Northern Ireland Housing Market Areas

No abstract available

Technology development for the over-expression, purification and crystallisation of human membrane proteins

Currently, the field of mammalian membrane protein structural biology is in its infancy. Existing technologies and experiences have shown that it is possible to obtain the structures of mammalian membrane proteins if sufficient work and thought has been invested. However, there is still an urgent need to develop new methodologies and approaches to improve all aspects of this important area of biological research. Here, a series of novel technologies for the overproduction, purification and crystallisation of human membrane proteins are described which have been tested with a representative member from each of the G-protein coupled receptor (adenosine 2a receptor (A2aR)) and membrane enzyme (sterol isomerase (SI)) superfamilies. The methylotrophic yeast Pichia pastoris is an excellent host cell for the overproduction of recombinant proteins including membrane proteins of mammalian origin. However, the commercially available expression vectors are far from what is required to maximise the production levels as well as simplify the detergent extraction and purification of human membrane proteins. Here, a series of related expression constructs were made that had different combinations of tags at both ends of the recombinant protein. The final optimised expression vectors had a C3 protease-iLOV-biotin acceptor-His10 (CLBH) tag fused to the C-terminus of the recombinant protein. The -CLBH vectors gave high level production of both test proteins (one Nin – hSI; one Nout – hA2aR) that could be rapidly purified to homogeneity using a generic protocol. The position of the His10 tag did not affect the expression level of the recombinant protein. In contrast, fusion of the biotin acceptor domain to the C-terminus of the recombinant protein increased its expression by a factor of between 2-4. The biotin acceptor domain could also be fully biotinylated in vitro using recombinantly expressed biotin ligase allowing purification/immobilisation of the target protein with streptavidin beads. Removal of the expression/ purification tags from the recombinant proteins with C3 protease occurred more efficiently than when TEV protease was used. An optimised protocol was developed that gave maximal production of our target proteins in fermenter culture at an induction temperature of 22°C. Care was taken to find a methanol feed rate that gave the highest levels of protein production without causing the accumulation of excess methanol in the culture (which is known to be toxic to the yeast). Using this protocol it was possible to make both hSI and hA2aR with a production level >10 mg of recombinant protein per litre of culture. As most MPs are colourless, target protein identification is usually performed by methods such as radioligand binding and/or Western blotting. However, these techniques can be time-consuming, use a lot of protein and do not give any information on the aggregation state of the protein in detergent solution. Previously, it has been shown that the processes of identifying and analysing membrane proteins in detergent solution can be accelerated by attaching green fluorescent protein to the C-terminus of the recombinant MP. Here, the potential of the recently described iLOV fluorescence tag for membrane protein applications was assessed. iLOV was shown to be an useful tool for optimising processes such as yeast clonal selection, protein production in fermenter culture, detergent and construct screening as well as tracking recombinant MPs through the purification process. Of note, the iLOV tag allowed a direct assessment of the stability and dispersity state of both target MPs in a range of detergents by fluorescence size exclusion chromatography (FSEC). Using this approach, it was shown that wild-type hA2aR solubilised using a combination of dodecyl-βDmaltoside (DDM) and cholesteryl-hemisuccinate (CHS) aggregated during purification on a Ni2+ column. Furthermore, it was shown that the hA2aR agonistconformationally-fixed mutant Rag23 is stable in DDM without any CHS present. Moreover, Rag23 was found to be monodisperse in a series of short-chain detergents (decyl-βD-maltoside, nonyl-βD-maltoside (NM) and β-octylglucoside) suggesting that this mutant is well-suited to structural studies. SI was remarkably robust in short chain detergents demonstrating a reasonable level of stability in the short chain detergent NM. The FSEC experiments showed that wild-type SI has considerably higher intrinsic stability than native hA2aR suggesting that membrane enzymes will prove to be more amenable to structural analysis than GPCRs. Rag23 and SI were both purified to homogeneity in a simple four-step procedure: i) Ni2+ purification, ii) cleavage with C3 protease, iii) reverse Ni2+ purification and iv) gel-filtration chromatography. A buffer/salt screen was devised that allowedthose conditions where SI had maximal thermostability in detergent-solution to be identified. SI was found to have greatest stability in sodium phosphate buffer at acidic pH. Using this information, it was possible to purify monodisperse SI in DM suggesting that this protein may make an excellent candidate for structural studies too. Crystallisation trials with SI were performed using the commercially available sparse matrix screen MemSys/MemStart. In addition, a lipidic-sponge phase sparse-matrix crystallisation screen that was developed in collaboration with Prof. Richard Neutze (University of Chalmers, Sweden) was tested using SI. Cholesterol could be incorporated into all of the sponges that make up the screen upto a concentration of 10%. (This is important as the activity of many mammalian membrane proteins is cholesterol-dependent). To date, no diffracting crystals of SI have been obtained with either the conventional or lipidic-sponge phase crystallisation approaches. In short, a series of novel technologies/methodologies have been developed that will act as a platform for future efforts to solve the structures of a wide-range of human membrane proteins

Telepresence: Joan Jonas and the Emergence of Performance and Video Art in the 1970s

