Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and flourescence evaluation

Aims: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. Methods and Results: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K d and relative Bmax values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, Kd and relative B max, which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. Conclusions: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. Significance and Impact of the Study: The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

Loss of chance and the evaluation of hypothetical's in contractual claims

Analyses how to calculate damages for the loss of an opportunity by reason of a breach of contract, in the light of the House of Lords judgment in Gregg v Scott concerning clinical negligence. Discusses whether different principles apply to contract claims and torts.

Phakometric measurement of ocular surface radius of curvature and alignment: evaluation of method with physical model eyes

Ophthalmophakometric measurements of ocular surface radius of curvature and alignment were evaluated on physical model eyes encompassing a wide range of human ocular dimensions. The results indicated that defocus errors arising from imperfections in the ophthalmophakometer camera telecentricity and light source collimation were smaller than experimental errors. Reasonable estimates emerged for anterior lens surface radius of curvature (accuracy: 0.02–0.10 mm; precision 0.05–0.09 mm), posterior lens surface radius of curvature (accuracy: 0.10–0.55 mm; precision 0.06–0.20 mm), eye rotation (accuracy: 0.00–0.32°; precision 0.06–0.25°), lens tilt (accuracy: 0.00–0.33°; precision 0.05–0.98°) and lens decentration (accuracy: 0.00–0.07 mm; precision 0.00–0.07 mm).

Symmetric and asymmetric evaluation of the Tokamap in comparison with direct symplectic integration

Comparing the Poincaré plots of the Tokamap and the underlying Hamiltonian system reveals large differences. This stems from the particular choice of evaluation of the singular perturbations present in the system (a series of δ functions). A symmetric evaluation approach is proposed and shown to yield results that almost perfectly match the Hamiltonian system. © 2005 The American Physical Society.

Solid dispersions: formulation, characterisation, permeability and genomic evaluation

Poor water solubility is characterised by low dissolution rate and consequently reduced bioavailability. Formulation of solid dispersion of the drug has attracted considerable interest as a means of improving dissolution process of a range of poorly water soluble drugs. This current study investigates the formulation of solid dispersion for a range of poorly water soluble drugs with varying physicochemical properties including paracetamol, sulphamethoxazole, phenacetin, indomethacin, chloramphenicol, phenylbutazone and succinylsulphathiazole. Solid dispersions were prepared using various drugs to polymer ratios. PEG 8000 was selected as a carrier in the solid dispersions. The study revealed that inclusion of drug within the polymeric matrix, ratio of drug to polymer and physicochemical properties of the drug molecules enhance the dissolution rate. Characterisations of the solid dispersions were performed using DSC, FTIR and SEM. These studies revealed that all seven drugs were present in the amorphous form within the solid dispersions and there was a lack of interaction between the PEG 8000 and drug. Stability studies for solid dispersions showed that all seven drugs studied were unstable at accelerated conditions (40°C±2°C/75%RH±5%RH) whereas, they were found to be stable for 12 months at room conditions. Permeability of indomethacin, phenacetin, phenylbutazone and paracetamol were higher for solid dispersions as compared to drug alone across Caco-2 cell monolayers. From the cell uptake studies it was shown that PEG 8000 enhanced rhodamine123 uptake which suggested that PEG 8000 may increase the permeability of these drugs in solid dispersions. Gene expression profiles analyzing the expression changes in the ABC and solute carrier transporter during permeability studies.ABCA10, ABCB4, ABCC12, SLC12A6, MCT13, SLC22A12 and SLC6A6 gene expression were increased by indomethacin alone whereas solid dispersion of indomethacin resulted in a slight increase in expression. ABCC12 and SAMC gene expression was increased in case of paracetamol alone but slightly increased when exposed to solid dispersion of paracetamol.

The ecology of paper mill by-product and its evaluation as a casing medium in the culture of Agaricus bisporus (Lange) Pilat

