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.

Projecto de formação contínua de professores de educação visual em regime b-learning

Tese de mestrado, Educação (Área de especialização em Educação e Tecnologias Digitais), Universidade de Lisboa, Instituto de Educação, 2014

Dynamic Network Mechanisms of Relational Integration

A prominent hypothesis states that specialized neural modules within the human lateral frontopolar cortices (LFPCs) support “relational integration” (RI), the solving of complex problems using inter-related rules. However, it has been proposed that LFPC activity during RI could reflect the recruitment of additional “domain-general” resources when processing more difficult problems in general as opposed to RI specifi- cally. Moreover, theoretical research with computational models has demonstrated that RI may be supported by dynamic processes that occur throughout distributed networks of brain regions as opposed to within a discrete computational module. Here, we present fMRI findings from a novel deductive reasoning paradigm that controls for general difficulty while manipulating RI demands. In accordance with the domain- general perspective, we observe an increase in frontoparietal activation during challenging problems in general as opposed to RI specifically. Nonetheless, when examining frontoparietal activity using analyses of phase synchrony and psychophysiological interactions, we observe increased network connectivity during RI alone. Moreover, dynamic causal modeling with Bayesian model selection identifies the LFPC as the effective connectivity source. Based on these results, we propose that during RI an increase in network connectivity and a decrease in network metastability allows rules that are coded throughout working memory systems to be dynamically bound. This change in connectivity state is top-down propagated via a hierarchical system of domain-general networks with the LFPC at the apex. In this manner, the functional network perspective reconciles key propositions of the globalist, modular, and computational accounts of RI within a single unified framework.

Identification of mechanisms controlling the transcriptional activity of nuclear factor kappa B (NF-κB) p65/RelA

Dissertação para a obtenção do grau de doutor em Biologia pelo Instituto de Tecnologia Química e Biológica. Universidade Nova de Lisboa.

Synaptic Integration of Adult-Born Hippocampal Neurons Is Locally Controlled by Astrocytes.

Adult neurogenesis is regulated by the neurogenic niche, through mechanisms that remain poorly defined. Here, we investigated whether niche-constituting astrocytes influence the maturation of adult-born hippocampal neurons using two independent transgenic approaches to block vesicular release from astrocytes. In these models, adult-born neurons but not mature neurons showed reduced glutamatergic synaptic input and dendritic spine density that was accompanied with lower functional integration and cell survival. By taking advantage of the mosaic expression of transgenes in astrocytes, we found that spine density was reduced exclusively in segments intersecting blocked astrocytes, revealing an extrinsic, local control of spine formation. Defects in NMDA receptor (NMDAR)-mediated synaptic transmission and dendrite maturation were partially restored by exogenous D-serine, whose extracellular level was decreased in transgenic models. Together, these results reveal a critical role for adult astrocytes in local dendritic spine maturation, which is necessary for the NMDAR-dependent functional integration of newborn neurons.

Development of techniques for testing the effect of the E1 region on adenovirus integration /

