The development of a model system and high throughput assay for the study of interactions between zinc finger proteins and DNA

It has been recognised for some time that a full code of amino acid-based recognition of DNA sequences would be useful. Several approaches, which utilise small DNA binding motifs called zinc fingers, are presently employed. None of the current approaches successfully combine a combinatorial approach to the elucidation of a code with a single stage high throughput screening assay. The work outlined here describes the development of a model system for the study of DNA protein interactions and the development of a high throughput assay for detection of such interactions. A zinc finger protein was designed which will bind with high affinity and specificity to a known DNA sequence. For future work it is possible to mutate the region of the zinc finger responsible for the specificity of binding, in order to observe the effect on the DNA / protein interactions. The zinc finger protein was initially synthesised as a His tagged product. It was not possible however to develop a high throughput assay using the His tagged zinc finger protein. The gene encoding the zinc finger protein was altered and the protein synthesised as a Glutathione S-Transferase (GST) fusion product. A successful assay was developed using the GST protein and Scintillation Proximity Assay technology (Amersham Pharmacia Biotech). The scintillation proximity assay is a dynamic assay that allows the DNA protein interactions to be studied in "real time". This assay not only provides a high throughput method of screening zinc finger proteins for potential ligands but also allows the effect of addition of reagents or competitor ligands to be monitored.

The interaction between the implementation of an occupational health and safety management system and safety culture:a case study in the rubber industry

A prominent theme emerging in Occupational Health and Safety (OSH) is the development of management systems. A range of interventions, according to a prescribed route detailed by one of the management systems, can be introduced into an organisation with some expectation of improved OSH performance. This thesis attempts to identify the key influencing factors that may impact upon the process of introducing interventions, (according to B88800: 1996, Guide to Implementing Occupational Health and Safety Management Systems) into an organisation. To help identify these influencing factors a review of possible models from the sphere of Total Quality Management (TQM) was undertaken and the most suitable TQM model selected for development and use in aSH. By anchoring the aSH model's development in the reviewed literature a range ofeare, medium and low level influencing factors were identified. This model was developed in conjunction with the research data generated within the case study organisation (rubber manufacturer) and applied to the organisation. The key finding was that the implementation of an OSH intervention was dependant upon three broad vectors of influence. These are the Incentive to introduce change within an organisation which refers to the drivers or motivators for OSH. Secondly the Ability within the management team to actually implement the changes refers to aspects, amongst others, such as leadership, commitment and perceptions of OSH. Ability is in turn itself influenced by the environment within which change is being introduced. TItis aspect of Receptivity refers to the history of the plant and characteristics of the workforce. Aspects within Receptivity include workforce profile and organisational policies amongst others. It was found that the TQM model selected and developed for an OSH management system intervention did explain the core influencing factors and their impact upon OSH performance. It was found that within the organisation the results that may have been expected from implementation of BS8800:1996 were not realised. The OSH model highlighted that given the organisation's starting point, a poor appreciation of the human factors of OSH, gave little reward for implementation of an OSH management system. In addition it was found that general organisational culture can effectively suffocate any attempts to generate a proactive safety culture.

Development of an in-vitro model system to investigate the mechanism of muscle protein catabolism induced by proteolysis-inducing factor

The mechanism of muscle protein catabolism induced by proteolysis-inducing factor, produced by cachexia-inducing murine and human tumours has been studied in vitro using C2C12 myoblasts and myotubes. In both myoblasts and myotubes protein degradation was enhanced by proteolysis-inducing factor after 24 h incubation. In myoblasts this followed a bell-shaped dose-response curve with maximal effects at a proteolysis-inducing factor concentration between 2 and 4 nM, while in myotubes increased protein degradation was seen at all concentrations of proteolysis-inducing factor up to 10 nM, again with a maximum of 4 nM proteolysis-inducing factor. Protein degradation induced by proteolysis-inducing factor was completely attenuated in the presence of cycloheximide (1 μM), suggesting a requirement for new protein synthesis. In both myoblasts and myotubes protein degradation was accompanied by an increased expression of the α-type subunits of the 20S proteasome as well as functional activity of the proteasome, as determined by the 'chymotrypsin-like' enzyme activity. There was also an increased expression of the 19S regulatory complex as well as the ubiquitin-conjugating enzyme (E214k), and in myotubes a decrease in myosin expression was seen with increasing concentrations of proteolysis-inducing factor. These results show that proteolysis-inducing factor co-ordinately upregulates both ubiquitin conjugation and proteasome activity in both myoblasts and myotubes and may play an important role in the muscle wasting seen in cancer cachexia. © 2002 Cancer Research UK.

