The lateral spinal nucleus (LSN), located in the dorsolateral funiculus, is an area that has been poorly understood, but has been implicated in nociception. To investigate the function of this nucleus, three broad areas were investigated: responses to nociceptive stimuli, neurochemical relations to the NK-1 receptor, and projections from this nucleus to several brain centres, to try to gain a greater understanding of the functions of this nucleus. The following conclusions can be drawn from the studies undertaken here: • A series of double-labelling experiments for confocal microscopy were carried out in the rat (Sprague-Dawley) to investigate the LSN responses to a variety of peripheral cutaneous noxious stimuli. It was found that the LSN responds to both thermal and chemical peripheral cutaneous noxious stimulation. However, unlike as previously thought, only a small number of neurons in the LSN are activated by a peripheral noxious stimulus, with hot water (55°C applied to the hind-paw) activating the most, as revealed by Fos immunoreactivity. Only 15% of LSN neurons showed response to this peripheral noxious stimulus. Interestingly, unlike the superficial dorsal horn (SDH), bilateral activation of LSN neurons after the application of a peripheral noxious stimulus was found in most of the experiments carried out. • Triple and quadruple-labelling experiments for confocal microscopy were carried out in the rat to investigate neurochemical relations at this site. It was found that although the LSN is abundant in staining for substance P, the number of LSN neurons showing immunoreactivity for the target of substance P (the NK-1 receptor) represented only one-third of all neurons at this site. However, substance P and nitric oxide synthase were associated with NK-1 neurons, and specifically nitric oxide synthase terminals were preferentially associated with NK-1 neuronal cell bodies. However, unlike the superficial dorsal horn, nitric oxide synthase terminals were not associated with inhibitory GABAergic neurons. • Using retrograde injection techniques (in the rat) combined with multiple immunolabelling for confocal microscopy, the LSN was shown to project to areas traditionally associated with nociception (caudal ventrolateral medulla and mediodorsal thalamus) but also projected to the hypothalamus and also the lateral globus pallidus. Indeed, the regions found to have the most projections from the LSN were the lateral and medial hypothalamus, with most of those neurons (>80%) possessing the NK-1 receptor. Interestingly, although numbers of retrogradely labelled neurons were low, they represented 30% of all labelled neurons that projected from the LSN to the lateral globus pallidus. In conclusion, the extent of involvement of the LSN in nociception is less than previously thought, but with projections to the hypothalamus, it could be postulated that the LSN functions as an integrative nucleus for autonomic and homeostatic functions, and related motivational and affective responses to autonomic function.

Membrane fusion in all eukaryotic cells is facilitated by the formation of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes; a process that is regulated by Sec1p/Munc18 (SM) proteins. Membrane fusion has been conserved through evolution and hence a lot or our knowledge about the molecular mechanism that regulates membrane traffic has come from experimentally tractable model organisms such as Saccharomyces cerevisiae. The work presented in this thesis demonstrates that the mammalian SNARE protein, syntaxin 16 (Sx16), is a functional homologue of the yeast SNARE protein, Tlg2p, as expression of Sx16 in tlg2Δ cells, fully complements trafficking defects displayed by these cells. This finding is supported by experiments demonstrating that Sx16 interacts both physically and functionally with the SM protein of Tlg2p, Vps45p, both in vivo and in vitro. Vps45p regulates Sx16 in a manner similar to the way that it regulates Tlg2p; controlling entry into functional SNARE complexes and regulating cellular levels. A model, in which Vps45p is required for regulating membrane fusion and regulating the cellular levels of Tlg2p, is also presented and discussed in this thesis.

