The Apicomplexan parasite Toxoplasma gondii is considered an extremely successful pathogen for its capacity to invade virtually any nucleated cell. Host cell invasion is an active process thought to be driven by the same acto-myosin machinery that drives gliding motility. The current model suggests that at the core of the complex is MyoA, a small unconventional class XIVa myosin, which, together with its molecular partner myosin light chain 1 (MLC1), produces mechanical force on short actin (ACT1) filaments to power gliding and invasion. However, efficient conditional removal of the key components of the acto-MyoA motor complex indicated that although these proteins were important, they were not essential for motility or invasion. Some plausible explanations of this surprising finding were: probable redundancy among motor complex proteins, presence of residual protein in the conditional mutant lines, and/or compensatory mechanisms for driving these essential steps of the T. gondii life cycle. Considering these hypotheses, and given that T. gondii encodes for 11 myosins and 7 myosin light chains, this study focused on different possibilities upon MyoA and MLC1 depletion, Therefore, overlapping subcellular localisations and functions had to be considered. Due to its structural similarity, and that it shares molecular partners with MyoA, myosin C (MyoC) was the first candidate considered to compensate for MyoA function in the myoA KO. In fact, a myoA/B/C KO was unable to grow in in vitro conditions due to a detrimental egress phenotype, although it could still glide and invade. Here, the mlc1 KO, myoB/C/mlc1 KO, and a set of MyoC complementation constructs in the myoA KO were analysed in deep detail to further investigate the redundancy hypothesis. The results obtained do not dismiss functional redundancies between MyoA and MyoC; on the contrary, they show that some mechanisms, such as egress, can be rescued in the myoA KO. However, this redundancy does not explain how mlc1 and myoB/C/mlc1 KO parasites remain motile and invasive in the absence of residual protein in these conditional mutants. These results make it necessary to critically revisit the current motor model and re-evaluate the functions of the proteins involved in it. This thesis presents evidence that the acto-MyoA motor complex is involved in substrate attachment rather than in force generation. Finally, a hypothetical model for gliding motility is proposed aimed at reconciling recent observations. Together, considering different interactors and mechanisms, these results highlight the complexity of T. gondii tachyzoite cellular biology during progression of the lytic cycle.

