Liposomes:Formulation and characterisation as contrast agents and as vaccine delivery systems

Liposome systems are well reported for their activity as vaccine adjuvants; however novel lipid-based microbubbles have also been reported to enhance the targeting of antigens into dendritic cells (DCs) in cancer immunotherapy (Suzuki et al 2009). This research initially focused on the formulation of gas-filled lipid coated microbubbles and their potential activation of macrophages using in vitro models. Further studies in the thesis concentrated on aqueous-filled liposomes as vaccine delivery systems. Initial work involved formulating and characterising four different methods of producing lipid-coated microbubbles (sometimes referred to as gas-filled liposomes), by homogenisation, sonication, a gas-releasing chemical reaction and agitation/pressurisation in terms of stability and physico-chemical characteristics. Two of the preparations were tested as pressure probes in MRI studies. The first preparation composed of a standard phospholipid (DSPC) filled with air or nitrogen (N2), whilst in the second method the microbubbles were composed of a fluorinated phospholipid (F-GPC) filled with a fluorocarbon saturated gas. The studies showed that whilst maintaining high sensitivity, a novel contrast agent which allows stable MRI measurements of fluid pressure over time, could be produced using lipid-coated microbubbles. The F-GPC microbubbles were found to withstand pressures up to 2.6 bar with minimal damage as opposed to the DSPC microbubbles, which were damaged at above 1.3 bar. However, it was also found that DSPC-filled with N2 microbubbles were also extremely robust to pressure and their performance was similar to that of F-GPC based microbubbles. Following on from the MRI studies, the DSPC-air and N2 filled lipid-based microbubbles were assessed for their potential activation of macrophages using in vitro models and compared to equivalent aqueous-filled liposomes. The microbubble formulations did not stimulate macrophage uptake, so studies thereafter focused on aqueous-filled liposomes. Further studies concentrated on formulating and characterising, both physico-chemically and immunologically, cationic liposomes based on the potent adjuvant dimethyldioctadecylammonium (DDA) and immunomodulatory trehalose dibehenate (TDB) with the addition of polyethylene glycol (PEG). One of the proposed hypotheses for the mechanism behind the immunostimulatory effect obtained with DDA:TDB is the ‘depot effect’ in which the liposomal carrier helps to retain the antigen at the injection site thereby increasing the time of vaccine exposure to the immune cells. The depot effect has been suggested to be primarily due to their cationic nature. Results reported within this thesis demonstrate that higher levels of PEG i.e. 25 % were able to significantly inhibit the formation of a liposome depot at the injection site and also severely limit the retention of antigen at the site. This therefore resulted in a faster drainage of the liposomes from the site of injection. The versatility of cationic liposomes based on DDA:TDB in combination with different immunostimulatory ligands including, polyinosinic-polycytidylic acid (poly (I:C), TLR 3 ligand), and CpG (TLR 9 ligand) either entrapped within the vesicles or adsorbed onto the liposome surface was investigated for immunogenic capacity as vaccine adjuvants. Small unilamellar (SUV) DDA:TDB vesicles (20-100 nm native size) with protein antigen adsorbed to the vesicle surface were the most potent in inducing both T cell (7-fold increase) and antibody (up to 2 log increase) antigen specific responses. The addition of TLR agonists poly(I:C) and CpG to SUV liposomes had small or no effect on their adjuvanticity. Finally, threitol ceramide (ThrCer), a new mmunostimulatory agent, was incorporated into the bilayers of liposomes composed of DDA or DSPC to investigate the uptake of ThrCer, by dendritic cells (DCs), and presentation on CD1d molecules to invariant natural killer T cells. These systems were prepared both as multilamellar vesicles (MLV) and Small unilamellar (SUV). It was demonstrated that the IFN-g secretion was higher for DDA SUV liposome formulation (p<0.05), suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.

