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.

Studies on Haemonchus contortus infections in Merino sheep

The work described in this thesis was initially directed towards the elucidation of the self-cure phenomenon as a flock phenomenon under natural grazing conditions. As a consequence of these studies it became apparent that several important aspects of the epidemiology and of the clinical syndromes associated with haemonchosis required further investigation. The thesis is therefore essentially a description of haemonchosis of sheep in an endemic area supported, where indicated, by observations obtained by experimental infections. Each of the separate aspects studied is prefaced by a review of the relevant literature and the details of many individual observations which are not included in the text may be found in the various appendices. Some results, for example the individual faecal egg counts and worm burdens at autopsy, were too voluminous to be included and these are available at The Veterinary School of the University of Glasgow.

Essays on firm behaviour in the euro area

This thesis examines firms' real decisions using a large panel of unquoted euro area firms over the period 2003-2011. To this end, this thesis is composed of five chapters in which three are the main empirical chapters. They assess the dimensions of firm behaviour across different specifications. Each of these chapters provide a detailed discussion on the contribution, theoretical and empirical background as well as the panel data techniques which are implemented. Chapter 1 describes the introduction and outline of the thesis. Chapter 2 presents an empirical analysis on the link between financial pressure and firms' employment level. In this set-up, it is explored the strength of financial pressure during the financial crisis. It is also tested whether this effect has a different impact for financially constrained and unconstrained firms in the periphery and non-periphery regions. The results of this chapter denote that financial pressure exerts a negative impact on firms' employment decisions and that this effect is stronger during the crisis for financially constrained firms in the periphery. Chapter 3 analyses the cash policies of private and public firms. Controlling for firm size and other standard variables in the literature of cash holdings, empirical findings suggest that private firms hold higher cash reserves than their public counterparts indicating a greater precautionary demand for cash by the former. The relative difference between these two type of firms decreases (increases) the higher (lower) is the the level of financial pressure. The findings are robust to various model specifications and over different sub-samples. Overall, this chapter shows the relevance of firms' size. Taken together, the findings of Chapter 3 are in line with the early literature on cash holdings and contradict the recent studies, which find that the precautionary motive to hold cash is less pronounced for private firms than for public ones. Chapter 4 undertakes an investigation on the relation between firms' stocks of inventories and trade credit (i.e. extended and taken) whilst controlling for the firms' size, the characteristics of the goods transacted, the recent financial crisis and the development of the banking system. The main findings provide evidence of a trade-off between trade credit extended and firms' stock of inventories. In other words, firms' prefer to extend credit in the form of stocks to their financially constrained customers to avoid holdings costly inventories and to increase their sales levels. The provision of trade credit by the firms also depends on the characteristics of the goods transacted. This impact is stronger during the crisis. Larger and liquid banking systems reduce the trade-off between the volume of stocks of inventories and the amount sold on credit. Trade credit taken is not affected by firms' stock of inventories. Chapter 5 presents the conclusions of the thesis. It provides the main contributions, implications and future research of each empirical chapter.