Dictyostelium discoideum is a model eukaryotic organism with many equivalent cellular mechanisms to humans. In this project Dictyostelium has been used to help investigate the role of rhomboid proteins. Bioinformatics tools were used to identify four potential rhomboids encoded within the Dictyostelium genome that contain the residues necessary for proteolytic activity. To identify their functions, three null mutants rhmA- , rhmB- and rhmC- have been constructed, and their growth and development observed. It was found that development was unaltered following deletion of rhmC, whereas attempts to knock out the putative mitochondrial rhmD were unsuccessful. rhmA and rhmB null mutants gave rise to pleiotropic effects, rhmA altering the response to chemoattractants and demonstrating decreased motility in general. It also showed altered mitochondrial morphology which is consistent with phototaxis defects in slugs. rhmB null cells had slower growth rates, reaching stationary phase at a reduced density; a delayed response to folic acid stimulation; larger cell size in the unicellular phase and altered stalks in the multicellular phase. These results correspond with qPCR analysis, in which RhmA and RhmB transcript levels are highest during the multicellular development phase. Dictyostelium is also a useful non-animal eukaryote for testing novel compounds and dissecting cell regulatory molecular networks. In this thesis I have used this social amoeba to test the effect of a series of arylboronic acids and a novel compound, cardiosulfa, on development, chemotaxis and viability of Dictyostelium. The arylboronic acids were investigated previously by collaborators for serine protease inhibition and urokinase-type plasminogen activator (uPA) inhibition, both in vitro and in vivo. In those biochemical assays, three compounds, BC11, SR3 and BC57 displayed micromolar inhibition of uPA with an excellent selectivity profile over related proteases (Smith et al., 2012). Previous studies suggested that cardiosulfa, disrupted heart development in the zebrafish. Gene expression profiling studies showed that cardiosulfa induces high transcription levels of members of the aryl hydrocarbon receptor (AhR) family (Ko et al. 2009). These test compounds reported to show effects in vivo also disturbed cell adhesion and migration, without having an effect on Dictyostelium viability. Networks of predicted activity were constructed to establish molecular mechanisms for cardiosulfa and arylboronic acids analogues. In conclusion this project provides the first insight into the molecular and cellular functions of rhomboid proteins, potential aryl hydrocarbon receptor and urokinase-type plasminogen activator by using Dictyostelium as an early biomedical model to study these effects in eukaryotes.

The identification of a blood-based diagnostic biomarker for breast cancer (BC) would be particularly beneficial to those at increased risk of developing BC and could result in earlier detection which may increase survival rates due to earlier treatment. Alterations in α-1-acid glycoprotein (AGP) glycosylation levels occur during disease and this study sought to determine the diagnostic potential of AGP glycan variation in triple negative breast cancer (TNBC) compared to BC of unknown type and healthy controls as well as women at increased risk of developing BC compared to age-matched healthy controls. AGP was isolated from blood of two different sample populations using low pressure chromatography. AGP purity was confirmed using SDS-PAGE and concentration determined using spectrophotometry. Structural analysis of AGP glycan monosaccharide and oligosaccharide content was undertaken using high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Increased AGP concentrations were observed, in comparison to their controls, in BC of unknown type, TNBC and “at-risk” samples. Quantitative alterations in monosaccharide composition were also present. N-acetylgalactosamine (GalNAc) was present in over 88% of TNBC samples and was inversely correlated with age. For the TNBC groups, GalNAc was also present at higher levels in samples of individuals with family history of BC. There was an overall increase in GlcNAc levels compared to age-matched healthy controls and GalNAc presence in 81% of “at risk” samples. Oligosaccharide analysis revealed increased branching in BC of unknown type and TNBC < 35 years of age, whereas the “normal” healthy population and TNBC >35 possessed less branching. A similar trend was observed between the “at risk” samples and the age-matched controls. These branching patterns aligned well with the corresponding monosaccharide data. Overall, this study indicated that alterations in AGP levels and glycosylation exist between TNBC compared to BC of unknown type and “normal” healthy controls as well as an “at risk” population and age-matched healthy controls. The data could underpin the development of a new diagnostic BC biomarker.

