Using elastin-like recombinamers as scaffolds for tissue engineering and regenerative medicine in the context of skin regeneration

Dissertação de mestrado integrado em Engenharia Biomédica (especialização em Biomateriais, Reabilitação e Biomecânica)As lesões crónicas resultam do comprometimento do processo de cicatrização. O envelhecimento da população e o aparecimento de comorbidades que prejudicam a regeneração da pele contribuem para o aumento da incidência de lesões crónicas, sendo agora considerado uma epidemia. Como as terapias atuais apresentam desvantagens relevantes no tratamento de lesões crónicas, existe a necessidade clínica para o desenvolvimento de soluções eficazes que tratem as lesões crónicas a curto prazo, reduzindo a falha e a carga económica destas terapias. Assim, esta dissertação visava a criação de um scaffold de acordo com os princípios da Engenharia de Tecidos (TE) para promover a regeneração da pele utilizando elastin-like recombinamers (ELRs). Estas proteínas são polímeros geneticamente modificados com base na sequência natural da elastina, a qual é uma importante proteína estrutural da pele. Como este biomaterial combina as vantagens das proteínas recombinantes e as propriedades da elastina, foram criados dois polímeros recombinantes, SKS-IKVAV e SKS-PPFLM, os quais resultam de duas modificações diferentes da proteína SKS. Os ELRs foram concebidos e expressos na Technical Proteins Nanobiotechnology (TPNBT) S.L., como parte de um estágio ERASMUS (Contrato Financeiro 2020-1-PT01-KA103-077707). Apesar dos resultados favoráveis em relação à expressão proteica, não foi possível desenvolver um protocolo de purificação destes polímeros durante o estágio ERASMUS na TPNBT, o que inviabilizou a construção de scaffolds e concluir se o SKS-IKVAV e/ou o SKS-PPFLM podem ser utilizados na TE da pele. Uma vez que as proteínas SKS-IKVAV e SKS-PPFLM não puderam ser utilizadas para a construção de scaffolds, uma nova abordagem, utilizando a proteína STAR, foi testada no Centro de Engenharia Biológica da Universidade do Minho para o desenvolvimento de soluções de TE da pele. Esta proteína demonstra características semelhantes às dos ELRs, e foi utilizada para a criação de filmes à base de proteínas. Estes materiais baseados na proteína STAR foram caracterizados fisicamente, quimicamente e in vitro e a potencial utilização dos mesmos na regeneração da pele foi favoravelmente concluída. As perspetivas futuras dos filmes à base de STAR incluem aprofundar o conhecimento do seu comportamento in vitro e a incorporação de substâncias bioativas com propriedades anti-inflamatórias, anti-oxidantes e antibacterianas, para combater as principais causas do desenvolvimentos de lesões crónicas.Chronic wounds result from a disruption in the wound healing process. The aging of population and the appearance of comorbidities that jeopardize skin regeneration contribute for an increase on chronic wounds incidence being now considered as an epidemic. Since current therapies present significant drawbacks in the treatment of chronic wounds, there is a clinical need to develop effective solutions that treat chronic wounds in the short term, reducing failure and the economic burden of these therapies. As a result, the goal of this dissertation was to design a scaffold based on Tissue Engineering (TE) principles to promote skin regeneration using elastin-like recombinamers (ELRs). These proteins are genetically modified polymers based on the sequence of natural elastin, which is a structural protein found in the skin. Since this biomaterial combines the benefits of recombinant proteins with the properties of elastin, two recombinant polymers, SKS-IKVAV and SKS-PPFLM, were created from two different modifications of the SKS protein. The elastin-like recombinamers were designed and expressed at Technical Proteins Nanobiotechnology (TPNBT) S.L., as part of an ERASMUS traineeship (Financial Agreement 2020-1- PT01-KA103-077707). Despite the promising results regarding protein expression, it was not possible to develop a purification protocol for these polymers during the ERASMUS trainee in TPNBT, making the construction of the scaffolds and determining whether SKS-IKVAV and/or SKS-PPFLM can be used in skin TE impossible. Since the SKS-IKVAV and SKS-PPFLM proteins could not be used for scaffold design, a new approach, using the STAR protein, was tested at the University of Minho's Centre of Biological Engineering, for the development of solutions for skin TE. This protein shows similar properties to ELRs and was used to create protein-based films. These STAR-based materials were physically, chemically, and in vitro characterized, and their potential use in skin regeneration was concluded favorably. The future prospects for STAR-based films will include greater understanding of their in vitro behavior, as well as the incorporation of bioactive substances with anti-inflammatory, antioxidant, and antibacterial properties to combat the primary causes of chronic injury development.I would also like to express my deepest gratitude to Professor José Carlos Rodríguez Cabello for allowing me to develop part of this project at Technical Proteins NanoBiotechnology, S.L. during my ERASMUS traineeship (Financial Agreement 2020-1-PT01-KA103-077707)

