The formulation of artificial reference standards for use within the ELISPOT assay

Whether to assess the functionality of equipment or as a determinate for the accuracy of assays, reference standards are essential for the purposes of standardisation and validation. The ELISPOT assay, developed over thirty years ago, has emerged as a leading immunological assay in the development of novel vaccines for the assessment of efficacy. However, with its widespread use, there is a growing demand for a greater level of standardisation across different laboratories. One of the major difficulties in achieving this goal has been the lack of definitive reference standards. This is partly due to the ex vivo nature of the assay, which relies on cells being placed directly into the wells. Thus, the aim of this thesis was to produce an artificial reference standard using liposomes, for use within the assay. Liposomes are spherical bilayer vesicles with an enclosed aqueous compartment and therefore are models for biological membranes. Initial work examined pre-design considerations in order to produce an optimal formulation that would closely mimic the action of the cells ordinarily placed on the assay. Recognition of the structural differences between liposomes and cells led to the formulation of liposomes with increased density. This was achieved by using a synthesised cholesterol analogue. By incorporating this cholesterol analogue in liposomes, increased sedimentation rates were observed within the first few hours. The optimal liposome formulation from these studies was composed of 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (Chol) and brominated cholesterol (Brchol) at a 16:4:12 µMol ratio, based on a significantly higher (p<0.01) sedimentation (as determined by a percentage transmission of 59 ± 5.9 % compared to the control formulation at 29 ± 12 % after four hours). By considering a range of liposome formulations ‘proof of principle’ for using liposomes as ELISPOT reference standards was shown; recombinant IFN? cytokine was successfully entrapped within vesicles of different lipid compositions, which were able to promote spot formation within the ELISPOT assay. Using optimised liposome formulations composed of phosphatidylcholine with or without cholesterol (16 µMol total lipid) further development was undertaken to produce an optimised, scalable protocol for the production of liposomes as reference standards. A linear increase in spot number by the manipulation of cytokine concentration and/or lipid concentrations was not possible, potentially due to the saturation that occurred within the base of wells. Investigations into storage of the formulations demonstrated the feasibility of freezing and lyophilisation with disaccharide cryoprotectants, but also highlighted the need for further protocol optimisation to achieve a robust reference standard upon storage. Finally, the transfer of small-scale production to a medium lab-scale batch (40 mL) demonstrated this was feasible within the laboratory using the optimised protocol.

Technological and organisational change in small- to medium-sized manufacturing companies:a learning organisation perspective

This paper examines the extent to which a learning organisation perspective is attainable in small- to medium-sized manufacturing companies. An audit tool is developed from the literature on organisational learning and recognised processes that lead towards becoming a learning organisation. The paper focuses on the application of the audit tool in three UK automotive component suppliers which are all experiencing pressures for change imposed by the major vehicle manufacturers. The main changes are concerned with tiering of the supply chain and substantial delegation of responsibilities to component suppliers including an increasing emphasis on innovation and continuous improvement. The companies presented in the paper are taken from a research project into the impact of changes in supply chain relationships on the operation of small- and medium-sized manufacturing firms in the West Midlands region of the UK. The ways in which the companies are responding to change are presented together with the results of a self-assessment using the developed audit tool. These results suggest that companies of this type tend to focus on change in those areas that involve least challenge to the established power and authority of management.

Biomass-based small and micro combined heat and power (CHP) systems:application and status in the United Kingdom

This chapter discusses the current state of biomass-based combined heat and power (CHP) production in the UK. It presents an overview of the UK's energy policy and targets which are relevant to the deployment of biomass-based CHP and summarises the current state for renewable, biomass and CHP. A number of small-scale biomass-based CHP projects are described while providing some indicative capital costs for combustion, pyrolysis and gasification technologies. For comparison purposes, it presents an overview of the respective situation in Europe and particularly in Sweden, Finland and Denmark. There is also a brief comment about novel CHP technologies in Austria. Finally it draws some conclusions on the potential of small-scale biomass CHP in the UK. © 2011 Woodhead Publishing Limited All rights reserved.

