Publications

Abstract Alzheimer's disease (AD) is a multifactorial age-related disease associated with oxidative stress (OS) and impaired cholinergic transmission. Accordingly, targeting mitochondrial OS and restoring cholinergic transmission can be an effective therapeutic strategy toward AD. Herein, we report for the first time dual-target hydroxybenzoic acid (HBAc) derivatives acting as mitochondriotropic antioxidants and cholinesterase (ChE) inhibitors. The studies were performed with twomitochondriotropic antioxidants AntiOxBEN(1) (catechol derivative), and AntiOxBEN(2) (pyrogallol derivative) and compounds 15-18, which have longer spacers. Compounds AntiOxBEN(1) and 15, with a shorter carbon chain spacer (six- and eight-carbon) were shown to be potent antioxidants and BChE inhibitors (IC50 = 85 +/- 5 and 106 +/- 5 nM, respectively), while compounds 17 and 18 with a 10-carbon chain weremore effective AChE inhibitors (IC50 = 7.7 +/- 0.4 and 7.2 +/- 0.5 mu M, respectively). Interestingly, molecular modeling data pointed toward bifunctional ChEs inhibitors. The most promising ChE inhibitors acted by a non-competitive mechanism. In general, with exception of compounds 15 and 17, no cytotoxic effects were observed in differentiated human neuroblastoma (SH-SY5Y) and human hepatocarcinoma (HepG2) cells, while A beta-induced cytotoxicity was significantly prevented by the new dual-target HBAc derivatives. Overall, due to its BChE selectivity, favorable toxicological profile, neuroprotective activity and drug-like properties, which suggested blood-brain barrier (BBB) permeability, themitochondriotropic antioxidant AntiOxBEN(1) is considered a valid lead candidate for the development of dual acting drugs for AD and other mitochondrial OS-related diseases.

Abstract The presented research explores four years of newcomer engineering students at FEUP, one of the largest faculties of engineering in Portugal. The students are surveyed in a mandatory course common to all engineering programs at the mentioned faculty, totalling an involvement of about four thousand. This research explores the perceptions of 1198 newcomer students regarding learning and satisfaction, workload, integration into academic work environment and institutional support whilst trying to find gender differences regarding the following variables: engineering program, academic year and change of residence. The questionnaire used in the presented research was validated and its internal consistency was excellent. The findings reveal that students' perceptions on learning and satisfaction as well as on institutional support (two out of four factors) are consistently similar between genders throughout the four years of the study. The differences found between male and female students on integration and workload, however consistent, are small if not marginal. The study is significant because it shows the relevance of the optimization efforts for integration (in the academic work environment) introduced in a mandatory course at the start of engineering degrees in order to bridge the gap between male and female students. This research shows that we are walking towards gender equality in engineering, but we feel that there is still some effort to be done, namely through inspiring scholars to analyse and act upon academic contexts and higher education governance without prejudice and with an open mind: a commitment that is as hard as necessary.

Abstract The extracellular pH (pHe) is a key determinant of the cellular (micro)environment and needs to be maintained within strict boundaries to allow normal cell function. Here we used HEK293 cells to study the effects of pHe acidification (24 h), induced by mitochondrial inhibitors (rotenone, antimycin A) and/or extracellular HCl addition. Lowering pHe from 7.2 to 5.8 reduced cell viability by 70% and was paralleled by a decrease in cytosolic pH (pHc), hyperpolarization of the mitochondrial membrane potential (Delta psi), increased levels of hydroethidineoxidizing ROS and stimulation of protein carbonylation. Co-treatment with the antioxidant a-tocopherol, the mitochondrial permeability transition pore (mPTP) desensitizer cyclosporin A and Necrostatin-1, a combined inhibitor of Receptor-interacting serine/threonine-protein kinase 1 (RIPKI) and Indoleamine 2,3-dioxygenase (IDO), prevented acidification-induced cell death. In contrast, the caspase inhibitor zVAD.fmk and the ferroptosis inhibitor Ferrostatin-1 were ineffective. We conclude that extracellular acidification induces necroptotic cell death in HEK293 cells and that the latter involves intracellular acidification, mitochondrial functional impairment, increased ROS levels, mPTP opening and protein carbonylation. These findings suggest that acidosis of the extracellular environment (as observed in mitochondrial disorders, ischemia, acute inflammation and cancer) can induce cell death via a ROS- and mPTP opening-mediated pathogenic mechanism.

Abstract Menadione, also known as vitamin K-3, is a 2-methyl-1,4 naphthoquinone with a potent cytotoxic activity mainly resulting from its quinone redox-cycling with production of reactive oxygen species (ROS). Although increased ROS generation is considered a relevant mechanism in cancer cell death, it may not be sufficiently effective to kill cancer cells due to phenotypic adaptations. Therefore, combining ROS-generating agents with other molecules targeting important cancer cell phenotypes can be an effective therapeutic strategy. As mitochondrial dysfunction has been implicated in many human diseases, including cancer, we describe here the discovery of a mitochondrial-directed agent (MitoK(3)), which was developed by conjugating a TPP cation to the C3 position of the menadione's naphthoquinone ring, increasing its selective accumulation in mitochondria, as well as led to alterations of its redox properties and consequent biological outcome. MitoK(3) disturbed the mitochondrial bioenergetic apparatus, with subsequent loss of mitochondrial ATP production. The combinatory strategy of MitoK3 with anticancer agent doxorubicin (DOX) resulted in a degree of cytotoxicity higher than those of the individual molecules, as the combination triggered tumour apoptotic cell death evident by caspase 3/9 activities, probably through mitochondrial destabilization or by interference with mitochondrial redox processes. The results of this investigation support the importance of drug discovery process in developing molecules that can be use as adjuvant therapy in patients with specific cancer subtypes.

