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Introduction: Sporadic CJD (sCJD) has a varied clinical and pathological phenotype. This variability is felt to be influenced by patients' genotype at codon 129 on the prion protein gene (PRNP); and has associations with PrPres protein type. There is a polymorphism at codon 129 of PRNP, which results in three genotypes (MM, MV, VV). Additionally, the protease resistant protein (PrPres) seen in patients with CJD can also be classified according to certain biochemical characteristics. The protein types are determined by their mobility on western blot electrophoresis and their degree of glycosylation (type 1, 2a, 2b). In 1996, Parchi and Gambetti categorised cases by sub-types relating to these findings (broadly speaking: MM1, MM2, MV1, MV2, VV1 and VV2) suggesting this as an explanation for the clinical and pathological diversity of the condition (Parchi 1996). Relatively recently a significant minority of patients have been found to have both PrPres protein types present in the brain. Two recent papers have proposed a revised classification system, taking into account the co-occurrence phenomenon, suggesting there are further distinct groups of patients with sCJD (Cali 2009, Parchi 2009). Methods: 108 patients referred to the UK CJD surveillance system, confirmed to have had sporadic CJD at post-mortem, were included in the study. All cases had PRNP genetic analysis to exclude genetic CJD and establish their codon 129 status. Biochemical analysis of disease-associated prion protein via western blot was undertaken in the temporal, parietal and occipital lobes and the thalamus in all patients. A structured assessment of the clinical features seen both at presentation and during the patients' illness was undertaken. A summary of the pathological findings for the patients was also recorded. The clinical and pathological features were then analysed for the relative influence of the codon 129 genotype, protein typing and the combination of the two as the revised sub-type, according to the 2009 Parchi classification system. Results: 26 out of 108 patients had mixed PrP isotypes on western blot analysis. On comparing the most common of these mixed groups [MM 1(+2)] with those patients with the same genotype and predominant protein type [MM1], there were subtle differences in the clinical phenotype and pattern of investigation results. However, the major clinical findings were the same. In general, there was a trend towards increased disease duration with increasing relative ratios of type 2 proteins. On reviewing the pathological findings there were no findings unique to any genotype-isotype sub-group. Conclusions: While there were differing trends, the lack of distinct clinical or pathological findings by genotype-isotype sub-type suggest that these groups do not represent unique strains of prions, but rather groupings over a spectrum of disease. Therefore, more detailed biochemical analysis leading to a revised classification may be more accurate, but currently adds limited useful extra clinical information.

The subject matter for this research work is the area of perioperative recovery for patients undergoing major abdominal surgery. During clinical studies we have investigated some of the factors influencing postoperative recovery as well as suggesting strategies to improve patient care. The main focus of the scientific work of this thesis is the role of intravenous fluids in the perioperative management of patients undergoing abdominal surgery. We found that restriction of intravenous fluid in the postoperative period does not significantly improve recovery in terms of gastrointestinal function (4.2 (3.2-6.9) versus 4.7 (3.7-6.1) days; p=0.80) or hospital stay (5.9 (4.0-7.9) versus 5.8 (4.1-7.3) days; p=0.90). Analysing our findings in the context of what is already known suggests that the immediate perioperative period when the effect of the metabolic-endocrine response is at its greatest is the most important period for fluid management. During this period fluid optimisation has an important role in patient recovery but following this period the body’s own homeostatic mechanisms are more able to cope with any fluid excess. We also found that using a ‘fast-track’ regime we could reduce hospital stay to levels comparable with other studies in the published literature. Our work using a multi-modal rehabilitation regime in association with both laparoscopic and open surgery suggests that it is the postoperative care package which has the more major influence on recovery. Our findings are in agreement with other small sized studies beginning to appear in the literature and indicate that further large scale studies are required to determine the role of laparoscopic surgery and any potential benefits. One of the most significant causes of morbidity for patients undergoing abdominal surgery is postoperative ileus. During the course of our studies we found that the extent of surgery and particularly handling and exposure of the intestines seems to have little effect on the duration of postoperative ileus. These findings add to the previously contradictory findings of other groups. Our experience with ‘fast-track’ postoperative programmes was also applied to liver surgery, an area where it has not previously been reported, to show that a variety of abdominal procedures may benefit from this approach. By comparing our results with series published in the medical literature we found that hospital stay can be significantly reduced (4 versus 5-8 days).

