Complexos de ruténio como potenciais agentes terapêuticos: estudos dos mecanismos de captação celular e modulação de enzimas metabólicas

Tese de mestrado em Química Inorgânica (Aplicações em Diagnóstico e Terapia), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2012Os complexos de metais de transição têm demonstrado serem bons candidatos como agentes antitumorais. Para que o seu efeito terapêutico seja eficaz, os complexos devem ser capturados pela célula e interagir com alvos intracelulares. A exploração dos mecanismos de transporte celular é de extrema importância na avaliação do seu potencial terapêutico. Uma das características das células tumorais é apresentarem disfunção metabólica, i.e., elevada actividade glicolítica conhecida como efeito de Warburg. A interacção de compostos metálicos antitumorais com proteínas/enzimas celulares tem constituído actualmente uma área de investigação de grande interesse, como uma tentativa de identificação de outros alvos celulares para além do ADN. Resultados recentes obtidos pela equipa FCUL-CTN/IST revelaram que alguns compostos de Ru (II) são citotóxicos em algumas linhas celulares tumorais humanas. Pretendeu-se então estudar os mecanismos de captação de compostos de Ru (II), como potenciais agentes antitumorais usando moduladores do transporte celular e estudaram-se as interacções desses compostos com alvos celulares relacionados com o metabolismo (enzimas glicolíticos, enzimas redox da membrana celular, sistemas de transporte de membrana).The transition metal complexes have shown to be good candidates as antitumor agents. For their therapeutic effect to be efficient, the complexes must enter the cell and interact with intracellular targets. The exploitation of the mechanisms of cellular transport is extremely important in evaluating the therapeutic potential. One of the characteristics of tumor cells is exhibiting metabolic dysfunction, i.e., high glycolytic activity known as Warburg effect. The interaction of antitumor metal compounds with cellular proteins/enzymes has constituted an area of research currently of great interest as an attempt to identify other cellular targets besides DNA. Recent results obtained by the team FCUL-CTN/IST revealed that some compounds of Ru (II) are cytotoxic for some human tumor cell lines. It was intended to study the mechanisms of uptake of Ru (II) compounds, as potential antitumor agents using modulators of cellular transport and the interactions of the compounds with cellular targets related to metabolism (glycolic enzymes, redox enzymes of cellular membrane, transport membrane systems) were also studied

O diálogo intercultural nas escolas portuguesas: o caso do Clube Europeu da Escola Secundária de São Pedro do Sul

