Nanostructured 3D constructs based on chitosan and chondroitin sulphate multilayers for cartilage tissue engineering

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Biomateriais, Reabilitação e Biomecânica)Articular cartilage damage is a persistent and increasing problem with the aging population, and treatments to achieve biological repair remain a challenge. The lack of efficient modalities of treatments has prompted research into tissue engineering (TE). TE approaches are a promising strategy for improving the rate of repair of articular cartilage lesions by combining cells, biomaterials and microenvironmental factors. The work developed at this thesis was aimed to study the potential of nanostructured scaffolds based on layer-by-layer (LbL) methodology to be used in articular cartilage TE. The polyelectrolytes used for producing these structures were two polysaccharides: chitosan (polycation) and chondroitin sulphate (polyanion). These polyelectrolytes were chosen due to its biocompatibility and positive influence on cartilage. The sequential combination of these polyelectrolytes led to a decrease in frequency and increase in dissipation as seen using quartz microbalance (QCM), indicating the development of a multilayered film. For the proof of concept, biological assays were first performed in multilayered surfaces with bovine chondrocytes (bch). No cytotoxic effects of the film were found. Thus, the formation of a nanostructured film was transposed to a three-dimensional (3D) level combining LbL and leaching of spherical sacrificial templates. The homogenous distribution of polysaccharides in the scaffolds was confirmed by Fourier transformed infrared spectroscopy (FTIR). Morphological analysis of the scaffolds was based on scanning electron microscope (SEM), optical microscopy, and histology. The results showed a high porosity, which leads to swollen structure when immersed in phosphate buffered saline (PBS). The enzymatic degradation tests, using PBS and enzymatic solution with lysozyme and hyaluronidase, showed that the scaffold has a gradual degradation, and as expected the rate of degradation was higher in the enzymatic solution. Mechanical properties of the scaffold were evaluated using dynamic mechanical analysis (DMA). The results revealed that scaffold exhibit viscoelastic behaviour which corroborates the results obtained at QCM. The applicability of the nanostructured scaffold for cartilage was evaluated in cellular assays with bch and human mesenchymal cells (hMSCs). Tests of cell viability, SEM and quantification of DNA revealed that scaffolds promote cell adhesion and proliferation. Differentiation studies demonstrated the production of glycosaminoglycans (GAGs) by both cells. These results confirmed the maintenance of phenotype of bch and the chondrogenic differentiation of hMSCs. Thus, we believe the scaffolds developed may have potential use in cartilage TE approaches.Os danos da cartilagem articular são um problema persistente e crescente com o envelhecimento da população, pelo que os tratamentos para obter a reparação biológica continuam a ser um desafio. A falta de tratamentos eficientes impulsionou a investigação de estratégias de engenharia de Tecidos (TE), as quais são estratégias promissoras para melhorar a taxa de reparação de lesões da cartilagem articular, combinando células, biomateriais e factores microambientais. O trabalho desenvolvido nesta tese teve como objectivo estudar o potencial de “scaffolds” nanoestruturados baseados na metodologia de “layer-by-layer” (LbL) para estratégias de TE da cartilagem articular. Os materiais utilizados para a produção destas estruturas foram dois polissacarídeos: o quitosano (policatião) e o sulfato de condroítina (polianião). Estes foram seleccionados devido à sua biocompatibilidade e influência positiva na cartilagem. A combinação sequencial destes polielectrólitos leva a uma diminuição da frequência e aumento da dissipação na microbalança de quartzo (QCM), indicando o desenvolvimento de um filme com multicamadas. Para comprovar a aplicabilidade destas estruturas, primeiramente foram realizados ensaios biológicos em superfícies com multicamadas, utilizando condrócitos bovinos (bch). O filme produzido não apresentou qualquer efeito citotóxico. Assim, a formação de um filme nanoestruturado foi transposta para o nível tridimensional (3D) combinando LbL com lixiviação de partículas de sacrifício esféricas. A presença dos polissacarídeos nos “scaffolds” foi confirmada pela espectroscopia de infravermelhos por transformadas de Fourier (FTIR). A análise morfológica dos “scaffolds” foi baseada em microscopia electrónica de varrimento (SEM), microscopia óptica, bem como cortes histológicos. Os resultados obtidos demonstraram uma elevada porosidade, o que leva à dilatação da estrutura quando imersa em tampão fosfato salino (PBS). Os testes de degradação enzimática realizados, usando PBS e uma solução enzimática composta por lisozima e hialuronidase, demonstraram que a degradação dos “scaffolds” ocorreu de forma gradual e tal como esperado foi mais acentuada na solução enzimática. As propriedades mecânicas dos “scaffolds” foram avaliadas usando análise dinâmica mecânica (DMA). Os resultados revelaram que os “scaffolds” exibem um comportamento viscoelástico o que corrobora os resultados obtidos na QCM. A aplicabilidade destas estruturas para a cartilagem avaliou-se em ensaios celulares com bch e células humanas mesenquimais (hMSCs). Os testes de viabilidade celular, SEM e quantificação de DNA revelaram que os “scaffolds” promovem a adesão celular e proliferação. Estudos de diferenciação demonstraram a produção de glucosaminoglicanos (GAGs) de ambas as células. Estes resultados confirmam a manutenção de fenótipo dos bch e a diferenciação condrogénica de hMSCs. Desta forma, acreditamos que os “scaffolds” desenvolvidos terão uma potencial utilização em estratégias de TE para a cartilagem

