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.

Desenho de modelos neuronais para a deteção de sismos

A performance dos detetores sísmicos atualmente utilizados pode e deve ser melhorada. Atualmente existem vários algoritmos para a deteção de sismos de forma automática, desde os sistemas simples baseados em STA/LTA, aos mais sofisticados baseados em reconhecimento de padrões. Este estudo pretende dar continuidade ao desenvolvimento de uma abordagem de deteção de eventos sísmicos ao nível da estação local, utilizando uma técnica bastante conhecida, chamada Máquina de Vetores de Suporte (SVM). SVM é amplamente utilizada em problemas de classificação, devido a sua boa capacidade de generalização. Nesta experiência, a técnica baseada em SVM é aplicada em diferentes modos de operações. Os resultados mostraram que a técnica proposta dá excelentes resultados em termos de sensibilidade e especificidade, além de exigir um tempo de deteção suficientemente pequeno para ser utilizado num sistema de aviso precoce (early-warning system). Começamos pela classificação de dados de forma Off-line, seguido da validação do classificador desenvolvido. Posteriormente, o processamento de dados é executado de forma contínua (On-line). Os algoritmos foram avaliados em conjuntos de dados reais, provenientes de estações sísmicas da Rede de Vigilância Sísmica de Portugal, e em aplicações reais da área de Sismologia (simulação de funcionamento em ambiente real). Apesar de apenas duas estações serem consideradas, verificou-se que utilizando a combinação de detetores, consegue-se uma percentagem de deteção idêntica para quando utilizado um único modelo (Abordagem OR) e o número de falsos alarmes para a combinação de modelos é quase inexistente (Abordagem AND). Os resultados obtidos abrem várias possibilidades de pesquisas futuras.

Tecnologia lítica da Middle Stone Age da Gruta da Leba (Huíla, SW Angola)

África é hoje o foco principal na investigação sobre a problemática da emergência da humanidade. O registo arqueológico actual sugere que também alinhagem do Homo sapiens surgiu na África subsaariana, tornando a Middle Stone Age no período chave no debate sobre a emergência do comportamento humano moderno. Os primeiros indícios de comportamento humano presumivelmente moderno provêm da África do Sul, mas há um enorme vazio arqueográfico sobre as áreas limítrofes desse território como é o caso de Angola que podem trazer novos dados à discussão. A presente dissertação debruça-se sobre a colecção arqueológica da Gruta da Leba (Huíla, SW de Angola), actualmente parte do Arquivo Científico Tropical (Instituto de Investigação Científica Tropical), que permanece inédita, apesar de recolhida em escavação arqueológica pela Junta de Investigações do Ultramar durante os anos 1950, no âmbito da Missão Antropobiológica de Angola. O conjunto é composto por uma indústria lítica com mais de 1500 peças, bem como restos faunísticos e amostras de sedimentos, de uma sequência estratigráfica com três níveis de ocupação da Middle Stone Age. O principal objectivo deste estudo é caracterizar a produção e utilização dos materiais líticos da Middle Stone Age da Gruta da Leba, através da sistematização da informação de cariz tecnológico e tipológico do conjunto artefactual. A partir da análise dos materiais líticos é possível definir padrões de variabilidade na exploração das matérias-primas e cadeias operatórias de produção do sítio. Pretende-se, assim, enquadrar regionalmente as indústrias líticas no panorama mais alargado da Pré-História da África subsaariana, para a validação dos modelos crono-culturais convencionais e avaliação dos parâmetros teórico-metodológicos aplicados na abordagem à tecnologia lítica africana em relação à problemática do comportamento humano moderno.

