Abordagens de engenharia de tecidos para reparação de lesões vertebromedulares: hydrogéis e células estaminais mesenquimais

Dissertação de mestrado em Ciências da SaúdePatients with Spinal Cord Injury (SCI) still face physical, psychological and economic problems, since current treatments are unsatisfactory regarding regenerative therapies that translate into a complete and functional recovery. Numerous strategies have been arising for the treatment of this pathology, but single therapy approaches demonstrated to be insufficient for the successful repair of SCI. Therefore, combinatorial approaches that act in a synergistic arrangement, may help to overcome this problem, as a combination of techniques will avoids the limitations of using a single individual technique. Thus, in the present study, it was evaluated the efficacy of three natural hydrogels: Gellan-Gum (functionalized with a fibronectin peptide), Collagen and NVR-Gel in promoting neuritogenesis, using an in vitro model of axonal regeneration based on Dorsal Root Ganglia explants (DRGs). Secondly, it was assessed the effects of combining the three hydrogels with Adipose tissue-MSCs (ASCs) and the effects on the regenerative properties of their secretome, using cocultures of ASCs with the DRGs explants, on the different hydrogels. Then, a biomolecular approach using GDNF (Glial cell-derived Neurotrophic Factor) covalently attached to iron oxide nanoparticles (NPs) was tested, to determine their effect on axonal growth and length, using the same in vitro model. Lastly, it was evaluated the combined use of the NPs with the ASCs and the hydrogels, to assess the sustainability of this tissue engineering approach for SCI. This work demonstrated that the use of hydrogels supports both axonal growth and ASCs survival, providing a permissive matrix for the regrowth of new axons and enhances the known regenerative potential of the Mesenchymal like Stem Cells isolated from the adipose tissue (ASCs). The results also reveal the importance of having biological cues, such as ECM molecules, to promote a more effective and pronounce effect on both the ASCs and DRGs cultures. Moreover, the interaction of the ASCs with the collagen affected positively the ASCs’ viability, morphology and secretome, besides promoting the highest axonal growth of the three matrices, indicating that this is a strong strategy for SCI applications. Concerning the biomolecular approach, results demonstrated an improvement by both GDNF-NPs and soluble GDNF, confirming the positive effects of using neurotrophic support. However, it was observed that the NPs had a deleterious effects on the ASCs paracrine actions, a fact that should be further investigated before their combined use. This thesis established that ASCs-Collagen interaction is sufficient to significantly improve the axonal growth, and that sometimes “less is more”, as the same group induced a higher neuritogenesis than the combinatorial approach with the NPs. A deeper understanding of the different interactions might give new insights for tissue engineering approaches, and perhaps getting one step closer to a successful and functional recovery from SCI.Pacientes com Lesões Vertebro-Medulares (LVM) ainda enfrentam problemas físicos, psicológicos e económicos, uma vez que os tratamentos atuais são insatisfatórios no que toca a terapias regenerativas que levem a uma recuperação completa e funcional. Várias estratégias têm surgido para o tratamento desta patologia, no entanto, terapias individuais mostraram-se insuficientes em reparar com sucesso as LVM. Por essa razão, abordagens combinatórias que atuam de forma sinergistica, ajudam a superar este problema, uma vez que uma combinação de técnicas melhora ou evita as limitações da utilização de uma técnica individual. Assim sendo, neste estudo foi avaliada a eficácia de três hidrogéis naturais: Gellan-Gum (funcionalizado com um peptídeo de fibronectina), colágenio e NVR-Gel na promoção de neuritogénese, usando um modelo in vitro de crescimento axonal baseado em explantes de Gânglios da Raiz Dorsal (DRGs). Em segundo lugar, foi avaliado os efeitos combinados dos hidrogéis com as células mesenquimais do tecido adiposo (ASCs), e os efeitos nas propriedades regenerativas do seu secretoma, usando co-culturas de ASCs com os explantes de DRGs, sobre os diferentes hidrogéis. Em seguida, foi testada uma abordagem biomecular, em que o GDNF (Factor Neurotrófico derivado de Células Gliais) foi covalentemente ligado a nanoparticulas (NPs) de óxido de ferro, para determinar o seu efeito no crescimento e comprimento axonal, usando o mesmo modelo in vitro. Por último, foi avaliado o uso combinado das NPs com as ASCs nos hidrogéis, para avaliar a sustentabilidade desta abordagem de engenharia de tecidos para reparar LVM. Este trabalho demonstrou que o uso de hidrogéis auxila o crescimento axonal e a sobrevivência das ASCs, fornece uma matriz que é permissiva para o crescimento de novos axónios e aumenta o conhecido potencial regenerativo das células estaminais mesenquimais. Os resultados também revelam a importância de ter pistas biológicas, tais como moléculas da matriz extracelular, para promover um efeito mais eficaz e pronunciado tanto nas culturas de ASCs como nas de DRGs. Além disso, a interação das ASC com o colágenio afetou positivamente a viabilidade das ASCs, a sua morfologia e o seu secretoma, além de promover o maior crescimento axonal das três matrizes, indicando que esta é uma forte estratégia para aplicações em LVM. Em relação à abordagem biomolecular, os resultados demonstraram uma melhoria do GDNF tanto nas NPs como na forma solúvel, confirmando os efeitos positivos de utilizar suporte neurotrófico. No entanto, observouse que as NPs tiveram efeitos prejudiciais nas ações parácrinas das ASCs, algo que deve ser investigado antes da sua utilização combinada. Esta tese estabeleceu que a interação ASC-colágenio é suficiente para melhorar significativamente o crescimento axonal, e que às vezes "menos é mais", visto que este último grupo induziu uma maior neuritogénese do que a abordagem combinatória com as NPs. Uma compreensão mais profunda das diferentes interações pode dar novas perspectivas para abordagens de engenharia de tecidos, e talvez ficar um passo mais perto de uma recuperação bem sucedida e funcional das LVM

