3D functionalized multi-supportive structures (FMSS): a novel route for spinal cord injury regeneration

Programa doutoral em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisUma lesão do sistema nervoso acarreta normalmente consequências devastadoras. No caso particular de lesões da medula espinal (LME), os pacientes têm que lidar com múltiplas insuficiências biológicas. Por este motivo, é urgente desenvolver terapias que possam resolver eficazmente este problema. Diferentes estratégias têm sido propostas para tratar esta condição, tais como, terapias baseadas em transplantação celular e/ou terapias moleculares. No entanto, os resultados não têm sido satisfatórios, reforçando a ideia que são necessárias abordagens alternativas. A engenharia de tecidos tem sido proposta como um novo método para enfrentar estes problemas. Esta estratégia implica o uso de uma estrutura 3D que é capaz de suportar o crescimento e a diferenciação celular de forma a criar um ambiente adequado para o desenvolvimento de um tecido funcional. Tendo por base os conceitos da engenharia de tecidos, o principal objectivo do trabalho descrito nesta tese foi o desenvolvimento de um sistema que possa ser aplicado facilmente e eficazmente no tratamento de LME. Neste trabalho, o polímero natural de poli-caprolactona de amido (SPCL) foi processado por prototipagem rápida de forma a criar estruturas tubulares porosas. Seguidamente, o interior das estruturas de SPCL foram preenchidos com o hidrogel gellan gum de forma a criar uma estrutura híbrida. Neste sistema, o SPCL tem como objectivo mimetizar as funções do osso vertebral. Por sua vez, o hidrogel tem como objectivo servir como um sistema de encapsulamento celular de forma a suportar a regeneração nervosa. As estruturas desenvolvidas possuem espessura, orientação e geometria do poro e características mecânicas configuráveis. Adicionalmente, as cytotoxic behavior and allowed the in vitro culturing of oligodendrocytes, olfactory ensheathing cells (OECs) and Schwann cells for periods up to three weeks. In order to overcome some of the drawbacks associated with the use of gellan gum, namely the absence of biological signals for cellular adhesion and proliferation, this hydrogel was chemically conjugated with the peptide sequence GRGDS, via Diels-Alder click chemistry. It was observed that the novel GRGDS-gellan gum had a profound effect on neural stem cell morphology and proliferation. These results demonstrated the importance of GRGDS for cell-gellan gum interaction. Subsequently, the in vivo experiments revealed that hybrid structures are biocompatible. However, more importantly, in vivo tests showed that SPCL are suitable structures to promote spine stabilization and that the stabilization of SCI rats was associated with functional motor recovery. Finally, in the scope of this thesis, it was also assessed the effects of OECs secretome on the growth of adult stem cells derived from adipose, bone marrow, umbilical cord and nervous tissue. Results revealed that OECs secretome increase the metabolic activity and/or proliferation of these adult stem cells. As a concluding remark, it can be stated that the work described in this thesis brings new knowledge to the cell biology, biomaterial and spinal cord injury fields. The results obtained in the in vivo experiments indicate that researchers currently testing treatments for SCI repair might have to take into account the use of spine stabilization in combination with their approaches. From the biomaterial point of view, the simplicity and broad applicability of the Diels-Alder click chemistry (used to functionalize the gellan gum) can be easily extended to other molecules to further improve this material. Finally, important understanding was herein created about biological/molecular interactions between OECs and adult stem cells from several origins.Uma lesão do sistema nervoso acarreta normalmente consequências devastadoras. No caso particular de lesões da medula espinal (LME), os pacientes têm que lidar com múltiplas insuficiências biológicas. Por este motivo, é urgente desenvolver terapias que possam resolver eficazmente este problema. Diferentes estratégias têm sido propostas para tratar esta condição, tais como, terapias baseadas em transplantação celular e/ou terapias moleculares. No entanto, os resultados não têm sido satisfatórios, reforçando a ideia que são necessárias abordagens alternativas. A engenharia de tecidos tem sido proposta como um novo método para enfrentar estes problemas. Esta estratégia implica o uso de uma estrutura 3D que é capaz de suportar o crescimento e a diferenciação celular de forma a criar um ambiente adequado para o desenvolvimento de um tecido funcional. Tendo por base os conceitos da engenharia de tecidos, o principal objectivo do trabalho descrito nesta tese foi o desenvolvimento de um sistema que possa ser aplicado facilmente e eficazmente no tratamento de LME. Neste trabalho, o polímero natural de poli-caprolactona de amido (SPCL) foi processado por prototipagem rápida de forma a criar estruturas tubulares porosas. Seguidamente, o interior das estruturas de SPCL foram preenchidos com o hidrogel gellan gum de forma a criar uma estrutura híbrida. Neste sistema, o SPCL tem como objectivo mimetizar as funções do osso vertebral. Por sua vez, o hidrogel tem como objectivo servir como um sistema de encapsulamento celular de forma a suportar a regeneração nervosa. As estruturas desenvolvidas possuem espessura, orientação e geometria do poro e características mecânicas configuráveis. Adicionalmente, as estruturas apresentaram um comportamento não tóxico e permitiram o crescimento in vitro de oligodendrócitos, células do bolbo olfactivo (OECs) e células de Schwann. De forma a ultrapassar algumas das desvantagens do gellan gum, nomeadamente o facto de este material não possuir na sua estrutura sinais biológicos, este material foi modificado com o péptido GRGDS. Foi observado que o GRGDS-gellan gum tem um forte efeito na morfologia e proliferação de células estaminais neuronais. Estes resultados demonstraram a importância do péptido GRGDS na interacção célula-gellan gum. Seguidamente, as experiencias in vivo revelaram que estas estruturas são biocompatíveis, mas, mais importante, os testes em animais demostraram que as estruturas de SPCL permitem a eficaz estabilização da coluna vertebral e que esta estabilização promove a recuperação da função motora de ratos com LME. Finalmente, no âmbito do trabalho descrito nesta tese, foi também estudado o efeito que os factores secretados pelas OECs exercem sobre a proliferação e/ou actividade metabólica de células estaminais adultas derivadas do tecido adiposo, da medula óssea, do cordão umbilical e do tecido nervoso. Os resultados revelaram que as OECs produzem factores que promovem o aumento da actividade metabólica e/ou proliferação dessas células estaminais adultas. O trabalho descrito nesta tese originou novo conhecimento para as áreas de biologia, de biomateriais e de regeneração de LME. As experiências in vivo revelaram que os investigadores que estejam actualmente a testar terapias para LME devem ter em conta a estabilização vertebral. Do ponto de vista dos biomateriais, a modificação do gellan gum é simples e de ampla aplicabilidade. Neste sentido, o gellan gum pode agora ser facilmente modificado com diferentes moléculas consoante a aplicação desejada. Finalmente, o entendimento sobre o efeito dos factores produzidos pelas OECs em células estaminais, poderá revelar-se importante para futuras terapias celulares

