ARAP3 is a phosphoinositide 3’-kinase- (PI3K) and Rap-regulated dual GTPase activating protein (GAP) for Arf6 and RhoA, that was identified from porcine leukocyte cytosol in a screen for phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P₃) binding proteins. Firstly, we show that ARAP3 binds its regulator, PtdIns(3,4,5)P₃, in an unusual PH domain-dependent manner. A point mutation in the most N-terminal of its five PH domains abrogates PtdIns(3,4,5)P₃ binding and interferes with ARAP3’s catalytic activities. However, we demonstrate that none of the five PH domains is sufficient to bind PtdIns(3,4,5)P₃ in isolation. We proposed a mechanism by which basic residues in two PH domains, the SAM domain and elsewhere in the protein synergise to mediate affinity PtdIns(3,4,5)P₃ binding. Secondly, we have investigated the role of ARAP3 <i>in vivo </i>using chemotaxing neutrophils as a model system. Previous work in the laboratory showed that neutrophils lacking ARAP3 were unable to undergo net movement towards the source of a local gradient of chemoattractant. We have used novel methodologies to analyse the local distribution of ARAP3, and the local distributions and global levels of phosphoinositides in primary neutrophils from ARAP3 mouse models. We show that ARAP3 localises to the leading edge, and directly or indirectly regulates the local distribution of PtdIns(3,4,5)P₃ during chemotaxis. We propose that PtdIns(3,4,5)P₃ recruits ARAP3 to the leading edge of a chemotaxing neutrophil, where it is part of a feedback loop that is responsible for the maintenance of polarisation in the direction of a chemoattractant gradient.

This dissertation presents structural data relevant to Ral’s role in endocytosis via its interaction with RLIP76. Using <i>in vitro</i> biochemical techniques and NMR spectroscopy the interaction between RalB and the Ral binding domain (RBD) of RLIP76 has been characterised. Binding experiments with the RLIP76 RBD and full length RLIP76 show that Ral binds with a 1 μm affinity for the full length protein, while a higher affinity is expected for the RLIP76 RBD. Binding of ATP to the RLIP76 RBD has also been tested, although results were not able to confirm binding. The solution structures of the free RLIP76 RBD and the complex of this domain with RalB are presented and discussed in the context of the other Ral complexes. The structure of the Ral/RLIP76 RBD complex shows that RLIP76 binds to Ral primarily via the C-terminal helix of the RBD which binds to switch II of the small G protein. The architecture of the complex is such that RLIP76 is in a position to bind D49 of switch I, although the data do not support more extensive contacts with switch I. The structure of the Ral/RLIP76 complex is contrasted with other effector interactions with the small G protein Ral and a review of al helical interactions of effectors with small G proteins has enabled a general binding model for all effectors binding via helical motifs to be established. To understand the role of the Ral/RLIP76 complex in the wider context of endocytosis and RLIP76 signalling, the RhoGAP and Ral binding domains were modelled together. This exploratory modelling of the RhoGAP and Ral binding domains together is presented and used as a basis for the discussion of future work.

In this thesis I have identified several chromatin associated proteins and transcription factors that can regulate. Notch target gene expression and suggested models of how they may function during transcription regulation of the Notch pathway. First, I have shown that a Histone chaperone protein, Asf1, is involved in Notch target gene repression. Based on data that Asf1 recruited by binding to components of the Su(H) repressor complex and that Asf1 exists in a timer with Histone H3/H4, a model was formulated in which Asf1 brings Histone dimers to form new nucleosides on Notch target genes to aid in repression. Second, a screen for potential chromatin associated proteins involved in Notch signalling was performed. Several chromatin related genes were found to genetically interact with the Notch pathway. Four of the best candidate genes were studied further with direct effects on Notch target genes shown. One of these genes was <i>taiman</i>, the co-activator of the Ecdysone receptor, which is part of the steroid signalling pathway in <i>Drosophila. </i>Further work on <i>taiman</i> revealed it was expressed in the wing disc but its role in regulation of Notch remains unclear. Third, the role of Ecdysone signalling in Notch target gene regulation was investigated further. A series of <i>in vivo</i> experiments were performed where Ecdysone signalling levels were manipulated by altering the activity of the Ecdysone receptor. Changes in Ecdysone signalling were able to regulate the expression of three Notch target genes in two different tissues. For some genes a synergistic response in expression was observed when both Notch and Ecdysone pathway were activated. Furthermore, one of the primary Ecdysone targets, the Broad complex, was also shown to affect Notch target gene expression, leading to model where both the Ecdysone receptor and the Broad isoforms may co-operate on the promoters of Notch target genes to regulate their temporal expression.

