Avaliação dos efeitos estruturais da lixiviação das argamassas de ligação dos blocos das barragens de alvenaria de pedra

As barragens de alvenaria de pedra são, em regra, estruturas antigas. Para garantir a funcionalidade e a segurança das obras em exploração, estas estruturas são sujeitas periodicamente a obras de beneficiação e reabilitação, essencialmente para assegurar o seu monolitismo estrutural e a impermeabilidade que progressivamente se perdem por diferentes causas, em geral associadas a efeitos térmicos, ações sísmicas e, sobretudo, à lixiviação das argamassas que ligam os blocos de pedra. Esta lixiviação provoca uma degradação progressiva da estrutura, ocasionando a perda de resistência e da rigidez mecânica, afetando o seu comportamento estrutural e hidráulico. No trabalho desenvolveu-se uma metodologia de avaliação dos efeitos estruturais da deterioração das alvenarias das barragens considerando a lixiviação das argamassas de ligação dos blocos, a partir de análises expeditas das águas realizadas nas inspeções periódicas e dos resultados da observação de deslocamentos e de caudais infiltrados pelo corpo das obras. A partir dos caudais infiltrados, avaliou-se a permeabilidade média das alvenarias e estimaram-se as perdas de massa associadas à referida lixiviação. Com os deslocamentos observados estimaram-se os módulos de elasticidade médios das estruturas e estabeleceu-se uma correlação entre as referidas perdas de massa e a parcela dos deslocamentos atribuídos aos efeitos do tempo. As análises apoiaram-se em modelos planos de percolação e estruturais, representativos do comportamento dos perfis transversais das obras, que foram resolvidos pelo programa comercial FLAC, que se baseia no método das diferenças finitas. Utilizaram-se como casos de estudo as quatro barragens de alvenaria portuguesas para as quais se dispõe dos elementos de observação requeridos, que são as barragens de Guilhofrei, Andorinhas, Freigil e Covão do Ferro. Os resultados obtidos são prometedores no que respeita à interpretação dos deslocamentos que se verificam ao longo do tempo nas obras, considerando a deterioração estrutural devida à lixiviação das argamassas de ligação dos blocos das barragens de alvenaria de pedra

Enginnering cardiac tissue using human induced pluripotent stem cell derivatives: Proteomic characterization of co-cultures of cardiomyocytes and endothelial cells

Prediction of cardiac toxicity effect is extremely relevant in the development of new drugs for different medical applications. In this way, it is important to develop more predictable human cell-based models which physiologically better mimic the human heart and allow the prediction of this toxic effect as well as establish the tools that enable the characterization of these complex cell models. To recreate engineered cardiac tissue, it is essential to reproduce the complexity of the heart by resorting to different cell types. Cardiomyocytes (CMs) are functional contractile units of the heart, and it is known that their communication with endothelial cells (ECs) is crucial for cardiac homeostasis. The aim of this study is to recreate a human pluripotent stem cells (hiPSC)-based cardiac tissue model and evaluate the impact of communication between both cell types on the phenotype of CMs. Co-cultures of hiPSC-CM and hiPSC-EC were established and maintained for 12 days as confirmed by immunofluorescence microscopy. Quantitative whole-proteome analysis was performed using SWATH Mass Spectrometry tools to compare the conditions of hiPSC-CM mono-culture and the co-culture of hiPSC-CM and hiPSC-EC. Our data showed relative increase of expression ratios of morphological maturation-related cardiac proteins in hiPSC-CM co-cultures. In particular, the expression ratios of MYH7/MYH6, MYL2/MYL7, TNNI3/TNNI1 increased 2.4-, 5.1-, and 5-fold, respectively, when compared to the mono-culture condition, indicating that in the presence of hiPSC-EC, hPSC-CM display a more adult- and ventricular- like phenotype. Changes in the extracellular matrix composition were also observed, especially related with the increased expression of ECM proteins in co-culture condition namely, collagens I and III (8.6-fold and 6-fold, respectively), fibronectin (3.5-fold) and thrombospondin-4 (2.5-fold). Other growth factors attributed to the extracellular space (e.g. CTGF, PAI1, CRTAP, IGFBP7, and NPPB) that may be responsible for the communication between both cell types have also shown to be up-regulated in the co-culture condition. The presence of a SMA+ (myofibroblast-like) population in the co-culture condition was observed by immunofluorescence microscopy images, which is in agreement with the more complex and fibrotic extracellular matrix found by whole proteome analysis. Ultrastructure characterization of CMs was carried out by transmission electron microscopy. In both conditions, hiPSC-CM displayed aligned myofibrils composed by sarcomeres with organized Z-disks, A- and I-bands, intercalated discs between adjacent cells as well as abundant mitochondria. Noteworthy, sarcomere length was higher in hiPSC-CM cultured with hiPSC-EC, suggesting structural changes associated with cardiomyocyte maturation. Calcium imaging is being performed to evaluate calcium handling of hiPSC-CMs and their response to drugs. All together our data revealed that promoting the communication of hiPSC-CM and hiPSC-EC induced structural changes in hiPSC-CM associated with maturation. This study provides important insights towards the development of more complex cardiac tissues and establishes potent analytical tools for the characterization of these models. This work was supported by Fundação para a Ciência e Tecnologia (FCT)-funded project CARDIOSTEM (MITP-TB/ECE/0013/2013); and iNOVA4Health UID/Multi/04462/2013, a program supported by FCT/Ministério da Educação e Ciência, through national funds and cofounded by FEDER under the PT2020 Partnership Agreement. BA. was supported by FCT Grant SFRH/BD/52475/201