Differentiating human NT2/D1 neurospheres as a versatile in vitro 3D model system for developmental neurotoxicity testing

Developmental neurotoxicity is a major issue in human health and may have lasting neurological implications. In this preliminary study we exposed differentiating Ntera2/clone D1 (NT2/D1) cell neurospheres to known human teratogens classed as non-embryotoxic (acrylamide), weakly embryotoxic (lithium, valproic acid) and strongly embryotoxic (hydroxyurea) as listed by European Centre for the Validation of Alternative Methods (ECVAM) and examined endpoints of cell viability and neuronal protein marker expression specific to the central nervous system, to identify developmental neurotoxins. Following induction of neuronal differentiation, valproic acid had the most significant effect on neurogenesis, in terms of reduced viability and decreased neuronal markers. Lithium had least effect on viability and did not significantly alter the expression of neuronal markers. Hydroxyurea significantly reduced cell viability but did not affect neuronal protein marker expression. Acrylamide reduced neurosphere viability but did not affect neuronal protein marker expression. Overall, this NT2/D1 -based neurosphere model of neurogenesis, may provide the basis for a model of developmental neurotoxicity in vitro.

Deschooling society? A lifelong learning network for sustainable communities, urban regeneration and environmental technologies

The complexity and multifaceted nature of sustainable lifelong learning can be effectively addressed by a broad network of providers working co-operatively and collaboratively. Such a network involving the third, public and private sector bodies must realise the full potential of accredited flexible and blended formal learning, contextual opportunities offered by enablers of informal and non formal learning and the affordances derived from the various loose and open spaces that can make social learning effective. Such a conception informs the new Lifelong Learning Network Consortium on Sustainable Communities, Urban Regeneration and Environmental Technologies established and led by the Lifelong Learning Centre at Aston University. This paper offers a radical, reflective and political evaluation of its first year in development arguing that networked learning of this type could prefigure a new model for lifelong learning and sustainable education that renders the city itself a creative medium for transformative learning and sustainability.

Bidirectional transport between the trans-Golgi network and the endosomal system

The exchange of proteins and lipids between the trans-Golgi network (TGN) and the endosomal system requires multiple cellular machines, whose activities are coordinated in space and time to generate pleomorphic, tubulo-vesicular carriers that deliver their content to their target compartments. These machines and their associated protein networks are recruited and/or activated on specific membrane domains where they select proteins and lipids into carriers, contribute to deform/elongate and partition membrane domains using the mechanical forces generated by actin polymerization or movement along microtubules. The coordinated action of these protein networks contributes to regulate the dynamic state of multiple receptors recycling between the cell surface, endosomes and the TGN, to maintain cell homeostasis as exemplified by the biogenesis of lysosomes and related organelles, and to establish/maintain cell polarity. The dynamic assembly and disassembly of these protein networks mediating the exchange of membrane domains between the TGN and endosomes regulates cell-cell signalling and thus the development of multi-cellular organisms. Somatic mutations in single network components lead to changes in transport dynamics that may contribute to pathological modifications underlying several human diseases such as mental retardation.

Spatially-multiplexed fibre-optic Bragg grating strain and temperature sensor system based on interferometric wavelength-shift detection

A prototype fibre-optic system using interferometric wavelength-shift detection, capable of multiplexing up to 32 fibre-optic Bragg grating strain and temperature sensors with identical characteristics, has been demonstrated. This system is based on a spatially multiplexed scheme for use with fibre-based low-coherence interferometric sensors, reported previously. Four fibre-optic Bragg grating channels using the same fibre grating have been demonstrated for measuring quasi-static strain and temperature.

Achieving cost competitiveness with an agent based integrated process planning and production scheduling system

From a manufacturing perspective, the efficiency of manufacturing operations (such as process planning and production scheduling) are the key element for enhancing manufacturing competence. Process planning and production scheduling functions have been traditionally treated as two separate activities, and have resulted in a range of inefficiencies. These include infeasible process plans, non-available/overloaded resources, high production costs, long production lead times, and so on. Above all, it is unlikely that the dynamic changes can be efficiently dealt with. Despite much research has been conducted to integrate process planning and production scheduling to generate optimised solutions to improve manufacturing efficiency, there is still a gap to achieve the competence required for the current global competitive market. In this research, the concept of multi-agent system (MAS) is adopted as a means to address the aforementioned gap. A MAS consists of a collection of intelligent autonomous agents able to solve complex problems. These agents possess their individual objectives and interact with each other to fulfil the global goal. This paper describes a novel use of an autonomous agent system to facilitate the integration of process planning and production scheduling functions to cope with unpredictable demands, in terms of uncertainties in product mix and demand pattern. The novelty lies with the currency-based iterative agent bidding mechanism to allow process planning and production scheduling options to be evaluated simultaneously, so as to search for an optimised, cost-effective solution. This agent based system aims to achieve manufacturing competence by means of enhancing the flexibility and agility of manufacturing enterprises.

