Purification of plasmid DNA for use in human gene therapy

Two key issues defined the focus of this research in manufacturing plasmid DNA for use In human gene therapy. First, the processing of E.coli bacterial cells to effect the separation of therapeutic plasmid DNA from cellular debris and adventitious material. Second, the affinity purification of the plasmid DNA in a Simple one-stage process. The need arises when considering the concerns that have been recently voiced by the FDA concerning the scalability and reproducibility of the current manufacturing processes in meeting the quality criteria of purity, potency, efficacy, and safety for a recombinant drug substance for use in humans. To develop a preliminary purification procedure, an EFD cross-flow micro-filtration module was assessed for its ability to effect the 20-fold concentration, 6-time diafiltration, and final clarification of the plasmid DNA from the subsequent cell lysate that is derived from a 1 liter E.coli bacterial cell culture. Historically, the employment of cross-flow filtration modules within procedures for harvesting cells from bacterial cultures have failed to reach the required standards dictated by existing continuous centrifuge technologies, frequently resulting in the rapid blinding of the membrane with bacterial cells that substantially reduces the permeate flux. By challenging the EFD module, containing six helical wound tubular membranes promoting centrifugal instabilities known as Dean vortices, with distilled water between the Dean number's of 187Dn and 818Dn,and the transmembrane pressures (TMP) of 0 to 5 psi. The data demonstrated that the fluid dynamics significantly influenced the permeation rate, displaying a maximum at 227Dn (312 Imh) and minimum at 818Dn (130 Imh) for a transmembrane pressure of 1 psi. Numerical studies indicated that the initial increase and subsequent decrease resulted from a competition between the centrifugal and viscous forces that create the Dean vortices. At Dean numbers between 187Dn and 227Dn , the forces combine constructively to increase the apparent strength and influence of the Dean vortices. However, as the Dean number in increases above 227 On the centrifugal force dominates the viscous forces, compressing the Dean vortices into the membrane walls and reducing their influence on the radial transmembrane pressure i.e. the permeate flux reduced. When investigating the action of the Dean vortices in controlling tile fouling rate of E.coli bacterial cells, it was demonstrated that the optimum cross-flow rate at which to effect the concentration of a bacterial cell culture was 579Dn and 3 psi TMP, processing in excess of 400 Imh for 20 minutes (i.e., concentrating a 1L culture to 50 ml in 10 minutes at an average of 450 Imh). The data demonstrated that there was a conflict between the Dean number at which the shear rate could control the cell fouling, and the Dean number at which tile optimum flux enhancement was found. Hence, the internal geometry of the EFD module was shown to sub-optimal for this application. At 579Dn and 3 psi TMP, the 6-fold diafiltration was shown to occupy 3.6 minutes of process time, processing at an average flux of 400 Imh. Again, at 579Dn and 3 psi TMP the clarification of the plasmid from tile resulting freeze-thaw cell lysate was achieved at 120 Iml1, passing 83% (2,5 mg) of the plasmid DNA (6,3 ng μ-1 10.8 mg of genomic DNA (∼23,00 Obp, 36 ng μ-1 ), and 7.2 mg of cellular proteins (5-100 kDa, 21.4 ngμ-1 ) into the post-EFD process stream. Hence the EFD module was shown to be effective, achieving the desired objectives in approximately 25 minutes. On the basis of its ability to intercalate into low molecular weight dsDNA present in dilute cell lysates, and be electrophoresed through agarose, the fluorophore PicoGreen was selected for the development of a suitable dsDNA assay. It was assesseel for its accuracy, and reliability, In determining the concentration and identity of DNA present in samples that were eleclrophoresed through agarose gels. The signal emitted by intercalated PicoGreen was shown to be constant and linear, and that the mobility of the PicaGreen-DNA complex was not affected by the intercalation. Concerning the secondary purification procedure, various anion-exchange membranes were assessed for their ability to capture plasmid DNA from the post-EFD process stream. For a commercially available Sartorius Sartobind Q15 membrane, the reduction in the equilibriumbinding capacity for  ctDNA in buffer of increasing ionic demonstrated that DNA was being.adsorbed by electrostatic  interactions only. However, the problems associated with fluid distribution across the membrane demonstrated that the membrane housing was the predominant cause of the .erratic breakthrough curves. Consequently, this would need to be rectified before such a membrane could be integrated into the current system, or indeed be scaled beyond laboratory scale. However, when challenged with the process material, the data showed that considerable quantities of protein (1150 μg) were adsorbed preferentially to the plasmid DNA (44 μg). This was also shown for derived Pall Gelman UltraBind US450 membranes that had been functionalised by varying molecular weight poly-L~lysine and polyethyleneimine ligands. Hence the anion-exchange membranes were shown to be ineffective in capturing plasmid DNA from the process stream. Finally, work was performed to integrate a sequence-specific DNA·binding protein into a single-stage DNA chromatography, isolating plasmid DNA from E.coli cells whilst minimising the contamination from genomic DNA and cellular protein. Preliminary work demonstrated that the fusion protein was capable of isolating pUC19 DNA into which the recognition sequence for the fusion-protein had been inserted (pTS DNA) when in the presence of the conditioned process material. Althougth the pTS recognition sequence differs from native pUC19 sequences by only 2 bp, the fusion protein was shown to act as a highly selective affinity ligand for pTS DNA alone. Subsequently, the scale of the process was scaled 25-fold and positioned directly following the EFD system. In conclusion, the integration of the EFD micro-filtration system and zinc-finger affinity purification technique resulted in the capture of approximately 1 mg of plasmid DNA was purified from 1L of E.coli  culture in a simple two stage process, resulting in the complete removal of genomic DNA and 96.7% of cellular protein in less than 1 hour of process time.