Global characterization of the Quercus suber ectomycorrhizal transcriptome using 454 pyrosequencing

In temperate forests, trees live in symbiosis with fungi and depend on them for their survival. Species from the Fagaceae family, including oaks, establish a symbiotic relationship in their roots with basidiomycetous fungi, called ectomycorrhizas (ECM). ECM symbiosis is essential for the life and health of trees in temperate and boreal forests where it plays a major role in nutrient cycling and in functioning of the forest ecosystem. Trees with well developed ECM root tips are more tolerant to environmental stresses, such as drought, and iotic stresses such as root pathogens. There is a general agreement that ECM fungi increase plant survival and productivity. Cork oak is well adapted to water scarcity typical of the Mediterranean summer, due to a root system that can reach several metres in depth, and to the abundance of roots at the soil surface associated with ECM. The development of ECM symbiosis is characterized by the successive development of three structural components: a mantle of fungal tissue that encloses the root, the intraradical network of hyphae (Hartig net), where plant and fungus exchange metabolites, and the extraradical mycelium, which extends into the soil and is responsible for nutrient and water uptake. All these processes are highly regulated and are accompanied by alterations on gene expression in both partners. The current project is integrated in the effort for covering the transcriptome of Quercus suber and is focused on the identification of ESTs involved in the development of ECM symbiosis in cork oak. Since only 236 ESTs from Quercus suber are currently known, the use of genomics approaches for gene discovery or functional studies is far from being achieved for this plant species.This work was supported by the FCT project SOBREIRO/0034/200

Involvement of reactive oxygen species during early stages of ectomycorrhiza establishment between Castanea sativa and Pisolithus tinctorius

Evidence for the participation of reactive oxygen species (ROS) and antioxidant systems in ectomycorrhizal(ECM) establishment is lacking. In this paper, we evaluated ROS production and the activities of superoxide dismutase (SOD) and catalase (CAT) during the early contact of the ECM fungus Pisolithus tinctorius with the roots of Castanea sativa (chestnut tree). Roots were placed in contact with P. tinctorius mycelia, and ROS production was evaluated by determining the levels of H2O2 and O2 I− during the early stages of fungal contact. Three peaks of H2O2 production were detected, the first two coinciding with O2 I− bursts. The first H2O2 production peak coincided with an increase in SOD activity, whereas CAT activity seemed to be implicated in H2O2 scavenging. P. tinctorius growth was evaluated in the presence of P. tinctorius-elicited C. sativa crude extracts prepared during the early stages of fungal contact. Differential hyphal growth that matched the H2O2 production profile with a delay was detected. The result suggests that during theearly stages of ECM establishment, H2O2 results from an inhibition of ROS-scavenging enzymes and plays a role in signalling during symbiotic establishment

Propriedade Intelectual e transferência de tecnologia

São analisadas as mudanças propostas nas relações comerciais e econômicas internacionais, com especial destaque a área da Propriedade Intelectual, atualmente debatidas no âmbito da OMPI e da GATT. Os novos temas trazidos a discussão e o ordenamento institucional defendido pelos países desenvolvidos afetara mio somente os governos como, principalmente, as empresas, representando serias ameaças ao fluxo de transferência de tecnologias para os países em desenvolvimento

Efficient adeno-associated virus serotype 5 capture with affinity functionalized nanofiber adsorbents

