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.

How to improve Sonae's processes of suppliers of services' management?

Sonae is one of the greatest retailers in Portugal. As the business got bigger, it centralized operations. Direcção de Serviços Administrativos processes all invoices sent by suppliers. However, there are many which deliver errors and are not processed automatically. As a result, there are lost invoices and suppliers who are not paid, while the company’s accountability becomes less transparent. Such is due to a lack of proactive attitude towards suppliers as well as to a lack of incentives for employees to perform well. Great savings may be achieved with little effort, if the right things are measured so that the right tools may be applied.

A qualitative case study of the processes of peer education in a young adult tobacco control initiative, Leave the Pack Behind

Peer education involves peers offering credible and reliable information about sensitive life issues through the means of an informal peer group setting (Topping & Ehly, 1998). The purpose of this instrumental case study was to examine the processes of peer education through the exploration of two teams within a young adult tobacco control initiative, Leave the Pack Behind (LTPB). This qualitative case study examined two peer education teams over an eight-month period. Interviews, focus groups and observations were conducted with 12 participants across two peer education teams. Findings show the complexities of the processes of peer education including a connection between the stages of change and the changing role of the peer educator across stages of the empowerment process. Peer education teams and factors in the macro environment were also found to impact the process of peer education. This study provides a new definition for the process of peer education: peer education is a fluid process of knowledge exchange in which peer educators adopt different styles of facilitation as people move through stages of empowerment and change. This study contributes to the academic hterature upon the processes of peer education by providing a definition, a model and an overall understanding through an ecological and empowerment framework. The findings from this study suggest peer educators can be further trained to: use specific peer educational approaches that fit with student smoker's stage of change; better understand their position as a peer educator on the LTPB team; understand the reciprocal relationship between the macro environment and the peer education teams having an effect on one another.

Teachers' processes of negotiating meaning of new curriculum

This qualitative study addresses the question of how teachers negotiate meaning of new curriculum to better understand how curriculum is transformed from a theoretical construct to a practical one. Through interviews with 5 teachers, their experiences were examined as they negotiated the process of implementing new curriculum. Three theoretical constructs provided the entry point into the study: epistemology, teacher knowledge, and teacher learning. Using inductive analysis, 4 points or attributes of negotiation emerged: reference, growth, autonomy, and reconciliation. These attributes provided a theoretical framework from which a constructivist conceptualization of teacher learning and teacher knowledge could serve to understand the process of how teachers negotiate meaning of curriculum. Studied and theorized in this way, teacher knowledge and teacher learning are seen to be inextricably linked in a relationship that is dynamically changed by forces of stability and instability. Theorizing the negotiation of meaning from a constructivist epistemology also strengthened the assertion that negotiating meaning is a unique structural process, and that knowledge construction is therefore unique to each knower and subject to experience in a particular time and place. The implications for such a theory are, first, that it questions the legitimacy of privatized teacher practice and, second, that it calls for a renewed conceptualization of collegial network and relationship to strengthen the capacity for negotiating meaning of curricular initiatives. Understanding the relationship of curricular theory and negotiating meaning also has implications for curriculum development. In particular, the study highlights the necessity of professional discretion and the generative process of negotiating meaning.

Exploring the Ethical Decision-Making Processes of Community Care Providers

While there has been a recent shift away from isolated, institutionalized living conditions, persons with Intellectual Disabilities (ID) may still experience restricted access to choice when it comes to making decisions about the basic aspects of their lives. A tension remains between protecting individuals from harm and promoting their right to independence and personal liberties. This tension creates complex questions and ethical concerns for care providers supporting persons with ID. This study explored the ethical decision-making processes of care providers and specifically, how care providers describe the balance of protecting supported individuals from harm while promoting their right to self-determination. Semi-structured interviews were conducted with six care providers employed by a local community agency that supports young and older adults with ID. Data were analysed using thematic analysis and broader themes were developed following phases of open and selective coding. Results indicated that care providers described ethical decision-making processes as frequent, complex, subjective, and uncomfortable. All participants described the importance of promoting independent decision-making among the individuals they support and assisting supported individuals to make informed decisions. Participants also reported work colleagues and supervisors as primary sources of information when resolving ethical concerns. This suggests that complex ethical decision-making processes are being taken seriously by care providers and supervising staff. The results of this study are well-positioned to be applied to the development of a training program for frontline care providing staff supporting individuals in community care settings.