The mast cell has been implicated in the aetiology of a range of allergic and inflammatory diseases including asthma. However, the regulatory processes which control the cell's activity remain unclear. In the present study, the effect of cyclic nucleotide modulation on mast cell function was assessed. It was hoped that this would not only indicate the importance of both cyclic adenosine and guanosine monophosphate in cellular regulation but also give further information into the mode of action of a range of anti-allergic agents particularly disodium cromoglycate (DSCG). Exogenously applied cyclic adenosine monophosphate produced negligible inhibition of histamine release from rat peritoneal mast cells (RPMC) unlike the cell permeable derivatives of the cyclic nucleotide which were effective inhibitors. Unexpectedly, cyclic guanosine monophosphate (cGMP) produced a dose-dependent attenuation of histamine release from RPMC. However, this inhibition was strikingly reduced if the cells were preincubated with cGMP before stimulation. Such inhibition and marked tachyphylaxis closely resembles the effects of DSCG on RPMC. Further comparison between cGMP and DSCG revealed that they shared parallel kinetics of tachyphylaxis, underwent cross-tachyphylaxis and were ineffective inhibitors of histamine release from mast cells isolated from the mouse, guinea-pig and human. These results suggest that cGMP and DSCG mediate mast cell stabilisation through similar mechanisms. Biochemical analysis of the RPMC phosphodiesterase (PDE) profile revealed that the predominant enzymes were PDE IV and PDE V although some PDE III was also present. Whole cell functional studies, using selective PDE inhibitors, demonstrated that the PDE V targeted drug, zaprinast, produced a particularly potent inhibition of RPMC secretion which paralleled the effects noted using both DSCG and cGMP. Zaprinast's attenuatory activity seems likely to be removed from an interaction with PDE V as a range of equipotent PDE V inhibitors had variable inhibitory effects on RPMC. Thus, modulation of cyclic nucleotide levels at both intra- and extra-cellular sites can have marked effects on the activation state of mast cells. Moreover, the results yield useful information to the elucidation of DSCG's mechanism of action. The uncovering of this mechanism may result in the development of more effective anti-allergic agents which share DSCG's low in vivo toxicity.

Supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) are used to study the presence of additives in polymers that are used in living systems. Representative samples are used to illustrate and develop the extraction capability of the system with extracts being analysed both by on-line and off-line SFC, gas chromatography-mass spectrometry (GC-MS), and gravimetrically. Different types of additives are employed illustrating their chromatographic behaviour and the uses of both flame ionisation (FID) and UV /Visible detection. The qualitative aspects of the analysis of polymer additives are augmented by on-line linking of a mass spectrometer (MS) to the SFC. The spectra obtained from this system are compared with other probe-MS, GC-MS and library spectra that are available to establish the quality of the spectra obtained. The development of on-line SFE-SFC-MS for the analysis of polymer additives allows for a quick qualitative analysis of the compounds present in polymer samples. Using a set of flame retardants, which are difficult to analyse by comparable methods, on-line SFE-SFC quantitative investigations are performed on polyurethane foams containing these compounds using short extraction times and varying pressures. The results are compared with the Soxhlet extracts and off-line SFE-SFC analysis of the same samples. An extraction model is used to explain the pattern of extraction of additives from both polyurethane foams and laboratory prepared polyvinyl chloride formulations. The studies are performed using different mobile phase strengths and particle sizes to establish the processes involved. The extracts are analysed quantitatively by SFC-FID and GC-FID.