Acute myeloid leukaemia (AML) encompasses a group of aggressive haematological neoplasms. It is a cancer stem cell (CSC) disorder. The Hedgehog (Hh) signalling pathway is one of the self-renewal pathways, highly conserved across species and important in determining stem cell fate, affecting a number of clinically important downstream targets including the Bcl-2 family. Abnormal Hh signalling has been associated with a diverse range of human malignancies. In myeloid malignancies, Hh signalling has been found to be vital in the maintenance and expansion of the CSC. Primary immotile cilia regulate canonical Hh signal transduction. These highly specialised organelles are present in single-celled eukaryotes through to humans, with defective primary cilia expression being linked to disease. It is unclear whether haematopoietic cells, normal or malignant, express primary cilia and the role the Hh pathway plays in the pathophysiology of AML. We sought to answer these questions, and further, to determine whether the Hh signalling pathway represented a therapeutic target in AML. Analysis of primary human AML mononuclear cells (MNCs) (n=76) showed the Hh pathway to be deregulated. SMO was significantly deregulated (p < 0.0001) with two divergent cohorts identified. GLI-1, indicative of pathway activity, was detected in 52.6% of samples. Sanger sequencing (n=36) did not identify a mutation in SMO to account for the pathway deregulation though a number of silent and missense mutations were identified (mean 1; range 1-3). 25% of our AML samples (9/37) showed expression of GLI-1 in >10% of cells by immunohistochemistry (IHC) on formalin fixed paraffin embedded (FFPE) primary human bone marrow trephines (BMTs) compared to none of our normal controls (n=10) (p < 0.0001). GLI-1 expression was independent of SMO and PTCH-1. SHH was significantly down-regulated (p < 0.001) within the blast population whilst secreted SHH, measured by enzyme-linked immunosorbant assay, was up-regulated suggesting paracrine activity. Impaired post-translational modification of SHH was demonstrated with protein located within the nuclei by IHC and immunocytochemistry (ICC). Nuclear expression of SHH was limited to primitive (CD34+) haematopoietic cells and absent from mature haematopoietic (CD14+, CD15+) cells. This correlated with a 20-fold reduction in HHAT, the acetyltransferase involved in Hh processing, in normal primitive haematopoietic cells compared to normal MNCs (p < 0.01). There was no correlation between subtype, mutational profile or clinical outcome and any of the components of the Hh pathway. Primary immotile cilia were identified in all AML (n=23), and 20% of normal (n=10) primary human FFPE BMTs by ICC. Primary cilia were not identified in AML cell lines (n=7) or primitive (n=4) or mature haematopoietic cells (n=6) isolated from peripheral blood, suggesting they are lost once cells migrate from the bone marrow (BM) microenvironment. In vitro, SMO inhibition with 20μM cyclopamine reduced cell proliferation by trypan blue exclusion in select, genetically diverse AML cell lines (HL-60, Kasumi-1, KG1a, MOLM-13, MV4-11, OCI-AML3 and THP-1). No change in early or late apoptosis was seen in HL-60, KG1a, MV4-11, OCI-AML3 and THP-1 by flow cytometry (FACS). In contrast an increase in the number of dead cells by trypan blue exclusion was seen in Kasumi-1 (p < 0.01) and MOLM-13 (p < 0.05) with apoptosis confirmed by FACS (both p < 0.05). Culture of the OCI-AML3 cell line with cyclopamine led to cell cycle arrest with an increase in G0-G1 cells (p < 0.05), and a 3-fold reduction in cell division by CFSE (p=ns); striking morphological changes were seen with an increase in cytoplasm, granules and vacuoles and loss of nucleoli, with FACS analysis demonstrating increased expression of CD11b (p < 0.001) and CD11c (p < 0.0001) consistent with a more mature phenotype. Early haematopoietic markers NAB2, GATA1, EGR2, SCL, IRF8 and EGR1 were down-regulated whilst PU1, GCSF and MPO involved in differentiation and maturation were up-regulated (p=ns) in cyclopamine treated cells. Colony forming cell (CFC) assays showed a statistically significant reduction in the more pluripotent CFU-GM (p=0.006) colonies and an increase in omnipotent CFU-G (p=0.013) colonies following culture with cyclopamine 20μM. There is extensive evidence supporting Bcl-2 is altered in malignancy; its role in AML cell maintenance and survival, is well recognised. Further, Bcl-2 is a key downstream target of the Hh signalling pathway. Complete linkage analysis found key members of the Bcl-2 family and cell cycle regulators to cluster with components of the Hh signalling pathway. Sensitivity to the BH3 mimetic ABT-199 was not solely dependent on the expression of Bcl-2, rather a complex interplay between the pro-apoptotic and anti-apoptotic family members. Targeting Bcl-2 had a variable effect on KG1a, MOLM-13, MV4-11, OCI-AML3 and THP-1 cells; combination treatment with ABT-199 and Ara-C showed a highly synergistic effect on cell death in MOLM-13 cells (Chou-Talalay CI < 0.47 (range 0.13-0.47) and DRI > 2 (range 2-70) for all dose combinations). We show the Hh signalling pathway to be deregulated in AML, expression of components of the pathway changing with myeloid maturation. We demonstrate primary cilia on haematopoietic cells within the BM, with an increased frequency observed in AML. Their absence when cells migrate from the BM fits with their function and suggests a ‘switching off’ of canonical signalling on maturation. In the absence of primary cilia, we show the Hh pathway remains active, suggesting a role for non-canonical signalling in AML. Pharmacological inhibition led to both apoptosis and differentiation; preliminary results suggest the effect is dependent upon the degree of SMO inhibition. We believe further work is required to determine the role of the Hh signalling pathway in normal and malignant haematopoiesis but that our data, considered especially in context with other recently published studies provides a rationale to continue to explore SMO or downstream Bcl-2 pathway inhibition as potential therapies in AML.