Doutoramento em Educação - Didática e Desenvolvimento CurricularEm sociedades cada vez mais abertas, permeáveis e multiculturais como as atuais, a escola e as suas atividades são fundamentais para a formação de futuros cidadãos, ao promover aspetos relevantes da cidadania ativa como aqueles relacionados com o diálogo intercultural e o conhecimento mútuo (pessoal e coletivo). Esta investigação perfila-se no entendimento de que a educação intercultural é uma abordagem educativa que favorece a abertura à diversidade cultural, assentando no pressuposto de que a valorização da diversidade e do diálogo intercultural promove conhecimentos, atitudes e competências interculturais considerados como relevantes para a atualidade, uma vez que levam a um melhor interconhecimento relativamente ao outro e por consequência, combatem a exclusão, a xenofobia, o racismo e a discriminação. Neste contexto, o presente estudo centra-se na análise das atividades do Clube Europeu (CE) da Escola Secundária de São Pedro do Sul (ESSPS) destinadas aos alunos do Ensino Básico regular, desenvolvidas ao longo do ano letivo de 2010-2011, procurando observar como estas promovem o diálogo intercultural nos alunos e facultam uma melhor informação acerca da Europa, contribuindo deste modo para uma maior compreensão do pluralismo europeu. No ano em causa, a investigadora acompanhou pessoalmente todas as atividades realizadas pelo CE para os alunos acima referidos, já que a investigação decorreu na escola onde leciona, e aplicou, ela própria, os instrumentos de recolha de dado no sentido de verificar os objetivos propostos. Trata-se portanto de um estudo de caso de características etnográficas, de cariz qualitativo, onde a investigadora assumiu o papel de observador participante. Após a realização de cada atividade do CE neste ano letivo, foram recolhidos os dados da investigação por meio da aplicação de um inquérito por questionário aos alunos que as realizaram, aos professores e encarregados de educação participantes numa delas (o “Projeto Quem Somos?”), às chefias intermédias e de topo da escola, à Coordenadora do CE da ESSPS e ao Coordenador Nacional dos Clubes Europeus. Pretendia-se, com estes questionários, conhecer as suas opiniões sobre a ação e as potencialidades e/ou constrangimentos do CE da escola estudada. Todos os dados foram analisados de acordo com a técnica de análise de conteúdo e com base num dispositivo de análise construído a partir do modelo de Competência Intercultural de Byram (1997). Os resultados obtidos parecem apontar para o facto de a ação deste CE ter dado a conhecer a diversidade cultural europeia, nomeadamente a sua composição e funcionamento, a sua diversidade linguística, as suas tradições e património construído. Ajudou também a formar e preservar a identidade cultural e a valorizar essa mesma diversidade, assim como a viver os valores europeus. Promoveu competências de sabercompreender e saber-fazer relativamente a outros povos e culturas, particularmente a compreender os problemas económicos e sociais, a importância das línguas na comunicação e os desafios da realidade atual, contribuindo também para que os alunos aprendessem a descentrar-se de si próprios colocando-se no lugar do outro, e, vivendo experiências de diálogo, aumentassem a sua autonomia pessoal, no exercício da sua cidadania. As chefias intermédias e de topo assim como os dirigentes do Clube Europeu da escola e dos clubes nacionais afirmaram, como potencialidades das atividades do CE da ESSPS, aspetos relacionados com a valorização do respeito pelo outro e sua cultura associada ao aumento do conhecimento histórico e cultural e a aprendizagens transversais dos alunos; referiram ainda um maior trabalho cooperativo entre professores e alunos e uma maior ligação escola-família-comunidade, dando cumprimento ao seu Projeto Educativo. Relativamente aos constrangimentos, afirmaram-se limitações de caráter temporal, financeiro e logístico, assim como de mobilização da escola e da comunidade e de interconhecimento e trabalho colaborativo entre Clubes Europeus, em particular entre os do distrito de Viseu. Tratando-se de um estudo bastante circunscrito e de cariz exploratório, não é possível fazer generalizações, dada a ausência de outras investigações sobre o funcionamento e atividades de outros Clubes Europeus, nomeadamente no mesmo distrito. No entanto, pelos resultados obtidos parecenos haver aspetos nas atividades dos Clubes Europeus (ou pelo menos nas do CE analisado) que reputamos como fundamentais para o crescimento dos jovens, tais como o conhecimento do pluralismo europeu, a valorização da diversidade cultural e