Combinatorial therapies for spinal cord injury: strategies to induce regeneration

The authors want to acknowledge the financial support from Prémios Santa Casa Neurociências ‐ Prize Melo e Castro for Spinal Cord Injury Research (MC-04/17); from Portuguese Foundation for Science and Technology [Doctoral fellowship (SFRH/BD/103075/2014) to EDG; Post‐ Doctoral fellowship (SFRH/BPD/97701/2013) to NAS; IF Development Grant to AJS]. This work is funded by national funds through FCT under the scope of grant reference TUBITAK/0007/2014 and 3599-PPCDT Project: PTDC/DTP-FTO/5109/2014. This article has been developed under the scope of the projects NORTE-01-0145- FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). This work has been funded by FEDER funds, through the Competitiveness Factors Operational Programme (COMPETE), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01- 0145-FEDER-007038.info:eu-repo/semantics/publishedVersio

Hydrogels and cell based therapies in spinal cord injury regeneration

Spinal cord injury (SCI) is a central nervous system- (CNS-) related disorder for which there is yet no successful treatment. Within the past several years, cell-based therapies have been explored for SCI repair, including the use of pluripotent human stem cells, and a number of adult-derived stem and mature cells such as mesenchymal stem cells, olfactory ensheathing cells, and Schwann cells. Although promising, cell transplantation is often overturned by the poor cell survival in the treatment of spinal cord injuries. Alternatively, the therapeutic role of different cells has been used in tissue engineering approaches by engrafting cells with biomaterials. The latter have the advantages of physically mimicking the CNS tissue, while promoting a more permissive environment for cell survival, growth, and differentiation. The roles of both cell- and biomaterial-based therapies as single therapeutic approaches for SCI repair will be discussed in this review. Moreover, as the multifactorial inhibitory environment of a SCI suggests that combinatorial approaches would be more effective, the importance of using biomaterials as cell carriers will be herein highlighted, as well as the recent advances and achievements of these promising tools for neural tissue regeneration.The authors would like to acknowledge the Portuguese Foundation for Science and Technology (Grant no. PTDC/SAU-BMA/114059/2009; IF Development Grant to António J. Salgado); Prémios Santa Casa Neurociências for funds attributed to António J. Salgado under the scope of the Prize Melo e Castro for Spinal Cord Injury Research; cofunded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER)

