early patient mortality. Growing evidence has revealed pulmonary macrophages’ role as modulators of the fibrotic processes. The proportion, phenotype, and function of alveolar (AM) and interstitial macrophages (IM) at the early stages of bleomycin-induced pulmonary fibrosis have not been clearly described. In this way, our study aimed to characterize these macrophage populations and investigate the effect on fibroblast activation. C57BL/6 mice were intratracheally injected with bleomycin and were sacrificed at day 3, 5, and 7 for the performance of flow cytometry and fluorescent-activated cell sorting analysis for protein and gene expression quantification. After bleomycin administration, the proportion of IM was significantly higher than that of AM, which showed a decay during the inflammatory phase, and peaked at day 7. At day 7 of the inflammatory phase, AM started shifting their phenotype from M1-like towards M2, while IM showed a M2-like phenotype. Conditioned medium derived from IM sorted at day 7 induced fibroblast activation and differentiation in myofibroblasts in vitro. Our findings indicate that IM are the largest macrophage population at the early stages of experimental pulmonary fibrosis and are secreted mediators able to activate fibroblasts, pointing to macrophage modulation as a potential therapeutic strategy to restrain progressive fibrosing lung disorders.This research was funded by the Programa Operacional Potencial Humano (POPH) and Programa Investigador Fundação para a Ciência e Tecnologia (FCT) (IF/00959/2014); by Programa Operacional Fatores de Competitividade (COMPETE) and national funds of FCT (POCI-01-0145-FEDER-028113); and by national funds of FCT (UIDB/50026/2020, UIDP/50026/2020)

Portuguese propolis antitumoral activity in melanoma involves ROS production and induction of apoptosis

Melanoma is the most aggressive and life-threatening skin cancer type. The melanoma genome is the most frequently mutated, with the BRAF mutation present in 40–60% of melanoma cases. BRAF-mutated melanomas are characterized by a higher aggressiveness and progression. Adjuvant targeted treatments, such as BRAF and MEK inhibitors, are added to surgical excision in BRAF-mutated metastatic melanomas to maximize treatment effectiveness. However, resistance remains the major therapeutic problem. Interest in natural products, like propolis, for therapeutic applications, has increased in the last years. Propolis healing proprieties offer great potential for the development of novel cancer drugs. As the activity of Portuguese propolis has never been studied in melanoma, we evaluated the antitumoral activity of propolis from Gerês (G18.EE) and its fractions (n-hexane, ethyl acetate (EtOAc), and n-butanol) in A375 and WM9 melanoma cell lines. Results from DPPH•/ABTS• radical scavenging assays indicated that the samples had relevant antioxidant activity, however, this was not confirmed in the cell models. G18.EE and its fractions decreased cell viability (SRB assay) and promoted ROS production (DHE/Mitotracker probes by flow cytometry), leading to activation of apoptotic signaling (expression of apoptosis markers). Our results suggest that the n-BuOH fraction has the potential to be explored in the pharmacological therapy of melanoma.Foundation for Science and Technology (FCT)—projects UIDB/04050/2020, UDBI/04033/2020, UIDB/50026/2020, UIDP/50026/2020, UIDB/50006/2020, UIDP/50006/2020, and by the project NORTE-01-0145-FEDER-000055, supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). S.P.C., C.B.M. and A.S.F. are recipients of FCT grants (2020.05779.BD, SFRH/BD/145955/2019, and PD/BD/128276/2017, respectively

3-bromopyruvate boosts the effect of chemotherapy in acute myeloid leukemia by reducing cell antioxidant defense

Single Cause Single Cure Foundation. Fundação para a Ciência e Tecnologia (FCT) through the PhD grant (SFR/BD/146065/2019)NewG Lab Pharm

A plumieridine-rich fraction from Allamanda polyantha inhibits chitinolytic activity and exhibits antifungal properties against Cryptococcus neoformans

