Evaluation of videokeratoscopy

The present thesis evaluates various aspects of videokeratoscopes, which are now becoming increasingly popular in the investigation of corneal topography. The accuracy and repeatability of these instruments has been assessed mainly using spherical surfaces, however, few studies have assessed the performance of videokeratoscopes in measuring convex aspheric surfaces. Using two videokeratoscopes, the accuracy and repeatability of measurements using twelve aspheric surfaces is determined. Overall, the accuracy and repeatability of both instruments were acceptable, however, progressively flatter surfaces introduced greater errors in measurement. The possible reasons for these errors are discussed. The corneal surface is a biological structure lubricated by the precorneal tear film. The effects of variations in the tear film on the repeatability of videokeratoscopes have not been determined in terms of peripheral corneal measurements. The repeatability of two commercially available videokeratoscopes is assessed. The repeatability is found to be dependent on the point of measurement on the corneal surface. Typically, superior and nasal meridians exhibit poorest repeatability. It is suggested that interference of the ocular adnexa is responsible for the reduced repeatability. This localised reduction in repeatability will occur for all videokeratoscopes. Further, comparison with the keratometers and videokeratoscopes used show that measurements between these instruments are not interchangeable. The final stage of this thesis evaluates the performance of new algorithms. The characteristics of a new videokeratoscope are described. This videokeratoscope is used to test the accuracy of the new algorithms for twelve aspheric surfaces. The new algorithms are accurate in determining the shape of aspheric surfaces, more so than those algorithms proposed at present.

myo-inositol pentakisphosphates. Structure, biological occurrence and phosphorylation to myo-inositol hexakisphosphate

1. Standard and high-performance anion-exchange-chromatographic techniques have been used to purify myo-[3H]inositol pentakisphosphates from various myo-[3H]inositol-prelabelled cells. Slime mould (Dictyostelium discoideum) contained 8 microM-myo-[3H]inositol 1,3,4,5,6-pentakisphosphate 16 microM-myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and 36 microM-D-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate [calculated intracellular concentrations; Stephens & Irvine (1990) Nature (London) 346 580-583]; germinating mung-bean (Phaseolus aureus) seedlings contained both D- and L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate (which was characterized by 31P and two-dimensional proton n.m.r.) and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate; HL60 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 500-fold excess over the other species), myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate; and NG-115-401L-C3 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 100-fold excess over the other species), D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate, myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate. 2. Multiple soluble ATP-dependent myo-inositol pentakisphosphate kinase activities have been detected in slime mould, rat brain and germinating mung-bean seedling homogenates. In slime-mould cytosolic fractions, the three myo-inositol pentakisphosphates that were present in intact slime moulds could be phosphorylated to myo-[3H]inositol hexakisphosphate: the relative first-order rate constants for these reactions were, in the order listed above, 1:8:31 respectively (with first-order rate constants in the intact cell of 0.1, 0.8 and 3.1 s-1, assuming a cytosolic protein concentration of 50 mg/ml), and the Km values of the activities for their respective inositol phosphate substrates (in the presence of 5 mM-ATP) were 1.6 microM, 3.8 microM and 1.4 microM. At least two forms of myo-inositol pentakisphosphate kinase activity could be resolved from a slime-mould cytosolic fraction by both pharmacological and chromatographic criteria. Rat brain cytosol and a soluble fraction derived from germinating mung-bean seedlings could phosphorylate myo-inositol D/L-1,2,4,5,6-, D/L-1,2,3,4,5-, 1,2,3,4,6- and 1,3,4,5,6-pentakisphosphates to myo-inositol hexakisphosphate: the relative first-order rate constants were 57:27:77:1 respectively for brain cytosol (with first-order rate constants in the intact cell of 0.0041, 0.0019, 0.0056 and 0.000073 s-1 respectively, assuming a cytosolic protein concentration of 50 mg/ml) and 1:11:12:33 respectively for mung-bean cytosol (with first-order rate constants in a supernatant fraction with a protein concentration of 10 mg/ml of 0.0002, 0.0022, 0.0024 and 0.0066 s-1 respectively).

