Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase-transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals. Copyright (C) 2004 John Wiley Sons, Ltd.

Gene transfer into genomes of human cells by the sleeping beauty transposon system

The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70-80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy.

Structure-function analysis of the inverted terminal repeats of the Sleeping Beauty transposon

Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates. (C) 2002 Elsevier Science Ltd. All rights reserved.

Society, theatre and Social Work: beauty will save us

Three different worlds, sometimes concentric and often intersecting —society, theatre and the art of performance— and social work. Diverse worlds that live, reflect and self-reflect and interact, and can also afford an opportunity for meeting, misunderstanding and confrontation, and above all offer the possibility of profound change.This article considers the experience of a theatre company that has spent more than three years moving at the limits of these three universes. To these three worlds can be added an infinite number of words that fill them with meaning and significance: territory, meeting, diversity and search. An artistic experience that has chosen to focus on creating scenarios for debate and to examine the difficulties, the human contradictions and the constant and inexhaustible confrontation with human experience. At the heart of this theatrical activity is all of this, seeking the balance between narration, meeting, investigation and the artistic dimension. This meeting between society, theatre and social work also contains the search for sustainability of this cultural business, in an Italy that has been destroyed by a crisis that is not merely economic, but also of values and, above all, of role models. The guiding theme, though not always made explicit, is always present and essential: the search for beauty.

Sexing up the city in the international beauty contest: the performative nature of spatial planning and the fictive spectacle of place branding

This paper responds to recent calls for more academic research and critical discussion on the relationship between spatial planning and city branding. Through the lens of Liverpool, the article analyses how key planning projects have delivered major transformations in the city's built environment and cultural landscape. More specifically, in concentrating on the performative nature of spatial planning it reveals the physical, symbolic and discursive re-imaging of Liverpool into a 'world class city'. Another aspect of the paper presents important socioeconomic datasets and offers a critical reading of the re-branding in showing how it presents an inaccurate representation of Liverpool. The evidence provided indicates that a more accurate label for Liverpool is a polarised and divided city, thereby questioning the fictive spectacle of city branding. Finally, the paper ends with some critical commentary on the role of spatial planning as an accessory to the sophistry of city branding.

“Something there that wasn’t there before”: representação do amor romântico em Beauty and the Beast (1991)

Dissertação apresentada à Escola Superior de Comunicação Social como parte dos requisitos para obtenção de grau de mestre em Audiovisual e Multimédia.

Young adult Brazilian beauty consumers perception of Lancôme and the effectiveness of social media relationhips with local celebrities on their purchase intention

The aim of this research is to evaluate if a premium beauty brand, in this case, Lancôme, can influence positively the purchase intention from Brazilian young adults, between 18 and 29 years old, consumers of beauty products, by initiating a relationship with a local celebrity or “it” girl on social media. This hypothesis has not been tested, and this research is a first attempt of evaluating it. Additionally, the consumer behavior, brand preferences and social media activeness of this age segment in Brazil are further studied as important insights for beauty brands to conquer these consumers. Results did not confirm the positive influence of local celebrities on this age segment’s purchase intention but several suggestions are made for future research to revisit this topic. Furthermore, there is a significant brand love for M.A.C., an international Lancôme competitor, amongst this target, as well as a probable price sensitivity facing premium beauty brands.

The consumption of beauty products on famale young adults in Portugal. Recommenrations for the brand Lancôme to improve its store trafic

This project aims to explore the Portuguese Beauty and Cosmetics market, and to discuss the usage and purchase behaviour of young female adults, between 18 and 26 years old. After a market analysis based on secondary data, it evaluates the results of qualitative and quantitative research based on 9 interviews and 126 online questionnaires to explore the consumers’ reasoning when choosing products from this category – fragrances, skin care or make-up – as well as their attitude towards brands, with a special focus on the premium cosmetics brand Lancôme. Contrary to our expectations there was no statistically significant positive influence of the online touchpoints within this age segment’s purchase intention. However, results indicate that Lancôme is already being perceived by some as young and modern, but is still suffering the threat of Mass Market brands that are valued by this target, mainly due to a price sensitivity towards premium beauty brands.