Tuberculosis (TB) has plagued humanity for centuries, and, despite being largely curable, it still claims thousands of lives daily. The causative agent, Mycobacterium tuberculosis (M. tb), shows resilience by evading immunological and antitubercular challenges. This is mainly due to its lipid-rich cell wall, whose main components are mycolic acids (MAs). MAs are long fatty acids with functional groups of precise stereochemistry. The MA chemical structure and composition balance in the cell wall modulates antigenicity, cell wall permeability, and virulence. MA chemical structure also steers MA folding. However, MA structure-function relationships are still not fully understood. In this work, exhaustive MA conformational determination studies were performed in order to generate systematic detail into the correlation of MA structure and conformations, which may in turn hold the key to understanding TB at the atomistic level. Atomistic molecular dynamics (MD) simulations, in different solvents and temperatures, were performed on 166 MAs covering all three M. tb MA classes, both the cis and trans stereochemistries of the proximal cyclopropyl group, and a wide range of chain lengths. Analysis focused on dihedral angle and principal component analysis (PCA) clustering, as well as intra-molecular distance matrix calculations. The results presented in this work confirmed the MA functional groups as folding points and the spontaneity of the MA folding. Three major conformations were identified, namely four-chain, knot, and five-chain folds, with the four-chain fold being the dominant conformation. The knot fold demonstrated definable subtypes, whose further characterisation may offer potential for refinement of current serodiagnostic methods, since MAs have been used as antigens in TB serodiagnosis.

No description available.

No description available.

No description available.

Mediation of biological functions occurs via tightly regulated signal transduction pathways. These complex cascades often employ crosstalk with other signalling pathways to exert strict control to allow for correct cellular responses. The cyclic AMP signalling pathway is involved in a wide range of cellular processes which require tight control, including cell proliferation and differentiation, metabolism and inflammation. Protein Kinase C (PKC) signalling is also involved in the regulation of many biological functions, due to the wide range of PKC isoforms, and there is emerging evidence that there are critical points of crosstalk between these two central signalling pathways. The aims of this research, therefore, are to identify the molecular basis underlying this pivotal cross-communication. The identification of the complex formed by Receptor for activated C Kinase 1 (RACK1), a scaffold protein for PKC, and the cyclic AMP-specific phosphodiesterase PDE4D5 demonstrated a potential area of crosstalk between the cyclic AMP and PKC signalling pathways although the function of the complex remained largely unknown. In this thesis I have outlined a role for RACK1 binding to PDE4D5 to control the enzymatic activity of the phosphodiesterase. Although RACK1 does not affect the intracellular localisation of PDE4D5, it does afford structural stability to PDE4D5, providing protection against denaturation. Furthermore, interaction with RACK1 facilitates high affinity binding of PDE4D5 to cyclic AMP and increases phosphodiesterase sensitivity to inhibition by rolipram, a PDE4-specific inhibitor that is a therapeutic treatment for depression and Alzheimer’s disease. Additionally, RACK1-bound PDE4D5 was found to be activated by PKCα, providing a route of negative regulation by PKC on cyclic AMP in HEK293 cells. The discovery of EPAC (Exchange Protein directly Activated by Cyclic AMP) has opened up the field of cyclic AMP research, providing an alternative route for the cyclic AMP signalling originally thought to occur solely through Protein Kinase A (PKA). Recent investigations have linked cyclic AMP signalling via EPAC to the control of inflammation, through the induction of Suppressor of Cytokine Signalling 3 (SOCS-3) to inhibit IL-6 signalling. Here I have further delineated this pathway in COS1 to show that induction of SOCS-3 by EPAC requires phospholipase C (PLC) ε. Investigation into downstream effectors of PLC action lead to the identification of PKCα and PKCδ as essential components of this pathway, further elucidating a mechanism by which cyclic AMP can affect inflammation and revealing a point of crosstalk between the two signalling pathways. Further elaborating on the identification of PKC isoforms α and δ as crucial components in the control of cytokine signalling by cyclic AMP via EPAC, investigations into the effect of cyclic AMP on PKC α and δ activation and autophosphorylation, and on downstream effectors, were carried out. It was revealed that cyclic AMP had no influence on PKCδ activity, although a role for cyclic AMP signalling through EPAC on the activation and autophosphorylation of PKCα was identified. Additionally, phosphorylation of the downstream kinase ERK was found to occur independently of PKC activation and required the presence of EPAC1 in COS1 cells. The work presented in this thesis therefore begins to delineate a novel pathway in which the cyclic AMP and PKC pathways work together to afford cell regulation, including the regulation of gene expression, through novel areas of crosstalk.

