Design and synthesis of potential antimicrobial agents

Chorismate mutase is one of the essential enzymes in the shikimate pathway and is key to the survival of the organism Mycobacterium tuberculosis. The x-ray crystal structure of this enzyme from Mycobacterium tuberculosis was manipulated to prepare an initial set of in silico protein models of the active site. Known inhibitors of the enzyme were docked into the active site using the flexible ligand / flexible active site side chains approach implemented in CAChe Worksystem (Fujitsu Ltd). The resulting complexes were refined by molecular dynamics studies in explicit water using Amber 9. This yielded a further set of protein models that were used for additional rounds of ligand docking. A binding hypothesis was established for the enzyme and this was used to screen a database of commercially available drug-like compounds. From these results new potential ligands were designed that fitted appropriately into the active site and matched the functional groups and binding motifs founds therein. Some of these compounds and close analogues were then synthesized and submitted for biological evaluation. As a separate part of this thesis, analogues of very active anti-tuberculosis pyridylcarboxamidrazone were also prepared. This was carried out by the addition and the deletion of the substitutions from the lead compound thereby preparing heteroaryl carboxamidrazone derivatives and related compounds. All these compounds were initially evaluated for biological activity against various gram positive organisms and then sent to the TAACF (USA) for screening against Mycobacterium tuberculosis. Some of the new compounds proved to be at least as potent as the original lead compound but less toxic.

Design and synthesis of potential antimicrobial agents

Tuberculosis is one of the most devastating diseases in the world primarily due to several decades of neglect and an emergence of multidrug-resitance strains (MDR) of M. tuberculosis together with the increased incidence of disseminated infections produced by other mycobacterium in AIDS patients. This has prompted the search for new antimycobacterial drugs. A series of pyridine-2-, pyridine-3-, pyridine-4-, pyrazine and quinoline-2-carboxamidrazone derivatives and new classes of carboxamidrazone were prepared in an automated fashion and by traditional synthesis. Over nine hundred synthesized compounds were screened for their anti mycobacterial activity against M. fortutium (NGTG 10394) as a surrogate for M. tuberculosis. The new classes of amidrazones were also screened against tuberculosis H37 Rv and antimicrobial activities against various bacteria. Fifteen tested compounds were found to provide 90-100% inhibition of mycobacterium growth of M. tuberculosis H37 Rv in the primary screen at 6.25 μg mL-1. The most active compound in the carboxamidrazone amide series had an MIG value of 0.1-2 μg mL-1 against M. fortutium. The enzyme dihydrofolate reductase (DHFR) has been a drug-design target for decades. Blocking of the enzymatic activity of DHFR is a key element in the treatment of many diseases, including cancer, bacterial and protozoal infection. The x-ray structure of DHFR from M. tuberculosis and human DHFR were found to have differences in substrate binding site. The presence of glycerol molecule in the Xray structure from M. tuberculosis DHFR provided opportunity to design new antifolates. The new antifolates described herein were designed to retain the pharmcophore of pyrimethamine (2,4- diamino-5(4-chlorophenyl)-6-ethylpyrimidine), but encompassing a range of polar groups that might interact with the M. tuberculosis DHFR glycerol binding pockets. Finally, the research described in this thesis contributes to the preparation of molecularly imprinted polymers for the recognition of 2,4-diaminopyrimidine for the binding the target. The formation of hydrogen bonding between the model functional monomer 5-(4-tert-butyl-benzylidene)-pyrimidine-2,4,6-trione and 2,4-diaminopyrimidine in the pre-polymerisation stage was verified by 1H-NMR studies. Having proven that 2,4-diaminopyrimidine interacts strongly with the model 5-(4-tert-butylbenzylidene)- pyrimidine-2,4,6-trione, 2,4-diaminopyrimidine-imprinted polymers were prepared using a novel cyclobarbital derived functional monomer, acrylic acid 4-(2,4,6-trioxo-tetrahydro-pyrimidin-5- ylidenemethyl)phenyl ester, capable of multiple hydrogen bond formation with the 2,4- diaminopyrimidine. The recognition property of the respective polymers toward the template and other test compounds was evaluated by fluorescence. The results demonstrate that the polymers showed dose dependent enhancement of fluorescence emissions. In addition, the results also indicate that synthesized MIPs have higher 2,4-diaminopyrimidine binding ability as compared with corresponding non-imprinting polymers.