Autoimmune inflammatory reactions occur in a number of disorders to a variety of self antigens but the precise cellular and molecular mechanisms resulting in pathology are largely unresolved. In some instances T cell mediated reactions are thought to be the main contributors to disease mechanisms but it is becoming increasingly evident that B cells, and their cognate antibodies, play a significant and contributory role in disease pathogenesis. Therefore establishing the occurrence of highly-specific B cell antigen-driven adaptive immune responses within autoimmune disorders would provide a valuable understanding into the roles of B cells within these disorders. The work of this study sought to determine the incidence of these B cell antigen-driven adaptive immune responses in the target tissues of the autoimmune disorders myositis and vasculitis, two autoimmune disorders characterised by a wide range of autoantibodies which have been implicated in the pathological mechanisms of these diseases. This study aimed to test the hypothesis that infiltrating B cells within the target tissue of myositis and vasculitis patients were being stimulated by antigen present within the tissue resulting in clonal diversification and affinity maturation mechanisms which would contribute to the pathological mechanisms of these disorders. Initially the cellular phenotypes and organisations of the inflammatory infiltrating cell population were characterised by immunohistochemical techniques, with particular interest on the infiltrating B cell and plasma cell populations. No typical ectopic germinal centre structures were observed in the target tissues from either disorder; cellular aggregations varied from loose aggregations to dense cellular follicles. Both B cell and plasma cell phenotypes were included in these infiltrating populations in correlation with varying numbers of cells positive for helper, cytotoxic and regulatory T cell, follicular dendritic cell, macrophage and proliferating cell markers. Double fluorescent labelling of B cells with the Ki67 proliferation marker indicated the possible expansion and clonal diversification of these cells within the target tissues. To address the main objective of this study and the possible antigen-driven mechanisms and active participation of these infiltrating B cells within the target tissue of these disorders, Ig gene repertoires, mutational characteristics, clonal diversification and affinity maturation were examined from infiltrating cells microdissected from areas of aggregation within the target tissues of both disorders. From the muscle infiltrating cells, Ig gene selections which were both patient and disease specific, mutational characteristics and oligoclonal expansion of B cells were observed establishing the involvement of muscle infiltrating B cells in the antigen-driven responses within inflamed muscle. Alternatively in the inflamed skin of vasculitis patients very few Ig gene rearrangements could be identified and no clonally related sequences or oligoclonal expansion of B cells were observed despite mutational characteristics indicating that antigen-driven diversification had occurred within these cells. Collectively the results indicate a role for B cells in the antigen-driven responses towards autoantigens within both disorders, either within an active antigen-driven response within the target tissue or at alternative sites resulting in cell migration into the target tissues. In the final part of this study recombinant antigens were used to identify antigen-specific B and plasma cells within the inflammatory infiltrating cell population in some cases of myositis. Pilot experiments were conducted to establish the sequence characteristics of Ig genes from these antigen-specific cells. Results of this study demonstrate the influence of B cell antigen-driven responses, either directly or indirectly, within the target tissues of myositis and vasculitis patients respectively, although the exact mechanisms leading to autoimmune reactions, and additional roles of B cells within these reactions, remain unresolved. The work presented from this study provides a foundation for further work to fully ascertain the role of B cells and antibodies within the target tissues of autoimmune disorders and in characterising the disease-associated antibodies, identifying stimulating antigens as well as assessing the effects of somatic hypermutation on the affinity and specificity of autoantigen specific antibodies. Increased understanding of B cells in these disorders will ultimately assist in the diagnosis and management of these diseases.

Actin polymerisation is a highly dynamic process which drives many cellular events, including endocytosis and cell motility. It is known that actin monomers are added to the filaments at the leading edge of a migrating cell and that this polymerisation is the driving force of protrusion. Much is known about the activation and regulation of this dynamic actin remodelling, but many questions about the exact nature of the interaction between the actin filaments and the plasma membrane remain. Weisswange et al (Weisswange et al., 2005) found that the leading edge of protruding fish keratocytes functions as a diffusion barrier for lipid dyes. The aim of the here presented thesis was to continue this project and to study the actin-membrane interaction at the leading edge, using the diffusion barrier as an initial read-out method. First it was investigated whether this diffusion barrier is present in other cell types and could therefore be seen as a general feature of protrusion. Using Fluorescence Recovery After Photo-bleaching (FRAP)in B16 F1 cells, it could be shown that the diffusion of membrane anchored GFP (GFP-F) is significantly inhibited at the leading edge compared to lamellar regions. No reduction in diffusion could be observed after destruction of the actin meshwork or at non-protruding sites of the lamellipodial periphery, showing that the diffusion barrier depends on active protrusion. The diffusion of cytoplasmic GFP was not altered near the leading edge compared to in the lamellipodium, indicating that only membrane bound proteins are affected. After showing that the diffusion barrier is a general feature of protrusion, the exact nature of the actin-membrane interaction causing this phenomenon was investigated. No direct interaction between actin and the membrane could be observed using FLIM-FRET, but FRAP experiments on fixed cells and a correlation between the strength of the diffusion barrier with the speed of protrusion indicate that the reason for the reduction in diffusion around the leading edge is the force created by the actin filaments pushing against the membrane. Further FRAP experiments indicate that actin regulating proteins such as IRSp53 are influenced by the restricted diffusion zone at the leading edge. We propose that the lipid diffusion barrier traps regulatory proteins at the leading edge and can therefore be seen as a positive feedback mechanism for actin polymerisation.

