Colloidal nanoparticles stabilised in an aqueous mixture were used for the rapid detection of disease biomarkers at high levels of sensitivity. Peptide substrates were used to functionalise the nanoparticle surfaces for targeted interaction with specific enzymes related to breast cancer and other maladies. These systems have potential clinical applications for disease detection and high-throughput drug screening. Semiconductor nanoparticles or quantum dots (QDs) were functionalised by a synthetic peptide substrate for the detection of the protease breast cancer biomarker urokinase plasminogen activator (uPA). A non-radiative energy pathway was created through the conjugation of the peptide to a 1.4 nm gold nanoparticle. The enzymatic dispersion of this system was quantified by fluorescence spectroscopy and used to determine the enzyme concentration. A materials-based characterisation of the uPA system was conducted using small angle X-ray scattering at the Australian Synchrotron. Results were used to determine the maximum number of peptide conjugates that could bind to the QD and how they spatially arranged themselves around the surface. Application of these results can be used in the design of new QD-based biosensors. The uPA detection assay was combined with a Förster resonance energy transfer based QD detection assay for the kinase breast cancer biomarker, human epidermal growth factor receptor - 2 (Her2). Multiplexed detection of the two enzymes was successfully shown in a proof-of-concept experiment. This assay demonstrates the potential for accurate biosensing by nanoparticles in a complex biological environment. A highly sensitive peptide assembled gold nanoparticle based assay for the detection of proteolytic enzymes was designed for use with absorbance spectroscopy and surface enhanced Raman spectroscopy (SERS). The protease thermolysin was detected at a concentration of 0.01 nM using SERS. The assay illustrated the usefulness of SERS for enzyme detection for the investigation of protein-peptide interactions.

This thesis describes the fabrication of biomimetic substrates, and their use as tools to probe cellular interactions of key immune cells. Nanoparticles of gold and zinc sulfide have been fabricated, and patterned into nanoarrays. Adaptive (T cell) and innate (NK cell) immune cell responses to nanoscale spacing of ligand-receptor pairs were measured, and the effect of presenting stimulatory ligands on substrates with varying mechanical properties has been tested for T cell responses. The advanced materials in this thesis act to create artificial immune synapses, and probe the effect of these stimuli on engagement and activation of human immune cells. Specifically, block co-polymers were used to form polymer micelles which encapsulate metal ions and form metal or metal compound nanoparticles. Micelles encapsulating metal ions or nanoparticles were formed and deposited onto substrates using Block Co-polymer Micellar Lithography (BCML) to form nanoparticle arrays with controlled inter-particle spacing. Well controlled gold nanoparticle arrays with spacing between 25-150nm have been produced. The technique has been further developed to include fabrication of zinc sulfide particles and nanoarrays. Zinc sulfide nanoparticles showed a unique internal structure with 5nm crystalline domains set in an amorphous matrix and an optical band gap of between 3.88-4.28eV. Nanoparticle arrays were then functionalised with biological ligands, notably antibodies that engage with the NK cell surface receptor CD16, or the T cell TCR/CD3 moiety. The cellular response to these materials was measured, and was sensitive to the nanoscale arrangement of stimulatory ligands; both cell types responded to ligands with 25nm, but not 104nm, inter-ligand spacing. In an alternative approach, spherical PEG hydrogel particles of diameter 5-50μm were formed with controlled rigidity between 3-2000kPa. T cell response as a function of substrate rigidity was tested, and cells showed maximal response to anti-CD3 functionalised substrates with rigidities of 3-5kPa.

The cell:cell bond between an immune cell and an antigen presenting cell is a necessary event in the activation of the adaptive immune response. At the juncture between the cells, cell surface molecules on the opposing cells form non-covalent bonds and a distinct patterning is observed that is termed the immunological synapse. An important binding molecule in the synapse is the T-cell receptor (TCR), that is responsible for antigen recognition through its binding with a major-histocompatibility complex with bound peptide (pMHC). This bond leads to intracellular signalling events that culminate in the activation of the T-cell, and ultimately leads to the expression of the immune eector function. The temporal analysis of the TCR bonds during the formation of the immunological synapse presents a problem to biologists, due to the spatio-temporal scales (nanometers and picoseconds) that compare with experimental uncertainty limits. In this study, a linear stochastic model, derived from a nonlinear model of the synapse, is used to analyse the temporal dynamics of the bond attachments for the TCR. Mathematical analysis and numerical methods are employed to analyse the qualitative dynamics of the nonequilibrium membrane dynamics, with the specic aim of calculating the average persistence time for the TCR:pMHC bond. A single-threshold method, that has been previously used to successfully calculate the TCR:pMHC contact path sizes in the synapse, is applied to produce results for the average contact times of the TCR:pMHC bonds. This method is extended through the development of a two-threshold method, that produces results suggesting the average time persistence for the TCR:pMHC bond is in the order of 2-4 seconds, values that agree with experimental evidence for TCR signalling. The study reveals two distinct scaling regimes in the time persistent survival probability density prole of these bonds, one dominated by thermal uctuations and the other associated with the TCR signalling. Analysis of the thermal fluctuation regime reveals a minimal contribution to the average time persistence calculation, that has an important biological implication when comparing the probabilistic models to experimental evidence. In cases where only a few statistics can be gathered from experimental conditions, the results are unlikely to match the probabilistic predictions. The results also identify a rescaling relationship between the thermal noise and the bond length, suggesting a recalibration of the experimental conditions, to adhere to this scaling relationship, will enable biologists to identify the start of the signalling regime for previously unobserved receptor:ligand bonds. Also, the regime associated with TCR signalling exhibits a universal decay rate for the persistence probability, that is independent of the bond length.