Free radicals as potential antitumour agents

The aim of this work was to use extremely low concentrations of free radical generating compounds as a 'catalyst' to trigger endogenous free radical chain reactions in the host and to selectively eliminate neoplastic cells in the host. To test the hypothesis, a number of free radical generating compounds were screened on several malignant cell lines in vitro to select model compounds that were used against tumour models in vivo. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and its derivatives were selected at the model compounds for in vivo experiments in view of their high cytotoxic potency against several malignant cell lines in vitro. The water soluble derivative, 2,2-diphenyl-1-(2', 4'-dinitro-6'-sulphophenyl) hydrazyl (DDSH) given by subcutaneous injections demonstrated significant antitumour activities against the MAC 16 murine colon adenocarcinoma implanted subcutaneously in male NMRI mice at nanomolar concentration range. 40-60% of long term survival of over 60 days was achieved (compared with control survival of 20 days) with total tumour elimination. This compound was also active against both P388 leukaemia in male BDF1 mice and TLX5 lymphoid tumour in male CBA/CA mice at a similar concentration range. However, some of these animals died suddenly after treatment with no evidence of disease present at post mortem. The cause of death was unknown but thought to be related to the treatment. There was significant increase in serum level of malondialdehyde (MDA) following treatment, but did not correlate to the antitumour activities of these compounds. Induction of supcroxide dismutase (SOD), and glutathione peroxidase (GPx) occurred around day 8 after the administration of DDSH. Histological sections of MAC16 tumours showed areas of extensive massive haemorrhagic necrosis and vascular collapse associated with perivascular cell death following the administration of nanomolar concentration of DDSH which was probably compatible with the effects of free radicals. It was concluded that the antitumour activities of these compounds may be related to free radical and cytokine production.

Lipophilic antifolates as potential antipsoriatic agents

The lipophilic dihydrofolate reductase (DHFR) inhibitor m-azidopyrimethamine (MZP) was investigated for suitability for development as a topical antipsoriatic agent. The clinical features and treatments for psoriasis were reviewed. High performance liquid chromatography (HPLC) was employed as the main analytical method, with UV spectroscopy being used in some cases. Reduction of the azido-group was proposed as a potential detoxification mechanism for MZP. The rates of reduction of a series of substituted phenyl azide compounds by dithiothreitol were investigated and found to depend on the substitution pattern of the aryl azide molecular, with electron deficient azides exhibiting faster rates of reduction in the system studied. The rates of reduction of MZP and analogous compounds were also studied using this model. The skin penetration of MZP was assessed using an in vitro hairless mouse skin model. The rate of permeation (flux) of MZP across hairless mouse skin was found to be dependent on the quantity of propylene glycol used as cosolvent in the vehicle and the pH. The use of a pretreatment regime of oleic acid in propylene glycol was shown to greatly increase the penetration of MZP through the hairless mouse skin as compared to application without pretreatment, or pretreatment with other penetration enhancers. The metabolism of MZP was studied in in vitro models comprising skin homogenates, SV-K14 human keratinocyte cell cultures and skin commensal bacterial cultures. No conversion of MZP to the corresponding amine was detected in any of the models. The growth inhibitory properties of MZP were investigated in an in vitro SV-K14 human keratinocyte cell culture model and compared with those of other DHFR inhibitors. [14C]-pyrimethamine was shown to be taken up by the SV-K14 keratinocytes.

Design, synthesis and biological evaluation of potential antibacterial oligonucleotides

Purine and pyrimidine triplex-forming oligonucleotides (TFOs), as potential antibacterial agents, were designed to bind by Hoogsteen and reverse Hoogsteen hydrogen bonds in a sequence specific manner in the major groove of genomic DNA at specific polypurine sites within the gyrA gene of E. coli and S. pneumoniae. Sequences were prepared by automated synthesis, with purification and characterisation determined by high performance liquid chromatograpy, capillary electrophoresis and mass spectrometry. Triplex stability was assessed using melting curves where the binding of the third strand to the duplex target, was assessed over a temperature range of 0-80°C, and at pH 6.4 and 7.2. The most successful of the unmodified TFOs (6) showed a Tm value of 26 °C at both pH values with binding via reverse Hoogsteen bonds. Binding to genomic DNA was also demonstrated by spectrofluorimetry, using fluorescein-labelled TFOs, from which dissociation constants were determined. Modifications in the form of 5mC, 5' acridine attachment, phosphorothioation, 2'-0-methylation and phosphoramidation, were made in order to. increase Tm values. Phosphoramidate modification was the most with increased Tm values of 42°C. However, the final purity of these sequences was poor due to their difficult syntheses. FACS (fluorescent activated cell sorting) analysis was used to determine the potential uptake of a fluorescently labelled analogue of 6 via passive, coJd shock mediated, and anionic liposome aided, uptake. This was established at 20°C and 37°C. At both temperatures anionic lipid-mediated uptake produced unrivalled fluorescence, equivalent to 20 and 43% at 20 and 37°C respectively. Antibacterial activity of each oligonucleotide was assessed by viable count anaJysis relying on passive uptake, cold shocking techniques, chlorpromazine-mediated uptake, and, cationic and anionic lipid-aided uptake. All oligonucleotides were assessed for their ability to enhance uptake, which is a major barrier to the effectiveness of these agents. Compound 6 under cold shocking conditions produced the greatest consistent decline in colony forming units per ml. Results for this compound were sometimes variable indicating inconsistent uptake by this particular assay method.