a promoção do diálogo intercultural, numa Europa a necessitar de unidade e refundação assentes em valores partilhados, coletivamente assumidos e para os quais a escola pode e deve contribuir.In increasingly open, permeable and multicultural societies like the present ones, the school and its activities are central to the formation of future citizens by promoting relevant aspects of active citizenship as the issues of intercultural dialogue and the mutual knowledge (personal and collective). This research profiles on the understanding that the intercultural education is an educational approach that suits openness to cultural diversity, relying on the assumption that the value of diversity and intercultural dialogue promote intercultural attitudes and skills considered relevant to the present, since this leads to a better inter knowledge concerning the other and therefore, combat exclusion, xenophobia, racism and discrimination. In this context, the actual study concentrates on the analysis of the European Club activities (EC) secondary school of São Pedro do Sul (ESSPS) for pupils of regular basic education, developed throughout the school year 2010-2011, trying to observe how it promotes intercultural dialogue among students and provides better information about Europe, thus contributing to a better understanding of European pluralism. The research took place in the school where the researcher teaches and in the year in question, personally followed all activities undertaken by the EC to the students mentioned above, applying itself analytical instruments in order to verify the proposed objectives. It is therefore, a case study of ethnographic characteristics with a qualitative perspective, where the researcher assumed the role of participant observer. After the accomplishment of each activity of the EC in this school year, the research data was collected through the applying of a survey to students involved in those activities, teachers and parents participated in one of them (Who we are Project?) and also to the middle and Top Management and Top of the school, the EC Coordinator of ESSPS and the National Coordinator of the European Club. With these questionnaires, we intended to know their views on the action and the potential and / or EC constraints of the studied school. All data was analyzed according to the content analysis technique and from an analysis device based on the Byram’s Intercultural Competence model (1997). The results seem to point out that this European Club’s action has made European cultural diversity known, namely its composition and functioning, its linguistic diversity, its traditions and heritage. It has also helped to shape and preserve cultural identity and to value diversity, as well as to live European values. It has promoted competency in the understanding of other people and other cultures, economic and social problems, the importance of languages in communication and the challenges of present reality. It has helped students to learn to focus on others and put themselves in the place of the other. Having lived experiences of dialogue has also increased students’ personal autonomy in the exercise of their citizenship. Intermediate and top leaders, as well as those responsible for the school’s European Club along with other national clubs mentioned the enhancement in respecting others, their culture, and in historical and cultural knowledge plus the transverse learning of students as potential of the ESSPS European Club’s activities. A noticeable cooperation between teachers and students was felt and a greater link between school-familycommunity in compliance with the Educational Project was also experienced. With regard to restriction, financial and logistical constraints were stated. Likewise, there were some limitations in time and in school/community mobilization, in sharing knowledge and working collaboratively among European Clubs, particularly among those in the district of Viseu. Since this is a very limited study and of exploratory nature, it is not possible to generalize, because of the lack of studies about the operating activities of other European Clubs, including the district of Viseu. However, the results obtained seem to have aspects in the activities of the European Clubs (or at least in the analyzed EC) which we consider fundamental for the growth of young people, such as knowledge of European pluralism, the valorization of cultural diversity and the promotion of intercultural dialogue in Europe in need of unity based on shared values and re-founding collectively assumed and for which the school can and should contribute

