Kainatc receptors (K Rs) arc members of the ionotroplc glutamatc receptor family and are tetramers made up of combinations of GluR5, 6 & 7 and KAI & 2 subunits. KARs arc highly expressed in the brain where they are located both pre- and postsynaptically and regulate network function. Recent work has shown that KARs function was closely related to their subunit composition, This is best described in hippocampus where KARs regulate circuit excitability and where GIuR6 subunit has been implicated in mediating temporal lobe epilepsy. KARs are also highly expressed early in development in sensory pathways and have been shown to be involved in regulating short-term synaptic plasticity during development; however little is known about their subunit specific roles in this mechanism.

Thrombospondins (TSPs) are conserved, multi-domain, extracellular glycoproteins that participate in many biological processes. TSP-1 is present in low levels in the extracellular matrix (ECM) of healthy vessel walls and is up-regulated during neointima and plaque formation. TSP-1 induces vascular smooth muscle cell (SMC) proliferation and migration upon vascular injury or during atherosclerosis. The aims of this' dissertation were: 1) to identify molecular mechanisms and interactions of the C-terminal, L-Iectin domain that mediates ECM deposition of TSP-1; 2) to examine the functional role of ECM-deposited TSP-1 in vessel wall ECM assembly. From cell culture experiments to identify how TSPs accumulate in ECM, a novel mechanism of trans inter-molecular interactions between TSP molecules within ECM is proposed. This mechanism depends on a specific motif in the L-Iectin domain and can act through either homophilic (TSP-1- TSP-1) or heterophilic (TSP-1-TSP-5) interactions to mediate ECM accumulation of TSPs as puncta. Fibrillar collagen may stabilise these puncta in ECM, as inhibition of fibrillar collagen production in fibroblasts decreased the level of TSP-1 in ECM but did not alter puncta patterning. Because only trimeric TSP-1 is retained in ECM, the basis of trimerisation was investigated by use of synthetic peptides derived from the coil-coiled region of TSP-1. Biophysical methodologies proved that a coiled-coil domain peptide, without any N-terminal cysteines, was stably trimeric in solution. Lastly, the role of TSP-1 in healthy aortae was examined with reference to the organisation of aortic walls in wild-type mice, Thbsl -/- mice and mice with transgenic overexpression of TSP-1 in vascular SMC. 10 to 12-week old mice of both genders were included in each set and measurements were made from the ascending and the thoracic aorta. The total width of elastin layers was increased in Thbs1 -/- mice and layers were more irregular. Similar perturbations were identified in aortae from the TSP-1 transgene overexpression mice. Thus, either increasing or decreasing TSP-1 perturbs the aortic wall ECM. The new mechanisms identified might also have implications for atherosclerosis and cardiovascular disease.

A rich gallery of novel systems based on the sequence KLVFF (namely KLVFF, FFKLVFF, AAKLVFF, AAKLVAA, βAβAKLVFF, KLVFF-PEG, FFKLVFF-PEG, AAKLVFF-PEG, AAKLVAA-PEG, FF-PEG, FFFF-PEG) was synthesized and characterized. For the first time structure-properties relationships of this class of materials were systematically explored, with emphasis on self-association properties in both bulk and solution. Such a comparative investigation, essentially absent from the literature, provides insights into the underlying mechanism of amyloid fibrogenesis, given that KL VFF has been identified as critical for amyloid fibril formation of the amyloid-β peptide. In this respect, this virtually unique, bottom-up approach contributes to the development of therapeutics for Alzheimer's disease. Despite the fact that all the peptides were found to organize into predominantly β-sheet conformations (in a variety of solvents and in bulk) they exhibit a versatile range of morphological features, such as fibrils, twisted assemblies of protofilaments, hollow tubes and single tapes. Interestingly, FFKLVFF was found to exhibit strong fibrillization properties, while helically twisted ribbons were obtained from βAβAKL VFF. The self-organization of KLVFF towards fibrillar symmetries gives rise, under certain conditions, to time-dependant hydrogels with potential for application in tissue engineering. Notably, only FFKLVFF-PEG and FFFF-PEG retain the β-sheet conformation of the peptide precursor, and they form fibrils bearing a peptidic core surrounded by a corona of PEG chains. At high volume fractions packing of the FFKLVFF-PEG fibrils leads to the evolution of nematic and hexagonal columnar phases. Owing to their inherent biocompatible character, those hybrids carry great promise to substitute a number of conventional polymers in applications such as tissue engineering, cell growth and drug delivery. In the solid state, the interplay between two competing factors, polymer crystallization and peptide fibrillization, was studied for several conjugates. Two distinct cases were identified; solidification of FFKL VFF-PEG controlled by peptide- peptide interactions, while KLVFF -PEG and AAKL V AA-PEG crystals reflected the characteristics of PEG spherulites.