A novel plasma etching and emission monitoring system (PEEMS) to assess protein and lipid spoilation for hydrogel contact lenses

The work presents a new method that combines plasma etching with extrinsic techniques to simultaneously measure matrix and surface protein and lipid deposits. The acronym for this technique is PEEMS - Plasma Etching and Emission Monitoring System. Previous work has identified the presence of proteinaceous and lipoidal deposition on the surface of contact lenses and highlighted the probability that penetration of these spoilants will occur. This technique developed here allows unambiguous identification of the depth of penetration of spoilants to be made for various material types. It is for this reason that the technique has been employed in this thesis. The technique is applied as a 'molecular' scalpel, removing known amounts of material from the target. In this case from both the anterior .and posterior surfaces of a 'soft' contact lens. The residual material is then characterised by other analytical techniques such as UV/visible .and fluorescence spectroscopy. Several studies have be.en carried out for both in vivo and in vitro spoilt materials. The analysis and identification of absorbed protein and lipid of the substrate revealed the importance of many factors in the absorption and adsorption process. The effect of the material structure, protein nature (in terms of size, shape and charge) and environment conditions were examined in order to determine the relative uptake of tear proteins. The studies were extended to real cases in order to study the. patient dependent factors and lipoidal penetration.

Design, modeling and construction of a novel ablative fast pyrolysis reactor and product collection system

The objective of this work was to design, construct and commission a new ablative pyrolysis reactor and a high efficiency product collection system. The reactor was to have a nominal throughput of 10 kg/11r of dry biomass and be inherently scalable up to an industrial scale application of 10 tones/hr. The whole process consists of a bladed ablative pyrolysis reactor, two high efficiency cyclones for char removal and a disk and doughnut quench column combined with a wet walled electrostatic precipitator, which is directly mounted on top, for liquids collection. In order to aid design and scale-up calculations, detailed mathematical modelling was undertaken of the reaction system enabling sizes, efficiencies and operating conditions to be determined. Specifically, a modular approach was taken due to the iterative nature of some of the design methodologies, with the output from one module being the input to the next. Separate modules were developed for the determination of the biomass ablation rate, specification of the reactor capacity, cyclone design, quench column design and electrostatic precipitator design. These models enabled a rigorous design protocol to be developed capable of specifying the required reactor and product collection system size for specified biomass throughputs, operating conditions and collection efficiencies. The reactor proved capable of generating an ablation rate of 0.63 mm/s for pine wood at a temperature of 525 'DC with a relative velocity between the heated surface and reacting biomass particle of 12.1 m/s. The reactor achieved a maximum throughput of 2.3 kg/hr, which was the maximum the biomass feeder could supply. The reactor is capable of being operated at a far higher throughput but this would require a new feeder and drive motor to be purchased. Modelling showed that the reactor is capable of achieving a reactor throughput of approximately 30 kg/hr. This is an area that should be considered for the future as the reactor is currently operating well below its theoretical maximum. Calculations show that the current product collection system could operate efficiently up to a maximum feed rate of 10 kg/Fir, provided the inert gas supply was adjusted accordingly to keep the vapour residence time in the electrostatic precipitator above one second. Operation above 10 kg/hr would require some modifications to the product collection system. Eight experimental runs were documented and considered successful, more were attempted but due to equipment failure had to be abandoned. This does not detract from the fact that the reactor and product collection system design was extremely efficient. The maximum total liquid yield was 64.9 % liquid yields on a dry wood fed basis. It is considered that the liquid yield would have been higher had there been sufficient development time to overcome certain operational difficulties and if longer operating runs had been attempted to offset product losses occurring due to the difficulties in collecting all available product from a large scale collection unit. The liquids collection system was highly efficient and modeling determined a liquid collection efficiency of above 99% on a mass basis. This was validated due to the fact that a dry ice/acetone condenser and a cotton wool filter downstream of the collection unit enabled mass measurements of the amount of condensable product exiting the product collection unit. This showed that the collection efficiency was in excess of 99% on a mass basis.