Motivation and Reading Achievement: Understanding the Needs and Motivation Processes of Adult Literacy Learners

Adult struggling readers are understudied and most evidence-based remedial approaches target youth. This thesis examined relationships among motivation constructs across typical and struggling adult readers. Age was also investigated as a moderator in these relationships. Participants included 198 adults in adult basic education and 138 undergraduate students. Examining the influence of self-efficacy on reading achievement, moderation analyses indicated there were stronger relationships for typical readers. Furthermore, stronger relationships were found for younger participants when moderated by age. Additional regression analyses identified positive relationships between two measures of intrinsic motivation and reading value. This relationship was replicated for avoidance and value. Though age was not uniformly sampled across ability grouping, age did not account for these effects. Despite difficulties with reading, adults still exhibited motivation to engage with texts with equal to greater levels of reading value. Value and intrinsic motivation may have unique developmental courses associated with longstanding reading challenges.

Activism and participation among people of migrant background : discourses and practices of inclusiveness in four italian cities

Alors que l’intérêt pour les processus d’intégration des immigrants et des minorités ethniques est en pleine croissance parmi les chercheurs européens, les facteurs qui expliquent les différentes formes de participation civique et politique doivent être examinés plus en profondeur. Prenant pour base la littérature sur l’immigration, cette étude examine la question de recherche suivante: Comment peut-on expliquer les variations des formes de participation civique et politique des activistes issus de l’immigration au niveau local? Afin de répondre à cette question, cette étude identifie les formes de participation de la part d’activistes issus de l’immigration dans quatre villes Italiennes et examine les discours et les pratiques de multiples acteurs impliqués dans le domaine de l’immigration dans un contexte national d’hostilité croissante. Cette thèse soutient que pour comprendre différentes formes de participation, il est important de considérer non seulement l’État et les acteurs institutionnels, mais aussi les acteurs non-institutionnels et examiner comment ces derniers influencent les opportunités ainsi que les restrictions à la participation. Par ailleurs, cette recherche examine les canaux conventionnels et non-conventionnels dans quatre villes italiennes et étudie les activistes issus de l’immigration comme des acteurs politiques pertinents, capables de se mobiliser et d’influencer la participation à travers leur interaction et alliances avec les acteurs de la société d’accueil. Cette recherche a permis de produire trois résultats. Le premier montre que les approches d’intégration adoptées par les acteurs sont importantes. Cette étude a identifié trois approches d’intégration: 1) « welfariste », basée sur l’idée que les immigrants sont dans le besoin et doivent donc recevoir des services; 2) interculturelle, basée sur l’idée que les immigrants sont de futurs citoyens et que l’intégration est réciproque; 3) promotion des droits politiques, basée sur l’idée que les immigrants ont des droits politiques fondamentaux ; et qui encourage l’ouverture des canaux de participation politique, surtout aux immigrants privés du droit de vote local. L’analyse empirique démontre que, alors que l’approche welfariste n’encourage pas la participation parce qu’elle conçoit les immigrants comme des acteurs passifs, les autres deux approches ont respectivement un impact sur les formes de participation civique et politique. La deuxième conclusion souligne le rôle des acteurs de gauche. En particulier, cette étude montre que les acteurs qui ouvrent de canaux pour la participation ne sont pas uniquement les acteurs de gauche modérée, comme les autorités locales, les partis politiques et les syndicats, mais aussi les groupes de gauche radicale et non-institutionnelle. Chaque acteur de gauche comprend et agit différemment par rapport aux sujets de l’immigration et de la participation et ce fait influence comment les activistes issues de l’immigration se mobilisent. La troisième conclusion met en évidence le rôle de la perception des opportunités par les activistes issus de l’immigration et la façon avec laquelle ils s’approprient les discours et les pratiques des acteurs de gauche. Ce travail démontre que l’ouverture de canaux est possible grâce à l’engagement de personnes issues de l’immigration qui agissent à travers les opportunités qui leurs sont offertes, créent des alliances avec la gauche et défient les discours et pratiques des acteurs locaux.