Optical fibre sensors with applications in gas and biological sensing

This thesis describes the study of various grating based optical fibre sensors for applications in refractive index sensing. The sensitivity of these sensors has been studied and in some cases enhanced using novel techniques. The major areas of development are as follows. The sensitivity of long period gratings (LPGs) to surrounding medium refractive index (SRI) for various periods was investigated. The most sensitive period of LPG was found to be around 160 µm and this was due to the core mode coupling to a single cladding mode but phase matching at two wavelength locations, creating two attenuation peaks, close to the waveguide dispersion turning point. Large angle tilted fibre gratings (TFGs) have similar behaviour to LPGs, in that they couple to the co-propagating cladding modes. The tilted structure of the index modulation within the core of the fibre gives rise to a polarisation dependency, differing the large angle TFG from a LPG. Since the large angle TFG couple to the cladding mode they are SRI sensitive, the sensitivity to SRI can be further increased through cladding etching using HF acid. The thinning of the cladding layer caused a reordering of the cladding modes and shifted to more SRI sensitive cladding modes as the investigation discovered. In a SRI range of 1.36 to 1.40 a sensitivity of 506.9 nm/URI was achieved for the etched large angle TFG, which is greater than the dual resonance LPG. UV inscribed LPGs were coated with sol-gel materials with high RIs. The high RI of the coating caused an increase in cladding mode effective index which in turn caused an increase in the LPG sensitivity to SRI. LPGs of various periods of LPG were coated with sol-gel TiO2 and the optimal thickness was found to vary for each period. By coating of the already highly SRI sensitive 160µm period LPG (which is a dual resonance) with a sol-gel TiO2, the SRI sensitivity was further increased with a peak value of 1458 nm/URI, which was an almost 3 fold increase compared to the uncoated LPG. LPGs were also inscribed using a femtosecond laser which produced a highly focused index change which was no uniform throughout the core of the optical fibre. The inscription technique gave rise to a large polarisation sensitivity and the ability to couple to multiple azimuthal cladding mode sets, not seen with uniform UV inscribed gratings. Through coupling of the core mode to multiple sets of cladding modes, attenuation peaks with opposite wavelength shifts for increasing SRI was observed. Through combining this opposite wavelength shifts, a SRI sensitivity was achieved greater than any single observed attenuations peak. The maximum SRI achieved was 1680 nm/URI for a femtosecond inscribed LPG of period 400 µm. Three different types of surface plasmon resonance (SPR) sensors with a multilayer metal top coating were investigated in D shape optical fibre. The sensors could be separated into two types, utilized a pre UV inscribed tilted Bragg grating and the other employed a post UV exposure to generate surface relief grating structure. This surface perturbation aided the out coupling of light from the core but also changed the sensing mechanism from SPR to localised surface plasmon resonance (LSPR). This greatly increased the SRI sensitivity, compared to the SPR sensors; with the gold coated top layer surface relief sensor producing the largest SRI sensitivity of 2111.5nm/URI was achieved. While, the platinum and silver coated top layer surface relief sensors also gave high SRI sensitivities but also the ability to produce resonances in air (not previously seen with the SPR sensors). These properties were employed in two applications. The silver and platinum surface relief devices were used as gas sensors and were shown to be capable of detecting the minute RI change of different gases. The calculated maximum sensitivities produced were 1882.1dB/URI and 1493.5nm/URI for silver and platinum, respectively. Using a DFB laser and power meter a cheap alternative approach was investigated which showed the ability of the sensors to distinguish between different gases and flow rates of those gases. The gold surface relief sensor was coated in a with a bio compound called an aptamer and it was able to detect various concentrations of a biological compound called Thrombin, ranging from 1mM to as low as 10fM. A solution of 2M NaCl was found to give the best stripping results for Thrombin from the aptamer and showed the reusability of the sensor. The association and disassociation constants were calculated to be 1.0638×106Ms-1 and 0.2482s-1, respectively, showing the high affinity of the Aptamer to thrombin. This supports existing working stating that aptamers could be alternative to enzymes for chemical detection and also helps to explain the low detection limit of the gold surface relief sensor.

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.