Depression and rheumatoid arthritis have an estimated co-morbidity of 13-20%. However, the mechanisms whereby peripheral inflammation might alter brain function are unknown. We hypothesised that pro-inflammatory cytokines released in the periphery will result in neurochemical, structural and functional changes related to depression. Therefore, the aim of this thesis was to investigate altered central nervous system function in a rodent model of rheumatoid arthritis, the murine model of collagen induced arthritis (CIA). The CIA model is an established model used to investigate novel anti-inflammatory agents and resembles rheumatoid arthritis as the model is chronic and involves an autoimmune response to type II collagen. To our knowledge brain function in the CIA model has not previously been examined. Therefore, we began by identifying key neurochemical, cellular and functional changes associated with depression which had the greatest likelihood of being influenced by pro-inflammatory cytokines. Serotonin and dopamine transporter densities. The serotonergic system is implicated in the pathology of depression and there is evidence that pro-inflammatory cytokines may influence the serotonin transporter (SERT) in vitro. In vitro autoradiography binding of [125I]-β-carbomethoxy-3-β-(4 iodophenyl)tropane ([125I]-β-CIT) in the presence of mazindol and [3H]-citalopram was used to determine SERT binding in mice with CIA and controls. Out of 15 regions of interest investigated a significant change in SERT binding was identified by [125I]-β-CIT binding, in the nucleus accumbens (58%), thalamus (62%), and dentate gyrus (-60%) in CIA mice compared to controls. However, no significant difference in SERT density was detected in any region by [3H]-citalopram binding. Dopamine transporter (DAT) binding sites were also examined using [125I]-βCIT in the presence of displacer fluoxetine and [3H]-WIN 35,428. Out of 14 regions investigated a significant difference in DAT binding was only observed in the caudate putamen (95%) in the CIA group in comparison to the control group. However, no significant difference in DAT binding was detected in any region by [3H]-WIN 35,428 binding. A limitation of this study was the small group sizes and the degree of clinical symptoms in the CIA group. The data suggest that SERT and DAT transporter densities are not altered by CIA. [14C]-2-Deoxyglucose autoradiographic study of local cerebral glucose utilisation. To investigate brain function the [14C]-2-deoxyglucose ([14C]-2-DG) autoradiographic technique and a challenge to the serotonergic system were employed to identify any abnormalities in regional cerebral glucose utilisation. Overall there was no significant difference in the index of cerebral glucose utilization (iLCMRglu) in mice with chronic clinical symptoms of CIA. To investigate altered serotonergic function in the CIA model fenfluramine, a drug which stimulates serotonin release and blocks serotonin re-uptake was employed. Fenfluramine challenge in the CIA group resulted in only 3 out of the 35 regions of interest examined being significantly different from fenfluramine challenged controls. The orbital cortex (-41%) and the molecular level of the hippocampus (-26%) demonstrated a significant difference in iLCMRglu Overall the data suggest minimal influence of CIA on brain function. Cell proliferation and cell survival in the hippocampus. Hippocampal atrophy is implicated in the pathology of depression and there is evidence to suggest that pro-inflammatory cytokines reduce cell proliferation in vitro. To investigate hippocampal cell proliferation mice were administered 5’ –bromo-2’-deoxyuridine (BrdU), a marker of proliferating cells, prior to and after developing chronic clinical symptoms of CIA. There was no significant difference in cell proliferation prior to the development of clinical symptoms. There was a statistically significant increase in cell proliferation after chronic clinical symptoms in the CIA model in one out of two separate experiments. The data has been interpreted cautiously due to the fact the significant increase in cell proliferation was not reproduced. Cell survival was also investigated during the onset of clinical symptoms and the data demonstrated no significant effect of CIA on cell survival. Conclusion. The data indicate minimal influence of peripheral inflammation on the central nervous system, at least in the murine CIA model. Two possible explanations are that the CIA murine model is not a suitable model to detect changes in brain function associated with rheumatoid arthritis or that uninvestigated neurochemical systems play a role. This thesis highlights our limited understanding of the CIA model and whether or not it represents the features associated with rheumatoid arthritis other then peripheral inflammation. Further characterisation of the brain and development of the CIA model is required to establish if it is a suitable model to investigate the association between depression and rheumatoid arthritis. This is important as understanding the cause of depression and how the cause influences the brain will allow for the development of more specific treatments.