Bilingualism and educational achievement:a study of young bilinguals in Birmingham schools and colleges

This study aimed firstly to investigate current patterns of language use amongst young bilinguals in Birmingham and secondly to examine the relationship between this language use and educational achievement. The research then focussed on various practices, customs and attitudes which would favour the attrition or survival of minority languages in the British situation. The data necessary to address this question was provided by a sample of three hundred and seventy-four 16-19 year olds, studying in Birmingham schools and colleges during the period 1987-1990 and drawn from the main linguistic minority communities in Birmingham. The research methods chosen were both quantitative and qualitative. The study found evidence of ethnolinguistic vitality amongst many of the linguistic minority communities in Birmingham: a number of practices and a range of attitudes indicate that linguistic diversity may continue and that a stable diglossic situation may develop in some instances, particularly where demographical and religious factors lead to closeness of association. Where language attrition is occurring it is often because of the move from a less prestigious minority language or dialect to a more prestigious minority language in addition to pressures from English. The educational experience of the sample indicates that literacy and formal language study are of key importance if personal bilingualism is to be experienced as an asset; high levels of oral proficiency in the L1 and L2 do not, on their own, necessarily correlate with positive educational benefit. The intervening variable associated with educational achievement appears to be the formal language learning process and literacy. A number of attitudes and practices, including the very close associations maintained with some of the countries of origin of the families, were seen to aid or hinder first language maintenance and second language acquisition.

The development of a market orientation in a turbulent, transitional environment:the case of Ukraine

This research is concerned with the case of Ukraine where there has been a fundamental change in the economic environment engendered by the transition from a centrally planned to a market economy and the resulting conditions of economic crisis. The way that Ukrainian marketers and consumers are reacting to this environmental change is the principal concern of the research together with the basic linkages between critical environmental elements and strategic marketing decisions. This research analyses the way in which marketing evolves in a turbulent, transitional environment. The study is based on eight case analyses within the retail sector of the Ukrainian market. The process of transition to a free market economy leads a researcher to question the validity of existing theories that have been based on empirical data that has been collected in the stable marketing environment of the West. For this reason an in-depth, theory building methodology is preferred. A number of broad research objectives were set. Firstly, to establish whether the sales - production - marketing stages model of marketing evolution, derived from empirical data collected in stable Western environments, is relevant in the context of the turbulent, transitional environment of Ukraine. Secondly, to establish if, in this turbulent, transitional environment, idiosyncratic approaches to marketing are emerging and thirdly to assess the development of attitudes to marketing, organisation for marketing and the development of marketing strategy in Ukraine. The main conclusions of this thesis are firstly, that the stages model of marketing orientation is not relevant in the context of Ukraine, secondly that there are idiosyncratic approaches to marketing emerging in the turbulent, transitional environment of Ukraine and finally that the above developments have a profound impact on the development of attitudes to marketing, organisation for marketing and the development of marketing strategy in Ukraine.