Cryptococcosis is a fungal infection caused mainly by the pathogenic yeasts Cryptococcus neoformans and Cryptococcus gattii. The infection initiates with the inhalation of propagules that are then deposited in the lungs. If not properly treated, cryptococci cells can disseminate and reach the central nervous system. The current recommended treatment for cryptococcosis employs a three-stage regimen, with the administration of amphotericin B, flucytosine and fluconazole. Although effective, these drugs are often unavailable worldwide, can lead to resistance development, and may display toxic effects on the patients. Thus, new drugs for cryptococcosis treatment are needed. Recently, an iridoid named plumieridine was found in Allamanda polyantha seed extract; it exhibited antifungal activity against C. neoformans with a MIC of 250 µg/mL. To address the mode of action of plumieridine, several in silico and in vitro experiments were performed. Through a ligand-based a virtual screening approach, chitinases were identified as potential targets. Confirmatory in vitro assays showed that C. neoformans cell-free supernatant incubated with plumieridine displayed reduced chitinase activity, while chitinolytic activity was not inhibited in the insoluble cell fraction. Additionally, confocal microscopy revealed changes in the distribution of chitooligomers in the cryptococcal cell wall, from a polarized to a diffuse cell pattern state. Remarkably, further assays have shown that plumieridine can also inhibit the chitinolytic activity from the supernatant and cell-free extracts of bacteria, insect and mouse-derived macrophage cells (J774.A1). Together, our results suggest that plumieridine can be a broad-spectrum chitinase inhibitor

Arbustus unedo essence: morphological and genetic characterization of the strawberry tree of Castelo de Paiva

O medronheiro é um arbusto da região mediterrânica que pode ser encontrada por todo o país. Ao contrário do que verifica na região sul do país, no concelho de Castelo de Paiva é atribuída uma reduzida importância económica a esta espécie. Com o intuito de preservar e potenciar a produção desta espécie e contribuir para a dinamização da economia do concelho, procedeu-se à caracterização morfológica e genética de uma amostra da população de medronheiros de Castelo de Paiva. A caracterização morfológica e genética foi realizada para um total de 10 genótipos. Para tal recolheram-se 70 folhas aleatoriamente em cada árvore. Em 40 folhas mediu-se o comprimento, largura, comprimento do pedúnculo, peso fresco, peso seco e determinou-se a área foliar. Dos caracteres morfológicos analisados, aqueles que se revelaram mais úteis na distinção dos vários genótipos foram: comprimento do pedúnculo, peso fresco e peso seco. As restantes 30 folhas foram utilizadas para a caracterização genética. Esta caracterização foi realizada recorrendo a um marcador de DNA, ISSR. Os 5 primeiros exemplaresutilizados na técnica de ISSR demonstraram-se polimórficos. Os resultados da caracterização genética sugerem que a variabilidade genética na população é média a alta.The strawberry tree is a shrub native in the Mediterranean region and it can be found throughout Portugal. Unlike the case in the southern region of the country, in Castelo de Paiva a minor economic importance is given to this species. In order to preserve, to enhance the production of this species and to contribute to the boosting of the economy of the region, we proceeded to the characterization of a small sample population of this fruit tree of Castelo de Paiva in what concerns to its morphology and genetics. The morphological and genetic characterization was performed for a total of 10 genotypes. For this, 70 leaves were randomly collected from each tree. For 40 leaves, it was measured the length, the width, the peduncle length, the wet weight, the dry weight and determined the leaf area. Of the morphological characteristics analyzed, the ones that proved most useful in distinguishing the various genotypes were: the length peduncle, the wet weight and the dry weight. The remaining 30 leaves were used in the genetic characterization. This characterization was performed using a DNA marker, the ISSR. The 5 primers used in the ISSR technique proved to be polymorphic. The results from the genetic characterization suggest that variability in population genetics is medium to high

Phagosomal removal of fungal melanin reprograms macrophage metabolism to promote antifungal immunity

Acknowledgements This work was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01- 0145-FEDER-000013), the Fundação para a Ciência e Tecnologia (FCT) (SFRH/BD/136814/2018 to S.M.G., SFRH/BD/141127/2018 to C.D.O., PD/BD/137680/2018 to D.A., IF/00474/2014 to N.S.O., IF/01390/2014 to E.T., IF/00959/2014 to S.C., IF/00021/2014 to R.S., PTDC/SAU-SER/29635/2017 and CEECIND/04601/2017 to C.C., and CEECIND/03628/2017 to A.C.), the Institut Mérieux (Mérieux Research Grant 2017 to C.C.), and the European Society of Clinical Microbiology and Infectious Diseases (ESCMID Research Grant 2017 to A.C.). M.G.N. was supported by a Spinoza grant of the Netherlands Organization for Scientific Research. A.A.B. was supported by the Deutsche Forschungsgemeinschaft Collaborative Research Center/Transregio TR124 FungiNet (project A1). G.D.B. was funded by the Wellcome Trust (102705), the MRC Centre for Medical Mycology and the University of Aberdeen (MR/N006364/1).Peer reviewedPublisher PD