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.

African Studies and the Postcolonial Challenge

Abrahamsen, Rita, 'African Studies and the Postcolonial Challenge', African Affairs (2003) 102(407) pp.189-210 RAE2008

African Pasts: Memory and History in African Literatures

Woods, T. (2007). African Pasts: Memory and History in African Literatures. Manchetser: Manchester University Press. RAE2008

China's performance in Sino-African cooperation and the impact in Africa's development

Trabalho de Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para a obtenção do grau de Mestre em Cooperação Internacional e Desenvolvimento

Ben Marais (1909-1999): The influences on and heritage of a South African Prophet during two periods of transformation

University of Pretoria / Dissertation / Department of Church History and Church Policy / Advised by Prof J W Hofmeyr

The African slave trade / by Thomas Fowell Buxton.

Digitized by Google

African missionary heroes and heroines / by H.K.W. Kumm.

http://www.archive.org/details/africanmissionar00kummuoft

African wastes reclaimed : illustrated in the story of the Lovedale Mission

http://books.google.com/books?vid=OCLC09108077

Singing Through Struggle: Music as a Mode of Cultural Exchange in African American Border City Churches After Emancipation, 1862-1890

This project investigates how religious music, invested with symbolic and cultural meaning, provided African Americans in border city churches with a way to negotiate conflict, assert individual values, and establish a collective identity in the post- emancipation era. In order to focus on the encounter between former slaves and free Blacks, the dissertation examines black churches that received large numbers of southern migrants during and after the Civil War. Primarily a work of history, the study also employs insights and conceptual frameworks from other disciplines including anthropology and ritual studies, African American studies, aesthetic theory, and musicology. It is a work of historical reconstruction in the tradition of scholarship that some have called "lived religion." Chapter 1 introduces the dissertation topic and explains how it contributes to scholarship. Chapter 2 examines social and religious conditions African Americans faced in Baltimore, MD, Philadelphia, PA, and Washington, DC to show why the Black Church played a key role in African Americans' adjustment to post-emancipation life. Chapter 3 compares religious slave music and free black church music to identify differences and continuities between them, as well as their functions in religious settings. Chapters 4, 5, and 6 present case studies on Bethel African Methodist Episcopal Church (Baltimore), Zoar Methodist Episcopal Church (Philadelphia), and St. Luke’s Protestant Episcopal Church (Washington, DC), respectively. Informed by fresh archival materials, the dissertation shows how each congregation used its musical life to uphold values like education and community, to come to terms with a shared experience, and to confront or avert authority when cultural priorities were threatened. By arguing over musical choices or performance practices, or agreeing on mutually appealing musical forms like the gospel songs of the Sunday school movement, African Americans forged lively faith communities and distinctive cultures in otherwise adverse environments. The study concludes that religious music was a crucial form of African American discourse and expression in the post-emancipation era. In the Black Church, it nurtured an atmosphere of exchange, gave structure and voice to conflict, helped create a public sphere, and upheld the values of black people.

Ubuntu, Jesus, and Earth: Integrating African Religion and Christianity in Ecological Ethics

Africa faces problems of ecological devastation caused by economic exploitation, rapid population growth, and poverty. Capitalism, residual colonialism, and corruption undermine Africa's efforts to forge a better future. The dissertation describes how in Africa the mounting ecological crisis has religious, political, and economic roots that enable and promote social and environmental harm. It presents the thesis that religious traditions, including their ethical expressions, can effectively address the crisis, ameliorate its impacts, and advocate for social and environmental betterment, now and in the future. First, it examines African traditional religion and Christian teaching, which together provide the foundation for African Christianity. Critical examination of both religious worldviews uncovers their complementary emphases on human responsibility toward planet Earth and future generations. Second, an analysis of the Gwembe Tonga of Chief Simamba explores the interconnectedness of all elements of the universe in African cosmologies. In Africa, an interdependent, participatory relationship exists between the world of animals, the world of humans, and the Creator. In discussing the annual lwiindi (rain calling) ceremony of Simamba, the study explores ecological overtones of African religions. Such rituals illustrate the involvement of ancestors and high gods in maintaining ecological integrity. Third, the foundation of the African morality of abundant life is explored. Across Sub-Saharan Africa, ancestors' teachings are the foundation of morality; ancestors are guardians of the land. A complementary teaching that Christ is the ecological ancestor of all life can direct ethical responses to the ecological crisis. Fourth, the eco-social implications of ubuntu (what it means to be fully human) are examined. Some aspects of ubuntu are criticized in light of economic inequalities and corruption in Africa. However, ubuntu can be transformed to advocate for eco-social liberation. Fifth, the study recognizes that in some cases conflicts exist between ecological values and religious teachings. This conflict is examined in terms of the contrast between awareness of socioeconomic problems caused by population growth, on the one hand, and advocacy of a traditional African morality of abundant children, on the other hand. A change in the latter religious view is needed since overpopulation threatens sustainable living and the future of Earth. The dissertation concludes that the identification of Jesus with African ancestors and theological recognition of Jesus as the ecological ancestor, woven together with ubuntu, an ethic of interconnectedness, should characterize African consciousness and promote resolution of the socio-ecological crisis.

