Introduction: Major recent advances in modern surgery have focussed on processes and pathways relating to perioperative recovery. Optimising patients perioperatively is essential in improving outcomes. The enhanced recovery programme is an integrated pathway that combines evidence based practice in a synergistic manner to improve outcomes. This research concerns perioperative recovery for patients undergoing major abdominal surgery. Aim: The aim of this study was to investigate the factors that influence the implementation of an enhanced recovery programme in patients undergoing elective colorectal surgery. Method: The study was done in four phases. The first phase was to assess the feasibility of introducing fast track surgery in our unit by recruiting patients undergoing reversal of loop ileostomy so as to reduce hospital stay. The second phase, compared laparoscopic colorectal surgery with open colorectal surgery with regards to hospital stay and complication rates. Both groups of patients were followed up over a two year period to compare incisional hernia rates. The third phase, compared the use of video education in the psychological preparation of patients undergoing elective colorectal resection with information leaflets and verbal information. The fourth phase, compared short term outcomes between patients undergoing elective colorectal resection early in the week(Monday to Wednesday) with those later in the week(Thursday to Friday). Results: Early discharge is safe and achievable following reversal of loop ileostomy. Laparoscopic surgery does not improve short term outcomes following colorectal surgery compared with open surgery. Long term outcomes (incisional hernia rates) are similar. Supplementing video education with oral and written information prepares patients better psychologically for surgery although it does not improve short term outcomes. Operating on patients earlier in the week improves short term outcomes. Conclusion: The enhanced recovery programme is feasible and safe and should be practiced by individual units offering colorectal surgery. Patients benefit from preconditioning using video education and being operated upon early in the week.

Serotonin (5HT) and nitric oxide (NO) are important signalling molecules with roles in behavioural control and various psychological conditions. Various lines of evidence suggest interactions between 5HT and NO signalling, however little is known about their interaction in modulating intrinsic properties of cortical neurons and the effects on the neuronal network activity in the cortex. This project studied the effects of 5HT and NO on cultured neurons isolated from Wistar rat cortices. 5HT and NO produced diverse responses in cortical neurons, reflecting the heterogeneity of cortical neuron types. Neither 5HT nor NO appeared to significantly modulate glutamate induced depolarisations. However, they affected the number of APs elicited by the glutamate response, hinting at the possible modulation of intrinsic membrane properties. Subsequently, k-Means clustering based on 10 electrophysiological parameters was used to divide cultured cortical neurons into 10 different cell types with consistent firing properties. 5HT and NO had diverse, cell-type specific effects on neuronal excitability and cellular parameters in these cell types. Furthermore, 5HT and NO showed complex interactions between each other, suggesting modulatory cross-talk. Using ODQ to block soluble guanylate cyclase activity suggests that NO effects are mediated not only by sGC/cGMP signalling, but also S-nitrosylation. Comparing results obtained in cell culture to data from acute cortical slices showed similarities in basic cellular properties. However, 5HT and NO effects on pyramidal cell properties differed markedly between slices and cultures. At the network level, 5HT increased the spontaneous EPSC frequency in pyramidal neurons through 5HT2 receptors, whereas NO had no effect. Interestingly, NO was able to reduce the 5HT induced increase in EPSC frequency when inhibitory synaptic interactions were blocked, possibly by modulating 5HT1 receptor function. The results obtained demonstrate the interaction between 5HT and NO in modulating both intrinsic membrane properties and neocortical network activity.

No description available.

