Different routes to pure food allergens: extraction, recombinant techniques and total chemical synthesis for obtaining the plant-food pan-allergen LTP

La richiesta di allergeni puri è in continuo aumento per scopi diagnostici, come standard per metodi di rilevamento e di quantificazione, per l'immunoterapia e per lo studio a livello molecolare dei meccanismi delle reazioni allergiche, al fine di facilitare lo sviluppo di possibili cure. In questa tesi di dottorato sono descritte diverse strategie per l’ottenimento di forme pure di non-specific Lipid Transfer Proteins (nsLTPs), le quali sono state riconosciute essere rilevanti allergeni alimentari in molti frutti e verdure comunemente consumati e sono state definite come modello di veri allergeni alimentari. Una LTP potenzialmente allergenica, non nota in precedenza, è stata isolata dalle mandorle, mentre una LTP dall’allergenicità nota contenuta nelle noci è stata prodotta mediante tecniche di DNA ricombinante. Oltre a questi approcci classici, metodi per la sintesi chimica totale di proteine sono stati applicati per la prima volta alla produzione di un allergene, utilizzando Pru p 3, la LTP prototipica e principale allergene della pesca nell'area mediterranea, come modello. La sintesi chimica totale di proteinepermette di controllarne completamente la sequenza e di studiare la loro funzione a livello atomico. La sua applicazione alla produzione di allergeni costituisce perciò un importante passo avanti nel campo della ricerca sulle allergie alimentari. La proteina Pru p 3 è stata prodotta nella sua intera lunghezza e sono necessari solo due passaggi finali di deprotezione per ottenere il target nella sua forma nativa. Le condizioni sperimentali per tali deprotezioni sono state messe a punto durante la produzione dei peptidi sPru p 3 (1-37) e sPru p 3 (38-91), componenti insieme l'intera proteina. Tecniche avanzate di spettrometria di massa sono state usate per caratterizzare tutti i composti ottenuti, mentre la loro allergenicità è stata studiata attraverso test immunologici o approcci in silico.

Advances in (poly)phenol research: novel sources, bioavailability, bioaccumulation and bioactivity

In the last decades, increasing scientific evidence has correlated the regular consumption of (poly)phenol-rich foods to a potential reduction of chronic disease incidence and mortality. However, epidemiological evidence on the role of (poly)phenol intake against the risk of some chronic diseases is promising, but not conclusive. In this framework a proper approach to (poly)phenol research is requested, using a step by step strategy. The plant kingdom produces an overwhelming array of structurally diverse secondary metabolites, among which flavonoids and related phenolic and (poly)phenolic compounds constitute one of the most numerous and widely distributed group of natural products. To date, more than 8000 structures have been classified as members of the phytochemical class of (poly)phenol, and among them over 4000 flavonoids have been identified. For this reason, a detailed food (poly)phenolic characterization is essential to identify the compounds that will likely enter the human body upon consumption, to predict the metabolites that will be generated and to unravel the potential effects of phenolic rich food sources on human health. In the first part of this work the attention was focused on the phenolic characterization of fruit and vegetable supplements, considering the increasing attention recently addressed to the so called "nutraceuticals", and on the main coffee industry by-product, namely coffee silverskin. The interest oriented toward (poly)phenols is then extended to their metabolism within the human body, paramount in the framework of their putative health promoting effects. Like all nutrients and non-nutrients, once introduced through the diet, (poly)phenols are subjected to an intense metabolism, able to convert the native compounds into similar conjugated, as well as smaller and deeply modified molecules, which in turn could be further conjugated. Although great strides have been made in the last decades, some steps of the (poly)phenol metabolism remain unclear and are interesting points of research. In the second part of this work the research was focused on a specific bran fraction, namely aleurone, added in feed pellets and in bread to investigate the absorption, metabolism and bioavailability of its phenolic compounds in animal and humans, with a preliminary in vitro step to determine their potential bioaccesibility. This part outlines the best approaches to assess the bioavailability of specific phenolics in several experimental models. The physiological mechanisms explaining the epidemiological and observational data on phenolics and health, are still far from being unraveled or understood in full. Many published results on phenolic actions at cell levels are biased by the fact that aglycones or native compounds have been used, not considering the previously mentioned chemical and biological transformations. In the last part of this thesis work, a new approach in (poly)phenol bioactivity investigation is proposed, consisting of a medium-long term treatment of animals with a (poly)phenol source, in this specific case resveratrol, the detection of its metabolites to determine their possible specific tissue accumulation, and the evaluation of specific parameters and/or mechanism of action at target tissue level. To conclude, this PhD work has contributed to advancing the field, as novel sources of (poly)phenols have been described, the bioavailability of (poly)phenols contained in a novel specific bran fraction used as ingredient has been evaluated in animal and in humans, and, finally, the tissue accumulation of specific (poly)phenol metabolites and the evaluation of specific parameters and/or mechanism of action has been carried out. For these reasons, this PhD work should be considered an example of adequate approach to the investigation of (poly)phenols and of their bioactivity, unavoidable in the process of unequivocally defining their effects on human health.

