Multiresponsive spiropyran-based copolymer: synthesis and characterization

Photochromic copolymers carrying spiropyran side groups have attracted particular attention for potential applications in optical devices, photonic memory and photosensing, as well as, fluorescence imaging.[1] These materials allow overcoming many of the limitations inherent to traditional spiropyran doped polymers, such as, phase separation of the colorant and retardation of the decoloration of the open form.[2] The introduction of light sensitive moieties within responsive polymers has led to the development of sophisticated multiresponsive systems.[3] Block copolymers are important self-assembling systems that can assume a diversity of nanometer-scale morphologies due to the incompatibility and the connectivity constrains between the chemically distinct segments. Therefore, self-assembly of photochromic copolymers in the solid state or in solution allows the development of nanostructured materials. [4] The aim of this research study is the synthesis of a multiresponsive spiropyran-based copolymer, poly(styrene)- co-poly([1´,3´,3´-trimethyl-6-methacryloyloxyspiro(2H-1-benzopyran-2,2´-indoline)]4-vinylbenzoic acid) 2. The functionalized block copolymer poly(styrene)-block-poly(4-vinylbenzoic acid) (PSt-b-P4VBA) 1 synthesized by reversible addition fragmentation chain-transfer (RAFT) polymerization was coupled with 1,3,3-trimethyl-6- hydroxyspiro(2H-1-benzopyran-2,2-indoline). The link between the functionalized block copolymer and spiropyran was successfully obtained by Steglich esterification using DMAP/DCC as catalysts (Scheme 1).n-STeP ProjectNORTE-07-0124-FEDER-000039Programa Operacional Regional do Norte (ON.2)PEst-C/CTM/LA0025/2013 (Strategic Project - LA 25 - 2013-2014

Effect of clay mineral addition on properties of bio-based polymer blends

The effect of clay mineral addition to bio-based blends on morphology and physical properties of thermoplastic starch (TPS) and polypropylene grafted with maleic anhydride (PP-g-MA) was investigated. Blends and nanocomposites containing organoclay, Cloisite 30B, were prepared by melt mixing and characterized by several techniques. X-ray diffraction (XRD), scanning and transmission electron microscopy (SEM, STEM) and dynamic mechanical analysis (DMA) demonstrate a very good dispersion of the clay mineral in the polymer matrix, an increase of polymer compatibility and an improvement of mechanical properties. Biodegradation studies performed in compost revealed that Cloisite 30B addition enhanced the matrix biodegradability. Therefore clay minerals, which can be obtained from natural resources, can be efficiently used to improve the properties of bio-based materials and contribute to sustainability.The authors acknowledge the n-STeP - Nanostructured systems for Tail, with reference NORTE-07-0124-FEDER-000039, supported by the Programa Operacional Regional do Norte (ON.2), PEst-C/CTM/LA0025/2013 (Strategic Project - LA 25 - 2013-2014)

Thermoplastic starch-polypropy/lene reinforced with clay

In this study, bio-based blends of thermoplastic starch (TPS) and polypropylene grafted with maleic anhydride (PP-g-MA) without and with organoclay, Cloisite 30B, were prepared in an internal mixer. A TPS-g-PP copolymer was successfully obtained in melt and its formation was confirmed by SEM. The establishment of this copolymer creates an interpenetrating network leading an intercalated/exfoliated clay nanocomposite. Several analytical, angle X-ray diffraction (XRD), scanning and transmission electron microscopy (SEM, STEM) and dynamic mechanical analysis (DMA) were used to characterize the prepared materials. The nanocomposites mechanical properties were improved, showing an increase of about 11% for the nanocomposite when compared with the neat blend. Biodegradation studies performed in compost revealed good percentage of weight loss for the copolymer with clay addition

Development of silver and clay-starch bio-nanocomposites

Starch, among biopolymers is that had the lowest production cost, wide availability, fully biodegradability and is a renewable agriculture resource. Starch due to its sensitivity to humidity and poor mechanical properties cannot be used in many applications. For that, the dispersion of clays in this material improves their physical and mechanical properties, at very low filler loadings. On the other hand, the incorporation of silver nanoparticles into biocompatible and biodegradable matrices gives antimicrobial properties opening a new field for packaging applications. [...