Biological activity and cellular uptake of [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complex

Anticancer activity of the new [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] (Me(2)bpy = 4,4'-dimethyl-2,2'-bipyridine) complex was evaluated in vitro against several human cancer cell lines, namely A2780, A2780CisR, HT29, MCF7, MDAMB231 and PC3. Remarkably, the IC50 values, placed in the nanomolar and sub-micromolar range, largely exceeded the activity of cisplatin. Binding to human serum albumin, either HSA (human serum albumin) or HSA(faf) (fatty acid-free human serum albumin) does not affect the complex activity. Fluorescence studies revealed that the present ruthenium complex strongly quench the intrinsic fluorescence of albumin. Cell death by the [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complex was reduced in the presence of endocytosis modulators and at low temperature, suggesting an energy-dependent mechanism consistent with endocytosis. On the whole, the biological activity evaluated herein suggests that the complex could be a promising anticancer agent. (C) 2013 Elsevier Inc. All rights reserved

A new family of iron(II)-cyclopentadienyl compounds shows strong activity against colorectal and triple negative breast cancer cells

The following is available online, Figure S1—1H NMR spectrum of complexes 6, in acetone-d6, Table S1. Bond lengths [Å] and angles [°] for [Fe(η5-Cp)(CO)(PhCN)(PPh3)][CF3SO3] 1, [Fe(η5-Cp)(CO)(p-NCPhNH2)(PPh3)][CF3SO3] 4 and [Fe(η5-Cp)(CO)(p-NCPhBr)(PPh3)][CF3SO3] 5, Table S2. Relevant TD-DFT (PBE0) excitation energies (λ), oscillator strengths (f) and compositions (only those > 5% are shown), for complexes 1–6, compared with experimental data (λexp). Both calculated and experimental values were obtained in dichloromethane, Figure S2—UV-Vis spectra of complexes 1–6 in DMSO along the 24 h study, Figure S3—UV-Vis spectra of complexes 1–6 in DMSO/DMEM mixture along the 24 h study and its variation plot (%) (bottom), Figure S4. ‘FeCp’ compounds affect the colony formation ability of SW480 cell line. Analysis of the colony formation ability, after 48 h of incubation with 1/4 IC50 and IC50, in SW480 cell line. Representative images of colony formation assay in SW480 cell line, Figure S5. ‘FeCp’ compounds induce apoptosis colorectal cancer-derived cell line. Apoptotic cell death was analyzed by Annexin V fluorescein isothiocyanate (AV-FITC) and propidium iodide (PI) assay in SW480 cells, after incubation with IC50 and 2×IC50 concentrations for 48 h. Representative histograms of SW480 cell line double stained with AV and PI, Table S4. Crystal data and structure refinement for [Fe(η5-Cp)(CO)(PhCN)(PPh3)][CF3SO3] 1, [Fe(η5-Cp)(CO)(p-NCPhNH2)(PPh3)][CF3SO3] 4 and [Fe(η5-Cp)(CO)(p-NCPhBr)(PPh3)][CF3SO3] 5.A family of compounds with the general formula [Fe(η5-C5H5)(CO)(PPh3)(NCR)]+ has been synthesized (NCR = benzonitrile (1); 4-hydroxybenzonitrile (2); 4-hydroxymethylbenzonitrile (3); 4-aminobenzonitrile (4); 4-bromobenzonitrile (5); and, 4-chlorocinnamonitrile (6)). All of the compounds were obtained in good yields and were completely characterized by standard spectroscopic and analytical techniques. Compounds 1, 4, and 5 crystallize in the monoclinc P21/c space group and packing is determined by short contacts between the phosphane phenyl rings and cyclopentadienyl (compounds 1 and 4) or π-π lateral interactions between the benzonitrile molecules (complex 5). DFT and TD-DFT calculations were performed to help in the interpretation of the experimental UV-Vis. data and assign the electronic transitions. Cytotoxicity studies in MDA-MB-231 breast and SW480 colorectal cancer-derived cell lines showed IC50 values at a low micromolar range for all of the compounds in both cell lines. The determination of the selectivity index for colorectal cells (SW480 vs. NCM460, a normal colon-derived cell line) indicates that the compounds have some inherent selectivity. Further studies on the SW480 cell line demonstrated that the compounds induce cell death by apoptosis, inhibit proliferation by inhibiting the formation of colonies, and affect the actin-cytoskeleton of the cells. These results are not observed for the hydroxylated compounds 2 and 3, where an alternative mode of action might be present. Overall, the results indicate that the substituent at the nitrile-based ligand is associated to the biological activity of the compounds.Centro de Química Estrutural acknowledges Fundação para a Ciência e Tecnologia (FCT) for the Project UIDB/00100/2020. This work was also funded in the scope of the project PTDC/QUI-QIN/28662/2017 (FCT) and by the strategic program UID/BIA/04050/2019 (FCT). A. Pilon and Ana Rita Brás thank FCT for their Ph.D. Grants (SFRH/BD/139412/2018 and SFRH/BD/139271/2018, respectively). A. Valente acknowledges the CEECIND 2017 Initiative (CEECIND/01974/2017). P. J Costa thank FCT for Investigador FCT Program IF/00069/2014, exploratory project IF/00069/2014/CP1216/CT0006, and strategic project UID/MULTI/04046/2019. P. J. Costa also acknowledges FCT, Programa Operacional Regional de Lisboa (Lisboa 2020), Portugal 2020, FEDER/FN, and the European Union for project LISBOA-01-0145-FEDER-028455 / PTDC/QUI-QFI/28455/2017

Studies on the mechanism of action of anti-tumor bis(aminophenolate) ruthenium(III) complexes

