O papel emergente da autofagia/mitofagia na toxicidade induzida por α-sinucleína: estudos no modelo de levedura de envelhecimento cronológico

Tese de doutoramento em Ciências da Saúdeα-Synuclein (α-syn) misfolding and aggregation is strongly associated with both idiopathic and familial forms of Parkinson disease (PD). Evidence suggests that α-syn has an impact on cell clearance routes and protein quality control systems, such as the ubiquitin-proteasome system (UPS) and autophagy. Recent advances in the key role of the autosomal recessive PARK2/PARKIN and PINK1 genes in mitophagy, highlighted this process as a prominent new pathogenic mechanism. Nevertheless, the role of autophagy/mitophagy in the pathogenesis of sporadic and autosomal dominant familial forms of PD is still enigmatic. The yeast Saccharomyces cerevisiae is a powerful “empty room” model that has been exploited to clarify different molecular aspects associated with α-syn toxicity, which combines the advantage of being an established system for aging research. Here, we showed that the heterologous expression of α-syn WT or the A53T mutant form induced toxicity, additionally the toxic phenotype was more aggravated when the α-syn expression was induced at the stationary cells growth phase. At this growth phase, it was demonstrated that the induction of the toxic α-syn variants led to the stimulation of the unfolded protein response (UPR), by the activation of the Ire-Hac1 signaling pathway. The activation of this pathway is probably responsible by the induction of the ATG6 and ATG8 mRNA levels, linked with autophagy and ATG32, ATG33 and DNM1 mRNA levels, which are genes specifically associated with mitophagy, suggesting that autophagy and mitophagy are stimulated under α-syn-induced toxicity. In fact modulation of autophagy, by its inhibition with chloroquine or 3 methyladenine, reverted the α-syn toxic phenotype detected by an extension of in the chronological life span (CLS) of cells expressing the α-syn WT or A53T mutant forms. Additionally, with the specific method to quantify the autophagy or mitophagy activities, the alkaline phosphatase assay, it was confirmed that the toxicity due to the heterologous expression of the toxic α-syn variants was accompanied by exacerbated stimulation of both autophagy and mitophagy. Modulation of mitophagy, by its impairment, achieved by deletion of ATG11 or ATG32 resulted in a CLS extension, accompanied by physiologic autophagic levels, further implicating mitophagy in the α-syn toxicity. It is established that reactive oxygen species, particularly superoxide anion (O22-), are associated with autophagy induction. In the experimented conditions, the stimulation of autophagy/mitophagy under α-syn expression was accompanied with O22- accumulation. Nevertheless, cells with impaired mitophagy display lower levels of O22-. Thus, the findings do not rule out O22- as inducer of autophagy in these conditions. Other molecules that have been associated with the regulation of autophagy in higher eukaryotic cells are the sirtuins, particularly the SIRT1. Deletion of SIR2, the yeast homologue of SIRT1, essential for α-syn-induced toxicity, abolished autophagy and mitophagy, thereby rescuing cells from α-syn toxicity. These data show that Sir2 functions as a regulator of autophagy, like its mammalian homologue, SIRT1, but also of mitophagy, mediating the ATG32 mRNA levels. Caloric restriction (CR) is a physiologic intervention known to promote life span extension, by reducing the activity of various signal transduction pathways either directly or through the decrease in the activity of nutrient-sensing pathways. Wild type cells submitted to CR experienced a CLS extension accompanied by physiological levels of autophagy/mitophagy, independently of the expression of α-syn toxic variants. In fact, in α-syn-induced toxicity conditions, autophagy/mitophagy induction was significant lower in comparison with the same cells submitted to non-CR conditions, supporting the hypothesis that induction of autophagy/mitophagy are directly related with the α-syn-induced toxicity. CR, like mitophagy abrogation, has the capacity to reduce α-syn-induced toxicity. The observed high CLS of atg11 and atg32 cells expressing α-syn WT or A53T mutant form in comparison with the wild type cells submitted to the same conditions, suggested that CR controls autophagy/mitophagy independently, or at least partially independent, of the pathways regulating mitophagy . Similar results were observed in sir2 cells, in which CR is able to further increase the CLS of sir2 cells expressing α-syn toxic variants, suggesting that under CR conditions, the activated signaling pathways are independent of Sir2. Altogether our work highlights that α-syn expression is associated with autophagy, particularly mitophagy stimulation mediated by the regulation of ATG32 by Sir2, an important phenomenon linked to α-syn-induced toxicity during aging. These findings open new insights to the study of the mechanism associated with PD pathogenesis in higher eukaryote organisms.A acumulação intracelular da proteína α-sinucleina (α-sin) com alterações conformacionais leva à formação de agregados que estão associados com as formas idiopática e familiar da doença de Parkinson (DP). Evidências sugerem que a α-sin interfere com as vias de degradação proteica e com o sistema de controlo de qualidade das proteínas, tais como o sistema ubiquitina-proteasoma e a autofagia. Um papel fundamental das proteínas PARK2/PARKIN e PINK1, cujos genes estão envolvidos em formas autossómicas recessivas da DP, na regulação da mitofagia, destacam este processo como um novo proeminente mecanismo patogénico da DP. No entanto, o papel da autofagia/mitofagia na patogénese das formas esporádica e familiar autossómica dominante da DP é ainda enigmático. A levedura Saccharomyces cerevisiae é um poderoso modelo, que tem sido explorado para esclarecer diferentes aspectos moleculares associados com a toxicidade da α-sin, combinando a vantagem de ser um sistema estabelecido para o estudo do envelhecimento. Este trabalho demonstra que a expressão heteróloga da α-sin selvagem ou da forma mutante A53T induz toxicidade, que é mais evidente quando a expressão da α-sin foi induzida em células envelhecidas (pós-mitóticas em fase estacionária de crescimento). A expressão das variantes tóxicas de α-sin levou à ativação de uma resposta específica da célula através da via de sinalização Ire-Hac1. A activação desta via é, provavelmente, responsável pelo aumento dos níveis de mRNA dos genes ATG6 e ATG8, associados com o processo autofágico, e também dos genes ATG32, ATG33 e DNM1, especificamente associados com a mitofagia, sugerindo que a autofagia e a mitofagia estão implicadas na toxicidade induzida pela expressão de α-sin. De fato, a modulação da autofagia, através da sua inibição com cloroquina ou 3 metiladenina reverteu o fenótipo tóxico da α-sin, detetado através do aumento da longevidade cronológica das células. Paralelamente, com o auxílio de um método específico para a quantificação da atividade autofágica ou mitofágica, confirmou-se que a toxicidade devida à expressão heteróloga das variantes tóxicas de α-sin foi acompanhada pela estimulação exacerbada do processo autofágico e mitofágico. A modelação da mitofagia, através da sua inibição, por deleção dos genes ATG11 ou ATG32, resultou numa extensão da longevidade cronológica, acompanhada por níveis autofágicos fisiológicos, implicando a mitofagia na toxicidade induzida pela α-sin. Está estabelecido que as espécies reactivas de oxigénio, em particular o anião superóxido (O22-), regulam a indução de autofagia. Nas condições em estudo, a estimulação da autofagia/mitofagia em células a expressar α-sin foi acompanhada pela acumulação de O22-. No entanto a acumulação de O22- não é observada em células incapazes de realizar mitofagia. Assim, os resultados não descartam a possibilidade de O22- estar a contribuir para a indução da autofagia, em condições de toxicidade induzida pela α-sin. Outras moléculas que têm sido associadas com a regulação da autofagia, em células eucariotas superiores, são as sirtuinas, particularmente SIRT1. A deleção de Sir2 (proteína de levedura homólogo de SIRT1), cujo seu papel foi previamente demonstrado como sendo crucial para a toxicidade da α-sin, resultou na supressão da autofagia e da mitofagia, e consequentemente numa melhor performance das células. Estes dados mostram, pela primeira vez, que Sir2 para além de ser uma molécula reguladora da autofagia, tal como o seu homólogo de mamífero, SIRT1, regula também a mitofagia, controlando os níveis de mRNA do gene ATG32. A restrição calórica (RC) é uma intervenção fisiológica que promove a extensão da longevidade, reduzindo diretamente a atividade de várias vias de transdução de sinal, ou diminuindo a actividade das vias de sinalização de nutrientes. As células da estirpe selvagem submetidas a RC demonstraram uma extensão na longevidade cronológica, acompanhada por níveis fisiológicos de autofagia/mitofagia, independentemente da expressão das variantes tóxicas de α-sin. No entanto, esta indução foi significativamente menor quando comparada com as mesmas células submetidas a condições de não-RC, corroborando a hipótese de que a RC tem a capacidade de reduzir a toxicidade induzida por expressão da α-sin. Adicionalmente, células deletadas no gene ATG11 ou ATG32, a expressar α-sin selvagem ou a forma mutante A53T, quando sujeitas a RC apresentaram uma maior longevidade cronológica em comparação com as células da estirpe selvagem submetidos às mesmas condições, o que sugere que a RC tem a capacidade de controlar a toxicidade, devido a expressão de α-sin, por mecanismos independentes, ou pelo menos parcialmente independentes, dos mecanismos que estão na base da regulação da mitofagia. Os mesmos resultados foram observados para as células deletadas no gene SiR2, sugerindo que a RC regula a autofagia/mitofagia por vias de sinalização independentes de Sir2. Em suma, o nosso trabalho sugere que a expressão de α-sin promove a estimulação da autofagia, particularmente da mitofagia, e que esta indução é regulada pelos níveis de mRNA do gene ATG32, através da acção da proteína Sir2, um evento relevante ligado à indução da toxicidade de α-sin, durante o envelhecimento. Estes resultados abrem novas perspectivas para o estudo de mecanismos associados com a patogénese da DP, em organismos eucariotas superiores.Fundação para a Ciência e Tecnologia (FCT) - project PTDC/BIAMIC/ 114116/2009 and SRFH/BD/41674/200

