Biomarcadores genéticos e circulantes associados às catecolaminas (COMT,β2AR e TMRS) na psoríase

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2012A Psoríase é uma doença inflamatória crónica mediada por processos imunitários, caracterizada por erupções cutâneas e que afecta cerca de 2-3% da população mundial. O tipo clínico mais comum é a psoríase vulgar, que tipicamente tem como fenótipo o aparecimento de placas por todo o corpo. Apesar de a sua etiologia ser desconhecida, sabe-se que esta é uma doença onde existe uma hiperproliferação dos queratinócitos, associada a uma desregulação imune. No geral os estudos genéticos de associação são importantes na medida em que permitem associar os polimorfismos ao risco de aparecimento de doenças, bem como à sua evolução. Os estudos bioquímicos dos componentes do sangue também são importantes, de modo a verificar se existem enzimas circulantes de células ou do plasma cujas alterações ao nível da sua actividade (aumento ou diminuição) sejam o reflexo (proxy) de distúrbios locais em proteínas envolvidas no metabolismo e mecanismos de acção que afectem o desenvolvimento da doença. Dadas as catecolaminas (CAs) estarem muito associadas com vias de sinalização do sistema imune, e particularmente com mecanismos de inflamação, característica da psoríase, o presente trabalho focou-se no estudo de duas variantes polimórficas de substituição (SNPs): 1) no codão 16 de gene do receptor β2-adrenérgico (ADRβ2), que está envolvido na resposta às catecolaminas; 2) no codão 158 da Catechol-O-Metiltransferase (COMT), enzima que metaboliza as catecolaminas. Foi ainda estudada a actividade da Redutase Transmembranar do Ferricianeto dependente de NADH intracelular do eritrócito, que reduz o ferricianeto a ferrocianeto, e cuja acção é modulada por receptores β2-adrenérgicos. Os resultados mostram que poderão existir diferenças significativas na actividade da Redutase Transmembranar (RTM) nos indivíduos Psoriásicos, apresentando estes, valores diminuídos. Parece ainda existir uma correlação entre os valores elevados do Índice de Área e Gravidade da Psoríase (PASI) e valores mais baixos da actividade da RTM. Foi encontrada uma tendência na associação da variante Val158 do gene da COMT e a doença. Sendo que no caso do polimorfismo Arg16Gly, parece existir uma tendência para o alelo Gly16 constituir um factor de risco para a psoríase. No entanto os resultados encontram-se condicionados pelo tamanho e características da amostragem.Psoriasis is a chronic, immune-mediated skin disease, which affects approximately 2-3% of the worldwide population. The most common clinical type is psoriasis vulgaris, which presents the appearance of plaques all over the body as the clinical phenotype. Despite its unknown etiology, it is well established that the physiopathology of this disease is associated with a hyper proliferation of keratinocytes and an immunologic deregulation. Genetic studies are important, because they allow us to associate polymorphisms with the risk for a disease, as well as the response to therapeutics. Biochemical studies are also important in order to verify the existence of modifications in enzymatic activities (up or down regulated) that are potentially involved in the progression of the disease. In psoriasis, catecholamines may have an important role, since thery are associated with imune pathways, particularly with inflamation mechanisms, a characteristic of this disease.In this context, association studies between the disease and two single nucleotide polymorphisms were performed. The polymorphisms were in: 1) codon 16 of the β2-adrenergic receptor a protein involved in response to catecholamines; 2) codon 158 of the Catechol-O-Methiltransferase (COMT), an enzyme that metabolizes catecholamines. It was also studied the effect of the variation of the activity of NADH-dehydrogenase of the erythrocytes in the disease. This enzyme reduces the ferricyanide to ferrocyanide, and its activity is modulated by β2-adrenergic receptors. Results show significant differences in the activity of the NADH-dehydrogenase in the erythrocytes of psoriatic patients, which present lower values. Also it appears to exist a correlation between high values of the Psoriasis Area and Severity Index (PASI) and lower values of the activity of the NADH-dehydrogenase in the erythrocyte of psoriatic patients. It was found a possible relation between the genotype COMTHH as well as the allele Gly16 of the ADRβ2 with the risk to develop psoriasis. Results suggest an association between the activity of the NADH-dehydrogenase as well as the Val158 of the COMT gene and the Gly16 of the ADRβ2 with the disease, although these results are conditioned by the size of the sampling

