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.

Depois de Orpheu : 1916-1935

O objetivo deste trabalho era verificar como, nos jornais e revistas, o Primeiro Modernismo tinha sido acolhido. Depois do escândalo de Orpheu e da forma agressiva como os seus colaboradores tinham sido recebidos, cabia verificar se tinha havido, ou não, uma evolução na análise da sua obra ao longo dos anos, de 1916 a 1935, tanto mais que as vidas de cada um destes tinha evoluído de forma diferenciada. As mortes prematuras de uns, a subida a postos de influência e poder, ou a longevidade de outros foram fatores que poderiam ter condicionado a evolução da crítica jornalística. Por um lado, os críticos poderiam ter alterado a sua visão face a estes criadores, podendo o tempo ter funcionado a favor destes. O que em 1915 era estranho e bizarro poderia em 1935 ser inédito e original. Por outro lado, a conjuntura política poderia condicionar não só a criação artística, mas também o seu enquadramento social. Em 1915, a guerra entre monárquicos e republicanos foi marcada pelas referências a Orpheu. Mais tarde com a ditadura militar e a ascensão do Estado Novo poderão os nossos artistas voltar a ser alvo de referências políticas. A verdade é que tal não acontece. O poder político menospreza-os, apesar de nalguns casos terem uma participação ativa nas suas fileiras. A verdade é que os autores mais importantes e significativos foram, pouco a pouco, e com o passar dos anos, integrados na sociedade literária e começaram a ser vistos como criadores a ter em conta, senão mesmo de referência. O seu aparecimento em jornais e revistas na qualidade de escritores, jornalistas, artistas plásticos ou mesmo críticos fez com que estivessem sob os holofotes da imprensa e como tal sujeitos a uma análise permanente. E esta foi-se alterando com o passar do tempo e com o reconhecimento dos mesmos. Hoje ninguém duvida do seu valor, mas entre 1915 (data da publicação de Orpheu e 1935 (ano da morte de Pessoa) todos eram vistos como loucos, jovens com alguma habilidade para a escrita, promessas do panorama literário e finalmente, em alguns casos, motivos de orgulho para o nosso património artístico. Só uma leitura dos periódicos mais relevantes permitiria estudar a receção do nosso Primeiro Modernismo. E foi esse o objetivo desta tese.

Banco invest: Understanding how to grow in teh portuguese individual clients segment competitors analysis

Field lab: Business project

Banco invest: Understanding how to grow in the portuguese individual clients segment benchnarking

Field lab: Business project

Banco Invest understanding the drivers of growth

Field lab: Business project

Banco invest: Understanding how to grow in the portuguese individual clients segment

Field lab: Business project

Understanding impact readiness in Portugal’s social sector and designing a framework for capacity-building for social impact

This project is based on the theme of capacity-building in social organisations to improve their impact readiness, which is the predictability of delivering intended outcomes. All organisations which have a social mission, non-profit or for-profit, will be considered to fall within the social sector for the purpose of this work. The thesis will look at (i) what is impact readiness and what are the considerations for building impact readiness in social organisations, (ii) what is the international benchmark in measuring and building impact readiness, (iii) understand the impact readiness of Portuguese social organisations and the supply of capacity building for social impact in Portugal currently, and (iv) provide recommendations on the design of a framework for capacity building for impact readiness adapted to the Portuguese context. This work is of particular relevance to the Social Investment Laboratory, which is a sponsor of this project, in its policy work as part of the Portuguese Social Investment Taskforce (the “Taskforce”). This in turn will inform its contribution to the set-up of Portugal Inovação Social, a wholesaler catalyst entity of social innovation and social investment in the country, launched in early 2015. Whilst the output of this work will be set a recommendations for wider application for capacity-building programmes in Portugal, Portugal Inovação Social will also clearly have a role in coordinating the efforts of market players – foundations, corporations, public sector and social organisations – in implementing these recommendations. In addition, the findings of this report could have relevance to other countries seeking to design capacity building frameworks in their local markets and to any impact-driven organisations with an interest in enhancing the delivery of impact within their work.