Funding Information: The authors acknowledge the funding of Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), projects PTDC/EQU-EQU/0142/2020 and EXPL/EQU-EQU/1567/2021; SN is the recipient of an FCT fellowship from the project PTDC/EQU-EQU/0142/2020. Publisher Copyright: Copyright © 2023 Neto, Mendes, Santos, Solbrand, Carrondo, Peixoto and Silva.Adeno-associated viruses (AAVs) are one of the most promising tools for gene therapy applications. These vectors are purified using affinity and ion exchange chromatography, typically using packed beds of resin adsorbents. This leads to diffusion and pressure drop limitations that affect process productivity. Due to their high surface area and porosity, electrospun nanofiber adsorbents offer mass transfer and flow rate advantages over conventional chromatographic media. The present work investigated the use of affinity cellulose-based nanofiber adsorbents for adeno-associated virus serotype 5 (AAV5) capture, evaluating dynamic binding capacity, pressure drop, and AAV5 recovery at residence times (RT) less than 5 s. The dynamic binding capacity was found to be residence time-dependent, but nevertheless higher than 1.0 × 1014 TP mL−1 (RT = 1.6 s), with a pressure drop variation of 0.14 MPa obtained after loading more than 2,000 column volumes of clarified AAV5 feedstock. The single affinity chromatography purification step using these new affinity adsorbents resulted in 80% virus recovery, with the removal of impurities comparable to that of bead-based affinity adsorbents. The high binding capacity, virus recovery and reduced pressure drop observed at residence times in the sub-minute range can potentially eliminate the need for prior concentration steps, thereby reducing the overall number of unit operations, process time and costs.publishersversionpublishe

Acessibilidade dos edifícios destinados à prática de desporto nas pessoas com deficiência - intervenção dos/as enfermeiros/as especialistas em reabilitação

Background: The nurse specialist in rehabilitation nursing is essential in the involvement of people with disabilities in sports practices. Its role is to intervene in deviations of health of the person and to evaluate the accessibility of the building. Objectives: Analyze architectural barriers in the building and analyze the differences taking into account the typology of the building and their location. Methodology: Quantitative paradigm of descriptive and transversal nature. A universe composed of 9 public sports buildings of a Municipality. The data collection instrument is an observation grid built from the legislation in force. The treatment of data using simple descriptive statistics and chi-square. Results: There are architectural barriers in buildings, but there have been no significant differences in accessibility conditions between pool and field buildings or between buildings located in the city or periphery Conclusion: In the municipality under study, people with disabilities have difficulty accessing suitable places to practice some sports activities. Rehabilitation nurses should evaluate the enclosures in relation to architectural barriers to make proper counseling and sensitization to the local authority in order to make it more inclusive.Enmarcado: El enfermero especialista en enfermería de rehabilitación es imprescindible en la participación de la persona con discapacidad en prácticas deportivas. Su papel es intervenir en desvíos de salud de la persona y aún evaluar las accesibilidades del edificado. Objetivos: Analizar las barreras arquitectónicas y comprobar si la tipología del edificio (con piscina y con campo) el la ubicación de los pueblos (ciudad y periferia) interfiere en las condiciones de accesibilidad. Metodología: Paradigma cuantitativo de naturaleza descriptiva y transversal. Universo compuesto por 9 edificios deportivos públicos de un Municipio. El instrumento de recolección de datos es una rejilla de observación construida a partir de la legislación vigente. El tratamiento de datos con la estadística descriptiva simple y chi-cuadrada Resultados: Existen barreras arquitectónicas en los edificios, pero no se observaron diferencias significativas con respecto a las condiciones de accesibilidad entre edificios con piscina y con campos ni entre edificios ubicados en la ciudad o periferia Conclusión: En el municipio en estudio las personas con discapacidad tienen dificultades de acceso a lugares adecuados para la práctica de algunas actividades deportivas. Las enfermeras de rehabilitación deben evaluar los recintos sobre las barreras arquitectónicas para hacer el debido asesoramiento y sensibilización ante el municipio, a fin de hacerla más inclusiva.Enquadramento: O/A enfermeiro/a especialista em enfermagem de reabilitação tem um papel fundamental no envolvimento da pessoa com deficiência em práticas desportivas. O seu papel é intervir em desvios de saúde da pessoa e ainda avaliar as acessibilidades do edificado. Objetivos: Analisar barreiras arquitetónicas no edificado e analisar as diferenças tendo em conta a tipologia do edifício e a localização dos mesmos. Metodologia: Paradigma quantitativo de natureza descritiva e transversal. Universo composto por 9 edifícios desportivos públicos dum Município Português. O instrumento de colheita de dados é uma grelha de observação construída a partir da legislação em vigor. O tratamento de dados fez-se com recurso a estatística descritiva simples e qui-quadrado. Resultados: Existem barreiras arquitetónicas nos edifícios como escadas com degraus e corrimãos inadequados, instalações sanitárias com lavatórios e sanitas sem alturas corretas, entre outras, mas não se verificaram diferenças significativas relativamente às condições de acessibilidade entre edifícios com piscina e com campos nem entre edifícios localizados na cidade ou periferia.  Conclusão: No município em estudo as pessoas com deficiência têm dificuldades de acesso a locais adequados para a prática de algumas atividades desportivas. As/os enfermeiros/as de reabilitação devem avaliar os recintos relativamente às barreiras arquitetónicas para fazer o devido aconselhamento e sensibilização junto da autarquia, a fim de a tornar mais inclusiva