Rhodococcus equi (R. equi) is a Gram positive, facultative and weakly acid-fast bacterium, which causes respiratory disease in foals worldwide resulting in pneumonia, often with fatal consequences. Human infections of R. equi that are resistant to antibiotics are increasingly reported. A novel approach of treating R. equi infections is needed. While phage/phage-derived proteins have been demonstrated for effective treatment and diagnoses of major pathogenic bacteria such as Staphylococcus aureus and Mycobacterium tuberculosis, little is known about the diversity and genomics of phages infecting R. equi. The objectives of this study were to isolate and characterise the diversity of phages infecting R. equi, to sequence a single R. equi phage genome and, based on detailed bioinformatic analysis, to identify and characterise the in vitro activity of selected R. equi phage proteins with potential lytic activity. The diversity of 26 lytic R. equi phages isolated from Scottish soil was assessed, resulting in a designation of 16 distinct groups by genome restriction profiles and with host range analysis. All belong to the Siphoviridae, with the exception of phage E3, the first Myoviridae known to infect Rhodococcus and capable of lysing 8 R. equi strains tested. Analysis of the genome termini of representatives of the 12 phage groups revealed the presence of cohesive ends in phages V6, V15, GV10, GV11, GV12 and GV22, while 6 phage genome terminal cohesive ends could not be established. The genome sequence of E3 was determined, revealing a dsDNA genome of 142,563 bp, encoding 221 ORFs. The E3 genome is globally similar to that of the Myoviridae of mycobacteriophages. Interestingly, a holin, 2 putative endolysins (lysA) and 3 putative lipolytic (lysB) genes were identified in the E3 genome. One putative lysA and lysB, encoding gp98 and gp84 respectively, were cloned and the in vitro activity investigated, revealing lytic activity against R. equi for gp98 while the activity of gp84 could not be established. The findings presented in this thesis represent a significant advance in R. equi and mycolata phage biology and provide interesting insight to the evolutionary context of mycolata-infecting Myoviridae, supporting the argument for taxonomy classification based on comparative genomics.

The alpha-2-macroglobulin superfamily consists of large multi-domain proteins that are activated by protease cleavage. One arm of this family consists of protease inhibitors that undergo a large conformational change upon protease cleavage, simultaneously physically trapping the cleaving protease and covalently linking to it via a thioester bond. However, there is little mechanistic understanding of how protease cleavage activates the conformational changes that lead to protease inactivation. These protease inhibitors are found in tetrameric, dimeric and monomer forms within eukaryotic blood/lymph fluid. The recently described Escherichia coli alpha-2-macroglobulin (ECAM) is a periplasmic, inner membrane anchored protease inhibitor. The gene encoding ECAM, yfhM, is found within an operon alongside pbpC, which encodes penicillin binding protein 1C. These two proteins have been proposed to function in defence and repair against host proteases with ECAM acting to inhibit proteases that have breached the outer membrane and Pbp1C repairing damage to the peptide linkages within the peptidoglycan layer. This thesis describes the structural and biophysical characterisation of ECAM and an investigation into the role of Pbp1C in ECAM function. In order to gain insight into the mechanism through which protease cleavage activates ECAM we used a combination of X-ray crystallography, small angle X-ray scattering and analytical ultracentrifugation to characterise the conformational changes that occur on protease cleavage. The X-ray structure of protease cleaved ECAM revealed a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native inter-domain interactions that maintain ECAM in the inactivated form. Owing to the similarity in structure and domain architecture of ECAM and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group. Further to this, it was shown that ECAM is processed in vivo, existing largely as truncated forms in growing E. coli cells. Interestingly, Pbp1C plays a key role in ECAM processing, with cell lacking pbpC showing an accumulation of full-length and dimeric forms of ECAM.

No description available.

