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(2022) Kokkinos, JohnThesisLess than 10% of patients with pancreatic ductal adenocarcinoma (PDAC) survive more than 5 years. One of the characteristic features that drive the aggressive nature of PDAC is its multicellular, heterogeneous, and fibrotic microenvironment. We previously identified a cytoskeletal protein, βIII-tubulin, as a novel therapeutic target in PDAC. However, the PDAC cell survival mechanisms controlled by βIII-tubulin were previously unknown. We also identified a major gap in the ability of human PDAC preclinical models to accurately mimic the 3D multicellular architecture and stroma of the disease. Thus, the aims of this work were (1) to evaluate the pro-survival role of βIII-tubulin in PDAC; (2) to establish a new patient derived tumour explant model that maintains all features of the PDAC microenvironment; and (3) to use the tumour explant model to test the clinical potential of silencing βIII-tubulin expression as well as two stromal targets that had been previously explored by our lab: solute carrier 7A11 (SLC7A11) and heat shock protein 47 (HSP47) Here, we identified that silencing βIII-tubulin in pancreatic cancer cells activated extrinsic apoptosis and increased their sensitivity to extrinsic apoptosis inducers including tumour necrosis factor-α (TNFα), Fas-ligand (FasL), and TNF-related apoptosis inducing factor (TRAIL). We next established the patient derived PDAC tumour explant model. We cultured whole-tissue tumour explants from PDAC patients for 12 days and demonstrated that explants maintained their 3D multicellular architecture, proliferative state, and collagen fibrosis. We also demonstrated the ability to deliver chemotherapeutics and siRNA-nanoparticles to the tumour explants. Finally, we tested the utility of this model to investigate the clinical potential of silencing three different therapeutic targets. We showed that therapeutic silencing of βIII-tubulin combined with TRAIL increased extrinsic apoptosis, decreased cell proliferation, and decreased tumour cell number. Inhibition of the stromal target SLC7A11 reduced tumour cell number and inhibited activity of stromal cancer-associated fibroblasts. Silencing of another target, HSP47, also led to a reduction in tumour cells and decreased cell proliferation. Overall, this work has discovered a previously unexplored role of βIII-tubulin as a brake on extrinsic cell death and has developed a new human PDAC preclinical model with utility in the drug development and precision medicine pipeline.
(2022) Gunasekera, SanjivThesisThe arteriovenous fistula (AVF) is a vasculature created for end-stage renal disease patients who undergo haemodialysis. This vasculature is often affected by stenosis in the juxta-anastomotic (JXA) region and the presence of disturbed haemodynamics within the vessel is known to initiate such diseased conditions. A novel treatment involving the implantation of a flexible stent in the JXA region has shown potential for retaining healthy AVFs. Only a limited number of experimental studies have been conducted to understand the disturbed flow conditions, while the impact of stent implantation on the haemodynamics within the AVF is yet to be explored. The study was initiated by developing a benchtop patient-specific AVF model to conduct a Tomographic Particle Image Velocimetry (Tomo-PIV) measurement. The subsequent temporally resolved volumetric velocity field was phase-averaged to quantify fluctuations occurring over the inlet pulsatile conditions. It was noted that high turbulent kinetic energy (TKE) was generated at the JXA region. To study the effects of the stent implantation, Large Eddy Simulations (LES) comparing the AVF geometry with and without the presence of the stent implantation were conducted. The trajectory of the flow in the stented case was funnelled within the stent encapsulated region which in turn, contained the disturbed flow within the stent lumen while mitigating the generation of turbulence. Consequently, the distribution of adverse wall shear stress (WSS) in the stented region was much lower compared to that of the `stent-absent' case. Simulations were also conducted on the diseased patient AVF, before the stent implantation, to make an overall assessment of the effect of treatment. Larger and persistent regions of high TKE were noted in the vessel downstream of the stenosis despite the lower velocity of flow in the diseased model. In summary, the stent implantation in the patient AVF showed the ability to funnel flow disturbances away from the vessel wall, thereby leading to lower adverse WSS distributions. The presence of the stent also mitigated turbulence generation. These findings provide valuable insight into the favourable haemodynamic effects of this novel endovascular procedure, thus, substantiating this treatment strategy to treat vascular disease in AVFs.
The use of patient-specific modelling in the assessment of a clinical indicator for arteriovenous fistula failure(2021) Ng, OliviaThesisThe arteriovenous fistula (AVF) is a surgically-made vascular structure connecting an artery to a vein. It is the optimal form of vascular access for haemodialysis-dependent end-stage renal disease patients. However, AVF are prone to access dysfunction through the formation of stenoses, which compromise the structure’s utility. To date, a plethora of clinical models are used to predict AVF formation failure based on patient factors and other models predicting late AVF failure by assessing haemodynamics and quantifying disturbed flow behaviours and wall shear stress metrics with stenosis formation. That said, inconsistencies were identified in the correlation between these metrics and diseased AVFs. This thesis aims to assess the suitability of another haemodynamic-related metric, resistance, derived from pressure drop and flow rates through patient-specific CFD modelling, for diagnosing and predicting AVF failure. A three-dimensional ultrasound scanning system was used to obtain patient-specific geometry and flow profiles, used for CFD models which were then analysed, with resistance calculated for each patient. The significance of patient-specific CFD modelling was demonstrated in its usefulness to generate a patient-targeted indicator of diseased AVF. To study the effectiveness of resistance as a metric, the relationship between CFD-derived resistance and the potential for AVF failure was evaluated, starting with classification of resistance results among patients who had undergone treatment for stenosis. An exploratory study into the suitability of CFD-derived resistance and its association with patients’ AVF conditions was further conducted by classifying data from a larger patient dataset and fitting the classified data to a multilevel regression model. CFD-derived resistance was found to be higher at the proximal vein of problematic AVF, however this figure was 76% lower among patients who had undergone stenosis treatment. Meanwhile, no correlation was found between resistance at the proximal artery and patency status. An area under curve of 92.1% was found from the receiver operating characteristic analysis, noting an outstanding discrimination of the classification. CFD-derived resistance appears to be a promising metric in the assessment of a suitable diagnostic marker for AVF failure. This research concludes with aspirations for clinical implementation of a related system, alongside routine surveillance of AVF.