Abstract Pharmacological interventions targeting mitochondria present several barriers for a complete efficacy. Therefore, a new mitochondriotropic antioxidant (AntiOxBEN(3)) based on the dietary antioxidant gallic acid was developed. AntiOxBEN(3) accumulated several thousand-fold inside isolated rat liver mitochondria, without causing disruption of the oxidative phosphorylation apparatus, as seen by the unchanged respiratory control ratio, phosphorylation efficiency, and transmembrane electric potential. AntiOxBEN(3) showed also limited toxicity on human hepatocarcinoma cells. Moreover, AntiOxBEN(3) presented robust iron-chelation and antioxidant properties in both isolated liver mitochondria and cultured rat and human cell lines. Along with its low toxicity profile and high antioxidant activity, AntiOxBEN(3) strongly inhibited the calcium-dependent mitochondrial permeability transition pore (mPTP) opening. From our data, AntiOxBEN(3) can be considered as a lead compound for the development of a new class of mPTP inhibitors and be used as mPTP de-sensitiser for basic research or clinical applications or emerge as a therapeutic application in mitochondria dysfunction-related disorders. [GRAPHICS] .

Abstract Background: Oncology drugs are only effective in a small proportion of cancer patients. Our current ability to identify these responsive patients before treatment is still poor in most cases. Thus, there is a pressing need to discover response markers for marketed and research oncology drugs. Screening these drugs against a large panel of cancer cell lines has led to the discovery of new genomic markers of in vitro drug response. However, while the identification of such markers among thousands of candidate drug-gene associations in the data is error-prone, an appraisal of the effectiveness of such detection task is currently lacking. Methods: Here we present a new non-parametric method to measuring the discriminative power of a drug-gene association. Unlike parametric statistical tests, the adopted non-parametric test has the advantage of not making strong assumptions about the data distorting the identification of genomic markers. Furthermore, we introduce a new benchmark to further validate these markers in vitro using more recent data not used to identify the markers. Results: The application of this new methodology has led to the identification of 128 new genomic markers distributed across 61% of the analysed drugs, including 5 drugs without previously known markers, which were missed by the MANOVA test initially applied to analyse data from the Genomics of Drug Sensitivity in Cancer consortium. Conclusions: Discovering markers using more than one statistical test and testing them on independent data is unusual. We found this helpful to discard statistically significant drug-gene associations that were actually spurious correlations. This approach also revealed new, independently validated, in vitro markers of drug response such as Temsirolimus-CDKN2A (resistance) and Gemcitabine-EWS-FLI1 (sensitivity). © 2018 The Author(s).

Abstract In this work, we report for the first time the accumulation activity by energized rat heart mitochondria and the ionic transfer process at a liquid liquid interface of a novel mitochondria-targeted antioxidant, named as AntiOxCIN(4), which is structurally based on a hydroxycinnamic acid. Lipophilicity studies conducted at the water/1,6-dichlorohexane (DCH) interface allowed the building up of an ionic partition diagram of AntiOxCIN(4) in accordance with the electrochemical data obtained. The partition coefficients of both positively charged (-2.3) and zwitterionic (0.2) forms of the antioxidant were determined. This study contributed to gaining an insight about the ability of the synthesized antioxidants to cross biomembrane barriers by using an interface between two immiscible electrolyte solutions (ITIES) as a model system.

Abstract L-tyrosine controlled release systems, based on imprinted polymers radically polymerized in modified organic solvents (dimethyl sulfoxide and methanol) were developed. A set of copolymers synthesized with different functional monomers and the crosslinker ethylene glycol dimethylacrylate were evaluated regarding both the imprinting and controlled release features. Those prepared in modified-DMSO were mesoporous (surface area ranging 370-690 m(2)/g) but not the ones prepared in modified methanol with the exception of the imprinted polymer prepared with the monomer vinylbenzoic acid (209 m(2)/g). The swelling degree ranged 32 to 64% and appeared to be mostly dependent on the functional group employed in the synthesis of the polymers. When tested in dynamic mode a clear imprinting effect has been observed in some of the imprinted polymers. However, selectivity for L-Tyr against similar compounds could not be found in these conditions. Selectivity could be observed when carrying out the isothermal analysis, with selectivity factors up to 2.7. A few polymers exhibited also other remarkable features such as high L-Tyr load capacity (up to 253 mg L-Tyr/g) and large binding strength (up to 55 L(m)mg((1-m)))/g). Therefore, imprinted polymers capable of a selective sorption of L-Tyr upon equilibration, containing stronger binding sites for L-Tyr, appeared promising vehicles for its controlled release. In fact, the release assays with L-Tyr impregnation by adsorption, showed profiles corresponding to controlled release systems, with high yield (40-50%) and continuous release (up to at least 7 h). In addition, release mechanisms recommended for controlled release systems were deduced from kinetic data fitting to the Korsmeyer and Order 0 models.