Família, trabalho e relações conjugais : perspectivas de dominação e de igualdade no feminino e no masculino

A família mantém-se a célula do indivíduo, onde este cresce e aprende a viver em sociedade. O trabalho é sinónimo de sobrevivência para os indivíduos. Entre as várias modalidades que coexistem nos dias de hoje, o trabalho assalariado é aprincipal fonte de rendimentos para metade dos portugueses. As relações conjugais são um espaço de intensas trocas afectivas que interferem com os indicadores de natalidade e com estabilidade emocional de cada indivíduo. Se é precsio aumentar o investimento profissional, como se organizam as famílias? Como é que são feitas as escolhas individuais e em que medida é que estas interferem no equilíbrio familiar? Porque é que a redistribuição de tarefas familiares é um assunto tabu, em que ninguém quer mexer? serve os interesses de quem? Os indivíduos têm consciência das desigualdades? Se têm, porque as mantêm? Par5a aprofundar estas questões efectuam-se entrevistas a alguns casais profissionalmente activos e com filhos. A partir de estudos de caso definem-se relações conjugais que se podem estabelecer em termos de dominação ou de igualdade

Evaluation by flow cytometry of Escherichia coli viability in lettuce after disinfection

Foodborne outbreaks due to the consumption of ready-to-eat vegetables have increased worldwide, with Escherichia coli (E. coli) being one of the main sources responsible. Viable but nonculturable bacteria (VBNC) retain virulence even after some disinfection procedures and constitute a huge problem to public health due to their non-detectability through conventional microbiological techniques. Flow cytometry (FCM) is a promising tool in food microbiology as it enables the distinction of the different physiological states of bacteria after disinfection procedures within a short time. In this study, samples of lettuce inoculated with E. coli were subject to disinfection with sodium hypochlorite at free chlorine concentrations of 5, 10, 25, 50, and 100 mg·L1 or with 35% peracetic acid at concentrations of 5, 10, 25, and 50 mg·L1. The efficiency of these disinfectants on the viability of E. coli in lettuce was evaluated by flow cytometry with LIVE/DEAD stains. Results from this study suggest that FCM can effectively monitor cell viability. However, peracetic acid is more effective than sodium hypochlorite as, at half the concentration, it is enough to kill 100% of bacteria and always induces a lower percentage of VBNC. Finally, we can conclude that the recommended levels of chemical disinfectants for fresh fruit and vegetables are adequate when applied in lettuce. More importantly, it is possible to ensure that all cells of E. coli are dead and that there are no VBNC cells even with lower concentrations of those chemicals. These results can serve as guidance for lettuce disinfection, improving quality and the safety of consumption.This research was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE‐01‐0145‐ FEDER‐000004) funded by the European Regional Development Fund under the scope of Norte2020‐Programa Operacional Regional do Norte. Pilar Teixeira acknowledges FCT for grant SFRH/BPD/86732/2012.info:eu-repo/semantics/publishedVersio