The MAPK cascade is a highly conserved signal transduction module that occurs throughout eukaryotic organisms. The control of the interactions between the various cascade components and their associated proteins is one level of control that exists to regulate the flow of information. The sterile alpha motif (SAM) was identified by searches for homologies between signal transduction proteins. Further database searches revealed that this 65-70 amino acid domain is found in approximately 70 proteins, whose function is involved in signal transduction or developmental regulation. SAM domains have been shown to mediate homo- and hetero-oligomerisation. For this report four SAM domains from the proteins STE50, STE11 (<I>Saccharomyces cerevisiae</I>), Ste4 and Byr2 (<I>Schizosaccharomyces pombe</I>) were cloned. All were expressed and purified successfully, and the solution structure of the STE50 SAM domain was determined. The homo- and heterotypic interactions of the STE50 SAM domain were also investigated using NMR, biophysical techniques such as analytical ultracentrifugation and dynamic light scattering, and by GST pull-down studies. Finally, residues important for the normal function of the STE50 SAM domain were identified by mutation.

Previous work in the lab identified potential Notch targets in a muscle related <i>Drosophila</i> cell line using a combination of genome wide expression array, chromatin immunoprecipitation (ChIP) and bioinformatics approaches. My project was focussed on investigating the regulation of EGFR pathway components by Notch signalling and unravelling mechanisms by which this crosstalk is dependent on context. Previous genetic studies have demonstrated extensive crosstalk between the Notch and EGFR pathways. The unexpected result from the genome wide studies was the overrepresentative of EGFR pathway components that were direct targets. This group of nine targets included both positive and negative regulators of the pathway. One of my first goals was to validate these as direct targets, for which I used a combination of luciferase and in-vivo reporter assays. To address the question of how activation and inhibition of the EGFR pathway is resolved into a final effect on EGFR output the temporal profiles of the different components was investigated. Initial results show that there are distinct temporal activation profiles for different EGFR related genes following Notch activation. This is predicted to lead to an initial inhibition of EGFR signalling (due to fast initial inhibitor accumulation) followed by an activation of signalling (due to inhibitor decrease and activator production). Current work on this project is focussed on identifying different classes of temporal response at the genome wide scale and asking whether there is any consistency in the types of genes that adopt specific profiles. One of the challenges in dissecting the response to Notch is understanding why genes respond only in certain contexts. <i>Argos</i>, which encodes an EGFR inhibitor, is one example of a gene whose response to Notch signalling is context dependent. My results show that <i>argos</i> is positively regulated in the muscle progenitors. However, in the wing pouch, <i>argos</i> expression is reduced upon Notch activation. I have mapped these contrasting responses to separable enhancers, one active in the muscle precursors that I have shown to be directly regulated by Notch and the other active in the wing pouch where it integrates both EGFR and Notch signals. The latter is mediated by the Notch target E(spl)mβ and is therefore indirectly affected by Notch. Further studies to distinguish the components acting through the different enhancers revealed that the bHLH protein Twist is required for Notch activation of the muscle precursor enhancer and the wing pouch enhancer activity appears to involve an interplay between general activation and restricted repressors.