ID helix-loop-helix (HLH) proteins play a central role in the regulation of mammalian cell growth, differentiation and tumourigenesis. In many human cancer types, the expression of one or more of the four 10 family members (ID1 to ID4) is deregulated. Extensive data supports a direct role for deregulated ID protein expression in conferring malignant properties on tumour cells, through their inhibition of basic HLH (bHLH) transcription factors, which highlights the ID proteins as candidate therapeutic targets. This research aimed to develop peptides capable of inhibiting ID protein function, and build on the current understanding of key residues within the HLH binding domain that govern the specificity of ID protein binding. Molecular modelling analysis facilitated the randomisation of several key residues at the HLH dimer interface of ID-binding transcription factor E47 to create peptide libraries. Using a semi-rational design approach, combined with a protein-fragment complementation screening strategy (PCA), two specific high-affinity ID1 and ID3-binding peptides were obtained, capable of blocking ID protein inhibition of E47. The ID proteins and their inhibitors were further characterised through thermodynamic analysis of their structure and stability using circular dichroism and a range of other biophysical techniques, and were found to display mostly helical structures that increased in stability on addition of PCA-selected inhibitors. The homo-and hetero-dimerisation of members of the ID family was also investigated, and complex size analysis revealed that 101, 3 and 4 can exist as tetrameric structures. In addition to this; heterodimeric interactions were observed between ID1 and the remaining ID proteins; the highest affinity of which was that between ID1 and ID4, which has not been previously reported. In future studies, ID peptide ligands can be explored for the development of anti-cancer and regenerative medicine therapeutics and used as experimental tools for dissecting molecular mechanisms of the ID proteins.

Bradykinin and related peptides (BRPs) are common components of amphibian defensive skin secretions, particularly in ranid and phyllomedusine frogs and in bombinid toads. BRPs are known to be highly vasoactive with some possessing potent effects on blood vessel formation (angiogenesis) - a process known to be initiated by direct effects on endothelial cells. Human microvessel endothelial (HMEC) cells are a stable laboratory cell line often used for preliminary screening in such studies of BRP function. Since these cells are the starting points for fundamental biological studies, we examined their catabolism of bradykinin (BK) and maximakinin (MK). MK represents an N-terminally extended version of the former but with higher potency. Both BK and MK were broken down by proteases present on HMEC cells with half-lives of 5h and 2h, respectively. However, as two major metabolites of MK retained the receptor-active site of BK, the true half-life of non- active metabolite generation was 5h (BK) and 9h (MK). Bradykinin antagonists, kinestatin and QUB919, both from amphibian skin, were not degraded by HMEC cells and their presence did not interfere with the degradation of BK or MK. Using primer sets designed for bradykinin Bland B2 receptors, transcripts of appropriate size were amplified from an HMEC cell cDNA library. Bradykinin-related peptides are thus catabolised in different ways by HMEC cells and the cells were shown to contain polyadenylated mRNAs for both bradykinin receptor sub-types, Bland B2. To examine other functions of BRPs, we screened reverse phase HPLC fractions of venoms and defensive skin secretions to identify, structurally characterise and ultimately chemically-synthesise novel peptides to examine their effects on bradykinin activity using smooth muscle bioassays. Many BRPs exhibited anti- cancer and anti-microbial functions in our experiments giving us a broader perspective for further research in the BRP field. · ';.

