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The work presented in this thesis describes the production and characterisation of recombinant streptavidin-cytokine fusion proteins. Immunoadjuvants can augment the weak or non-existent antitumour response of a patient with cancer to immunotherapy. The response of breast cancer patients to cancer vaccines directed against tumour associated antigens, such as HER2/neu, is often weak and it has been reported that additional immunostimulation, such as that provided by an immunoadjuvant, is required in order to achieve effective therapeutic results. One approach to the development of novel immunoadjuvants is the production of fusion proteins which are designed to perform the dual functions of general activation of the immune system as well as directing the immune response specifically towards a TH1-type response, which is widely regarded as the optimal patient response for cancer immunotherapy.Streptavidin is a bacterial protein and an immunogenic molecule that induces an unspecific immune response. The cytokines IL-2 and IL-18 both promote TH1-type responses; IL-2 is already in clinical use as a cancer therapy, while investigation of the role of IL-18 as an immunotherapy is ongoing.A strategy was devised for the production of novel recombinant fusion proteins designed for use as immunoadjuvants; these proteins comprised an N-terminal truncated streptavidin core protein sequence and a C-terminal cytokine, specifically either IL-2 or IL-18, separated by a short polypeptide linker region. Molecular cloning techniques were used to generate DNA expression constructs encoding the recombinant fusion proteins which were expressed in an inducible bacterial system using plasmid expression vector pCR(R)T7/NT-TOPO(R). The expressed recombinant proteins were found to accumulate within insoluble bacterial inclusion bodies and protocols were developed and optimised for the isolation and solubilization of these proteins. Protein solubilization required the use of buffers at high pH (pH 12.5) which resulted in disrupting protein structural integrity; a pulsed dilution method was subsequently employed to achieve refolding of the proteins prior to further analysis.Characterization of fusion proteins STV/IL-2 (streptavidin-IL-2) and STV/IL-18 (streptavidin-IL-18) was conducted using native and dissociating PAGE and Western analysis. Antibody binding studies provided preliminary confirmation of the identity of the STV/IL-2 fusion protein. Similar studies to characterize STV/IL-18 were initially encouraging but proved inconclusive and further analysis of this protein is required.These initial investigations have validated this approach using the expression systems established here for the production of recombinant streptavidin-cytokine fusion proteins. A number of issues to be addressed have been highlighted regarding problems encountered with protein yields, solubilisation and maintenance of structural integrity. It is therefore concluded that further modification and optimisation of the expression system and protein isolation procedures employed is necessary to provide an appropriate system for the production of these fusion proteins; this will subsequently permit the investigation of their potential for use in therapeutic applications.

Heart Failure is the state in which the heart is unable to pump enough blood to meet the needs of the body, resulting from events that progressively damage the myocardium and/or its ability to contract normally. Several compensatory mechanisms allow for short-term maintenance of adequate delivery of blood to the body. Progression from the initial events to heart failure is caused by the accumulation of pathologic changes and their effects, many of which result from chronic activation of these compensatory mechanisms. One of these mechanisms is the activation of the neurohormonal β-adrenergic system, to which the β₃-adrenergic receptor (β₃-AR) belongs. Research on the role of the β₃-AR in the heart has focused on the short-term protective role of the receptor, based on its ability to reduce the effects of over-stimulation of the heart's other adrenergic-system components. However, during the course of heart failure, its up-regulation and functional persistence may also contribute to the progression of the disease. There have been few large-animal studies, no human trials, and no long-term studies of β₃-AR agonists for use in the treatment of heart failure. Ultimately, individualized treatment strategies that, over time, modulate the relative levels of agonistic and antagonistic effects across each of the three β₃-AR subtypes, may be most effective for management of heart failure. However, further research and trials with β₃-AR agonists are realistic and necessary places to start.

The epidemiology of head and neck squamous cell carcinomas (HNSCC), primarily those of the oropharynx, has changed dramatically over the last two decades. Specifically, HNSCC appears to be a distinct entity that is related to infection by human papilloma virus (HPV)(Vokes et al., 2015) (Fakhry et al., 2008). Moreover, the incidence of HPV-associated oropharyngeal (OP) cancers is rising, likely as a consequence of changing life styles and sexual behaviors. These tumors appear to be biologically and clinically distinct from other HNSCC tumors affecting predominantly middle-aged white men having no or only a brief history of tobacco consumption. The cell cycle regulatory protein, p16, is usually over expressed in HPV-OPSCC, and its detection using immunohistochemistry and in situ hybridization is a reliable surrogate marker for the disease (Ang et al., 2012). When compared to traditional head and neck cancer that is associated with the repeated insult of tobacco use, HPV-related OPSCC has a favorable natural history and is more responsive to treatment. As a result, patients with this cancer have improved long-term survival and consequently are more likely to experience chronic therapy-induced morbidity (Ang et al., 2012). The purpose of this thesis is to provide a comprehensive review of the molecular mechanisms that underlie HPV-mediated OPSCC, and the licensed prophylactic HPV vaccinations available, and to discuss the current thoughts on whether to deescalate potentially damaging treatments in these patients.