Little is known about the fundamental mechanisms required for cargo sorting into discrete intracellular trafficking pathways. A large body of evidence places opposing polarity microtubule motor proteins in the endosomal network. With specific roles for different family members, microtubule motors are suggested to play a central role in defining the high specificity of cargo sorting. The role of microtubule-based motor proteins in membrane trafficking pathways is first investigated in this thesis using total internal reflection fluorescence (TI RF) imaging techniques and high-resolution object tracking assays. Sorting nexins (SNX) are a group of cytoplasmic and membrane-associated proteins involved in trafficking, driving membrane deformation and coupling to microtubule-based motors hence their key role in endosomal dynamics. Coordination of opposite motors applies longitudinal tension to facilitate SNX-labelled endosome scission. The same cohort of motors ensures SNX-coated subdomain segregation and motility therefore participating in the maintenance of the functional architecture of these organelles. The cytoplasmic dynein interactome is subsequently established by stable-isotope by amino-acids labelling proteomics to identify novel dynein interacting proteins and allocate any prospective new adaptors within discrete intracellular trafficking pathways. A novel dynein adaptor, TIP47, required for lipid droplet biogenesis, is identified and characterised, driving the motility of these organelles; and crucial for their formation. Other proteins were identified as promising candidates to elucidate the variety of cargos transported by the unique minus-end direct motor cytoplasmic dynein. Microtubule-based motor protein functions in bacterial pathogen trafficking is later dissected using Salmonella enterica serovar Typhimurium infection of mammalian cells and confocal imaging. Opposing polarity motors dynein-l and kinesin-l are both required for efficient bacterial intracellular trafficking and replication. Recruited from the onset of bacterial invasion, dynein-l interaction with the Salmonella-containing vacuole (SCV) persists after its localisation near the Golgi apparatus indicating that at least one of the SCV positioning mechanisms requires dynein function. HOOK3, also identified as a dynein interacting protein in the proteomic assays undertaken in this thesis, is proposed to be the dynein adaptor at the SCV. Its Golgi -binding domain suggests its potential role in tethering the SCV to the Golgi apparatus, hence potentially mediating both dynein activity and Golgi tethering to explain SCV positioning. HOOK3 may also have a physiological function in docking endosomal subdomains destined to the Golgi to the microtubule network via interaction with dynein. This new body of evidence brings the scientific community a step closer to understanding the fundamental role of microtubule motors in membrane trafficking in physiological and pathological conditions.

Endocytosis is the active process by which useful biomolecules cross the plasma membrane into the cell. There are several mechanisms, carried out by an extensive machinery of lipids and proteins, the most well-characterised of which is clathrin-mediated endocytosis (CME). The mechanism of endocytosis influences the trafficking pathway and terminal destination of a cargo once it has entered the cell. When attempting to deliver nanoparticles carrying drugs or RNA to cells, or when assessing the toxic effect of unintentional exposure to industrial nanoparticles, it is crucial to have knowledge of these pathways, specific to each cargo, to enhance the design and understanding of nanoparticle efficacy. In this thesis, two novel techniques for studying the uptake of nanoparticles, both quantitative and qualitative, were developed. These were used to study and characterise a model system of 50 nm silica particles endocytosed by retinal pigment epithelial cells. The methods were: • A MATLAB® programme to analyse uptake data obtained using confocal microscopy; • A particle tracking technique, based on optical tweezers, used on a single cell level. The uptake mechanism of 50 nm silica particles, analysed using confocal imaging of cells exposed to the particles under inhibition conditions, and quantified using the MATLAB® programme, was found to be mainly CME. Intracellular trafficking, analysed using confocal and electron microscopy, took place via the early and late endosomes, and most of the particles ended up in the lysosomes. The optical-tweezers based tracking technique was developed with the aim of following the endocytosis and trafficking pathway of a particle in a live cell, and although this was not achieved for 50 nm silica, single particle measurements were made of 500 nm latex on the surface live cells, to show proof-of-principle of the use of the technique. In summary, this thesis presents a holistic approach to studying the uptake and trafficking of nanoparticles, and demonstrates the design and implementation of widely-applicable techniques for this purpose

No description available.