The Caliciviridae are a group of small, non-enveloped viruses with a positive sense, single stranded RNA genome. Caliciviruses include the noroviruses, responsible for winter vomiting disease, as well as several important veterinary pathogens. Feline calicivirus (FCV) is an excellent model for studying calicivirus entry, having a known protein receptor and being readily propagated in cell culture. Here we explore calicivirus entry, using FCV. Virus entry is the critical first step of infection and is therefore an important area of study. Both alpha 2-6 linked sialic acid and feline junctional adhesion molecule A (fJAM-A) have been identified as receptors for FCV. The attachment of FCV to fJAM-A, is followed by uptake via clathrin mediated endocytosis. Little is known, however, on the viral escape mechanism leading to delivery of the viral RNA into the cytoplasm. We set out to explore the nature of FCV attachment and uncoating using structural, biochemical and biophysical analyses. By cryogenic electron microscopy we have characterized the virus-receptor interaction at high-resolution. Using electron microscopy and an RNA release assay, we have investigated virion uncoating. Finally, we have explored the importance of receptor glycosylation, and oligomerisation. Our analysis has allowed us to construct an atomic model of the major capsid protein VP1. Upon binding to fJAM-A, FCV undergoes a conformational change (rotation and tilting of the capsomeres). Flexibility in the receptor decorated virion has prevented high-resolution structure analysis of the conformational change or the virus-receptor interaction. We have, however, seen that the structural changes are limited to the capsid spikes. We hypothesised that the conformational change may be a priming step that would prepare the virus for uncoating upon internalisation. We found that upon lowering the pH below 5, receptor decorated virions disassembled, supporting this hypothesis. Disassembly of the virus-receptor complex at low pH presented a tool for estimating the quantity of receptor needed to prime the capsid for uncoating. Cryo-EM studies reveal that FCV bound fJAM-A is monomeric although the receptor was found to be dimeric in solution as previously described for the human and murine homologues. Furthermore, it is hypothesised that this is the form found at tight junctions between cells. We propose that disruption of fJAM-A homodimers may be the mechanism by which induction of viral uptake by endocytosis is triggered. Finally, we have confirmed the presence of an N-linked glycosylation on fJAM-A and show that the removal of this carbohydrate moiety does not affect viral binding in vitro.

SP-D is a large hydrophilic protein, consisting of four collagenous trimers, which is secreted by alveolar type II and non-ciliated bronchiolar epithelial cells and participates in calcium-dependant agglutination of inhaled microorganisms. Known high-resolution crystal structures of mono- and disaccharide bound recombinant human SP-D include a heptose disaccharide that mimics the inner core fragment of bacterial lipopolysaccharide. This thesis focuses on the structural characterisation of SP-D binding to lipopolysaccharides from Gram-negative bacteria. Intact LPS and several hydrolysed smooth and rough mutants were soaked into native crystals of a recombinant head and neck fragment of SP-D. Those soaked with hydrolysed Escherichia coli J5 and Salmonella minnesota R7 LPS showed electron density corresponding to ligand in the ligand-binding site. All crystals processed conformed to spacegroup P21, all with unit cells close to a= 55, b= 108, c= 55 Å, = 91°. The maximum resolution diffraction observed was 1.63 Å. The R7-soaked structure was refined at 1.77 Å, with a final R-factor of 18.71% and R-free of 22.05%. The structure reveals a well-defined trisaccharide unit of two heptose saccharides and a single Kdo residue in protein chains B and C. The Kdo is present as a five-membered, anhydro residue known to form during mild acid hydrolysis. The third, outermost heptose of the R7 inner core is not visible in the electron density. The refined structure demonstrates binding of LPS via the O6 and O7 hydroxyl groups of the glycerol sidechain of HepI, the innermost heptose, demonstrating for the first time structurally not only the binding of a physiologically relevant bacterially derived ligand but also the recognition of a non-terminal monosaccharides by SP-D. No direct interaction is observed between the second heptose and the protein, but two hydrogen bonds are seen between the anhydro-Kdo and the amino acids Arg343 and Asp325 which flank the SP-D ligand binding pocket.

