Importance of tissue transglutaminase in repair of extracellular matrices and cell death of dermal fibroblasts after exposure to a solarium ultraviolet A source

Investigations were undertaken to study the role of the protein cross-linking enzyme tissue transglutaminase in changes associated with the extracellular matrix and in the cell death of human dermal fibroblasts following exposure to a solarium ultraviolet A source consisting of 98.8% ultraviolet A and 1.2% ultraviolet B. Exposure to nonlethal ultraviolet doses of 60 to 120 kJ per m2 resulted in increased tissue transglutaminase activity when measured either in cell homogenates, "in situ" by incorporation of fluorescein-cadaverine into the extracellular matrix or by changes in the epsilon(gamma-glutamyl) lysine cross-link. This increase in enzyme activity did not require de novo protein synthesis. Incorporation of fluorescein-cadaverine into matrix proteins was accompanied by the cross-linking of fibronectin and tissue transglutaminase into nonreducible high molecular weight polymers. Addition of exogenous tissue transglutaminase to cultured cells mimicking extensive cell leakage of the enzyme resulted in increased extracellular matrix deposition and a decreased rate of matrix turnover. Exposure of cells to 180 kJ per m2 resulted in 40% to 50% cell death with dying cells showing extensive tissue transglutaminase cross-linking of intracellular proteins and increased cross-linking of the surrounding extracellular matrix, the latter probably occurring as a result of cell leakage of tissue transglutaminase. These cells demonstrated negligible caspase activation and DNA fragmentation but maintained their cell morphology. In contrast, exposure of cells to 240 kJ per m2 resulted in increased cell death with caspase activation and some DNA fragmentation. These cells could be partially rescued from death by addition of caspase inhibitors. These data suggest that changes in cross-linking both in the intracellular and extracellular compartments elicited by tissue transglutaminase following exposure to ultraviolet provides a rapid tissue stabilization process following damage, but as such may be a contributory factor to the scarring process that results.

Integration of the Manakov-PMD Equation with Precomputed M(w) Matrices for PMD Simulation

A novel direct integration technique of the Manakov-PMD equation for the simulation of polarisation mode dispersion (PMD) in optical communication systems is demonstrated and shown to be numerically as efficient as the commonly used coarse-step method. The main advantage of using a direct integration of the Manakov-PMD equation over the coarse-step method is a higher accuracy of the PMD model. The new algorithm uses precomputed M(w) matrices to increase the computational speed compared to a full integration without loss of accuracy. The simulation results for the probability distribution function (PDF) of the differential group delay (DGD) and the autocorrelation function (ACF) of the polarisation dispersion vector for varying numbers of precomputed M(w) matrices are compared to analytical models and results from the coarse-step method. It is shown that the coarse-step method achieves a significantly inferior reproduction of the statistical properties of PMD in optical fibres compared to a direct integration of the Manakov-PMD equation.

Towards a polymeric binding mimic for cytochrome CYP2D6

A series of fluorescent molecularly imprinted polymers has been prepared with a view to generating material capable of mimicking the binding characteristics of the metabolically important cytochrome isoform CYP2D6. Such polymers would have the possibility to form the sensing element in a high-throughput assay for the prediction of CYP2D6 affinity. The imprinted polymers possessed binding-dependent fluorescence. They re-bound their templates and various cross-reactivities were encountered for test compound/drug recognition. One polymer in particular exhibited a rational discrimination amongst the related synthetic templates and was reasonably successful in recognising CYP2D6 substrates from a drug panel. © 2005 Elsevier B.V. All rights reserved.

Numerical implementation of the Manakov-PMD equation with precomputed M(Ω) matrices

The Manakov-PMD equation can be integrated with the same numerical efficiency as the coarse-step method by using precomputed M(Ω) matrices, which entirely avoids the somewhat ad-hoc rescaling of coefficients necessary in the coarse-step method.

