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  • 84 Reads
Effects of Doxorubicin and Mitoxantrone in the brain of differently aged mice: in vivo chemobrain study
Published: 01 November 2019 by MDPI in 5th International Electronic Conference on Medicinal Chemistry session Posters

Doxorubicin (DOX) and mitoxantrone (MTX) are two chemotherapeutic drugs, inhibitors of topoisomerase II that are used against several types of cancer. Regardless of their efficacy in cancer treatment, these drugs can cause long-term toxicity in several organs. The chemotherapy-induced cognitive impairment or ‘chemobrain’ has been widely demonstrated in neuropsychological testing in cancer patients, even several years after treatment. Nonetheless, the underlying mechanisms of chemobrain are still not fully understood. Our work aimed to evaluate the effects of clinically relevant cumulative doses of DOX or MTX on the brain of male CD-1 mice at 3 different ages [juvenile (4 weeks), adult (3 months) and old (18-20 months)]. Mice received intraperitoneal injections of the drugs or a saline solution (control groups), twice a week, for 3 weeks. Between different aged groups, MTX-groups received a total cumulative dose of MTX 6 mg/kg. Regarding DOX, infant and adults received a cumulative dose of DOX 18 mg/kg, while the old group received a total cumulative dose of DOX 9 mg/kg. During the entire experimental procedure mice well-being was monitored, as well as food and water consumption. Mice were euthanized one week (adults and old mice) or seventeen days (juvenile mice) after the last injection and several determinations were done.

Concerning the brain’s redox status, total glutathione (GSHt), reduced glutathione (GSH) and GSH/oxidized glutathione (GSSG) ratio decreased in DOX adults, but in DOX infant brains despite receiving an equal cumulative dose no changes were seen. Nevertheless, DOX increased brain ATP levels in juvenile mice. MTX did not cause significant changes neither in glutathione nor in ATP brain levels in the groups tested. An extra experimental procedure was done with adult mice, where the left brain hemisphere was fixated in 4% paraformaldehyde, mounted in a vibratome and sectioned in the coronal plane throughout its extent. A systematic random sampling of the coronal sections was done at 480 µm interval. The sections obtained are being used for immunofluorescent detection of phosphorylated Tau, glial fibrillary acidic protein, BAX and p53 proteins in the hippocampus formation.

In conclusion, our work showed that DOX and MTX differentially impact the brain and that DOX evoked damage might be dependent on animals’ age.


ARM and VMC acknowledge Fundação da Ciência e Tecnologia (FCT) for their grants (SFRH/BD/129359/2017 and SFRH/BPD/110001/2015). Work supported by FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE and by national funds by the FCT within the project “PTDC/DTP-FTO/1489/2014 – POCI-01-0145-FEDER-016537”.

  • Open access
  • 82 Reads
Anticancer drugs-induced toxicity in different age male CD-1 mice
Published: 01 November 2019 by MDPI in 5th International Electronic Conference on Medicinal Chemistry session Posters

Anticancer drugs like doxorubicin (DOX) and mitoxantrone (MTX) are used to treat cancer. Nevertheless, they have worrying long-term toxicity. MTX and DOX cause cardiotoxicity and several risk factors could aggravate it. The aim of this work was to study the toxicity of clinically relevant MTX and DOX doses in infant (4 weeks), adult (3 months), and old (18-20 months) male CD-1 mice. All animals received biweekly intraperitoneal administrations, for 3 weeks. MTX-treated groups received a total cumulative dose of 6 mg/kg, DOX-treated infants and adults received a total cumulative dose of 18 mg/kg, while a second group of adults and old mice received a total cumulative DOX dose of 9 mg/kg. The animals’ weight, food and water consumptions and general welfare were recorded throughout the experiment. Mice were sacrificed 7 days (adults and old animals) or 17 days (infants) after the last injection. Blood, brain, heart, kidneys, spleen, and liver were collected. The heart, kidneys, and liver were analyzed through light microscopy and blood plasma was used for biochemical determinations. Regarding the results, significant body weight loss was observed in the adult population treated with MTX and DOX 18 mg/kg, and in DOX-treated infants’ animals, when compared to the respective controls. In addition, food and water consumptions decreased in the infant and adult populations treated with MTX and DOX 18mg/kg. Regarding organ weight/ brain weight ratios at sacrifice, the ratio of all the organs were significantly decreased in the MTX and DOX-treated adults, except in adult DOX 9 mg/kg. Alanine aminotransferase (ALT) plasma levels were significantly increased in the MTX and DOX-treated infant. Moreover, in DOX-treated adult (18 mg/kg), aspartate aminotransferase (AST)/ALT ratio and total creatine-kinase (CK) plasma levels increased, when compared to controls. Regarding histology, all the MTX and DOX-treated populations presented lesions in heart, kidneys, and liver as did the old control animals. Histological staining using Sirius Red identified a significant increase in interstitial collagen deposition in the myocardium, liver, and kidneys in all drug-treated adult mice. Therefore, we can conclude that although cumulative dose is considered a main factor when studying anticancer drugs toxicity, the age of administration is also determinant in the toxicity observed. In our study, adult mice seemed to be more prone to MTX and DOX-induced toxicity; still, further data needs to be gathered to determine the underlying factors for that susceptibility.