Effluent from pulp and paper production at the Kemsley mill of Bowaters U.K. Paper Company Limited passes through two treatment stages before its discharge into the Swale estuary. Suspended material removed during treatment is deposited on wasteground as a thin sludge. The solids it contains are mainly wood components lost during pulp production, whilst it also has a high salt content, derived from chemicals used in pulping processes. After deposition the sludge undergoes an ageing process during which it dries out and its salt content is reduced. This ageing can be reproduced and accelerated by improved drainage under controlled conditions. The paper mill sludge was investigated as a casing medium in the culture of Agaricus bisporus (Lange) Pilat, the cultivated mushroom. It was unsuitable up to one year from deposition due largely to the inhibitory effect of its salt content on fruiting. Material eighteen months or more in age gave yields comparable to standard peat casing. Before use as a casing the material must be shredded to a satisfactory structure, neutralised with chalk, and pasteurised to eliminate organisms harmful to the crop. The prepared medium has a high water holding capacity and a structure resilient to management procedures, important requirements of a good casing. A passive movement of salts from the compost to the casing was shown to occur during culture, capable of enhancing the natural decline in cropping if sufficiently great. The ions chloride, potassium, sodium and sulphate were shown to be responsible, their damaging effects being due to high conductivity created in the casing. Studies of elements available during culture suggested phosphate availability in the compost could limit crop potential, whilst iron released by mycelium of A.bisporus in the casing may be utilised by associated micro-organisms.

Environmental and nature conservation : a facet study of concern for the quality of the natural environment

A multi-variate descriptive model of environmental and nature conservation attitudes and values is proposed and empirically supported. A mapping sentence is developed out of analysis of data from a series of Repertory Grid interviews addressing conservation employees' attitudes towards their profession's activities. The research is carried out within the meta-theoretical framework of Facet Theory. A mapping sentence is developed consisting of 9 facets. From the mapping sentence 3 questionnaires were constructed viewing the selective orientations towards environmental concern. A mapping sentence and facet model is developed for each study. Once the internal structure of this model had been established using Similarity Structure Analysis, the elements of the facets are subjected to Partial Order Scalogram Analysis with base coordinates. A questionnaire was statistically analysed to assess the relationship between facet elements and 4 measures of attitudes towards, and involvement with, conservation. This enabled the comparison of the relative strengths of attitudes associated with each facet element and each measure of conservation attitude. In general, the relationship between the social value of conservation and involvement pledges to conservation were monotonic; perceived importance of a conservation issue appearing predictive of personal involvement. Furthermore, the elements of the life area and scale facets were differentially related to attitude measures. The multi-variate descriptive model of environmental conservation values and attitudes is discussed in relation to its implications for psychological research into environmental concern and for environmental and nature conservation.

Herbal medicines:physician's recommendation and clinical evaluation of St.John's Wort for depression

Why some physicians recommend herbal medicines while others do not is not well understood. We undertook a survey designed to identify factors, which predict recommendation of herbal medicines by physicians in Malaysia. About a third (206 out of 626) of the physicians working at the University of Malaya Medical Centre ' were interviewed face-to-face, using a structured questionnaire. Physicians were asked about their personal use of, recommendation of, perceived interest in and, usefulness and safety of herbal medicines. Using logistic regression modelling we identified personal use, general interest, interest in receiving training, race and higher level of medical training as significant predictors of recommendation. St. John's wort is one of the most widely used herbal remedies. It is also probably the most widely evaluated herbal remedy with no fewer than 57 randomised controlled trials. Evidence from the depression trials suggests that St. John's wort is more effective than placebo while its comparative efficacy to conventional antidepressants is not well established. We updated previous meta-analyses of St. John's wort, described the characteristics of the included trials, applied methods of data imputation and transformation for incomplete trial data and examined sources of heterogeneity in the design and results of those trials. Thirty randomised controlled trials, which were heterogeneous in design, were identified. Our meta-analysis showed that St. John's wort was significantly more effective than placebo [pooled RR 1.90 (1.54-2.35)] and [Pooled WMD 4.09 (2.33 to 5.84)]. However, the remedy was similar to conventional antidepressant in its efficacy [Pooled RR I. 0 I (0.93 -1.10)] and [Pooled WMD 0.18 (- 0.66 to 1.02). Subgroup analyses of the placebo-controlled trials suggested that use of different diagnostic classifications at the inclusion stage led to different estimates of effect. Similarly a significant difference in the estimates of efficacy was observed when trials were categorised according to length of follow-up. Confounding between the variables, diagnostic classification and length of trial was shown by loglinear analysis. Despite extensive study, there is still no consensus on how effective St. lohn's wort is in depression. However, most experts would agree that it has some effect. Our meta-analysis highlights the problems associated with the clinical evaluation of herbal medicines when the active ingredients are poorly defined or unknown. The problem is compounded when the target disease (e.g. depression) is also difficult to define and different instruments are available to diagnose and evaluate it.

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.

