The Natural Killer (NK) cell activating receptor DNAM-1 (CD226) is stimulated through recognition of CD112 (nectin-2) and CD155 (nectin-like molecule 5; PVR) on target cells. HCMV UL141 elicits protection from NK-cells by down-regulating CD155 from the cell surface and sequestering it in the ER (Tomasec, 2005). Here, HCMV UL141 was shown to be involved in the down-regulation of CD112. Interestingly, UL141 appeared necessary but not sufficient to modulate CD112 expression. This thesis therefore focused on a hypothesis whereby UL141 was acting with one or more additional HCMV genes to target CD112 for degradation. This project was the first to utilise an entire recombinant adenovirus (RAd) library expressing individual HCMV ORFs (RAd-HCMV-ORF library) to screen for function. The RAd-HCMV-ORF library clearly provided an extremely powerful tool for the screening of HCMV gene function as results were highly repeatable and robust. The co-infection of RAd-UL141 and RAd-US2 resulted in a single, clear, positive hit in the final screening process. This hit was further verified by immunoblot where CD112 appeared to be down-regulated in cells infected with both RAd-UL141 and RAd-US2, compared to controls. While a Hela-US2 cell line which stably expressed US2 also down-regulated CD112 when infected with RAd-UL141. A RCMVΔUS1-11 virus was constructed, which failed to down-regulate CD112 from the cell surface of RCMVΔSU1-11 infected cells. The addition of proteasome inhibitors was able to partially restore CD112 expression in HCMV infected cells (Prod'homme et al., 2010). It therefore appeared that US2 and UL141 act to degrade CD112 via the proteasome during HCMV infection. CD112 downregulation may have the potential to prevent DNAM-1:CD112 interaction between HCMV infected targets and effector cells of the immune system, providing another facet to HCMV’s ability to avoid the human immune response.

No description available.

Aim of this thesis was to evaluate mixtures of indigestible carbohydrates in vitro to predict their effects on gut function. In this study, I investigate the effect of combining carbohydrates with different fermentative properties and their interactive influences, reflected in the end products from in vitro fermentation. The study focused on the rate of fermentation and fermentability of such mixtures and the SCFA produced to gain an index of the likely site of fermentation in the colon. The main aim of the thesis was to produce a mixture of carbohydrates which would delay but preserve butyrate production from rapidly fermenting carbohydrates such as raftilose. This was achieved in several mixtures but mostly those containing raftilose and ispaghula. In general, mixtures of carbohydrates were fermented more slowly than raftilose alone. Overall, ispaghula was the most effective in slowing the rate of fermentation compared with pectin or gums. Mixing raftilose with ispaghula or guar gum gave the best preservation of n-butyrate and propionate production. The rate of n-butyrate production was less rapid in mixed cultures of three carbohydrates (raftilose, ispaghula and pectin) than cultures of 100mg raftilose but production of n-butyrate was preserved. In summary, ispaghula and raftilose in two-carbohydrate mixtures and ispaghula, pectin and raftilose in three-carbohydrate mixtures delayed the release of butyrate with no loss in butyrate production and may move butyrate further round the colon, at the same time reducing the potential adverse effects of raftilose. Moreover, the addition of pectin (or guar gum) may add the therapeutic effect of delaying nutrient absorption in the small intestine was well. These studies have identified at least two mixtures (raftilose & ispaghula; raftilose, ispaghula & pectin) worthy of study in more detail in man.

