In this thesis regulation of amino acid transport system A in the placenta is investigated. Firstly, a suitable in vitro placental model is identified from two clones of the BeWo choriocarcinoma cell line.  Both the ATCC and b30 clones show the phenotypic and genotypic characteristics of the parent cell line.  The b30 clone forms a polarised monolayer when cultured on permeable filters suitable for studying transcellular transport.  The b30 clone was chosen as the model to be used for all further studies in this thesis. The regulation of amino acid transport system A by substrate restriction was demonstrated over 30 years ago in the placenta.  However, studies were limited to MeAIB uptake and did not include transcellular or molecular studies.  This thesis demonstrates that transcellular ⁴C-MeAIB transport is increased following amino acid restriction and that this increase is linked to the relocalisation of existing system A SNAT2 transporters to the plasma membrane and increased SNAT2 expression following chronic restriction. In contrast, SNAT1 does not appear to be involved in this response and is down regulated by non-essential amino acid deprivation. Amino acid transport system A is highly regulated by many factors and it was suggested that increased system A activity may be a general nutrient stress response.  This thesis tested this hypothesis by studying the effects of micronutrient deprivation on transcellular ⁴C-MeAIB transfer.  Both iron and copper deficiency caused significant increases in transcellular transfer that could be reversed upon micronutrient replenishment. Finally, the effects of endocrine stress on amino acid transport system A were investigated.

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The aim of this thesis was to test whether cross-linking ATP analogues could be used to provide direct evidence of the amino acid residues which contribute to the ATP binding domain of P2X receptors. The UV irradiation of 2-azido ATP caused the compound to cross-link to the P2X1 receptor and caused a significant reduction in the response to ATP. The reduction of cross-linking following the pre-treatment of cells in excess ATP suggested competition between ATP and 2-azido ATP for occupancy of the P2X receptor binding site. This was proven with the radio-labelling of the P2X1 receptor with 2-azido [y32p] ATP. Similar techniques were used to identify other photo-reactive compounds with activity at the P2X1 receptor. These photo-reactive compounds had reactive sites at different coordinates around the ATP molecule and can potentially cross-link to different regions within the ATP binding domain. In an attempt to localize the ATP binding site, the P2X1 receptor was tagged with flag and his epitopes and purified. Protein mass fingerprinting showed that the digestion of the P2X1 protein did not provide enough coverage of the protein to guarantee successful analysis by mass spectrometry. Additionally, initial studies using digestion to identify peptide fragments which had bound to 2-azido [y32P] ATP showed that experimental conditions caused the cleavage of the label from the peptide. This thesis has demonstrated that photo-reactive ATP analogues can be used to label the P2X receptors. Further work should identify a suitable combination of enzymes to efficiently fragment the protein and allow spectral analysis to identify the amino acid residues which covalently bind to each photo-reactive compound. This would give the first direct evidence of the contributing amino acid residues in the ATP binding domain of the P2X receptors.

A number of N-acetyl-S-glycosyl cysteine derivatives have been prepared through the development of a general and simply applicable synthetic pathway, by modifying existing literature methods. The coupling of N-acetyl-L-cysteine and a carbohydrate is desirable as it may improve the efficacy of the labile N-acetyl-L-cysteine as a drug. The S-glycosyl cysteines prepared are as follows: N-acetyl-S-D-glucopyranosyl-L-cysteine, alpha and beta-anomers, N-acetyl-S-beta-D-ribopyranosyl-L-cysteine, N-acetyl-S-alpha-D-mannopyranosyl-L-cysteine and N-acetyl-O-methyl-S-(2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl)-L-cysteine. The coupling reaction was designed to yield both a and beta-anomers in the same step, and this was observed in the synthesis of the glucose derivatives. However, the other carbohydrates chosen appear to couple more selectively. The preparation of N-acetyl-O-methyl-S-alpha-D-glucopyranosyl-L-cysteine was also carried out by a different method, but this proved to be more involved and resulted in lower yields. The stability of N-acetyl-S-beta-D-glucopyranosyl-L-cysteine towards the thioglycosidase enzyme myrosinase was studied. N-acetyl-S-beta-D-glucopyranosyl-L- cysteine was found to be stable to hydrolysis by myrosinase, but some inhibition of a standard sinigrin-myrosinase hydrolysis was observed. The thioglycosidic linkage (-SH) of N-acetyl-S-beta-D-glucopyranosyl-L-cysteine is stable to hydrolysis in acidic media, which is contrary to previous work reported in the literature. However, the hydrolysis of the amide group of the cysteine side chain occurred in acidic solution giving the apparently acid-stable S-beta-D-glucopyranosyl-L-cysteine. A kinetic study of this acetyl cleavage was undertaken and a second order rate constant of 5.96 +/- 0.24 x 10-4 dm3 mol-1 hours-1 was obtained.