Typical kernel size and number of sparse random matrices over Galois fields: a statistical physics approach

Using methods of statistical physics, we study the average number and kernel size of general sparse random matrices over GF(q), with a given connectivity profile, in the thermodynamical limit of large matrices. We introduce a mapping of GF(q) matrices onto spin systems using the representation of the cyclic group of order q as the q-th complex roots of unity. This representation facilitates the derivation of the average kernel size of random matrices using the replica approach, under the replica symmetric ansatz, resulting in saddle point equations for general connectivity distributions. Numerical solutions are then obtained for particular cases by population dynamics. Similar techniques also allow us to obtain an expression for the exact and average number of random matrices for any general connectivity profile. We present numerical results for particular distributions.

Properties of sparse random matrices over finite fields

Typical properties of sparse random matrices over finite (Galois) fields are studied, in the limit of large matrices, using techniques from the physics of disordered systems. For the case of a finite field GF(q) with prime order q, we present results for the average kernel dimension, average dimension of the eigenvector spaces and the distribution of the eigenvalues. The number of matrices for a given distribution of entries is also calculated for the general case. The significance of these results to error-correcting codes and random graphs is also discussed.

Modified hydrogel matrices in fibre optic sensors

Common problems encountered in clinical sensing are those of non-biocompatibility, and slow response time of the device. The latter, also applying to chemical sensors, is possibly due to a lack of understanding of polymer support or membrane properties and hence failure to optimise membranes chosen for specific sensor applications. Hydrogels can be described as polymers which swell in water. In addition to this, the presence of water in the polymer matrix offers some control of biocompatibility. They thus provide a medium through which rapid transport of a sensed species to an incorporated reagent could occur. This work considers the feasibility of such a system, leading to the design and construction of an optical sensor test bed. The development of suitable membrane systems and of suitable coating techniques in order to apply them to the fibre optics is described. Initial results obtained from hydrogel coatings implied that the refractive index change in the polymer matrix, due to a change in water content with pH is the major factor contributing to the sensor response. However the presence of the colourimetric reagent was also altering the output signal obtained. An analysis of factors contributing to the overall response, such as colour change and membrane composition were made on both the test bed, via optical response, and on whole membranes via measurement of water content change. The investigation of coatings with low equilibrium water contents, of less than 10% was carried out and in fact a clearer signal response from the test bed was noted. Again these membranes were suprisingly responding via refractive index change, with the reagent playing a primary role in obtaining a sensible or non-random response, although not in a colourimetric fashion. A photographic study of these coatings revealed some clues as to the physical nature of these coatings and hence partially explained this phenomenon. A study of the transport properties of the most successful membrane, on a coated wire electrode and also on the fibre optic test bed, in a series of test environments, indicated that the reagent was possibly acting as an ion exchanger and hence having a major influence on transport and therefore sensor characteristics.

Synthesis and characterisation of imprinted polymeric receptor mimics

Molecularly imprinted polymers (MIPs) are crosslinked polymers containing bespoke functionalised cavities arising from the inclusion of template molecules in the polymerisation mixture and their later extraction. When the polymers are prepared functional polymerisable monomers are included which become part of the polymer matrix and serve to decorate the cavities with functionality appropriate to the template molecules. Overall, binding sites are created which have a memory for the template both in terms of shape and matching functionality. Fluorescent molecularly imprinted polymers have the benefit of a fluorophore in their cavities that may respond to the presence of bound test compound by a change in their fluorescence output. The work presented falls into three main areas. A series of fluorescent MIPs was prepared with a view to generating material capable of mimicking the binding characteristics of the metabolically important cytochrome isoform CYP2D6. The MIPs re-bound their templates and various cross-reactivities were encountered for test compound/drug recognition. One MIP in particular exhibited a rational discrimination amongst the related synthetic templates and was reasonably successful in recognising CYP2D6 substrates from the drug set tested. In order to give some insights into binding modes in MIPs, attempts were made to produce functional monomers containing two or more fluorophores that could be interrogated independently. A model compound was prepared which fitted the dual-fluorophore criteria and which will be the basis for future incorporation into MIPs. A further strand to this thesis is the deliberate incorporation of hydrophobic moieties into fluorescent functional monomers so that the resulting imprinted cavities might be biomimetic in their impersonation of enzyme active sites. Thus the imprinted cavities had specific hydrophobic regions as well as the usual polar functionality with which to interact with binding test compounds.