The effect of antifoams upon recombinant protein production in yeast

Foaming during fermentation reduces the efficiency of the process leading to increased costs and reduced productivity. Foaming can be overcome by the use of chemical antifoaming agents, however their influence upon the growth of organisms and protein yield is poorly understood. The objective of this work was to evaluate the effects of different antifoams on recombinant protein production. Antifoam A, Antifoam C, J673A, P2000 and SB2121 were tested at different concentrations for their effect on the growth characteristics of Pichia pastoris producing GFP, EPO and A2aR and the yield of protein in shake flasks over 48 h. All antifoams tested increased the total GFP in the shake flasks compared to controls, at higher concentrations than would normally be used for defoaming purposes. The highest yield was achieved by adding 1 % P2000 which nearly doubled the total yield followed by 1 % SB2121, 1 % J673A, 0.6 % Antifoam A and lastly 0.8 % Antifoam C. The antifoams had a detrimental effect upon the production of EPO and A2aR in shake flasks, suggesting that their effects may be protein specific. The mechanisms of action of the antifoams was investigated and suggested that although the volumetric mass oxygen transfer coefficient (kLa) was influenced by the agents, their effect upon the concentration of dissolved oxygen did not contribute to the changes in growth or recombinant protein yield. Findings in small scale also suggested that antifoams of different compositions such as silicone polymers and alcoxylated fatty acid esters may influence growth characteristics of host organisms and the ability of the cells to secrete recombinant protein, indirectly affecting the protein yield. Upon scale-up, the concentration effects of the antifoams upon GFP yield in bioreactors was reversed, with lower concentrations producing a higher yield. These data suggest that antifoam can affect cells in a multifactorial manner and highlights the importance of screening for optimum antifoam types and concentrations for each bioprocesses.

The production, harvest and adsorptive recovery of an infectious herpes simplex virus vaccine

At present there is not a reliable vaccine against herpes virus. Viral protein vaccines as yet have proved unsuccessful to meet the challenge of raising an appropriate immune response. Cantab Pharmaceuticals has produced a virus vaccine that can undergo one round of replication in the recipient in order to produce a more specific immune reaction. This virus is called Disabled Infectious Single Cycle Herpes Simplex Virus (DISC HSV) which has been derived by deleting the essential gH gene from a type 2 herpes virus. This vaccine has been proven to be effective in animal studies. Existing methods for the purification of viruses rely on laboratory techniques and for vaccine production would be on a far too small a scale. There is therefore a need for new virus purification methods to be developed in order to meet these large scale needs. An integrated process for the manufacture of a purified recombinant DISC HSV is described. The process involves culture of complementing Vero (CR2) cells, virus infection and manufacture, virus harvesting and subsequent downstream processing. The identification of suitable growth parameters for the complementing cell line and optimal limes for both infection and harvest are addressed. Various traditional harvest methods were investigated and found not to be suitable for a scaled up process. A method of harvesting, that exploits the elution of cell associated viruses by the competitive binding of exogenous heparin to virus envelope gC proteins, is described and is shown to yield significantly less contaminated process streams than sonication or osmotic approaches that involve cell rupture (with> 10-fold less complementing cell protein). High concentrations of salt (>0.8M NaCl) exhibit the same effect, although the high osmotic strength ruptures cells and increase the contamination of the process stream. This same heparin-gC protein affinity interaction is also shown to provide an efficient adsorptive purification procedure for herpes viruses which avoids the need to pre-treat the harvest material, apart from clarification, prior to chromatography. Subsequent column eluates provide product fractions with a 100-fold increase in virus titre and low levels of complementing cell protein and DNA (0.05 pg protein/pfu and 1.2 x 104 pg DNA/pfu respectively).