Linking cellular proteostasis to yeast longevity

Proteostasis is a cellular housekeeping process that refers to the healthy maintenance of the cellular proteome that governs the fate of proteins from synthesis to degradation. Perturbations of proteostasis might result in protein dysfunction with consequent deleterious effects that can culminate in cell death. To deal with the loss of proteostasis, cells are supplied with a highly sophisticated and interconnected network that integrates as major players the molecular chaperones and the protein degradation pathways. It is well recognized that the ability of cells to maintain proteostasis declines during ageing, although the precise mechanisms are still elusive. Indeed, genetic or pharmacological enhancement of the proteostasis network has been shown to extend lifespan in a variety of ageing models. Therefore, an improved understanding of the interventions/mechanisms that contribute to cellular protein quality control will have a huge impact on the ageing field. This mini-review centers on the current knowledge about the major pathways that contribute for the maintenance of Saccharomyces cerevisiae proteostasis, with particular emphasis on the developments that highlight the multidimensional nature of the proteostasis network in the maintenance of proteostasis, as well as the age-dependent changes on this network.This work was developed under the scope of the project NORTE01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). BSM is supported by the fellowship SFRH/BPD/90 533/2012 funded by the Fundac¸ao para a Ci ˜ encia e ˆ Tecnologia (FCT, Portugal)

Targeting metabolic reprogramming in acute myeloid leukemia

The cancer metabolic reprogramming allows the maintenance of tumor proliferation, expansion and survival by altering key bioenergetics, biosynthetic and redox functions to meet the higher demands of tumor cells. In addition, several metabolites are also needed to perform signaling functions that further promote tumor growth and progression. These metabolic alterations have been exploited in different cancers, including acute myeloid leukemia, as novel therapeutic strategies both in preclinical models and clinical trials. Here, we review the complexity of acute myeloid leukemia (AML) metabolism and discuss how therapies targeting different aspects of cellular metabolism have demonstrated efficacy and how they provide a therapeutic window that should be explored to target the metabolic requirements of AML cells.This research was funded by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, by the European Regional Development Fund (FEDER), through the Competitiveness Factors Operational Programme (COMPETE), grant number NORTE-01-0145-FEDER-000013 and by the projects POCI-01-0145-FEDER-028159 and POCI-01-0145-FEDER-030782 by FEDER, through the COMPETE, and by National funds, through the Foundation for Science and Technology (FCT)

Evaluation of the protective effect of Hypericum perforatum phenolics compounds, in the toxicity induced by heterologous expression of α-synuclein