While Human Leukocyte Antigen (HLA) typing remains the central means of selecting stem cell donors and determining stem cell transplantation (SCT) outcome, non-HLA molecules are also involved in the process. HLA compatibility is the main factor determining the occurrence of graft-versus-host disease (GvHD) in patients. It has also been shown that minor histocompatibility antigen differences as well as genetic polymorphisms that are not sequenced by standard methodology for HLA typing can play a role. As early predictive markers for the occurrence of GvHD are still lacking and a better understanding of the mechanisms and pathways involved are still needed to provide insight into the pathogenesis of the disease, this study aims to identify novel targets for identifying allogeneic responses predictive of such a serious complication of this therapeutic treatment. The mixed lymphocyte culture (MLC) model was used under different conditions in order to identify potential genetic markers associated with allogeneic responses. Initially, this model using the mononuclear cell fraction of blood samples from mismatched individuals was validated by comparing cell activation and proliferation and time course studies, as well as using irradiation of cells and lipopolysaccharide (LPS) in an effort to mimic the in vivo conditions occurring during the infusion of allogeneic cells to patients during SCT. Through a limited gene expression analysis of immune related genes, of both HLA matched and HLA-mismatched mononuclear cell fractions, we further validated the time and conditions used for global gene expression profiling and observed different gene expression changes between the two HLA settings. The investigation of global gene expression changes, of the HLA matched and HLA mismatched MLC, enabled us to identify differentially regulated genes and pathways. A great number of these genes were related to immune function. These genes were also found to be associated with GvHD and graft rejection in previous studies. By investigating global gene expression changes in mismatched MLC we aimed to identify genes that may constitute good targets to study further as markers for donorrecipient alloresponses. The most highly upregulated genes were IDO1, CXCL9, CXCL10 and CCL8. The majority of differentially regulated genes were IFN-γ inducible genes and IFN-γ neutralisation in MLCs abrogated their induction. The microRNA-155, a recently identified target for GvHD, was also found to be significantly induced in HLA mismatched MLC but not in the matched setting and its induction was not diminished by IFN-γ blockade. In this proof-of-principle study we show gene expression changes in mismatched MLC that represent alloreactive responses and correlate with markers involved in GvHD and can potentially be useful in further studies of the biological processes involved in this disease but also in the search of early predictive markers for the disease.

The human bacterial pathogen Neisseria meningitidis (Nm) is one of the world’s leading causative agents of systemic meningitis, which is characterised by inflammation of the meninges and more rarely, septicaemia. The high mortality rate and debilitating long term effects found in infants and young adults is due to the mounting of a large scale immune response by the host leading to severe inflammation and tissue death. The reasons as to how the meningococcus is able to breach innate immune barriers to cause this kind of infection, however, is still unclear. One important innate immune defence mechanism is the production of acute phase proteins such as C-Reactive Protein (CRP), which is known to opsonise pathogens such as N. meningitidis and enhance its uptake into human phagocytic cells. This investigation has demonstrated the enhanced uptake of CRP opsonised Nm into human macrophages and dendritic cells (DCs); two important innate immune cells which are some of the first to encounter the bacterium upon its dissemination through mucosal epithelium into the blood stream. Not only are these cells important phagocytes needed for the clearance of this pathogen, but dendritic cells in particular are the crucial link to the adaptive immune system, and play an important role in mounting effective, long term immunity to pathogens such as Nm. The enhanced uptake of CRP opsonised Nm was shown to occur by Fc gamma (γ) receptors I and II, which are expressed by both macrophages and dendritic cells. This was demonstrated by the blocking of Fcγ receptors with human IgG and specific antibodies to either receptor, which significantly reduced CRP enhanced uptake. The effect of CRP enhanced uptake of Nm into DC downstream immune responses was investigated to assess if CRP exacerbates or abrogates the hosts inflammatory response to Nm. Both unopsonised and CRP opsonised Nm were able to activate DCs and stimulate the up regulation of co-stimulatory and antigen presentation molecules, which is of significant importance in order to present processed Nm antigens to T cells and mount effective adaptive immune responses. Additionally, unopsonised Nm and CRP opsonised Nm were equally capable of up regulating the secretion of inflammatory cytokines from DCs. This suggests that CRP enhanced uptake of Nm by these phagocytic cells may be beneficial towards the host as despite the increased uptake of Nm by both cell types (potentially for phagocytic clearance), the pro-inflammatory cytokine response by DCs is not further exacerbated, thereby limiting inflammatory damage, which is known to the be the major contributor to the pathogenesis of meningococcal meningitis.