Time scale analysis for fluidizedbedmeltgranulation III: binder solidification rate

In series I and II of this study ([Chua et al., 2010a] and [Chua et al., 2010b]), we discussed the time scale of granule–granule collision, droplet–granule collision and droplet spreading in Fluidized Bed Melt Granulation (FBMG). In this third one, we consider the rate at which binder solidifies. Simple analytical solution, based on classical formulation for conduction across a semi-infinite slab, was used to obtain a generalized equation for binder solidification time. A multi-physics simulation package (Comsol) was used to predict the binder solidification time for various operating conditions usually considered in FBMG. The simulation results were validated with experimental temperature data obtained with a high speed infrared camera during solidification of ‘macroscopic’ (mm scale) droplets. For the range of microscopic droplet size and operating conditions considered for a FBMG process, the binder solidification time was found to fall approximately between 10-3 and 10-1 s. This is the slowest compared to the other three major FBMG microscopic events discussed in this series (granule–granule collision, granule–droplet collision and droplet spreading).

The metamorphic environments of the Strontian, Foyers and Dalbeattie intrusions, Scotland

Three metamorphic aureoles around intrusions of the Caledonian 'Newer Granite' suite are described. Each represents a different orogenic environment. The Strontian complex is intruded into sillimanite grade Moinian metasediments at the core of the orogen. The aureole comprises three zones; a transitional muscovite + sillimanite + K-feldspar zone, a sillimanite + K-feldspar zone and an inner cordierite + K-feldspar zone. Contact migmatization occurs in the inner part of the aureole. Zoning profiles from garnets in both regional and aureole assemblages show retrograde Mn-rich rims. Fe and Mg compositions are re-equilibrated to contact conditions. Apparent re-equilibration of Ca compositions results from increasingly ideal solid solution behaviour of Ca in plagioclase and garnet with increasing temperature. Temperatures of 690°C at 4.1 kbar (XH2O = 0.53) are estimated in the cordierite + K-feldspar zone, dropping to 630°C (XH2O = 0.69) at the sillimanite + K-feldspar isograd. The zones increase in width to the east, influenced by the regional thermal gradient at the time of intrusion. The timer-scale of the contact event, t2, relative to the regional, tl, - is estimated as t2/t1 = 101.1+ -0.7 and is consistent with Intrusion at an early stage of regional uplift and cooling. The Foyers complex intrudes Moinian rocks at a higher structural level. Regional assemblages range from garnet to sillimanite grade. Three contact zones are recognised; a sillimanite zone, a sillimanite + K-feldspar zone and an inner cordierite + K-feldspar zone. The limit of the aureole is marked by the breakdown of garnet which shows disequilibrium, both texturally, and in complex zoning profiles, within it. Temperatures of 660°C at 3.9 kbar (XH20 = 0.14) are estimated in the cordierite + K-feldspar zone? The Dalbeattie complex is at the margin of the orogen, intruded into low grade Silurian metasediments. Two zones are recognised; a biotite zone and an inner hornblende zone. Cordierite and diopside are present in the inner zone.