The principle aim of this project is the investigation of metabolism and mechanisms of pentamidine resistance in trypanosomatids. An understanding of these mechanisms may allow the development of novel drugs to treat Leishmaniasis and human African trypanosomiasis (HAT), caused by the protozoan parasites Leishmania spp and Trypanosoma brucei. In this study a pentamidine resistance L. mexicana promastigote cell line was generated in vitro. This cell line was 20-fold resistant to pentamidine when compared to the parental wild type cells. Furthermore, these lines were cross resistant to other diamidine compounds. A proteomic analysis of these cell lines revealed numerous changes to the proteome, with the down regulation of several flagellar proteins. A hypothesis to investigate a role of the voltage dependent anion channel (VDAC) in pentamidine resistance was also explored. The metabolomic approach involved the investigation of transketolase and the pentose phosphate pawthway. A previous study involving a transketolase knockout T. brucei cell line indicated that an increased sensitivity to pentamidine and methylene blue. A transketolase deficient L. mexicana cell line was generated to test this hypothesis in Leishmania, however the differences were minimal. A metabolomic analysis of the L. mexicana tkt null cell line (lmtkt-/-) revealed an increase in ribose 5-phosphate, a key substrate of transketolase. Erythrose 4-phosphate also increased in the lmtkt-/- cells, indicating a source of this metabolite independent of TKT. It appears that the deletion of TKT prevents any flux through the oxidative branch of the PPP returning to the glycolytic pathway. Interestingly, the lmtkt-/- cells do not acidify the medium to the same extent as the wild type cells; however a glucose assay indicated that both cell lines used similar quantities of glucose. This would suggest that there is a change in the metabolites excreted by the lmtkt-/- cell line. Finally, a global metabolomics approach was investigated using high resolution mass spectrometry. Metabolomics is a rapidly developing field in systems biology, and whilst significant improvements have been made in mass spectrometry; the ability to analyse and interpret raw metabolomic datasets on a global scale has been largely neglected. Consequently, a database program to query these complex datasets was constructed.

Myocardial ischaemia is characterised by a reduction in blood flow sufficient to cause a pathological change in myocardial function. One of the myocardial adaptive responses to ischaemic stress involves a change in gene expression which is regulated by the interaction of DNA binding proteins with specific DNA sequences. Identifying intracellular signalling pathways that link the changes in the extracellular environment during ischaemia to alterations in gene expression may provide important information on how the myocardium adapts to ischaemic stress. Specific regulatory sequences that interact with DNA binding proteins in response to ischaemic stress may be used as part of a gene therapy protocol to regulate foreign gene expression in the ischaemic myocardium. One of the aims of this study was to identify control regions that may play a role in the myocardial response to ischaemic stress. A hybrid promoter, containing myosin heavy chain basal regulatory sequences plus four copies of the erythropoietin HIF-1 binding site, conferred inducible expression of a luciferase reporter gene in response to 15 minutes of ischaemia followed by reperfusion. This induction was rapid and reversible upon reperfusion of the ischaemic myocardium. The human skeletal α-actin (SkAct) promoter was also investigated but showed no significant activation in response to 15 minutes of ischaemia followed by reperfusion in rat and rabbit myocardium at the time points examined. The SkAct promoter was further characterised by analysing the relative expression of mutant and wild-type SkAct promoters in the rat heart after direct DNA injection. This study indicated possible regulatory sequences that may be involved in the cardiac specific regulation of the SkAct gene and indicated for some promoter constructs a possible discordance between in vivo and in vitro data. The extent and severity of the pathological loss of function caused by myocardial ischaemia is critically dependent on the collateral circulation perfusing the ischaemic region. Stimulating angiogenesis in the ischaemic heart by adenovirus mediated overexpression of angiogenic factors may offer the possibility of increasing collateral perfusion to ischaemic areas, improving myocardial function. Adenoviral vectors are capable of delivering foreign genes to the myocardium with high efficiency and low toxicity. With a view to limiting the extent of myocardial ischaemia and preventing further expansion of the ischaemic areas, the present study also aimed to develop a potentially therapeutic adenoviral vector expressing an angiogenic factor. A replication deficient recombinant adenovirus expressing the highly specific and potent angiogenic factor, vascular endothelial growth factor (VEGF), was constructed. Using this viral vector for infection of cell lines in culture, evidence was obtained of foreign expression of the recombinant gene product.