Research aiming to understand pathogens in infection is shifting rapidly towards considering not only the individual pathogen but the whole microbial community. Therefore, understanding microbial communities through exploring the key questions in community ecology, such as the relationship between diversity and stability, are relevant here also. Research has made considerable progress in characterising microbial communities of different body sites but the human oropharynx microbiome is still among the less well known despite its importance in hosting various commensal bacteria and being an important entry site for pathogenic intrusion. Determining the healthy oropharynx microbiome will allow comparison to various disease scenarios and the attributes that change a community from a healthy to diseased state. This thesis represents the most comprehensive survey of looking at the longitudinal bacterial community structure in the oropharynx. Here, analysis was done on the bacterial oropharynx microbiome composition, its natural fluctuations and stability, and relating these to the changes that occur to the microbiome before, during and after an infection. This involved initial swabbing of the oropharynx of eighteen baseline-healthy, non-smoking participants weekly for a total period of 9 months and sequencing the V1-V2 region of the 16S rRNA gene using Illumina MiSeq sequencing. This would determine the community make up that is representative of a healthy state. This was then directly compared to oropharyngeal samples taken weekly from 12 smokers within the same age range for a total period of 6 months to observe the community differences between smokers and non-smokers. Looking at the healthy participants (non-smokers) alone, the key taxa recovered were Firmicutes at phylum level and Streptococcus, Prevotella and Veillonella at genus level; these were the most abundant taxa in healthy samples. There was variation in taxa within and between participants, but this variability in microbial community structure occurred more at genus and OTU level. Variability was influenced by changes in health status, although environmental factors were also likely to play a role even though they were not investigated here. Disturbances to the oropharynx microbiome were shown in participants that had cold-related symptoms (negative for viruses) and antibiotic treatment. These communities had decreased diversity (as opposed to high diversity healthy communities) and changes in abundances of certain taxa. However, participants recovered quickly from these disturbances (within one week after the disturbance) in that the microbiome returned to a state similar in community composition prior to the disturbance. This showed the oropharynx microbiome of baseline-healthy participants to be relatively resilient and stable as samples from the same participants were similar on a weekly basis. Looking at smokers, they had distinct changes in the bacterial community of the oropharynx in comparison to non-smoking healthy participants. This included changes in abundance of taxa with increased Bacteroidetes, Proteobacteria and Actinobacteria at phylum level and Streptococcus at genus level and increased abundances in pathogenic microorganisms such as S. pneumoniae which overall affected the functions associated with the bacterial community. These communities also appeared stable (regardless of having an altered state) in that samples from smoking participants were also similar on a weekly basis, but interestingly, were only disrupted during antibiotic treatment and not during an infection from samples with cold related symptoms. Therefore this thesis provides insight into the oropharynx microbiome of healthy participants (non-smokers) and smokers. It examines the stability and resilience of the oropharynx microbiome during specific scenarios and identifies the key and important taxa in a healthy and unhealthy community. By continuing to develop this research it may be possible to identify, treat and restore respiratory diseases by examining the oropharynx microbiome through identification of taxa and functions.

No description available.

Neurodegenerative conditions such as Multiple Sclerosis (MS) and spinal cord injury (SCI) affect hundreds of thousands of people each year worldwide, and numerous cell transplant-based therapeutic strategies are being investigated to aid in the repair and regeneration of the central nervous system. Of particular interest are mesenchymal stem cells (MSCs), due to their differentiation potential, their immunomodulatory effects, and their ability to stimulate various biological properties due to the substantial variety of growth factors, chemokines, and other signalling molecules secreted by these cells. MSCs taken from the bone marrow (BM-MSCs) have demonstrated significant reparative potential in animal models of both MS and SCI. The question I address throughout this thesis however, is whether MSCs from another niche; the olfactory mucosa (OM-MSCs), are a preferable or at least alternative candidate for such therapies, compared to BM-MSCs, and if they are, why are they? Previous studies have shown that OM-MSCs can be purified and grown from human olfactory mucosa and when incubated with rat glial/neuronal co-cultures are capable of increasing axonal myelination, an effect not elicited by BM-MSCs. This potentially has great therapeutic benefit for a range of neurodegenerative conditions, as a significant part of the regenerative process involves replacing the protective myelin membrane which ensheaths axons. A comparative study of the two types of MSCs shows a number of similarities, including the expression of the same panel of MSC markers, a 64% homology in miRNA expression, an ability to differentiate towards bone and fat, and a propensity for bone formation when cultured on osteogenic nanotographies. This thesis also outlines a number of differences between each phenotype which suggest that OM-MSCs could even be a preferred alternative, especially in neuroregenerative therapies. OM-MSCs were shown to express significantly more Nestin than BM-MSCs, and to proliferate at a significantly higher rate, two observations which may be related. This increased proliferation would have enormous benefit for their use, as BM-MSCs are mitotically quite slow, and any MSC-based therapies would require very large numbers of cells. Twenty six different miRNA were shown to be differentially expressed between BM-MSCs and OM-MSCs. Three of these; miR-140-5p, miR-146a-5p, and miR-335-5p were linked to three important biological functions; myelination, cell survival, and cell proliferation respectively. These three biological functions, importantly, are ones which were observed as being behavioural differences between OM-MSCs and BM-MSCs. OM-MSCs were also shown to secrete significantly more of the pro-myelinating chemokine, CXCL12, which was confirmed as being regulated by the microRNA, miR-140-5p. This offered a potential mechanism for the pro-myelinating effect of OM-MSCs, and also opens up new research potential for investigating therapeutic targets to regulate myelination. The data presented in this thesis shows many similarities between BM-MSCs and OM-MSCs, but it also highlights some profound differences which suggest that either they originate from a different lineage entirely, or that the cellular niche that they reside in does indeed affect the differentiation and behaviour of mesenchymal stem cells.