Adenoviral vectors are currently the most widely used gene therapeutic vectors, but their inability to integrate into host chromosomal DNA shortened their transgene expression and limited their use in clinical trials. In this project, we initially planned to develop a technique to test the effect of the early region 1 (E1) on adenovirus integration by comparing the integration efficiencies between an E1-deleted adenoviral vector (SubE1) and an Elcontaining vector (SubE3). However, we did not harvest any SubE3 virus, even if we repeated the transfection and successfully rescued the SubE1 virus (2/4 transfections generated viruses) and positive control virus (6/6). The failure of rescuing SubE3 could be caused by the instability of the genomic plasmid pFG173, as it had frequent intemal deletions when we were purifying It. Therefore, we developed techniques to test the effect of E1 on homologous recombination (HR) since literature suggested that adenovirus integration is initiated by HR. We attempted to silence the E1 in 293 cells by transfecting E1A/B-specific small interfering RNA (siRNA). However, no silenced phenotype was observed, even if we varied the concentrations of E1A/B siRNA (from 30 nM to 270 nM) and checked the silencing effects at different time points (48, 72, 96 h). One possible explanation would be that the E1A/B siRNA sequences are not potent enough to Induce the silenced phenotype. For evaluating HR efficiencies, an HR assay system based on bacterial transfonmatJon was designed. We constmcted two plasmids ( designated as pUC19-dl1 and pUC19-dl2) containing different defective lacZa cassettes (forming white colonies after transformation) that can generate a functional lacZa cassette (forming blue colonies) through HR after transfecting into 293 cells. The HR efficiencies would be expressed as the percentages of the blue colonies among all the colonies. Unfortunately, after transfonnation of plasmid isolated from 293 cells, no colony was found, even at a transformation efficiency of 1.8x10^ colonies/pg pUC19, suggesting the sensitivity of this system was low. To enhance the sensitivity, PCR was used. We designed a set of primers that can only amplify the recombinant plasmid fomied through HR. Therefore, the HR efficiencies among different treatments can be evaluated by the amplification results, and this system could be used to test the effect of E1 region on adenovirus integration. In addition, to our knowledge there was no previous studies using PCR/ Realtime PCR to evaluate HR efficiency, so this system also provides a PCR-based method to carry out the HR assays.

Measuring curriculum integration in the junior grades: the design of an instrument

This study \Alas initiated in response to the Junior Division Review (1985) publ ished by the Ministry of Education for the Province of Ontario. Curriculum integration is an element used within the educational paradigm designed by the Ontario Ministry of Education. It is a term frequent1y verbal ized b>' educators in this province, but because of 1 imi ted resource support regarding this methodology, it was open to broad interpretation resulting in an extreme v ar i at i on i nit simp 1 eme n tat i on • I n de ed, the Min i s try intimated that it was not occurring to any significant degree across the province. The objective of this thes is was· to define integration in the junior classroom and de-:.ign a meas.ur·ement in-:.tr-ument which would in turn high 1 i gh t indicators of curriculum integration. The :.tudy made a prel iminary, field-based survey of educa tiona 1 professionals in order to generate a relevant description of integrated curr-iculum programm i ng as def i ned in the j un i or classroom. The description was a compilation of views expressed by a random selection of teachers, consultants, supervisory officers and principals. The survey revea 1 ed a much more comprehens i ve vi et·<,l of the attributes of integrated programming than tradition would dictate and resulted in a functional definition tha t was broader than past prac t ices. Based on the information generated by this survey, an instrument ou t 1 in i ng program cr iter i a of was devised. an integrated junior cla~·sroom Th i s measuremen t i nstrumen t , designed for all levels of educators, was named uThe Han~.son I nstrumen t for the Measuremen t of Program Integrat ion in the Jun i or Cl assroom". It refl ected five categories intrinsic to the me thodol ogy of integration: Teacher Behaviour, Student Behaviour, Classroom Layout, Cl as~·r oom Environment and Progr amm i ng. Each category and the items therein were successfully tested in val idi ty and rel iabi 1 i ty checKs. Interestingly, the individual class was found to be the major variable programming in in the measuremen t the j un i or d i vis i on • of The integrated instrument demonstrated potential not onl)' a~· an initial measure of the degree of integrated curriculum, but as a guide to strategies to implement such a methodology.