Strategy making process, its content and context in small professional football clubs

This thesis examines the phenomenon of strategy. Making as practised by small professional football clubs. The study was undertaken because football clubs were perceived to have problems with strategy-making and because it was believed that the specific circumstances of football clubs could be outside the range of views covered by conventional views of strategy-making. The characteristics of the club environment are its uncertainty and unpredictability, simultaneous competition and co--operation, strong regulations, and a not-for-profit orientation. Small clubs in particular face a constant struggle for financial viability and survival, due in part to split business and playing objectives. The study was designed to establish the extent and nature of the difficulties clubs experience with a view to preparing the way for creating practical guidance on ways to overcome them. Clearly, in order to survive in the long term, small professional football clubs require very effective strategic decisions. This study has addressed this issue by inquiring into the nature of strategy making for these organisations with the objective to establish the general direction in which the football clubs in question should be moving. As a result, the main research question to guide this investigation was determined as: Why do small professional football clubs have difficulties making strategies. The investigation was based on an analysis the concept of strategy and its elements, the strategic vision and objectives, the process by which strategic action comes about, the strategic action itself, and the context within which this action occurs. Data has been collected, analysed and interpreted in relation to each of these elements. Together with a wide variety of published material, 20 small football clubs have been sampled and personal interviews were conducted with board members of those clubs. The findings indicate that small football clubs do indeed experience considerable difficulties in making strategies, the reasons for which lie both in the characteristics of their competitive environment and their approaches to strategy-making. The competitive environment is characterised by a cartel-like structure with a high degree of regulation, high levels of uncertainty, little control over the core product or the production process, short-term business cycles and a close geographical link between a club with its local market. The management of clubs is characterised by the need to balance conflicting sporting and business objectives. Formal planning techniques are of little use in the small football club context as decision-making processes have a strong political character and the development of novel strategies is hindered by a strong conservative, industry paradigm and a lack of financial and managerial resources. It is concluded that there is no simple advice to be given to clubs, as they must re-examine the relationship between their playing and business objectives to create a unified and workable approach.

Marketing in indigenous and Asian small firms in the West Midlands.

This study is concerned with examining the application of marketing during the start-up, development and growth of small firms in the West Midlands. As an exploratory study, it provides evidence to support the central hypothesis of the thesis that whilst many small firms have the potential to progress through to the successful growth stage of development, they fail to do so because of their owner-managers' orientation towards production and selling and because they do not apply formal marketing during the initial stages of business development. A comparative approach to studying marketing in indigenous and Asian firms is adopted in an attempt to fill a gap in the literature on the characteristics and differences in the formation and development processes of these two groups of enterprises. The study has three main objectives and is based on qualitative research techniques of in-depth interviews, case studies and longitudinal studies among sixty-six firms representing the key activities of the small firms sector of the local economy. Firstly, it investigates owner-managers' orientation in developing and managing new and established businesses and explores the sources of, and changes in their orientation during the various stages of development. Secondly, it assesses the owner-manager's awareness and understanding of what constitutes the marketing function and investigates what aspects of marketing are applied during the different stages of business development. Finally, the study monitors and evaluates the outcomes and implications of applying formal marketing techniques in a small sample of firms over a period of two years. The thesis concludes by using the findings of the study to contribute additions to existing models of growth and by proposing new models of evolution and application of marketing in small firms.

Cost effectiveness of production control systems

This thesis deals with the problems associated with the planning and control of production, with particular reference to a small aluminium die casting company. The main problem areas were identified as: (a) A need to be able to forecast the customers demands upon the company's facilities. (b) A need to produce a manufacturing programme in which the output of the foundry (or die casting section) was balanced with the available capacity in the machine shop. (c) The need to ensure that the resultant system enabled the company's operating budget to have a reasonable chance of being achieved. At the commencement of the research work the major customers were members of the automobile industry and had their own system of forecasting, from which they issued manufacturing schedules to their component suppliers, The errors in the forecast were analysed and the distributions noted. Using these distributions the customer's forecast was capable of being modified to enable his final demand to be met with a known degree of confidence. Before a manufacturing programme could be developed the actual manufacturing system had to be reviewed and it was found that as with many small companies there was a remarkable lack of formal control and written data. Relevant data with regards to the component and the manufacturing process had therefore to be collected and analysed. The foundry process was fixed but the secondary machining operations were analysed by a technique similar to Component Flow Analysis and as a result the machines were arranged in a series of flow lines. A system of manual production control was proposed and for comparison, a local computer bureau was approached and a system proposed incorporating the production of additional management information. These systems are compared and the relative merits discussed and a proposal made for implementation.

