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.

Using an LED as a sensor and visible light communication device in a smart illumination system

The need for more efficient illumination systems has led to the proliferation of Solid-State Lighting (SSL) systems, which offer optimized power consumption. SSL systems are comprised of LED devices which are intrinsically fast devices and permit very fast light modulation. This, along with the congestion of the radio frequency spectrum has paved the path for the emergence of Visible Light Communication (VLC) systems. VLC uses free space to convey information by using light modulation. Notwithstanding, as VLC systems proliferate and cost competitiveness ensues, there are two important aspects to be considered. State-of-the-art VLC implementations use power demanding PAs, and thus it is important to investigate if regular, existent Switched-Mode Power Supply (SMPS) circuits can be adapted for VLC use. A 28 W buck regulator was implemented using a off-the-shelf LED Driver integrated circuit, using both series and parallel dimming techniques. Results show that optical clock frequencies up to 500 kHz are achievable without any major modification besides adequate component sizing. The use of an LED as a sensor was investigated, in a short-range, low-data-rate perspective. Results show successful communication in an LED-to-LED configuration, with enhanced range when using LED strings as sensors. Besides, LEDs present spectral selective sensitivity, which makes them good contenders for a multi-colour LED-to-LED system, such as in the use of RGB displays and lamps. Ultimately, the present work shows evidence that LEDs can be used as a dual-purpose device, enabling not only illumination, but also bi-directional data communication.

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.

Desenvolvimento de um sensor para utilização em fixadores externos em ortopedia

Nesta dissertação é apresentada a metodologia adoptada no desenvolvimento de um sensor capaz de monitorizar a evolução da consolidação óssea proveniente de patologias tratadas com a utilização de aparelhos de fixação externa. O sensor tem como finalidade medir o deslocamento relativo entre os componentes do fixador externo que são proporcionais aos níveis de consolidação do osso. Surge como alternativa aos processos convencionais (raios-X, ultra-sons, etc.) que não possibilitam a detecção de imperfeições no processo de regeneração óssea numa fase precoce. Uma análise a vários materiais e geometrias revelaram que um sensor fabricado em latão e sob a forma de anel, após uma deformação de compressão de 4

Electrical characterization and modification of low dimensional oxide semiconductors for sensor applications

This work reviews the recent research on ion and UV irradiation of β-

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

Contém resumo

Electro-Optical/Infrared sensor turret integration on an aircraft - structural im-pact on LOCKHEED MARTIN C-130H and design methodology

The growing need to patrol and survey large maritime and terrestrial areas increased the need to integrate external sensors on aircraft in order to accomplish those patrols at increasingly higher altitudes, longer range and not depending upon vehicle type. The main focus of this work is to elaborate a practical, simple, effective and efficient methodology for the aircraft modification procedure resulting from the integration of an Elec-tro-Optical/Infra-Red (EO/IR) turret through a support structure. The importance of the devel-opment of a good methodology relies on the correct management of project variables as time, available resources and project complexity. The key is to deliver a proper tool for a project de-sign team that will be used to create a solution that fulfils all technical, non-technical and certi-fication requirements present in this field of transportation. The created methodology is inde-pendent of two main inputs: sensor model and aircraft model definition, and therefore it is in-tended to deliver the results for different projects besides the one that was presented in this work as a case study. This particular case study presents the development of a structure support for FLIR STAR SAPHIRE III turret integration on the front lower fuselage bulkhead (radome) of the LOCKHEED MARTIN C-130 H. Development of the case study focuses on the study of local structural analysis through the use of Finite Element Method (FEM). Development of this Dissertation resulted in a cooperation between Faculty of Science and Technology - Universidade Nova de Lisboa and the company OGMA - Indústria Aeronáutica de Portugal

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.

PHB-HV 3D Scaffold supports osteoblast growth

Bone tissue engineering requires a biocompatible scaffold that supports cell growth and enhances the native repair process. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV) is a biodegradable 3D scaffold with 88.1 â 0.3% porosity and pore size of 163.5 â 0.1 mm. Previous studies demonstrated the potential of PHB-HV as a scaffold in spinal cord repair. The aim of this study was to evaluate PHB-HV as a scaffold for bone regeneration by assessing the cytocompatability of this scaffold.