The results obtained from the experiments presented in this study aimed to further explore the role of cyclin dependent kinases and cyclins in the protozoan parasite Leishmania major. Cdks in kinetoplastids, CRKs, are the key regulators that allow cells to progress through different cell cycle phases and promote parasite proliferation during infection. In chapter 3 of this study, the results presented showed that L. major CYCA is capable of activating CRK3 in an in vitro kinase assay using histone H1 as substrate. The CRK3/CYCA active complex was then used to analyse the effect of the phosphorylation at the CRK3 activation threonine using a kinase activating kinase (yeast CAK or Civ-1). Phosphorylated CRK3 activity was compared to non-phosphorylated CRK3 and it was found that the phosphorylation promotes a 5-fold increase in kinase activity of the complex. The accessory protein Cks1 was assayed in vitro with the active CRK3/CYCA complex and it was shown that Cks1 might have an inhibitory effect when histone H1 substrate is used. The IC50 for two different kinase inhibitors (Flavopiridol and Indirubin) was determined for the in vitro CRK3/CYCA complex and compared with the values found for the in vivo purified CRK3. Similar values were obtained suggesting that the in vivo complex is indeed represented by the recombinant complex. In the following chapter 4, yeast Civ-1 purified from E. coli, was used to try to phosphorylate, in a similar manner, the activation of threonine/serine residues from other L. major CRKs. The kinases assessed were CRK1, CRK2, CRK4, CRK6 and CRK7. None of these were phosphorylated by Civ-1 suggesting that the only CRK under this type of regulation is CRK3. L. major CRK1-4 and CRK6-8 were tested in kinase assays by mixing under described conditions with L. major CYC9 and kinase activities towards three different substrates were assessed. L. major CYC9 was not able to activate the above kinases and the kinase subunit that interacts with this cyclin could not be identified. In chapter 5, the L. major CYCA was used to elucidate the characteristics of this cyclin in vivo. A gene disruption strategy aimed to replace the two genomic alleles of this protein gene by homologous recombination. Plasmids were developed with flanking regions of this gene placed in association with two different drug resistance genes, one for each of the allele’s disruption. These constructs were not able to produce the first allele knock out suggesting that not only this gene might be essential but the levels of expression may also be important. Tagging L. major CYCA was also attempted in vivo using two different strategies (i.e. two different tagging systems). The first tag employed was the TAP tag syste. Although drug resistant transfected cell lines were obtained, no tag detection could be observed by western blot using different tag-specific antibodies (α-protein-A and α-calmodulin antibodies). The second tag employed was HA, the 9-amino acid sequence YPYDVPDYA, derived from the human influenza hemagglutinin (HA) protein. Plasmids that contained C and N-terminal HA tagged L. major CYCA were used to transfect WT cells and cells extracts of resistant cell lines analysed by western blot. Both C and N-terminal HA tagged CYCA were detected by the α-HA antibody. Following the confirmation of the presence of the tagged CYCA in the cell extracts an affinity purification using an HA affinity matrix was attempted and the matrix binding material was used in in vitro kinase assays. The presence of kinase activity towards Histone H1 confirmed that CYCA was being succesfully immunoprecipitated in complex with a kinase partner. The identity of the co-eluted CRK could be confirmed using specific α-CRK3 antibody that detected CRK3 in the eluted material.