Teachers' conceptions of their role

An analysis is made of the conceptions which serving teachers have of their role, though no attempt is made to relate this to their practice of teaching. A series of role items was collected to afford a description of the teacher's role in terms of school and society expectations as well as classroom behaviours. These were taken from the literature and from interviews with teachers, and confirmed in a preliminary survey. Presented as a questionnaire, replies to the main investigation were made by 881 teachers, working in a variety of schools from nurseries to comprehensives. Two attempts have been made to construct a role model. The first, depending on the judgement of items fitting theoretically derived roles, failed, due to diffuseness in the role of teacher. The second used factor analysis; six factors were extracted which represent meaningful and distinct areas of role. The analysis has depended largely on examination of scores taken from these factors. Teachers in all types of school have similar conceptions of discipline. Nursery-infant and junior staff generally agree on the other areas investigated, but the concepts of secondary teachers are distinct. They are more conservative and less child-centered. When the class being taught is held constant, few differences in role conception are found to be related to sex, being a parent, graduate status, or personality, as measured in terms of the extrovert and neurotic dimensions. The first few years of teaching bring considerable changes in role conception, and further changes occur with prolonged experience. Deputy heads in junior schools and nursery nurses have quite distinct role conceptions; those of all other teachers, including those holding senior posts in secondary schools, are similar. The perception of school climate influences the role conception of primary teachers directly, but it does not influence that of secondary teachers. The greatest variation in role conception is related to scores on the radical scale of Oliver and Butcher. Primary school teachers experience little constraint, but that reported by secondary school teachers is considerable, especially that coming from the head. Despite difficulties caused by the wide division between primary and secondary education, teachers have an accurate perception of the roles their colleagues adopt. A few misunderstandings may be due to a feeling of idealism amongst nursery and infant teachers. There is evidence in their conception of role that would enhance the professional standing of teachers, but this is not in a form which is likely to be recognised by the public.

Reduction of noise levels in vaccum cleaners

The work described in this thesis is directed towards the reduction of noise levels in the Hoover Turbopower upright vacuum cleaner. The experimental work embodies a study of such factors as the application of noise source identification techniques, investigation of the noise generating principles for each major source and evaluation of the noise reducing treatments. It was found that the design of the vacuum cleaner had not been optimised from the standpoint of noise emission. Important factors such as noise `windows', isolation of vibration at the source, panel rattle, resonances and critical speeds had not been considered. Therefore, a number of experimentally validated treatments are proposed. Their noise reduction benefit together with material and tooling costs are presented. The solutions to the noise problems were evaluated on a standard Turbopower and the sound power level of the cleaner was reduced from 87.5 dB(A) to 80.4 db(A) at a cost of 93.6 pence per cleaner.The designers' lack of experience in noise reduction was identified as one of the factors for the low priority given to noise during design of the cleaner. Consequently, the fundamentals of acoustics, principles of noise prediction and absorption and guidelines for good acoustical design were collated into a Handbook and circulated at Hoover plc.Mechanical variations during production of the motor and the cleaner were found to be important. These caused a vast spread in the noise levels of the cleaners. Subsequently, the manufacturing processes were briefly studied to identify their source and recommendations for improvement are made.Noise of a product is quality related and a high level of noise is considered to be a bad feature. This project suggested that the noise level be used constructively both as a test on the production line to identify cleaners above a certain noise level and also to promote the product by `designing' the characteristics of the sound so that the appliance is pleasant to the user. This project showed that good noise control principles should be implemented early in the design stage.As yet there are no mandatory noise limits or noise-labelling requirements for household appliances. However, the literature suggests that noise-labelling is likely in the near future and the requirement will be to display the A-weighted sound power level. However, the `noys' scale of perceived noisiness was found more appropriate to the rating of appliance noise both as it is linear and therefore, a sound level that seems twice as loud is twice the value in noys and also takes into consideration the presence of pure tones, which even in the absence of a high noise level can lead to annoyance.

Strategic development of transport systems: a study of the physical constraints on planning processes

Investment in transport infrastructure can be highly sensitive to uncertainty. The scale and lead time of strategic transport programmes are such that they require continuing policy support and accurate forecasting. Delay, cost escalation and abandonment of projects often result if these conditions are not present. In Part One the physical characteristics of infrastructure are identified as a major constraint on planning processes. The extent to which strategies and techniques acknowledge these constraints is examined. A simple simulation model is developed to evaluate the effects on system development of variations in the scale and lead time of investments. In Part Two, two case studies of strategic infrastructure investment are analysed. The absence of a policy consensus for airport location was an important factor in the delayed resolution of the Third London Airport issue. In London itself, the traffic and environmental effects of major highway investment ultimately resulted in the abandonment of plans to construct urban motorways. In both cases, the infrastructure implications of alternative strategies are reviewed with reference to the problems of uncertainty. In conclusion, the scale of infrastructure investment is considered the most important of the constraints on the processes of transport planning. Adequate appraisal of such constraints may best be achieved by evaluation more closely aligned to policy objectives.

