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This book includes the program, the abstracts and all remaining details pertaining to the 2nd IEF International Colloquium ROADS TO CARE,held at the Faculty of Arts and Humanities of the University of Coimbra between October 19 and 21, 2022,organized by the R&D Unit IEF - Institute for Philosophical Studies, funded by the FCT -Foundation for Science and Technology, I.P., of the Ministry of Science, Technology and Higher Education of the Government of the Portuguese Republic, under the UIDB/00010/2020 project.

Dissertação de mestrado integrado em Engenharia de Materiais, Atualmente, as principais causas para a rejeição de implantes dentários são a lenta osteointegração e consequente adesão e crescimento bacteriano, o que pode levar ao aparecimento de doença peri-implantar. A osteointegração e a inibição da adesão bacteriana podem ser melhoradas através da modificação das propriedades da superfície do implante. Esta dissertação foca-se numa primeira fase na modificação de superfícies metálicas de tântalo (Ta), através de oxidação por plasma eletrolítico (PEO). A modificação por PEO, pretende promover a bioatividade da superfície e acelerar a osteointegração através da mimetização da morfologia e química do osso humano, com a formação de estruturas micro/nanoporosas e incorporação de elementos osteocondutores (cálcio (Ca) e o fósforo (P)). Numa segunda fase, sobre esta superfície bioativa, nanopartículas (NPs) de zinco (Zn), com e sem camada de carbono (C), são depositadas por pulverização catódica em magnetrão para dotar esta superfície com atividade antimicrobiana. Foram testadas diferentes quantidades de NPs depositadas nas superfícies de Ta oxidadas por PEO. Aumentando o tempo de deposição e/ou diminuindo a pressão de trabalho, foi possível depositar maior quantidade de NPs de Zn com diferentes morfologias. A libertação de iões zinco aumentou com o aumento da quantidade de NPs e diminuiu com a deposição da camada de carbono. As superfícies otimizadas, a com maior e a com menor quantidade de NPs, foram estudadas quanto à atividade antimicrobiana e resistência à corrosão. A presença de NPs de Zn teve grande impacto na melhoria da atividade antimicrobiana, com a maior quantidade de NPs a mostrar maior inibição do crescimento de C. Albicans do que a superfície porosa de Ta2O5. Os resultados de corrosão revelaram que a formação de Ta2O5 promovida pela técnica de PEO conduziu à melhoria do comportamento à corrosão, em comparação com o Ta não modificado. Em contrapartida a incorporação das NPs metálicas de Zn promoveu uma degradação da resistência à corrosão das amostras, que foi melhorando com o aumento do tempo de imersão. Estes resultados demonstraram que a incorporação de NPs de Zn nas superfícies porosas de Ta2O5 inibiu eficazmente o crescimento microbiano. Assim, as técnicas de PEO e pulverização catódica de magnetrão são técnicas de modificação de superfícies promissoras para a funcionalização de superfícies de Ta para implantes dentários., Nowadays, the main causes of the rejection of dental implants are their poor osseointegration and the bacterial adhesion and growth, which may lead to peri-implantitis disease. The osseointegration and the bacteria adhesion inhibition can be enhanced by modifying implant surface properties. This thesis is focused, in a first phase, on the surface modification of metallic tantalum (Ta), by plasma electrolytic oxidation (PEO). The modification by PEO intends to promote the surface bioactivity and accelerate the osseointegration by mimetic the morphology and chemical from the bone, with the formation of micro/nano-porous structures and the incorporation of osteoconductive elements (calcium (Ca) and phosphorus (P)). In a second phase, over these bioactive surfaces, zinc (Zn) nanoparticles (NPs) with and without a thin carbon (C) layer are deposited by magnetron sputtering to endow this surface with antimicrobial activity. Different quantities of NPs deposited on the bioactive surfaces were tested. Increasing the deposition time and/or decreasing the working pressure, it was possible to deposit a higher amount of Zn NPs with different morphologies and sizes. The zinc ions release increased with the amount of the NPs and decreased when the carbon layer covered the NPs. The chosen optimized surfaces, the one with lower and the one with higher ionic release, were further studied regarding the antimicrobial activity and corrosion resistance. The presence of Zn NPs had a great impact on the improvement of the antimicrobial capacity, as the higher quantity of Zn NPs (and higher zinc ions release) showed the highest C. albicans growth inhibition compared to the porous Ta2O5 surface. The C layer did not reveal a significant difference when compared to the respective surface. The corrosion results revealed that the formation of Ta2O5 by the PEO lead to improvement of the corrosion behavior, compared to untreated Ta surfaces. On the other hand, the incorporation of metallic Zn NPs promoted the degradation of corrosion resistance, which improved as a function of immersion time and became closer to the porous Ta2O5 surface corrosion behavior. These results demonstrated that the deposition of Zn NPs onto porous Ta2O5 surfaces efficiently inhibits the microbial growth. Thus, the PEO and magnetron sputtering are promising surface modification techniques for functionalize Ta surfaces for dental implants.

