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The electron-nuclear Overhauser effect on spin ½ nuclei in solutions containing free radicals has been calculated from a method based on treatment used by Solomon. The magnitude and sign of the enhancement of the nuclear signal, which may be represented by a factor ρ depends on the nature and time dependence of the the electron-nuclear signal, which is assumed to be either dipolar or scalar. The time dependence for the dipole-dipole interactions is assumed to arise from the translational diffusion of the free radicals and the molecule containing the nuclei. three models are employed for the scalar interactions. In the first of these, termed the Sticking Model, the scalar interaction is assumed to be finite only while a molecule and free radical are "stuck" together. Thus sticking time is assumed to be short in comparison with the diffusional correlation time. However, an alternative model in which this assumption is not made has been postulated to explain certain experimental results. This is termed the Modified Sticking Model. In the third model, called the Diffusion Model, the hypothesis is made that the scalar interaction is a very short range interaction with magnitude ℏ B(d/R) exp. [-λ(r-d)]. where λ d ≫ 1, B and λ are constants, R is the distance between a nucleus and an electron, and d is the distances of the closest approach, inside of which the interaction is zero. In this model the time dependence of the scalar interaction, as in the case of the dipolar interactions, arises from the relative diffusion of the molecules and free radicals in the solution. For all the models of the scalar interaction, ρ may be plotted as a function of wsτc where ws is the electron Larmor frequency and τc the correlation time and it is found that the sign of ρ may change. The product wsτc may be altered by working at different magnetic fields or changing ρc. From measurements, at two different magnetic fields, of the nuclear spin-lattice relaxation times, the concentration of free radical in the solution and ρ, the theories allow the calculation of the mean diffusion coefficient of the radicals and molecules in solution, the correlation times and the measure of the relative importance of scalar and dipolar interactions. Experiments on fluorinated and protonic solvents containing the radical tri-tert-butyl phenoxyl (T.T.B.P) have been carried out at two different magnetic fields. The proton-free radical interactions may be explained in terms of dipole-dipole interactions. On the other hand with fluorine nuclei scalar coupling does seem to be important. The Sticking Model does not successfully describe the effect of this coupling. The other models give reasonable values for the calculable parameters, but it is difficult to decide unambiguously between them. If scalar coupling does occur, a scalar shift of the nuclear resonance is expected. this has been observed for hexaflourobenzene solutions containing T.T.B.P. The theoretical and experimental results are compared, and while either theory might explain the observed shift, less assumptions have to be made in using the Diffusion Model. The correlation time dependence of the Overhauser effect has also been studied for two systems, hexafluorobenzene and 1-1-1-trichloro-2-2-2-trifluoro-ethane, at two magnetic fields. The correlation times were altered by addition of carbon disulphide or a chlorinated hydrocarbon or by changing the temperature of the sample. Qualitatively the Diffusion Model was in best agreement with the experimental results, but quantitatively, neither model was satisfactory. In the Diffusion Model, the assumption λd≫1 is made and in an attempt to obtain better agreement with the experimental results ρ has been calculated using different values of λd in thescalar interaction function. The best agreement between experiment and theory was for values of λd≫ 1000. Measurements of the proton electron Overhauser effect can usually be interpreted in terms of dipolar interactions (positive ρ) between the electrons and the protons. However for the resonances of the tert-butyl groups of tri-tert-butyl phenol in solutions of T.T.B.P. ρ is negative. This has been shown to be associated with the proton exchange between the radical and phenol molecules. It is postulated that in the radical molecule the scalar coupling between the electron and the tert-buyl protons, occurs at least in part by a hyperconjugative mechanism. the random rotation of these tert-butyl groups could modulate this scalar coupling. This would lead to a positive polarization of the tert-butyl protons, which persists when the radical accepts a proton and becomes a tri-tert-butyl phenol molecule.