“A beautiful mind” – Promoção da literacia em saúde mental

Introdução: Associado à iliteracia do que fazer para prevenir, retardar e quando procurar ajuda, perante a esquizofrenia, requer-se ao enfermeiro especialista em saúde mental um agir ético, alicerçado no conhecimento empírico, técnico-científico, envolvendo os deveres profissionais: dignidade, individualidade e autonomia. Objetivo: Promover a literacia em saúde mental, através da análise do adoecer de uma pessoa com esquizofrenia e das questões éticas associadas ao cuidar em enfermagem de saúde mental. Metodologia: Estudo de caso - filme “Uma Mente Brilhante”, de natureza qualitativa, exploratória-descritiva. Resultados e Discussão: A esquizofrenia transforma a forma de pensar, de sentir e de relação com as pessoas, conduzindo à estigmatização associada a mitos conectivos entre a doença mental e a violência, contribuindo para a dificuldade/ exclusão no emprego, apoio social e procura de ajuda. Conclusão: Salienta-se a promoção da literacia do agir perante uma pessoa com esquizofrenia, e do cuidado ético em enfermagem inerente.info:eu-repo/semantics/publishedVersio

Oak root response to ectomycorrhizal symbiosis establishment: RNA-Seq derived transcript identification and expression profiling

Ectomycorrhizal symbiosis is essential for the life and health of trees in temperate and boreal forests where it plays a major role in nutrient cycling and in functioning of the forest ecosystem. Trees with ectomycorrhizal root tips are more tolerant to environmental stresses, such as drought, and biotic stresses such as root pathogens. Detailed information on these molecular processes is essential for the understanding of symbiotic tissue development in order to optimize the benefits of this natural phenomenon. Next generation sequencing tools allow the analysis of non model ectomycorrhizal plant-fungal interactions that can contribute to find the "symbiosis toolkits" and better define the role of each partner in the mutualistic interaction. By using 454 pyrosequencing we compared ectomycorrhizal cork oak roots with non-symbiotic roots. From the two cDNA libraries sequenced, over 2 million reads were obtained that generated 19,552 cork oak root unique transcripts. A total of 2238 transcripts were found to be differentially expressed when ECM roots were compared with non-symbiotic roots. Identification of up- and down-regulated gens in ectomycorrhizal roots lead to a number of insights into the molecular mechanisms governing this important symbiosis. In cork oak roots, ectomycorrhizal colonization resulted in extensive cell wall remodelling, activation of the secretory pathway, alterations in flavonoid biosynthesis, and expression of genes involved in the recognition of fungal effectors. In addition, we identified genes with putative roles in symbiotic processes such as nutrient exchange with the fungal partner, lateral root formation or root hair decay. These findings provide a global overview of the transcriptome of an ectomycorrhizal host root, and constitute a foundation for future studies on the molecular events controlling this important symbiosis.This work was funded by the Portuguese Foundation for Science and Technology (www.fct.pt) in the frame of the project Cork Oak EST Consortium SOBREIRO/0034/2009. Post-doc grant to MS was supported by the Portuguese Foundation for Science and Technology (SFRH/BPD/25661/2005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript