Development of a novel screening method for the isolation of precursor RNA processing mutants of Saccharomyces cerevisiae

The availability of conditional-lethal yeast (Saccharomyces cerevisiae) mutants that are defective in the process of pre-mRNA splicing (precursor RNA processing; prp) has greatly facilitated the characterisation of components of the splicing machinery. When this project was initiated nine pip complementation groups had been defined (prp2-prpll), all of which accumulate pre-mRNA at the expense of mRNA when incubated at the non-permissive temperature. Pre-mRNA splicing is a complex and dynamic process relying on many gene products for its completion and therefore many more complementation groups remained to be identified. Determination of a prp phenotype has relied on the direct measurement of protein to RNA ratios and on the Northern blot analysis of conditional-lethal mutants incubated at the restrictive temperature. Such analyses are both time-consuming and labour-intensive. In this thesis I present the development of a novel screening procedure which positively identifies prp mutants. I have fused a yeast intron-containing gene to the lacZ gene of E.coli such that only the pre-mRNA generated from this fusion can encode an active β-galactosidase fusion-protein. This gene-fusion has been introduced into a prp2 strain and the encoded pre-mRNA has been shown to accumulate on incubation at the non-permissive temperature. This pre-mRNA accumulation results in an increase in β-galactosidase activity. Exploiting this observation a simple plate assay has been developed and used to screen a pool of temperature-sensitive mutants for defects in pre-mRNA splicing. A number of prp mutants have been identified and I present the results of their initial characterisation

An exploration of neglected themes in the development of domestic violence perpetrator programmes in the UK

This thesis is based on a body of published work which critically examines the major influences on the development of domestic violence perpetrator programmes in the UK. The thesis explores how a series of evolving theoretical frameworks, social policy, and organisational contexts have influenced approaches to practice with perpetrators, and how programme effectiveness has been variously determined and assessed. The origins of the papers and the linking narrative developed from a sense of professional dissatisfaction that several important themes concerning programmes and the potential for them to engage more effectively with perpetrators have frequently been overlooked or marginalised in the research literature. These themes concern the wider social contexts in which perpetrator programmes in the UK emerged, the explanations for men’s violence and abuse which have variously prevailed, and the practices adopted in programmes as a consequence. They note the extent to which various protagonists including feminist activists, social policy professionals, academic researchers and probation and social work managers have often conceptualised perpetrator programmes as abstract entities. Consequently, this thesis addresses a number of important and original themes. It addresses and emphasises the importance of relationships between programme practitioner and participant, significantly acknowledging the emotional impact upon practitioners of undertaking complex work in an innovative and demanding area. It takes into account the significance of the wider social, structural and cultural circumstances in which programmes function. It also examines the neglected question of what desisting from domestic abuse might actually entail as well the rewards and challenges involved. It explores how men who have perpetrated violence and abuse might better be enabled to desist from this behaviour and live more positive lives and discusses the implications for programmes and for practice

Rejecting and retaining aspects of selfhood: Constructing desistance from abuse as a 'masculine' endeavour

Evaluative studies of men who have attended domestic violence perpetrator programmes have, thus far, paid attention to the question of what they are expected to desist from. This is entirely appropriate. However, the question of what they are expected to achieve, or ‘become’, is less clearly articulated, indeed often overlooked. Based on a series of interviews with men who had completed perpetrator programmes, the narratives explored in this articles suggest that their abusive behaviour was underpinned by fears about how to ‘perform masculinity’ satisfactorily in the past. Consequentially, the programme experience was perceived as threatening or as ‘feminising’. However, the accounts of these men suggest that in desisting from abusive behaviour, issues of identity and processes of behaviour change remain profoundly gendered. Indeed, committing to desistance is perceived as something of an ‘heroic struggle’ in which qualities associated with being a ‘proper man’ are harnessed and utilised in the process.Output Status: Forthcoming/Available Onlin

Isolation of plant transcription factors using a modified yeast one-hybrid system

BACKGROUND: The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontech™ product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination. Unfortunately, such cDNA libraries prepared directly in yeast can not be used for the efficient recovery of purified plasmids and thus are incompatible with existing yeast one-hybrid systems, which use yeast transformation for the library screen. RESULTS: Here we propose an adaptation of the yeast one-hybrid system for identification and cloning of transcription factors using a MATCHMAKER cDNA library. The procedure is demonstrated using a cDNA library prepared from the liquid part of the multinucleate coenocyte of wheat endosperm. The method is a modification of a standard one-hybrid screening protocol, utilising a mating step to introduce the library construct and reporter construct into the same cell. Several novel full length transcription factors from the homeodomain, AP2 domain and E2F families of transcription factors were identified and isolated. CONCLUSION: In this paper we propose a method to extend the compatibility of MATCHMAKER cDNA libraries from yeast two-hybrid screens to one-hybrid screens. The utility of the new yeast one-hybrid technology is demonstrated by the successful cloning from wheat of full-length cDNAs encoding several transcription factors from three different families

Review of the effectiveness of current community ownership mechanisms and of options for supporting the expansion of community ownership in Scotland

This report presents the findings of research commissioned by the Scottish Land Commission to review the effectiveness of community ownership mechanisms and options for simplifying or improving these mechanisms to enable and support the expansion of community ownership in Scotland. This included reviewing processes relating to negotiated sales or transfers of land and/or assets to communities, as well as legislative mechanisms including the Community Right to Buy (CRtB), Crofting Community Right to Buy, the Transfer of Crofting Estates (Scotland) Act 1997 and Asset Transfer measures under the Community Empowerment (Scotland) Act 2015

Generation of drought-resistant transgenic cereals using transcription factors isolated from wheat grain

Tese de doutoramento em Ciências da Saúde, ramo de Medicina, na especialidade de Medicina Interna (Cardiologia), apresentada à Faculdade de Medicina da Universidade de Coimbr

Synthetic auxin herbicides : finding the lock and key to weed resistance

Synthetic auxin herbicides are designed to mimic indole-3-acetic acid (IAA), an integral plant hormone affecting cell growth, development, and tropism. In this review, we explore target site genes in the auxin signaling pathway including SCFTIR1/AFB, Aux/IAA, and ARFs that are confirmed or proposed mechanisms for weed resistance to synthetic auxin herbicides. Resistance to auxin herbicides by metabolism, either by enhanced cytochrome P450 detoxification or by loss of pro-herbicide activation, is a major non-target-site resistance pathway. We speculate about potential fitness costs of resistance due to effects of resistance-conferring mutations, provide insight into the role of polyploidy in synthetic auxin resistance evolution, and address the genetic resources available for weeds. This knowledge will be the key to unlock the long-standing questions as to which components of the auxin signaling pathway are most likely to have a role in resistance evolution. We propose that an ambitious research effort into synthetic auxin herbicide/target site interactions is needed to 1) explain why some synthetic auxin chemical families have activity on certain dicot plant families but not others and 2) fully elucidate target-site cross-resistance patterns among synthetic auxin chemical families to guide best practices for resistance management