Synthesis, crystallography and biological activity of myo-inositol phosphates

The antioxidant property of myo-inositol hexakisphosphate is important in the prevention of hydroxyl radical formation which may allow it to act as a 'safe' carrier of iron within the cell. Here, the hypothesis that the recently discovered natural product, myo-inositol 1,2,3-trisphosphate represents the simplest structure to mimic phytate's antioxidant activity has been tested. The first synthesis of myo-inositol 1,2,3-trisphosphate has been completed, along with its X-ray structure determination and that of key synthetic intermediates. Iron binding studies of myo-inositol 1,2,3-trisphosphate demonstrated that phosphate groups with the equatorial-axial-equatorial conformation are required for complete inhibition of hydroxyl radical formation. myo-Inositol monophosphatase is a key enzyme in recycling myo-inositol from its monophosphates in the brain and its inhibition is implicated in lithium's antimanic properties. Current synthetic strategies require inositol compounds to be protected (often with more than one group), resolved, phosphorylated and deprotected to produce the desired optically active myo-inositol phosphates. Here, the synthesis of myo-inositol 3-phosphate has been achieved in only 4 steps from myo-inositol. The stereoselective addition of the chiral phosphorylating agent (2R,4S,5R)-2-chloro-3,4-dimethyl-5-phenyl-1,3,2-oxazaphospholidin-2-one to a protected inositol intermediate allowed separation of diastereoisomers and easy deprotection to myo-inositol 3-phosphate. This strategy also allows the possible introduction of labels of oxygen and sulphur to give a thiophosphate of known stereochemistry at phosphorus which would be useful for the analysis of the stereochemical course of phosphate hydrolysis catalysed by inositol monophosphatase.

A biological-inspired support frame for an artificial cornea

Bilateral corneal blindness represents a quarter of the total blind, world-wide. The artificial cornea in assorted forms, was developed to replace opaque non-functional corneas and to return sight in otherwise hopeless cases that were not amenable to corneal grafts; believed to be 2% of corneal blind. Despite technological advances in materials design and tissue engineering no artificial cornea has provided absolute, long-term success. Formidable problems exist, due to a combination of unpredictable wound healing and unmanageable pathology. To have a solid guarantee of reliable success an artificial cornea must possess three attributes: an optical window to replace the opaque cornea; a strong, long term union to surrounding ocular tissue; and the ability to induce desired host responses. A unique artificial cornea possesses all three functional attributes- the Osteo-odonto-keratoprosthesis (OOKP). The OOKP has a high success rate and can survive for up to twenty years, but it is complicated both in structure and in surgical procedure; it is expensive and not universally available. The aim of this project was to develop a synthetic substitute for the OOKP, based upon key features of the tooth and bone structure. In doing so, surgical complexity and biological complications would be reduced. Analysis of the biological effectiveness of the OOKP showed that the structure of bone was the most crucial component for implant retention. An experimental semi-rigid hydroxyapatite framework was fabricated with a complex bone-like architecture, which could be fused to the optical window. The first method for making such a framework, was pressing and sintering of hydroxyapatite powders; however, it was not possible to fabricate a void architecture with the correct sizes and uniformity of pores. Ceramers were synthesised using alternative pore forming methods, providing for improved mechanical properties and stronger attachment to the plastic optical window. Naturally occurring skeletal structures closely match the structural features of all forms of natural bone. Synthetic casts were fabricated using the replamineform process, of desirable natural artifacts, such as coral and sponges. The final method of construction by-passed ceramic fabrication in favour of pre-formed coral derivatives and focused on methods for polymer infiltration, adhesion and fabrication. Prototypes were constructed and evaluated; a fully penetrative synthetic OOKP analogue was fabricated according to the dimensions of the OOKP. Fabrication of the cornea shaped OOKP synthetic analogue was also attempted.