Strategies for performance improvement in the Chinese chemical industry:evidence from case study investigations

Purpose – This paper sets out to explore the proposition that building competences is more effective than privatisation and restructuring to improve performance in the Chinese chemical industry. Design/methodology/approach – Case study research has been undertaken in the Chinese chemical industry. The two case companies provide representative data on the factors under investigation. The case investigations that are described were complemented by a survey, the results of which have been reported elsewhere. Findings – Results obtained from the research show that privatisation of Chinese state-owned enterprises is not always an effective strategy to improve performance. In the case study companies, the development of core competences was more effective in enhancing performance. Research limitations/implications – The research results are limited by the scope of the study, which was carried out in the Chinese chemical industry. They are also based on in-depth case investigations in only two companies, but are supported by a large-scale survey reported elsewhere. The results have implications for academic researchers interested in China's privatisation programme. Practical implications – The research has practical implications for companies outside China that are considering collaborative operations with Chinese companies or investing in joint ventures. It also has implications for suppliers or customers of Chinese companies. Originality/value – The paper is based on original case study investigations carried out in Chinese enterprises and is supported by a survey of representative companies in China's chemical sector. Value is derived from understanding the basis of improved performance in the companies studied.

Workers' understanding of chemical risks: electroplating case study

BACKGROUND: There is limited research concerning how small companies in particular, respond to health and safety messages. AIMS: To understand individuals' knowledge and beliefs about chemical risks and to compare these with those of experts. METHODS: The use of chromic acid in particular, and also other chemicals associated with chrome plating were studied. All chromium plating firms were based in the West Midlands. The methodology involved initial face to face interviews (n = 21) with chromium platers, structured questionnaires (n = 84) to test the prevalence of beliefs identified in the interviews, an expert questionnaire, and a workshop to discuss findings. The responses of platers were compared with those of occupational health and safety experts. RESULTS: Although chromium platers appeared to understand the short term adverse effects of the chemicals to which they are exposed, their understanding of long term, or chronic effects appeared to be incomplete. They had good knowledge of acute effects based primarily on experience. Platers were aware of the hazardous nature of the chemicals with which they work, but did not draw distinction between the terms "hazards" and "risks". They had difficulties articulating the effects of the chemicals and how exposure might occur; although it is inappropriate to equate this with lack of knowledge. A significant minority of platers displayed deficiencies in understanding key technical terms used in Safety Data Sheets. CONCLUSIONS: This study provides a method which can be used to gain some understanding of workers' knowledge and beliefs about risks that they are exposed to in the workplace. The study also identifies gaps between the platers' knowledge and beliefs and those of experts. New risk information needs to be designed which addresses the information needs of platers using language that they understand.

Nitrogen cycle of effluent-irrigated energy crop plantations:from wastewater treatment to thermo-chemical conversion processes

This paper reviews nitrogen (N) cycle of effluent-irrigated energy crop plantations, starting from wastewater treatment to thermo-chemical conversion processes. In wastewater, N compounds contribute to eutrophication and toxicity in water cycle. Removal of N via vegetative filters and specifically in short-rotation energy plantations, is a relatively new approach to managing nitrogenous effluents. Though combustion of energy crops is in principle carbon neutral, in practice, N content may contribute to NOx emissions with significant global warming potential. Intermediate pyrolysis produces advanced fuels while reducing such emissions. By operating at intermediate temperature (500°C), it retains most N in char as pyrrolic-N, pyridinic-N, quaternary-N and amines. In addition, biochar provides long-term sequestration of carbon in soils.