Chapter 1 introduces the concept of enzyme and provides a general overview of biodegradation of aromatic compounds by enzymes. A characterisation of Toluene Dioxygenase (TDO) and the use of arene cis-dihydrodiols in synthesis are also discussed. Finally, the chapter 1 gives a brief description of carbasugars, their occurrence in nature and some of the literature syntheses. Chapter 2 contains the general information associated with the discovery, isolation and biological activities of pericosines, a review of structure elucidation of pericosine A and some of the recent literature syntheses of pericosine A and C. The chemoenzymatic syntheses of pericosines A and C using arene cis-dihydrodiols as starting materials is also discussed. Chapter 3 gives a brief description of isolation and bioactivity of gabosines and a review of the chemical syntheses of MK7607. The chemoenzymatic approach to MK7607 and gabosines D and E, discussion of experimental results are also provided. Chapter 4 details the biological activities of pinitol, isolation and structure elucidation of chlorosphaeropsidone. The chemoenzymatic synthesis of pinitol and the effort made towards the total synthesis of the chlorosphaeropsidone are presented. This chapter also examines the strategies for synthesis of opposite enantiomer of bromobenzene-cis-dihydrodiol. Chapter 5 contains full experimental details and references.

Current methodologies for recognizing similar protein motifs are predominantly based upon establishing a homology relationship between the sequences. These methods are widely exploited to annotate new genomes and assign putative functions to new genes. However they are usually based on sequence data alone. More recent approaches have incorporated structural data into methods to improve the predictions compared to just sequence based methods alone. So far these approaches have not been widely exploited in bioinformatics for identifying common, small motifs. A test system was examined containing such a degenerate but short, repeating motif, the tetratricopeptide repeat (TPR). Sequence analysis was done to assess the effectiveness of common search tools for finding TPR motifs. These methods included BLAST, PSI-BLAST and Hidden Markov Models and found the latter to be easily the most effective search strategy. Further sequence analysis of the TPR motif was carried out to demonstrate the extent to which TPRs with similar sequences are related in functional terms. In addition a full structural analysis was also performed. The results of the sequence and structural analysis of the TPR allowed structural information to be obtained and structurally conserved features in TPRs comprising conserved interacting residues pair positions were revealed. Comparative models were built and evaluated for all annotated TPR sequences with unknown structures to assess their compatibility with the TPR motif structure. From these and other models the interaction energy of structurally adjacent residues pairs has been calculated. These models were generated by mutating residues in key conserved positions to all possible amino acid combinations. The energy is then evaluated for each of these 20x20 pair combinations. This energy is then integrated into sequence based methods such as Hidden Markov Models with the aim of improving TPR prediction. An improvement in search sensitivity and specificity is demonstrated which should allow improved identification and annotation of this motif in sequence databases.

Peroxisome Proliferator-Activated Receptor-y (PPARy) is a ligand-activated nuclear hormone receptor. PPARy is activated by naturally-occurring (eg. oxidized low-density lipoprotein (oxLDL), and 15-deoxy-delta-12,14-prostaglandinJ2 (15dPGJ2)) and pharmacological compounds (eg. the anti-hyperglycemic agent Rosiglitazone). However, disturbance of ER functions by PPARy ligands causes ER stress, and activation of the unfolded protein response (UPR). In the current study, the impacts of natural and synthetic PPARy ligands on ER stress/UPR were investigated. Treatment (0.5h) of HL-1 cardiomyocytes or MM6 monocytes with Rosiglitazone (0- 10µM)/15dPGJ2 (0-3µM) disrupted ER homeostasis via inhibition of the ER 'housekeeping Ca2+ pump' SERCA2b. Inhibition of SERCA2b-catalysed Ca2+ pumping activity led within 4h to unchecked Ca2+ leakage from the ER, and activation of UPR transcription factor XBP-1. Within 24-72h, this caused up-regulation of UPR genes, Bip and SERCA2b. However, severe ER stress impairs the ability of BiP/SERCA2b to restore ER homeostasis; hence, UPR can lead to apoptosis via activation of proapoptotic CHOP/caspase pathways. Interestingly, this was seen at lower Rosiglitazone levels in HL-l than in MM6 cells. Thus, Rosiglitazone and 15dPGJ2 can cause apoptosis, particularly in HL-1 cells, via a mechanism involving ER stress/UPR. oxLDL intenalization by monocyte/macrophages leads to incorporation of cholesterol molecules into the ER membrane, SERCA2b inhibition, unchecked Ca2+ leakage from the ER, ER stress and UPR activation. Different monocyte/macrophage subsets were investigated: with regard to activation of XBP-I; upregulation of UPR target genes (BiP/CHOP); and apoptosis/cell viability, the effects of oxLDL (1-40µg/ml; 24h) exhibited a consistent pattern: non-polarised monocyte/macrophages were less sensitive to oxLDL than M2-polarised macrophages. Such enhanced susceptibility of anti-inflammatory M2-macrophages could, over time, result in a shift similar to that seen in obese individuals in vivo; i.e. relative increases in proportion of non-M2 cells within each macrophage population, and thus contribute to the development of a proinflammatory milieu in the tissues of obese individuals.

