Edward Conway


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Graduate Student Supervision

Master's Student Supervision (2010 - 2018)
Polyphosphate as a modulator of the complement system (2013)

Coagulation and complement are evolutionarily related, with several well-described mechanisms of cross-talk. Recently, it was established that polyphosphate (polyP) is a physiologic activator and promoter of coagulation. I hypothesized that polyP also plays a role in regulating complement, and thereby acts as an additional molecular bridge between coagulation and complement. Evidence to support this was provided by studies in bacteria, where defects in polyP synthesis and degradation alter its resistance to serum-mediated killing. In this thesis, I show that polyP suppresses total complement-mediated lytic activity and the terminal pathway, resulting in decreased lysis of foreign erythrocytes by the membrane attack complex (MAC). In contrast, monophosphate exhibits 10-fold less inhibition in total hemolytic activity, and has no effect on the terminal pathway. I also provide evidence that polyP destabilizes the C5b,6 complex to prevent functional MAC formation. Implications of the role of polyP in complement modulation are discussed.

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Murine CD248 and its Cytoplasmic Domain: Characterising a Novel Tumor and Inflammation Marker (2012)

CD248 is a member of a family of transmembrane glycoproteins containing an N-terminal C-type lectin-like domain. This family includes thrombomodulin and CD93, proteins known to modulate immunity, cell proliferation and homeostasis. CD248 is expressed in perivascular and stromal cells, during embryonic development and post-natally during inflammation and cancer. In the mesenchymal compartment of most normal adult tissues, CD248 expression is not detectable. It is believed that CD248 is involved in cross-talk between endothelial cells and pericytes, thereby playing a role in growth, metastasis and angiogenesis associated with the development of tumours and inflammatory lesions. Intracellular signaling, mediated via the cytoplasmic domain of CD248, promotes tumour growth and inflammation. CD248 knock-out mice and mice expressing CD248 lacking the cytoplasmic domain were resistant to tumour growth and inflammatory arthritis. Although signaling pathways have not been delineated, examination of the cytoplasmic domain of CD248 reveals three highly conserved putative phosphorylation sites and a PDZ-binding motif. We hypothesised that these structural features are important for CD248 function.We generated a variety of murine CD248 pcDNA constructs that encode CD248 with mutations in the cytoplasmic domain. We confirmed that all the mutants were transcribed and translated. Mutant proteins were expressed on the cell surface, in a similar manner to wildtype CD248. Introduction of some mutant forms CD248 into cells caused CD248 to exhibit different intracellular localisation and induced changes in cellular morphology compared to wildtype. Limited functional studies demonstrated CD248-dependent alterations in cellular MMP-9 production. The findings underline an important role of CD248’s cytoplasmic domain in regulating cellular morphology and function that may impact its role in health and disease. Extracellular interacting partners for CD248 have been previously described. Searches for intracellular partners interacting with the cytoplasmic domain have been less successful. Our results strongly suggest that these exist. Co-immunoprecipitation studies have revealed several putative interacting proteins that set the stage for future confirmatory and functional analyses. The cytoplasmic domain of CD248 is important to study as it holds much promise as a therapeutic target for proliferative disorders. The information gathered in this project may be used to delineate clinically relevant CD248 signaling pathways.

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Surface Properties of Crandallite in Relation to Froth Floatation (2012)

The surface properties of crandallite relevant to the anionic flotation of salt-type minerals were investigated through electrokinetic, turbidity, and wettability techniques. All the tests were performed on fine crandallite particles as a function of pH and reagent concentration. The selected reagents included starch and oleic acid.From the zeta potential measurements, the iso-electric point of crandallite was found to be at pH 5.5 and the mineral surface can be expected to be negatively charged under typical flotation conditions (pH 9-11). The value of the iso-electric point correlated well with the stability of crandallite particles towards aggregation. Suspensions of fine crandallite showed minimum turbidity at pH 5.5 suggesting that the aggregation of the mineral was most pronounced at the iso-electric point. Calcium ions behaved as potential determining ions by increasing the iso-electric point to pH 6.5. In addition, calcium ions also acted as specifically adsorbing ions at higher pH.In the presence of starch, crandallite particles were strongly flocculated producing supernatants with the clarity of tap water. At the same time, the effect of starch on the zeta potential of crandallite was rather weak, consistent with the non-ionic character of the polysaccharide. The stability of crandallite towards aggregation was not strongly affected by oleic acid suggesting that the surfactant did not interact with the mineral. Wettability measurements on crandallite particles showed that oleic acid did not render the mineral particles hydrophobic at pH 10.5 (under normal flotation conditions) or at pH 7, which was in strong contrast to the wettability response of apatite. Under these conditions, crandallite surface was negatively charged and the adsorption of the anionic surfactant seemed to involve physical electrostatic forces as opposed to chemical interactions known to occur between apatite and oleic acid.Overall, the experimental results strongly suggest that crandallite remains hydrophilic during apatite flotation at pH 10.5 and the main mechanism of contamination of apatite concentrates by crandallite is through mechanical entrainment in the froth rather than by true flotation. In this respect, the role of starch as a flocculant should be beneficial in reducing the amount of fine crandallite reporting to the apatite concentrate.

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