Synthesis and evaluation of polystyrene based soluble polymeric supports for organic synthesis

Several copolymers of linear polystyrene were prepared for evaluation as soluble polymeric supports for organic synthesis. These polymers were utilized for the synthesis of ?2-isoxazoline compounds. The target compounds were synthesized via 1,3-dipolar cycloaddition reactions between polymer bound alkenes and nitrile oxides generated in situ from their corresponding aldoximes. The cleaved ?2-isoxazoline compounds were tested for biological activity against Mycobacterium fortuitum. To compare the success of these linear polystyrene copolymers, some of the ?2-isoxazoline compounds synthesized on soluble polymeric supports were also prepared via traditional crosslinked polymer supports. The polymer-bound ?2-isoxazolines were also tested for antimicrobial activity. In addition attempts were made to prepare polymers containing the ?2-isoxazolines but anchored by non-hydrolysable bonds. Although the copolymers of polystyrene gave good loading capacity in mmol/g, and being soluble in chlorinated solvents it was possible to monitor the reactions by 1H NMR spectroscopy, the cleavage of the polymer bound products proved to be quite troublesome. Product purification was not as straightforward as it was anticipated. Isolation of the cleaved target compounds proved to be time consuming and laborious when compared to the traditional organic synthesis and solid phase organic synthesis (SPOS). Polymer-bound ?2-isoxazolines close to the polymer backbone exhibited some biological activity against Staphylococcus aureus. Polymers with substitution at the para-position of the aryl substituent at position 3 of isoxazoline ring showed antimicrobial activity.

A study in radiopaque polymeric materials

The problems associated with x-ray-transparent denture base are defined and conventional approaches to their solution are assessed. Consideration of elemental absorption parameters leads to the postulation that atoms such as zinc, and bromine, may be effective radiopacifiers over at least part of the clinical x-ray spectrum. These elements had hitherto been considered too light to be effective. Investigation of copolymers of methylmethacrylate and p-bromostyrene revealed no deleterious effects arising from the aromatically brominated monomer (aliphatic bromination caused UV destabilisation). For effective x-ray absorption a higher level of bromination would be necessary, but the expense of suitable compounds made further study unjustifiable. Incorporation of zinc atoms into the polymer was accomplished by copolymerisation of zinc acrylate with methylmethacrylate in solution. At high zinc levels this produced a powder copolymer convenient for addition to dental polymers in the dough moulding process. The resulting mouldings showed increasing brittleness at high loadings of copolymer. Fracture was shown to be through the powder particles rather than around them, indicating the source of weakness to be in the internal structure of the copolymer. The copolymer was expected to be cross-linked through divalent zinc ions and its insolubility and infusibility supported this. Cleavage of the ionic cross links with formic acid produced a zinc-free linear copolymer of high molecular weight. Addition of low concentrations of acrylic acid to the dough moulding monomer appeared to 'labilise' the cross links producing a more homogeneous moulding with adequate wet strength. Toxicologically the zinc-containing materials are satisfactory and though zinc is extracted at a measurable rate in an aqueous system, this is very small and should be acceptable over the life of a denture. In other respects the composite is quite satisfactory and though a marketable product is not claimed the system is considered worthy of further study.