Oxygen is a vital molecule for life and its precise quantification is pivotal to ensure correct functioning of cells. Instruments able to quantify accurately the oxygen levels are extremely important to understand cells behaviour especially in a three-dimension (3D) environment, where uneven distribution of oxygen can occur leading to zones of low cell viability. Optical oxygen sensing is a relatively recent method to monitor oxygen concentration. This approach exploits the emissive behaviour of a broad family of molecules (organic species and organometallic complexes), for which the luminescence emission is proportional to the quantity of oxygen present in the environment. Unlike methods based on the use of electrodes, optical oxygen sensing allows monitoring of oxygen levels without causing oxygen depletion in the observed sample and can be more easily applied in miniaturised devices. Metalloporphyrins are widely employed as optical oxygen sensors thanks to their favourable physico-chemical properties. The aim of this project was to synthesise a library of oxygen-sensing porphyrins to monitor variation of oxygen in 3D cell cultures. For this specific application, the chromophore needed to (1) be a platinum or palladium complex to ensure oxygen response, (2) present a suitable functional group to allow conjugation to a support (e.g., a biomacromolecule supporting the 3D cell construct) to avoid leaching and cell uptake, and (3) water-solubility to allow conjugation in aqueous solutions. We first focused our attention on the synthesis of tetra meso-substituted porphyrins bearing a carboxylic group as the conjugatable function on one of the beta-positions of the macrocycle. This approach was overall successful in terms of yield and reaction conditions, but the side-chain proved insufficiently stable to allow further manipulation of the species. Attention was then directed to the synthesis of asymmetric porphyrin A3B, bearing the conjugable group on one of the meso-substituted aryl ring. Novel positively and negatively charged water-soluble conjugatable porphyrins were developed during this work, obtained from a single high-value intermediate. The new species presented overall good yields employing relatively mild reaction conditions, via a synthetic procedure with gram-scale applicability. In addition, in this work the synthesis of two known water-soluble conjugatable porphyrin was improved and scaled-up. Platinum (II) and palladium (II) insertion in the water-soluble macrocycles was achieved employing a microwave-assisted reaction, which ensured reaction completion in short times. Finally, a high potential purification approach based on ion exchange was developed for cationic and anionic water-soluble derivatives. This method allows obtaining highly pure compounds employing cheap reagents and without the use of expensive purification instruments. The conjugation of the species to the proteins chosen as matrices for hydrogels (silk fibroin and collagen) was performed via N-hydroxysuccinimide chemistry. Oxygen response of the complexes synthesised was tested both in aqueous solution and supported on protein-based hydrogels. Most species showed oxygen-dependent fluorescence and linear Stern-Volmer plot supported on solid support, indicating their suitability to monitor the oxygen levels in a heterogeneous system such as a hydrogel. To the best of our knowledge, this work represents the first example of optical oxygen measurements in silk fibroin hydrogels.

No description available.

Human disease due to food-borne pathogens remains a medical problem worldwide. Campylobacter jejuni is the most common cause of bacterial food-borne diarrhoeal disease, but the mechanisms by which it causes disease remain unclear. This bacterium is also the most common antecedent to the peripheral neuropathies Guillain-Barre syndrome (GBS) and Miller-Fisher syndrome (MFS). The main aim of this project was to compare cytotoxicity of isolates of C. jejuni towards mammalian cells in culture, to purify any cytotoxic activity and to compare strains by plasmid and proteomic analysis. Using the API Campy identification kit, 37 of the 39 strains were identified as C. jejuni jejuni serotype 1 or 2 with identification (ID) scores > 98.3%; and two strains were identified as C. jejuni jejuni 1 with ID scores of 88.3%. GOT (Gamma Glutamyl Transferase) test was the most important test to differentiate between C. jejuni jejuni 1 and 2 which showed negative and positive results, respectively. The cytotoxic activity against Vero and Caco-2 cells of some of the typed strains of C. jejuni was tested. Cytotoxic activity was clearly demonstrated using Vero and Caco-2 cells and these activities peaked after incubation of the cell culture with sample for 16h. Comparison of wild-type strains 81-176 and 11168 with mutants of these strains lacking cytolethal distending toxin (CDT) or putative haemolysin or phospholipase toxins indicated that the toxicity observed was not due to any of these proteins, as cytotoxicity was unaffected by their absence. Some isolates (e.g. C0L12) showed low cytotoxicity while others, especially C. jejuni 81-176, consistently produced high cytotoxin activity which was heat-labile and lethal to tissue culture cells. Cytotoxicity comparisons of inner and outer membrane preparations with a cytoplasmic fraction of C. jejuni strain 81-176 and C0L12 indicated that greatest activity was found in the inner membrane fraction. Proteins in the membrane preparation could not be easily fractionated and cell-free extracts of strains with low (COL12) and high (81-176) cytotoxic effects were chosen for further purification and characterisation studies. Although some differences were detected after DEAE- Sepharose fractionation and two-dimensional electrophoretic (2-DE) separation of proteins, no individual proteins could be clearly related to cytotoxicity. However, proteomic analysis did reveal some interesting properties of the C. jejuni strains. Of the proteins characterised by mass spectrometry after 2-DE, oxidoreductases were the most frequently identified in the virulent strain 81-176, but not in the type strain 11168. There was no apparent correlation between toxin production and clinical symptoms in patients from whom the strains were isolated. The study indicated that not all C. jejuni strains tested produced cytotoxin (s), but that cytotoxin producers seemed to be predominantly human strains. The presence of virB11 and tetO genes carried by plasmids was examined by PCR in the 39 C. jejuni isolates together with strains 81-176 and NCTC 11168. Detection rates for the virB11 and tetO genes in the clinical isolates were 28.2% and 12.82%, respectively.