Inovação e equidade no acesso ao medicamento

Independentemente da sua condição socioeconómica, todos os países possuem políticas de saúde e medicamento que têm como objetivo promover a equidade na acessibilidade aos cuidados de saúde e medicamentos, e também nos resultados de saúde por parte dos seus cidadãos. Porém, face a um cenário de crise financeira, aliado a um aumento da despesa em medicamentos e ao aparecimento de medicamentos inovadores muito caros, faz com que os orçamentos de saúde estabelecidos para os diferentes países, principalmente os mais afetados pela crise, não consigam suportar de forma sustentável os custos resultantes da introdução das novas terapias nos seus serviços de saúde. Assim sendo, a inovação terapêutica pode ser causadora de iniquidade irá sempre favorecer aqueles que possuam mais recursos financeiros, a menos que seja feita uma nova abordagem ética em que a prevenção é a chave fundamental para a promoção da saúde e obtenção de equidade entre os grupos socioeconómicos e também entre os países.Regardless of their socioeconomic status, all countries have Health and Drug Policies aiming to promote equity in accessibility to health care and medicines and also in health outcomes for its citizens. However, a financial crisis scenario coupled with an increasing pharmaceutical spending and the emergence of very expensive innovative medicines, causes the stablished health budget not being able to bear the costs from the introduction of these new therapies on their health services, particularly for the countries afected the most by the financial crisis. Therefore, therapeutic innovation may cause inequity in favor of those who have ore financial resources unless a new ethical approach in which prevention is the key to promote healtch and also achieve equity between socioeconomic groups and countries

Small molecule fisetin modulates alpha-Synuclein aggregation

Funding Information: iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), which is co-funded by Funda??o para a Ci?ncia e Tecnologia (FCT)/Minist?rio da Ci?ncia e do Ensino Superior, through national funds, and by FEDER under the PT2020 Partnership Agreement, is acknowledged. Authors would like to acknowledge FCT for financial support of RR (SFRH/BD/116597/2016). JP, RR, GG, and CNS acknowledges funding via BacHBerry (Project No. FP7-613793; www.bachberry.eu). RM is funded by FCT Scientific Employment Stimulus Contract CEEC/04567/CBIOS/2020. TFO was supported by the DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB) and is currently supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany?s Excellence Strategy?EXC 2067/1-390729940. It is also acknowledged the European Research Council (ERC) under the European Union?s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 804229. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Phenolic compounds are thought to be important to prevent neurodegenerative diseases (ND). Parkinson’s Disease (PD) is a neurodegenerative disorder known for its typical motor features, the deposition of α-synuclein (αsyn)-positive inclusions in the brain, and for concomitant cellular pathologies that include oxidative stress and neuroinflammation. Neuroprotective activity of fisetin, a dietary flavonoid, was evaluated against main hallmarks of PD in relevant cellular models. At physiologically relevant concentrations, fisetin protected SH-SY5Y cells against oxidative stress overtaken by tert-butyl hydroperoxide (t-BHP) and against methyl-4-phenylpyridinuim (MPP+)-induced toxicity in dopaminergic neurons, the differentiated Lund human Mesencephalic (LUHMES) cells. In this cellular model, fisetin promotes the increase of the levels of dopamine transporter. Remarkably, fisetin reduced the percentage of cells containing αsyn inclusions as well as their size and subcellular localization in a yeast model of αsyn aggregation. Overall, our data show that fisetin exerts modulatory activities toward common cellular pathologies present in PD; remarkably, it modulates αsyn aggregation, supporting the idea that diets rich in this compound may prove beneficial.publishersversionpublishe