Cartografia solar: estimativa do potencial solar no município de Albufeira

A dependência energética dos combustíveis fósseis é, reconhecidamente, uma das principiais inquietações das economias mundiais. Questões como a concentração de gases de efeito de estufa, poluição atmosférica e o facto de estes combustíveis serem esgotáveis estão cada vez mais no centro das atenções globais, ao que se associa um aumento significativo da população mundial e por conseguinte, dos consumos. Contudo, começa a ser notório um empenho na procura de soluções viáveis que possam contornar esta dependência, nomeadamente no desenvolvimento de tecnologia que permita o aproveitamento de fontes de energias renováveis. Neste sentido, Portugal encontra-se numa posição privilegiada sendo, dos países da europa, o que apresenta índices mais elevados de radiação solar por unidade de superfície, em média cerca de 1500kWh/m2/ano. O sucesso do aproveitamento de fontes de energia renováveis vai depender, em muito, da análise e compreensão do potencial determinado pelas realidades locais, pelos dados meteorológicos, pelas tecnologias existentes e por fatores económicos. (WITTMAN et al., 1997). O processo para estimativa o potencial não é simples e implica uma interação disciplinar; no entanto tendem a aparecer modelos que, a montante do processo, simplificam a análise espacial da energia solar. Neste campo, o contributo dos Sistemas de Informação Geográfica tem sido fundamental, oferecendo um conjunto de ferramentas de processamento de dados bastantes úteis para a modelação solar. A presente dissertação combina a utilização dos SIG com a análise do potencial solar, apresentando como objetivos principais: i) a estimativa do potencial solar no Município de Albufeira e ii) a criação de uma aplicação que disponibilize os resultados através da web, facilitando a consulta e análise espacial dos mesmos.

Marine foundation species shifting ranges and genetic baselines

This thesis revealed the most importance factors shaping the distribution, abundance and genetic diversity of four marine foundation species. Environmental conditions, particularly sea temperatures, nutrient availability and ocean waves, played a primary role in shaping the spatial distribution and abundance of populations, acting on scales varying from tens of meters to hundreds of kilometres. Furthermore, the use of Species Distribution Models (SDMs) with biological records of occurrence and high-resolution oceanographic data, allowed predicting species distributions across time. This approach highlighted the role of climate change, particularly when extreme temperatures prevailed during glacial and interglacial periods. These results, when combined with mtDNA and microsatellite genetic variation of populations allowed inferring for the influence of past range dynamics in the genetic diversity and structure of populations. For instance, the Last Glacial Maximum produced important shifts in species ranges, leaving obvious signatures of higher genetic diversities in regions where populations persisted (i.e., refugia). However, it was found that a species’ genetic pool is shaped by regions of persistence, adjacent to others experiencing expansions and contractions. Contradicting expectations, refugia seem to play a minor role on the re(colonization) process of previously eroded populations. In addition, the available habitat area for expanding populations and the inherent mechanisms of species dispersal in occupying available habitats were also found to be fundamental in shaping the distributions of genetic diversity. However, results suggest that the high levels of genetic diversity in some populations do not rule out that they may have experienced strong genetic erosion in the past, a process here named shifting genetic baselines. Furthermore, this thesis predicted an ongoing retraction at the rear edges and extinctions of unique genetic lineages, which will impoverish the global gene pool, strongly shifting the genetic baselines in the future.

The activity of mouse Cerberus like 2 during cardiogenesis - genetic and morphogenetic studies

Cardiogenesis is a delicate and complex process that requires the coordination of an intricate network of pathways and the different cell types. Therefore, understanding heart development at the morphogenetic level is an essential requirement to uncover the causes of congenital heart disease and to provide insight for disease therapies. Mouse Cerberus like 2 (Cerl2) has been defined as a Nodal antagonist in the node with an important role in the Left-Right (L/R) axis establishment, at the early embryonic development. As expected, Cerl2 knockout mice (Cerl2-/-) showed multiple laterality defects with associated cardiac failure. In order to identify the endogenous role of Cerl2 during heart formation independent of its described functions in the node, we accurately analyzed animals where laterality defects were not present. We thereby unravel the consequences of Cerl2 lossof- function in the heart, namely increased left ventricular thickness due to hyperplasia of cardiomyocytes and de-regulated expression of cardiac genes. Furthermore, the Cerl2 mutant neonates present impaired cardiac function. Once that the cardiac expression of Cerl2 is mostly observed in the left ventricle until around midgestration, this result suggest a specific regulatory role of Cerl2 during the formation of the left ventricular myoarchitecture. Here, we present two possible molecular mechanisms underlying the cardiac Cerl2 function, the regulation of Cerl2 antagonist in activation of the TGFßs/Nodal/Activin/Smad2 signaling identified by increased Smad2 phosphorilation in Cerl2-/- hearts and the negative feedback between Cerl2 and Wnt/ß-catenin signaling in heart formation. In this work and since embryonic stem cells derived from 129 mice strain is extensively used to produce targeted mutants, we also present echocardiographic reference values to progressive use of juveniles and young adult 129/Sv strain in cardiac studies. In addition, we investigate the cardiac physiology of the surviving Cerl2 mutants in 129/Sv background over time through a follow-up study using echocardiographic analysis. Our results revealed that Cerl2-/- mice are able to improve and maintain the diastolic and most of systolic cardiac physiologic parameters as analyzed until young adult age. Since Cerl2 is no longer expressed in the postnatal heart, we suggest that an intrinsic and compensatory mechanism of adaptation may be active for recovering the decreased cardiac function found in Cerl2 mutant neonates. Altogether, these data highlight the role of Cerl2 during embryonic heart development in mice. Furthermore, we also suggest that Cerl2-/- may be an interesting model to uncover the molecular, cellular and physiological mechanisms behind the improvement of the cardiac function, contributing to the development of therapeutic approaches to treat heart failures.