Drying of biomass for second generation synfuel production

Drying is a major and challenging step in the pre-treatment of biomass for production of second generation synfuels for transport. The biomass feedstocks are mostly wet and need to be dried from 30 to 60 wt% moisture content to about 10-15 wt%. The present survey aims to define and evaluate a few of the most promising optimised concepts for biomass pre-treatment scheme in the production of second generation synfuels for transport. The most promising commercially available drying processes were reviewed, focusing on the applications, operational factors and emissions of dryers. The most common dryers applied now for biomass in bio-energy plants are direct rotary dryers, but the use of steam drying techniques is increasing. Steam drying systems enable the integration of the dryer to existing energy sources. In addition to integration, emissions and fire or explosion risks have to be considered when selecting a dryer for the plant. In steam drying there will be no gaseous emissions, but the aqueous effluents need often treatment. Concepts for biomass pre-treatment were defined for two different cases including a large-scale wood-based gasification synfuel production and a small-scale pyrolysis process based on wood chips and miscanthus bundles. For the first case a pneumatic conveying steam dryer was suggested. In the second case the flue gas will be used as drying medium in a direct or indirect rotary dryer.

Which factors drive firm survival within an industry boundary over time?:the UK onshore oil and gas production industry 1984-1999

This thesis looks at the UK onshore oil and gas production industry and follows the history of a population of firms over a fifteen-year period following the industry's renaissance. It examines the linkage between firm survival, selection pressures and adaptation responses at the firm level, especially the role of discretionary adaptation, specifically exploration and exploitation strategies.Taking a Realist approach and using quantitative and qualitative methods for triangulation on a new data base derived from archival data, as well as informant interviews, it tests seven hypotheses' about post-entry survival of firms. The quantitative findings suggest that firm survival within this industry is linked to discretionary adaptation, when measured at the firm level, and to a mixture of selection and adaptation forces when measured for each firm for each individual year. The qualitative research suggests that selection factors dominate. This difference in views is unresolved. However the small, sparse population and the nature of the oil and gas industry compared with other common research contexts such as manufacturing or service firms suggests the results be treated with caution as befits a preliminary investigation. The major findings include limited support for the theory that the external environment is the major determinant of firm survival, though environment components affect firms differentially; resolution of apparent literature differences relating to the sequencing of exploration and exploitation and potential tangible evidence of coevolution. The research also finds that, though selection may be considered important by industry players, discretionary adaptation appears to play the key role, and that the key survival drivers for thispopulation are intra-industry ties, exploitation experience and a learning/experience component. Selection has a place, however, in determining the life-cycle of the firm returning to be a key survival driver at certain ages of the firm inside the industry boundary.

Low severity Fischer-Tropsch synthesis for the production of synthetic hydrocarbon fuels