ARM and VMC acknowledge Fundação da Ciência e Tecnologia (FCT) for their grants (SFRH/BD/129359/2017 and SFRH/BPD/110001/2015, respectively). This work is funded by national funds through FCT – Fundação para a Ciência e a Tecnologia, I.P., under the Norma Transitória – DL57/2016/CP1334/CT0006. This work was supported by FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE and by national funds by the Fundação para a Ciência e Tecnologia (FCT) within the project “PTDC/DTP-FTO/1489/2014 – POCI01-0145-FEDER-016537”.

  • Open access
  • 69 Reads
The main products of cyclophosphamide bioactivation exert a cardiotoxic effect at clinical important concentrations in AC16 cardiac cells
Published: 01 November 2019 by MDPI in 5th International Electronic Conference on Medicinal Chemistry session ECMC-5

Cyclophosphamide is used against lymphomas, solid tumors, namely breast, ovarian, bone and soft tissue tumors, in bone marrow transplant conditioning regimens and also in the treatment of autoimmune diseases. Despite its broad use, the application of cyclophosphamide is dose limited by its cardiotoxic effects, which have been linked to its intricate bioactivation process. In this study, we evaluated the cytotoxicity of cyclophosphamide (100 to 10000 µM) and two of its main metabolites, 4-hydroxycyclophosphamide (1 to 25 µM) and acrolein (5 to 100 µM) in AC16 cells, a human cardiomyocyte cell line. Two distinctive cellular states of AC16 cells were used: differentiated with DMEM/F12 medium + 2% horse serum and proliferative, using DMEM/F12 medium + 12.5% fetal bovine serum. The tetrazolium-based colorimetric (MTT) and the neutral red (NR) uptake assays were performed at different time-points (24, 48 or 72h) for both cell states and experimental conditions. Furthermore, metabolomic evaluation was conducted in proliferative and differentiated cells after their incubation for 24h with subtoxic concentrations LC05 of cyclophosphamide, 4-hydroxycyclophosphamide and acrolein.

In AC16 proliferative cells, cyclophosphamide caused toxicity already at 2 500 µM in the MTT reduction assay after a 72-h incubation, while in the NR assay significant toxicity was only seen at 5 000 µM. For the same time-point, both tests revealed that the differentiated state showed significant toxicity only at 7 500 µM. For 4-hydroxycylophoshamide, the 1 µM concentration caused a marked cytotoxic effect in the MTT assay at 72h in the proliferative cellular state. Meanwhile, in the differentiated cells, a clear cytotoxic effect was only seen at 5 µM of 4-hydroxycylophoshamide for both tests at 48h. On the other hand, in acrolein-exposed cells, the differentiated cells were more sensitive as a concentration of 15 µM caused a cytotoxic effect in the MTT assay at 24h, in comparison to 25 µM for the proliferative state. Additionally, metabolomic profiling was performed in proliferative and differentiated cells after their incubation for 24h with subtoxic concentrations LC05 of cyclophosphamide (2.5 mM for both cellular states) and 4-hydroxycyclophosphamide (5 and 1 µM for the proliferative and the differentiated cells, respectively). Since acrolein is formed from the spontaneous degradation of the 4-hydroxycyclophosphamide in a straight pathway, the concentrations of acrolein used in this study were the same as the LC05 of the aforementioned metabolite and not the LC05 itself of acrolein. The intracellular metabolome of the cells was examined using gas chromatography/mass spectrometry (GC/MS). A multivariate analysis of the intracellular data showed a distinctive metabolic pattern between the control and the metabolites in proliferative cells, while no changes were seen in cells in differentiated state.