The concept of endothelial progenitor cells (EPCs) has attracted considerable interest in cardiovascular research, but despite a decade of research there are still no specific markers for EPCs and results from clinical trials remain controversial. To identify membrane proteins, which could serve as potential markers for EPCs, an alternative proteomic method was adopted to obtain sufficient membrane material for proteomic analysis. Microparticles (MPs), the intact vesicles formed from the plasma membrane, were harvested from the conditioned medium of EPC cultures and their protein composition was analysed by liquid chromatography-tandem mass spectrometry. Surprisingly, the platelet-specific integrin alpha lib emerged as the most abundant integrin in EPC cultures. Subsequent experiments confirmed that the conventional methods for isolating peripheral blood-derived mononuclear cells (PBMNCs) lead to a substantial contamination with platelets. Notably, platelets readily disintegrate into platelet MPs when activated during PBMNCs isolation conditions. These platelet MPs are taken up by the PBMNCs, which acquire "endothelial" characteristics (CD31, von Willebrand factor [VWF], lectin-binding), and angiogenic properties. In a large population-based study (n =526), platelets emerged as a positive predictor for the number of colony-forming units and early outgrowth EPCs. This study provides the first evidence that the cell type consistent with current definitions of an EPC phenotype may arise from an uptake of platelet MPs by mononuclear cells resulting in a gross misinterpretation of their cellular progeny. In addition, the release of platelet MPs at sites of vascular injury may play a role in orchestrating tissue repair and contribute to the activation of an angiogenic programme in monocytes by inducing the expression of non-coding regulatory RNA, known as microRNAs, which act as translational repressers. The uptake of platelet MPs by mononuclear cells induced the release of the CXCL7 chemokine which in turn guided a de novo induction of miR-885-5p in mononuclear cells. The increased expression of miR-885-5p resulted in the targeted reduction of the actin-bundling protein LCP-1, facilitating the adhesive and migratory ability of mononuclear cells. These findings demonstrate the advantage of using an unbiased proteomic approach to assess cellular phenotypes and advise caution in attributing the benefits inclinical trials using unselected bone marrow mononuclear cells (BMCs) to stem cell-mediated repair.

Activated EGF receptors (EGFR) are sorted onto internal vesicles of multivesicular endosomes/bodies (MVBs), thus removing receptors from the recycling pathway and targeting them for degradation. The EGFR tyrosine kinase has two major substrates within MVBs, the EGFR itself and annexin 1. Annexins have been proposed to play multiple roles in membrane traffic and both annexins 1 and 2 have been localised to early transferrin positive endosomes and to MVBs. In these studies a combination of gene knockout and RNAi-induced protein depletion was used to investigate the effect of loss of annexins 1 and 2 on the formation of MVBs and on EGFR trafficking. MVBs form constitutively in unstimulated cells but EGF stimulation significantly increased both the number of MVBs formed and the number of internal vesicles per MVB. Neither annexin is required for MVB formation, but EGF- stimulated inward vesiculation, within a sub-population of EGFR-containing MVBs, is mediated through annexin 1. Consistent with a role for annexin 1 in internal vesicle formation, annexin 1 and EGFR were present on the same internal vesicles within MVBs from EGF stimulated cells, but annexin 2 was not. In annexin 1 -/- cells there was no effect on EGF degradation, but a small reduction in EGFR degradation was seen. Prolonged MAPK signalling was observed in annexin 1 -/- cells, which also exhibited enhanced EGF-stimulated cell motility. Additionally, loss of annexin 1 was found to alter the shape of mouse lung fibroblasts. EGF- stimulated phosphorylation of annexin 1 was required for annexin 1-mediated inhibition of cell motility, but not for annexin 1-mediated regulation of cell shape. Therefore, annexin 1 is involved in specific EGF-stimulated effects, including internal vesicle formation, downregulation of EGFR signalling and inhibition of cell motility.

