Development of a Piggybac based direct reprogramming system for derivation of integration free induced pluripotent stem cells

Induced pluripotent stem cells (iPSc) have great potential for applications in regenerative medicine, disease modeling and basic research. Several methods have been developed for their derivation. The original method of Takahashi and Yamanaka involved the use of retroviral vectors which result in insertional mutagenesis, presence in the genome of potential oncogenes and effects of residual transgene expression on differentiation bias of each particular iPSc line. Other methods have been developed, using different viral vectors (adenovirus and Sendai virus), transient plasmid transfection, mRNA transduction, protein transduction and use of small molecules. However, these methods suffer from low efficiencies; can be extremely labor intensive, or both. An additional method makes use of the piggybac transposon, which has the advantage of inserting its payload into the host genome and being perfectly excised upon re-expression of the transposon transposase. Briefly, a policistronic cassette expressing Oct4, Sox2, Klf4 and C-Myc flanked by piggybac terminal repeats is delivered to the cells along with a plasmid transiently expressing piggybac transposase. Once reprogramming occurs, the cells are re-transfected with transposase and subclones free of tranposon integrations screened for. The procedure is therefore very labor intensive, requiring multiple manipulations and successive rounds of cloning and screening. The original method for reprogramming with the the PiggyBac transposon was created by Woltjen et al in 2009 (schematized here) and describes a process with which it is possible to obtain insert-free iPSc. Insert-free iPSc enables the establishment of better cellular models of iPS and adds a new level of security to the use of these cells in regenerative medicine. Due to the fact that it was based on several low efficiency steps, the overall efficiency of the method is very low (<1%). Moreover, the stochastic transfection, integration, excision and the inexistence of an active way of selection leaves this method in need of extensive characterization and screening of the final clones. In this work we aime to develop a non-integrative iPSc derivation system in which integration and excision of the transgenes can be controlled by simple media manipulations, avoiding labor intensive and potentially mutagenic procedures. To reach our goal we developed a two vector system which is simultaneously delivered to original population of fibroblasts. The first vector, Remo I, carries the reprogramming cassette and GFP under the regulation of a constitutive promoter (CAG). The second vector, Eneas, carries the piggybac transposase associated with an estrogen receptor fragment (ERT2), regulated in a TET-OFF fashion, and its equivalent reverse trans-activator associated with a positive-negative selection cassette under a constitutive promoter. We tested its functionality in HEK 293T cells. The protocol is divided in two the following steps: 1) Obtaining acceptable transfection efficiency into human fibroblasts. 2) Testing the functionality of the construct 3) Determining the ideal concentration of DOX for repressing mPB-ERT2 expression 4) Determining the ideal concentration of TM for transposition into the genome 5) Determining the ideal Windows of no DOX/TM pulse for transposition into the genome 6) 3, 4 and 5) for transposition out of the genome 7) Determination of the ideal concentration of GCV for negative selection We successfully demonstrated that ENEAS behaved as expected in terms of DOX regulation of the expression of mPB-ERT2. We also demonstrated that by delivering the plasmid into 293T HEK cells and manipulating the levels of DOX and TM in the medium, we could obtain puromycin resistant lines. The number of puromycin resistant colonies obtained was significantly higher when DOX as absent, suggesting that the colonies resulted from transposition events. Presence of TM added an extra layer of regulation, albeit weaker. Our PCR analysis, while not a clean as would be desired, suggested that transposition was indeed occurring, although a background level of random integration could not be ruled out. Finally, our attempt to determine whether we could use GVC to select clones that had successfully mobilized PB out of the genome was unsuccessful. Unexpectedly, 293T HEK cells that had been transfected with ENEAS and selected for puromycin resistance were insensitive to GCV.

IP/optical integration in access network infrastructures: key issues on resource provisioning

This thesis contributes to the advancement of Fiber-Wireless (FiWi) access technologies, through the development of algorithms for resource allocation and energy efficient routing. FiWi access networks use both optical and wireless/cellular technologies to provide high bandwidth and ubiquity, required by users and current high demanding services. FiWi access technologies are divided in two parts. In one of the parts, fiber is brought from the central office to near the users, while in the other part wireless routers or base stations take over and provide Internet access to users. Many technologies can be used at both the optical and wireless parts, which lead to different integration and optimization problems to be solved. In this thesis, the focus will be on FiWi access networks that use a passive optical network at the optical section and a wireless mesh network at the wireless section. In such networks, two important aspects that influence network performance are: allocation of resources and traffic routing throughout the mesh section. In this thesis, both problems are addressed. A fair bandwidth allocation algorithm is developed, which provides fairness in terms of bandwidth and in terms of experienced delays among all users. As for routing, an energy efficient routing algorithm is proposed that optimizes sleeping and productive periods throughout the wireless and optical sections. To develop the stated algorithms, game theory and networks formation theory were used. These are powerful mathematical tools that can be used to solve problems involving agents with conflicting interests. Since, usually, these tools are not common knowledge, a brief survey on game theory and network formation theory is provided to explain the concepts that are used throughout the thesis. As such, this thesis also serves as a showcase on the use of game theory and network formation theory to develop new algorithms.