The human body contains a variety of different cell types that share a common genome, but differ in how they use the information encoded therein. Variation in molecular content exists even at the level of individual cells, and to provide deeper insight into complex cellular processes methods that permit analysis of each cell on its own are needed. This thesis presents molecular methods for localized detection of individual nucleic acid molecules. The developed methods are based on padlock probes and target-primed rolling circle amplification. Single-molecule detection sensitivity in combination with single-nucleotide genotyping selectivity enables detection of allelic DNA variants and closely related target sequences directly in cells. Padlock probes further enable multiplex detection of targets, and in combination with image analysis quantitative molecular data for individual cells can be acquired for large cell populations at a resolution that no other in situ detection method can provide at present.   In this thesis, the in situ target-primed rolling circle amplification technique was first used for genotyping of a point mutation in the mitochondrial genome with padlock probes. This displayed mitochondrial DNA heterogeneity in cell populations. Application of the method on comet assay preparations showed that mitochondrial genomes are lost from these samples prior to analysis. Nuclear DNA targets, however, can be efficiently detected in corresponding samples. Padlock probes and rolling circle amplification are thus an attractive alternative to FISH analysis for localized DNA detection in comet assay samples. A method was also developed for localized detection of individual mRNA molecules with padlock probes and rolling circle amplification. This method provides unique possibilities to genotype allelic variants of transcripts in situ. mRNA expression is associated with substantial cell-to-cell variation and our presented method permits simultaneous visualization of multiple transcripts directly in complex tissue samples. Application of the methods presented in this thesis will enable new types of studies of biological samples from both normal and disease states.

In early life the immature immune system has a reduced ability to control infection. This susceptibility is offset by transfer of protective immune components from the mother. Helminth infections are widespread and can have a long lasting influence on host immunity. Children of mothers exposed to helminth infections may display T cell sensitization to endemic helminth infections and associations have been made between maternal helminth infection and altered immune responses to childhood diseases and vaccinations. This shows that helminth-modified maternal immunity may imprint on early offspring immune development in-utero or through breast milk in the form of transfer of, for example, antibodies, cytokines and lymphocytes. Our study shows that, in mice, maternal infection with the helminth Nippostrongylus brasiliensis is not only associated with a passive transfer of antigen specific antibody(IgG1) but also inherently alters offspring immunity, increasing offspring cytokine production, alveolar macrophages, lung neutrophils and B cell population development and proliferation. Pups born to N. brasiliensis exposed mothers also had increased populations of lung and spleen CD4+ cells and higher subpopulations of central memory and effector CD4+ cells compared to pups born to naive mothers.

Includes abstract. / Includes bibliographical references (leaves 170-210). / Human immunodeficiency virus (HIV) infected children are at greater risk of developing tuberculosis disease, and might benefit from vaccination with novel TB vaccines. However, little is known about the effect of HIV-infection on function and phenotype of T cell responses to mycobacterial antigens in children. This study compares both CD4 and CD8 T cell cytokine expression and memory phenotype in children, following in vitro stimulation with mycobacterial antigens, also contained in novel anti-TB vaccines that are currently undergoing clinical trials.

We proposed that the development of a new high capacity polymer microsphere technology, termed ReSyn, could be developed as viable detection reagents for lateral flow technology. This body of work outlines the development of this new high capacity polymer microsphere technology for suitability to flow on lateral flow membranes, and highly specific biomarker detection for immunoassay development. Proof-of-concept was achieved using hCG (pregnancy biomarker) and validated for detection of pLDH and HRP2 as biomarkers of malaria. The sensitivity, stability and multiplex capability of these microspheres were further explored and compared against the current ‘gold' standard detection agent for lateral flow, colloidal gold. Malaria was selected as a suitable target for evaluation of the microsphere technology since it is considered to be a global epidemic that can benefit greatly from improved point-of-care diagnostics. Malaria affects almost half of the world's population and is responsible for causing approximately 655 000 deaths per annum in 2010, with 90% of these deaths occurring in Africa and 85% of these deaths occurring in children under 5 years of age (del Prado et al., 2014; Kokwaro, 2009; White et al., 2014; WHO, 2009). Febrile disease diagnosis at point-of-care is often based on symptomatic diagnosis rather than on the use of validated diagnostic technologies, and is considered one of the major contributing factors for the high morbidity and mortality rate of malaria (Chandler et al., 2008; Kain et al., 1998; Kokwaro, 2009). Improved diagnostic technologies, allowing for sensitive and accurate diagnosis at the point-of-care, could assist alleviating these problems through the improved management of disease (Bell et al., 2006). Lateral flow rapid diagnostic tests are the preferred method for point-of-care diagnostics in resource constrained areas but have several limitations including sensitivity and stability in resource constrained settings (Bell et al., 2006). Improvements in detection agents are seen as a viable approach to improving these features of diagnostic assays. The results of this study show that the polymer microspheres provide improved stability to immobilised antibodies, with potential for translation into improved stability for diagnostic assays in tropical malaria endemic regions. The polymer microspheres offered high specificity and comparable visual sensitivity to the market leader colloidal gold and is therefore considered as alternate detector agents in lateral flow assays. Additionally, the microspheres can be dyed various colours (red and blue in this study), allowing for specific and sensitive multiplex detection of multiple analytes in a single sample. This increases the versatility of the microspheres for lateral flow diagnostic application, and improves the interpretation of lateral flow diagnostic technology at the point-of-care.