Pathogenic protozoa of the genus Leishmania pose a significant burden on global health. Control relies on a limited range of chemotherapeutic options, with amphotericin B of increasing importance. Understanding how resistance may emerge to this drug is therefore of some concern. As amphotericin B acts through its affinity for leishmanial sterols, altered sterol composition has been described. However, little is known about the genetic basis of these changes. In this thesis, selection and characterisation of four novel amphotericin B-resistant L. mexicana lines are described. Changes to parasite drug sensitivity and fitness were profiled, as well as alterations in metabolism. This revealed heterogeneity between lines, suggesting that many changes arise stochastically during selection of resistance. In one line, no fitness costs to infectivity and replication in primary macrophages were found. Hypersensitivity to the drug pentamidine, however, was a consistent phenotype. All parasites demonstrated altered sterol composition. Genetic and transcriptomic profiling revealed associated changes in sterol biosynthesis genes, with mutation in sterol C5-desaturase observed in one line and loss of sterol C24-methyltransferase expression apparent in the other three. Broader analysis revealed extensive and apparently stochastic transcriptome remodelling, associated with chromosomal copy number changes. Deletion of the miltefosine transporter was found in one line, associated with miltefosine cross-resistance. Through reintroduction of candidate genes into resistant lines, a role for these changes was demonstrated in both amphotericin B resistance and pentamidine hypersensitivity. Reintroduction of sterol biosynthesis genes, but not the miltefosine transporter, was associated with at least partial restoration of wild-type sterol composition. Finally, changes to the sterol C24-methyltransferase locus were investigated, revealing that structural variations are the basis of expression changes, possibly mediated by repetitive sequences at this locus. These data demonstrate multiple routes to drug resistance, and suggest that at least some of these routes allow retention of parasite infectivity. Evidence of structural instability at the locus of sterol C24-methyltransferase, associated with altered sterol composition and drug resistance, is of particular concern.