Signal transducers and activators of transcription (STAT) 5a and 5b, are key signalling proteins activated by the cytokine interleukin-2 (IL-2), and therefore critically regulates important immunological processes such as T cell homeostasis and immune-regulation. While the biological functions of STAT5 are well established from murine genetic studies, the downstream mediators of these proteins are poorly understood. In this study, an improved chromatin immunoprecipitation (ChIP)-cloning method, using magnetic microbeads for immunocapture of chromatin was developed to identify in-vivo STAT5a and STAT5b binding sites in fresh and activated primary human CD4 and CD8 peripheral T cells. Six libraries were generated, which identified 329 STAT5a and/or STAT5b-specific binding sites of which 87% contained canonical GAS motifs, TTCN3GAA and/or TTN5AA. Genomic mapping of sites revealed the striking observation that the majority of STAT5-binding sites identified here mapped to intergenic (>50kb upstream) or intronic, rather than promoter proximal regions. Bioinformatic analyses, using Gene Ontology programmes to annotate and functionally classify the genes associated with binding sites, predicted novel functions for STAT5 such as transport and metabolism, in addition to previously known functions such as cell differentiation, proliferation, signal transduction, apoptosis and development. Additionally, several target-genes were identified, whose aberrant functions are associated with malignant transformation of cells, consistent with the frequent dysregulation of STAT5 noted in various cancers. ChIP-PCR validation studies on a subset of sites from each library, demonstrated that 98% were bonafide STAT5 binding sites. Kinetic gene expression analyses performed on 31 annotated genes, by qRT-PCR revealed 17 novel target-genes that were upregulated (76%) or downregulated (24%) following IL-2 stimulation, and included two lineage-specification factors, c-MAF and RORA. Given the importance of IL-2 in facilitating CD4 Th2 cell differentiation, the regulation of c-MAF by STAT5 was functionally characterised further using biochemical and molecular techniques. These studies revealed that the epigenetic regulation of c-MAF is dependent on STAT5 and IL-2 signalling. In conclusion, this study has identified a number of novel STAT5 regulated genes downstream of IL-2 activation of T cells, and provides an insight into the various cellular functions regulated by these proteins.

Antibodies used for cancer treatment and immune therapy have, to date, been of the IgG class. Efficacy might be improved by using the IgE class of antibodies as these have higher affinity for their Fc-receptors. IgE has a greater tissue penetration and longer half-life in tissue and IgE bound to IgE-receptor-expressing effector cells is thought to actively infiltrate tumours. IgE has not been subject of in vivo imaging studies to date. Objectives: The aim was to radiolabel both anti-CSPG4-IgE and MOV18-IgE, and their IgG counterparts targeted to the same antigen, while maintaining the functionality of the antibodies; and subsequently, to compare IgE with IgG in in vivo imaging and biodistribution studies in a disease model. Methods: IgE’s and their antigen-matched IgG counterparts were engineered against two different tumour antigens. Tumour models were developed to assess targeting in vivo and imaging and biodistribution studies using radiolabelled IgE were carried out. Six antibodies were engineered for comparison: MOv18-IgE and -IgG antibody targeted against the folate receptor alpha (FR ) expressed on ovarian cancer cells; A MOv18-IgE and -IgG chimeric rat/mouse were engineered for evaluation in a new syngeneic immunocompetent rat model to mimic the immunotherapeutic antibody (human/mouse chimeric MOv18-IgE and -IgG) planned for a clinical study; and a second antibody, anti-CSPG4-IgE, targeted against the chondroitin sulfate proteoglycan 4 (CSPG4), expressed in melanoma cancer was compared to its IgG counterpart. The antibodies were analysed in a NOD/SCID xenograft mouse model with splenic engraftment of human peripheral blood lymphocytes. All antibodies were labelled with 111In using the same bifunctional chelator and labelling conditions (p-SCN-CHX-A"-DTPA) and radiolabelled with 111In at room temperature. Functional assays using FACS were carried out to assure binding to the target and high- and low-affinity Fc-binding to Fc"R expressing immune effector cells. NanoSPECT/CT imaging and biodistribution studies were used to determine targeting and clearance of IgE in vivo. Results: 111In-CHX-A"-DTPA-IgE and -IgG antibodies were labelled with high efficiency (>98%). Binding of the conjugated antibody to the target antigen and Fc"R expressing immune effector cells was identical to that of the native antibody. IgE showed a higher liver uptake in biodistribution (up to 75% ID IgE vs 5% ID IgG 8 h post injection) compared with the IgG counterpart as well as more than 10 times faster blood clearance where as IgG showed prolonged half-life (2.5 days) in blood. Tumour-to-blood and tumour-to-muscle ratios of IgE and IgG showed significant (tbr: P<0.05; tmr: P<0.0005) differences. Conclusion: Similar conjugation and radiolabelling of all antibodies as well as the in vivo assessment allowed the evaluation of targeting, biodistribution and clearance of IgE compared with its IgG counterpart. Observed characteristics of IgE like the rapid blood clearance and the higher liver uptake were found to be fundamentally different compared to its IgG counterpart and suggest a different and mostly unknown mode of action.