Advancements in sequencing technologies have now set the pace on applying the resulting information of our genome into a more clinical relevant output. Gene editing, with the ability to change genomic DNA in a targeted manner, has gained an important spot in the nextgeneration of advanced therapies. Moreover, spatio-temporal control of gene expression may provide an unprecedent understanding of the relevance of specific genes in biological processes as well as a triggerable tool to modulate cell activity. The main objective of this thesis was to develop nanoformulations for the light-responsive delivery of gene editing enzymes, particularly Cre recombinase. During the last years, the scientific community has done considerable efforts in the development of delivery strategies for the clinical translation of gene editing systems. From the different possibilities, non-viral delivery of protein or mRNA cargos is likely a safer and more efficient strategy. With this purpose we have developed two different nanocarriers each with different specificities. The first nanocarrier was based on lanthanide-doped upconversion nanocrystals (UCNPs) that converts NIR into blue light. A photocleavable linker able to attach in one end the UCNP and in the other end the Cre recombinase enzyme, while maintaining its enzymatic activity, allowed the NIR-light mediated delivery of the enzyme. In addition to Cre, the surface of UCNPs was modified with hydroxychloroquine to facilitate NP endolysosomal escape. The formulation was more efficient than conventional transfection agents (RNAiMAX) and other reported nanocarriers for the intracellular delivery of Cre, requiring less protein to achieve similar functional protein delivery levels. This nanocarrier was evaluated in vivo, in the setting of the brain, as a transcranial NIR gene editing system for deep brain regions including the subventricular zone and the ventral tegmental area. Results showed that the level of recombination obtained was sufficient to induce functional responses in the living animals. Although mRNA offers several advantages over protein or DNA delivery (straightforward production and transient expression), the successful delivery of this large anionic molecule is still very challenging. To obtain a biocompatible delivery of Cre mRNA, we took advantage of a lightsensitive polymeric library of 160 polymers that was previously reported by us for the identification of agents with transfections comparable to the commercial agents. We performed a highthroughput study for the identification of vectors with chemical diversity that could transfect mRNA. We could identify 7 candidates that, although not yet optimized for light-mediated expression of Cre enzyme, are UV/blue-light responsive. The candidates were able to rapidly transfect (10 min) a reporter cell line with Cre mRNA and obtain, in this condition, recombination levels up to 50% for the tested formulations.