Neuroprotection in a 3D Cell Model of Parkinson's Disease

Funding Information: This work was supported by European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program under grant agreement No. 804229; iNOVA4Health – UIDB/04462/2020 and UIDP/04462/2020, a program financially supported by Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES), through national funds. FCT/MCTES, through the project PTDC/BTM‐ORG/29580/2017. Authors would like to acknowledge FCT for financial support of R.C. (PD/BD/135492/2018) and J.G.‐P. (SFRH/BD/145522/2019). M.L. was supported by the Land‐BW (NAM‐ACCEPT) and funding by the European Union's Horizon 2020 research and innovation program under grant agreements No. 964537 (RISK‐HUNT3R), No. 964518 (ToxFree), and No. 825759 (ENDpoiNTs). Publisher Copyright: © 2022 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbHScope: Diets rich in (poly)phenols have been associated with positive effects on neurodegenerative disorders, such as Parkinson's disease (PD). Several low-molecular weight (poly)phenol metabolites (LMWPM) are found in the plasma after consumption of (poly)phenol-rich food. It is expected that LMWPM, upon reaching the brain, may have beneficial effects against both oxidative stress and neuroinflammation, and possibly attenuate cell death mechanisms relate to the loss of dopaminergic neurons in PD. Methods and Results: This study investigates the neuroprotective potential of two blood-brain barrier permeant LMWPM, catechol-O-sulfate (cat-sulf), and pyrogallol-O-sulfate (pyr-sulf), in a human 3D cell model of PD. Neurospheroids were generated from LUHMES neuronal precursor cells and challenged by 1-methyl-4-phenylpyridinium (MPP+) to induce neuronal stress. LMWPM pretreatments were differently neuroprotective towards MPP+ insult, presenting distinct effects on the neuronal transcriptome. Particularly, cat-sulf pretreatment appeared to boost counter-regulatory defense mechanisms (preconditioning). When MPP+ is applied, both LMWPM positively modulated glutathione metabolism and heat-shock response, as also favorably shifting the balance of pro/anti-apoptotic proteins. Conclusions: Our findings point to the potential of LMWPM to trigger molecular mechanisms that help dopaminergic neurons to cope with a subsequent toxic insult. They are promising molecules to be further explored in the context of preventing and attenuating parkinsonian neurodegeneration.publishersversionepub_ahead_of_prin

Obesity, acute kidney injury and mortality in patients with sepsis: a cohort analysis

Although the prognostic effect of obesity has been studied in critically ill patients its impact on outcomes of septic patients and its role as a risk factor for acute kidney injury (AKI) is not consensual. We aimed to analyze the impact of obesity on the occurrence of AKI and on in-hospital mortality in a cohort of critically ill septic patients. This study is retrospective including 456 adult patients with sepsis admitted to the Division of Intensive Medicine of the Centro Hospitalar Lisboa Norte (Lisbon, Portugal) between January 2008 and December 2014. Obesity was defined as a body mass index of 30 kg/m2 or higher. The Kidney Disease Improving Global Outcomes classification was used to diagnose and classify patients developing AKI. AKI occurred in 87.5% of patients (19.5% with stage 1, 22.6% with stage 2 and 45.4% with stage 3). Obese patients developed AKI more frequently than non-obese patients (92.8% versus 85.5%, p = .035; unadjusted OR 2.2 (95% CI: 1.04-4.6), p = .039; adjusted OR 2.31 (95% CI: 1.07-5.02), p = .034). The percentage of obese patients, however, did not differ between AKI stages (stage 1, 25.1%; stage 2, 28.6%; stage 3, 15.4%; p = .145). There was no association between obesity and mortality (p = .739). Of note, when comparing AKI patients with or without obesity in terms of in-hospital mortality there were also no significant differences between those groups (38.4% versus 38.4%, p = .998). Obesity was associated with the occurrence of AKI in critically ill patients with sepsis; however, it was not associated with in-hospital mortality.info:eu-repo/semantics/publishedVersio

Rituximab in Steroid-Dependent Podocytopathies

Introduction: Rituximab (RTX) has been reported as an effective treatment alternative in primary forms of minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) associated with steroid dependence and frequent relapses. However, the optimal RTX regimen and the outcomes of further doses of RTX remain unclear. This study aimed to evaluate the use of induction and maintenance RTX therapy for adults with primary podocytopathies. Methods: We performed a retrospective case series on adult patients with steroid-dependent podocytopathies who received an induction RTX therapy. Maintenance therapy was performed at physician’s discretion. Remission and relapse rates, concomitant corticosteroids and immunosuppressants use, B-cell depletion and adverse events were analyzed. Results: Fourteen patients (mean age at start of RTX 29.1 ± 21.9 years) with MCD (n = 7) or FSGS (n = 7) were treated with 2 doses of 1,000 mg 2 weeks apart (n = 13) or four doses of 375 mg/m2 (n = 1) of RTX. At last follow-up (mean 47.3 ± 101.7 months), 10 patients were in complete remission and two remained in partial remission. A reduction in the number of relapses, number of patients under corticosteroids and immunosuppressants, and dose of prednisolone was observed when compared to baseline (14 [100%] vs. 5 [35.7%]; 8/14 [57.1%] vs. 4/12 [33.3%]; 13/14 [92.9%] vs. 7/12 [58.3%]; 20 mg/day vs. 5.25 mg/day, respectively). Maintenance RTX therapy was used in 6 patients, with sustained complete remission. Infusion reactions were observed in 4 patients (one required treatment withdrawal). Conclusions: Our findings support the use of RTX for a steroid-free remission in podocytopathies and suggest that maintenance RTX is well-tolerated and associated with prolonged remission. Further studies are needed to confirm its efficacy and safety and establish the optimal induction and maintenance RTX regimen in steroid-dependent podocytopathies