Background: Neutrophils are reported to be the dominant phagocytes in sputum from tuberculosis (TB) patients and may explain the distinctive pattern of gene expression of Mycobacterium tuberculosis (Mtb) in these samples. Moreover, neutrophils play an important role during tuberculosis both in collaboration with macrophages as well as antimicrobial functions such as NETosis. Therefore, understanding the transcriptional changes of mycobacteria in response to neutrophils is of general interest. Methods: Following preliminary experiments to develop appropriate methods using BCG and a neutrophil cell line, primary human neutrophils were infected with stationary phase H37Rv preconditioned in RPMI overnight then sampled at 4 and 24 hours. Bacterial RNA samples from these experiments and from TB sputum were subjected to RNA-seq analysis by Illumina NextSeq-500. Reads were normalised by two different methods and differentially expressed genes were detected at q-value < 0.01. Selected results were confirmed by qRT-PCR. Transcription factors associated with detected genes were identified. Results: The colony counts of H37Rv incubated with human neutrophils reduced ~10-fold at 24 hours. Striking up regulation of Mtb tgs1, hspX and icl1 was observed during incubation in RPMI relative to exponentially growing bacilli. RNA-seq revealed 90 genes differentially expressed (28 repressed and 62 induced) in neutrophil encounters at 4 hours relative to RPMI controls. DosR and sigK were the most significantly associated transcription factors with the induced genes. Discussion: Comparison of the neutrophil-stimulated and sputum Mtb transcriptomes did not support the hypothesis that the phagocytes provide a key stimulus underpinning the sputum pattern. SigK regulated expression appears to be a significant aspect of the neutrophil stimulated Mtb transcriptome.

No description available.

The Wnt signalling pathway is of central importance in embryogenesis and adult tissue homeostasis, as well as numerous cancers and other diseases. Despite the developmental and potential therapeutic significance of this pathway many aspects of Wnt signalling, including the role of the master transcriptional co-activator, J3-catenin, remain poorly understood. This thesis describes the derivation of VHH intracellular antibodies, with specificity for β-catenin, and their application to the study of the canonical Wnt signalling pathway. Intracellular antibodies, intrabodies, are engineered antibody fragments capable of binding and modifying the function of intracellular proteins, and represent valuable tools for the study of intracellular interactions. The aim of the work was to further the understanding of the regulation of β-catenin, and potentially to use the intrabodies as tools to identify sites on β-catenin for future therapeutic intervention. A diverse immune llama VIM phagemid library was constructed, from which five antibodies with 3-6nM affinity for β-catenin were selected, and demonstrated function-modifying intracellular activity in a luciferase based Wnt signalling reporter bioassay. Control Ala-substituted CDR3 mutants were produced for each intrabody, and used to demonstrate the specific inhibition of β-catenin activity by the parent intrabodies. Further characterisation of intrabody LL3 demonstrated that it bound endogenous 13- catenin specifically and inhibited the Wnt signalling pathway downstream of the destruction complex. The binding location of LL3 on β-catenin Was shown to reside between residues 164 and 390, which overlaps with the binding domains of several transcription factors on β-catenin. This work is thought to represent the first report of functional inhibition of β-catenin-mediated co-activation of transcription by an intrabody, and further study of the method of action of function-modifying VHH intrabodies to β-catenin may enable the identification of novel sites for therapeutic intervention on the Wnt signalling pathway.