Initial studies involved the development of a novel polymer microarray platform which would provide unsurpassed miniaturisation for polymer screening. This required substantial development and optimisation of several parameters and the best results were achieved by printing polymers dissolved in 1-methyl-2-pyrrolidinone and patterned using a contact microarrayer with solid pins. Microarray platforms were developed that used different substrates. The first one used a gold-coated substrate that quenched non-specifically bound fluorescently-labelled proteins, whereas the second utilised a hydrogel coating that prevented non-specific cellular adhesion. The platform using the gold coated substrate was ideally suited to the high throughput study of the physico-chemical properties of the arrayed polymer libraries, via scanning electron microscopy, FT-IR and TOF-SIMS. These polymer microarray platforms provided high throughput while minimising the amount of both polymers and expensive reagents used. The polymer microarrays were used with both adherent and non-adherent immortalised cell lines. In both cases, polymers could be selected that provided selective cellular immobilisation. Such methodologies were subsequently utilised to identify novel materials that allowed gentle immobilisation of human primary renal tubular epithelial cells and mouse bone marrow dendritic cells. This platform was applied to the identification of polymers with potential applications in stem cell biology. In one project polymers were screened for the selective immobilisation of multipotent mesenchymal stromal cell populations from human bone marrow. Several poly(urethanes) with large soft segments provided unexpectedly high selective adhesion of the stromal population. The second project investigated the use of novel substrates that maintained mouse embryonic stem cell cultures in their undifferentiated phenotype state. Finally, the polymer microarray platform was optimised for the study of protein adhesion.

Sensitivity analysis studies how changes in the parameters affect the system's variables. Its application to metabolic systems (Metabolic Control Analysis, MCA) was traditionally developed under certain assumptions:i) the steady state is stable (the effect on the steady state values only is studied).ii) each reaction is catalyzed by one enzyme, the rates being proportional to the corresponding enzyme concentration.iii) the parameters are changed by a small (strictly speaking infinitesimal) amount. In the present work MCA is extended to deal with the instantaneous values of time-dependent metabolite concentrations and fluxes. Their summation and connectivity relationships are derived. In some cases it is more convenient to characterize the time courses by time-invariant variables (such as period and amplitude in oscillating systems). Summation relationships for time-invariant variables are also derived. Stability analysis shows that a linear chain of four enzyme-catalized reactions, where the third metabolite is a negative effector of the first enzyme constitutes a 'minimal' oscillator. The model is used to gain insight into the control of oscillations. The control exerted by enzyme concentrations and other parameters that are not proportional to the rate is appropriately described by parameter-unspecified coefficients (C<SUB>v</SUB>). A proof of the theorems of steady-state MCA in terms of C<SUB>v</SUB> is given. By a similar procedure an attempt is made to derive the theorems in terms of C<SUB>v</SUB> for time-dependent systems, which is only successful for the particular case of constant π-matrix. The effect that a simultaneous change in all the enzyme concentrations by the same factor α (Coordinate-Control Operation. CCO) has on the variables of time-dependent metabolic systems is investigated. This factor α can have any arbitrary large value. The metabolic variables are classified according to the relationships they fulfil when the CCO is applied. A method is given to test these relationships in experimental systems and quantify deviations from the predicted behaviour.

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The yolk proteins (YPs) in <i>Drosophila</i> have been shown to enter the oocyte via receptor mediated endocytosis (Mahowald, 1972; Butterworth <i>et al</i>., 1991). The YPs are sorted and specifically stored in the α-granules of the oocyte (Mahowald and Kambysellis, 1980). The aim of this project is to determine the YP domains involved in the specific interactions resulting in uptake into the oocyte. An evolutionary approach was chosen to attempt to determine these domains, since the YPs were assumed to have few evolutionary constraints and thus diverge readily. A functional assay was designed to investigate the uptake of foreign YPs in <i>D. melanogaster</i> and used to test YPs from different <i>Drosophila</i> and non-<i>Drosophila</i> species. The results indicated that the functional domains imvolved in YP uptake were conserved for up to 100 MYR (Million years) of Dipteran evolution. The molecular cloning and DNA sequence determination of <i>Calliphora yp</i> genes demonstrated that the YPs were very well conserved. Not only had the <i>Calliphora</i> YPs failed to diverge as expected but these also displayed the similarity to the vertebrate lipases shown in <i>Drosophila</i> YPs (Bownes <i>et al</i>., 1988; Perssons <i>et al</i>., 1989). Therefore suggesting that this is a structural constraint within the YPs of Diptera. The conservation of the amino acid sequence of the YPs did not suggest any domains which may have been involved in uptake; however, computer based database searches with five different regions of the protein suggested putative sites of importance for YP uptake. Their origin and possible functions are discussed. The <i>Calliphora yp</i> genes were also found to be expressed in a coordinate manner by both carcasses and ovaries, between stages 8 and 10b of oogenesis. These <i>yp</i> genes were found to be expressed in the follicle cells of the oocyte thus suggesting that the regulation of the <i>yp</i> gene expression in <i>Calliphora</i> and <i>Drosophila</i> is also conserved and thus possibly regulatory sequences and factors will be similar in these two species.

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No description available.

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Several fluorescent labels were studied to develop simple luminescence immunoassay methods for both macromolecules and low molecular weight analytes. Initially, the 'Fluram (fluorescamine) enhancement phenomenon' was investigated by extending its use for assay of the-thyroid hormone, triiodothyronine (T3). The observed enhanced fluorescence permitted the development of a homogeneous assay capable of detecting nanogram concentrations of the hormone in pure solution and hormone depleted serum. The technique was however, complicated by the overlapping Raman scatter peak and attempts to overcome this problem included the use of derivative spectroscopy [continued]…