Currently, the main source for the production of liquid transportation fuels is petroleum, the continued use of which faces many challenges including depleting oil reserves, significant oil price rises, and environmental concerns over global warming which is widely believed to be due to fossil fuel derived CO2 emissions and other greenhouse gases. In this respect, lignocellulosic or plant biomass is a particularly interesting resource as it is the only renewable source of organic carbon that can be converted into liquid transportation fuels. The gasification of biomass produces syngas which can then be converted into synthetic liquid hydrocarbon fuels by means of the Fischer-Tropsch (FT) synthesis. This process has been widely considered as an attractive option for producing clean liquid hydrocarbon fuels from biomass that have been identified as promising alternatives to conventional fossil fuels like diesel and kerosene. The resulting product composition in FT synthesis is influenced by the type of catalyst and the reaction conditions that are used in the process. One of the issues facing this conversion process is the development of a technology that can be scaled down to match the scattered nature of biomass resources, including lower operating pressures, without compromising liquid composition. The primary aims of this work were to experimentally explore FT synthesis at low pressures for the purpose of process down-scaling and cost reduction, and to investigate the potential for obtaining an intermediate FT synthetic crude liquid product that can be integrated into existing refineries under the range of process conditions employed. Two different fixed-bed micro-reactors were used for FT synthesis; a 2cm3 reactor at the University of Rio de Janeiro (UFRJ) and a 20cm3 reactor at Aston University. The experimental work firstly involved the selection of a suitable catalyst from three that were available. Secondly, a parameter study was carried out on the 20cm3 reactor using the selected catalyst to investigate the influence of reactor temperature, reactor pressure, space velocity, the H2/CO molar ratio in the feed syngas and catalyst loading on the reaction performance measured as CO conversion, catalyst stability, product distribution, product yields and liquid hydrocarbon product composition. From this parameter study a set of preferred operating conditions was identified for low pressure FT synthesis. The three catalysts were characterized using BET, XRD, TPR and SEM. The catalyst selected was an unpromoted Co/Al2O3 catalyst. FT synthesis runs on the 20cm3 reactor at Aston were conducted for 48 hours. Permanent gases and light hydrocarbons (C1-C5) were analysed in an online GC-TCD/FID at hourly intervals. The liquid hydrocarbons collected were analyzed offline using GC-MS for determination of fuel composition. The parameter study showed that CO conversion and liquid hydrocarbon yields increase with increasing reactor pressure up to around 8 bar, above which the effect of pressure is small. The parameters that had the most significant influence on CO conversion, product selectivity and liquid hydrocarbon yields were reactor temperature and catalyst loading. The preferred reaction conditions identified for this research were: T = 230ºC, P = 10 bar, H2/CO = 2.0, WHSV = 2.2 h-1, and catalyst loading = 2.0g. Operation in the low range of pressures studied resulted in low CO conversions and liquid hydrocarbon yields, indicating that low pressure BTL-FT operation may not be industrially viable as the trade off in lower CO conversions and once-through liquid hydrocarbon product yields has to be carefully weighed against the potential cost savings resulting from process operation at lower pressures.

In vivo and ex vivo regulation of visfatin production by leptin in human and murine adipose tissue: role of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways

Visfatin is an adipogenic adipokine with increased levels in obesity, properties common to leptin. Thus, leptin may modulate visfatin production in adipose tissue (AT). Therefore, we investigated the effects of leptin on visfatin levels in 3T3-L1 adipocytes and human/murine AT, with or without a leptin antagonist. The potential signaling pathways and mechanisms regulating visfatin production in AT was also studied. Real-time RT-PCR and Western blotting were used to assess the relative mRNA and protein expression of visfatin. ELISA was performed to measure visfatin levels in conditioned media of AT explants, and small interfering RNA technology was used to reduce leptin receptor expression. Leptin significantly (P<0.01) increased visfatin levels in human and murine AT with a maximal response at leptin 10(-9) M, returning to baseline at leptin 10(-7) M. Importantly, ip leptin administration to C57BL/6 ob/ob mice further supported leptin-induced visfatin protein production in omental AT (P<0.05). Additionally, soluble leptin receptor levels rose with concentration dependency to a maximal response at leptin 10(-7) M (P<0.01). The use of a leptin antagonist negated the induction of visfatin and soluble leptin receptor by leptin. Furthermore, leptin-induced visfatin production was significantly decreased in the presence of MAPK and phosphatidylinositol 3-kinase inhibitors. Also, when the leptin eceptor gene was knocked down using small interfering RNA, eptin-induced visfatin expression was significantly decreased. Thus, leptin increases visfatin production in AT in vivo and ex vivo via pathways involving MAPK and phosphatidylinositol 3-kinase signaling. The pleiotropic effects of leptin may be partially mediated by visfatin.

Decision support for lifecycle planning and risk management of small-scale biomass combined heat and power (bCHP) projects in the UK