Development of conjugated kefiran-chondroitin sulphate cryogels with enhanced properties for biomedical applications

Hydrogels based on natural polysaccharides can have unique properties and be tailored for several applications, which may be mainly limited by the fragile structure and weak mechanical properties of this type of system. We successfully prepared cryogels made of newly synthesized kefiran exopolysaccharide-chondroitin sulfate (CS) conjugate via carbodiimide-mediated coupling to overcome these drawbacks. The freeze-thawing procedure of cryogel preparation followed by lyophilization is a promising route to fabricate polymer-based scaffolds with countless and valuable biomedical applications. The novel graft macromolecular compound (kefiran-CS conjugate) was characterized through 1H-NMR and FTIR spectroscopy—which confirmed the structure of the conjugate, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA)—which mirrored good thermal stability (degradation temperature of about 215 °C) and, finally, gel permeation chromatography–size exclusion chromatography (GPC-SEC)—which proved an increased molecular weight due to chemical coupling of kefiran with CS. At the same time, the corresponding cryogels physically crosslinked after the freeze-thawing procedure were investigated by scanning electron microscopy (SEM), Micro-CT, and dynamic rheology. The results revealed a prevalent contribution of elastic/storage component to the viscoelastic behavior of cryogels in swollen state, a micromorphology with micrometer-sized open pores fully interconnected, and high porosity (ca. 90%) observed for freeze-dried cryogels. Furthermore, the metabolic activity and proliferation of human adipose stem cells (hASCs), when cultured onto the developed kefiran-CS cryogel, was maintained at a satisfactory level over 72 h. Based on the results obtained, it can be inferred that the newly freeze-dried kefiran-CS cryogels possess a host of unique properties that render them highly suitable for use in tissue engineering, regenerative medicine, drug delivery, and other biomedical applications where robust mechanical properties and biocompatibility are crucial.This research was funded by the Foundation for Science and Technology (FCT) from Portugal, with references CEECIND/00111/2017 and SFRH/BPD/94277/2013, respectively.H.R. and C.G. were supported by the Foundation for Science and Technology (FCT) from Portugal, with references CEECIND/00111/2017 and SFRH/BPD/94277/2013, respectively. The authors would like to express their sincere gratitude to Emanuel Fernandes for his invaluable contribution to this research by performing the STA experiment. The authors thank the financial support provided under the project “HEALTH-UNORTE: Setting-up biobanks and regenerative medicine strategies to boost research in cardiovascular, musculoskeletal, neurological, oncological, immunological and infectious diseases”, reference NORTE-01-0145-FEDER-000039, funded by the Norte Portugal Regional Coordination and Development Commission (CCDR-N), under the NORTE2020 Program