Bioprocess intensification for the continuous expansion of 3D human induced pluripotent stem cell aggregates in bioreactors

Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. Firstly, we demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4% oxygen) and perfusion at 1.3 day-1 dilution rate to improve hiPSC growth as 3D aggregates in xeno-free medium (Cellartis® DEF-CS™ 500 Xeno-Free Culture Medium). This strategy allowed for increased cell specific growth rate, maximum volumetric cell concentrations (4.7x106 cell/mL) and expansion factors (approximately 19), resulting in an overall improvement of 2.6-fold in cell yields. Extensive cell characterization, including whole proteomic analysis was performed to confirm that the pluripotent phenotype was maintained during culture. Secondly, we have tested different chemical and mechanical strategies for hiPSC aggregate dissociation, revealing similar viable cell recovery yields (approximately 50%). However, only the mechanical dissociation strategies enabled the re-aggregation of hiPSC in stirred conditions, with the mechanical dissociation using a 70 μm pore size nylon mesh allowing a higher expansion factor after dissociation. Finally, a scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using the mechanical dissociation for aggregate disruption/passaging. A total expansion factor of 1100 in viable cells was obtained in 11 days of culture after 3 sequential passages in bioreactors, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability. To our knowledge, this is the highest expansion factor reported for hiPSC for such a short culture time frame. The strategy described herein for continuous expansion of hiPSC provides important insights towards up-scaling the production of hiPSC. Integrative biomanufacturing processes using this continuous strategy are now being pursued for hiPSC expansion and differentiation towards cardiac lineages in order to recreate cardiac models for drug discovery, toxicity testing and disease modeling. Acknowledgments: This work was supported by Fundação para a Ciência e Tecnologia (FCT)-funded projects CARDIOSTEM (MITP-TB/ECE/0013/2013) and CardioRegen (HMSP-ICT/0039/2013); and iNOVA4Health UID/Multi/04462/2013, a program supported by FCT/Ministério da Educação e Ciência, through national funds and cofounded by FEDER under the PT2020 Partnership Agreement. BA. was supported by FCT Grant SFRH/BD/52475/2013. The work was also funded by a Vinnova Grant, registration number 2014-00310 (Cell therapies via large scale expansion of pluripotent stem cells)

Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches

Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4% oxygen) and perfusion at 1.3 day-1 dilution rate to improve hiPSC growth as 3D aggregates in xeno-free medium (Cellartis® DEF-CS™ 500 Xeno-Free Culture Medium). This strategy allowed for increased cell specific growth rate, maximum volumetric cell concentrations (4.7x106 cell/mL) and expansion factors (approximately 19), resulting in an overall improvement of 2.6-fold in cell yields. Extensive cell characterization, including whole proteomic analysis was performed to confirm that the pluripotent phenotype was maintained during culture. Furthermore, a scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation protocols for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11 days of culture (Figure 1), while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors. To our knowledge, this is the highest expansion factor reported for hiPSC for such a short culture time frame. The strategy described herein for continuous expansion of hiPSC provides important insights towards up-scale production of hiPSC. This will strengthen the utility of hiPSC in cell therapy, drug discovery, toxicity testing and disease modeling. Please click Additional Files below to see the full abstract

Toward a Microencapsulated 3D hiPSC-Derived in vitro Cardiac Microtissue for Recapitulation of Human Heart Microenvironment Features