Investigation of potential respiratory adverse effects of micro/nanofibrillated cellulose and cellulose nanocrystals using human lung cell lines.

Abstract publicado em: Environ Mol Mutagen. 2022 Aug;63(Suppl 1):72-73. (Abstracts from the 13th International Conference on Environmental Mutagens and 53rd Annual Meeting of the Environmental Mutagenesis and Genomics Society). https://onlinelibrary.wiley.com/toc/10982280/2022/63/S1Micro/nanofibrillated (CMF/CNF) and nanocrystalline (CNC) celluloses are innovative materials with enormous potential for industrial and biomedical applications. Their expanding production/application urges the investigation of their safety for human health. This study aimed at investigating the potential respiratory outcomes of two CMF/CNF and one CNC produced from bleached Eucalyptus globulus kraft pulp using human alveolar epithelial (A549) cells grown in monoculture or co-cultured with THP-1 monocyte-derived macrophages, by assessing their cellular uptake, cytotoxic, immunotoxic, genotoxic, and epigenetic effects. The nanocelluloses were characterized for their physicochemical properties: CMF displays a low percentage of nanofibrils while CNF comprises 100% fibrils with a diameter (D) circa 11 nm; CNC consists of nanorods with D of 4-5 nm and aspect ratio around 42. TEM analysis evidenced that CMF and CNF were internalised into A549 cells whereas CNC were not. Neither cytotoxicity (colorimetric and clonogenic assays) nor ROS induction was observed for any of the nanocelluloses. CMF caused chromosomal alterations (in vitro micronucleus assay) in A549 cells while negative results were obtained in co-culture and for the other micro/nanocelluloses in mono- or co-culture. Results in progress of DNA damage and gene mutation analyses will complement mutagenesis assessment. Additionally, potential inflammatory and epigenetic effects are being evaluated. These results contribute to the weight of evidence of nanocelluloses biological effects and knowledge of the underlying molecular mechanisms. Such information will drive the synthesis of the safest nanocelluloses,thus minimising potential negative impacts of their use on human and environmental health.FCT/MCTES through nacional funds (PTDC/SAU-PUB/32587/2017; UIDP/00009/2020; UIDB/00009/2020; 020.07168.BD)info:eu-repo/semantics/publishedVersio

Centrosome amplification arises before neoplasia and increases upon p53 loss in tumorigenesis

The uploaded article version is the Epub Ahead of Print version of the article, posted online 8 May 2018. It has been submitted to peer-review.The deposited article version contains attached the supplementary materials within the pdf.Centrosome abnormalities are a typical hallmark of human cancers. However, the origin and dynamics of such abnormalities in human cancer are not known. In this study, we examined centrosomes in Barrett's esophagus tumorigenesis, a well-characterized multistep pathway of progression, from the premalignant condition to the metastatic disease. This human cancer model allows the study of sequential steps of progression within the same patient and has representative cell lines from all stages of disease. Remarkably, centrosome amplification was detected as early as the premalignant condition and was significantly expanded in dysplasia. It was then present throughout malignant transformation both in adenocarcinoma and metastasis. The early expansion of centrosome amplification correlated with and was dependent on loss of function of the tumor suppressor p53 both through loss of wild-type expression and hotspot mutations. Our work shows that centrosome amplification in human tumorigenesis can occur before transformation, being repressed by p53. These findings suggest centrosome amplification in humans can contribute to tumor initiation and progression.Fundação para a Ciência e a Tecnologia–Harvard Medical School Program Portugal grant: (HMSP-CT/SAU-ICT/0075/2009); Liga Portuguesa Contra o Cancro; European Molecular Biology Organization Installation; Sociedade Portuguesa de Gastroenterologia.N/