The need for continued monitoring of antibiotic resistance patterns in clinical isolates of Staphylococcus aureus from London and Malta

Antibiotic resistance is an increasing problem in isolates of Staphylococcus aureus (S. aureus) worldwide. In 2001 The National Health Service in the UK introduced a mandatory bacteraemia surveillance scheme for the reporting of S. aureus and methicillin-resistant S. aureus (MRSA). This surveillance initiative reports on the percentage of isolates that are methicillin resistant. However, resistance to other antibiotics is not currently reported and therefore the scale of emerging resistance is currently unclear in the UK. In this study, multiple antibiotic resistance (MAR) profiles against fourteen antimicrobial drugs were investigated for 705 isolates of S. aureus collected from two European study sites in the UK (London) and Malta.

The economic impact of high growth start-ups:understanding the challenge for policy in the UK

Objectives The creation of more high-growth firms continues to be a key component of enterprise policy throughout the countries of the OECD. In the UK the developing enterprise policy framework highlights the importance of supporting businesses with growth potential. The difficulty, of course, is the ability of those delivering business support policies to accurately identify those businesses, especially at start-up, which will benefit from interventions and experiences an enhanced growth performance. This paper has a core objective of presenting new data on the number of high growth firms in the UK and providing an assessment of their economic significance. Approach This paper uses a specially created longitudinal firm-level database based on the Inter-Departmental Business Register (IDBR) held by the Office of National Statistics (ONS) for all private sector businesses in the UK for the period 1997-2008 to investigate the share of high-growth firms (including a sub-set of start-up more commonly referred to as gazelles) in successive cohorts of start-ups. We apply OECD definitions of high growth and gazelles to this database and are able to quantify for the first time their number (disaggregated by sector, region, size) and importance (employment and sales). Prior Work However, what is lacking at the core of this policy focus is any comprehensive statistical analysis of the scale and nature of high-growth firms in cohorts of new and established businesses. The evidence base in response to the question “Why do high-growth firms matter?” is surprisingly weak. Important work in this area has been initiated by Bartelsman et al., (2003),Hoffman and Jünge (2006) and Henreksen and Johansson (2009) but to date work in the UK has been limited (BERR, 2008b). Results We report that there are ~11,500 high growth firms in the UK in both 2005 and 2008. The share of high growth start-ups in the UK in 2005 (6.3%) was, contrary to the widely held perception in policy circles, higher than in the United States (5.2%). Of particular interest in the analysis are the growth trajectories (pattern of growth) of these firms as well as the extent to which they are restricted to technology-based or knowledge-based sectors. Implications and Value Using hitherto unused population data for the first time we have answered a fundamental research and policy question on the number and scale of high growth firms in the UK. We draw the conclusion that this ‘rare’ event does not readily lend itself to policy intervention on the grounds that the significant effort needed to identify such businesses ex ante would appear unjustified even if it was possible.

Attitudes towards emotional expression mediate the relationship between childhood invalidation and adult eating concern

Previous research has suggested that invalidating childhood environments are positively related to the symptoms of eating disorders. However, it is unclear how childhood environments might impact upon the development of eating disorder symptoms. This study examined the relationship between parental invalidation and eating disorder-related attitudes in a nonclinical sample and tested the mediating effect of attitudes towards emotional expression. Two hundred women, with a mean age of 21 years, completed measures of invalidating childhood environments, attitudes towards emotional expression, and eating pathology. Eating concerns were positively associated with recollections of an invalidating parental environment. The belief that the expression of emotions is a sign of weakness fully mediated the relationship between childhood maternal invalidation and adult eating concern. Following replication and extension to a clinical sample, these results suggest that targeting the individual's attitude towards emotional expression might reduce eating attitudes among women who have experienced an invalidating childhood environment. © 2012 John Wiley & Sons, Ltd and Eating Disorders Association.