The effects of the fungicides fenhexamid and myclobutanil on SH-SY5Y and U-251 MG human cell lines

Mixtures of pesticides in foodstuffs and the environment are ubiquitous in the developed world and although agents are usually exhaustively tested individually, the toxicological implications of pesticide mixtures are underreported. In this study, the effects of two fungicides, fenhexamid and myclobutanil were investigated individually and in combination on two human cell lines, SH-SY5Y neuronal cells and U-251 MG glial cells. After 48. h of incubation with increasing concentrations of pesticides ranging from 1 to 1000. μM, gene expression profiles were studied in addition to toxicity end points, including cell viability, mitochondrial depolarisation as well as cellular glutathione maintenance. There were no significant differences between the susceptibility of the two cell lines in terms of cell viability assessment or mitochondrial membrane potential, when agents were administered either individually or in combination. By contrast, in the presence of the fungicides, the SH-SY5Y cells showed significantly greater susceptibility to oxidative stress in terms of total thiol depletion in comparison with the astrocytic cells. Treatment with the two pesticides led to significant changes in the cell lines' expression of several genes which regulate cell cycle control and growth (RB1, TIMP1) as well as responses to DNA attrition (ATM and CDA25A) and control of apoptosis (FAS). There was no evidence in this study that the combination of fenhexamid and myclobutanil was significantly more toxic than individual exposure, although gene expression changes suggested there may be differences in the sub-lethal response of both cell lines to both individual and combined exposure.

Spray drying lactoferrin produces inhalable antimicrobial particles

The use of antimicrobial peptides and proteins as potential therapeutic agents in the management of multi-drug resistant infections is considered an attractive concept especially since such compounds should theoretically have low immunogenicity, high bioavailability with negligible toxicity. In this study we investigated the potential of developing a dry powder inhaler formulation of lactoferrin (a multifunctional iron binding protein). To achieve this, the protein was spray dried from a water only feedstock with suitably adjusted spray drying parameters. The particle size, degree of crystallinity, moisture content and yield of the spray dried powders along with the minimum bactericidal concentration (MBC) against Pseudomonas aeruginosa strain PAO1, were assessed. Dry powder inhaler formulations were prepared, and in vitro assessment studies using the multistage impinger were carried out to assess the aerosolisation performance of the formulations. Data obtained indicate that spray dried lactoferrin retains activity against biofilms and may be successfully employed in the treatment of chronic airway infections.

Synthesis and biological evaluation of potential anti-cancer agents

The new technology of combinational chemistry has been introduced to pharmaceutical companies, improving and making more efficient the process of drug discovery. Automated combinatorial chemistry in the solution-phase has been used to prepare a large number of compounds of anti-cancer screening. A library of caffeic acid derivatives has been prepared by the Knoevenagel condensation of aldehyde and active methylene reagents. These products have been screened against two murine adenocarcinoma cell lines (MAC) which are generally refractive to standard cytotoxic agents. The target of anti-proliferative action was the 12- and 15-lipoxygenase enzymes upon which these tumour cell lines have been shown to be dependent for proliferation and metastasis. Compounds were compared to a standard lipoxygenase inhibitor and if found to be active anti-proliferative agents were tested for their general cytotoxicity and lipoxygenase inhibition. A solid-phase bound catalyst, piperazinomethyl polystyrene, was devised and prepared for the improved generation of Knoevenagel condensation products. This piperazinomethyl polystyrene was compared to the traditional liquid catalyst, piperidine, and was found to reduce the amount of by-products formed during reaction and had the advantage of easy removal from the reaction. 13C NMR has been used to determine the E/Z stereochemistry of Knoevenagel condensation products. Soluble polymers have been prepared containing different building blocks pendant to the polymer backbone. Aldehyde building blocks incorporated into the polymer structure have been subjected to the Knoevenagel condensation. Cleavage of the resultant pendant molecules has proved that soluble linear polymers have the potential to generate combinatorial mixtures of known composition for biological testing. Novel catechol derivatives have been prepared by traditional solution-phase chemistry with the intention of transferring their synthesis to a solid-phase support. Catechol derivatives prepared were found to be active inhibitors of lipoxygenase. Soluble linear supports for the preparation of these active compounds were designed and tested. The aim was to develop a support suitable for the automated synthesis of libraries of catechol derivatives for biological screening.