The chemical modification of polymer blends by reactive processing

The primary objective of this research was to examine the concepts of the chemical modification of polymer blends by reactive processing using interlinking agents (multi-functional, activated vinyl compounds; trimethylolpropane triacrylates {TRIS} and divinylbenzene {DVD}) to target in-situ interpolymer formation between immiscible polymers in PS/EPDM blends via peroxide-initiated free radical reactions during melt mixing. From a comprehensive survey of previous studies of compatibility enhancement in polystyrene blends, it was recognised that reactive processing offers opportunities for technological success that have not yet been fully realised; learning from this study is expected to assist in the development and application of this potential. In an experimental-scale operation for the simultaneous melt blending and reactive processing of both polymers, involving manual injection of precise reactive agent/free radical initiator mixtures directly into molten polymer within an internal mixer, torque changes were distinct, quantifiable and rationalised by ongoing physical and chemical effects. EPDM content of PS/EPDM blends was the prime determinant of torque increases on addition of TRIS, itself liable to self-polymerisation at high additions, with little indication of PS reaction in initial reactively processed blends with TRIS, though blend compatibility, from visual assessment of morphology by SEM, was nevertheless improved. Suitable operating windows were defined for the optimisation of reactive blending, for use once routes to encourage PS reaction could be identified. The effectiveness of PS modification by reactive processing with interlinking agents was increased by the selection of process conditions to target specific reaction routes, assessed by spectroscopy (FT-IR and NMR) and thermal analysis (DSC) coupled dichloromethane extraction and fractionation of PS. Initiator concentration was crucial in balancing desired PS modification and interlinking agent self-polymerisation, most particularly with TRIS. Pre-addition of initiator to PS was beneficial in the enhancement of TRIS binding to PS and minimisation of modifier polymerisation; believed to arise from direct formation of polystyryl radicals for addition to active unsaturation in TRIS. DVB was found to be a "compatible" modifier for PS, but its efficacy was not quantified. Application of routes for PS reaction in PS/EPDM blends was successful for in-situ formation of interpolymer (shown by sequential solvent extraction combined with FT-IR and DSC analysis); the predominant outcome depending on the degree of reaction of each component, with optimum "between-phase" interpolymer formed under conditions selected for equalisation of differing component reactivities and avoidance of competitive processes. This was achieved for combined addition of TRIS+DVB at optimum initiator concentrations with initiator pre-addition to PS. Improvements in blend compatibility (by tensiles, SEM and thermal analysis) were shown in all cases with significant interpolymer formation, though physical benefits were not; morphology and other reactive effects were also important factors. Interpolymer from specific "between-phase" reaction of blend components and interlinking agent was vital for the realisation of positive performance on compatibilisation by the chemical modification of polymer blends by reactive processing.

Dynamic simulation of chemical processes

This thesis describes the design and implementation of an interactive dynamic simulator called DASPRII. The starting point of this research has been an existing dynamic simulation package, DASP. DASPII is written in standard FORTRAN 77 and is implemented on universally available IBM-PC or compatible machines. It provides a means for the analysis and design of chemical processes. Industrial interest in dynamic simulation has increased due to the recent increase in concern over plant operability, resiliency and safety. DASPII is an equation oriented simulation package which allows solution of dynamic and steady state equations. The steady state can be used to initialise the dynamic simulation. A robust non linear algebraic equation solver has been implemented for steady state solution. This has increased the general robustness of DASPII, compared to DASP. A graphical front end is used to generate the process flowsheet topology from a user constructed diagram of the process. A conversational interface is used to interrogate the user with the aid of a database, to complete the topological information. An original modelling strategy implemented in DASPII provides a simple mechanism for parameter switching which creates a more flexible simulation environment. The problem description generated is by a further conversational procedure using a data-base. The model format used allows the same model equations to be used for dynamic and steady state solution. All the useful features of DASPI are retained in DASPII. The program has been demonstrated and verified using a number of example problems, Significant improvements using the new NLAE solver have been shown. Topics requiring further research are described. The benefits of variable switching in models has been demonstrated with a literature problem.