Recreational shore fishing in southern Portugal: biological and socio-economic aspects and perspectives for management

Although overfishing is a concern for many fish stocks, it was for a long time only associated with commercial fishing exploitation, with less or no attention being given to the recreational fisheries. Recent research has shown however that the impact of recreational fishing on particular species can be considerable, and that the recreational harvest needs to be taken into account if fisheries are to be accurately assessed and effectively managed. In Portugal, the first recreational fishing regulations were only recently implemented. However, mirroring other European countries, regulations lacked scientific support, and specific knowledge of the activity was limited to a few studies with limited coverage. This thesis aimed to characterize the biological and socioeconomic aspects of the recreational shore angling activity in southern Portugal, to investigate whether the regulations in place were adequate and effective, and to provide recommendations for improved management and conservation of the inshore fisheries resources. A combined aerial-roving survey was conducted to gather data on fishing effort, catch, fishing trips and socioeconomic aspects (including anglers’ perceptions of regulations) of the recreational angling activity. The analysis of anglers’ catches suggested that compliance with daily bag limits was high, with less than 0.5% of creels exceeding the 10 kg angler-1 day-1 bag limit. Overall, 11.5% of the retained fishes were undersized, but non-compliance with minimum size limits was found to be high for some species (e.g. seabass, 73% undersized). In terms of the impact of recreational shore angling, the total estimated catches corresponded to less than 1% of the commercial landings for the same period (shared species). However, shore angling catches for white sea bream (Diplodus sargus) were found to be considerable, corresponding to 65% of the commercial landings (39.4% of total catch). In terms of anglers’ perceptions about the recreational fishing regulations in Portugal, the present study has shown that the majority of anglers accepted the existence of some kind of SRF regulations, but in general there was a partial or total disagreement with the recreational fishing restrictions recently put in place. Most anglers perceived themselves as not being involved in the decision-making process and claimed that some restrictions lacked a meaningful rationale (e.g. prohibition of fishing from piers/jetties). Fishers’ awareness with regard to specific aspects of the restrictions (such as the rationale for minimum size limits) was found to be very limited. During the same period, catches from sport fishing competitions were examined to test for differences with the recreational activity in terms of catches, and evaluate long term trends in catch and mean size of fish. Catches of the sport fishing competitions were found to be different from those observed for recreational fishing, being dominated by different species (e.g. garfish, mullets), and suggesting different fishing strategies of the the two types of anglers. High percentages of undersized fish were observed to be captured (and retained) during the competitions (in particular seabass, with 100% undersized), probably as a result of a single allowable minimum size (AMS) of 15 cm for all species in use in competitions. Lastly, catch and release fishing experiments were carried out to assess post-release mortality of three recreationally important species: two banded sea bream Diplodus vulgaris; black sea bream Spondyliosoma cantharus; and gilthead sea bream Sparus aurata. Post-release mortalities were found to be low (0-12%). The main predictor of mortality for Sparus aurata was anatomical hooking location, with 63% of the fishes that died being deeply hooked. The results support the release of fish, either from mandatory (e.g. minimum landing sizes) or voluntary practices. In summary, this thesis has demonstrated that the impact of recreational fishing for particular species is significant and needs to be taken into account for more effective management and stock assessment purposes. It has also highlighted several management issues that should be addressed in order to promote more adequate regulations in the future and prevent noncompliance issues. A periodic monitoring of the recreational fishing activity including all fishing modes (i.e. spear fishing, boat, and shore angling) would also be beneficial to ensure a timely knowledge on the global recreational fishing activity and support future management actions.