Management of fungal biofilms represents a significant challenge to oral healthcare. As a preventive approach, minimising adhesion between intra-oral devices and microorganisms would be an important step forward. Denture stomatitis (DS) is a multifactorial denture-associated inflammation of the oral mucosa where candidal biofilms are one of the contributing factors. Therefore, understanding candidal biofilms on dentures and finding novel strategies to control these biofilms are of significance. Interference with the adhesion step of biofilm formation is hypothetically effective strategy to control biofilms. To understand the relationship between denture candidal load, denture material type and C. albicans biofilm forming heterogeneity in DS, quantitative polymerase chain reaction (qPCR) molecular method and crystal violet (CV) assay were used. This study investigated two novel strategies to control C. albicans biofilms through interfering with adhesion: natural polyphenol curcumin (CUR) and modifying the topography of the denture material surface. Based on the optimised effective CUR concentrations, CUR adsorption to PMMA denture material was spectrophotometrically analysed. Based on these data, the effect of adsorbed CUR to PMMA and CUR pre-exposure on adhesion of C. albicans were assessed. The effect of CUR on Candida-Candida adhesion was investigated and the expression profile of selected adhesion and aggregation-associated genes was assessed using qPCR method. Micro/nano-fabricated polycarbonate and PMMA materials were replicated using injection and compression moulding techniques, respectively and were characterised using scanning electron microscopy (SEM). Adhesion of C. albicans on the micro and nano-scaled patterns was assessed using microscopic and qPCR molecular methods, respectively. The physical characteristics of the materials were assessed using theta tensiometer and a white light profiler. The data demonstrated that although C. albicans was detected in greater quantities in diseased individuals, it was not associated with increased biofilm biomass. Denture substrata were shown to influence biofilm biomass, with poly(methyl methacrylate) providing the most suitable environment for C. albicans to reside. Subsequent studies showed that CUR concentrations of 50 μg/ml could prevent adhesion to PMMA. This effect was enhanced by the CUR pre-treatment of yeast cells (>90% inhibition, p < 0.001). Investigation of the biological impact of CUR showed that it preferentially affected immature morphological forms (yeast and germlings), and actively promoted aggregation of the cells. Transcriptional analyses showed that CUR temporally modulated adhesion and aggregation associated genes. Finally, PMMA denture material was replicated to show nano features. These topographies influenced adhesion of C. albicans, depending on the candidal morphological form and the shape. Nano-pit spatial arrangements variably affect the adhesion of C. albicans, where SQ arrangement demonstrated a significant anti-adhesive capacity. Differential adhesin expression was observed on these surfaces, which were affected by the wettability and roughness of surfaces tested. In summary, C. albicans is an important determinant of denture disease, so preventing its adhesion and biofilm formation were worthwhile objectives. This thesis has shown that CUR molecules and SQ nano-pit topographies reduced C. albicans adhesion, demonstrating that chemical and physical inhibition strategies are useful. The data presented in this thesis showed the high potential of the novel strategies to be used against C. albicans biofilms, and encourages the further investigation of these approaches against polymicrobial denture biofilms.

GM-CSF is an important haematopoietic growth factor and immune modulator. Studies on T cells revealed that efficient activation of the human GM-CSF gene is dependent upon the activation of an enhancer located 3 kb upstream of the promoter, inducible by phorbol myristate acetate and calcium ionophore (PMNI) via kinase-and Ca\(^2\)\(^+\) -dependent signalling pathways, respectively. This enhancer is often aberrantly remodelled as a constitutive DNase hypersensitive site (DHS) in acute myeloid leukaemia (AML). To investigate the role of MAPKs in enhancer activity and chromatin remodelling, I used activated T blasts and human leukaemic cell lines as inducible model systems. The combination of MEK and p38 MAPK inhibitors reduced PMNI-induced GM-CSF gene expression and the DHS at the enhancer. This was associated with a reduction in DNA-binding activity for the MAPK-inducible AP-1 and in the phosphorylation of MSK1, which in turn stimulates NF-κB transcriptional activity by phosphorylating p65 at Ser276. The combination of MEK and p38 inhibitors also reduced the PMNI-mediated recruitment of AP-1, MSK1 and NF-κB at the enhancer. These data demonstrate a cross-talk between the MAPK and NF-κB signalling pathways in regulating GM-CSF gene transcription and therefore represent potential targets for the treatment of AML cases where aberrant chromatin remodelling occurs.