Dissertação de mestrado em Bioquimica apresentada ao Departamento Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra O cancro da mama é uma doença heterogénea e é a maior causa de morte por cancro entre as mulheres. A quimioterapia é um tipo de tratamento essencial à sobrevivência e à qualidade de vida dos pacientes com cancro da mama. Contudo, a quimioterapia pode estar associada a uma toxicidade em células não cancerígenas sendo, deste modo, limitada por efeitos secundários severos. Por outro lado, o seu efeito terapêutico pode ser temporário e, muitas vezes, os pacientes acabam por desenvolver resistência a muitos agentes de quimioterapia. Os sistemas de transporte e entrega de fármacos foram propostos como uma estratégia para combater estas limitações. Os nanosistemas, quando desenvolvidos com base em estratégias de direcionamento passivo e activo, têm o potencial de aumentar a concentração intracelular dos fármacos no tumor e evitar a toxicidade nos outros tecidos. Neste contexto, vários estudos têm sido realizados por forma a desenvolver formulações de lipossomas que possam ser utilizadas como sistemas de transporte e entrega de agentes de quimioterapia. O objectivo deste trabalho consistiu no desenvolvimento de uma nova formulação de lipossomas, para transporte e entrega de fármacos, que tivesse a capacidade de direcionar e libertar a epirrubicina especificamente nas células do cancro da mama, através da utilização de um Affibody® contra o EGFR. Desta forma, esta estratégia poderia aumentar substancialmente a eficiência dos tratamentos do cancro da mama disponíveis actualmente. Para isso, foram desenvolvidas novas formulações de lipossomas, a partir de lipossomas convencionais compostos por HSPC e colesterol, aos quais se acrescentou DSPG (lípido aniónico) e DSPE-PEG (lípido modificado com um polímero hidrofílico). Após a encapsulação do fármaco, realizou-se a caracterização das formulações tendo em conta a eficiência de encapsulação do fármaco, o tamanho e estabilidade dos lipossomas. Os resultados obtidos mostraram que a incorporação de DSPG nos lipossomas aumentou a eficiência de encapsulação da epirrubicina. Por sua vez, a inclusão de DSPE-PEG nos lipossomas resultou num aumento de libertação do fármaco durante o armazenamento. Contudo, este efeito foi anulado na presença de 10% de FBS. A formulação de lipossomas composta por HSPC:Chol:DSPG:DSPE-PEG (6:3:0,6:0,4 razão molar) combina a elevada capacidade de encapsulação de epirrubicina com características de superfície e tamanho (perto de 150nm) favoráveis ao seu direcionamento passivo e acumulação nos tumores sólidos. Os estudos de actividade antitumoral in vitro, realizados numa linha celular de cancro da mama do tipo triplo-negativa (células MDA-MB-231), mostraram que existiam apenas pequenas diferenças, em termos de morte celular, entre as diferentes formulações de lipossomas desenvolvidas. Verificou-se também que a epirrubicina livre apresentou uma citotoxicidade muito superior à epirrubicina encapsulada nas formulações de lipossomas. Este facto deve-se, muito provavelmente, à associação celular reduzida das formulações de lipossomas desenvolvidas. Neste sentido, uma estratégia de vectorização específica para o cancro da mama poderá ser solução para aumentar a internalização celular dos liposomas contendo epirrubicina e, consequentemente, a sua eficácia terapêutica. Por outro lado, o conteúdo do lipossoma pode ser libertado no microambiente tumoral em resposta a um estímulo específico, nomeadamente em resposta à degradação causada pela enzima sPLA2 (sobrexpressa no cancro da mama). Os resultados obtidos demostraram que a sPLA2 é capaz de induzir a libertação da epirrubicina contida nos lipossomas, sendo a sua actividade dependente da composição dos lipossomas. A incorporação de DSPG nos lipossomas resultou num aumento significativo da actividade da sPLA2. Os resultados obtidos sugerem que os lipossomas HSPC:Chol:DSPG:DSPE-PEG (6:3:0,6:0,4) apresentam um grande potencial para serem utilizados no desenvolvimento de um sistema de transporte e entrega de fármacos que tenha a capacidade de libertar a epirrubicina, nas células de cancro da mama, de forma específica e eficiente. Breast Cancer is a heterogeneous disease and a leading cause of death in women. Chemotherapy is a crucial treatment to improve survival and quality of life of breast cancer patients. However, chemotherapy is limited by severe and harmful toxic effects to normal cells and its effect is not long lasting. Moreover, patients can develop resistance to many different types of chemotherapeutic agents. Regarding this, drug delivery systems have been attempted to overcome these problems. By using both passive and active targeting strategies, nanocarriers can enhance the intracellular concentration of chemotherapeutic agents in cancer cells while avoiding toxic effects on healthy tissues. Moreover, they have the potential to overcome drug resistance. In this context, liposome-based technology has been widely studied and evolved to overcome chemotherapeutic needs. The purpose of this work was to develop a novel liposomal-based drug delivery approach that had the ability to target and release epirubicin specifically to breast tumors, by using an anti-EGFR Affibody® as a targeting moiety, in order to improve the current breast cancer chemotherapeutic strategies. Regarding this goal, several liposomal formulations were developed. A conventional liposome formulation of HSPC and cholesterol was enriched with a steric stabilizer, DSPE-PEG, and an anionic lipid, DSPG. Formulations were characterized according to their size, entrapment efficiency and drug release. The obtained results showed that the incorporation of DSPG into liposomes improved the epirubicin entrapment efficiency. On the other hand, inclusion of DSPE-PEG in liposomes resulted in an increased release of epirubicin during storage. However, this effect was abolished in the presence of 10% FBS. The HSPC:Chol:DSPG:DSPE-PEG (6:3:0.6:0.4 molar ratio) liposome formulation combined high epirubicin encapsulation efficiencies with the surface characteristics and the size (near 150nm) favorable to passively target solid tumors. The in vitro antitumoral activity studies, performed in a triple-negative breast cancer cell line (MDA-MB-231 cells), showed only slight differences, in terms of cell death, between the developed formulations. It was also observed that free epirubicin presented a higher cytotoxicity than the developed liposomal-epirubicin formulations. This fact is most probably due to the reduced cell association registered with these formulations. Therefore, an active targeting strategy would be a promising approach to enhance cellular association and, consequently, the therapeutic efficacy of the epirubicin-containing liposomes. On the other hand, epirubicin could be released from liposomes specifically in the tumor microenvironment in response to a sPLA2 (an enzyme overexpressed in breast tumors) stimulus. The obtained results demonstrated that sPLA2 has the ability to induce the leakage of epirubicin, this sPLA2-mediated drug release being dependent on the liposomes composition. The incorporation of DSPG in the liposomes resulted in a significant increase in the sPLA2 activity. Overall, the obtained results suggest that HSPC:Chol:DSPG:DSPE-PEG (6:3:0.6:0.4 molar ratio) liposomes present a great potential to be used in the development of a drug delivery system that has the ability to specifically and efficiently release epirubicin in breast cancer cells.

Formal lecture given on the 21st of October 2022 by Teresa Toldy, in the capacity of keynote speaker, and hosted by José Manuel Beato during the 2nd IEF International Colloquium ROADS TO CARE, held at the Faculty of Arts and Humanities of the University of Coimbra between October 19 and 21, 2022, organized by the R&D Unit IEF - Institute for Philosophical Studies, funded by the FCT - Foundation for Science and Technology, I.P., of the Ministry of Science, Technology and Higher Education of the Government of the Portuguese Republic, under the UIDB/00010/2020 project. Abstract: Kristen Dunphy’s presentation of TV series “Wakefield” (2020) includes a sentence decisive for my presentation: “There’s a fine line between sanity and madness”. Every and each chapter of this series starts with a psych nurse standing at the edge of an abyss in the Australian Blue Mountains. Actually, the psych ward where he works is near to that abyss. Sanity and madness are presented as something that goes far beyond a simple clinic situation: they may be considered almost as a symbol of a vulnerability that is common to every and each human being: we are all at the edge of an abyss – the abyss of mortality. And yet, at the same time, the notion that, even when you have to take care of someone, you are also hit by your own fragility results in an interpretation of human existence as a complex web of caring and being cared. Our common vulnerability may lead human beings to be drawn into the abyss or to dance on the edge of it, even if that dance is precarious. My presentation will focus on the awareness of our common vulnerability as the ground to the construction of an ethics of care. Bionote: Teresa Maria Leal de Assunção Martinho Toldy, PhD in Theology (feminist theology) at the Philosophisch-Theologische Hochschule Sankt Georgen (Frankfurt/Germany), Master in Theology (Catholic University, Lisbon) and 1st grade in Theology at the same University. Postdoctorate in CES. Full Professor at Fernando Pessoa University. Teaches in the field of Ethics. Researcher at CES (Thematic Line: Democracy, Justice and Human Rights). Co-coordinator of GT-POLICREDOS together with Júlia Garraio and Luciane Lucas Santos and co-coordinator of GPS, with Ana Cristina Santos e Madalena Duarte. Former Vice-Chair of the Portuguese Association on Women's Studies (2009-2014). Member of the Editorial Board of the ESWTR Studies in Religion and member of ESWTR. Fields of specialization: religion; feminist studies. Other fields of interest: Ethics. Publishes in the fields of religion, gender, and feminist studies.