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.

Salmonella species can cause wide-ranging disease from mild food-poisoning enteritis to a systemic, sometimes fatal typhoid infection. These bacteria have evolved to survive in different environments within and outside the host and do so through the regulation of differential gene expression following activation of certain stress response systems. In gram negative bacteria such as Salmonella, envelope stress responses (ESR) are response systems that target stresses affecting components of the cell envelope such as the periplasm and outer membrane proteins. The two best characterised ESRs are the RpoE stress response system and the CpxAR two-component signal transduction system. Two further ESRs, the BaeSR response and the phage shock response have also recently been identified. The intention of this thesis was to characterise the ESR systems of S. Typhimurium to widen our current knowledge of genes involved in these systems and their role in the pathogenesis of S. Typhimurium with the ultimate aim of identifying possible candidate vaccine genes that may be used in future therapeutics against Salmonella infection. Firstly, extensive mutagenesis and phenotypic analysis studies were undertaken to characterise genes thought to be members of the RpoE regulon. Study of the phage shock response was initiated through mutagenesis, characterisation and regulation studies. A microarray experiment was designed in collaboration with colleagues at the Sanger Centre to identify members of the S. Typhimurium CpxAR regulon, with several members of this regulon being characterised further. The structural components of HtrA, an important ESR protein in S. Typhimurium, were analysed and finally work within this thesis was involved in the investigation of potential overlaps between both the RpoE and CpxAR systems. This led to the establishment of preliminary studies to investigate the vaccine potential of the tol - pal genes in S. Typhimurium.

The vascular endothelium contributes to the maintenance of vascular health by regulating vascular tone and leukocyte adhesion, amongst others. The vasoregulatory actions of the endothelium are mediated through coordinated release of vasodilators such as nitric oxide (NO) and prostacyclin, and vasoconstrictors such as endothelin-1 and thromboxane A2. Endothelial NO is the principal vasodilator in the vasculature and is produced by endothelial nitric oxide synthase (eNOS). Insulin is a vasoactive hormone that exerts its vasodilatory effects through eNOS-mediated NO production. Endothelial function is impaired in a number of disorders, including insulin resistance, diabetes and atherosclerosis, leading to dysregulated vasodilation as well as increased monocyte adhesion and plaque formation (atherosclerosis). The underlying molecular mechanisms leading to endothelial dysfunction are still in question. The work presented in this thesis addressed this question by investigating how insulin signalling and eNOS-mediated NO and superoxide production in human vascular endothelial cells are affected under experimental hyperinsulinaemia (chapter 3) and experimental hyperglycaemia (chapter 4). Atherogenic processes in human aortic endothelial cells (HAEC) were investigated by assessing monocyte adhesion under experimental hyperinsulinaemia (chapter 3), and by determining the contribution of NO and AMP-dependent kinase (AMPK) activity to the regulation of endothelial chemokine production (chapter 6). The potential of insulin to modify the subcellular distribution of eNOS was investigated in chapter 5. Clinical hyperinsulinaemia correlates with attenuated NO-mediated vasodilation, but it is not clear how hyperinsulinaemia impairs eNOS-mediated NO production. The present study modelled hyperinsulinaemia in HAEC and demonstrated a blunted response of hyperinsulinaemic cells to Ca2+-stimulated, but not insulin-stimulated eNOS-mediated NO synthesis. To address the underlying mechanisms responsible, the protein expression levels of components of the metabolic and mitogenic insulin signalling pathways, and of the metabolic energy sensor, AMPK, were quantified. Experimental hyperinsulinaemia slightly and non-significantly increased basal and insulin-stimulated eNOSS1177 phosphorylation in a time-dependent manner, and the levels of eNOST495 increased following acute insulin stimulation under these conditions. No marked dysregulation of individual insulin signalling pathway components was identified as a potential cause, but increased activating AMPKT172 phosphorylation was found to be a potential cause of increased unstimulated eNOSS1177 phosphorylation under experimental hyperinsulinaemia. Monocyte adhesion to hyperinsulinaemic HAEC did not differ from control HAEC, indicating that experimental hyperinsulinaemia did not act as a proatherogenic factor in the present study. Overt diabetes was simulated by experimental hyperglycaemia in human umbilical vein endothelial cells (HUVEC) and its effect on insulin-stimulated eNOS phosphorylation and endothelial superoxide production assessed. Insulin tended to stimulate phosphorylation of eNOSS615 and eNOSS1177, and decrease phosphorylation of eNOSS114, eNOST495 and eNOSS633 under control conditions. Experimental hyperglycaemia slightly reduced basal phosphorylation of Ser633 and significantly reduced insulin-stimulated phosphorylation of Ser114, but mildly increased basal Ser615 phosphorylation, indicating some dysregulation of eNOS phosphorylation. The upstream components of the metabolic insulin signalling pathway were not impaired in hyperglycaemic conditions. The subcellular localisation of eNOS is thought to have implications for its function. This study showed that eNOS localises to the plasma membrane, the nucleus, the cytoplasm and, primarily, the perinuclear area of HAEC. Insulin stimulation did not affect this distribution. Phospho-eNOS species were found primarily at the plasma membrane, and insulin may modulate the abundance of an intracellular eNOST495 species. Previous work in our laboratory on AMPK-mediated reduction of adhesion molecule expression has lead to the investigation of AMPK- and NO-mediated regulation of chemokine production in the present study. Inhibition of NO synthesis increased the production of monocyte chemoattractant protein (MCP)-1 in HAEC. AMPK activity downregulated TNFα-stimulated MCP-1 expression, and this was NO-dependent in the short-term, but may be NO-independent during prolonged AMPK activation. These data implicate NO and AMPK as antiatherogenic mediators in vascular endothelial cells in vitro. Taken together, the data in this thesis provide further insight into some of the molecular mechanisms involved in endothelial function and their response to hyperinsulinaemia, hyperglycaemia and proatherogenic stimulation.