The social and entrepreneurial orientation of small businesses:mutually exclusive or reinforcing? (INTERACTIVE PAPER)

Principal Topic - As argued by Acs and Phillips (2002) it is not only “the creation of wealth (entrepreneurship)” but also “the reconstitution of wealth (philanthropy)”, which has been essential for the inherent dynamism of the market economy (Ibid., p.201). However, we understand little about the entrepreneurship – philanthropy link in institutional contexts that differ from that of leading developed market economies. Accordingly our research agenda is to investigate the entrepreneurship-philanthropy nexus in a very different context of Lithuania, a country which shed a command economy system twenty years ago. In particular, we are interested to see if the cluster of attitudes and strategies of firms conducive to entrepreneurship, i.e. their entrepreneurial orientation (Covin & Slevin, 1989), is consistent or contradictory with philanthropy? In other words, is philanthropy strongly associated with some core components of entrepreneurship, or is it an entrepreneurial anomaly, relying on a minority of economic actors that provide important links with wider, non-economic communities. Method - The study draws on 270 randomly sampled, phone interviews with owners and ownermanagers of small and medium-sized enterprises (SMEs), i.e. firms with less than 250 employees. Interviews were conducted in Lithuania during January- March, 2008. Our results are based on confirmatory factor analysis combined with regression analysis. Results and Implications - Despite the legacy of informal institutions that is conducive neither to entrepreneurship nor to civic society, we found that by now, (i) the companies that score highest on entrepreneurial orientation construct, (ii) that perform best and those (iii) that have foreign owners are also most likely to declare their commitment to philanthropy. Our findings that most entrepreneurial firms are also involved in philanthropy are consistent with the perspective on the pattern of development in an entrepreneurial economy as outlined by Acs and Phillips (2002).

Accounting for derivatives:an evaluation of reporting practice by UK banks

In 1998 the Accounting Standards Board (ASB) published FRS 13, ‘Derivatives and other Financial Instruments: Disclosures’. This laid down the requirements for disclosures of an entity’s policies, objectives and strategies in using financial instruments, their impact on its risk, performance and financial condition, and details of how risks are managed. FRS 13 became effective in March 1999, and this paper uses the 1999 annual reports of UK banks to evaluate the usefulness of disclosures from a user’s perspective. Usefulness is measured in terms of the criteria of materiality, relevance, reliability, comparability and understandability as defined in the ASB’s Statement of Principles (ASB, 1999). Our findings suggest that the narrative disclosures are generic in nature, the numerical data incomplete and not always comparable, and that it is difficult for the user to combine both narrative and numerical information in order to assess the banks’ risk profile. Our overall conclusion is therefore that current UK financial reporting practices are of limited help to users wishing to assess the scale of an institution’s financial risk exposure.

Market risk reporting by the world's top banks:evidence on the diversity of reporting practice and the implications for international accounting harmonisation

The increasing adoption of international accounting standards and global convergence of accounting regulations is frequently heralded as serving to reduce diversity in financial reporting practice. In a process said to be driven in large part by the interests of international business and global financial markets, one might expect the greatest degree of convergence to be found amongst the world’s largest multinational financial corporations. This paper challenges such claims and presumptions. Its content analysis of longitudinal data for the period 2000-2006 reveals substantial, on going diversity in the market risk disclosure practices, both numerical and narrative, of the world’s top-25 banks. The significance of such findings is reinforced by the sheer scale of the banking sector’s risk exposures that have been subsequently revealed in the current global financial crisis. The variations in disclosure practices documented in the paper apply both across and within national boundaries, leading to a firm conclusion that, at least in terms of market risk reporting, progress towards international harmonisation remains rather more apparent than real.

A computational model of intonation for yorùbá text-to-speech synthesis:design and analysis

In this paper we present the design and analysis of an intonation model for text-to-speech (TTS) synthesis applications using a combination of Relational Tree (RT) and Fuzzy Logic (FL) technologies. The model is demonstrated using the Standard Yorùbá (SY) language. In the proposed intonation model, phonological information extracted from text is converted into an RT. RT is a sophisticated data structure that represents the peaks and valleys as well as the spatial structure of a waveform symbolically in the form of trees. An initial approximation to the RT, called Skeletal Tree (ST), is first generated algorithmically. The exact numerical values of the peaks and valleys on the ST is then computed using FL. Quantitative analysis of the result gives RMSE of 0.56 and 0.71 for peak and valley respectively. Mean Opinion Scores (MOS) of 9.5 and 6.8, on a scale of 1 - -10, was obtained for intelligibility and naturalness respectively.