PAMAM dendrimers functionalised with an anti-TNF α antibody and chondroitin sulphate for treatment of rheumatoid arthritis

"Available online 6 January 2021"Rheumatoid arthritis is a chronic autoimmune disease characterised by joint synovial inflammation, along with cartilage and bone tissue destruction. Dendrimers can offer new opportunities as drug delivery systems of molecules of interest. Herein we aimed to develop poly(amidoamine) dendrimers (PAMAM), functionalised with chondroitin sulphate (CS), lined with anti-TNF α antibodies (Abs) to provide anti-inflammatory properties. Physicochemical characterisation demonstrated that anti-TNFα Abs-CS/PAMAM dendrimer NPs were successfully produced. The in vitro studies revealed that CS/PAMAM dendrimer NPs did not affect the ATDC5 and THP-1 cell lines' metabolic activity and proliferation, presenting good cytocompatibility and hemocompatibility. Moreover, anti-TNFα Abs-CS/PAMAM dendrimer NPs showed suitable TNF α capture capacity, making them appealing for new immunotherapies in RA patients.The authors thank the financial support under the Norte2020 project (“NORTE-08-5369-FSE-000044”) and BD/137726/2018/J6 21340zkMF. The FCT distinction attributed to J. M. O. under the Investigator FCT program (number IF/01285/2015) is also greatly acknowledged. C. G. also wished to acknowledge FCT for supporting her research (No. SFRH/BPD/94277/2013). RS and AG-F thank Xunta de Galicia (Grupo de Referencia Competitiva, ED431C 2016041) and Centro de Investigaciones Biom ́edicas (CINBIO), Vigo, Spain, for sup-porting their research

Physiological characterization of Colletotrichum gloeosporioides isolates from Mangifera indica

A antracnose tem sido relatada como a principal doença pré e pós-colheita da manga, atacando folhas e frutos. Este trabalho objetivou realizar a caracterização fisiológica e patogênica de cinco isolados de Colletotrichum gloeosporioides da mangueira cv. Amrapali. Para a caracterização fisiológica, os isolados foram cultivados sob diferentes regimes de luz (0h, 12h e 24h) a 25°C, em que medições diárias até o 6 DAI (Dias após inoculação) foram feitas para obtenção de medias de crescimento micelial, e aos 6 DAI quantificação de esporos. Em seguida foi avaliado o potencial patogênico dos isolados através de medições de lesões na face adaxial das folhas aos 2, 4, 6, 8 e 10 DAI. Ambas as avaliações foram conduzidas em delineamento inteiramente casualizado. Isolados de C. gloeosporioides cultivados sob o regime de 12 horas luz e sob a temperatura de 25°C obtiveram melhor desenvolvimento fisiológico e o maior desenvolvimento patogênico no regime de 0 horas de luz.Anthracnose comprises in the main pre- and post-harvest disease of mango, causing damage on the leaves and fruits. This work aimed to carry out a physiological and pathogenic characterization of five isolates of Colletotrichum gloeopsorioides from mango trees cv. Amrapali. For the physiological characterization, the isolates were cultivated under different light regimes (0h, 12h and 24h) at 25°C, wherein daily measurements were taken up to 6 DAI (Days after inoculation) to obtain mycelial growth averages, and at 6 DAI spore quantification. Then, the pathogenic potential of the isolates was evaluated, through measurements of lesions on the top surface of the leaves at 2, 4, 6, 8 and 10 DAI. Both evaluations were carried out in a completely randomized design. Isolates of C. gloeosporioides cultivated under 12 hours light at temperature of 25°C obtained the best physiological development and the highest pathogenic development was observed in the regime of 0 hours light