Enhancing student learning experience through a novel electronic coursework assessment and feedback management system

Recent National Student Surveys revealed that many U.K. university students are dissatisfied with the timeliness and usefulness of the feedback received from their tutors. Ensuring timeliness in marking often results in a reduction in the quality of feedback. In Computer Science where learning relies on practising and learning from mistakes, feedback that pin-points errors and explains means of improvement is important to achieve a good student learning experience. Though suitable use of Information and Communication Technology should alleviate this problem, existing Virtual Learning Environments and e-Assessment applications such as Blackboard/WebCT, BOSS, MarkTool and GradeMark are inadequate to support a coursework assessment process that promotes timeliness and usefulness of feedback while maintaining consistency in marking involving multiple tutors. We have developed a novel Internet application, called eCAF, for facilitating an efficient and transparent coursework assessment and feedback process. The eCAF system supports detailed marking scheme editing and enables tutors to use such schemes to pin-point errors in students' work so as to provide helpful feedback efficiently. Tutors can also highlight areas in a submitted work and associate helpful feedback that clearly links to the identified mistakes and the respective marking criteria. In light of the results obtained from a recent trial of eCAF, we discuss how the key features of eCAF may facilitate an effective and efficient coursework assessment and feedback process.

De-industrialisation:Rowthorn and Wells' Model revisited

This paper presents a simple three-sector model that seeks to establish links between structural change in employment and the level of economic development. The model is a modified version of that of Rowthorn - Wells (1987). The theoretical analysis is supplemented with simple econometric tests which illustrate how the modified Rowthorn - Wells model can be used (i) to motivate empirical estimates of the link between the level of development and structures of employment, and (ii) to illustrate structural distortions under command economies.

De-industrialisation and the post-communist transition:Rowthorn and Wells' model revisited

In this paper we present a simple three-sector model explaining the structural change in employment, which is a modified version of Rowthorn-Wells (1987). We supplement the theoretical analysis with simple econometric tests, which illustrate how the modified Rowthorn-Wells model can be used to (i) motivate empirical estimates of the link between the level of development and structures of employment, (ii) illustrate structural distortions under the command economies, and the structural adjustment that happened during the post-Communist transition. We demonstrate that in the case of these economies, the transition process leads to an adjustment to the employment structures predicted by the model.

Uncatalysed and potassium-catalysed pyrolysis of the cell-wall constituents of biomass and their model compounds

Cell-wall components (cellulose, hemicellulose (oat spelt xylan), lignin (Organosolv)), and model compounds (levoglucosan (an intermediate product of cellulose decomposition) and chlorogenic acid (structurally similar to lignin polymer units)) have been investigated to probe in detail the influence of potassium on their pyrolysis behaviours as well as their uncatalysed decomposition reaction. Cellulose and lignin were pretreated to remove salts and metals by hydrochloric acid, and this dematerialized sample was impregnated with 1% of potassium as potassium acetate. Levoglucosan, xylan and chlorogenic acid were mixed with CHCOOK to introduce 1% K. Characterisation was performed using thermogravimetric analysis (TGA) and differential thermal analysis (DTA). In addition to the TGA pyrolysis, pyrolysis-gas chromatography-mass spectrometry (PY-GC-MS) analysis was introduced to examine reaction products. Potassium-catalysed pyrolysis has a huge influence on the char formation stage and increases the char yields considerably (from 7.7% for raw cellulose to 27.7% for potassium impregnated cellulose; from 5.7% for raw levoglucosan to 20.8% for levoglucosan with CHCOOK added). Major changes in the pyrolytic decomposition pathways were observed for cellulose, levoglucosan and chlorogenic acid. The results for cellulose and levoglucosan are consistent with a base catalysed route in the presence of the potassium salt which promotes complete decomposition of glucosidic units by a heterolytic mechanism and favours its direct depolymerization and fragmentation to low molecular weight components (e.g. acetic acid, formic acid, glyoxal, hydroxyacetaldehyde and acetol). Base catalysed polymerization reactions increase the char yield. Potassium-catalysed lignin pyrolysis is very significant: the temperature of maximum conversion in pyrolysis shifts to lower temperature by 70 K and catalysed polymerization reactions increase the char yield from 37% to 51%. A similar trend is observed for the model compound, chlorogenic acid. The addition of potassium does not produce a dramatic change in the tar product distribution, although its addition to chlorogenic acid promoted the generation of cyclohexane and phenol derivatives. Postulated thermal decomposition schemes for chlorogenic acid are presented. © 2008 Elsevier B.V. All rights reserved.

Urban politics and the social practices of critique and justification:conceptual insights from French pragmatism

Recent times have witnessed a growing belief in urban spaces as 'assemblages' produced through interwoven and spatially differentiated forces that converge at particular sites. There is also continuing interest in the nature of neoliberal tendencies and the rise of post-politics and democracy in urban governance. These accounts typically lack attention towards the comprehensive conceptualization of the heterogeneous logics and mechanics of relations and negotiations between actors. This paper seeks to advance these perspectives by exploring the potential contribution of French pragmatism thinking to how social life is produced through practical dialogue between actors through critique, argumentation and justification. © The Author(s) 2012.