Automated synthesis and evaluation of potential new anti-microbial agents

A series of N1-benzylideneheteroarylcarboxamidrazones was prepared in an automated fashion, and tested against Mycobacterium fortuitum in a rapid screen for antimycobacterial activity. Many of the compounds from this series were also tested against Mycobacterium tuberculosis, and the usefulness as M.fortuitum as a rapid, initial screen for anti-tubercular activity evaluated. Various deletions were made to the N1-benzylideneheteroarylcarboxamidrazone structure in order to establish the minimum structural requirements for activity. The N1-benzylideneheteroarylcarbox-amidrazones were then subjected to molecular modelling studies and their activities against M.fortuitum and M.tuberculosis were analysed using quantitative structure-analysis relationship (QSAR) techniques in the computational package TSAR (Oxford Molecular Ltd.). A set of equations predictive of antimycobacterial activity was hereby obtained. The series of N1-benzylidenehetero-arylcarboxamidrazones was also tested against a multidrug-resistant strain of Staphylococcus aureus (MRSA), followed by a panel of Gram-positive and Gram-negative bacteria, if activity was observed for MRSA. A set of antimycobacterial N1-benzylideneheteroarylcarboxamidrazones was hereby discovered, the best of which had MICs against m. fortuitum in the range 4-8μgml-1 and displayed 94% inhibition of M.tuberculosis at a concentration of 6.25μgml-1. The antimycobacterial activity of these compounds appeared to be specific, since the same compounds were shown to be inactive against other classes of organisms. Compounds which were found to be sufficiently active in any screen were also tested for their toxicity against human mononuclear leucocytes. Polyethylene glycol (PEG) was used as a soluble polymeric support for the synthesis of some fatty acid derivatives, containing an isoxazoline group, which may inhibit mycolic acid synthesis in mycobacteria. Both the PEG-bound products and the cleaved, isolated products themselves were tested against M.fortuitum and some low levels of antimycobacterial activity were observed, which may serve as lead compounds for further studies.

Transglutaminases as tanning agents for the leather industry

This study was aimed at determining whether the protein crosslinking enzymes, transglutaminases, had the potential to be used as tanning agents, using native bovine hide and purified soluble rat tail collagen as real and model substrates, respectively. We demonstrate that transglutaminases (TGs) were capable of covalently crosslinking collagen molecules together such that on average every collagen molecule contained at least one epsilon(gamma-glutamyl)lysine crosslink. However, transglutaminase-mediated crosslinking did not affect the denaturation temperature of either native bovine hide or soluble rat tail collagens when used in isolation or together with other proteins and bifunctional diamines as crosslinking facilitators. In an initial study into the effect of TG-mediated crosslinking on the tensile strength of chrome-tanned bovine hide, such crosslinking led to a 30 per cent decrease in tensile strength. Despite a change in the gel melting point mediated by epsilon(gamma-glutamyl)lysine crosslinking, the use of transglutaminases as alternative tanning agents seems unlikely given the present data.

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

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

Increased potential of a cationic liposome-based delivery system:enhancing stability and sustained immunological activity in pre-clinical development

The combination of dimethyl dioctadecyl ammonium bromide (DDA) and the synthetic cord factor trehalose dibehenate (TDB) with Ag85B-ESAT-6 (H1 fusion protein) has been found to promote strong protective immune responses against Mycobacterium tuberculosis. The development of a vaccine formulation that is able to facilitate the requirements of sterility, stability and generation of a vaccine product with acceptable composition, shelf-life and safety profile may necessitate selected alterations in vaccine formulation. This study describes the implementation of a sterilisation protocol and the use of selected lyoprotective agents in order to fulfil these requirements. Concomitantly, close analysis of any alteration in physico-chemical characteristics and parameters of immunogenicity have been examined for this promising DDA liposome-based tuberculosis vaccine. The study addresses the extensive guidelines on parameters for non-clinical assessment, suitable for liposomal vaccines and other vaccine delivery systems issued by the World Health Organisation (WHO) and the European Medicines Agency (EMEA). Physical and chemical stability was observed following alteration in formulations to include novel cryoprotectants and radiation sterilisation. Immunogenicity was maintained following these alterations and even improved by modification with lysine as the cryoprotective agent for sterilised formulations. Taken together, these results outline the successful alteration to a liposomal vaccine, representing improved formulations by rational modification, whilst maintaining biological activity.

