The aim of this thesis was the analysis of the role of nucleocytoplasmic transport factors in <i>Drosophila </i>development. Potential nucleocytoplasmic transport factors in <i>Drosophila</i> were identified in database searches by their homology to known factors from yeast and humans. For candidate factors the sequence and mRNA expression pattern was further analysed. While most mRNAs tested were ubiquitously expressed, one newly identified factor, called <i>Drosophila</i> <i>cas </i>(<i>dcas</i>), because of its homology to human CAS (cellular apoptosis susceptibility factor), showed a specific expression pattern, Zygotic <i>dcas</i> mRNA was highly enriched in the embryonic central nervous system, although the protein showed a generalised distribution.  Mutations in <i>dcas</i> were identified and analysed. A P-element insertion in the <i>dcas</i> gene causes lethality, whereas several hypomorphic alleles showed specific phenotypes at different developmental stages. Detailed analysis revealed that Dcas functions in the export of importin-α in <i>Drosophila, </i>as previously reported for humans and yeast. However, mutations in <i>dcas </i>also resulted in highly specific defects fate determination in the peripheral nervous system during the development of the external sensory organ (commonly known as “bristles”). Similar phenotypes have been reported in mutants that increase signalling by the Notch pathway. Several components of this pathway were therefore tested for localisation defects in <i>dcas </i>mutants and one protein showed an altered nuclear/cytoplasmic distribution. These findings confirm that Dcas has the same basic function in the export of importin-α in intact <i>Drosophila </i>as in single cells of humans and yeast. In addition to this “house-keeping” function, mutations in <i>dcas</i> cause specific phenotypes, indicating that some pathways are particularly sensitive to a perturbation in nuclear protein important. This is very likely applicable to other transport factors in other multicellular organisms.

In this study, the domain movement of AnxAl in response to Ca²⁺ in the presence of phospholipid membrane was studied using the intrinsic fluorophore Tryptophan 12 on its N-terminal domain. Increases in fluorescence intensity and blue shift of emission peak wavelength show that the N-terminal domain has a hydrophobic interaction with phospholipids. Neutron diffraction profiles of multi-stack phospholipid bilayers in the presence of AnxAl N-terminal peptides indicate that the peptides lie parallel to the surface of phospholipid bilayers. These results support the notion that the N-terminal domain interacts with membranes in a calcium-independent manner. A model where membrane aggregation is mediated simultaneously by the two membrane binding sites of one AnxAl molecule is thus possible. Plant annexins, annexin Ghl from <i>Gossypium hirsutum </i>and annexin 24 from <i>Capsicum annuum </i>(Anx(Ghl) and Anx24(Ca32), respectively), have been reported to possess a calcium-dependent membrane binding behaviour similar to animal annexins. Interestingly, hydrophobic and positively charged residues are well conserved among plant annexins, and fully exposed on the convex side (canonical membrane binding side) of the C-terminal core surface. In this study, AnxAl(Gh1) and Anx24(Ca32) are demonstrated to also possess calcium-independent membrane binding activity, which is directly attributed to these conserved surface residues. Anx(Gh1) was successfully crystallised using high concentration of CaCl₂ in a phosphate crystallisation buffer. Three Ca²⁺ ions are localised in the Ca²⁺-bound structure presenting typical Ca²⁺ coordination geometries observed in animal annexins. Two adjacent cysteine residues, Cys116 and Cys243, have been speculated to carry a putative RedOx function of Anx(Ghl). The RedOx activity of Cys116 and Cys243 was probed using trypsin digest mapping of Anx(Ghl) with H₂O₂ treatment. These experiments indicate that the two cysteine residues remain a reduced state.

The neurotrophic protein Nerve Growth Factor (NGF) represents a new target for the development of potential therapies for neurological disorders such as Alzheimer's disease (AD). Peptides spanning the length of the hNGF sequence were assembled using both manual and automated solid-phase procedures. These sequences were tested in a competitive assay and their ability to inhibit <SUP>125</SUP>I-GF binding to a recombinant form of the gp75 NGF-receptor assessed. Considerable levels of inihbitory activity were observed from peptides selected from the C-terminal fragment of the protein. The shortest sequence demonstrating this activity was found to span the amino acid residues Arg 100 to Arg 114. Removal of either residue from the sequence resulted in the inactivation of the peptide in the assay. This would appear to implicate both these basic side-chains in the receptor binding recognition process. Several peptides were investigated by high-field nuclear magnetic resonance spectroscopy (NMR) and a complete assignment of the spectra were made. Analysis of the through-space interactions indicated that these peptides adopt a random conformation in solution.