CD31 mediates homophilic interactions between leukocytes and macrophages during inflammation, apoptotic cells remain attached and are engulfed whereas viable cells actively detach. We hypothesised that differential recruitment of signalling and adapter molecules to the cytoplasmic domain were responsible for the disengagement of the viable leukocyte from macrophages. Investigation with a static attachment assay using THP-1 as a macrophage model showed the ITIM of CD31 on the leukocyte was important for viable cell detachment. Our data also implicated a role for the recruitment of SHP-2 which we attempted to knock-down by siRNA delivered by lentivirus. SHP-2, in addition to its phosphatase activity, also acts as a docking protein. To examine for potential interacting partners we fused the cytoplasmic domain of CD31 to GST which we used in pulldown assays from lysates of viable and apoptotic leukocytes. We demonstrated that the recruitment of SHP-1 and SHP-2 were dependent on an intact ITIM (immunoreceptor tyrosine-based inhibitory motif). Interestingly, apoptotic cell lysates promoted dephosphorylation of the in vitro phosphorylated GST-CD31, suggesting an increase in phosphatase activity in aged neutrophils. We were unable to demonstrate an interaction between the cytoplasmic domain of CD31 with putative binding partners β-catenin, src, RasGAPp120, RhoGAPp190, talin or calmodulin. A proteomic approach by MALDI-TOF and MS/MS identified Hsp90 as a novel binding partner of CD31 irrespective of the phosphorylation state. In contrast, 14-3-3ε bound to phosphorylated CD31, whereas eIF3 specifically bound to an ITIM double tyrosine mutant. The binding of Hsp90 to CD31 was proposed to occur via a TPR motif within the cytoplasmic domain of CD31 which comprises a surface fold of basic amino acids complexing a highly acidic carboxy tail of Hsp90. Truncation and site directed mutagenesis of the cytoplasmic domain revealed multiple binding sites for Hsp90; specifically two regions containing the sequences (KAFYLRKAKAK), previously shown to be the calmodulin binding region, and a novel area (SNNEKMSDMEANSHY) which has significant homology with other TPR-containing proteins. Systematic mutagenesis of the putative basic charged amino acids within the cytoplasmic domain of CD31 which may mediate the interaction with Hsp90 also supports the presence of a TPR motif. The importance of Hsp90, 14-3-3ε and eIF3 is currently unknown, although it is interesting to note that CD31 was recently found within this laboratory to associate with the voltage-gated potassium channel HERG which also binds 14-3-3ε and Hsp90. eIF3 is an RNA helicase that may link CD31 and leukocyte motility to spreading initiation centres where motility can be viewed as a rapid turnover of focal adhesion complexes. Together, these studies have identified novel binding partners of CD31 may form a macromolecular complex to promote CD31-dependent leukocyte motility and detachment from macrophages.

Background: Adherence to antiretroviral therapy (ART) is critical: only 70% achieve viral suppression at a year. Current adherence methodologies, with slow reaction to missed dosing, inadequately predict virological outcomes. Ideal adherence methods would be cheap, easy to use, and allow rapid response to missed doses to improve outcomes. We explored ideal adherence monitoring methodology for a large public sector ART clinic in Cape Town. Methods: We designed a randomised controlled study for ART-naïve individuals to determine whether text messaging after a missed dose would improve adherence recorded by an electronic adherence monitoring device (EAMD), reduce treatment interruptions or impact on virological outcome (using regression modelling). Five other measures of adherence were captured prospectively during the study: selfrecall (SR), clinic-based pill count (CPC), pharmacy refill data (PR-average or PR-gaps) and efavirenz concentration. The predictive value of each adherence methodology on virological and HIV-1 resistance outcomes was compared by calculating the area under the receiver operating characteristic curve, from logistic regression models. The impact of efavirenz concentration and CYP2B6 metaboliser genotype data on failure was examined using Cox proportion hazard modelling; and the most predictive lower limit for EFV concentration was determined. Antiretroviral cohort and pharmacy refill data were compared, using simple statistics, to determine which provided the best method of determining those retained in care.