Determination of viral load and integration status of HPV 16 in normal and LSIL exfoliated cervical cells

L’intégration du génome du virus papilloma humain (VPH) a été reconnu jusqu’`a récemment comme étant un événnement fréquent mais pourtant tardif dans la progression de la maladie du col de l’utérus. La perspective temporelle vient, pourtant, d’être mise au défi par la détection de formes intégrées de VPH dans les tissus normaux et dans les lésions prénéoplasiques. Notre objectif était de déterminer la charge virale de VPH-16 et son état physique dans une série de 220 échantillons provenant de cols uterins normaux et avec des lésions de bas-grade. La technique quantitative de PCR en temps réel, méthode Taqman, nous a permis de quantifier le nombre de copies des gènes E6, E2, et de la B-globine, permettant ainsi l’évaluation de la charge virale et le ratio de E6/E2 pour chaque spécimen. Le ratio E6/E2 de 1.2 ou plus était suggestif d’intégration. Par la suite, le site d’intégration du VPH dans le génome humain a été déterminé par la téchnique de RS-PCR. La charge virale moyenne était de 57.5±324.6 copies d'ADN par cellule et le ratio E6/E2 a évalué neuf échantillons avec des formes d’HPV intégrées. Ces intégrants ont été amplifiés par RS-PCR, suivi de séquençage, et l’homologie des amplicons a été déterminée par le programme BLAST de NCBI afin d’identifier les jonctions virales-humaines. On a réussi `a identifier les jonctions humaines-virales pour le contrôle positif, c'est-à-dire les cellules SiHa, pourtant nous n’avons pas detecté d’intégration par la technique de RS-PCR dans les échantillons de cellules cervicales exfoliées provenant de tissus normaux et de lésions de bas-grade. Le VPH-16 est rarement intégré dans les spécimens de jeunes patientes.

Exercises B

Exercises and solutions in LaTex

Exercises B pdf

Exercises and solutions in PDF

Securitización en el desierto: Una respuesta al terrorismo en Malí y en Níger, 2009 - 2013.

Este es un estudio sobre las dinámicas de seguridad en Malí durante el periodo de 2009 a 2013. La investigación busca explicar de qué manera se ha dado un proceso de securitización de los grupos insurgentes frente a la amenaza generada por la proliferación de grupos armados no estatales en el territorio comprendido entre Malí y Níger. Se toma a Níger con el ánimo de ver la existencia de un subcomplejo regional de seguridad entre este país y Malí. De esta manera se afirma que el aumento de las actividades insurgentes y terroristas en la zona compuesta por Malí y Níger se da por la proliferación de actores armados no estatales, entre los cuales se encuentran los grupos seculares e insurgentes Tuareg, las agrupaciones islamistas fundamentalistas y los grupos que se componen entre rebeldes Tuareg, criminales e islamistas, éstos actores han afectado la percepción que tiene Malí sobre su seguridad.

Stock return comovements and integration within the Latin American integrated market

Financial integration has been pursued aggressively across the globe in the last fifty years; however, there is no conclusive evidence on the diversification gains (or losses) of such efforts. These gains (or losses) are related to the degree of comovements and synchronization among increasingly integrated global markets. We quantify the degree of comovements within the integrated Latin American market (MILA). We use dynamic correlation models to quantify comovements across securities as well as a direct integration measure. Our results show an increase in comovements when we look at the country indexes, however, the increase in the trend of correlation is previous to the institutional efforts to establish an integrated market in the region. On the other hand, when we look at sector indexes and an integration measure, we find a decreased in comovements among a representative sample of securities form the integrated market.

Towards a Eurasian Economic Union: the challenge of integration and unity. CEPS Special Report No. 75/December 2012

In the lead-up to the creation of a Eurasian Economic Union in 2015, the Customs Union and the Common Economic Space between Russia, Belarus and Kazakhstan represent two elements of the most ambitious regional integration project launched in the post-Soviet era since 1991. This CEPS Special Report examines both the potential and the limits of Eurasian economic integration. For the purpose of assessing the Eurasian integration process, CEPS applied a modified version of a framework first developed by Ernest B. Haas and Philippe C. Schmitter in 1964 to project whether economic integration of a group of countries automatically engenders political unity. Taking the data available for the early stages of the European integration process as a benchmark, the results for the Customs Union and the Common Economic Space point to a rather unfavourable outlook for Eurasian economic integration.