The emerging strategic partnership between the European Union and the African Union

Many among the emerging generation of political elites in Africa see the role the European Union (EU) plays in the maintenance of an unprecedented period of peace in Western Europe as an inspirational example of the manner in which the African Union (AU) can contribute to peace and stability in Africa. This doctoral thesis examines security cooperation between the EU and the AU, with a particular focus on the nature and substance of that cooperation. It suggests that despite the establishment of various EU–AU institutions and ties with a role in security policy and cooperation, such security cooperation is limited in substance. This study argues that EU–AU security cooperation is especially constrained by the emergence of alternative partners, most notably China, and by failures of implementation and follow-through. Two case studies, the first dealing with EU–AU cooperation in peacekeeping, and the second addressing the silent water crisis along with the link between water and security, have been analysed in detail to determine the effectiveness and sustainability of the EU–AU partnership. A number of important lessons for regionalism, interregionalism and multilateralism are drawn from the bond between the EU and the AU. This doctoral thesis will prove that, despite an emphasis on the problematic term ‘strategic’ by both EU and AU policymakers, EU–AU cooperation is limited and somewhat lacking in strategic direction. The cooperation between the EU and the AU focuses mainly on EU financial support for AU peacekeeping and specific projects in Africa (e.g. in the water sector), as well as on a limited political dialogue. Nonetheless, the EU–AU link represents the most comprehensive partnership the AU has with any non-African actor. This study will furthermore demonstrate that the United Nations (UN) is an indispensable third-party to their relationship and it is therefore more appropriate to speak of the AU–EU–UN nexus. This doctoral thesis concludes that the AU–EU–UN nexus is an important example of interregionalism in a global context and that such interregionalism is an important emerging part of global governance.

Psychosocial well-being of African migrant children in Ireland: a cultural orientation

This thesis explores the psychosocial wellbeing of sub-Saharan African migrant children in Ireland. A sociocultural ecological (Psychosocial Working Group, 2003) and resilience lens (Masten & Obradovic, 2008; Ungar, 2011) is used to analyse the experiences of African migrant children in Ireland. The research strategy employs a mixed-methods design, combining both an etic and emic perspective. Grounded theory inquiry (Strauss and Corbin, 1994) explores the experiences of African migrant children in Ireland by drawing on multi-sited observations over a period of six months in 2009, and on interviews and focus group discussions conducted with African children (aged 13-18), mothers and fathers. An emically derived ‘African Migrant Child Psychosocial Well-being’ scale was developed by drawing on data gathered through rapid ethnographic (RAE) free listing exercises carried out in Cork, Dublin and Dundalk with sixty-one participants (N=21 adults, N=28 15-18-year-olds, N=12 12-14-year-olds) and three African community key informants to elicit local understandings of psychosocial well-being. This newly developed scale was used alongside standardised measures of well-being to quantitatively measure the psychosocial adjustment of 233 African migrant children in Cork, Dublin and Dundalk aged 11-18. Findings indicate that the psychosocial wellbeing of the study population is satisfactory when benchmarked against the psychosocial health profile of Irish youth (Dooley & Fitzgerald, 2012). These findings are similar to trends reported in international literature in this field (Georgiades et al., 2006; Gonneke, Stevens, Vollebergh, 2008; Sampson et al., 2005). Study findings have implications for advancing psychosocial research methods with non-Western populations and on informing the practice of Irish professionals, mainly in the areas of teaching, psychology and community work.