Schizophrenia is a severe, chronic and debilitating psychiatric disorder. Current therapies have no efficacy in treating the cognitive impairments which are largely responsible for the poor quality of life of schizophrenia patients and contribute to the massive economic burden that is associated with the disorder. Although it is known that schizophrenia is highly heritable, the underlying genetic basis is still poorly understood due to the complex polygenetic nature of the disorder. Several candidate genes which are thought to increase risk for the incidence of schizophrenia have been identified. Two such schizophrenia candidate genes are neuregulin 1 (NRG1) and v-erb-a erythroblastic leukaemia viral oncogene homolog 4 (ERBB4). As well as the genetic evidence from genetic association studies, studies of animal models and the endogenous biological functions of NRG1 and ERBB4 in the CNS suggest that these genes may play an important role in the pathophysiology of schizophrenia. However, very little is known about the functions of these genes in specific brain regions in adulthood with respect to cognition. To address this, I have utilised recombinant adeno-associated viral particles (rAAVs) as a vehicle to mediate knockdown of the expression of Erbb4 specifically within the medial prefrontal (mPFC) cortex of adult rats. This allows for a spatially and temporally controlled investigation of the role that Erbb4 signalling may play in prefrontal cortex-dependent behaviours in adulthood. Following initial in vitro and in vivo validation of the functionality of the rAAVs, further in vivo studies confirmed that, five weeks after stereotaxic injection of rAAVs encoding a short hairpin sequence corresponding to Erbb4 (shErbb4.rAAV), into the mPFC of rats, there was significant Erbb4 protein knockdown, as analysed by ELISA. Subsequent western blot analysis revealed that Erbb4 knockdown consequently increased the level of Nrg1 expression and decreased the activity of Akt signalling, but had no effect on Erk signalling. Erbb4 knockdown specifically within the mPFC increased performance accuracy in the 5-choice serial reaction time task at 5 weeks post-surgery. Furthermore, viral mediated Erbb4 knockdown specifically within the mPFC heightened the II sensitivity to the locomotor inducing effects of amphetamine. There were, however, no effects of Erbb4 knockdown on pre-pulse inhibition at any time points assessed. These results indicate that Nrg1-Erbb4 signalling in the PFC modulates cognitive performance but not sensorimotor gating, and that dopaminergic transmission may be regulated by Nrg1-Erbb4 signalling. In conclusion, this study highlights the ability of viral mediated gene manipulation to investigate regionally specific roles of schizophrenia candidate genes in adulthood in terms of cognition and downstream signalling pathways. This may translate to a better understanding of how these genes may exert potentially pathophysiological effects in patients and ultimately lead to improved treatments.