Drug/Water interactions in hydrogel matrices

The primary aim of this research has been the investigation of the role of water structuring effects in the widely different extents of irritancy displayed by certain antibiotics. The compounds involved were members of the Lincomycin group of antibiotics. The aqueous solution behaviour of these co~pounds was studied using techniques such as vapour pressure osmometry end differential scanning calorimetry (D.S.C.). The effects of the antibiotics on water structure in hydrogel membrane preparations In which the equilibrium water content (E.W.C.) and constituent amounts of freezing and non-freezing water ware varied were also investigated using D.S.C. The permeability of water swollen hydrogel preparations to aqueous antibiotic solutions as well as other solutes were studied. A series of hydrogel preparations into which the antibiotics had been incorporated during polymerisation were developed and used in studies of the effects of the antibiotics end their water structure modifications on the permeation of a range of solutes.

The controlled release of macromolecules from macroporous hydrophyllic polymer matrices

This work has used novel polymer design and fabrication technology to generate bead form polymer based systems, with variable, yet controlled release properties, specifically for the delivery of macromolecules, essentially peptides of therapeutic interest. The work involved investigation of the potential interaction between matrix ultrastructural morphology, in vitro release kinetics, bioactivity and immunoreactivity of selected macromolecules with limited hydrolytic stability, delivered from controlled release vehicles. The underlying principle involved photo-polymerisation of the monomer, hydroxyethyl methacrylate, around frozen ice crystals, leading to the production of a macroporous hydrophilic matrix. Bead form matrices were fabricated in controllable size ranges in the region of 100µm - 3mm in diameter. The initial stages of the project involved the study of how variables, delivery speed of the monomer and stirring speed of the non solvent, affectedthe formation of macroporous bead form matrices. From this an optimal bench system for bead production was developed. Careful selection of monomer, solvents, crosslinking agent and polymerisation conditions led to a variable but controllable distribution of pore sizes (0.5 - 4µm). Release of surrogate macromolecules, bovine serum albumin and FITC-linked dextrans, enabled factors relating to the size and solubility of the macromolecule on the rate of release to be studied. Incorporation of bioactive macromolecules allowed retained bioactivity to be determined (glucose oxidase and interleukin-2), whilst the release of insulin enabled determination of both bioactivity (using rat epididymal fat pad) and immunoreactivity (RIA). The work carried out has led to the generation of macroporous bead form matrices, fabricated from a tissue biocompatible hydrogel, capable of the sustained, controlled release of biologically active peptides, with potential use in the pharmaceutical and agrochemical industries.

The durability of glass fibre reinforced cements made with new cementitious matrices

Widespread use of glass fibre reinforced cement (GRC) has been impeded by concerns over its durability. Three degradation mechanisms are proposed - fibre corrosion, Ca(OHh precipitation and matrix densification - although their relative importance is debated. Matrices with reduced alkalinities and Ca(OH)2 contents are being developed; the aim of this study was to investigate their hydration and interaction with alkali-resistant fibres to determine the factors controlling their long-term durability, and assess the relevancy of accelerated ageing. The matrices studied were: OPC/calcium-sulphoaluminate cement plus metakaolin (C); OPC plus metakaolin (M); blast-furnace slag cement plus a micro-silica based additive (D); and OPC (O). Accelerated ageing included hot water and cyclic regimes prior to tensile testing. Investigations included pore solution expression, XRD, DTA/TG, SEM and optical petrography. Bond strength was determined from crack spacings using microstructural parameters obtained from a unique image analysis technique. It was found that, for the new matrices - pore solution alkalinities were lower; Ca(OH)2 was absent or quickly consumed; different hydrates were formed at higher immersion temperatures; degradation under 65°C immersion was an order of magnitude slower, and no interfilamental Ca(OH)2 was observed .It was concluded that: fibre weakening caused by flaw growth was the primary degradation mechanism and was successfully modelled on stress corrosion/static fatigue principles. OPC inferiority was attributed partly to its higher alkalinity but chiefly to the growth of Ca(OH)2 aggravating the degradation; and hot water ageing although useful in model formulation and contrasting the matrices, changed the intrinsic nature of the composites rather than simply accelerating the degradation mechanisms.