Soluble proteins can be targeted and inserted into the Endoplasmic Reticulum (ER) co-translationally or post-translationally, Co-translational translocation requires a ribonucleoprotein complex. Signal Recognition Particle (SRP), to target the ribosome-nascent chain complex to the ER-localised translocon. The targeting and insertion of membrane-bound proteins into the ER is more complex. One class of membrane proteins have the transmembrane domain near their C-terminus (tail-anchor proteins), and whilst the other classes of membrane proteins have had their mode of insertion elucidated, the insertion mechanism of these proteins remains unknown. This thesis deals with two areas of protein targeting in <i>Saccharomyces cerevisiae</i>. First the mode of insertion of Ufe1p, a tail-anchor protein, was examined by use of a variety of reporters. This work identified a region N-terminal to the transmembrane domain that is required for correct localisation of the protein. Second, the role of the SRP component, Srp72p was examined, Srp72p is essential for SRP function, but its role has not been determined. A screen was set up to identify conditional mutants of <i>SRP72</i>. In addition, deletions and point mutations were created to analyse the roles of conserved residues within the protein. Mutations affecting a conserved region of the protein towards the C-terminus were found to confer slight defects in SRP-dependent translocation. Intracellular localisation of the mutant proteins was identical to that of the wild-type protein. A mini-screen performed on two mutants identified multicopy suppressors of translocation defects. Finally, preliminary comparison of the binding affinities of SRP containing mutated Srp72p or wild-type SRP with the SRP receptor revealed a subtle difference.

Aquaporins (AQPs) are membrane channel proteins which facilitate rapid water transport across cell membranes. It is believed these channels are involved in many physiological processes including renal water conservation, neuro-homeostasis, digestion, regulation of body temperature and reproduction. At least 11 mammalian AQPs (AQP0 – AQP10) have been identified to date, making them potential therapeutic targets for drug intervention. Initial studies have shown that Hg²⁺ irreversibly blocks water transport in AQP-1 by the formation of a mercaptide covalent bond with cysteine residue 189 deep within the pore. AQP-1 function can also be modified by tetraethylammonium chloride (TEA) (100 μM), which reversibly blocks water permeability in <i>Xenopus laevis</i> oocytes injected with AQP-12 and AQP-2 by 44 ± 11% and 49 ± 18% respectively. However, a specific reversible AQP inhibitor has so far not been reported. A 2.2 Å high resolution crystal structure of <i>Escherichia coli</i> glycerol facilitator (G1pF) was employed as a model for water transport through AQP-1. A number of compound libraries, based on the lead compounds of glycerol and TEA have been synthesised using a range of automated techniques. Several of these compounds have been screened as putative aquaplugs on <i>Xenopus laevis</i> oocytes injected with AQP-1, 2, 3, 4 and 5. Initial results suggest that it is possible to selectively block water throughput I AQP-1, using dimethyl ethyl hexadecanyl ammonium bromide (100 μM), by 80 ± 7% and AQP-2, using dimethyl ethyl decanyl ammonium bromide (100 μM), by 58 ± 16%. To our knowledge these are the first known selective blockers of AQP-1 and 2. These results have provided the basis to develop more focused lead compound optimisation which in turn should establish further qualitative structure-activity-relationship data to aid in our understanding of the mechanisms associated with water transport throughout the AQP family.

This thesis consists of three relative independent projects all of which concentrate on the syntheses of biologically active natural products. In the first project, a convenience route for synthesis of [8-<SUP>3</SUP>H]-berberine sulfate has been established and two routes for preparation of <SUP>14</SUP>C labelled berberine are explored. A series of compounds containing a methylenedioxyphenyl functionality have been synthesised for using as antigens in immunological studies. The second project concerns development of chemistry directed towards the stereocontrolled synthesis of Baogongteng A, a novel 2,6-disubstituted tropane alkaloid which is a powerful M-cholinergic agonist used in the treatment of glaucoma. By exploiting 4-hydroxy-L-proline as the chiral synthon, useful methods such as preparation of 2-formyl 4-hydroxy-L-proline derivatives, and the stereoselective bromination of the C-3 of 4-hydroxy-L-proline derivatives, have been established. About forty new organic compounds have been synthesised and identified. The final project concerns exploration of new routes for the syntheses of biotin biosynthetic precursors and their fluorined analogues which are types of compounds potentially valuable in agricultural chemistry. This work has led to the development of methods of wide application, such as the preparation of highly purified N-Boc-L-alaninal, which should prove to be of use in future studies in this and related fields.