A study of adaptive load balancing algorithms for distributed systems

With the advent of distributed computer systems with a largely transparent user interface, new questions have arisen regarding the management of such an environment by an operating system. One fertile area of research is that of load balancing, which attempts to improve system performance by redistributing the workload submitted to the system by the users. Early work in this field concentrated on static placement of computational objects to improve performance, given prior knowledge of process behaviour. More recently this has evolved into studying dynamic load balancing with process migration, thus allowing the system to adapt to varying loads. In this thesis, we describe a simulated system which facilitates experimentation with various load balancing algorithms. The system runs under UNIX and provides functions for user processes to communicate through software ports; processes reside on simulated homogeneous processors, connected by a user-specified topology, and a mechanism is included to allow migration of a process from one processor to another. We present the results of a study of adaptive load balancing algorithms, conducted using the aforementioned simulated system, under varying conditions; these results show the relative merits of different approaches to the load balancing problem, and we analyse the trade-offs between them. Following from this study, we present further novel modifications to suggested algorithms, and show their effects on system performance.

Aspects of the design and control of manufacturing systems subject to demand uncertainty

The recent explosive growth in advanced manufacturing technology (AMT) and continued development of sophisticated information technologies (IT) is expected to have a profound effect on the way we design and operate manufacturing businesses. Furthermore, the escalating capital requirements associated with these developments have significantly increased the level of risk associated with initial design, ongoing development and operation. This dissertation has examined the integration of two key sub-elements of the Computer Integrated Manufacturing (CIM) system, namely the manufacturing facility and the production control system. This research has concentrated on the interactions between production control (MRP) and an AMT based production facility. The disappointing performance of such systems has been discussed in the context of a number of potential technological and performance incompatibilities between these two elements. It was argued that the design and selection of operating policies for both is the key to successful integration. Furthermore, policy decisions are shown to play an important role in matching the performance of the total system to the demands of the marketplace. It is demonstrated that a holistic approach to policy design must be adopted if successful integration is to be achieved. It is shown that the complexity of the issues resulting from such an approach required the formulation of a structured design methodology. Such a methodology was subsequently developed and discussed. This combined a first principles approach to the behaviour of system elements with the specification of a detailed holistic model for use in the policy design environment. The methodology aimed to make full use of the `low inertia' characteristics of AMT, whilst adopting a JIT configuration of MRP and re-coupling the total system to the market demands. This dissertation discussed the application of the methodology to an industrial case study and the subsequent design of operational policies. Consequently a novel approach to production control resulted. A central feature of which was a move toward reduced manual intervention in the MRP processing and scheduling logic with increased human involvement and motivation in the management of work-flow on the shopfloor. Experimental results indicated that significant performance advantages would result from the adoption of the recommended policy set.

Aspects of the design and operation of production control systems in manufacturing industry

Computerised production control developments have concentrated on Manufacturing Resources Planning (MRP II) systems. The literature suggests however, that despite the massive investment in hardware, software and management education, successful implementation of such systems in manufacturing industries has proved difficult. This thesis reviews the development of production planning and control systems, in particular, investigates the causes of failures in implementing MRP/MRP II systems in industrial environments and argues that the centralised and top-down planning structure, as well as the routine operational methodology of such systems, is inherently prone to failure. The thesis reviews the control benefits of cellular manufacturing systems but concludes that in more dynamic manufacturing environments, techniques such as Kanban are inappropriate. The basic shortcomings of MRP II systems are highlighted and a new enhanced operational methodology based on distributed planning and control principles is introduced. Distributed Manufacturing Resources Planning (DMRP), was developed as a capacity sensitive production planning and control solution for cellular manufacturing environments. The system utilises cell based, independently operated MRP II systems, integrated into a plant-wide control system through a Local Area Network. The potential benefits of adopting the system in industrial environments is discussed and the results of computer simulation experiments to compare the performance of the DMRP system against the conventional MRP II systems presented. DMRP methodology is shown to offer significant potential advantages which include ease of implementation, cost effectiveness, capacity sensitivity, shorter manufacturing lead times, lower working in progress levels and improved customer service.