Gamification applied to autism spectrum disorder

Gamification is a recent technique in software development that allows the application of game principles to non-game contexts and environments. In an increasingly technological world, gamification has now higher popularity, and it is currently used in several technologies. One of the health conditions where gamification can bring great benefits is in autism spectrum disorder (ASD), which is a persistent neurodevelopmental disorder that can be characterized briefly by deficits in verbal and non-verbal communication, difficulties in interaction, and manifestation of stereotyped movements or interests. In the case of ASD, the programs, software, or the mobile applications should focus on the development of intrapersonal (such as motivation) and interpersonal (social skills) skills. Therefore, gamification can be useful in cases of ASD, but it is necessary to increase the analysis of the potentialities and needs for improvement of technologies and applications available on the market.info:eu-repo/semantics/publishedVersio

A Novel Frameshift CHD4 Variant Leading to Sifrim-Hitz-Weiss Syndrome in a Proband with a Subclinical Familial t(17;19) and a Large dup(2)(q14.3q21.1)

This article belongs to the Section Molecular Genetics and Genetic Diseases.The genetic complexity of neurodevelopmental disorders (NDD), combined with a heterogeneous clinical presentation, makes accurate assessment of their molecular bases and pathogenic mechanisms challenging. Our purpose is to reveal the pathogenic variant underlying a complex NDD through identification of the "full" spectrum of structural genomic and genetic variants. Therefore, clinical phenotyping and identification of variants by genome and exome sequencing, together with comprehensive assessment of these and affected candidate genes, were carried out. A maternally-inherited familial translocation [t(17;19)(p13.1;p13.3)mat] disrupting the GSG1 like 2 gene (GSG1L2), a 3.2 Mb dup(2)(q14.3q21.1) encompassing the autosomal dominant OMIM phenotype-associated PROC and HS6ST1 gene, and a novel frameshift c.4442del, p.(Gly1481Valfs*21) variant within exon 30 of the Chromodomain helicase DNA binding protein 4 (CHD4) have been identified. Considering the pathogenic potential of each variant and the proband's phenotype, we conclude that this case basically fits the Sifrim-Hitz-Weiss syndrome or CHD4-associated neurodevelopmental phenotype. Finally, our data highlight the need for identification of the "full" spectrum of structural genomic and genetic variants and of reverse comparative phenotyping, including unrelated patients with variants in same genes, for improved genomic healthcare of patients with NDD.If this article is accepted for publication, Open Access publication will be funded by UMIB—Unidade Multidisciplinar de Investigação Biomédica, ICBAS—Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal/ITR—Laboratory for Integrative and Trans lational Research in Population Health, Porto, Portugal (https://umib.icbas.up.pt/, accessed on 14 December 2022), both supported by FCT—Fundação para a Ciência e a Tecnologia in the frameworks of UIDP/00215/2020; LA/P/0064/2020. This research was supported by national funds through FCT—Fundação para a Ciência e a Tecnologia, Research Grant HMSP-ICT/0016/2013 of the Harvard Medical School—Portugal Program in Translational Research and Information.info:eu-repo/semantics/publishedVersio

Fatores de risco para ideação suicida e suicídio na população de adolescentes e adultos jovens transgênero