Cell encapsulated hydrogel microspheres as "building blocks" for producing 3D constructs

Cell encapsulation within hydrogel microspheres shows great promise in the field of tissue engineering and regenerative medicine (TERM). However, the assembling of microspheres as building blocks to produce complex tissues is a hard task because of their inability to place along length scales in space. We propose a proof-of-concept strategy to produce 3D constructs using cell encapsulated as building blocks by perfusion based LbL technique. This technique exploits the â bindingâ potential of multilayers apart from coating

Evaluating ubiquitous computing environments using 3D simulation

Human activity is very dynamic and subtle, and most physical environments are also highly dynamic and support a vast range of social practices that do not map directly into any immediate ubiquitous computing functionally. Identifying what is valuable to people is very hard and obviously leads to great uncertainty regarding the type of support needed and the type of resources needed to create such support. We have addressed the issues of system development through the adoption of a Crowdsourced software development model [13]. We have designed and developed Anywhere places, an open and flexible system support infrastructure for Ubiquitous Computing that is based on a balanced combination between global services and applications and situated devices. Evaluation, however, is still an open problem. The characteristics of ubiquitous computing environments make their evaluation very complex: there are no globally accepted metrics and it is very difficult to evaluate large-scale and long-term environments in real contexts. In this paper, we describe a first proposal of an hybrid 3D simulated prototype of Anywhere places that combines simulated and real components to generate a mixed reality which can be used to assess the envisaged ubiquitous computing environments [17].

Mapeamento do uso e cobertura da terra a partir da segmentação e classificação de imagens-fração solo, sombra e vegetação derivadas do modelo linear de mistura aplicado a dados do sensor TM/Landsat5, na região do reservatório de Tucuruí - PA

Técnicas de sensoriamento remoto são fundamentais para o monitoramento das mudanças de uso da terra, principalmente em áreas extensas como a Amazônia. O mapeamento de uso da terra, geralmente é realizado por métodos de classificação manual ou digital pixel a pixel, os quais consomem muito tempo. Este estudo aborda a aplicação do modelo linear de mistura em uma imagem Landsat-TM segmentada para o mapeamento das classes de uso da terra na região do reservatório de Tucuruí-PA para os anos de 1996 e 2001.

Detecção de cicatrizes de áreas queimadas baseada no modelo linear de mistura espectral e imagens índice de vegetação utilizando dados multitemporais do sensor MODIS/TERRA no estado do Mato Grosso, Amazônia brasileira

O objetivo desta pesquisa foi avaliar os dados do sensor MODIS para detectar e monitorar cicatrizes de áreas recém queimadas. Utilizamos imagens da reflectância de superfície do sensor MODIS: produto MOD09 (dia 5 de outubro) e produto MOD13A1 (meses de outubro e novembro). Foi avaliada também uma série temporal de um ano dos índices de vegetação (IV) EVI e NDVI (produto MOD13A1). Uma imagem do sensor ETM+ (dia 5 de outubro) foi utilizada como base para a delimitação dos polígonos amostrais e avaliação dos dados MODIS devido a sua melhor resolução espacial. A metodologia focou na aplicação do modelo linear de mistura espectral nas imagens reflectância para a geração das imagens fração sombra. Análises de regressão foram efetuadas para comparação entre o percentual de sombra derivado da imagem ETM+ e das imagens MODIS. As alterações multitemporais nas imagens IV foram avaliadas com base no teste de Tukey. Os resultados mostraram que a imagem fração sombra gerada a partir do produto MOD09 apresentou um R² = 0,66 (p < 0,01) em relação aos dados ETM+. Para as imagens do produto MOD13A1 não foram identificadas relações significativas. Os IV dentro dos mesmos polígonos apresentaram uma variação sazonal durante o ano. No entanto, não houve uma diminuição significativa dos valores destes índices nos meses onde foram observadas as cicatrizes de áreas recém queimadas. Portanto, o produto MOD09 mostrou-se mais eficiente que o produto MOD13A1 para a detecção de cicatrizes de áreas recém queimadas. A análise multitemporal dos IV sugeriu que não foi possível detectar este mesmo padrão na área de estudo.

Development of a new 3D OpenFOAM® solver to model the cooling stage in profile extrusion

[Excerpt] The advantages resulting from the use of numerical modelling tools to support the design of processing equipment are almost consensual. The design of calibration systems in profile extrusion is not an exception . H owever , the complex geome tries and heat exchange phenomena involved in this process require the use of numerical solvers able to model the heat exchange in more than one domain ( calibrator and polymer), the compatibilization of the heat transfer at the profile - calibrator interface and with the ability to deal with complex geometries. The combination of all these features is usually hard to find in commercial software. Moreover , the dimension of the meshes required to ob tain accurate results, result in computational times prohibitive for industrial application. (...)