Effects of tillage on processes of organic matter sequestration

To increase the organic matter (OM) content in the soil is one main goal in arable soil management. The adoption of tillage systems with reduced tillage depth and/or frequency (reduced tillage) or of no-tillage was found to increase the concentration of soil OM compared to conventional tillage (CT; ploughing to 20-30 cm). However, the underlying processes are not yet clear and are discussed contradictorily. So far, few investigations were conducted on tillage systems with a shallow tillage depth (minimum tillage = MT; maximum tillage depth of 10 cm). A better understanding of the interactions between MT implementation and changes in OM transformation in soils is essential in order to evaluate the possible contribution of MT to a sustainable management of arable soils. The objectives of the present thesis were (i) to compare OM concentrations, microbial biomass, water-stable aggregates, and particulate OM (POM) between CT and MT soils, (ii) to estimate the temporal variability of water-stable aggregate size classes occurring in the field and the dynamics of macroaggregate (>250 µm) formation and disruption under controlled conditions, (iii) to investigate whether a lower disruption or a higher formation rate accounts for a higher occurrence of macroaggregates under MT compared to CT, (iv) to determine which fraction is the major agent for storing the surplus of OM found under MT compared to CT, and (v) to observe the early OM transformation after residue incorporation in different tillage systems simulated. Two experimental sites (Garte-Süd and Hohes Feld) near Göttingen, Germany, were investigated. Soil type of both sites was a Haplic Luvisol. Since about 40 years, both sites receive MT by a rotary harrow (to 5-8 cm depth) and CT by a plough (to 25 cm depth). Surface soils (0-5 cm) and subsoils (10-20 cm) of two sampling dates (after fallow and directly after tillage) were investigated for concentrations of organic C (Corg) and total N (N), different water-stable aggregate size classes, different density fractions (for the sampling date after fallow only), microbial biomass, and for biochemically stabilized Corg and N (by acid hydrolysis; for the sampling date after tillage only). In addition, two laboratory incubations were performed under controlled conditions: Firstly, MT and CT soils were incubated (28 days at 22°C) as bulk soil and with destroyed macroaggregates in order to estimate the importance of macroaggregates for the physical protection of the very labile OM against mineralization. Secondly, in a microcosm experiment simulating MT and CT systems with soil <250 µm and with 15N and 13C labelled maize straw incorporated to different depths, the mineralization, the formation of new macroaggregates, and the partitioning of the recently added C and N were followed (28 days at 15°C). Forty years of MT regime led to higher concentrations of microbial biomass and of Corg and N compared to CT, especially in the surface soil. After fallow and directly after tillage, a higher proportion of water-stable macroaggregates rich in OM was found in the MT (36% and 66%, respectively) than in the CT (19% and 47%, respectively) surface soils of both sites (data shown are of the site Garte-Süd only). The subsoils followed the same trend. For the sampling date after fallow, no differences in the POM fractions were found but there was more OM associated to the mineral fraction detected in the MT soils. A large temporal variability was observed for the abundance of macroaggregates. In the field and in the microcosm simulations, macroaggregates were found to have a higher formation rate after the incorporation of residues under MT than under CT. Thus, the lower occurrence of macroaggregates in CT soils cannot be attributed to a higher disruption but to a lower formation rate. A higher rate of macroaggregate formation in MT soils may be due to (i) the higher concentrated input of residues in the surface soil and/or (ii) a higher abundance of fungal biomass in contrast to CT soils. Overall, as a location of storage of the surplus of OM detected under MT compared to CT, water-stable macroaggregates were found to play a key role. In the incubation experiment, macroaggregates were not found to protect the very labile OM against mineralization. Anyway, the surplus of OM detected after tillage in the MT soil was biochemically degradable. MT simulations in the microcosm experiment showed a lower specific respiration and a less efficient translocation of recently added residues than the CT simulations. Differences in the early processes of OM translocation between CT and MT simulations were attributed to a higher residue to soil ratio and to a higher proportion of fungal biomass in the MT simulations. Overall, MT was found to have several beneficial effects on the soil structure and on the storage of OM, especially in the surface soil. Furthermore, it was concluded that the high concentration of residues in the surface soil of MT may alter the processes of storage and decomposition of OM. In further investigations, especially analysis of the residue-soil-interface and of effects of the depth of residue incorporation should be emphasised. Moreover, further evidence is needed on differences in the microbial community between CT and MT soils.

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.

Component processes of early reading, spelling, and narrative writing skills in Turkish: a longitudinal study

The study examined: (a) the role of phonological, grammatical, and rapid automatized naming (RAN) skills in reading and spelling development; and (b) the component processes of early narrative writing skills. Fifty-seven Turkish-speaking children were followed from Grade 1 to Grade 2. RAN was the most powerful longitudinal predictor of reading speed and its effect was evident even when previous reading skills were taken into account. Broadly, the phonological and grammatical skills made reliable contributions to spelling performance but their effects were completely mediated by previous spelling skills. Different aspects of the narrative writing skills were related to different processing skills. While handwriting speed predicted writing fluency, spelling accuracy predicted spelling error rate. Vocabulary and working memory were the only reliable longitudinal predictors of the quality of composition content. The overall model, however, failed to explain any reliable variance in the structural quality of the compositions

Use of the statistical properties of the wavelet-transform coefficients for optimization of integration time in Fourier transform spectrometry

We show that an analysis of the mean and variance of discrete wavelet coefficients of coaveraged time-domain interferograms can be used as a specification for determining when to stop coaveraging. We also show that, if a prediction model built in the wavelet domain is used to determine the composition of unknown samples, a stopping criterion for the coaveraging process can be developed with respect to the uncertainty tolerated in the prediction.