Synthesis and biological properties of 2-phenylbenzothiazole derivatives

2-Phenylbenzothiazoles have structural similarities to the antioestrogenic 2-phenylindole, zindoxifene and to the oestrogenic isoflavone, genistein which also inhibits tyrosine kinases. Hydroxylated 2-phenylbenzothiazole derivatives were therefore produced and tested for oestrogenic and tyrosine kinase inhibitory activity. Synthesis of methoxy substituted 2-phenylbenzothiazoles was via the Jacobson method, demethylation being effected by boron tribromide at -70oC. Three amino substituted 2-phenylbenzothiazoles were also synthesised and tested for activity. Data is presented for oestrogen receptor binding activity, aromatase inhibitory activity, epidermal growth factor receptor tyrosine kinase (EGFRTK) inhibitory activity and cytotoxicity to ANN-1, 3T3, MCF-7 and WIDR cells. Oestrogen receptor binding affinity (RBA) was shown by five of the nine compounds tested. 2-(4-hydroxy)-6-hydroxybenzo-thiazole was the most active of the benzothiazoles tested (RBA 0.7). This is low but comparable to that of genistein. EGFRTK inhibitory activity was shown by four of the six benzothiazole derivatives tested; activity was comparable to that of genistein. Cytotoxicity assays have shown no selective toxicity of 2-phenylbenzothiazoles to any of the cell lines tested. Toxicity to MCF-7 cells was similar to that for other cell lines despite some compounds showing oestrogen receptor binding capacity. Amino-substituted 2-phenylbenzothiazoles showed selective toxicity towards transformed ANN-1 cells compared to normal 3T3 cells but the mechanism of this selectivity has not been established. Molecular modelling techniques, including CHEM-X, QUANTA and MOPAC were used to compare known ATP-competitive tyrosine kinase inhibitors with a model of ATP built from the crystal structure of the ATP-phosphoglycerate kinase complex. Structural features thought to be important to kinase inhibition were found and used to suggest further 2-phenylbenzothiazole analogues which may have improved activity.

Structure-activity relationships for α-calcitonin gene-related peptide

Calcitonin gene-related peptide (CGRP) is a member of the calcitonin (CT) family of peptides. It is a widely distributed neuropeptide implicated in conditions such as neurogenic inflammation. With other members of the CT family, it shares an N-terminal disulphide-bonded ring which is essential for biological activity, an area of potential α-helix, and a C-terminal amide. CGRP binds to the calcitonin receptor-like receptor (CLR) in complex with receptor activity-modifying protein 1 (RAMP1), a member of the family B (or secretin-like) GPCRs. It can also activate other CLR or calcitonin-receptor/RAMP complexes. This 37 amino acid peptide comprises the N-terminal ring that is required for receptor activation (residues 1-7); an α-helix (residues 8-18), a region incorporating a β-bend (residues 19-26) and the C-terminal portion (residues 27-37), that is characterized by bends between residues 28-30 and 33-34. A few residues have been identified that seem to make major contributions to receptor binding and activation, with a larger number contributing either to minor interactions (which collectively may be significant), or to maintaining the conformation of the bound peptide. It is not clear if CGRP follows the pattern of other family B GPCRs in binding largely as an α-helix. Linked Articles This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7 © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Assessment of physical and hydrological properties of biological soil crusts using x-ray microtomography and modelling

Biological soil crusts (BSCs) are formed by aggregates of soil particles and communities of microbial organisms and are common in all drylands. The role of BSCs on infiltration remains uncertain due to the lack of data on their role in affecting soil physical properties such as porosity and structure. Quantitative assessment of these properties is primarily hindered by the fragile nature of the crusts. Here we show how the use of a combination of non-destructive imaging X-ray microtomography (XMT) and Lattice Boltzmann method (LBM) enables quantification of key soil physical parameters and the modeling of water flow through BSCs samples from Kalahari Sands, Botswana. We quantify porosity and flow changes as a result of mechanical disturbance of such a fragile cyanobacteria-dominated crust. Results show significant variations in porosity between different types of crusts and how they affect the flow and that disturbance of a cyanobacteria-dominated crust results in the breakdown of larger pore spaces and reduces flow rates through the surface layer. We conclude that the XMT–LBM approach is well suited for study of fragile surface crust samples where physical and hydraulic properties cannot be easily quantified using conventional methods.

Quantitative structure-activity relationships and the prediction of MHC supermotifs

The underlying assumption in quantitative structure–activity relationship (QSAR) methodology is that related chemical structures exhibit related biological activities. We review here two QSAR methods in terms of their applicability for human MHC supermotif definition. Supermotifs are motifs that characterise binding to more than one allele. Supermotif definition is the initial in silico step of epitope-based vaccine design. The first QSAR method we review here—the additive method—is based on the assumption that the binding affinity of a peptide depends on contributions from both amino acids and the interactions between them. The second method is a 3D-QSAR method: comparative molecular similarity indices analysis (CoMSIA). Both methods were applied to 771 peptides binding to 9 HLA alleles. Five of the alleles (A*0201, A* 0202, A*0203, A*0206 and A*6802) belong to the HLA-A2 superfamily and the other four (A*0301, A*1101, A*3101 and A*6801) to the HLA-A3 superfamily. For each superfamily, supermotifs defined by the two QSAR methods agree closely and are supported by many experimental data.