Development of 3D in vitro models for prediction of hepatic metabolism and toxicity

The development of human cell models that recapitulate hepatic functionality allows the study of metabolic pathways involved in toxicity and disease. The increased biological relevance, cost-effectiveness and high-throughput of cell models can contribute to increase the efficiency of drug development in the pharmaceutical industry. Recapitulation of liver functionality in vitro requires the development of advanced culture strategies to mimic in vivo complexity, such as 3D culture, co-cultures or biomaterials. However, complex 3D models are typically associated with poor robustness, limited scalability and compatibility with screening methods. In this work, several strategies were used to develop highly functional and reproducible spheroid-based in vitro models of human hepatocytes and HepaRG cells using stirred culture systems. In chapter 2, the isolation of human hepatocytes from resected liver tissue was implemented and a liver tissue perfusion method was optimized towards the improvement of hepatocyte isolation and aggregation efficiency, resulting in an isolation protocol compatible with 3D culture. In chapter 3, human hepatocytes were co-cultivated with mesenchymal stem cells (MSC) and the phenotype of both cell types was characterized, showing that MSC acquire a supportive stromal function and hepatocytes retain differentiated hepatic functions, stability of drug metabolism enzymes and higher viability in co-cultures. In chapter 4, a 3D alginate microencapsulation strategy for the differentiation of HepaRG cells was evaluated and compared with the standard 2D DMSO-dependent differentiation, yielding higher differentiation efficiency, comparable levels of drug metabolism activity and significantly improved biosynthetic activity. The work developed in this thesis provides novel strategies for 3D culture of human hepatic cell models, which are reproducible, scalable and compatible with screening platforms. The phenotypic and functional characterization of the in vitro systems performed contributes to the state of the art of human hepatic cell models and can be applied to the improvement of pre-clinical drug development efficiency of the process, model disease and ultimately, development of cell-based therapeutic strategies for liver failure.

Produção e caracterização de espumas 3D obtidas por electrofiação para aplicação em engenharia de tecidos

A produção de estruturas tridimensionais poliméricas tem sido foco de estudo por parte da Engenharia de Células e Tecidos, pelo que mimetizam melhor as condições in vivo dos tecidos. A conjugação das propriedades eléctricas com arquitectura 3D permite uma regeneração tecidual mais eficaz. Desta forma este estudo incidiu na construção de scaffolds, que conjugasse as propriedades mecânicas, eléctricas e biológicas num só suporte. O processo utilizado para produção de scaffolds baseou-se na electrofiação de soluções poliméricas de PCL (8% m/m) com incorporação de óxido de grafeno em diferentes concentrações: 0.01%, 0.1% e 0.25% (m/V). Foram avaliados os parâmetros de electrofiação que permitiram a organização tridimensional. A composição química e a morfologia das membranas foram avaliadas por FTIR-ATR e por microscopia electrónica de varrimento (MEV), respectivamente. Através de ensaios de tracção e de permeabilidade estudou-se a influência de óido de grafeno na matriz polimérica. Foram feitas experiências de redução de óxido de grafeno nas fibras electrofiadas, tanto nas membranas como das espumas, através do uso de vapores de hidrazina. Este mecanismo mostrou-se ineficaz, uma vez que afectou a sua morfologia. As espumas foram avaliadas quanto à sua bioactividade e propriedades mecânicas (ensaios de compressão). Também foram realizados testes de viabilidade celular nas membranas e de adesão celular nas espumas, de forma a avaliar o seu potencial para aplicação biomédica. Os resultados destes ensaios revelaram que óxido de grafeno não apresenta efeitos citotóxicos para o organismo e a sua presença promove a adesão celular ao scaffold.

Jogo 3D online para reabilitação de pacientes afásicos

Esta plataforma foi projetada como auxílio complementar ao processo de reabilitação de doentes afásicos lusófonos. Uma gama de exercícios são disponibilizados de forma a induzir diferentes estímulos (compreensão escrita e auditiva e expressão escrita) sendo a grande maioria destes realizados dentro de um ambiente virtual em três dimensões onde o utilizador (dependendo da tarefa) pode interagir com objetos presentes dentro de uma casa. A principal particularidade desta plataforma reside no facto desta estar alojada online, dispensando instalações e permitindo um acompanhamento mais próximo por parte do terapeuta da fala do progresso feito pelo paciente. A ferramenta desenvolvida está disponível para visualização e teste no endereço www.weblisling.net.

Numerical simulation of blast effects on fibre grout RC panels

The main purpose of the present dissertation is the simulation of the response of fibre grout strengthened RC panels when subjected to blast effects using the Applied Element Method, in order to validate and verify its applicability. Therefore, four experimental models, three of which were strengthened with a cement-based grout, each reinforced by one type of steel reinforcement, were tested against blast effects. After the calibration of the experimental set-up, it was possible to obtain and compare the response to the blast effects of the model without strengthening (reference model), and a fibre grout strengthened RC panel (strengthened model). Afterwards, a numerical model of the reference model was created in the commercial software Extreme Loading for Structures, which is based on the Applied Element Method, and calibrated to the obtained experimental results, namely to the residual displacement obtained by the experimental monitoring system. With the calibration verified, it is possible to assume that the numerical model correctly predicts the response of fibre grout RC panels when subjected to blast effects. In order to verify this assumption, the strengthened model was modelled and subjected to the blast effects of the corresponding experimental set-up. The comparison between the residual and maximum displacements and the bottom surface’s cracking obtained in the experimental and the numerical tests yields a difference of 4 % for the maximum displacements of the reference model, and a difference of 4 and 10 % for the residual and maximum displacements of the strengthened model, respectively. Additionally, the cracking on the bottom surface of the models was similar in both methods. Therefore, one can conclude that the Applied ElementMethod can correctly predict and simulate the response of fibre grout strengthened RC panels when subjected to blast effects.