Amphibian skin secretions are known to contain numerous peptides with a large array of biological activities. Among the anuran amphibians, the Neotropical hylid frogs belonging to the subfamily Phyllomedusinae, are an excellent source of such bioactive peptides with antimicrobial and pharmacological activities. To date, more than 200 peptides from this frog taxon have been reported in the scientific literature and their structures have been deposited in genomic and proteomic data banks such as the Universal Protein Resource Consortium (UniProt). In this thesis, we have identified four novel tryptophyllins (TPHs) from the skin secretions of four different phyllomedusine species (Phyllomedusa sauvagei, Phyllomedusa hypocondrialis, Agalyehnis eallidryas and Phyllomedusa bieolor). The primary structures of these peptides were determined by combinations of Edman degradation and mass spectrometry techniques. Molecular cloning of respective cDNA sequences encoding the precursors of these TPHs was achieved from skin cDNA libraries of each species. The amino acid sequences deduced from the nucleotide sequences of cloned precursor cDNAs corresponded exactly with those obtained by chemical/mass spectrometric techniques. Some of these novel TPH peptides were found to have potent effects on mammalian smooth muscle and here we report for the first time that TPHs can antagonise the bradykinin-induced relaxation responses in rat tail artery smooth muscle preparations and that they have anticancer effects on human prostate cancer (LNCaPIPC3IDU145) cell lines. The catabolism of these TPHs by prostate cancer cells was analysed and all were found to be highly-resistant to degradation under the experimental conditions employed. The polyadenylated mRNAs encoding both bradykinin receptor subtypes, Bland B2, were also shown to be expressed in all three prostate cancer cell lines through molecular cloning of specific receptor cDNA fragments from cDNA libraries constructed from each. These data suggest that bradykinin receptors could be a hitherto uninvestigated target of potential utility in the treatment of prostate cancer. The data generated through the discrete studies reported in this thesis may aid in the understanding of possible biological roles played by amphibian skin TPHs and the contributions made by specific structural features within this heterogenous and largely unstudied family of amphibian skin peptides. The peptides described here for the first time may represent novel lead compounds for the design/development of new therapeutics for human neoplastic disease.

Amphibian skin secretions, ansmg from specialised granular glands, are often complex peptidomes containing many components of entirely novel structure and unique site-substituted analogues of known peptide families. Following the discovery that the granular gland transcriptome is present in such secretions in a PCR amenable form, we have designed a strategy for peptide structural characterisation involving the integration of "shotgun" cloning of peptide precursor encoding cDNAs, deduction of putative bioactive peptide structures and confirmation of these using tandem MSIMS sequencing. Here, we illustrate this strategy by means of the elucidation of the primary structures of antimicrobial peptides from the defensive skin secretions of the Chinese smelly frog, Odorrana livida. Synthetic replicates of the peptides were found to possess antimicrobial activity, anticancer cell activity and trypsin inhibition activity. These peptides are thus of widespread occurrence in the skin secretions of Asian ranid frogs and in those of the Odorrana group and they are particularly well-represented and of diverse structure in some species. The integration of the molecular analytical technologies described can thus provide 'a means of rapid structural characterisation of novel peptides from complex natural libraries in the absence of systematic on-line database data.

Rationally designed self-assembling f3-sheet fonning peptides have been previously shown to undergo nucleated one-dimensional self-assembly above a critical concentration in solution, from monomeric random coil into a hierarchy of well-defined f3-sheet . . nanotapes, ribbons and fibrils. The interest in the application of self-assembling peptides in m~dicine e.g. tissue engineering, and in bios~nsors dictates the need to correlate peptide molecular structure with membrane nanotoxicity. Here a series of parameters are examined that could affect the peptide interactions with phospholipid monolayersusing electrochemical techniques on a model DOPC-Hg supported system. These parameters are namely peptide aggregation state, polarity, net charge, applied voltage and amphiphilicity. For this study a total of 16 systematically altered self-assembling tape-forming peptides have been employed. It is ,shown _ that in the absence of applied voltage, amphiphilic peptides, with aromatic amino acids, a zero net charge and in monomeric rather than aggregated state in solution, have the highest propensIty to bind to DOPC. monolayers and give rise to discrete conducting structures. Positively charged amphiphilic peptides interact more strongly with Dope than negatively charged ones, whilst polar peptides show little interaction with DOPC. Applied negative voltage across the lipid layer drives the binding ofamphiphilic and polar positively charged peptides to the lipid layer, however such - .peptides are less able to undergo self-assembly into pore-like structures in the lipid environment. Introduction of serine amino acid from the choice of the available polar I amino acids in the peptide primary seq~ence facilitates significantly the peptide-DOPC interaction. This class of pep tides are shown to be considerably less membrane-disruptive compared to antimicrobial peptides. This research provides not only a greater insight into the rational design of new peptide-based biomaterials but also a further understanding of the interactions of natural antimicrobial and amyloid-forming peptides with biological membranes.