Objetivo: Essa revisão sistemática da literatura tem como objetivo trazer os fatores de risco para suicídio em adolescentes e jovens adultos mais relevantes na literatura. Métodos: Foi realizado uma busca de alta sensibilidade nas principais bases de dados: PubMed, Web of Science, Cinahl, EMBASE; Cochrane; BVS. Os critérios de inclusão foram: Estudos observacionais ou Ensaios Clínicos Randomizados que relacionam a ideação suicida e o suicídio de jovens e adolescentes com potenciais fatores de risco. Resultados: Os estudos apontaram diversos fatores de risco para ideação e suicídio na população estudada, dentre eles os mais citados foram rejeição familiar, não conformidade de gênero na infância, intimidação na escola, vtimização, não conformidade por ser transgênero e "depressão, ansiedade, outros transtornos mentais". Conclusão: A população de jovens e adolescentes transgêneros possuem maiores índices de suicídio, fato que está diretamente atrelado a maior quantidade de fatores de risco, associados a alta discriminação e violência e a própria fase da vid

Enhancing functionalization of health care textiles with gold nanoparticle-loaded hydroxyapatite composites

Hospitals and nursing home wards are areas prone to the propagation of infections and are of particular concern regarding the spreading of dangerous viruses and multidrug-resistant bacteria (MDRB). MDRB infections comprise approximately 20% of cases in hospitals and nursing homes. Healthcare textiles, such as blankets, are ubiquitous in hospitals and nursing home wards and may be easily shared between patients/users without an adequate pre-cleaning process. Therefore, functionalizing these textiles with antimicrobial properties may considerably reduce the microbial load and prevent the propagation of infections, including MDRB. Blankets are mainly comprised of knitted cotton (CO), polyester (PES), and cotton-polyester (CO–PES). These fabrics were functionalized with novel gold-hydroxyapatite nanoparticles (AuNPs-HAp) that possess antimicrobial properties, due to the presence of the AuNPs’ amine and carboxyl groups, and low propensity to display toxicity. For optimal functionalization of the knitted fabrics, two pre-treatments, four different surfactants, and two incorporation processes were evaluated. Furthermore, exhaustion parameters (time and temperature) were subjected to a design of experiments (DoE) optimization. The concentration of AuNPs-HAp in the fabrics and their washing fastness were critical factors assessed through color difference (∆E). The best performing knitted fabric was half bleached CO, functionalized using a surfactant combination of Imerol® Jet-B (surfactant A) and Luprintol® Emulsifier PE New (surfactant D) through exhaustion at 70 ◦C for 10 min. This knitted CO displayed antibacterial properties even after 20 washing cycles, showing its potential to be used in comfort textiles within healthcare environments

Tuning the Biological Activity of Camphorimine Complexes through Metal Selection

This research was funded by FCT—Fundação para a Ciência e a Tecnologia, through projects CQE (UIDB/00100/2020 and UIDP/00100/2020) and C2TN (UID/MULTI/04349/2019), the projects of the Research Unit Institute for Bioengineering and Biosciences—iBB (UIDB/04565/2020 and UIDP/04565/2020), the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB, and a PhD grant to J.P.C. (UI/BD/152244/2021).The cytotoxic activity of four sets of camphorimine complexes based on the Cu(I), Cu(II), Ag(I), and Au(I) metal sites were assessed against the cisplatin-sensitive A2780 and OVCAR3 ovarian cancer cells. The results showed that the gold complexes were ca. one order of magnitude more active than the silver complexes, which in turn were ca. one order of magnitude more active than the copper complexes. An important finding was that the cytotoxic activity of the Ag(I) and Au(I) camphorimine complexes was higher than that of cisplatin. Another relevant aspect was that the camphorimine complexes did not interact significantly with DNA, in contrast with cisplatin. The cytotoxic activity of the camphorimine complexes displayed a direct relationship with the cellular uptake by OVCAR3 cells, as ascertained by PIXE (particle-induced X-ray emission). The levels of ROS (reactive oxygen species) formation exhibited an inverse relationship with the reduction potentials for the complexes with the same metal, as assessed by cyclic voltammetry. In order to gain insight into the toxicity of the complexes, their cytotoxicity toward nontumoral cells (HDF and V79 fibroblasts) was evaluated. The in vivo cytotoxicity of complex 5 using the nematode Caenorhabditis elegans was also assessed. The silver camphorimine complexes displayed the highest selectivity coefficients (activity vs. toxicity).publishersversionpublishe