Endothelial inflammation leading to vascular dysfunction is a major contributor to the development of atherosclerosis. The release of adenosine at sites of inflammation represents an endogenous mechanism for limiting excessive inflammation and tissue damage. The majority of the anti-inflammatory effects of adenosine are mediated by signalling through the A2AAR and activation of the A2AAR has been shown to be protective in numerous models of inflammatory disease. Little is known about the molecular mechanisms behind these effects. However, in vitro studies using cultured endothelial cells indicate that signalling through the A2AAR can suppress activation of the NF kappa B and JAK/STAT proinflammatory signalling pathways. NF kappa B appears to be primed for activation in atherosclerosis-prone regions of the aorta indicating that suppression of NF kappa B signalling may protect against the development of atherosclerosis. In this study, the role of the A2AAR in regulating NF kappa B and JAK/STAT signalling pathway activation in the aorta was studied using A2AAR-deficient mice subjected to an LPS-induced model of septic shock. In response to LPS treatment, these mice displayed markedly elevated plasma levels of the pro-inflammatory cytokines TNF-alpha, IL-6, IL-1 beta and GMCSF compared to wild-type mice. Consistent with this finding, heightened activation of the NF kappa B and JAK/STAT pathways was detected in aortic protein samples from A2AAR-deficient mice as demonstrated by increased levels of the phosphorylated forms of I kappa B alpha and STAT1. However, expression of the NF kappa B and STAT1-regulated genes ICAM-1, VCAM-1 and TAP-1 was unaffected indicating the involvement of compensatory negative feedback mechanisms. These findings confirm a role for the A2AAR in regulation of pro-inflammatory signalling in the aorta. Further analysis of mechanisms which mediate this response may reveal new targets for therapeutic intervention to suppress inflammation in inflammatory disorders such as atherosclerosis. While the wide range of anti-inflammatory and tissue-protective responses elicited by the A2AAR have been well documented, the molecular regulation of the A2AAR has been less well studied. The presence of several serine and threonine residues in the extended C-terminal tail of the A2AAR suggests that it may be regulated by phosphorylation events occurring in this region. Indeed, the canine A2AAR is phosphorylated in response to PKC activation. Interestingly, several proteins have recently been identified as being able to interact with the C-terminal tail of the A2AAR. However, how these interactions are regulated is not known. One of the aims of this study was to characterise phosphorylation of the human A2AAR and to determine whether this could provide a means for regulating the binding of C-terminal interacting proteins. This was examined using human umbilical vein endothelial cells infected with recombinant adenovirus encoding the human A2AAR. It was found that phosphorylation of the human A2AAR could be induced in HUVECs by treatment with PMA or by stimulation of endogenous histamine H1 receptors. This was due to activation of PKC, as phosphorylation was inhibited by the PKC inhibitor GF109203X and by depletion of PKC following chronic treatment with PMA. Treatment of cells with the calcium-chelating agent BAPTA/AM did not inhibit PMA-induced phosphorylation indicating that a calcium-insensitive isoform of PKC was responsible. Meanwhile an siRNA-mediated gene silencing approach confirmed that PKC epsilon was not responsible indicating the involvement of either PKC delta or PKC theta. Previously reported interactions between the A2AAR and TRAX and 14-3-3 tau were confirmed in vitro by GST pull-down assay. Binding of 14-3-3 tau to the A2AAR appeared to be unaffected by treatment of HUVECs with PMA. However, A2AAR complex formation with TRAX was significantly reduced in samples from PMA-stimulated cells. These findings indicate that PKC-mediated phosphorylation may represent a means of regulating which proteins can interact with the C-terminal tail of the A2AAR. This may allow the A2AAR to initiate distinct signalling pathways depending on the cellular context in order to achieve the appropriate response.