Parkinson’s disease (PD), first described by James Parkinson in 1817, is a chronic, progressive neurodegenerative disorder. The pathologic hallmark is a deterioration of the substantia nigra of yet unknown causes, resulting in a deficiency of dopamine, an important neurotransmitter for the basal ganglia circuit and the presence of cytoplasmic eosinophilic inclusions named Lewy bodies, in which α-synuclein is the major constituent. Recent work implicates abnormal protein accumulation, protein phosphorylation, mitochondrial dysfunction and oxidative stress as common pathways implicated in PD pathogenesis. Polyphenolic compounds are commonly found in both edible and medicinal plants, and they have been reported to have multiple biological effects, including antioxidant activity. The budding yeast Saccharomyces cerevisiae has been used as a model to study several neurodegenerative diseases, including biological function of α-synuclein, as well as its toxicity. The heterologous expression of wild-type and A53T mutant form of α-synuclein causes toxicity in cells. Therefore, the aim of this study was to evaluate the possible protective effect of Hypericum perforatum phenolic compounds (quercetin, kaempferol and biapigenine), in the toxicity induced by the heterologous expression of α-synuclein, using the yeast Saccharomyces cerevisiae as a model. Preliminary results indicate that the presence of these phenolic compounds decrease the protein accumulation in cells expressing α-synuclein. We concluded that these phenolic compounds apparently have beneficial biological properties that consequently could have a potential use in preventing Parkinson’s disease.Fundação para a Ciência e a Tecnologia (FCT

Hypericum perforatum phenolics compounds: protective role in the toxicity induced by heterologous expression of α-synuclein

Parkinson’s disease (PD) is a neurodegenerative disorder with high prevalence and is characterized by the loss of dopaminergic neurons and the presence of cytoplasmic eosinophilic inclusions named Lewy bodies, in which α-synuclein is the major constituent. Several studies implicate abnormal protein accumulation, protein phosphorylation, mitochondrial dysfunction and oxidative stress as common pathways implicated in PD pathogenesis. Polyphenolic compounds are commonly found in both edible and medicinal plants, and they have been reported to have multiple biological effects, including antioxidant activity. The budding yeast Saccharomyces cerevisiae has been used as a model to study several neurodegenerative diseases, including biological function of α-synuclein, as well as its toxicity. The heterologous expression of wild-type and A53T mutant form of α-synuclein causes toxicity in cells. Therefore, the aim of this study was to evaluate the possible protective effect of Hypericum perforatum phenolic compounds (quercetin, kaempferol and biapigenine), in the toxicity induced by the heterologous expression of α-synuclein, using the yeast Saccharomyces cerevisiae as a model. Preliminary results indicate that the presence of these phenolic compounds decrease the toxicity observed in cells expressing α-synuclein without the presence of the compounds. We concluded that these phenolic compounds apparently have beneficial biological properties that consequently could have a potential use in preventing Parkinson’s disease.Fundação para a Ciência e Tecnologia (FCT) - (PTDC/BIA-MIC/114116/2009) e (SFRH/BD/41674/2007

Effects of intermittent fasting on regulation of metabolic homeostasis: a systematic review and meta-analysis in health and metabolic-related disorders

Intermittent fasting (IF) is an emerging dietetic intervention that has been associated with improved metabolic parameters. Nowadays, the most common IF protocols are Alternate-Day Fasting (ADF) and Time-Restricted Fasting (TRF), but in this review and meta-analysis we have also considered Religious Fasting (RF), which is similar to TRF but against the circadian rhythm. The available studies usually include the analysis of a single specific IF protocol on different metabolic outcomes. Herein, we decided to go further and to conduct a systematic review and meta-analysis on the advantages of different IF protocols for metabolic homeostasis in individuals with different metabolic status, such as with obesity, diabetes mellitus type 2 (T2D) and metabolic syndrome (MetS). Systematic searches (PubMed, Scopus, Trip Database, Web of Knowledge and Embase, published before June 2022) of original articles in peer-review scientific journals focusing on IF and body composition outcomes were performed. Sixty-four reports met the eligibility criteria for the qualitative analysis and forty-seven for the quantitative analysis. Herein, we showed that ADF protocols promoted the major beneficial effects in the improvement of dysregulated metabolic conditions in comparison with TRF and RF protocols. Furthermore, obese and MetS individuals are the most benefited with the introduction of these interventions, through the improvement of adiposity, lipid homeostasis and blood pressure. For T2D individuals, IF impact was more limited, but associated with their major metabolic dysfunctions—insulin homeostasis. Importantly, through the integrated analysis of distinct metabolic-related diseases, we showed that IF seems to differently impact metabolic homeostasis depending on an individual’s basal health status and type of metabolic disease.This work has been funded by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020