Proteomics can help to discover and quantify novel biomarkers and we show how this is achieved using liver fibrosis as an example. Liver biopsy is the reference standard for assessing liver fibrosis and serum biomarkers are less invasive. The severity of liver fibrosis can be determined using immunoassays. However biomarkers may be degraded due to sample storage conditions and therefore may not be detected using these antibody-based assays. Detection of biomarkers by mass spectrometry overcomes this disadvantage and the approach can be applied to all diseases. Two dimensional gel electrophoresis (2DE) was used to find differences in proteins in plasma samples from patients with varying liver fibrosis stages. The identified proteins were potential liver fibrosis biomarkers. Mass spectrometry can assay for biomarkers by detecting their tryptic peptides and fragments and was used to develop an antibody-free assay. We are the first and only lab in the UK to use a novel absolute quantitation method which is the only biomarker assay using a universal calibration mix. Using 2DE, we identified several potential biomarkers for liver fibrosis which were analysed by Western blotting using plasma samples from patients with varying stages of liver fibrosis. Our novel biomarkers were promising when compared to the markers used in current liver fibrosis tests. A mass spectrometry assay was developed for the best novel liver fibrosis biomarker. We are working towards the first ever antibody-free biomarker assay for liver fibrosis. Our assay is nine times faster than conventional quantitation by mass spectrometry making our approach for absolute biomarker quantitation applicable for clinical use. This is also the only assay which can analyse all points of the calibration curve and determine the absolute concentration of the biomarker in a single acquisition. Our assay may help reduce the need for invasive liver biopsies and the approach could be used for any other disease.

Mucosal pathogens target sites of infection through specific adherence to host glycoconjugate receptors. As a consequence, depletion of such receptors from the cell surface may be expected to inhibit attachment, impair bacterial colonization and reduce the activation of mucosal inflammation. We have used the glucose analogue and glycosphingolipid (GSL) biosynthesis inhibitor N-butyldeoxynojirimycin (NB-DNJ) to deplete human uroepithelial cells and the murine urinary tract mucosa of receptors for P-fimbriated Escherichia coli. NB-DNJ blocks the ceramide-specific glucosyltransferase, which catalyses the formation of glucosyl ceramide (GlcCer), the precursor for GSLs. The inhibitor was shown to decrease the GSL content in a dose-dependent way, and depletion markedly inhibited P-fimbriated bacterial attachment in vitro. NB-DNJ-fed C3H/HeN mice were depleted of GSLs in vivo and showed reduced susceptibility to experimental urinary tract infection with P-fimbriated E. coli. The mucosal inflammatory response was impaired, as shown by reduced chemokine secretion and lower neutrophil recruitment, and the bacteria colonized the urinary tract less efficiently than in normal mice. These results confirmed the role of P fimbriae-mediated adherence for colonization and inflammation and point to an interesting alternative to antibiotic treatment for urinary tract infection.

Immunologically self carbohydrates protect the human immunodeficiency virus type -1 (HIV-1) surface glycoprotein, gp120 from antibody recognition. However, one broadly neutralising antibody, 2G12, can protect against primary viral challenge by direct recognition of these "self" glycans on gp120.

The morphogenesis of HCV belongs to steps of the viral cycle that have not yet been targeted by antiviral strategies. With the lack of an efficient and reliable culture system able to produce and secrete HCV virions, we use two complementary approaches to study the effect of c~-gluensidase inhibitors on HCV morphogenesis, secretion and entry. These two approaches have allowed us to demonstrate tile mechanism of tile antiviral effect of O,-glucosidase inhibitors on HCV glyenprotein folding and assembly, morphogenesis, viral secretion, and viral infectivity.

The antiviral properties of iminosugars have been reported previously in vitro and in small animal models against Ebola virus (EBOV); however, their effects have not been tested in larger animal models such as guinea pigs. We tested the iminosugars N-butyl-deoxynojirimycin (NB-DNJ) and N-(9- methoxynonyl)-1deoxynojirimycin (MON-DNJ) for safety in uninfected animals, and for antiviral efficacy in animals infected with a lethal dose of guinea pig adapted EBOV. 1850 mg/kg/day NB-DNJ and 120 mg/kg/day MON-DNJ administered intravenously, three times daily, caused no adverse effects and were well tolerated. A pilot study treating infected animals three times within an 8 hour period was promising with 1 of 4 infected NB-DNJ treated animals surviving and the remaining three showing improved clinical signs. MON-DNJ showed no protective effects when EBOV-infected guinea pigs were treated. On histopathological examination, animals treated with NB-DNJ had reduced lesion severity in liver and spleen. However, a second study, in which NB-DNJ was administered at equally- spaced 8 hour intervals, could not confirm drug-associated benefits. Neither was any antiviral effect of iminosugars detected in an EBOV glycoprotein pseudotyped virus assay. Overall, this study provides evidence that NB-DNJ and MON-DNJ do not protect guinea pigs from a lethal EBOV-infection at the dose levels and regimens tested. However, the one surviving animal and signs of improvements in three animals of the NB-DNJ treated cohort could indicate that NB-DNJ at these levels may have a marginal beneficial effect. Future work could be focused on the development of more potent iminosugars