With the results obtained so far, we can conclude that cyclophosphamide per se only exerts cardiotoxic effect at high concentrations, while its metabolites were cytotoxic at clinically relevant concentrations. Moreover, µM concentrations of the metabolites caused changes in metabolic pathways in AC16 cells that are being further analyzed.

AMA and VMC acknowledge FCT for their grants (SFRH/BD/107708/2015 and SFRH/BPD/110001/2015). VMC’s grant is funded by national funds through FCT – Fundação para a Ciência e a Tecnologia, I.P., under the Norma Transitória – DL57/2016/CP1334/CT0006. This work was supported by FEDER funds [Operational Program for Competitiveness Factors – COMPETE and by FCT within the project “PTDC/DTP-FTO/1489/2014 – POCI-01-0145-FEDER-016537”].

  • Open access
  • 43 Reads
Use of Aptamers to deliver therapeutic genetic sequences in muscle

There are several types of muscular dystrophy which have as a common characteristic muscle weakness and wasting.

Antisense oligonucleotide approaches hold promise for the development of rational therapeutic approaches against muscular dystrophy.

There is need to improve the delivery of such oligonucleotides in a tissue such as the muscle which is highly abundant in the human body.

During the presentation, I am describing the identification and use of aptamers to enhance delivery in muscle.

  • Open access
  • 155 Reads
Drug (re-)design guided by biophysical characterization of interactions with biomimetic membranes

Successful drug development requires not only the optimization for specific and potent recognition by its pharmacodynamical targets, but also efficient delivery to these target sites. Drug-biomembrane reciprocal interactions are a key determinant to understand how a compound performs at a barrier with relevant implications in its pharmacokinetic behaviour especially in Absorption, Distribution, Metabolism and Excretion (ADME). Concerning this, a rational drug design, where medicinal chemists can envision how a structure can be optimized aiming an improved pharmaceutical profile, can be the solution to avoid bigger investments in drugs that might not be effective. Lipid biomimetic membrane models with different lipid constitution are increasingly employed as alternative platforms with very well defined and controlled conditions to predict structural, biophysical and chemical aspects involved in the compounds’ penetration and/or interaction with biomembranes. As a proof-of-concept, in this study several biomimetic membrane models (cell membrane and epithelial membrane of blood-brain barrier) were used and different biophysical techniques (derivative spectroscopy; quenching of steady-state and time-resolved fluorescence; dynamic light scattering; differential scanning calorimetry and small and wide angle x-ray diffraction) were applied to characterize the pharmacokinetic profile of a newly synthesized drug in order to support drug screening process decisions.

  • Open access
  • 55 Reads
Drugs and PAINs: A DrugBank analysis of pan-assay interference compounds
Published: 01 November 2019 by MDPI in 5th International Electronic Conference on Medicinal Chemistry session Posters

Background: Many successful drugs have been developed in the pharmaceutical industry starting from hits discovered by High-throughput Screening (HTS) methods, through various types of assays testing thousands of compounds against targets of interest in the therapeutic field. Unfortunately, several chemical motifs have been found to confer non-specificity against different targets, rendering compounds with such substructures to determine false-positive results in screening assays. These structures, widely known as PAINS (Pan-assay interference compounds), are often avoided by medicinal chemists when selecting hits from HTS assays, for toxicity- and potency-related reasons (Baell & Holloway, 2010, J Med Chem). However, several drugs classifiable as PAINS have already been approved, with many more being present in the drug development stages.

Objectives: Our study aims to investigate the frequency of use and utility of PAINs among the currently approved or in-development drugs by analyzing different types of PAINs comprised in the DrugBank chemical database, in order to better understand the impact of a pan-assay interference.

