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.

Du roman au théâtre: adaptations théâtrales dans la dramaturgie franco-portugaise au XIXe siècle

L’adaptation de romans à la scène au XIXe siècle dans la dramaturgie franco-portugaise passe par l’étude des relations et des contaminations intergénériques et interculturelles qui s’établissent entre les genres romanesque et dramatique dans les deux cultures. Pour ce faire, nous nous sommes consacrés dans une première partie aux questions théoriques qu’un tel exercice soulève. Effectivement, nous ne pourrions pas prétendre étudier et cerner un tel phénomène littéraire et artistique sans un abordage pluridimensionnel qui nous mène vers son approche à la lumière des études de Traduction et de Réception, d’un côté et, de l’autre, des études sociologiques. Questionner les fondements théoriques de l’adaptation théâtrale, sa production et sa circulation dans les deux pays supposait, pour nous, dès le départ, délimiter non seulement l’horizon d’attente d’une telle pratique artistique mais aussi sa place et son impact dans le champ littéraire et le polysystème culturel de l’époque. En contrepoint, nous nous sommes ensuite intéressés à la question de l’adaptation théâtrale et des contact zones, cette fois en passant par la problématique de la transculturation qu’engendre le contact de deux littératures différentes, s’affrontant dans le champ culturel et littéraire d’arrivée. Dans une deuxième partie, nous nous sommes concentrés sur la contextualisation historico-générique de l’adaptation théâtrale. Le recours à la transposition scénique de romans révèle un désir de gloire au niveau financier et artistique. La pratique est courante, voire généralisée dans le champ littéraire français du XIXe siècle. Parmi les adeptes de l’exercice de transposition, Dumas père est certainement l’auteur le plus prolifique. En outre, la transmodalisation générique s’insère dans un mouvement de création collective, puisque la collaboration entre auteurs dramatiques est très répandue. Par la suite, nous nous sommes intéressés à la genèse des textes à travers la transmodalisation du roman au théâtre : du roman-feuilleton au roman naturaliste. Nous avons constaté que le genre romanesque comme le genre dramatique sont empreints de contaminations intergénériques qui passent par la théâtralisation du roman puis par la romanisation du théâtre. Ainsi, l’adaptation apparaît comme la concrétisation de la fusion des genres romanesque et dramatique qui donnent lieu à un genre hybride. L’adaptation est alors le résultat d’une série de procédés et de techniques : la transmodalisation du roman s’adapte aux nécessités génériques du théâtre. De la sorte, nous avons distingué cinq types d’adaptations : l’adaptation fidèle, l’adaptation partielle, l’adaptation libre, l’adaptation pastichée et l’adaptation de l’adaptation. Dans une troisième partie, nous avons abordé le phénomène de l’adaptation dans le contexte littéraire portugais au XIXe siècle. À travers la présence accrue du théâtre français sur les scènes lisboètes, nous avons observé les rouages de la transposition d’un modèle étranger qui peut passer par différents processus, comme la traduction ou l’appropriation par l’adaptation théâtrale. Cela nous a amené à présenter cinq modèles d’adaptations théâtrales issues d’hypotextes français : l’adaptation française, la traduction fidèle de l’adaptation française, la traduction libre de l’adaptation française, l’adaptation à la couleur locale de l’adaptation française et l’adaptation portugaise d’un roman français. Nous avons également présenté quelques exemples de cas concrets, notamment Le Comte de Monte-Cristo adapté au répertoire lisboète. L’étude de ce phénomène culturel nous a permis de mieux cerner son impact sur le champ théâtral lisboète du XIXe siècle, dans la mesure où il contribue à la transculturation de la dramaturgie portugaise de l’époque.

Design of neuro-fuzzy models by evolutionary and gradient-based algorithms

All systems found in nature exhibit, with different degrees, a nonlinear behavior. To emulate this behavior, classical systems identification techniques use, typically, linear models, for mathematical simplicity. Models inspired by biological principles (artificial neural networks) and linguistically motivated (fuzzy systems), due to their universal approximation property, are becoming alternatives to classical mathematical models. In systems identification, the design of this type of models is an iterative process, requiring, among other steps, the need to identify the model structure, as well as the estimation of the model parameters. This thesis addresses the applicability of gradient-basis algorithms for the parameter estimation phase, and the use of evolutionary algorithms for model structure selection, for the design of neuro-fuzzy systems, i.e., models that offer the transparency property found in fuzzy systems, but use, for their design, algorithms introduced in the context of neural networks. A new methodology, based on the minimization of the integral of the error, and exploiting the parameter separability property typically found in neuro-fuzzy systems, is proposed for parameter estimation. A recent evolutionary technique (bacterial algorithms), based on the natural phenomenon of microbial evolution, is combined with genetic programming, and the resulting algorithm, bacterial programming, advocated for structure determination. Different versions of this evolutionary technique are combined with gradient-based algorithms, solving problems found in fuzzy and neuro-fuzzy design, namely incorporation of a-priori knowledge, gradient algorithms initialization and model complexity reduction.