Transcriptomic and chemogenomic analyses unveil the essential role of Com2-regulon in response and tolerance of Saccharomyces cerevisiae to stress induced by sulfur dioxide

During vinification Saccharomyces cerevisiae cells are frequently exposed to high concentrations of sulfur dioxide (SO2) that is used to avoid overgrowth of unwanted bacteria or fungi present in the must. Up to now the characterization of the molecular mechanisms by which S. cerevisiae responds and tolerates SO2 was focused on the role of the sulfite efflux pump Ssu1 and investigation on the involvement of other players has been scarce, especially at a genome-wide level. In this work, we uncovered the essential role of the poorly characterized transcription factor Com2 in tolerance and response of S. cerevisiae to stress induced by SO2 at the enologically relevant pH of 3.5. Transcriptomic analysis revealed that Com2 controls, directly or indirectly, the expression of more than 80% of the genes activated by SO2, a percentage much higher than the one that could be attributed to any other stress-responsive transcription factor. Large-scale phenotyping of the yeast haploid mutant collection led to the identification of 50 Com2-targets contributing to the protection against SO2 including all the genes that compose the sulfate reduction pathway (MET3, MET14, MET16, MET5, MET10) and the majority of the genes required for biosynthesis of lysine (LYS2, LYS21, LYS20, LYS14, LYS4, LYS5, LYS1 and LYS9) or arginine (ARG5,6, ARG4, ARG2, ARG3, ARG7, ARG8, ORT1 and CPA1). Other uncovered determinants of resistance to SO2 (not under the control of Com2) included genes required for function and assembly of the vacuolar proton pump and enzymes of the antioxidant defense, consistent with the observed cytosolic and mitochondrial accumulation of reactive oxygen species in SO2-stressed yeast cells.This work was funded by INNOVINE&WINE, Norte-01-0145-FEDER-000038, co-financed by the European Region-al Development Fund (ERDF) through Norte 2020 and by ERFD through POCI-COMPETE 2020. Support received by FCT-Portuguese Foundationfor Science and Technology(PTDC/EXPL/AGR-TEC/1823/2013 and PTDC/AGR-TEC/3315/2014) and by INTERACT project –“Integrated Research in Environment, Agro-Chain and Technology”, no. NORTE-01-0145-FEDER-000017, in its line of research enti-tled VitalityWine. Supportreceived by Biosystems and In-tegrative Sciences Institute (BioISI; FCT/UID/Multi/04046/2018) and iBB-Institute for Bioengi-neering and Biosciences (UID/BIO/04565/2019) by FCT and from Programa Operacional Regional de Lisboa 2020 (pro-ject no. 007317 and PTDC/AGR-TEC/3315/2014_LISBOA-01-0145-FEDER-016834) is also acknowledged.The authors thank Professor Isabel Sá-Correia for the help and guidance in conducting the chemogenomic analysi

Signalling mechanisms that regulate metabolic profile and autophagy of acute myeloid leukaemia cells

Acute myeloid leukaemia (AML) comprises a heterogeneous group of hematologic neoplasms characterized by diverse combinations of genetic, phenotypic and clinical features representing a major challenge for the development of targeted therapies. Metabolic reprogramming, mainly driven by deregulation of the nutrient-sensing pathways as AMPK, mTOR and PI3K/AKT, has been associated with cancer cells, including AML cells, survival and proliferation. Nevertheless, the role of these metabolic adaptations on the AML pathogenesis is still controversial. In this work, the metabolic status and the respective metabolic networks operating in different AML cells (NB-4, HL-60 and KG-1) and their impact on autophagy and survival was characterized. Data show that whereas KG-1 cells exhibited preferential mitochondrial oxidative phosphorylation metabolism with constitutive co-activation of AMPK and mTORC1 associated with increased autophagy, NB-4 and HL-60 cells displayed a dependent glycolytic profile mainly associated with AKT/mTORC1 activation and low autophagy flux. Inhibition of AKT is disclosed as a promising therapeutical target in some scenarios while inhibition of AMPK and mTORC1 has no major impact on KG-1 cells' survival. The results highlight an exclusive metabolic profile for each tested AML cells and its impact on determination of the anti-leukaemia efficacy and on personalized combinatory therapy with conventional and targeted agents.This work was developed under the scope of the project NORTE‐01‐0145‐FEDER‐000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), and by FEDER, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI‐01‐0145‐FEDER‐007038. OP and BSM are supported by fellowships from the Fundação para a Ciência e Tecnologia (FCT, Portugal) (SFRH/BD/52292/2013 and SFRH/BPD/90533/2012, respectively)info:eu-repo/semantics/publishedVersio