This dissertation is a study of the early career of the American artist Joan Jonas that spans the years 1970-1984. At the turn of the 1970s, Jonas was one of the first artists to pick up a video camera. Exploring “live” video’s unique capacity to mediate the present moment, Jonas actively integrated the technology into her live pieces, which are some of the earliest examples of what was then first called “performance art.” Performance art has often been aligned with presence. In contrast, I argue that what at stake in the proliferation of live artworks by Jonas and others that merged performance and video was not a reserve of unmediated experience, but a presence that was newly technologized: telepresence. As Jonas investigated the novel ability to perform at a distance enabled by electronic media, her work led somewhere surprising: to telegraphy, telepathy, and the earliest telephones—“tele”-technologies that appear long obsolete (or completely fantastical). Evoking optical telegraphs, spirit mediums, speaking trumpets, and science fictional prostheses, Jonas’s early oeuvre reactivates the historical contexts and unrealized potentials surrounding these dead media. In so doing, she illuminates enduring formations of the body, subjectivity, and teletechnology underlying not only the twinned emergence of performance and video art in the 1970s, but also telepresence as a seemingly very contemporary (and increasingly pervasive) category of experience

An Evaluation of Rent Regulation Measures within Scotland's Private Rented Sector

Throughout Europe there is widespread concern about private rents, both from tenants and governments, especially in Europe’s larger cities and their 'hot spot' neighbourhoods. This report examines how the Scottish Government has responded to this issue as part of the development of the new Private Residential Tenancy which came into effect in December 2017, thus setting this analysis within a broader review of European 'rent regulation' measures. The term 'rent regulation' is commonly applied across Europe to refer to measures which seek to limit 'in-tenancy' rent increases, whilst leaving the rents for new tenancies free to find their place within the market. In looking to balance the interests of tenants and landlords, the Scottish Government rejected rent control across the rental market, favouring instead measures to ensure that 'in-tenancy' rent increases are not excessive and do not exceed market rates. The Act which emerged in 2015 set out a new open-ended tenancy to replace the short-assured tenancy along with its typical fixed terms of six months. This led to concerns that unscrupulous landlords might use excessive rent rises as a means to repossess their property. The Act therefore sought to protect tenants from excessive rent increases in two ways: firstly, by allowing tenants who believe their proposed rent increase is out of step with the market to seek a formal review by the Rent Officer, and secondly through area-wide inflation-linked restrictions on rent increases through Rent Pressure Zones. High and rapidly rising rents in Aberdeen, at the time of the Bill's passage, helped to garner political support for Rent Pressure Zones. Whilst Aberdeen’s rents have now fallen back in the wake of the sharp decline in oil related activity, rents in both Edinburgh and Glasgow continue to cause concerns. The Rent Pressure Zone measures emerged relatively late in the policy-making process and therefore were not considered in much detail when the Bill was debated in Parliament. This may have contributed to the challenges now faced by local authorities seeking to utilise this measure. After scoping out and discussing these challenges, the report offers some suggestions as to how these might be best overcome

A Model to Incorporate Meaningful Community Engaged Learning Opportunities into Medium

Community engaged learning (CEL) has been identified as a high impact educational practice that can have profound influence on learning and improve student engagement (Kuh, 2008). Despite the potential to provide a meaningful learning experience, CEL opportunities are not widespread at large research institutions, and most examples arise from optional co-curricular activities or small classes (Holander, 2011). Current realities of increasing class sizes and decreasing resources can make implementing CEL challenging. Creative thinking is required to modify the critical elements of successful CEL to suit broader educational needs. This paper provides a tangible model for CEL assignments that can be adapted to suit medium to large classes, with an honest discussion of the lessons learned in the process from student, faculty and community perspectives. Based on key concepts of reciprocity, shared decision-making and mutual benefit we designed a novel CEL assignment in a large 4th year course (\u3e100 students). Briefly, student teams researched one of five priority areas identified by Wellington-Dufferin-Guelph Public Health (WDGPH) to write an evidence-based literature review. Based on these findings, students worked with WDGPH experts to translate their research into practical recommendations and tools to advance WDGPH programming. An end-of-semester showcase was used to highlight these applied projects. Students identified real world relevance and the opportunity to be creative as the main advantages of the assignment. Surprisingly, community partners identified the opportunity for leadership and mentorship as an unintended but welcomed benefit to the program. From a faculty perspective, the time required to coordinate and grade the projects during the teaching semester was manageable although the quality of student projects varied significantly. Future offerings should consider strategies to provide more tailored feedback to all students and to encourage a balance of effort between the research and applied aspects of the CEL project

Research exercise: ETHOS - Appropriate Solar Technology for Bihar, India

An investigation of the potential for appropriate solar technology in Bihar, India, including solar thermal refrigeration systems. The feasibility of solar PV cells within a micro grid system was studied for applications to existing refrigerators. This is pre-work for an upcoming ETHOS immersion in Bihar, India.https://ecommons.udayton.edu/stander_posters/1572/thumbnail.jp

Process Evaluation of Community Energy Development Programme Projects

This report sets out the findings, conclusions and recommendations of a Process Evaluation, conducted between January and March 2015, of the Community Energy Development Programme (CEDP) projects as part of the Malawi Renewable Energy Acceleration Programme (MREAP). It was commissioned by the Scottish Government (SG) as a product of the Institutional Support Programme Component (ISP) of MREAP. Its main purpose is to assess what has been delivered, how this has been achieved and to compile learning from the process for policy and future projects. The agreed scope for the process evaluation was the portfolio of 46 CEDP projects implemented across the 3 regions of Malawi and the relevant processes and systems in place to design, implement and manage these projects. Due consideration was also given to framing the scope of the evaluation through the choice of evaluation questions and the feasibility of what was possible