The importance of the ubiquitin-proteasome pathway in eukaryotic cellular regulation has become increasingly apparent during the last decade. In plants, regulated degradation by the ubiquitin/26S proteasome has been implicated in diverse signalling events including embryogenesis, hormone signalling and disease resistance. Ubiquitin moieties are ligated to target proteins through the sequential activities of E1, E2 and E3 enzymes leading either to proteasomal degradation or other regulatory outcomes in the cell. It is now established that ubiquitination is a reversible process and that removal of ubiquitin from target proteins by deubiquitinating enzymes (also termed ubiquitin proteases) can also serve a regulatory function. Deubiquitinating enzymes are proteases with specificity for the isopeptide linkages formed during ubiquitin ligation events. Current understanding of deubiquitinating enzyme function in plants is relatively limited and the aim of this project was to establish novel findings in this emerging field. This study reports an extensive analysis of the deubiquitinating enzymes in the Arabidopsis thaliana genome and functional characterisation of two closely related Arabidopsis Ubiquitin Specific Proteases: AtUBP12 and AtUBP13 and their respective orthologs in the solanaceous plants tobacco (Nicotiana tabacum) and Nicotiana benthamiana. Previous work suggested the potential involvement of NbUBP12 in disease resistance, in this study, established methodologies in Arabidopsis, tobacco and Nicotiana benthamiana were applied to investigate this possibility. Transcript induction studies in Arabidopsis reported the induction of both AtUBP12 and AtUBP13 by avirulent Pseudomonas and exogenously applied Salicylic acid (SA). Pathology assays in single allele Arabidopsis ubp12 and ubp13 mutants reported no alteration in resistance against virulent and avirulent strains of Pseudomonas, raising the possibility that AtUBP12 and AtUBP13 are functionally redundant. Investigations into redundancy between AtUBP12 and AtUBP13 were conducted using transgenic RNAi based cosupression and the isolation of genetic crosses between ubp12 and ubp13 mutant alleles. Collectively these approaches provide the first report that AtUBP12 and AtUBP13 are functionally redundant and are required for normal plant development with homozygous ubp12 ubp13 double mutants exhibiting a seedling lethal phenotype. Phenotypic analysis of ubp12 and ubp13 mutants indicated that functional redundancy between these genes was not complete with the novel observation of early flowering in ubp12 alleles under both long and short day photoperiods. Short day early flowering in ubp12 mutants was accompanied by the development aerial rosettes and suggests the crucial involvement of deubiquitination in the floral transition. The cDNA sequence of the tobacco AtUBP12 ortholog NtUBP12 was determined and utilised for VIGS based NbUBP12 gene silencing studies during disease resistance signalling in N. benthamiana. Loss of function studies indicated that NbUBP12 functions as a negative regulator of hypersensitive cell death (HR) induced by the Cladisporium fulvum elicitor Avr9 and R gene independent viral resistance against TMV. These findings represent the first reported link between deubiquitination and plant disease resistance. Respective cDNAs for AtUBP12 and NtUBP12 were cloned and expressed to demonstrate the function of their gene products by in vitro ubiquitin protease activity assays. Ubiquitin protease activity of UBP12 was directly implicated in C.fulvum Avr9 elicited cell death during tobacco transient overexpression assays. This experimental approach confirmed that UBP12 activity negatively regulates the Avr9 elicited HR with overexpression of AtUBP12 causing HR suppression and the corresponding AtUBP12 C208S active site mutant conferring a dominant negative HR promotion effect. Overall the presented data reports several novel insights which implicate Arabidopsis UBPs: AtUBP12 and AtUBP13 in plant development and suggests they also may stabilise common substrates which regulate disease resistance. AtUBP12 is also specifically implicated as a floral suppressor and in vitro assays have demonstrated that AtUBP12 and NtUBP12 encode functional ubiquitin proteases. In solanaceous plants, UBP12 activity negatively regulates the defence associated HR and virus resistance.

Nematodes, both free-living and parasitic, are dependant upon their Extra Cellular Matrix (ECM) for multiple aspects of functionality. Two distinct ECMs are present in Caenorhabditis elegans, the basement membrane and the cuticle. The cuticle of C. elegans, like other nematodes is composed largely of collagen-like proteins, with the trimeric collagenous proteins forming approximately 80% of the cuticle. Cuticle collagens are believed to be highly processed in a manner similar to vertebrate collagen maturation, with collagens being; co-translationanly modified, folded into triple helices and proteolytically cleaved at the C- and N- termini. Cross-linking of mature triple helical collagens into higher order structures leads to the generation of a flexible yet robust cuticle. Disulphide bonding is crucial in the formation of the cuticle, with cysteine cross-linking mutants having been shown to produce severely disrupted cuticles and associated lethal phenotypes. During the life cycle, C. elegans progresses through four moults during which a new cuticle is synthesised and the old cuticle is shed. Moulting occurs by proteolytic digestion and shedding of an anterior cuticular cap which provides an opening for the nematode to