Os principais tipos e manifestações da Cirrose Hepática: uma atualização clínica

Introdução: A cirrose hepática é um processo patológica crônico, considerado a hepatopatia mais comum, definido como a conversão difusa morfoestrutural por nódulos de arquitetura anômalo envoltos por fibrose. Objetivou-se descrever os tipos mais relevantes de cirrose e suas devidas manifestações. Metodologia: Trata-se de uma revisão bibliográfica, fundamentada nas plataformas do SciELO, PubMed, Scopus, utilizando os termos “hepatical cirrhosis”, “liver disease” e “hepatocellular insufficiency” a qual através da revisão narrativa, abordou amplamente a respeito da contextualização da cirrose e as principais etiologias. Resultados e Discussão: Foi analisado que tal condição afeta qualquer faixa etária, sexo, etnia e independe da classe socioeconômica, mas as diversas etiologias impõem um perfil epidemiológico específico conforme a aparição. As principais origens abordam o tipo alcoólico, hepatite, aplicação crônica de alguns fármacos e esteatose gordurosa ou não. Ademais, estima-se que estas afetam a anatomofuncionalidade do órgão responsável por grande parte da homeostase, culminando em diversas manifestações clínicas.  Conclusão: A cirrose é uma consequência grave de fatores de base em estágio avançado, a qual devido ao seu curso geralmente silencioso culmina no desenvolvimento e progressão clínica. Neste contexto, a atenção aos fatores predisponentes como alimentação rica em lipídios, estilismo, negligência a exames de rotina, sedentarismo e obesidade contribuem constituem medidas eficazes de prevenção primária.&nbsp

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Educomunicação, Transformação Social e Desenvolvimento Sustentável

Esta publicação apresenta os principais trabalhos dos GTs do II Congresso Internacional de Comunicação e Educação nos temas Transformação social, com os artigos que abordam principalmente Educomunicação e/ou Mídia-Educação, no contexto de políticas de diversidade, inclusão e equidade; e, em Desenvolvimento Sustentável os artigos que abordam os avanços da relação comunicação/educação no contexto da educação ambiental e desenvolvimento sustentável

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Manganese-labeled alginate hydrogels for image-guided cell transplantation

Cell transplantation has been studied extensively as a therapeutic strategy for neurological disorders. However, to date, its effectiveness remains unsatisfactory due to low precision and efficacy of cell delivery; poor survival of transplanted cells; and inadequate monitoring of their fate in vivo. Fortunately, different bio-scaffolds have been proposed as cell carriers to improve the accuracy of cell delivery, survival, differentiation, and controlled release of embedded stem cells. The goal of our study was to establish hydrogel scaffolds suitable for stem cell delivery that also allow non-invasive magnetic resonance imaging (MRI). We focused on alginate-based hydrogels due to their natural origin, biocompatibility, resemblance to the extracellular matrix, and easy manipulation of gelation processes. We optimized the properties of alginate-based hydrogels, turning them into suitable carriers for transplanted cells. Human adipose-derived stem cells embedded in these hydrogels survived for at least 14 days in vitro. Alginate-based hydrogels were also modified successfully to allow their injectability via a needle. Finally, supplementing alginate hydrogels with Mn ions or Mn nanoparticles allowed for their visualization in vivo using manganese-enhanced MRI. We demonstrated that modified alginate-based hydrogels can support therapeutic cells as MRI-detectable matrices.This work was supported by NanoTech4ALS (ref. ENMed/0008/2015, 13/EuroNanoMed/ 2016), funded under the EU FP7 M-ERA.NET program, and European Social Fund (POWR.03.02.00- 00-I028/17-00). Eduarda P. Oliveira acknowledges the Ph.D. scholarship (SFRH/BD/137726/2018), attributed by the Portuguese Foundation for Science and Technology (FCT). Authors also acknowledge the R&D Project KOAT—Kefiran exopolysaccharide: Promising biopolymer for use in regenerative medicine and tissue engineering, with reference PTDC/BTMMAT/29760/2017, financed by FCT and co-financed by FEDER and POCI.