Numerical study on the performance of improved masonry-to-timber connections in traditional masonry buildings

This paper deals with a numerical study on the structural performance of masonry-to-timber connections in ancient buildings. The work is supported on an experimental campaign carried out at University of Minho, which aims at characterising a strengthening solution based on the use of injected anchors for the improvement of the connection between masonry and timber frame walls. The numerical study resorts to a detailed 3D finite element model, which reproduces the experimental test setup and proce- dure. The modelling approach adopted allows an accurate characterisation of the behaviour of all struc- tural elements, in terms of stress field and displacement distribution. The 3D model was validated against the available experimental results, which was then used to perform parametric analyses in order to eval- uate the influence of key parameters. Finally, simplified analytical approaches to estimate the strength capacity of injected anchors on masonry are presented and discussed.This work was partly funded by Project FP7-ENV-2009-1244123-NIKER. The first author also acknowledges the financial support from the Portuguese Science Foundation (Fundacao de Ciencia e Tecnologia, FCT), through Grant SFRH/BD/71599/2010

Development of non-viral vectors for gene therapy for pathologies of the retina

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015The success of gene therapy relies on efficient gene transfer and stable transgene expression. Our goal was to develop non-viral vectors optimized for retinal gene therapy with continued gene expression. Polymers, chitosan and hyaluronic acid or the modified polymers (thiolated chitosan and aminated hyaluronic acid) were chosen considering their biocompatibility and biodegradability to prepare several formulations. Vectors were formulated and characterized regarding their physical properties, biocompatibility and gene transfer efficiency in vitro on both retinal pigment epithelial and HEK293 cells and gene expression in the mouse retina. Our results show that our vectors exhibit size and surface charge consistent with gene delivery. They also present long-term stability in both storage and physiological conditions, remain stable after several freeze-thaw cycles and are capable of efficiently protecting DNA from nuclease degradation. Transfection studies show that transfection efficiency and transgene expression is affected by cell type, polymer molecular weight and mode of integrase delivery with the polyplexes. The incorporation of hyaluronic acid affected formulation stability, as expected, but it did not affect DNA loading and protection. The combination of chitosan and hyaluronic acid in polyplexes showed a significant improvement of transfection efficiency compared to chitosan-based vectors. In order to achieve sustained gene transfer vectors were combined with phiC31-integrase to promote transgene integration. The combined strategy of chitosan-based delivery and integrase demonstrate prolonged gene expression of both small (GFP, 1 Kb) and large genes (CEP290, 8Kb) several weeks post-transfection. In vivo sub-retinal administration of our vectors showed efficient transfection and sustained transgene expression in RPE cells at least 6 months post- injection. Our results indicate this chitosan-based approach may overcome size limitations found in commonly used adeno-associated viruses mediated gene transfer, while maintaining a high safety profile and prolonged, sustained gene expression, thus constituting an alternative for retinal gene delivery.A terapia génica é uma abordagem terapêutica que tem mostrado grande potencial para o tratamento de doenças genéticas hereditárias ou adquiridas. Geralmente envolve a substituição ou inibição do gene mutado ou ainda a inserção de um novo gene. O sucesso de uma estratégia de terapia génica depende da eficiência da transferência genética e da expressão estável do gene transferido. Atualmente existem duas abordagens que são utilizadas no desenvolvimento de terapias génicas: a viral e a não viral. A abordagem viral, embora seja bastante eficiente, levanta preocupações com a segurança a nível imunológico e mutagénico. Por outro lado a abordagem não-viral caracteriza-se pela sua fácil utilização, capacidade de empacotamento de genes ilimitada e ausência de resposta imunitária. Apesar destas vantagens a aplicabilidade da abordagem não-viral encontra-se limitada pela sua baixa eficiência de transfeção quando comparada com a abordagem viral; deste modo, a optimização dos vetores não-virais visa sobretudo a melhoria da sua eficiência de transfeção. O nosso objectivo era desenvolver vetores não-virais para terapia génica ocular, em particular da retina. Considerando o órgão alvo, o olho apresenta características únicas para terapia génica como por exemplo o seu reduzido tamanho, relativo isolamento em relação à circulação sistémica e facilidade de acesso a diferentes tipos de tecidos por vias de administração diferentes. Grande parte das estratégias de terapia génica não-viral focam-se na utilização de lípidos ou polímeros catiónicos, mas até à data poucos estudos foram feitos sobre a sua utilização no olho e em particular na retina. Os polímeros, quitosano e ácido hialurónico ou os polímeros modificados (quitosano tiolado e ácido hialurónico aminado) foram escolhidos considerando a sua biocompatibilidade e biodegradabilidade para a preparação de diversas formulações de vectores. Estes polímeros já foram utilizados com bastante sucesso em aplicações como a entrega de fármacos, o que, aliado às propriedades referidas anteriormente, os torna bastante atrativos para aplicações de terapia génica, embora seja necessária a sua otimização. Esta otimização engloba a modificação química dos polímeros suprareferidos, tal como a modificação com cistamina que permite introduzir uma ponte dissulfeto, que pode ser clivada no meio intracelular pela glutationa, permitindo assim uma libertação do ADN mais facilitada. No caso do ácido hialurónico permite ainda adicionar grupos amina, que poderiam interagir electrostaticamente com o ADN. Experimentalmente podemos dividir este trabalho em 3 secções: i) a preparação de diversas formulações de vetores com quitosano e ácido hialurónico, que foram caracterizadas em relação às suas propriedades físicas, nomeadamente tamanho, polidispersão, carga superficial, eficiência da complexação de ADN, capacidade de proteção contra nucleases e estabilidade dos vectores em diferentes condições; ii) a caraterização dos vectores in vitro em relação à sua citotoxicidade e eficiência de transfeção em células epiteliais pigmentadas de retina e células HEK293; iii) a administração in vivo dos vectores com melhores resultados in vitro e a sua caracterização em relação à sua expressão génica na retina de ratinhos através de injeção subretiniana. Os nossos resultados mostram que os vetores têm tamanho e carga superficial adequados à entrega de genes, embora variem consoante a formulação. Apresentam também estabilidade a longo prazo tanto em condições de armazenamento como em condições fisiológicas de temperatura e pH. Os vetores mantêm-se estáveis após vários ciclos de congelação e descongelação e são capazes de proteger eficazmente o ADN da degradação por nucleases. Observouse também um efeito do peso molecular do quitosano nas propriedades dos vetores: a utilização de um polímero com maior peso molecular resultou em vetores de maiores dimensões com tendência para uma carga superficial superior, mas com uma estabilidade mais reduzida comparativamente aos vetores preparados com o polímero de menor peso molecular. Este efeito do peso molecular do polímero estendeu-se à eficiência de transfeção e vectores preparados com o polímero de menor peso molecular obtiveram melhores resultados. Os ensaios de transfeção mostram também que a eficiência de transfeção e a expressão do transgene é afectada pelo tipo de células e modo de entrega da integrase com os poliplexos. A eficiência de transfeção foi superior em células HEK293 do que em células pigmentadas da retina. A elevada estabilidade dos poliplexos tem sido associada com uma baixa eficiência de transfeção e a utilização de polímeros aniónicos tem sido usada como uma das opções para solucionar essa questão. A incorporação de ácido hialurónico nas formulações afectou a estabilidade das formulações, como era esperado, mas não afectou a complexação nem a proteção do ADN no poliplexo. A combinação de quitosano e ácido hialurónico nos poliplexos mostrou uma melhoria significativa na eficiência de transfeção comparada com vectores baseados apenas em quitosano. Os ensaios de transfeção usando vectores preparados com os polímeros modificados (quitosano tiolado e ácido hialurónico aminado) não revelaram melhorias significativas em relação aos polímeros não modificados. Pensa-se que isto poderá estar associado a interferências do contra-ião de grandes dimensões – tosilato no caso do quitosano tiolado - e a uma baixa percentagem de modificação no caso do ácido hialurónico aminado. Seria necessário repetir as reações de modificação de modo a substituir o contra-ião por cloreto e conseguir uma percentagem de modificação mais elevada do ácido hialurónico, respetivamente. De modo a obter uma expressão génica continuada os vectores foram combinados com a integrase do fago phiC31 para promover a integração do transgene no genoma da célula de forma segura e eficaz. A estratégia combinada de entrega baseada em vectores de quitosano e integrase demonstrou expressão génica prolongada tanto de genes de pequena dimensão, como o gene que codifica para a proteína verde fluorescente (com aproximadamente 1 kb) como de genes de maiores dimensões como o gene da proteína centrossomal CEP290 (com aproximadamente 8 kb) várias semanas após a transfeção. A administração subretiniana in vivo dos nossos vetores revelou transfeção eficiente e expressão do transgene continuada em células epiteliais pigmentadas de retina pelo menos 6 meses após a transfeção. Os nossos resultados indicam que esta abordagem baseada em vetores de quitosano pode ultrapassar as limitações de empacotamento encontradas nas técnicas de transferência de genes mediadas por vírus adeno-associados, mantendo um elevado perfil de segurança e expressão génica continuada, constituindo assim uma alternativa para a terapia génica na retina.FCT - Fundação para a Ciência e Tecnologi

Study of the antimicrobial and antioxidant effects of propolis extracts, a type of Algarve portuguese origin

Dissertação de mest., Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Univ. do Algarve, 2009In this work, extracts (aqueous, ethanolic and methanolic) of propolis harvested at two different times (winter and spring) from several locations of the Algarve region (B. N. Arrodeios, B. N. Pé da Serra, B. S. Arneijoafra and T. N. Madeira) were tested for their biological activities and composition. Results showed that propolis extracts were active against both Gram-positive and Gramnegative bacteria. All tested strains of bacteria showed susceptibility to the diluted propolis extracts (1:10) and in the majority of cases in a dose-dependent way. Most propolis samples collected at springtime showed higher antibacterial activity, in comparison with samples harvested at wintertime. There were also observed differences between collection sites and type of extract. These results correlate to HPLC results, where the same differences where observed. Regarding propolis citotoxicity, results showed that aqueous propolis extracts have no effect and ethanolic propolis extracts causes a small decrease in cell viability. Concerning antioxidant enzymatic activities, the superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GP) activities of samples of propolis were determined. Results showed that SOD activity was dependent on the collection time and decreased drastically from winter to spring in samples from B.N. Arrodeios and B.S. Arneijoafra. The opposite was observed in samples from B.N. Pé da Serra and T.N. Madeira. We expected that the samples with higher SOD activities would have higher CAT activities also. Such was not observed, which may suggest that there could be other antioxidant enzymes involved, different to the ones tested. In respect with protein contents, major differences were not observed when comparing samples collected at different times, except for samples from B.S. Arneijoafra, where a decrease occurred from the sample collected at winter to sample collected at spring time. This was the first study of the biological activities of Portuguese propolis from the Algarve region

Sustained gene expression in the retina by improved episomal vectors

Gene and cellular therapies are nowadays part of therapeutic strategies for the treatment of diverse pathologies. The drawbacks associated with gene therapy-low levels of transgene expression, vector loss during mitosis, and gene silencing-need to be addressed. The pEPI-1 and pEPito family of vectors was developed to overcome these limitations. It contains a scaffold/matrix attachment region, which anchors its replication to cell division in eukaryotic cells while in an extrachromosomal state and is less prone to silencing, due to a lower number of CpG motifs. Recent success showed that ocular gene therapy is an important tool for the treatment of several diseases, pending the overcome of the aforementioned limitations. To achieve sustained gene delivery in the retina, we evaluated several vectors based on pEPito and pEPI-1 for their ability to sustain transgene expression in retinal cells. These vectors stably transfected and replicated in retinal pigment epithelial (RPE) cells. Expression levels were promoter dependent with constitutive promoters cytomegalovirus immediate early promoter (CMV) and human CMV enhancer/human elongation factor 1 alpha promoter yielding the highest levels of transgene expression compared with the retina-specific RPE65 promoter. When injected in C57Bl6 mice, transgene expression was sustained for at least 32 days. Furthermore, the retina-specific RPE65 promoter showed higher efficiency in vivo compared to in vitro. In this study, we demonstrate that by combining tissue-specific promoters with a mitotic stable system, less susceptible to epigenetic silencing such as pEPito-based plasmids, we can achieve prolonged gene expression and a sustained therapeutic effect.Fundacao para a Ciencia e Tecnologia, Portugal [PEst/OE/EQB-LA 0023/2013, SFRH/BD/76873/2011, SFRH/BD/70318/2010, PTDC/SAU/BEB/098475/2008]; European Union [PIRG-GA-2009-249314

Do difficulties in emotion regulation impact self-esteem and adult attachment? – the role of trauma

Abstract in proceedings of the Fourth International Congress of CiiEM: Health, Well-Being and Ageing in the 21st Century, held at Egas Moniz’ University Campus in Monte de Caparica, Almada, from 3–5 June 2019.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.info:eu-repo/semantics/publishedVersio