ISePorto Robotic Soccer Team: A New Player Generation

Proceedings of the Scientific Meeting of the Portuguese Robotics Open 2004This paper describes the recent modifications in ISePorto MSL robotic football team and future improvements concerning the development and evolution of the team. The robot was substantially redesigned in order to achieve high reliability, allow better control and coordination capabilities and substantial increase in perception. New mechanical and hardware redesign is presented. Motion control subsystems, new vision hardware sensor and overall architecture are described. The team redesign is done for preparation for participating in the Robocup 2004. The main goal is to achieve not only an important evolution in the team control and coordination but also increased overall reliability

Soil stabilisation using alkaline activation of fly ash for self-compacting rammed earth construction

This paper studies the effectiveness of alkaline activation of low-calcium fly ash on the improvement of residual granitic soils to be used on rammed-earth construction. Different liquid:solid ratios, alkali concentrations and Na2O : ash ratios were tested. Effect of calcium hidroxide, sodium chloride and concrete superplasticiser is also reported. Compressive strength up to 7 days at 60ºC was determined. Results show that, in terms of mechanical strength, there is an optimum value for the activator:solids ratio and the alkali concentration, and that a decrease in the Na2O:ash ratio results in strength increase. No improvement was observed with the sodium chloride or the superplasticiser, while the calcium produced only a short term increase in strength. SEM/EDS analysis were used to analyse microstructural development, showing that strength is fairly related to the Si:Al and Na:Si ratios

Rammed earth: feasibility of a global concept applied locally

Rammed earth is an ancient building technique that has been continuously reinvented in the dynamic movement of people all over the world, where it has been used to build from dwellings to enormous fortresses and city walls. In the particular case of Portugal, the inhabitants have been closely related to earth construction. From one region to another, rammed earth, adobe and wattle-and-daub buildings are frequently found. The rammed earth construction is mainly found in the southern part of Portugal and is almost absent from the north. However, the relatively low seismic hazard of the north of Portugal plus the sustainability of earth as a building material encourages the development of this technique in the region. The suitability of the typical granite residual soils from the Minho region for rammed earth construction was assessed by means of an experimental program, in which three representative soils were subjected to expeditious and laboratory tests that evaluated the characteristics of the soils and the performance of rammed earth built with them. The results showed that the granite residual soils tested fulfil most of the requirements needed for rammed earth construction, being the low compressive strength its main limitation. In this way, an innovative and sustainable stabilization technique based on alkaline activation of fly ash is proposed

Comportamento de blocos de terra comprimida estabilizados com resíduos industriais

The construction of masonry with compressed earth blocks (CEBs) is a modern earth construction technique with current growing interest, but its environmental sustainability is many times depreciated by the use of traditional chemical stabilisation techniques (lime and cement addition). This paper presents an experimental program where the possibility of manufacturing CEBs using granitic residual soils (GRS) from northern Portugal is addressed. The GRS were shown to be inadequate and their use for manufacturing CEBs requires chemical stabilisation. A composition study involving stabilisation by addition of cement and addition of geopolymeric binders resulting from the alkaline activation of fly ash or of calcinated sludge was carried out. Then, CEBs stabilised with alkaline activation of fly ash were manufactured and their mechanical properties were tested. In addition, the compressive behaviour of masonry built with these CEBs was also characterized. In general, the stabilisation with alkaline activation of fly ash revealed excellent results with respect to the improvement in strength.A construção em alvenaria de blocos de terra comprimida (BTC) é uma técnica de construção em terra moderna com um atual interesse crescente, mas a sua sustentabilidade ambiental é muitas vezes desvalorizada pelo recurso a técnicas de estabilização química tradicionais (adição de cal e cimento). Este artigo apresenta um programa experimental onde é avaliada a possibilidade de se manufaturarem BTC com solo residual granítico (SRG) do Norte de Portugal. Mostrou-se que os SRG não são adequados e que o seu uso na produção de BTC requer estabilização química. Realizou-se um estudo de composição envolvendo estabilização por adição de cimento e de ligantes geopoliméricos à base da ativação alcalina de cinza volante ou lamas calcinadas. Em seguida, manufaturam-se BTC estabilizados com ativação alcalina de cinzas volantes e testaram-se as suas propriedades mecânicas. Além disto, caraterizou-se o comportamento em compressão da alvenaria construída com estes BTC. Em geral, a estabilização com ativação alcalina de cinzas volantes revelou excelentes resultados no que diz respeito ao melhoramento da resistência

Structural and functional properties of the capsid protein of Dengue and related Flavivirus