The purpose of this project was to generate low temperature active esterases for a constituent of laundry detergent, to permit the hydrolysis of fat and oil stains in cold water washing. To determine how well the esterase could work against substrate bound to a surface at low temperatures, it was decided to utilise the esterases as ligands in atomic force microscopy to measure affinity of said esterases against non-emulsified substrates. Early work involved the identification and characterisation of a novel family of esterases from extremophilic origins as possible ligands for use in the method described. Additional work was performed on testing the theory behind atomic force microscopy by developing ligands from thoroughly understood lipolytic enzymes and measuring any changes in activity. Finally, work was performed on making a psychrophilic variant through mutagenesis from an esterase that had not been previously studied in this project. Results indicated that there was one candidate from the novel family which showed great activity against long-chain substrates at the desired temperature, but experiments indicated that no member of the novel family were monomers, and were thus deemed unsuitable for use with atomic force spectroscopy. For this reason, other psychrophilic, monomeric esterase were explored. One such candidate was found, variants were made through directed evolution which were found to have increased activity over the wild-type form. Results from collaborators working with atomic force microscopy using ligands designed in this project indicated that it was valid as a method for analysing esterase affinity to substrate.

Escherichia coli is a diverse bacterial species found largely as a harmless commensal in the gastrointestinal tract of warm-blooded mammals. However, an array of highly adapted E. coli pathotypes have evolved over time capable of causing a variety of niche specific diseases, both intestinally and extraintestinally. This ability to cause disease results from the adaptation of the core genome to the host and the acquisition of horizontally acquired virulence factors. Furthermore, these foreign virulence factors are integrated into the regulatory network of the cell allowing niche specific competitive advantages for the emerged pathogen. Enterohaemorrhagic E. coli O157:H7 is a dangerous pathogen capable of causing haemorrhagic colitis and the potentially fatal haemolytic uremic syndrome. This pathogen is a harmless coloniser of ruminants whereas humans are extremely susceptible with transmission via the faecal-oral route, commonly associated with contaminated food products. It is becoming apparent that the carriage of virulence factors is only one element to the adaptive nature of E. coli pathogens, with the specific response to niche specific signals governing when and where these virulence factors are utilised. O157:H7 utilise a type 3 secretion system (T3SS) as their major colonisation factor, which facilitates subversion of and subsequent intimate attachment to the human intestinal epithelium. This T3SS is encoded on a pathogenicity island known as the locus of enterocyte effacement (LEE) that encodes all the necessary components of a T3SS and a number of effector proteins. The LEE is tightly regulated in a temporal manner that is highly responsive to components of the intestinal physiology - namely nutrients, metabolites and hormone-like signals. Interestingly, despite the LEE encoded T3SS being capable of mediating attachment to a diverse variety of cell types in vitro, this colonisation factor is exclusively utilised by intestinal E. coli pathotypes but the mechanistic reasoning behind this is unknown. In this work, the impact of the host metabolite D-serine on O157:H7 is explored in detail. D-serine is highly abundant in sites commonly colonised by extraintestinal E. coli pathotypes, such as the urinary tract and the brain. Furthermore, it acts as a positive fitness trait and regulator of virulence factors within these niches. Conversely, intestinal strains of E. coli are largely unable to metabolise D-serine but its effects on their gene expression has not previously been investigated. Here, it is demonstrated that D-serine selectively affects gene expression in O157:H7 by repressing the LEE encoded T3SS and activating the SOS stress response. The toxicity of D-serine was entirely dependent on intracellular accumulation of this metabolite however the ability of D-serine to repress the LEE was found to be independent of its ability to be metabolised. Comparative genomic analysis revealed that carriage of both the LEE and the D-serine tolerance locus (dsdCXA) is an extremely rare event attributed to the apparent incompatibility between the two loci. It is proposed that the negative effects of D-serine on the LEE limit pathotypes such as O157:H7 to the gastrointestinal tract by forcing the evolutionary loss of dsdCXA, demonstrating the importance of co-operation between horizontally acquired and core pathogenic elements in defining niche specificity. A novel D-serine sensing system was also identified and characterised in O157:H7. This system includes a D-serine transporter, YhaO, and a LysR-type transcriptional regulator, YhaJ, which are absolutely required for expression of the LEE in O157:H7. This system is highly conserved in all E. coli and further demonstrates the adaptive ability of the core genome to perceive and respond to important environmental signals that define specific niches. Collectively, this thesis describes the mechanistic basis of D-serine sensing in O157:H7 and the physiological relevance of D-serine sensing for diverse E. coli pathotypes. This work provides a strong framework for further research both by revealing novel insights into bacterial evolution and also creating potential targets for anti-bacterial therapeutics.