Chemical studies of plasma etchants used in integrated circuit manufacture

Plasma or "dry" etching is an essential process for the production of modern microelectronic circuits. However, despite intensive research, many aspects of the etch process are not fully understood. The results of studies of the plasma etching of Si and Si02 in fluorine-containing discharges, and the complementary technique of plasma polymerisation are presented in this thesis. Optical emission spectroscopy with argon actinometry was used as the principle plasma diagnostic. Statistical experimental design was used to model and compare Si and Si02 etch rates in CF4 and SF6 discharges as a function of flow, pressure and power. Etch mechanisms m both systems, including the potential reduction of Si etch rates in CF4 due to fluorocarbon polymer formation, are discussed. Si etch rates in CF4 /SF6 mixtures were successfully accounted for by the models produced. Si etch rates in CF4/C2F6 and CHF3 as a function of the addition of oxygen-containing additives (02, N20 and CO2) are shown to be consistent with a simple competition between F, 0 and CFx species for Si surface sites. For the range of conditions studied, Si02 etch rates were not dependent on F-atom concentration, but the presence of fluorine was essential in order to achieve significant etch rates. The influence of a wide range of electrode materials on the etch rate of Si and Si02 in CF4 and CF4 /02 plasmas was studied. It was found that the Si etch rate in a CF4 plasma was considerably enhanced, relative to an anodised aluminium electrode, in the presence of soda glass or sodium or potassium "doped" quartz. The effect was even more pronounced in a CF4 /02 discharge. In the latter system lead and copper electrodes also enhanced the Si etch rate. These results could not be accounted for by a corresponding rise in atomic fluorine concentration. Three possible etch enhancement mechanisms are discussed. Fluorocarbon polymer deposition was studied, both because of its relevance to etch mechanisms and its intrinsic interest, as a function of fluorocarbon source gas (CF4, C2F6, C3F8 and CHF3), process time, RF power and percentage hydrogen addition. Gas phase concentrations of F, H and CF2 were measured by optical emission spectroscopy, and the resultant polymer structure determined by X-ray photoelectron spectroscopy and infrared spectroscopy. Thermal and electrical properties were measured also. Hydrogen additions are shown to have a dominant role in determining deposition rate and polymer composition. A qualitative description of the polymer growth mechanism is presented which accounts for both changes in growth rate and structure, and leads to an empirical deposition rate model.

Factors affecting human reliability in the chemical process industry

This research was concerned with identifying factors which may influence human reliability within chemical process plants - these factors are referred to as Performance Shaping Factors (PSFs). Following a period of familiarization within the industry, a number of case studies were undertaken covering a range of basic influencing factors. Plant records and site `lost time incident reports' were also used as supporting evidence for identifying and classifying PSFs. In parallel to the investigative research, the available literature appertaining to human reliability assessment and PSFs was considered in relation to the chemical process plan environment. As a direct result of this work, a PSF classification structure has been produced with an accompanying detailed listing. Phase two of the research considered the identification of important individual PSFs for specific situations. Based on the experience and data gained during phase one, it emerged that certain generic features of a task influenced PSF relevance. This led to the establishment of a finite set of generic task groups and response types. Similarly, certain PSFs influence some human errors more than others. The result was a set of error type key words, plus the identification and classification of error causes with their underlying error mechanisms. By linking all these aspects together, a comprehensive methodology has been forwarded as the basis of a computerized aid for system designers. To recapitulate, the major results of this research have been: One, the development of a comprehensive PSF listing specifically for the chemical process industries with a classification structure that facilitates future updates; and two, a model of identifying relevant SPFs and their order of priority. Future requirements are the evaluation of the PSF listing and the identification method. The latter must be considered both in terms of `useability' and its success as a design enhancer, in terms of an observable reduction in important human errors.