Two recently published Ru(III) comp lexes bearing ( N 2 O 2 ) tetradentate bis(aminophenolate) ligands, 20 formulated as [Ru(III)( s alan )(PPh 3 )Cl] ( where s alan is the tetradentate ligand 6,6' - (1S,2S) - 21 cyclohexane - 1,2 - diylbis(azanediyl)bis(met hylene)bis(3 - methoxyphenol) in c omplex 1 , or 2,2' - (1S,2S) - 22 cyclohexane - 1,2 - diylbis(azanediyl)bis(meth ylene)bis(4 - methoxyphenol) in c omplex 2 , and PPh 3 is 23 triphenylphosphane ) were studied herein to outline their antitumor mode of action. Previously, it was 24 found that they were very active against human ovar ian and bre a st adenocarcinoma cells (on a 72 h 25 challenge ) . In this work, the human cisplatin - sensitive o varian adeno carcinoma line A2780 was used 26 as the cell model for further studies a t a 24 h challenge . B oth complexes are active , their cytotoxicity 27 being c omparable to that of c isplatin in the same conditions. 28 A s a possible target in the cell for their action, the int eraction of 1 and 2 with DNA was assessed 29 through displacement of well - established DNA fluorescent probes ( ethidium bromide , EB, and 4',6 - 30 d iamidino - 2 - p henylindole, DAPI ) through s teady - state and time - resolved fluorescence spectroscopy . 31 The complete emission spectra were analysed globally for the binary DNA ‒ p robe and ternary DNA ‒ 32 p robe ‒ Ru(III) complex systems . B oth Ru(III) complexes can displace EB and bind to DNA with 33 similar and moderate strong affinity with conditional stability constants of log K’ = ( 5.05 ± 0.01 ) for 1 34 and log K’= ( 4.79 ± 0.01 ) for 2 . The analysis of time - domain fluorescence intensity decays confirmed 35 both qualitatively and quantitatively the model used to describe the binding and competition processes. 36 Cell studies indicated that apoptosis is t he major mechanism of cell death for both complexes , with 2 37 (the mo re active complex ) promoting that process more efficiently than 1 . Transmission electron 38 micro graphs revealed clear a lteration s on intracellular organization consistent with the induction of 39 programmed cell death processes

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

Polymer-metal based compounds to target (non)hormone-responsive cancers : synthesis and mechanistic evaluation

Cancer is the second leading cause of death worldwide being responsible for 9.6 million deaths of a total of 18.1 million new cases in 2018. In particular, breast cancer is the most commonly diagnosed and the leading cause of death in women. Among the several types of breast cancers, the triple negative breast cancer, which is estrogen receptor negative (ER-), progesterone receptor negative (PR-) and human epidermal growth factor receptor 2 negative (HER2-) is known for its typical highly metastatic profile, with inferior prognosis and for which there is still no available effective targeted therapy. Thus, the investment in the research of new chemotherapeutic agents in this area is crucial and imperative. Within this PhD project, six new organic ligands and eighteen new ruthenium(II) cyclopentadienyl compounds were synthesized and fully characterized. These complexes were divided in three different families with the general formula [Ru(Cp)(P)(bipy)]+, where cyclopentadienyl (Cp), phosphane (P) and bipyridyl (bipy) moieties were functionalized with different groups in order to include a macromolecular ligand (polylactide - PLA and/or polyethylene glycol - PEG) and a biomolecule (biotin or curcumin) to enhance the selectivity of compounds to cancer cells, exploring both passive and active cancer targeting strategies. To assess the biological potential of the new compounds an array of in vitro studies aiming at establishing structure-activity relations was performed. The studies in hormone and nonhormone-responsive cancer cell lines encompassed the assessment of cytotoxicity, cell death, intracellular distribution (drug internalization) and anti-metastatic ability (colony formation assay) of compounds. Multidrug resistance for selected compounds on the main ABC transporters was also studied by flow cytometry. Finally, compounds’ toxicity was assessed using a zebrafish model and the lead compounds were subjected to preliminary in vivo studies using a nude mice animal model bearing triple negative breast cancer orthotopic tumors where their toxicity and efficacy on primary tumors and metastases was evaluated.Fulbright com o apoio da FC