The first part of this thesis examined by comparative analysis the phenotype, tissue distribution and functional profile of the human DC subsets, myeloid (m) DC and plasmacytoid (DC), in RA and PsA. This study demonstrated that circulating pDC and mDC are reduced in the peripheral blood (PB) of RA and PsA patients, but are accumulated to the inflammatory synovial fluid (SF) and synovial membrane (SM), commensurate with altered migration. Furthermore, it was demonstrated that the majority of pDC and mDC within the synovial compartment displayed a predominantly immature phenotype as identified by the low to absent expression of specific co-stimulation and maturation markers including CD80, CD83, CD86 and dendritic cell lysosome-associated membrane protein (DC-LAMP). However, pDC and mDC purified from SF underwent ex vivo phenotypic maturation and released cytokines to TLR stimulation at comparable levels to their normal PB purified counterparts. Commensurate with that observation, cytokine profiling of SM localized pDC and mDC revealed expression of the pro-inflammatory cytokines IL-12p70, IL-23p19, IL-15, IL-18 and IFN-α/β. Together these data indicated no apparent intrinsic cellular defect, suggesting that extrinsic factors in the local synovial environment may be culpable for inhibition of DC maturation. In addition mDC in SM expressed predominantly IL-12p70 and IL-23p19, suggesting a central role in the regulation and expansion of T cells, in particular Th17, which have been ascribed a pathologic role in arthritis. In contrast, pDC expressed predominantly IL-15 and IL-18, cytokines that can enhance IL-12 induced IFN-γ release and inhibit T cell apoptosis, thus indicating a role in the amplification and prolongation of inflammatory arthritis. Of particular interest it was demonstrated that pDC also expressed IFN-α/β in abundance. As pDC constituted the dominant DC subset in the SM in both RA and PsA, this may have pathological implications as IFN-α has been critically linked to pathology in other autoimmune diseases, including systemic lupus erythematosus (SLE) and, of particular import to PsA, psoriasis. In conclusion, these studies indicate that mDC and pDC possess multi-faceted roles in inflammatory arthritis, involved in both the initiation and perpetuation of arthritis, but also the potential suppression thereof. The data provide a firm basis upon which to build rational tolerance induction regimes in due course as the appropriate biological targeting agents become available in the clinical environment.