The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).

From recession to renewal:the impact of the financial crisis on public services and local government

Book review of From Recession to Renewal: The Impact of the Financial Crisis on Public Services and Local Government, Joanna Richardson (Ed). Bristol, Policy Press, 2010, ISBN 9781847426994

Action research at the Sea Fish Industry Authority:improving the quality of our knowing- how do practices achieve organisational learning and renewal and how does this change overtime?

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

Driving licensing renewal policy using neural network-based probabilistic decision support system

This paper investigates neural network-based probabilistic decision support system to assess drivers' knowledge for the objective of developing a renewal policy of driving licences. The probabilistic model correlates drivers' demographic data to their results in a simulated written driving exam (SWDE). The probabilistic decision support system classifies drivers' into two groups of passing and failing a SWDE. Knowledge assessment of drivers within a probabilistic framework allows quantifying and incorporating uncertainty information into the decision-making system. The results obtained in a Jordanian case study indicate that the performance of the probabilistic decision support systems is more reliable than conventional deterministic decision support systems. Implications of the proposed probabilistic decision support systems on the renewing of the driving licences decision and the possibility of including extra assessment methods are discussed.

Creative thinking experimentations for entrepreneurship with a disruptive, personalised and mobile game-based learning ecosystem

Based on an unprecedented need of stimulating creative capacities towards entrepreneurship to university students and young researchers, this paper introduces and analyses a smart learning ecosystem for encouraging teaching and learning on creative thinking as a distinct feature to be taught and learnt in universities. The paper introduces a mashed-up authoring architecture for designing lesson-plans and games with visual learning mechanics for creativity learning. The design process is facilitated by creativity pathways discerned across components. Participatory learning, networking and capacity building is a key aspect of the architecture, extending the learning experience and context from the classroom to outdoor (co-authoring of creative pathways by students, teachers and real-world entrepreneurs) and personal spaces. We anticipate that the smart learning ecosystem will be empirically evaluated and validated in future iterations for exploring the benefits of using games for enhancing creative mindsets, unlocking the imagination that lies within, practiced and transferred to multiple academic tribes and territories.

Action research: minimising the impact of disruptive incidents for rail passengers through improved sharing of information

This paper reports on an action research project based in the UK rail industry; it used a novel type of Soft Systems Methodology (known as PrOH Modelling) to facilitate change in a major Train Operating Company (TOC). The project looked at a number of different disruptive incidents to compare and contrast practice via the Mitigate, Prevent, React and Recover (MPRR) Framework. One incident is detailed in depth. The paper also looks at the general process of conducting action research. This work will be of interest for researchers in the rail sector and for those conducting operations action research projects.