Methods: DrugBank version 5.0, containing 10631 structures, has been analyzed using OSIRIS DataWarrior 4.4.4 software, filtering for PAIN substructures indicated by Baell & Holloway. Selected compounds representing PAINs set were managed and analyzed using DataWarrior, Microsoft Excel and SPSS Statistics, regarding approval status, pharmacology, toxicology, etc.

Results: From 10631 compounds extracted from DrugBank, more than 200 substances were identified as being PAINS. 10 types of problematic substructures have been identified in DrugBank, the most common structures to be found as PAINs being substances containing catechols, quinones, rhodanine-related groups, and phenolic mannich bases.

Conclusion: Although selectivity of a compound labeled as PAIN is indeed deficient, these problematic compounds seem not to be as problematic as originally predicted in terms of toxicity and pharmacological profile, as numerous substances found to be PAINs are successfully used in current therapy.

  • Open access
  • 627 Reads
Design, synthesis and in vitro biological evaluation of acrylamide derivatives against Chikungunya virus

The emergence and re-emergence of new- and old-diseases represent the significant challenge for humankind, where this is not prepared for it.1–3 Among these, zoonotic diseases account for billions of cases of human illness and thousands of deaths around the world, constituting a global public health problem.4,5 Alphavirus genus belongs to the Togaviridae family and includes more than 30 species of enveloped viruses.6–8 Chikungunya virus (CHIKV) is composed of (+)ssRNA, structural, and non-structural proteins.9–11 Frequently, infectious diseases caused by CHIKV are characterized by inflammation and pain of the musculoskeletal tissues, which often these are accompanied by swelling in the joints, resulting in cartilage damage.12,13 Currently, there are no licensed vaccines or chemotherapeutic agents able to prevent or treat CHIKV infections.14–16 Furthermore, the major part of the available antiviral agents and researches is focused on small synthetic molecules against Herpes, HIV, and Influenza viruses.17 However, several acrylamide compounds have been synthesized in different anti-CHIKV studies, where they have demonstrated promising antiviral activity, suggesting which this chemical class could be used as a scaffold for designing of new antiviral agents.18–20 Considering these facts, this research aims to explore the potential in vitro anti-CHIKV activity of several substituted acrylamide derivatives. In sense, a meticulous virtual screening was performed by using molecular dynamics simulations and docking for 132 acrylamide analogs toward the six more essential biological targets from CHIKV, such as nsP2 (PDB: 3TRK), nsP2/helicase (PDB: 6JIM), nsP3 (PDB: 3GPO), immature E protein (PDB: 3N40), E protein (PDB: 3N41), and capsid (PDB: 5H23). Subsequently, the ten most promising acrylamides were selected and synthesized by amide-coupling reactions, employing TBTU and DIPEA as coupling-agent and base, respectively. All derivatives were characterized by ¹H and ¹³C NMR, HLPC, purity (≥ 96%), and melting point. Then, the cytotoxicity MTT assay was performed for ten acrylamides (GP01-10) toward Vero E6 line cells at 20 and 40 µM concentrations. For the antiviral activity assays, CHIKV adsorption was performed for 2h in Vero E6 cells followed by the treatment with promising compounds at 40 µM for 72h and cell viability analysis (MTT assay). As result, it was observed that acrylamides GP03 and 09 exhibited weak viral inhibition values (49 and 32% at 40 µM, respectively). In contrast, the acrylamide GP07 displayed a significant in vitro anti-CHIKV activity, with an inhibition value of 81%. Based on this result, various molecular docking simulations was performed to suggest a potential CHIKV-target for GP07. From these, it was observed that the GP07 has a high affinity towards E protein, where it was able to interact with Cys470, Lys471, Asp472, Tyr478, Ser479, Cys480, Lys481, Leu623, and Val622 amino acid residues by van der Waals interactions. To confirm the antiviral activity, the intracellular labeling of virus was performed by using a monoclonal anti-CHIKV antibody (A54Q)/mouse anti-IgG Alexa Fluor 488, and the percentage of CHIKV-positive cells was detected by flow cytometry. As result, GP07 was able to reduce the percentage of CHIKV-positive cells from 74.8 to 0.91%, 48h post-treatment. In conclusion, all virtual simulations were corroborated by experimental results and the compound GP07 was identified as a promising anti-CHIKV agent for designing new drugs in the future.

