Studies on the synthesis of novel PP2A inhibitors and a GPCR detergent

Chapter 1 While targeting kinases in oncology research has been explored extensively, targeting protein phosphatases is currently in its infancy. However, a number of pharmaceutical companies are currently looking to expand their research efforts in this area. PP2A has been shown to down-regulate ERK5, a mitogen-activated protein kinase (MAPK) that has been shown to be important in driving the invasive phenotype of prostate cancer. Fostriecin and its related structural analogues PD 113,270 and 113,271 have been shown to inhibit a mitotic entry checkpoint in cell growth through the potent and selective inhibition of protein phosphatases PP1, PP2A, and PP4 (IC50 of 45 μM, 1.5 nM, and 3 nM respectively). Fostriecin is one of the most selective protein phosphatase inhibitors disclosed to date with a 104 fold selectivity for PP2A/PP4 versus PP1. Unfortunately, fostriecin and its analogues are very unstable, and this instability has effectively prevented them from being used as effective therapeutic leads. The microcystins and nodularins on the other hand, exhibit significant inhibitory activity against PP1 and PP2A (IC50 = 26 pM and 1.8 nM respectively), but their high toxicity has prevented any therapeutic application. Truncation of the ADDA chain from these polypeptides completely attenuates PP inhibitory activity. Simpler analogues incorporating the N-acylated ADDA chain and D-Ala retain moderate activity against PP1 and PP2A (IC50 = 1.0 μM and 0.17 μM respectively). The generation of a new series of fostriecin analogues to further expand its structure-activity relationship is envisaged with a view to creating new more stable PP2A inhibitors. It was hoped that by incorporating some of the more stable structural features of ADDA into fostriecin that stability and activity could be reconciled. With that in mind a series of PP2A inhibitors were synthesised and biologically evaluated. Chapter 2 GPCRs are an important area of research and are the targets of a quarter of the drugs on the market (2005). As a result, GPCRs continue to be at the forefront of research in both small and large drug companies. However one of the difficulties in studying this diverse class of membrane proteins is their tendency to denature in aqueous solution. As a result there is a pressing need to develop new detergents to solubilise, stabilise and crystallise GPCRs in their native form for further study. Cholesterol analogues have been shown to be important for stabilising membrane proteins and preventing their thermal inactivation. In addition the β2-adrenergic receptor, a GPCR membrane protein, has been crystallised in the active state with two cholesterol molecules bound between the I, II, III and IV helices of the protein. This appears to represent a distinct cholesterol binding pocket on the membrane protein that is speculated to be conserved across up to 44% of the rhodopsin class of GPCRs. CHOBIMALT is a cholesterol-based detergent that has been shown to exhibit promising GPCR-stabilising properties. When benchmarked against other cholesterol based detergents it was found to be superior to all others tested except for cholesteryl hemisuccinate.1 CHOBIMALT has an aggregation number of roughly 200 and forms 210 ± 30 kDa micelles, which are significantly larger than those of most detergents used for biological systems which is likely due to the packing constraints associated with CHOBMALT’s large polar headgroup.2 As a result, CHOBIMALT is used mostly as an additive to other commercially available detergents in order to decrease micelle size. A branched dimaltoside motif is common in recently synthesised detergents by Chae and co-workers. These detergents have shown promising detergent properties, for example the maltose neopentyl glycol (MNG) detergent synthesised by Chae. This branched dimaltoside detergent was shown to be able to solubilise and stabilise the very labile light harvesting complex I (LHI) from Rhodopsin capsulatus in its active form for 20 days with little loss of protein conformation.3 A cholesterol-based detergent was envisaged that combines the cholesterol framework of CHOBIMALT but replaces its linear tetrasaccharide with a branched dimaltoside. This detergent would then be investigated to assess its ability to solubilise, stabilise and crystallise GPCR proteins. This cholesterol-based detergent (shown below) was eventually synthesised in 9 linear steps from cholesterol.

'Don't stop': re-thinking the function of endings in narrative television

This thesis argues that the study of narrative television has been limited by an adherence to accepted and commonplace conceptions of endings as derived from literary theory, particularly a preoccupation with the terminus of the text as the ultimate site of cohesion, structure, and meaning. Such common conceptions of endings, this thesis argues, are largely incompatible with the realities of television’s production and reception, and as a result the study of endings in television needs to be re-thought to pay attention to the specificities of the medium. In this regard, this thesis proposes a model of intra-narrative endings, islands of cohesion, structure, and meaning located within television texts, as a possible solution to the problem of endings in television. These intra-narrative endings maintain the functionality of traditional endings, whilst also allowing for the specificities of television as a narrative medium. The first two chapters set out the theoretical groundwork, first by exploring the essential characteristics of narrative television (serialisation, fragmentation, duration, repetition, and accumulation), then by exploring the unique relationship between narrative television and the forces of contingency. These chapters also introduce the concept of intra-narrative endings as a possible solution to the problems of television’s narrative structure, and the medium’s relationship to contingency. Following on from this my three case studies examine forms of television which have either been traditionally defined as particularly resistant to closure (soap opera and the US sitcom) or which have received little analysis in terms of their narrative structure (sports coverage). Each of these case studies provides contextual material on these televisual forms, situating them in terms of their narrative structure, before moving on to analyse them in terms of my concept of intra-narrative endings. In the case of soap opera, the chapter focusses on the death of the long running character Pat Butcher in the British soap EastEnders (BBC, 1985-), while my chapter on the US sitcom focusses on the varying levels of closure that can be located within the US sitcom, using Friends (NBC, 1993-2004) as a particular example. Finally, my chapter on sports coverage analyses the BBC’s coverage of the 2012 London Olympics, and focusses on the narratives surrounding cyclists Chris Hoy and Victoria Pendleton. Each of these case studies identifies their chosen events as intra-narrative endings within larger, ongoing texts, and analyses the various ways in which they operate within those wider texts. This thesis is intended to make a contribution to the emerging field of endings studies within television by shifting the understanding of endings away from a dominant literary model which overwhelmingly focusses on the terminus of the text, to a more televisually specific model which pays attention to the particular contexts of the medium’s production and reception.