The work reported in this thesis is concerned with understanding aspects of temporal behaviour. A large part of the thesis is based on analytical studies of temporal properties and interface and architectural concerns. The main areas covered include: i. analysing long-term human processes and the impact of interruptions and delays ii. investigating how infrastructures can be designed to support synchronous fast pace activity iii.design of the Getting-to-Know (GtK) experimental notification server The work is motivated by the failure of many collaborative systems to effectively manage the temporal behaviour at the interface level, as they often assume that the interaction is taking place over fast, reliable local area networks. However, the Web has challenged this assumption and users are faced with frequent network-related delays. The nature of cooperative work increases the importance of timing issues. Collaborative users require both rapid feedback of their own actions and timely feedthrough of other actions. Although it may appear that software architectures are about the internals of system design and not a necessary concern for the user interface, internal details do show up at the surface in non-functional aspects, such as timing. The focus of this work is on understanding the behavioural aspects and how they are influenced by the infrastructure. The thesis has contributed to several areas of research: (a)the study of long-term work processes generated a trigger analysis technique for task decomposition in HCI (b)the analysis of architectures was later applied to investigate architectural options for mobile interfaces (c)the framework for notification servers commenced a design vocabulary in CSCW for the implementation of notification services, with the aim of improving design (d)the impedance matching framework facilitate both goal-directed feedthrough and awareness In particular, (c) and (d) have been exercised in the development of the GtK separable notification server.

The aims of this European Commission funded project, carried out at the University of Glasgow, were to develop an approach for the inclusion of ethics in a science undergraduate curriculum and to evaluate the success of that approach. The moral nature of science as an academic discipline and as a professional career justifies the resources spent on novel ethics teaching within a science course. Choices in science - allocation of research funds, selection of research topics, interaction with research subjects (animals, environment, other humans) etc. - often, if not always, include some elements of morality. The dilemmas involved require decision-making which cannot, and should not, be made without reflection on the values that govern science and society at large. From the student perspective, the ethics curriculum aims to promote and accelerate moral development. In the context of ethics teaching in a science curriculum, moral development consists of two components: moral sensitivity and moral cognitive skills. Moral sensitivity is an ability to understand that moral aspects are as valid as factual data, and to distinguish between the two. Moral cognitive skills consist of an ability to 1) analyse the moral aspects of a situation, 2) differentiate the significant from the insignificant, 3) foresee the moral consequences of actions, and 4) to make moral decisions, in particular when it is necessary to choose between two or more incompatible values. The minimal ethics teaching intervention used in this study was a success as it captured students' motivation and interest and supported moral sensitivity development, which is the first step of moral development. The results show that ethics education is needed to support students' search for adequate moral decision-making tools and their ability to include moral considerations in their general decision-making.

No description available.

Recent years have seen a huge proliferation in the use of content in distributed applications. This observation has been exploited by researchers to construct a new paradigm called content-centric networking. Within this paradigm, applications interact with the network using a simple content request/reply abstraction. The network is then responsible for routing this request towards the 'nearest' provider that can offer the content. This process therefore exploits the observation that applications rarely have a vested interest in where their content is obtained from. However, it currently ignores the fact that many applications similarly have no interest in how their content is obtained, as long as it falls within certain requirement bounds (e.g. performance, security etc.). Consequently, existing content-centric interfaces offer no support for stipulating such abstract requirements. This thesis therefore proposes an extension of the content-centric abstraction to include the concept of delivery-centricity. A delivery-centric abstraction is one that allows applications to associate (high-level) delivery requirements with content requests. The purpose of this is to offer access to content in a specialised and adaptable way by exploiting an application’s ambivalence towards the underlying means by which it is acquired. Through this, applications can simply issue abstract requirements that are satisfied by the underlying system. These requirements can range from performance needs to more diverse aspects such as overheads, anonymity, monetary cost and the ethical policies of providers. Using the above principles, this thesis proposes the design of a new system that can offer a delivery-centric abstraction. This process is guided by key design goals, which dictate a solution should be interoperable with existing sources, highly deployable and extensible. Specifically, a middleware approach is taken, which results in the development of the Juno middleware. Juno operates by using configurable protocol plug-ins that can interact with various third party providers to discover and access content. Using these plug-ins, Juno can dynamically (re-)configure itself to deliver content from the sources that are most conducive with the application’s requirements. The thesis is evaluated using a number of techniques; first, a detailed study of real-world delivery protocols is performed to motivate and quantify the benefits of using delivery-centricity. Alongside this, Juno’s functional aspects (discovery and delivery) are also evaluated using both simulations and a prototype deployment to understand the performance, overheads and feasibility of using a delivery-centric abstraction. Throughout the thesis, performance is focussed on as the primary delivery requirement. It is shown that utilising a delivery-centric abstraction can dramatically increase the ability to satisfy this requirement, and that Juno’s approach fully supports such improvements. It is then concluded that placing delivery-centricity in the middleware-layer is a highly effective approach, and that it can be performed in a feasible manner to ensure that delivery requirements are met. The key contributions of this thesis are therefore, (i) the introduction of a new delivery-centric abstraction, (ii) the design and implementation of a middleware solution to realise this abstraction, and (iii) the development of novel technologies to enable content-centric interoperation with existing (and future) content providers.