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Dengue, West Nile and Zika, closely related viruses of the Flaviviridae family, are an increasing global threat, due to the expansion of their mosquito vectors. They present a very similar viral particle with an outer lipid bilayer containing two viral proteins and, within it, the nucleocapsid core. This core is composed by the viral RNA complexed with multiple copies of the capsid protein, a crucial structural protein that mediates not only viral assembly, but also encapsidation, by interacting with host lipid systems. The capsid is a homodimeric protein that contains a disordered N-terminal region, an intermediate flexible fold section and a very stable conserved fold region. Since a better understanding of its structure can give light into its biological activity, here, first, we compared and analyzed relevant mosquito-borne Flavivirus capsid protein sequences and their predicted structures. Then, we studied the alternative conformations enabled by the N-terminal region. Finally, using dengue virus capsid protein as main model, we correlated the protein size, thermal stability and function with its structure/dynamics features. The findings suggest that the capsid protein interaction with host lipid systems leads to minor allosteric changes that may modulate the specific binding of the protein to the viral RNA. Such mechanism can be targeted in future drug development strategies, namely by using improved versions of pep14-23, a dengue virus capsid protein peptide inhibitor, previously developed by us. Such knowledge can yield promising advances against Zika, dengue and closely related Flavivirus.This work was supported by “Fundação para a Ciência e a Tecnologia–Ministério da Ciência, Tecnologia e Ensino Superior” (FCT-MCTES, Portugal) project PTDC/SAU-ENB/117013/2010, Calouste Gulbenkian Foundation (FCG, Portugal) project Science Frontiers Research Prize 2010. A.F.F., A.S.M. and J.C.R. also acknowledge FCT-MCTES fellowships SFRH/BD/77609/2011, PD/BD/113698/2015 and SFRH/BD/95856/2013, respectively. I.C.M. acknowledges FCT-MCTES Programs “Investigador FCT” (IF/00772/2013) and “Concurso de Estímulo ao Emprego Científico” (CEECIND/01670/2017). This work was also supported by UID/BIM/50005/2019, project funded by Fundação para a Ciência e a Tecnologia (FCT)/ Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through Fundos do Orçamento de Estado.info:eu-repo/semantics/publishedVersio

Compressed earth blocks stabilized with glass waste and fly ash activated with a recycled alkaline cleaning solution

Sustainable alternatives are increasingly demanded as a sound response, from the construction industry, to the worldwide growing concerns with the environment. Such effort is justifiable by the degree of the contribution of this human activity to the problem, and it has thus propelled the development of a major trend in terms of funded research. The study reported in this paper focused on the physical-mechanical properties of compacted earth blocks formed by a common Portuguese silty clay (as the mineral skeleton), stabilized with a sustainable alkali activated cement exclusively produced from wastes and residues, including coal fly ash and glass waste, in a 50/50 wt ratio combination, and activated with an alkaline solution from the aluminium industry, using activator/precursor weight ratios of 0.50, 0.57 and 0.75. After optimising the alkaline activated cement (AAC), the AAC/Soil blocks were fabricated, using the response surface method to define their composition based on curing periods of 28 and 180 days at controlled ambient temperature. Uniaxial compressive strength (UCS) and several durability tests were performed, and the material was characterised using FTIR and SEM. The results evidenced the effectiveness of the alkaline cementing agent in forming a binding matrix for the soil particles. An average compressive strength of 17.23 MPa, in unsaturated conditions, was obtained for the blocks. The newly formed soil-binder structure was very capable to withstand wetting and drying cycles, ice-thaw cycles and erosion. The microstructure of the material was further analysed, using scanning electron microscopy and energy dispersive spectroscopy. The results demonstrated the real possibility of using this type of cement as a viable alternative to traditional soil stabilisation binders used in earth construction.This work was funded by the R&D Project JUSTREST-Development of Alkali Binders for Geotechnical Applications Made Exclusively from Industrial Waste, with reference PTDC/ECM-GEO/0637/2014, financed by the Foundation for Science and Technology - FCT/MCTES (PIDDAC).The research was supported by the GEO-DESIGN project, no17501, co-financed by the European Regional Development Fund (ERDF) through NORTE 2020 (North Regional Operational Program, 2014/2020)

Mechanical characterization of dry-stack masonry made of CEBs stabilised with alkaline activation

The increasing interest on earth construction as a sustainable building solution led to the development of modern earth construction techniques, in particular of masonry made of compressed earth blocks (CEBs). The traditional chemical stabilisation of the soil is a frequently used improvement process. However, such process increases significantly the embodied energy of the CEBs. This paper presents an alternative technique for the stabilisation of CEBs, based on alkali activation of fly ash. The mechanical behaviour of the CEBs and of the respective dry stack masonry is comprehensively investigated through an experimental program, during which this technique proved to be highly effective.The authors wish to express their gratitude to Mr. Pedro Esteves for his support in the experimental work and to Eng. Jorge Henriques, on behalf of the company PEGOP – Energia Eléctrica SA, for providing the fly ash. The financial support provided by the Portuguese Science and Technology Foundation through the project FCOMP-01-0124-FEDER-028864 (FCT-PTDC/ECM-EST/2396/2012) is also gratefully acknowledged.Fundação para a Ciência e a Tecnologia (FCT