SAICTPAC/0047/2015 PTDC/BTMSAL/32566/ 2017 PTDC/MEC-CAR/29590/2017 UIDB/04462/2020 UIDP/04462/2020 H2020, ID:874827 SFRH/BD/52475/2013 SFRH/BPD/120595/2016The combination of cardiomyocytes (CM) and non-myocyte cardiac populations, such as endothelial cells (EC), and mesenchymal cells (MC), has been shown to be critical for recapitulation of the human heart tissue for in vitro cell-based modeling. However, most of the current engineered cardiac microtissues still rely on either (i) murine/human limited primary cell sources, (ii) animal-derived and undefined hydrogels/matrices with batch-to-batch variability, or (iii) culture systems with low compliance with pharmacological high-throughput screenings. In this work, we explored a culture platform based on alginate microencapsulation and suspension culture systems to develop three-dimensional (3D) human cardiac microtissues, which entails the co-culture of human induced pluripotent stem cell (hiPSC) cardiac derivatives including aggregates of hiPSC–CM and single cells of hiPSC–derived EC and MC (hiPSC–EC+MC). We demonstrate that the 3D human cardiac microtissues can be cultured for 15 days in dynamic conditions while maintaining the viability and phenotype of all cell populations. Noteworthy, we show that hiPSC–EC+MC survival was promoted by the co-culture with hiPSC–CM as compared to the control single-cell culture. Additionally, the presence of the hiPSC–EC+MC induced changes in the physical properties of the biomaterial, as observed by an increase in the elastic modulus of the cardiac microtissue when compared to the hiPSC–CM control culture. Detailed characterization of the 3D cardiac microtissues revealed that the crosstalk between hiPSC–CM, hiPSC–EC+MC, and extracellular matrix induced the maturation of hiPSC–CM. The cardiac microtissues displayed functional calcium signaling and respond to known cardiotoxins in a dose-dependent manner. This study is a step forward on the development of novel 3D cardiac microtissues that recapitulate features of the human cardiac microenvironment and is compliant with the larger numbers needed in preclinical research for toxicity assessment and disease modeling.publishersversionpublishe

Human Extracellular-Matrix Functionalization of 3D hiPSC-Based Cardiac Tissues Improves Cardiomyocyte Maturation

The work here presented was funded by Fundacao para a Ciencia e Tecnologia (FCT) projects NETDIAMOND (SAICTPAC/0047/2015), financially supported by FEEI-Lisboa2020 and FCT/POCI-01-0145-FEDER-016385, and MetaCardio (PTDC/BTM-SAL/32566/2017); iNOVA4-Health -UIDB/04462/2020 and UIDP/04462/2020, a program financially supported by FCT/Ministerio da Ciencia, Tecnologia e Ensino Superior, through national funds is acknowledged; Funding from INTERFACE Programme, through the Innovation, Technology and Circular Economy Fund (FITEC), is gratefully acknowledged; and EU-funded projects BRAV3 (H2020, ID:874827) and ERAatUC (ref. 669088). HVA, AFL, and DS were financed by FCT Grants SFRH/BPD/120595/2016 and PD/BD/139078/2018 and PD/BD/106051/2015, respectively.Human induced pluripotent stem cells (hiPSC) possess significant therapeutic potential due to their high self-renewal capability and potential to differentiate into specialized cells such as cardiomyocytes. However, generated hiPSC-derived cardiomyocytes (hiPSC-CM) are still immature, with phenotypic and functional features resembling the fetal rather than their adult counterparts, which limits their application in cell-based therapies, in vitro cardiac disease modeling, and drug cardiotoxicity screening. Recent discoveries have demonstrated the potential of the extracellular matrix (ECM) as a critical regulator in development, homeostasis, and injury of the cardiac microenvironment. Within this context, this work aimed to assess the impact of human cardiac ECM in the phenotype and maturation features of hiPSC-CM. Human ECM was isolated from myocardium tissue through a physical decellularization approach. The cardiac tissue decellularization process reduced DNA content significantly while maintaining ECM composition in terms of sulfated glycosaminoglycans (s-GAG) and collagen content. These ECM particles were successfully incorporated in three-dimensional (3D) hiPSC-CM aggregates (CM+ECM) with no impact on viability and metabolic activity throughout 20 days in 3D culture conditions. Also, CM+ECM aggregates displayed organized and longer sarcomeres, with improved calcium handling when compared to hiPSC-CM aggregates. This study shows that human cardiac ECM functionalization of hiPSC-based cardiac tissues improves cardiomyocyte maturation. The knowledge generated herein provides essential insights to streamline the application of ECM in the development of hiPSC-based therapies targeting cardiac diseases.publishersversionpublishe

Intensifying the manufacture of hiPSC therapy products through metabolic and process understanding