Mass spectrometry imaging (MSI) is a powerful tool for mapping the spatial distribution and relative abundance of molecules across a sample surface. The distribution of proteins, metabolites, lipids and drugs can be determined and unlike other molecular imaging techniques, such as immunohistochemistry (IHC), MSI is completely label-free. Therefore, this technique does not require prior knowledge of the molecule to be imaged and thousands of molecules can be imaged at once. This is particularly useful for untargeted imaging, to discover molecules which are important for a certain condition. For example, a healthy tissue sample can be compared to a diseased sample in the same imaging run to help understand the molecules involved in the disease process. MSI has become a popular technique in fields such as neuroscience, drug distribution studies and as a biomarker discovery tool in cancer. However, the use of MSI in microbiology has to date been limited. In the present study MSI was used to investigate molecular host-microbe interactions of both beneficial and pathogenic bacteria of the gastrointestinal tract. In the initial part of this thesis, MSI was employed to discover molecular changes in the host, caused by Salmonella enterica serovar Typhimurium infection. S. Typhimurium is a Gram-negative facultative intracellular bacterium and is a leading cause of food-borne infection worldwide in humans. The symptoms include abdominal cramps and diarrhoea, and although this infection is usually self-limiting with individuals recovering without the requirement for treatment, it can be more serious in young, old, malnourished, or immunocompromised people. S. Typhimurium is transmitted by ingestion of contaminated food or water. The bacteria infect cells of the gastrointestinal tract, causing inflammation, and cross the epithelial layer of the gut to enter underlying specialised immune tissue, the Peyer’s patches. S. Typhimurium can be taken up by immune cells, but can survive within these cells and are transported to another specialised immune location, the mesenteric lymph nodes (MLNs). In the present study, an S. Typhimurium infected, gastroenteritis mouse model was used to investigate host-pathogen interaction. Various tissue types were collected from 72 h infected, 48 h infected and uninfected mice including colon, Peyer’s patches and MLN. IHC staining was used to locate tissue types and regions where S. Typhimurium were present and MSI was employed to find molecular changes caused by the infection. This preliminary analysis highlighted 73 molecules that differed in abundance or distribution between infected and uninfected samples, across all three tissue types. These molecules could be investigated further in future, however, subsequent analysis focused on one molecule, which was identified as palmitoylcarnitine, a molecule involved in fatty acid metabolism. This molecule was present at high abundance in areas of the MLNs 72 h post infection, where both bacteria and infection induced tissue damage were present. This molecule was also present in uninfected samples and areas of infected tissue where no bacteria were present at lower levels, therefore this molecule was deemed to be host derived. It was hypothesised that this molecule could either be; produced by the immune system to directly damage S. Typhimurium, be produced by the immune system to enhance the immune response, or be a by-product of tissue damage and could further damage host cells. Therefore, subsequent analysis focused on testing the effects of palmitoylcarnitine to investigate these hypotheses. No effects were found when testing this molecule on bacterial growth or virulence. Palmitoylcarnitine localised to areas of immune cell, T cell, B cell and macrophage, disruption in the MLNs. Cells from MLNs were isolated and cultured in the presence of palmitoylcarnitine to investigate any effects of this molecule on immune cell death or activation. Palmitoylcarnitine was found to cause cell death by apoptosis of a particular subset of immune cells, CD4+CD25+ T cells. These immune cells are mostly likely regulatory T cells, which protect the host against excess damage during an immune response. Therefore, the overall hypothesis was S. Typhimurium infection could be disrupting fatty acid metabolism, leading to accumulation of palmitoylcarnitine. This in turn causes death of CD4+CD25+ T cells, which could be responsible for causing the excess tissue damage found in the MLNs during infection. The second part of this thesis employed MSI to investigate the interaction between the beneficial bacteria of the gastrointestinal tract, the microbiota, and the host brain. An altered microbiota has recently been linked to diseases throughout the body and differences in the microbiota have been found in patients with neurological disorders, such as autism, Parkinson’s disease and depression. There is still little known about the links between the brain and microbiota and how the microbiota may be influencing the brain. In the present study the colons and brains of mice with absent or depleted microbiota were compared to conventionally colonized mice to investigate possible links between the gut and the brain. MSI was demonstrated to be an effective technique to image known molecules, as well as previously unknown molecules which changed between microbiota depleted and conventionally colonized mice. A previously unknown microbiota derived molecule was chosen for further identification and analysis. This study demonstrates the capabilities of MSI as a discovery tool to find molecules important for host-microbe interactions. This study also aids in advancing the use of this technique in the field of microbiology, which would be highly beneficial in future to help understand immune evasion strategies of pathogenic bacteria and how the microbiota is interacting and crucial to the host.

No description available.

Macrophages are a dynamic cell type and represent a key component of the immune response, with a broad range of activities throughout the body. They respond to external cues, including microbes, alarmins, and growth factors, to eliminate invading pathogens through initiation of inflammation. Subsequently, they carry out several regulatory roles, including clearance of cellular debris, the resolution of inflammation, and wound healing to restore tissue homeostasis after an inflammatory response. The ability of macrophages to change their phenotype in this manner must be tightly regulated, as dysregulated macrophage activity is central to the pathogenesis of both inflammatory and fibrotic autoimmune disorders, such as Rheumatoid Arthritis (RA) and Idiopathic Pulmonary Fibrosis (IPF), respectively. One of the mechanisms by which regulation of macrophages occurs is the microRNA network. MicroRNAs (miRNAs) are a class of short, non-protein coding RNAs that post-transcriptionally regulate gene expression through translational repression, destabilisation or degradation of target mRNA. miR-155 is a multi-functional miRNA that has roles in regulating the development and function of many immune cells, including macrophages. Abnormal expression of miR-155 is associated with a number of autoimmune disorders and cancers. We reported previously that miR-155 is elevated in RA and contributes to the chronic, pro- inflammatory response of macrophages by repressing key anti-inflammatory proteins. However, the role of miR-155 in the regulation of remodelling responses by macrophages is less well characterised. Modulation of miRNA activity in cells through the use of mimics and inhibitors has emerged as a potential therapeutic strategy for the treatment of diseases. Technologies involving the use of lipid vesicles as delivery agents for introducing therapeutics into target cells have shown potential in increasing the drug efficacy.

No description available.