Cysteine is a thiol containing amino acid that readily undergoes oxidation by reactive oxygen species (ROS) to form sulphenic (R-SOH) sulphinic (RSO2H) and sulphonic (RSO3H) acids. Thiol modifications of cysteine have been implicated as modulators of cellular processes and represent significant biological modifications that occur during oxidative stress and cell signalling. However, the different oxidation states are difficult to monitor in a physiological setting due to the limited availability of experimental tools. Therefore it is of interest to synthesise and use a chemical probe that selectively recognises the reversible oxidation state of cysteine sulphenic acid to understand more about oxidative signalling. The aim of this thesis was to investigate a synthetic approach for novel fluorescent probe synthesis, for the specific detection of cysteine sulphenic acids by fluorescence spectroscopy and confocal microscopy. N-[2-(Anthracen-2-ylamino)-2-oxoethyl]-3,5-dioxocyclohexanecarboxamide was synthesised in a multistep synthesis and characterised by nuclear magnetic resonance spectroscopy. The optimisation of conditions needed for sulphenic acid formation in a purified protein using human serum albumin (HSA) and the commercially available biotin tagged probe 3-(2,4-dioxocyclohexyl)propyl-5-((3aR,6S,6aS)-hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-6-yl)pentanoate (DCP-Bio1) were identified. This approach was extended to detect sulphenic acids in Jurkat T cells and CD4+ T cells pre- and post-stimulus. Buthionine sulfoximine (BSO) was used to manipulate the endogenous antioxidant glutathione (GSH) in human CD4+ T cells. Then the surface protein thiol levels and sulphenic acid formation was examined. T cells were also activated by the lectin phytohaemagglutinin-L (PHA-L) and formation of sulphenic acid was investigated using SDS-PAGE, western blotting and confocal microscopy. Resting Jurkat cells have two prominent protein bands that have sulphenic acid modifications whereas resting CD4+ T cells have an additional band present. When cells were treated with BSO the number of bands increased whereas activation reduced the number of proteins that were modified. The identities of the protein bands containing sulphenic acids were explored by mass spectrometry. Cysteine oxidation was observed in redox, metabolic and cytoskeletal proteins. In summary, a novel fluorescent probe for detection of cysteine sulphenic acids has been synthesised alongside a model system that introduces cysteine sulphenic acid in primary T cells. This probe has potential application in the subcellular localisation of cysteine oxidation during T cell signalling.

Transferrin, a widespread vertebrate protein, is responsible, along with its specific receptor, for all of the iron transported into the cell. Despite its ubiquity and structural similarity across species, heterologous molecules display different binding affinities for human cells. In general transferrin binds more avidly to the homologous receptor whatever the species. The impetus for the first part of this thesis came from these observations, coupled with research, which showed that rat conceptuses grown in human serum were anaemic and malformed, conditions rectified by supplementation with rat but not human transferrin. The second part of the thesis concerns the development of an intravitelline injection system to by-pass the rat visceral yolk sac which surrounds the embryo in its amniotic sac; in the human the yolk sac is situated externally to both. Due to this arrangement and also because of its known degradative abilities, the rat yolk sac presents toxicologists with a problem when using the rat model in teratological studies because they cannot be sure what quantity of a compound or even what metabolite the rat embryo is receiving. Thus if the extraembryonic membranes could be by-passed it would be possible to perform experiments which might provide more information regarding acute toxicity and help in the accurate assessment of teratological risk. This improved injection method allows such experiments to be carried out in a reproducible manner with greater accuracy and with more discrete quantities than was possible with the earlier system. The two parts have been drawn together in experiments which compare the uptake of human and rat transferrin in embryos cultured in serum containing, and injected with, these two molecules. Over all, the results indicate that the rat yolk sac has an affinity three times greater for rat transferrin than it does for human transferrin. Furthermore, it appears that the embryo cannot differentiate between the two molecules and that it probably does not possess a specific transferrin receptor. The practical aspects of this technique in relation to experimental embryology and acute reproductive toxicology have been explored in chapter 6 of this thesis. From the point of view of acute toxicity testing the results have been promising enough to elicit industrial sponsorship for developing the procedure further.

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