  • Open access
  • 26 Reads
Nanotechnology mediated bee venom: Applications in rheumatoid arthritis
Published: 08 November 2019 by MDPI in 5th International Electronic Conference on Medicinal Chemistry session ECMC-5

Bee venom has made medicinal progress, specifically, with the Human Immunodeficiency Virus (HIV) and cancer treatment. However, in spite of this therapy being rooted in the treatment of inflammatory disease, bee venom has made little progress in treating Rheumatoid Arthritis. This substance is both simple and complex in chemical structure, and a few significant obstacles are limiting the effectiveness of the venom in arthritic patients. Primarily, the non-specific cytotoxicity of the venom can negatively affect the surrounding cells of the target, and the known degradation of bee venom, before it reaches the target cells, reduces the potency. One promising way to circumvent these issues would be through nanotechnology. Nanoparticles have a high surface area and, in conjunction with proper functionalization, can be used to derive the Melittin and other beneficial components of bee venom into an effective treatment for Rheumatoid Arthritis. The primary goal of this work is to study contemporary nanoparticles used in drug delivery and do a comparative study on the bee venom and the nanoparticles, helping to develop bee venom into a viable clinical treatment for Rheumatoid Arthritis.

  • Open access
  • 122 Reads
Stories from Staudinger: Synthesis of chiral beta-lactams
Published: 08 November 2019 by MDPI in 5th International Electronic Conference on Medicinal Chemistry session Posters

Combretastatin A-4 (CA-4) is the most potent antimitotic agent of the Combretastatin A series; a group of diaryl stilbenes isolated from the wood of the South African tree Combretum caffrum. [i] It has significant anticancer activity through inhibition of tubulin polymerization and microtubule assembly. [ii] While cis-stilbene structures demonstrate superior biological activity[iii], the corresponding trans derivatives are inherently more stable. Isomerization of cis CA-4 to the trans form is observed both during storage and in vivo during metabolism which dramatically reduces antitumour activity. [iv] Our group has previously employed the Staudinger reaction to synthesize novel 3-hydroxy-1,4-diaryl-2-azetidinones. The problem of CA-4’s Cis- Trans isomerization has been overcome via chemical manipulation of CA-4’s alkene bridge; utilizing a beta-lactam ring to induce cis restriction. A number of analogues have shown potent nanomolar antiproliferative activity in MCF-7 and HL-60 cells with enhanced activity relative to CA-4. [v] Typical Staudinger reactions form mixtures of cis and trans isomers depending on reaction conditions employed, and additionally; at the 3-hydroxy position’s chiral center; racemic mixtures of R&S enantiomers. Trans isomers of 3-substituted-2-azetidinones have been shown to be up to 50 times more potent than the corresponding cis derivatives[vi] emphasizing the requirement to optimize the Staudinger approach to minimize yields of the undesirable cis isomer. Levo- and dextro-rotatory enantiomers hold potential to display lesser or greater biological activity relative to one another.

Our current work aims to:

  1. Improve the available yield for chiral resolution by determining the necessary conditions to achieve stereoselective synthesis of trans 3-hydroxy-1,4-diaryl-2-azetidinones in the Staudinger reaction;
  2. Purification of racemic mixtures using N-(tert-butoxycarbonyl)-L-Proline as a chiral resolving agent to afford optically pure enantiomers for further biological evaluation.

Trans 3-hydroxy b-lactams have been separated from cis derivatives using chromatographic purification. We have since optimized the Staudinger reaction to return relative yields of 95:% relative ratio of trans: cis isomers; as indicated by integration of protons at position 3 and 4 of the beta-lactam on 1 H-NMR. Diastereomeric resolution using flash column chromatography followed by hydrolysis of chiral resolving agents has successfully yielded enantiomers of EMCL001&2. Preliminary biochemical data for the enantiomers in breast cancer cells will be reported.


[i] Watt, J. M.; Gerdina, M., The Medicinal and Poisonous Plants of Southern and Eastern Africa. E. & S. Livingstone Ltd: Edinburgh and London, U.K., 1962.