escape the previous stage cuticle. Both free-living and parasitic nematodes shed and exsheath their cuticles in this manner. Two distinct phases of cuticle processing become apparent: cuticle synthesis and cuticle degradation. Of the enzymes involved with processing of cuticular collagens, the quiescin sulfhydryl oxidases (QSOX), and the nematode astacins (NAS) are of particular interest with regard to cuticle synthesis and proteolytic cleavage of cuticular collagens respectively. QSOX have been shown to be linked directly to the generation of disulphide bonds, and have also been shown to associate with other essential proteins of cuticle formation, namely the protein disulphide isomerases. There are three distinct QSOX family members found within the C. elegans genome, which have been shown to temporally coincide with lethargus (cuticle synthesis) and have been proven to spatially localise to the C. elegans hypodermis, the tissue responsible for cuticle secretion. Characterisation of qsox mutants reveals weak cuticular phenotypes when disrupted singly; but, in combination, silencing of qsox-1 and qsox-2 resulted in blistered cuticles and lethality, by RNA mediated interference and double knockouts respectively. This demonstrates the essential nature of the cuticle associated QSOX enzymes, and to my knowledge represents the first loss-of-function mutant in a QSOX enzyme. xv Investigation of the NAS enzymes focused on the group V astacins, members of which exhibit the only notable defects associated with disruption of C. elegans nas genes, namely: dumpy body shape, nas-35/dpy-31; hatching, nas-34/hch-1; and moult defects, nas-36 and 37. With regard to proteolytic degradation of cuticular components, NAS-36 and NAS-37 were of specific interest as mutants resulted in moult defective nematodes unable to digest and fully escape their previous stage cuticles; in addition, spatial expression illustrated an association of these gene products with regions of cuticle attachment and degradation. C. elegans NAS-36 and NAS-37 were also shown to digest isolated L3(2M) trichostrongylid cuticles of parasites of veterinary importance, suggesting that the metalloprotease and cuticle substrates involved in exsheathment is conserved between trichostrongylid and free-living nematodes. Conservation is poor between ecdysozoan and non-moulting organisms, meaning that proteins such as NAS-36 and 37 could become specific novel targets for anti-nematode drug development.

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults, characterised by multiple tissue involvement and caused by an expansion of a (CTG)n repeat within the 3’-UTR of the DMPK gene (19q13.3). Normal individuals contain between 5 and 35 CTG repeats, whereas the repeats in DM1 patients expand in the range of 50 to several thousands. Longer alleles are very unstable and generally always increase in size when transmitted from parent to child, explaining the phenomenon of anticipation defined by earlier age of onset and an increase in the severity of the symptoms. Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous, hereditary motor and sensory neuropathy of the peripheral nervous system. To date, 30 different loci have been mapped and mutations have been identified in more than 20 different genes. The DM1+CMT++ family is a very unusual three generation family in which all patients co-segregate both DM1 and CMT (LOD score = 7.03). It was postulated that either a single or two closely linked mutations near the APOC2 marker must be the cause of DM1 and CMT. Southern blot analysis of restriction digested genomic DNA revealed a fragment equivalent to a small CTG expansion (~200-400) at the DM1 locus, but an expanded allele could not be amplified by PCR. We postulated that the expanded repeats may have predisposed the repeat tract and the flanking regions to further DNA instability, leading to a secondary deletion, insertion and/or rearrangement. These novel mutations might modify the expression of DMPK and/or nearby genes explaining the unusual clinical presentation. To identify the lesion in the DM1+CMT++ family, a variety of molecular approaches was performed. The molecular lesion identified was an insertion of a GC rich region within the CTG repeats. The allele was comprised of a variable number of CTGs at the 5'-end followed by (GGC)3 G (CCG)20 (CCGCTG)14 (CTG)35. Analysis of single molecule separated alleles revealed 3 that the interrupted 3'-end of the array was stable, while the CTG repeats at the 5'-end were unstable. Postulated mechanisms to explain the DM1 and CMT symptoms in the family were: a novel RNA gain-of-function, and/or a novel effect on the downstream genes. Finding an imperfect CTG repeat allele in the DM1+CMT++ family led us to suggest that imperfect CTG repeat alleles may not be unique events and other DM1 patients may also contain similar alleles. To investigate this DNA samples from 14 DM1 patients with an unusual molecular diagnosis were analysed. The majority of these patients presented with an imperfect CTG repeat allele containing CCGCTG hexamers and/or CCG repeats. Five patients contained two or three higher order repeats containing between 18 and 30 bp such as ((CTG)5 (CCG)5), ((CTG)2 (CCGCTG)4) and ((CTG)5 (CCG)2 (CCGCTG)). These findings further suggest that imperfect CTG repeat alleles might not be as rare as was previously believed. The results of this project point out the importance of performing a more detailed molecular characterisation of the DM1 patients, which could lead to the provision of more accurate prognoses and the development of effective therapies.