Chemical synthesis of DNA and RNA containing thio-substituted bases and their post-synthetic modifications

Modified oligonucleotides containing sulphur group have been useful tools for studies of carcinogenesis, protein or nucleic acid structures and functions, protein-nucleic acid interactions, and for antisense modulation of gene expression. One successful example has been the synthesis and study of oligodeoxynucleotides containing 6-thio-2'-deoxyguanine. 6-Thio-2-deoxyguanosine was first discovered as metabolic compound of 6- mercaptopurine (6-MP). Later, it was applied as drug to cure leukaemia. During the research of its toxicity, a method was developed to use the sulphur group as a versatile position for post-synthetic modification. The advantage of application of post-synthetic modification lies in its convenience. Synthesis of oligomers with normal sequences has become routine work in most laboratories. However, design and synthesis of a proper phosphoramidite monomer for a new modified nucleoside are always difficult tasks even for a skilful chemist. Thus an alternative method (post-synthetic method) has been invented to overcome the difficulties. This was achieved by incorporation of versatile nucleotides into oligomers which contain a leaving group, that is sufficiently stable to withstand the conditions of synthesis but can be substituted by nucleophiles after synthesis, to produce, a series of oligomers each containing a different modified base. In the current project, a phosphoramidite monomer with 6-thioguanine has been successfully synthesised and incorporated into RNA. A deprotection procedure, which is specific for RNA was designed for oligomers containing 6-thioguanosine. The results were validated by various methods (UV, HPLC, enzymatic digestion). Pioneer work in utilization of the versatile sulphur group for post-synthetic modification was also tested. Post-synthetic modification was also carried out on DNA with 6- deoxythioguanosine. Electrophilic reagents with various functional groups (alphatic, aromatic, fluorescent) and bi-functional groups have been attached with the oligomers.

The development of chemical exposure limits for the workplace

The thesis examines and explains the development of occupational exposure limits (OELs) as a means of preventing work related disease and ill health. The research focuses on the USA and UK and sets the work within a certain historical and social context. A subsidiary aim of the thesis is to identify any short comings in OELs and the methods by which they are set and suggest alternatives. The research framework uses Thomas Kuhn's idea of science progressing by means of paradigms which he describes at one point, `lq ... universally recognised scientific achievements that for a time provide model problems and solutions to a community of practitioners. KUHN (1970). Once learned individuals in the community, `lq ... are committed to the same rules and standards for scientific practice. Ibid. Kuhn's ideas are adapted by combining them with a view of industrial hygiene as an applied science-based profession having many of the qualities of non-scientific professions. The great advantage of this approach to OELs is that it keeps the analysis grounded in the behaviour and priorities of the groups which have forged, propounded, used, benefited from, and defended, them. The development and use of OELs on a larger scale is shown to be connected to the growth of a new profession in the USA; industrial hygiene, with the assistance of another new profession; industrial toxicology. The origins of these professions, particularly industrial hygiene, are traced. By examining the growth of the professions and the writings of key individuals it is possible to show how technical, economic and social factors became embedded in the OEL paradigm which industrial hygienists and toxicologists forged. The origin, mission and needs of these professions and their clients made such influences almost inevitable. The use of the OEL paradigm in practice is examined by an analysis of the process of the American Conference of Governmental Industrial Hygienists, Threshold Limit Value (ACGIH, TLV) Committee via the Minutes from 1962-1984. A similar approach is taken with the development of OELs in the UK. Although the form and definition of TLVs has encouraged the belief that they are health-based OELs the conclusion is that they, and most other OELs, are, and always have been, reasonably practicable limits: the degree of risk posed by a substance is weighed against the feasibility and cost of controlling exposure to that substance. The confusion over the status of TLVs and other OELs is seen to be a confusion at the heart of the OEL paradigm and the historical perspective explains why this should be. The paradigm has prevented the creation of truly health-based and, conversely, truly reasonably practicable OELs. In the final part of the thesis the analysis of the development of OELs is set in a contemporary context and a proposal for a two-stage, two-committee procedure for producing sets of OELs is put forward. This approach is set within an alternative OEL paradigm. The advantages, benefits and likely obstacles to these proposals are discussed.