Caloric restriction rescues yeast cells from alpha-synuclein toxicity through autophagic control of proteostasis

α-Synuclein (SNCA) is a presynaptic protein that is associated with the pathophysiology of synucleinopathies, including Parkinson's disease. SNCA is a naturally aggregation-prone protein, which may be degraded by the ubiquitin-proteasome system (UPS) and by lysosomal degradation pathways. Besides being a target of the proteolytic systems, SNCA can also alter the function of these pathways further, contributing to the progression of neurodegeneration. Deterioration of UPS and autophagy activities with aging further aggravates this toxic cycle. Caloric restriction (CR) is still the most effective non-genetic intervention promoting lifespan extension. It is known that CR-mediated lifespan extension is linked to the regulation of proteolytic systems, but the mechanisms underlying CR rescue of SNCA toxicity remain poorly understood. This study shows that CR balances UPS and autophagy activities during aging. CR enhances UPS activity, reversing the decline of the UPS activity promoted by SNCA, and keeps autophagy at homeostatic levels. Maintenance of autophagy at homeostatic levels appears to be relevant for UPS activity and for the mechanism underlying rescue of cells from SNCA-mediated toxicity by CR.BSM and HP are supported by fellowships from the Fundação para a Ciência e Tecnologia (FCT, Portugal) (SFRH/BPD/90533/2012 and SFRH/BD/133087/2017, respectively).info:eu-repo/semantics/publishedVersio

Current and emerging techniques for diagnosis and MRD detection in AML: a comprehensive narrative review

Acute myeloid leukemia (AML) comprises a group of hematologic neoplasms characterized by abnormal differentiation and proliferation of myeloid progenitor cells. AML is associated with poor outcome due to the lack of efficient therapies and early diagnostic tools. The current gold standard diagnostic tools are based on bone marrow biopsy. These biopsies, apart from being very invasive, painful, and costly, have low sensitivity. Despite the progress uncovering the molecular pathogenesis of AML, the development of novel detection strategies is still poorly explored. This is particularly important for patients that check the criteria for complete remission after treatment, since they can relapse through the persistence of some leukemic stem cells. This condition, recently named as measurable residual disease (MRD), has severe consequences for disease progression. Hence, an early and accurate diagnosis of MRD would allow an appropriate therapy to be tailored, improving a patient’s prognosis. Many novel techniques with high potential in disease prevention and early detection are being explored. Among them, microfluidics has flourished in recent years due to its ability at processing complex samples as well as its demonstrated capacity to isolate rare cells from biological fluids. In parallel, surface-enhanced Raman scattering (SERS) spectroscopy has shown outstanding sensitivity and capability for multiplex quantitative detection of disease biomarkers. Together, these technologies can allow early and cost-effective disease detection as well as contribute to monitoring the efficiency of treatments. In this review, we aim to provide a comprehensive overview of AML disease, the conventional techniques currently used for its diagnosis, classification (recently updated in September 2022), and treatment selection, and we also aim to present how novel technologies can be applied to improve the detection and monitoring of MRD.This work was supported by European Regional Development Fund (ERDF) through COMPETE2020, under the IMPAct-L project (030782); by the Foundation for Science and Technology (FCT) projects UIDB/50026/2020 and UIDP/50026/2020; and by the project NORTE-01-0145-FEDER-000055, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This project also received funding of the project Health From Portugal (C630926586-00465198), supported by Component C5–Capitalisation and Business Innovation, under the Portuguese Resilience and Recovery Plan, through the NextGenerationEU Fund. A.T. acknowledges the FCT studentship SFRH/BD/148091/2019. B.S.-M. acknowledges funding by FCT, grant number DL 57/2016