Mechanisms of antifatigue agents used in natural rubber

A large number of compounds containing quinonoid or hindered phenol functions were examined for their roles as antifatigue agents. Among the evaluated quinones and phenols expected to have macroalkyl radical scavenging ability, BQ, αTOC, γTOC and GM showed relatively good performance for fatigue resistance (although their performance was slightly less effective than the commercial aromatic amine antioxidants, IPPD and 6PPD). The compounds which were shown to have higher reactivity with alkyl radicals (via calculated reactivity indices) showed better fatigue resistance. This fact supports the suggestion that strong alkyl radical scavengers should be also effective antifatigue agents. Evidence produced based on calculation of reactivity indices suggests that the quinones examined react with alkyl radicals on the meta position of the quinone rings producing phenoxyl radicals. The phenoxyl radicals are expected either to disproportionate, to recombine with a further alkyl radical, or to abstract a hydrogen from another alkyl radical producing an olefine. The regeneration of quinones and formation of the corresponding phenols is expected to occur during the antifatigue activity. The phenol antioxidant, HBA is expected to produce a quinonoid compound and this is also expected to function in a similar way to other quinones. Another phenol, GM, which is also known to scavenge alkyl radicals showed good antifatigue performance. Tocopherols had effective antifatigue activity and are expected to have different antifatigue mechanisms from that of other quinones, hence αTOC was examined for its mechanisms during rubber fatiguing using HPLC analysis. Trimers of αTOC which were produced during vulcanisation are suggested to contribute to the fatigue activity observed. The evidence suggests that the trimers reproduce αTOC and a mechanism was proposed. Although antifatigue agents evaluated showed antifatigue activity, most of them had poor thermoxidative resistance, hence it was necessary to compensate for this by using a combination of antioxidants with the antifatigue agents. Reactive antioxidants which have the potential to graft on the polymer chains during reactive processing were used for this purpose. APMA was the most effective antioxidant among other evaluated reactive antioxidants. Although high ratio of grafting was achieved after optimisation of grafting conditions, it is suggested that this was achieved by long branches of APMA due to large extent of polymerisation. This is expected to cause maldistribution of APMA leading to reducing the effect of CB-D activity (while CB-A activity showed clear advantages for grafting). Further optimisation of grafting conditions is required in order to use APMA more effectively. Moreover, although synergistic effects between APMA and antifatigue agents were expected, none of the evaluated antifatigue agents, BQ, αTOC, γTOC and TMQ, showed significant synergism both in fatigue and thermoxidative resistance. They performed just as additives.

The evolving role of information specialists:change agents in process redesign

The research investigates the past, present and potential future role of Information Specialists (ISps) in process oriented companies. It tests the proposition that ISps in companies that have undertaken formal process reengineering exercises are likely to become more proactive and more business oriented (as opposed to technically oriented) than they had previously been when their organisations were organised along traditional, functional lines. A review of existing literature in the area of Business Process Reengineering and Information Management reveals a lack of consensus amongst researchers concerning the appropriate role for ISps during and after BPR. Opinion is divided as to whether IS professionals should reactively support BPR or whether IT/IS developments should be driving these initiatives. A questionnaire based ‘Descriptive Survey’ with 60 respondents is used as a first stage of primary data gathering. This is followed by follow-up interviews with 20 of the participating organisations to gather further information on their experiences. The final stage of data collection consists of further in-depth interview with four case study companies to provide an even richer picture of their experiences. The results of the questionnaire are analysed and displayed in the form of simple means, frequencies and bar graphs. The ‘NU-DIST’ computer based discourse analysis package was tried in relation to summarising the interview findings, but this proved cumbersome and a visual collation method is preferred. Overall, the researcher contends that the supposition outlined above is proven, and she concludes the research by suggesting the implications of these findings. In particular she offers a ‘Framework for Understanding and Action’ which is deemed to be relevant to both practitioners and future researchers.