Chemical constituents associated with sewage settling velocity profiles

A methodology has been developed to measure the chemical constituents associated with the settling velocity fractions that comprise a wastewater settling velocity profile (SVP). 31 wastewater samples were collected from fifteen different catchments in England and Wales. For each catchment, settling velocity and associated chemical constituent profiles were determined. The results are mainly for Suspended Solids (SS), Chemical Oxygen Demand (COD), Phosphorus (P) and Total Kjeadahl Nitrogen (TKN), however these are supplemented by the results from 5 events for a suite of heavy metals. COD, P, Hg, Mn and Pb were found to be predominantly associated with the solid phase and TKN, Al, Cu and Fe with the liquor phase of the wastewater samples. The results in the thesis are expressed as mass of pollutant (g) per mass total SS (kg). COD and P were found to be mainly associated with the sinkers and had a particular affinity for solids with settling velocities in the range 0.9-9.03mm/sec. TKN was mainly associated with the soluble phase, however of the solids that did settle, a peak was found to be associated within the settling velocity range 0.9-9.03mm/sec. The relationships identified for COD and P were generally found to be unaffected by flow conditions and catchment characteristics. However, TKN was found to be affected by catchment type. Data on the distribution of heavy metals was limited, and no specific relationships with solids were identified. 16 mean pollutant profiles are presented in the thesis. Presentation of the data in this form will enable the results to be of use in the design of sedimentation devices to predict removal efficiencies for solids and associated pollutants. The findings of the research may also be applied to modelling tools to provide further characteristics on the solids that are modelled than is currently used. This would enhance the overall performance of tools used in integrated catchment modelling.

Characterisation of a cavity transfer mixer as a chemical reactor

This research project examined the feasibility of using a cavity transfer mixer (CTM) as a continuous reactor to perform reactions between either solid or liquid reagents and polymer melt; reactions which have previously been typically carried out in batch reactor systems. Equipment has been developed to allow uniform and reproducible introduction of reagents into the polymer melt. Reactions have also been performed using batch processing equipment to enable comparison with the performance of the CTM. It was concluded that: a) there are certain reactions which cannot be carried out in a CTM, but which can be performed in a batch system such as a mill or a sigma blade mixer. This was found to be the case for some neutralisation reactions where the product was quasi crosslinked. b) the reactions that can be carried out in a CTM are performed more efficiently in a CTM than on a batch process. For example, when monomers were to be grafted onto polymers, this was more safely and efficiently performed in the CTM than in a mill or a sigma blade mixer. Residence time distributions (RTDs) for three CTMs were studied in order to gain an insight into the effect of CTM geometry on RTD, polymer melt flow pattern and reactor performance. A mathematical model has been developed to predict the influence of process parameters on RTD and the results compared with experimentally observed trends. The comparison was good. A programme of research has been drawn up to form the basis of an industrially based sponsored development project of the CTM reactor. This work programme was successfully marketed to companies with commercial interest in modified rubber and plastics as an integral part of the research programme of this thesis and the sponsored research programme has paralleled the work reported here.

Insect pest control research : the analysis of historical trends with special reference to scientometric analysis

The thesis investigates the value of quantitative analyses for historical studies of science through an examination of research trends in insect pest control, or economic entomology. Reviews are made of quantitative studies of science, and historical studies of pest control. The methodological strengths and weaknesses of bibliometric techniques are examined in a special chapter; techniques examined include productivity studies such as paper counts, and relational techniques such as co-citation and co-word analysis. Insect pest control is described. This includes a discussion of the socio-economic basis of the concept of `pest'; a series of classifications of pest control techniques are provided and analysed with respect to their utility for scientometric studies. The chemical and biological approaches to control are discussed as scientific and technological paradigms. Three case studies of research trends in economic entomology are provided. First a scientometric analysis of samples of chemical control and biological control papers; providing quantitative data on institutional, financial, national, and journal structures associated with pest control research fields. Second, a content analysis of a core journal, the Journal of Economic Entomology, over a period of 1910-1985; this identifies the main research innovations and trends, in particular the changing balance between chemical and biological control. Third, an analysis of historical research trends in insecticide research; this shows the rise, maturity and decline of research of many groups of compounds. These are supplemented by a collection of seven papers on scientometric studies of pest control and quantitative techniques for analysing science.