Endothelium forms an interface between vasculature and cellular sub endothelial environment responding to the changes in the immediate environment in health and disease. During vasculitic glomerulonephritis (VGN), autoantibodies raised against neutrophil serine proteases (SP) activate the neutrophils resulting in the release of neutrophilic products onto the endothelial surface changing its ability to support neutrophil recruitment and leading to its damage. We have hypothesized that endothelial functions are modulated by intra and extracellular environments of the vasculature and that the glomerular epithelial cells (podocytes) regulate glomerular endothelial cell (GEnC) functions. This work investigated the effects of SP (Proteinase-3 (PR3) and human neutrophil elastase (HNE)) in mediating neutrophil recruitment on EnC prior to the development of injury in a concentration and time dependent fashion. Most of the effects were inhibitable by the presence of α1-antitrypsin (α1-AT). Further, using an in vitro static co-culture system, this work demonstrates an endogenous mechanism by which podocytes modulate endothelial response to inflammatory stimuli in the glomerulus under healthy and disease conditions. Overall, these studies elucidate that GEnC presents an adhesive phenotype attracting neutrophils under the influence of inflammatory cytokines. During disease conditions as in VGN, exposure of endothelium to SPs results in a pre-activation stage leading to damage with high and/or prolonged exposures. Podocytes modulate glomerular endothelial responses to cytokine stimulation in health and disease. Thus, endothelial functions are tightly regulated by interacting cells and their molecules and could be an important phenomenon in the disease process of VGN where neutrophil recruitment plays a central pathogenic role.

Escherichia coli is one of the most studied model organisms in biology. Even with decades of research, there are a substantial number of genes with an as yet unknown function. Previously, to determine the link between gene function and phenotype took significant experimental effort. However, newer methods are capable of providing large amounts of biological data in short timeframes. One such method, transposon insertion sequencing, is a powerful research tool, which couples transposon mutagenesis and next generation sequencing to identify genes that have important or essential functions. Here, three transposon insertion sequencing methods were compared. The techniques were adapted from previously published literature. Based on a number of metrics one technique was shown to be superior for data generation. This method was chosen for application in further transposon-insertion sequencing experiments. Subsequently, the optimised method was used to assess which genes were essential for the viability of the model organism E. coli K12. The results of this work were compared with the literature and other databases of gene essentiality. A high degree of concordance was observed between our datasets and those generated previously through other methods. Indeed, the method described here was shown to have several benefits over previously used approaches. Finally, genes involved with maintenance of the outer membrane were identified by using markers for membrane permeability in tandem with the chosen method. In keeping with previous literature multiple genes involved with many aspects of the cell envelope were reported. Many of the reported genes were shown to be involved with metabolic processes related to the biogenesis and maintenance of the cell envelope.

The desmosome is an inter-cellular complex required for strong cell-cell adhesion in skin and heart tissue. The plakin family of proteins is important to the desmosome as their main function is to act as linkers between the cell surface and cytoskeleton. The structures, dynamics, impact of heart disease causing mutations and interactions of these component domains have remained largely unknown. In order to better understand this, biophysical techniques were used to shed light into the specific effects of pathological mutations using complementary techniques including nuclear magnetic resonance spectroscopy, small angle X-ray scattering and X-ray crystallography. Novel insights included the modular organization and flexibility of serial spectrin repeat constructs in the N-terminal plakin domain was established. Biophysical characterization of a variety of plakin family C-terminal tail constructs was successfully conducted in addition to understanding the molecular consequences of disease causing point mutations linked to Arryhthmogenic Right Ventricular Cardiomyopathy. Together this provides a better understanding of the specific roles of the plakin family’s N- and C- termini in linking the desmosomal machinery on the cell membrane to the intermediate filaments.