This study examines the interaction of fatty acids with nucleotide binding to UCP1, functional regulation of uncoupling in skeletal muscle mitochondria - which contain uncoupling protein 3 (UCP3) - and the role of the novel uncoupling proteins in ageing and ROS attenuation by expressing human UCP3 in <i>Drosophila melanogaster.</i> GDP binding to UCP1 in BAT mitochondria was studied using the fluorescent probe N-methylanthraniloyl guanosine-5-diphosphate (mant-GDP). Palmitate did not alter the affinity for mant-GDP or [³H]-GDP, but did cause a two-fold increase in the maximum number of binding sites (B_max) of mant-GDP, inconsistent with a simple competitive interaction. Fatty acids also increased the susceptibility of UCP1 to trypsin digestion. A model was developed to explain these results: fatty acids induce a transition in UCP1 from a “protected” to an “open” conformation that is more accessible to mant-GDP. The “open” conformation binds but is not inhibited by nucleotides. Contrary to published reports, superoxide and HNE induced GDP-sensitive proton conductance equally in skeletal muscle mitochondria from wild type and UCP3 knockout mice suggesting other carriers can be induced to conduct protons. Interestingly, palmitate prevented GDP from inhibiting the induced proton conductance. These results indicate that an interaction between fatty acids and nucleotides exists for other members of the mitochondrial carrier family. Interestingly, hUCP3 expression in <i>Drosophila </i>decreased lifespan despite increasing mitochondrial uncoupling when targeted to neuronal tissue. Unexpectedly, targeting hUCP3 to neuronal secretory cells increased levels of insulin-like peptides (DILPs) suggesting that an up-regulation of insulin signalling is the cause of the shortened lifespan and demonstrating for the first time that increasing DILP levels can decrease lifespan in <i>Drosophila.</i>

In this thesis, I test the hypothesis that a group of adhesion molecules, known as integrins, regulates the behaviour of neuroepithelial precursors. Integrins are a major group of receptors for extracellular matrix proteins and cell surface adhesion molecules. An enriched population of neuroepithelial precursors can be grown <I>in vitro</I> as free floating aggregates of cells under the influence of epidermal growth factor (EGF). I have used these aggregates, known as 'neurospheres', to model <I>in vitro</I> the development of neuroepithelial precursors <I>in vivo</I>. Neurosphere cells <I>in vitro</I> will undergo cell death, division, differentiation and migration in a similar manner to neuroepithelial precursors <I>in vivo</I>. I have characterised the integrins expressed by neurosphere cells and using integrin ligands from the extracellular matrix and integrin antagonists I have determined the role of these integrins in regulating neuroepithelial precursor migration and division. I found that neurosphere cells express a specific and limited set of integrins in culture. Specifically they express α5β1, α6Aβ1, α6Bβ1, αvβ1, αvβ5 and αvβ8 but do not express α1β1, α2β1, α3β1, α4β1, αvβ3. Furthermore, different integrins on these cells regulated cell division and migration. Specifically, α6β1 regulated cell migration but did not regulate cell division. In contrast, a second as yet uncharacterised, β1 integrin did regulate the division of these cells. These <I>in vitro</I> results emphasise the important role of integrin-extracellular matrix interactions in the development of neuroepithelial precursors. Finally, I discuss the role of an extracellular matrix molecule, tenascin-C, in the development of neuroepithelial cells <I>in vivo</I>. Taken together, these results support the idea that signalling through integrins is important in controlling the development of neuroepithelial precursors.