TBP1 expression increased around day 10 gestation in the mammary gland. Analysis of protein levels confirmed that the increase in TBP1 expression observed was due to an overall increase in 26S proteasome. The increase in TBP1 expression observed by differential display was at the stage when the mammary gland begins to respond to lactogenic hormone by differentiating and expressing the milk protein β casein. Studies in mammary epithelial cells (MEC) revealed the increase in the proteasome level was not lactogenic hormone dependent as the expression profile from the display results implied. We further cloned murine homologues of two other ATPases, TBP7 and Sug2 and performed a yeast two hybrid assay to determine if these ATPases interacted with one another. In order to address the role of TBP1 and the 26S proteasome in the mammary gland, a series of studies using a peptide inhibitor of the proteasome were performed on MECs. These results suggested that proteasome function was essential for MECs, and inhibition of the proteasome leads to the induction of apoptosis. This study showed that death was occurring from a specific stage in the cell cycle and that this was a p53 dependent response. To address the question of TBP1 function more specifically, a dominant negative TBP1 construct was generated. Constitutive expression studies suggested that TBP1 function was indeed essential for cell survival. A greater understanding of the function of the 26S proteasome in the processes of proliferation, differentiation and apoptosis should lead to an enhanced perception of the role proteolysis plays in normal mammary gland development.

The experiments described in this thesis investigated the growth of bovine cells infected with Theileria sp. macroschizonts in various strains of immunodeficient mice. It was hoped that it would be possible to create a system for the growth of Theileria infected cells in vivo without using bovine hosts. Previous work had shown that T.parva infected cells would establish as subcutaneous tumours in irradiated Swiss and nude mice (Irvin et al, 1977; Veterinary Parasitology 3, p.141-160). Most of the work in this thesis concerned the related parasite, T.annulata. T.anmilata infected cells were found to grow as subcutaneous tumours in irradiated Balb/c, irradiated NIMR and Balb/c nude, and unirradiated C.B-17 scid (severe combined immunodeficiency) mice. Infected cells failed to establish in C57 beige mice, with or without irradiation. The growth and survival of the tumours was dependent on the degree of immunosuppression of the host, and the size of the cell dose administered. In Balb/c mice, T.annulata tumours regressed as mice recovered from irradiation. Analysis of lymphocyte subsets using a fluorescence-activated cell sorter (FACS) showed differential susceptibility of B-cells, T-helper and cytotoxic T-cells to a sublethal dose of ionising radiation (46y). The numbers of these lymphocytes increased more rapidly after irradiation in tumour bearing mice than in those without tumours. In irradiated (46y) Balb/c nude and scid mice, subcutaneous T.annulata tumours failed to regress, despite the development of general haemorrhage and central necrosis. In scid mice, high doses (2xl07) of T.annulata infected cells injected intraperitoneally gave rise to ascites. However, low doses (2xl06 cells) did not. The presence of macroschizont infected cells in the peritoneal cavity was accompanied by a proliferation of macrophages. Several lines of evidence indicated that natural killer (NK) cells were not effective against T.annulata-infected cells in scid mice, but that macrophages were capable of controlling and eliminating the cells, particularly in the peritoneal cavity. In all the strains of mice examined, subcutaneous T.annulata tumours appeared to be damaged by natural immune mechanisms (macrophages) and neutrophils leading to haemorrhage and necrosis. However, the ability of the mice to completely reject the tumours depended on the presence of T and B-cells. Tumour Necrosis (TNF) was not detected in serum, or in tumour extracts. Attempts to induce tumour rejection using a preparation of rabbit TNF were not successful. Attempts to affect the growth of r.annu/ato-infected cells, injected i/p into scid mice, with human alpha and gamma interferons (HulFN-g, were also unsuccessful. The neutralisation of endogenous murine IFN-g with a monoclonal antibody allowed increased growth of T.annulata and 7".parva-infected cells in the intraperi-toneal site in scid mice. Of the mouse strains examined, the scid mouse was the most favourable host for Theileria-infected cells. Attempts to establish uninfected bovine cells in scid mice were not successful, and these results cast doubt on the use of scid mice as hosts for uninfected cells.