In vitro differentiation of human induced pluripotent stem cells into specific lineages such as cardiomyocytes (hPSC-CM) and hepatocytes (hPCS-Hep) is a crucial process to enable their application in cell therapy and drug discovery. Nevertheless, despite the remarkable efforts over the last decade towards the implementation of protocols for hPSC expansion and differentiation, there are some technological challenges remaining include the low scalability and differentiation yields. Additionally, generated cells are still immature, closely reminiscent of fetal/embryonic cells in what regards phenotype and function. In this study, we aim to overcome this hurdle by devising bioinspired and integrated strategies to improve the generation and functionality of these hiPSC-derivatives. We also applied robust multi-parametric techniques including proteomics, transcriptomics, metabolomics and fluxomics as complementary analytical tools to support bioprocess optimization and product characterization. We cultured hiPSC as 3D aggregates in stirred-tank bioreactors (STB) operated in perfusion and used a capacitance probe for in situ monitoring of cell growth/differentiation. After cell expansion, the hepatic differentiation step was integrated by addition of key soluble factors and controlling the dissolved oxygen concentration at various stages of the process to generate populations enriched for definitive endoderm, hepatocyte progenitors and mature hepatocytes. The analyses of hepatic markers expression throughout the stages of the differentiation confirmed that hepatocyte differentiation was improved in 3D spheroids when compared to 2D culture. Noteworthy, these hiPSC-HLC exhibited functional characteristics typical of hepatocytes (albumin production, glycogen storage and CYP450 activity). We also demonstrate the potential of dielectric spectroscopy to monitor cell expansion and hepatic differentiation in STB. For CM differentiation, we relied on the aggregation of hPSC-derived cardiac progenitors to establish a scalable differentiation protocol capable of generating highly pure CM aggregate cultures. We assessed if alteration of culture medium composition to mimic in vivo substrate usage during cardiac development improved further hPSC-CM maturation in vitro. Our results showed that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. “-Omics” analyses showed that addition of galactose to culture medium and culturing the cells under perfusion improves total oxidative capacity of the cells and ameliorates fatty acid oxidation. This study demonstrated that metabolic shifts during differentiation/maturation of hPSC-CM are a cause, rather than a consequence, of the phenotypic and functional alterations observed. The metabolic-based strategy established herein holds technical and economic advantages over the existing protocols due to its scalability, simplicity and ease of application. Funding: This work was supported by FCT-funded projects NETDIAMOND (SAICTPAC/0047/2015), MetaCardio (Ref.032566) and FCT/ERA-Net (ERAdicatPH; Ref. E-Rare3/0002/2015). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) is also acknowledged

Serological screening in a large-scale municipal survey in Cascais, Portugal, during the first waves of the COVID-19 pandemic: lessons for future pandemic preparedness efforts

BackgroundSerological surveys for SARS-CoV-2 were used early in the COVID-19 pandemic to assess epidemiological scenarios. In the municipality of Cascais (Portugal), serological testing combined with a comprehensive socio-demographic, clinical and behavioral questionnaire was offered to residents between May 2020 and beginning of 2021. In this study, we analyze the factors associated with adherence to this municipal initiative, as well as the sociodemographic profile and chronic diseases clinical correlates associated to seropositivity. We aim to contribute with relevant information for future pandemic preparedness efforts.MethodsThis was a cross-sectional study with non-probabilistic sampling. Citizens residing in Cascais Municipality went voluntarily to blood collection centers to participate in the serological survey. The proportion of participants, stratified by socio-demographic variables, was compared to the census proportions to identify the groups with lower levels of adherence to the survey. Univariate and multivariate logistic regression were used to identify socio-demographic, clinical and behavioral factors associated with seropositivity.ResultsFrom May 2020 to February 2021, 19,608 participants (9.2% of the residents of Cascais) were included in the study. Based on the comparison to census data, groups with lower adherence to this survey were men, the youngest and the oldest age groups, individuals with lower levels of education and unemployed/inactive. Significant predictors of a reactive (positive) serological test were younger age, being employed or a student, and living in larger households. Individuals with chronic diseases generally showed lower seroprevalence.ConclusionThe groups with low adherence to this voluntary study, as well as the socio-economic contexts identified as more at risk of viral transmission, may be targeted in future pandemic situations. We also found that the individuals with chronic diseases, perceiving higher risk of serious illness, adopted protective behaviors that limited infection rates, revealing that health education on preventive measures was effective for these patients

Identification of 12 new susceptibility loci for different histotypes of epithelial ovarian cancer.

To identify common alleles associated with different histotypes of epithelial ovarian cancer (EOC), we pooled data from multiple genome-wide genotyping projects totaling 25,509 EOC cases and 40,941 controls. We identified nine new susceptibility loci for different EOC histotypes: six for serous EOC histotypes (3q28, 4q32.3, 8q21.11, 10q24.33, 18q11.2 and 22q12.1), two for mucinous EOC (3q22.3 and 9q31.1) and one for endometrioid EOC (5q12.3). We then performed meta-analysis on the results for high-grade serous ovarian cancer with the results from analysis of 31,448 BRCA1 and BRCA2 mutation carriers, including 3,887 mutation carriers with EOC. This identified three additional susceptibility loci at 2q13, 8q24.1 and 12q24.31. Integrated analyses of genes and regulatory biofeatures at each locus predicted candidate susceptibility genes, including OBFC1, a new candidate susceptibility gene for low-grade and borderline serous EOC