[ii] Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; Garcia-Kendall, D., Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin Experientia 1988, (45).

[iii] Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; Garcia-Kendall, D., Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin A-4 ~. Experientia 1988, (45).

[iv] Ohsumi, K.; Hatanaka, T.; Fujita, K.; Nakagawa, R.; Fukuda, Y.; Nihei, Y.; Suga, Y.; Morinaga, Y.; Akiyama, Y.; Tsuji, T., Synthesis and antitumor activity of cis-restricted combrestatins: 5-membered heterocyclic analogues. YNTHESES AND ANTITUMOR ACTIVITY OF CISRESTRICTED COMBRETASTATINS: 5-MEMBERED HETEROCYCLIC ANALOGUES Bioorg. Med. Chem. Lett. 1998, 8, 3153-3158

[v]N.M. O’Boyle, M. Carr, L.M. Greene, O. Bergin, S.M. Nathwani, T.McCabe, D.G. Lloyd, D.M. Zisterer, M.J. Meegan, Synthesis and evaluation of azetidinone analogues of combretastatin A-4 as tubulin targeting agents, J. Med. Chem., 53 (2010), p. 8569

[vi] Azizah M. Malebari Lisa M. Greene, S. M. N., Darren Fayne, Niamh M. O'Boyle , Shu Wang , Brendan Twamley , Daniela M. Zisterer , Mary J. Meegan beta-Lactam analogues of combretastatin A-4 prevent metabolic inactivation by glucuronidation in chemoresistant HT-29 colon cancer cells. Eur. J. Med. Chem. 2017, 130, 261-285

  • Open access
  • 122 Reads
Tryptophanol-derived oxazoloisoindolinones: Novel small molecule p53 activators with promising antitumor activity

The tumour suppressor p53 is a pivotal target in cancer therapy as this protein is inactive in all human cancers. In the last years, our research group has been working on the design and synthesis of novel small molecules that are able to reactivate p53.[1] Of these, novel scaffolds containing the oxazoloisoindolinone moiety in their chemical structure emerged with very promising anti-cancer properties.[2] In this communication an overview about the therapeutic potential of a tryptophanol-derived oxazoloisoindolinone chemical library as selective p53 activators will be given. Based on the hit tryptophanol-derived small molecule SLMP53-1, identified as a wild-type and mutant p53 reactivator, a second series of compounds was prepared leading to DIMP53-1 (a p53-MDM2/X interactions dual inhibitor) and to SLMP53-2 (small molecule able to restore the wild-type function of mut p53Y220C). The tryptophanol-derived oxazoloisoindolinone chemical family was prepared by a stereoselective cyclocondensation reaction of enantiopure aminoalcohol tryptophanol with several commercially available oxoacids. From the screening of this library, several very promising molecules emerged with potent anticancer activity against aggressive cancers. The anticancer activity and mechanism of action of the target molecules was studied in human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53+/+) and the corresponding p53-null isogenic derivative cells (HCT116 p53-/-), as well as in several cancer cell lines with different p53 status. The most promising molecules were also evaluated in vivo.

REFERENCES: 1. a) J. D. Amaral et al. Chem. 2019, 7, 15; b) L. Raimundo et al. British J. Pharmacol. 2018, 175, 3947; c) R. C. Nunes et al. Eur. J. Med. Chem. 2017, 139, 168. 2. a) S. Gomes et al., Cancers 2019, 11, 1151; b) J. Soares et al. Mol. Oncol. 2017; 11; 612; c) J. Soares et al. Oncotarget 2016, 7, 4326; d) J. Soares et al. Eur. J. Pharm. Sci. 2015, 66, 138.

ACKNOWLEDGMENTS: We thank PT national funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) the funding through grants PTDC/QUI-QOR/29664/2017, UID/DTP/04138/2019 (iMed.ULisboa), UID/QUI/00100/2019 (CQE) and UID/QUI/50006/2019, the principal investigator grants CEECIND/02001/2017 (A. M. M. Antunes) and CEECIND/01772/2017 (M. M. M. Santos), and the PhD fellowships PD/BD/143126/2019 (V. Barcherini), SFRH/BD/96189/2013 (S. Gomes) and SFRH/BD/117931/2016 (M. Espadinha).