This thesis describes studies on the last two enzymes on the biotin biosynthetic pathway. The penultimate enzyme, dethiobiotin synthetase (DTBS), catalyses the <I>ureido</I> ring closure of dethiobiotin from (7<I>R,</I>8<I>S</I>)-7,8-diaminononanoate (DAPA), carbon dioxide and ATP. The final step, catalysed by biotin synthase, involves the chemically difficult insertion of sulphur into the unacativated carbon skeleton of dethiobiotin to yield biotin. A range of analogues of DAPA differing in chain length and C-8 substitution were synthesised and found to act as slow substrates of DTBS. Steady-state kinetic and binding parameters were evaluated for these modified substrates and their relevance to the mechanism of the DTBS reaction is discussed. Crystallographic and modelling studies of one analogue bound in the active site of DTBS suggest that the enzyme may employ an alternative mechanism of <I>ureido</I> ring closure to that of the native substrate. Biotin production was demonstrated in cell-free extracts of an <I>E. coli bio</I>B transformant and the origin of the sulphur atom in biotin was investigated. A novel cysteine desulphurase enzyme, which catalyses the of L-cysteine to give L-alanine and sulphur, was discovered and its possible role in the biotin synthase reaction is discussed. [6,6-<SUP>2</SUP>H<SUB>2</SUB>]-(±)-dethiobiotin and [6,6-F<SUB>2</SUB>]-(±)-dethiobiotin were identified as target compounds in intermediate trapping experiments and as selective biocides. A synthetic route to these compounds was devised and the introduction of label was explored using labelled catalysts and carbonyl transformation chemistry.

No description available.

The complement system is part of our innate immune response and is tightly regulated to prevent damage to host cells. Complement receptor type 1 (CR1) is one of the main regulators of complement activation and is also the immune adherance receptor on erythrocytes, important for clearance of immune complexes from the bloodstream. CR1s functions arise from its ability to bind complement proteins C3b and C4b. CR1 is a multimodular glycoprotein (220 kDa) that is too large to study in its intact form by NMR. Recently, the solution structure of functional site 2, which consists of three contiguous complement control protein (CCP) modules, has been solved. However, the structural information alone does not complete the story as dynamic motions within CR1 are likely to have implications for its functions. This thesis describes the assignment of 15N and 1H NMR data for the central CCP module (module 16) of functional site 2. The resonance assignments subsequently allowed the solution 3D structure to be determined and the Modelfree analysis of the module’s isotropic dynamics. The structure and dynamics of the lone module, when compared with previous work on larger fragments of functional site 2 allowed assessment of the importance, for their structure and flexibility, of the context of CCP modules. Following this, NMR 13C, 15N and 1H spectra for CR1 modules 2-3, which correspond to the C-terminal two thirds of functional site 1, were acquired. The resonance assignment of this double module was then performed to near completion. In parallel, a homology-based model of the structure of modules 2-3 was built using the structure of site 2 as a starting point. The isotropic dynamics were also analysed using Modelfree formalism.

During the course of this study, the role of <i>Drosophila </i>homologues of Golgi matrix proteins in the organisation of the early exocytic pathway (tER and Golgi) was investigated. dp115, dGM130 and dGRASP were depleted from <i>Drosophila </i>S₂cells by RNA interference. Using electron and immunofluorescence microscopy, dp115 depletion was shown to result in Golgi stack breakdown and tER dispersion. The effect on tER organisation was specific for dp115, as depletion of the other Golgi matrix proteins did not have a similar effect. Although, dGRASP or dGM130 single depletion did not lead to a quantitative disruption of the Golgi morphology, dGM130/dp115 and dGM130/dGRASP double depletions pointed to genetic interactions for building and/or maintenance of the Golgi stacks. Considering the key role of the Golgi apparatus in protein transport along the exocytic pathway, anterograde transport of plasma membrane protein Delta, from its site of synthesis (ER) to its final destination, was monitored in cell depleted of the above mentioned Golgi matrix proteins. Surprisingly, none of the depletions, including dp115 that alters considerably the organisation of the exocytic pathway, led to a significant inhibition of intracellular transport. Anterograde intracellular transport in the absence of Golgi stacks exists physiologically during <i>Drosophila </i>development. In a morphological study performed in imaginal discs during the transition from early 3^rd instar larva to white pupa, the biogenesis of the Golgi stacks from vesicular-tubular larval clusters was described. These larval clusters were shown to contain several Golgi-specific markers and, therefore seem capable of carrying out proper Golgi functions.