Integration of numerical models in geographic databases: the case of Sado estuary management

Geographic information systems (GIS) are now widely applied in coastal resource management. Their ability to organise and interface information from a large range of public and private data sources, and their ability to combine this information, using management criteria, to develop a comprehensive picture of the system explains the success of GIS in this area. The use of numerical models as a tool to improve coastal management is also widespread. Less usual is a GIS-based management to ol implementing a comprehensive management model and integrating a numerical modelling system into itself. In this paper such a methodology is proposed. A GIS-based management tool based on the DPSIR model is presented. An overview of the MOHID numerical modelling system is given and the method of integrating this model in the management tool is described. This system is applied to the Sado Estuary (Portugal). Some preliminary results of the integration are presented, demonstrating the capabilities of the management system.

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.