A combinatorial approach to the chemical synthesis and biological evaluation of 3,4,5-trisubstiituted furan-2(5H)-ones

Many important natural products contain the furan-2(5H)-one structure. The structure of this molecule lends itself to manipulation using combinatorial techniques due to the presence of more than one site for the attachment of different suhstituents. By developing different reaction schemes at the three sites available for attachment on the furan-2(5H)-one scaffold, combinatorial chemistry techniques can be employed to assemble libraries of novel furan 2(5H)-ones. These libraries can then be entered into various biological screening programmes. This approach will enable a vast diversity or compounds to be examined, in the hope or finding new biologically active Iead structures. The work in this thesis has investigated the potential that combinatorial chemistry has in the quest for new biologically active lead structures based on the furan-2(5H)-one structure. Different reactions were investigated with respect to their suitability for inclusion in a library. Once sets of reactions at the various sites had been established, the viability of these reactions in the assembly of combinatorial libraries was investigated. Purification methods were developed, and the purified products entered into suitable biological screening tests. Results from some of these tests were optimised using structure activity relationships, and the resulting products re-screened. The screening tests performed were for anticancer and antimicrobial activity, cholecystokinin (CCK-B) antagonism and anti-inflammatory activity (in the quest for novel cyclo-oxygenase (COX-2) selective non-steroidal anti-inflammatory drugs). It has been shown that many reactions undergone by the furan-2(5H)-one structure are suitable for the assembly of a combinatorial library. Investigation into the assembly of different libraries has been carried out with initial screening results included. From this work, further investigation into combinatorial library assembly and structure activity relationships of screened reaction products can be undertaken.

An economic, environmental and functional study of buildings erected for educational purposes

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Migraine, A study of environmental and intrapersonal factors

A study was carried out of 45 migrainous patients with visually induced migraine (VIM), and 25 migrainous students, each having an age and sex matched control. The study utilised questionnaires, interviews, electroencephalography (EEG) and visual evoked potentials (VEP). The experimental work and analysis was carried out in the Neuropsychology Unit in collaboration with the Birmingham Migraine Clinic, over a period of five years. The study suggests: 1. The literature on a possible relationship between migraine and epilepsy hitherto published is unreliable (supporting evidence is given). 2. That a much greater precision is needed in defining migraine for research purposes. 3. A revised methodology for the selection of controls is needed and this is proposed. 4. That despite what are now seen to be superficial similarities, there are clear distinctions of a fundamental nature between photo-sensitive epilepsy (PSE) and VIM. 5. Caution be used when taking headache as a symptom, since many of the precipitants of migrainous headache can also precipitate non-migrainous headache (NMH). 6. The list of visual precipitants of migraine is expanded (particularly flicker and pattern). 7. That colour (principally red) is a previously unreported precipitant of migraine. 8. The extended range of responses to flicker (the 'H' response) has no significant difference in its frequency of occurrence in patients and normal controls, which contradicts previous literature. 9. The mechanisms thought to underlie migraine serve to explain previously unexplained EEG findings. 10. Further research is needed and proposed.

Theoretical and empirical evaluation of phakic and pseudophakic accomodation

The binding theme of this thesis is the examination of both phakic and pseudophakic accommodation by means of theoretical modelling and the application of a new biometric measuring technique. Anterior Segment Optical Coherence Tomography (AS-OCT) was used to assess phakic accommodative changes in 30 young subjects (19.4 2.0 years; range, 18 to 25 years). A new method of assessing curvature change with this technique was employed with limited success. Changes in axial accommodative spacing, however, proved to be very similar to those of the Scheimpflug-based data. A unique biphasic trend in the position of the posterior crystalline lens surface during accommodation was discovered, which has not been alluded to in the literature. All axial changes with accommodation were statistically significant (p < 0.01) with the exception of corneal thickness (p = 0.81). A two-year follow-up study was undertaken for a cohort of subjects previously implanted with a new accommodating intraocular lens (AIOL) (Lenstec Tetraflex KH3500). All measures of best corrected distance visual acuity (BCDVA; +0.04 0.24 logMAR), distance corrected near visual acuity (DCNVA; +0.61 0.17 logMAR) and contrast sensitivity (+1.35 0.21 log units) were good. The subjective accommodation response quantified with the push-up technique (1.53 0.64 D) and defocus curves (0.77 0.29 D) was greater than the objective stimulus response (0.21 0.19 D). AS-OCT measures with accommodation stimulus revealed a small mean posterior movement of the AIOLs (0.02 0.03 mm for a 4.0 D stimulus); this is contrary to proposed mechanism of the anterior focus-shift principle.

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.