Biofilm formation is one of several significant virulence factors associated with life threatening pulmonary infections in immunocompromised individuals caused by Aspergillus fumigatus. Previous studies have demonstrated phase dependant antifungal activity against A. fumigatus biofilms. Antifungal resistance associated with fungal biofilms is a complex multifactorial phenomenon, and it remains unclear specifically how this manifests itself in A. fumigatus. This study therefore aimed to investigate adaptive resistance mechanisms in A. fumigatus biofilms. Different phases of A. fumigatus biofilms were grown for 8, 12, 24 and 48h in polystyrene plates in RPMI media. Functional efflux pump activity was subsequently assessed using an Ala-Nap fluorescent uptake assay. Extracellular material was extracted from each phase and the level of extracellular DNA (eDNA) was quantifiedusing a microplate fluorescence assay. The minimum inhibitory concentrations (MIC) of different classes of antifungals were assessed in the presence and absence of different inhibitors using a checkerboard assay, or with a fixed concentration, by the broth microdilution method to assess synergism, antagonism, or otherwise. The presence of eDNA and phenotypic changes in biofilm caused by antifungal agents and inhibitors were assessed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) techniques. The resultant biofilm biomass for different experiments was evaluated using a crystal violet assay. SYBR green qRT-PCR was used to assess the expression of different genes implicated in biofilm resistance (AfuMDR 1-4, ChiA-E, HSP90 and Fks1) over the period of multicellular development, using a diffusion chamber in a murine model and a Galleria mellonella infection model. The results from this study demonstrated phase dependant expression of efflux pumps in A. fumigatus biofilm populations, which actively contributes to azole resistance. Moreover, voriconazole treatment induced efflux pump expression in both in vitro and in vivo models.These data suggest that A. fumigatus efflux pump proteins, which evolved to become integral to their natural physiological function, have inadvertently induced resistance to azole drugs, albeit in the early phases of biofilm development. Assessment of A. fumigatus biofilm extracellular matrix (ECM), associated with maturing biofilms, showed that eDNA is an important architectural component of the biofilm, helping to maintain its stability. The antifungal sensitivity of different phases of A. fumigatus growth decreased significantly in the presence of DNase, indicating that decreased susceptibility to antifungals in the A. fumigatus is mediated in part by eDNA.Its release was shown to correlate withchitinase activity, a marker of autolysis, suggestive that autolysis was associated with eDNA release. It was hypothesised that heat shock protein 90 (HSP90) was involved in this autolytic pathway. Therefore, when HSP90 was pharmacologically inhibited this led to a decrease in matrix eDNA level, providing a compelling mechanism through which HSP90 might regulate biofilm antifungal resistance. To test whether these mechanisms of adaptive resistance had any bearing clinically, a G. mellonella model was developed. It was shown that each of the key genes were expressed during infection, both in control and antifungal treated larvae. This validates the potential use of this insect model for resistance and virulence studies. Overall, this study establishes several novel adaptive resistance mechanisms regulating biofilm drug resistance in A. fumigatus biofilms. Moreover, it highlights the potential to target these mechanisms as a therapeutic strategy for managing and improving clinical outcomes in these hard-to-treat infections.

The human Ureaplasma species are among the smallest and simplest self-replicating bacteria known to date. These microbes cause infection in humans, particularly in the upper genital tract during pregnancy, leading to several adverse outcomes including preterm birth, chorioamnionitis, and respiratory diseases of neonates. Little is known about the pathogenesis of Ureaplasma and mechanisms by which they avoid recognition and killing by the complement system. In this thesis, some mechanisms underlying serum resistance of Ureaplasma spp. were investigated. This goal was achieved by creating serum-resistant models of serum-sensitive laboratory Ureaplasma strains and developing and using some proteomic and molecular biology methods to study the role of potential factors, which mediate serum resistance and play a role in pathogenesis of Ureaplasma. My original contribution to the knowledge in this work was the development of transposon mutagenesis method that can now be used to study virulence genes of Ureaplasma. This method will also allow genetic manipulation of Ureaplasma for future studies. Monitoring and investigating induced serum-resistant strains using immunoblot analysis and proteomics revealed significant changes in two candidate proteins coincident with serum resistance. The first was the elongation factor Tu protein that found to be immunogenic and had altered pI isoforms. The observed change in this protein was consistent in all serum-resistant strains, which suggests a possible role in mechanism of serum resistance, possibly as a mediator for binding complement regulators, such as factor H and C4BP, at the cell surface of Ureaplasma. The second candidate protein was a novel 41 kDa protein that was uniquely expressed in all induced serum-resistant strains. Expression of this protein in all resistant strains strongly indicates its involvement in mechanism(s) of serum resistance of Ureaplasma. The possible gene that encodes for this 41 kDa protein has putatively been identified as UUR10_0137 in the genome of U. urealyticum serovar 10 (strain ATCC 33699) using the transposon mutagenesis method developed in this study. Although the gene product of UUR10_0137 gene is not known (hypothetical protein), this protein is now identified and proposed to have a role in serum resistance of Ureaplasma. The product of the UUR10_0137 gene could function as a complement regulator or inhibitor that prevents the activation of complement system, protecting Ureaplasma from the complement attack. The contribution of the multiple- banded antigen, MBA, was proven to be unimportant to serum resistance. Sole antigenic variations in this major surface antigen of Ureaplasma did not play any role in mediating serum resistance. Confirmation of a gene that mediates complement resistance would dramatically increase our understanding of Ureaplasma pathogenicity and provide a target for future human studies with preterm birth and Ureaplasma infection.