Janus kinases (JAKs) and Signal Transducer and Activators of Transcription (STATs) are essential for signalling in response to the Interferons (IFNs) and most cytokines. Aberrant activation of JAKs and/or STATs has been observed in many human cancer cells, and several recent studies have suggested that STAT3 oncogenic pathways are also associated with intrinsic drug resistance. Chemoresistance is a major cause of failure of cancer treatment. In lung cancer cells chemoresistance can be mediated by Fibroblast Growth Factor 2 (FGF-2). In small cell lung cancer (SCLC) cell lines FGF-2 induces the expression of anti-apoptotic proteins via a MEK-ERK-dependent mechanism, which involves the formation of a multi-protein complex constituted by B-RAF, PKCε and S6K2. We investigated the generality of this phenomenon by analysing additional types of cells. Since there have been reports suggesting that activation of JAKs and/or STATs can occur downstream of FGF-2 we hypothesised that JAKs and/or STATs may also play a role in FGF-2 mediated chemoresistance pathway(s). In U2OS osteosarcoma cells, FGF-2 offers protection against cisplatin-induced cell death. As with SCLC cells this involves B-RAF, PKCε, S6K2, MAPKs and, most notably, JAKs (JAK1, JAK2, or TYK2), but not the STATs. Whereas the non-small lung cancer cells (NSCLC) HCC95 respond to FGF-2, HCC78 do not. In neither case, however, did FGF-2 mediate chemoresistance against cisplatin. Using RNA interference (RNAi), we have observed that ERK2 knockdown caused a concomitant reduction of STAT3 levels. The data suggest that ERK2, but not ERK1, levels can regulate STAT3 expression, and that ERK1 and ERK2 have non-overlapping functions. It also indicates that ERK2 protein levels can regulate STAT3, an important signal transducer and activator of transcription. The identification of a novel inter-play between the JAKs and MAPKs in the context of chemoresistance opens new avenues for treatment, which urgently warrant additional studies.

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Neuropeptide Y (NPY) is widely expressed in both the central and peripheral nervous system and has an important role in the regulation of adult hippocampal neurogenesis by mediating the proliferation of neural precursor cells in both health and disease. The mechanisms underlying this neuroproliferative effect of NPY, however, are unknown. The aim of this project was to investigate these cellular pathways and the possible involvement of nitric oxide (NO) in NPY-mediated neuroproliferation using postnatal rat hippocampal cultures in vitro. NPY was found to have a purely proliferative effect on hippocampal neural precursor cells. The role of NO was explored by inhibiting the NO synthesising enzyme, nitric oxide synthase (NOS), which abolished the proliferative effect of NPY and supported the involvement of NO in NPY-mediated proliferation. Pharmacological analyses using subtype-selective inhibitors suggested that the neuronal isoform of NOS is the sole NOS subtype involved, which was expressed by both nestin+ precursors and class III β-tubulin+ neurons, the cell types previously shown to be responsive to NPY. The involvement of NO was further verified through loading hippocampal cells with an NO indicator, diaminofluorescein diacetate, where an increase in NO/N2O3 production was observed in nestin+ precursors and class III β-tubulin+ neurons in response to NPY treatment. The downstream signalling pathways coupling NPY-mediated NO synthesis to cell proliferation were identified, through the use of selective pharmacological agonists and antagonists, as soluble guanylate cyclase, cGMP-dependent protein kinase (PKG) and the extracellular signal-regulated kinases (ERK) 1/2. By assessing levels of NPY-mediated ERK 1/2 phosphorylation in response to NOS inhibition, it was found that ERK 1/2 activation was mediated only via NOS/NO mechanisms. This proliferative cGMP-PKG-ERK 1/2 signalling cascade appears to be mediated by intracellularly released NO, while on the other hand, the addition of extracellular NO through the application of NO donors exerted an inhibitory effect on neural precursor cell proliferation. In addition to demonstrating the dual nature of NO, this is the first time that the signalling mechanisms underlying the proliferative effect of NPY on neural precursor cells have been described. Understanding the mechanisms underlying the proliferation of neural precursor cells will ultimately be beneficial by allowing the development of novel therapeutic interventions for promoting hippocampal neurogenesis. To analyse the role of NO in the NPY-mediated neuroproliferation of hippocampal cells in three-dimensional (3D) cultures, Laponite, a novel synthetic silica hydrogel, was used. Culture medium-based Laponite hydrogels were developed before cell viability within the hydrogels were assessed by culturing hippocampal monolayers under gel cover. Hydrogel cover, however, resulted in cell behaviour reminiscent of preservation/fixation as monolayers showed no spatial or morphological changes over time, with one possible explanation being the high gel osmolarity. Although attempts at cell seeding showed more positive results, with cells adhering to a low heavy metal content variation of the hydrogel, determination of cell viability remained a problem due to prominent dye-gel binding. Although the rheological properties of Laponite make its use attractive, the biocompatibility of the hydrogels with hippocampal cells still require further optimisation if they are to be used as cell culture matrices.