Inclusão de uma criança com síndrome de turner numa escola do ensino regular: um estudo de caso

Dissertação de mestrado, Educação Especial - Domínios Cognitivo e Motor, Escola Superior de Educação e Comunicação, Universidade do Algarve, 2015

Integration von Nichtrationalität in die Praxis des Managements : ein zeitgemäßer unternehmensbezogener Erfolgsfaktor

Im Artikel wird Zunahme der Komplexität im Entscheidungsumfeld untersucht und auf zeitgemäße, auf Kreativität basierende Problemlösungsstrategien hingewiesen.

Integration der Klebflächenbehandlung in die Fertigung

In vielen Fällen muss vor dem Kleben eine Klebflächenbehandlung durchgeführt werden, da Klebverbindungen mit unvorbehandelten Teilen häufig zu geringe Klebfestigkeiten und/oder eine unzureichende Alterungsbeständigkeit aufweisen. Zur Klebflächenbehandlung stehen unterschiedliche Verfahren zur Verfügung. Wenn mit mehreren Behandlungen klebtechnisch einwandfreie Verbindungen hergestellt werden können, gilt es, das Verfahren zu ermitteln, welches am besten in den Fertigungsfluss integriert werden kann und die geringsten Kosten verursacht. Dabei muss auch die Arbeitssicherheit und der Umweltschutz mit beachtet werden. Zur Beurteilung der Verfahren werden Bewertungskriterien gegeben. Die Verfahren werden abschließend kurz charakterisiert.

Espaços dinâmicos em plataformas LMS

Tese de mestrado, Educação (Tecnologias da Informação e Comunicação e Educação), Universidade de Lisboa, Instituto de Educação, 2010

As TIC na criação de ambientes motivadores no processo de ensino e aprendizagem da economia no ensino secundário : um estudo sobre a implementação das TIC em sala de aula na unidade didática 11 na turma C do 11º ano da Escola Secundária Anselmo de Andrade

Relatório da prática de ensino supervisionada, Ensino da Economia e da Contabilidade, Universidade de Lisboa, 2012

Miniprojetos interculturais na aula de matemática : quatro estudos de caso

Tese de doutoramento, Educação (Didática da Matemática), Universidade de Lisboa, Instituto de Educação, 2014

Knowledge representation for data integration and exploration in translational medicine

Tese de doutoramento, Informática (Bioinformática), Universidade de Lisboa, Faculdade de Ciências, 2014

Environmental governance in China: weakness and potential from an environmental policy integration perspective

Interest in China’s capacity for environmental governance is growing, in line with its environmental woes and exponential economic growth. Environmental policy efforts have lacked effectiveness, confirming the persistence of a disjuncture between promise and performance. This article contributes to the debate through the analytical lens of Environmental Policy Integration (EPI): a normative concept and governance regime indispensable to sustainable development. It finds that China,like most OECD countries, falls short of the concept. Despite encouraging recent changes, driven by the Hu-Wen regime, and encapsulated in the idea of scientific development, the analysis reveals weaknesses in all three EPI-type responses: normative, organisational and procedural. The disjuncture is confirmed, but drawing on EPI’s normative perspective, it is suggested that the reasons for this lie as much in the framing of the promise, as in the performance, or implementation, itself. Based on this interpretation and on China’s unique extreme characteristics, it is recommended that environmental policy objectives be given principled priority status, as a condition for effective governance.

Comportamentos autolesivos em adolescentes : características epidemiológicas e análise de fatores psicopatológicos, temperamento efetivo e estratégias de coping

Tese de doutoramento, Medicina (Psiquiatria e Saúde Mental), Universidade de Lisboa, Faculdade de Medicina, 2014

A utilização das novas tecnologias de informação e comunicação na aprendizagem : o caso da turma do 2º ano do ensino profissional do curso técnico de receção

Relatório da prática de ensino supervisionada, Mestrado em Ensino da Economia e Contabilidade, Universidade de Lisboa, 2014

Migrações : novas realidades : viver num mundo em movimento

Supervisionada, Educação (Mestrado em Ensino de História e Geografia no 3º Ciclo do Ensino Básico e Ensino Secundário), Universidade de Lisboa, Instituto de Educação, 2014