Biomass is projected to account for approximately half of the new energy production required to achieve the 2020 primary energy target in the UK. Combined heat and power (CHP) bioenergy systems are not only a highly efficient method of energy conversion, at smaller-scales a significant proportion of the heat produced can be effectively utilised for hot water, space heating or industrial heating purposes. However, there are many barriers to project development and this has greatly inhibited deployment in the UK. Project viability is highly subjective to changes in policy, regulation, the finance market and the low cost incumbent; a high carbon centralised energy system. Unidentified or unmitigated barriers occurring during the project lifecycle may not only negatively impact on the project but could ultimately lead to project failure. The research develops a decision support system (DSS) for small-scale (500 kWe to 10 MWe) biomass combustion CHP project development and risk management in the early stages of a potential project’s lifecycle. By supporting developers in the early stages of project development with financial, scheduling and risk management analysis, the research aims to reduce the barriers identified and streamline decision-making. A fuzzy methodology is also applied throughout the developed DSS to support developers in handling the uncertain or approximate information often held at the early stages of the project lifecycle. The DSS is applied to a case study of a recently failed (2011) small-scale biomass CHP project to demonstrate its applicability and benefits. The application highlights that the proposed development within the case study was not viable. Moreover, further analysis of the possible barriers with the DSS confirmed that some possible modifications to be project could have improved this, such as a possible change of feedstock to a waste or residue, addressing the unnecessary land lease cost or by increasing heat utilisation onsite. This analysis is further supported by a practitioner evaluation survey that confirms the research contribution and objectives are achieved.

Sustainable agricultural production:an investigation in Brazilian semi-arid livestock farms

This paper investigates the environmental sustainability and competitiveness perceptions of small farmers in a region in northern Brazil. The main data collection instruments included a survey questionnaire and an analysis of the region's strategic plan. In total, ninety-nine goat and sheep breeding farmers were surveyed. Data analysis methods included descriptive statistics, cluster analysis, and chi-squared tests. The main results relate to the impact of education, land size, and location on the farmers' perceptions of competitiveness and environmental issues. Farmers with longer periods of education have higher perception scores about business competitiveness and environmental sustainability than those with less formal education. Farmers who are working larger land areas also have higher scores than those with smaller farms. Lastly, location can yield factors that impact on farmers' perceptions. In our study, farmers located in Angicos and Lajes had higher perception scores than Pedro Avelino and Afonso Bezerra, despite the geographical proximity of these municipalities. On the other hand, three other profile variables did not impact on farmers' perceptions, namely: family income, dairy production volume, and associative condition. The authors believe the results and insights can be extended to livestock farming in other developing countries and contribute generally to fostering effective sustainable development policies, mainly in the agribusiness sector. © 2013 Elsevier Ltd. All rights reserved.

Improving recombinant human adenosine A2A receptor production in yeast

Over 50% of clinically-marketed drugs target membrane proteins; in particular G protein-coupled receptors (GPCRs). GPCRs are vital to living cells, performing an active role in many processes, making them integral to drug development. In nature, GPCRs are not sufficiently abundant for research and their structural integrity is often lost during extraction from cell membranes. The objectives of this thesis were to increase recombinant yield of the GPCR, human adenosine A2A receptor (hA2AR) by investigating bioprocess conditions in large-scale Pichia pastoris and small-scale Saccharomyces cerevisiae cultivations. Extraction of hA2AR from membranes using novel polymers was also investigated. An increased yield of hA2AR from P. pastoris was achieved by investigating the methanol feeding regime. Slow, exponential feed during induction (μlow) was compared to a faster, exponential feed (μhigh) in 35 L pilot-scale bioreactors. Overall hA2AR yields were increased for the μlow cultivation (536.4pmol g-1) compared to the μhigh148.1 pmol g-1. hA2AR levels were maintained in cytotoxic methanol conditions and unexpectedly, pre-induction levels of hA2AR were detected. Small-scale bioreactor work showed that Design of Experiments (DoE) could be applied to screen for bioprocess conditions to give optimal hA2AR yields. Optimal conditions were retrieved for S. cerevisiae using a d-optimal screen and response surface methodology. The conditions were 22°C, pH 6.0, 30% DO without dimethyl sulphoxide. A polynomial equation was generated to predict hA2AR yields if conditions varied. Regarding the extraction, poly (maleic anhydride-styrene) or PMAS was successful in solubilising hA2AR from P. pastoris membranes compared with dodcecyl-β-D-maltoside (DDM) detergent. Variants of PMAS worked well as solubilising agents with either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or cholesteryl hemisuccinate (CHS). Moreover, esterification of PMAS improved solubilisation, suggesting that increased hydrophobicity stabilises hA2AR during extraction. Overall, hA2AR yields were improved in both, P. pastoris and S. cerevisiae and the use of novel polymers for efficient extraction was achieved.