Building 3D City Models: Testing and Comparing Laser Scanning and Low-Cost UAV Data Using FOSS Technologies

Contém resumo

Minimal computation structures for visual information applications based on printed electronics

In the early nineties, Mark Weiser wrote a series of seminal papers that introduced the concept of Ubiquitous Computing. According to Weiser, computers require too much attention from the user, drawing his focus from the tasks at hand. Instead of being the centre of attention, computers should be so natural that they would vanish into the human environment. Computers become not only truly pervasive but also effectively invisible and unobtrusive to the user. This requires not only for smaller, cheaper and low power consumption computers, but also for equally convenient display solutions that can be harmoniously integrated into our surroundings. With the advent of Printed Electronics, new ways to link the physical and the digital worlds became available. By combining common printing techniques such as inkjet printing with electro-optical functional inks, it is starting to be possible not only to mass-produce extremely thin, flexible and cost effective electronic circuits but also to introduce electronic functionalities into products where it was previously unavailable. Indeed, Printed Electronics is enabling the creation of novel sensing and display elements for interactive devices, free of form factor. At the same time, the rise in the availability and affordability of digital fabrication technologies, namely of 3D printers, to the average consumer is fostering a new industrial (digital) revolution and the democratisation of innovation. Nowadays, end-users are already able to custom design and manufacture on demand their own physical products, according to their own needs. In the future, they will be able to fabricate interactive digital devices with user-specific form and functionality from the comfort of their homes. This thesis explores how task-specific, low computation, interactive devices capable of presenting dynamic visual information can be created using Printed Electronics technologies, whilst following an approach based on the ideals behind Personal Fabrication. Focus is given on the use of printed electrochromic displays as a medium for delivering dynamic digital information. According to the architecture of the displays, several approaches are highlighted and categorised. Furthermore, a pictorial computation model based on extended cellular automata principles is used to programme dynamic simulation models into matrix-based electrochromic displays. Envisaged applications include the modelling of physical, chemical, biological, and environmental phenomena.

MarketOpolis: A market simulation based on investors decisions

This study aims to replicate Apple’s stock market movement by modeling major investment profiles and investors. The present model recreates a live exchange to forecast any predictability in stock price variation, knowing how investors act when it concerns investment decisions. This methodology is particularly relevant if, just by observing historical prices and knowing the tendencies in other players’ behavior, risk-adjusted profits can be made. Empirical research made in the academia shows that abnormal returns are hardly consistent without a clear idea of who is in the market in a given moment and the correspondent market shares. Therefore, even when knowing investors’ individual investment profiles, it is not clear how they affect aggregate markets.

Development of human central nervous system 3D in vitro models for preclinical research

Neurological disorders are a major concern in modern societies, with increasing prevalence mainly related with the higher life expectancy. Most of the current available therapeutic options can only control and ameliorate the patients’ symptoms, often be-coming refractory over time. Therapeutic breakthroughs and advances have been hampered by the lack of accurate central nervous system (CNS) models. The develop-ment of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of novel therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmentally, anatomically and physiologically) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity). The in vitro recapitulation of CNS phenotypic and functional features requires the implementation of advanced culture strategies that enable to mimic the in vivo struc-tural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. This thesis aimed at the development of novel human 3D in vitro CNS models by integrat-ing agitation-based culture systems and a wide array of characterization tools. Neural differentiation of hNSC as 3D neurospheres was explored in Chapter 2. Here, it was demonstrated that human midbrain-derived neural progenitor cells from fetal origin (hmNPC) can generate complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Chapter 3 focused on the development of cellular characterization assays for cell aggregates based on light-sheet fluorescence imaging systems, which resulted in increased spatial resolu-tion both for fixed samples or live imaging. The applicability of the developed human 3D cell model for preclinical research was explored in Chapter 4, evaluating the poten-tial of a viral vector candidate for gene therapy. The efficacy and safety of helper-dependent CAV-2 (hd-CAV-2) for gene delivery in human neurons was evaluated, demonstrating increased neuronal tropism, efficient transgene expression and minimal toxicity. The potential of human 3D in vitro CNS models to mimic brain functions was further addressed in Chapter 5. Exploring the use of 13C-labeled substrates and Nucle-ar Magnetic Resonance (NMR) spectroscopy tools, neural metabolic signatures were evaluated showing lineage-specific metabolic specialization and establishment of neu-ron-astrocytic shuttles upon differentiation. Chapter 6 focused on transferring the knowledge and strategies described in the previous chapters for the implementation of a scalable and robust process for the 3D differentiation of hNSC derived from human induced pluripotent stem cells (hiPSC). Here, software-controlled perfusion stirred-tank bioreactors were used as technological system to sustain cell aggregation and dif-ferentiation. The work developed in this thesis provides practical and versatile new in vitro ap-proaches to model the human brain. Furthermore, the culture strategies described herein can be further extended to other sources of neural phenotypes, including pa-tient-derived hiPSC. The combination of this 3D culture strategy with the implemented characterization methods represents a powerful complementary tool applicable in the drug discovery, toxicology and disease modeling.