Amyloid assemblies consist of an organised cross ~-sheet structure and can be formed by many proteins or peptides regardless of peptide sequence. Amyloid has been utilised by many species for a variety of functions, however, inappropriate amyloid formation is associated with a spectrum of devastating amyloid diseases. The nature of the primary cytotoxic species in amyloid disease is widely debated, with the consensus favouring pre-fibrillar, oligomeric entities over mature end-stage fibrils, despite an increasing body of evidence that suggest at least some fibrils may be associated with cytotoxicity. Amyloid fibrils can be considered as nanomaterials and, as the physical dimensions of nanomaterials can strongly influence their biological activity, this raises the possibility that fibril length may influence their cytotoxic potential. This thesis examines the role of fibril fragmentation on amyloid fibril-associated cytotoxicity and the cellular mechanisms involved. First, having introduced the themes of amyloidosis in Chapter 1 and the methods employed in this thesis in Chapter 2, in Chapter 3, ~2microglobulin amyloid fibrils are used as a model to investigate the effect of fibril fragmentation on cytotoxicity and membrane disruption. The fibrils were fragmented, reducing fibril length, with a consequent increase in cytotoxicity and membrane disruptive capacity. Amyloid fibrils generated from a range of disease specific, functional, de novo designed, and model systems were then formed and fragmented and their cytotoxicity was analysed using different cell lines. Strikingly, in each case, the amyloid fibrils were more cytotoxic following fragmentation. In Chapter 4 the cellular mechanisms associated with amyloid fibril-associated cytotoxicity were investigated. Using amyloid fibrils formed from ~2microglobulin (~2m) as a model, it was established that ~2m amyloid fibrils do "not cause cytotoxicity by plasma membrane disruption but, following internalisation, localise to and disrupt Iysosomes leading to cathepsin-- dependent cell death. Inhibition of fibril uptake or cathepsin activity was shown to protect VI against ~2m fibril-associated cytotoxicity, as well as the cytotoxicity associated with an array of other amyloid fibrils. Taken together the data suggest that amyloid fibrils cause cytotoxicity through disruption of Iysosomes, a mechanism common to many fibril types and increased by fibril fragmentation. The significance of these findings and the potential for future studies are then discussed in Chapter 5.

The aim of this study was to elucidate the immunoregulatory role of the neuropeptide, nociceptin (N/OFQ). The effects on human peripheral blood mononuclear cells (PBMCs), CD4⁺ T cells, T regulatory (Treg) cells and dendritic cells (DCs) were investigated. N/OFQ (10⁻¹⁴M and 10⁻¹⁰M) suppressed the proliferative response of PBMCs to the superantigen, staphylococcal enterotoxin B (SEB) and 004⁺ T cells stimulated with CHO(DR4/SEB/CD80) (OHO cells transfected with DR4 and MHO class II and pulsed with SEB). N/OFQ-induced decreased proliferation of SEB-activated T cells, could be blocked by the putative N/OFQ antagonist, UFP-101, indicating the response is specific to N/OFQ. Expression of the co-stimulatory receptor, glucocorticoid-!nduced tumour necrosis factor receptor (GITR) decreased In response to N/OFQ (10⁻¹⁰M) and expression of CD80 increased in response to N/OFQ (10⁻¹⁴M). Functional studies revealed an inhibitory role for CD80 in modulating T cell proliferation. Moreover, CD80 and to a lesser extent, CD86 are involved in the suppressive effect mediated by N/OFQ. N/OFQ can therefore modulate T:T cell communication.