Empatia e reconhecimento de emoções: o efeito do género e a relação da empatia com o alvo

A literatura mostra que a empatia se relaciona com o reconhecimento de emoções (e.g., Dimberg, Andréasson, & Thunberg, 2011) e é influenciada pelo alvo percecionado (Rueckert, Branch, & Doan, 2011). Uma das justificações para o efeito do alvo parece dever-se ao self-construto interdependente (Davis, 2004). As diferenças de género são relevantes, uma vez que as mulheres demonstram ser superiores aos homens na capacidade empática (e.g., Davis, 1980), no reconhecimento de emoções (e.g., Hall & Matsumoto, 2004), no grau de empatia sentida face a relações próximas (Rueckert et al., 2011) e na tendência para ser interdependente (e.g., Gore & Cross, 2011). O objetivo deste estudo foi observar a relação da empatia com a capacidade de reconhecer emoções e com o alvo de empatia, tendo em consideração as diferenças de género do observador. Para a realização do estudo utilizou-se uma amostra de conveniência, constituída por 150 participantes (50% mulheres), com idades compreendidas entre os 18 e os 63 anos. Os resultados mostram que a empatia não se relaciona, no global, com a capacidade de reconhecer expressões faciais emocionais. Observou-se também que existe um efeito do alvo no grau de empatia sentida pelo participante. Os participantes empatizam mais quando o alvo é um amigo do que quando é um indivíduo desconhecido ou com o qual não se identificam. Ambas as escalas de empatia se correlacionaram positivamente com a Escala de Self-Construto Interdependente. As diferenças de género foram observadas na capacidade empática e limitadas a algumas emoções no reconhecimento de expressões faciais. Não se observaram diferenças no grau de empatia sentida pelo alvo de empatia nem no índice de interdependência dos participantes. Quanto ao reconhecimento de emoções, a alegria, a surpresa e a raiva foram as mais facilmente reconhecidas, sendo a alegria e a raiva as mais rápidas. O medo foi a emoção com um índice de acertos mais baixo e um maior tempo despendido com o seu reconhecimento. Os resultados foram discutidos com base na literatura já existente.

The Rathbone Hotel: internship

The main objective of the internship is to complete the requirement of the Master program as prescribed by the University of the Algarve. An intern has to prepare an internship report at the end of the internship period but the main objective of the internship is to get experience of the real world organisation. The internship was concluded with the objective of getting practical knowledge in all departments at The Rathbone Hotel. The first responsibility I was assigned during these 12 months of internship period was to assist the Housekeeping Department. Secondly, I was allotted the work of F&B, Accounts, Reservations, Front Office, Back Office and assisting the Assistant Manager was the last task I was assigned during my Internship program. As an intern, I realised that I was successful to gather a lot of significant learning experiences which would be helpful in my future career. Work in all departments of The Rathbone Hotel offered me ample space and opportunities, not only to learn but also to exhibit my skills as a Rathbone team member. I could use my theoretical knowledge in real practice while participating in many discussions. I was actively involved in the revenue meetings where I shared my knowledge and views regarding the performance in Reservations of The Rathbone Hotel. I successfully completed all the assigned duties and passed them over to the supervisor at the end of the internship. I thoroughly enjoyed the challenges that came along every single day. These lessons that I have learned will be a valuable one for my future activities as well.

Traços psicopáticos em delinquentes juvenis: investigações sobre início na atividade criminal, etnicidade e género

Tese de doutoramento, Psicologia, Universidade do Algarve, Faculdade de Ciências Humanas e Sociais, 2013

Papéis de género, homofobia internalizada e autoestima sexual numa amostra de lésbicas e gays

Dissertação de mestrado, Psicologia (Psicologia Clínica e da Saúde), Faculdade de Ciências Humanas e Sociais, Universidade do Algarve, 2015

O apego ao lugar como determinante das intenções comportamentais no turismo: o caso do Alentejo

Tese de doutoramento, Turismo, Faculdade de Economia, Universidade do Algarve, 2015

New competitors and their intra-industry effects: the case of the hotel industry

Dissertação de Mestrado, Finanças Empresariais, Faculdade de Economia, Universidade do Algarve, 2015

Bairros de génese ilegal: compreender - intervir - reabilitar

Dissertação de mestrado, Arquitetura Paisagista, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Ciganos em Portugal : educação e género

Tese de mestrado, Ciências da Educação (Educação Intercultural), Universidade de Lisboa, Instituto de Educação, 2010

Espaço e perspectivas na representação visual

Relatório da prática de ensino supervisionada, Mestrado em Ensino de Artes Visuais, Universidade de Lisboa, 2011

Problemáticas de classificação da obra dramática de Nelson Rodrigues: críticas, reflexões e propostas

Tese de mestrado, Tradição Clássica e Cultura Europeia, Universidade de Lisboa, Faculdade de Letras, 2011

De como o pícaro chegou a Portugal e aí se apresentou:contributo para a história da recepção do romance picaresco espanhol no sistema literário português

Tese de doutoramento, Tradução (História da Tradução), Universidade de Lisboa, Faculdade de Letras, 2012

Entre Zêzere e Tejo propriedade e povoamento : (séculos XII-XIV)

Tese de doutoramento, História (História Medieval), Universidade de Lisboa, Faculdade de Letras, 2014