Molecular characterization of Gla-rich protein (GRP) gene expression and function

Gla-rich protein (GRP) is a vitamin K-dependent protein related to bone and cartilage recently described. This protein is characterized by a large number of Gla (γ-carboxyglutamic acid) residues being the protein with the highest Gla content of any known protein. It was found in a widely variety of tissues but highest levels was found in skeletal and cartilaginous tissues. This small secreted protein was also expressed and accumulated in soft tissues and it was clearly associated with calcification pathologies in the same tissues. Although the biological importance of GRP remains to be elucidated, it was suggested a physiological role in cartilage development and calcification process during vertebrate skeleton formation. Using zebrafish, an accepted model to study skeletal development, we have described two grp paralog genes, grp1 and grp2, which exhibited distinct patterns of expression, suggesting different regulatory pathways for each gene. Gene synteny analysis showed that grp2 gene is more closely related to tetrapod grp, although grp1 gene was proposed to be the vertebrate ortholog by sequence comparison. In addition, we identified a functional promoter of grp2 gene and using a functional approach we confirmed the involvement of transcription factors from Sox family (Sox9b and Sox10) in the regulation of grp2 expression. In an effort to provide more information about the function of grp isoforms, we generated two zebrafish transgenic lines capable to overexpress conditionally grp genes and possible roles in the skeleton development were studied. To better understand GRP function a mammalian system was used and the analysis of knockout mice showed that GRP is involved in chondrocyte maturation and the absence of GRP is associated to proteoglycans loss in calcified articular cartilage. In addition, we detected differences in chondrogenesis markers in articular chondrocyte primary culture. Overall, our data suggest a main role for GRP on chondrocyte differentiation.

Predicting intention to work with social robots

The growing number of robotic solutions geared to interact socially with humans, social robots, urge the study of the factors that will facilitate or hinder future human robot collaboration. Hence the research question: what are the factors that predict intention to work with a social robot in the near future. To answer this question the following socio-cognitive models were studied, the theory of reasoned action, the theory of planned behavior and the model of goal directed behavior. These models purport that all the other variables will only have an indirect effect on behavior. That is, through the variables of the model. Based on the research on robotics and social perception/ cognition, social robot appearance, belief in human nature uniqueness, perceived warmth, perceived competence, anthropomorphism, negative attitude towards robots with human traits and negative attitudes towards interactions with robots were studied for their effects on attitude towards working with a social robot, perceived behavioral control, positive anticipated emotions and negative anticipated emotions. Study 1 identified the social representation of robot. Studies 2 to 5 investigated the psychometric properties of the Portuguese version of the negative attitude towards robots scale. Study 6 investigated the psychometric properties of the belief in human nature uniqueness scale. Study 7 tested the theory of reasoned action and the theory of planned behavior. Study 8 tested the model of goal directed behavior. Studies 7 and 8 also tested the role of the external variables. Study 9 tested and compared the predictive power of the three socio-cognitive models. Finally conclusion are drawn from the research results, and future research suggestions are offered.

Connectivity patterns and early life history of the black-faced blenny Tripterygion delaisi (Cadenat and Blache, 1970)

Tese de doutoramento, Ciências do Mar, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Importância da informação no controlo da Diabetes Mellitus: estudo caso numa farmácia comunitária

Dissertação de mestrado, Ciências Farmacêuticas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

A influência da viscosidade dos solos argilosos na consolidação do dique nº 3 da Represa de Lebrija

Dissertação de mestrado, Engenharia Civil (Construção), Instituto Superior de Engenharia, Universidade do Algarve, 2014

Sistemas de custeio aplicados à atividade agrícola: o caso vitacress

Dissertação de Mestrado, Contabilidade, Faculdade de Economia, Universidade do Algarve, 2015

Relevância do resultado integral: caso da Euronext Lisbon

Dissertação de Mestrado, Contabilidade, Faculdade de Economia, Universidade do Algarve, 2015

A influência da batimetria no regime de impacte de tempestades. Estudo de um caso prático com recurso a SIG

Dissertação de Mestrado, Geomática – Ramo de Ciências da Informação Geográfica, Faculdade de Ciências e Tecnologia, Instituto Superior de Engenharia, Universidade do Algarve, 2015