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(2021)ThesisAlternative-lengthening-of-telomeres (ALT) is a telomere maintenance mechanism activated in approximately 10% cancers to counteract telomere shortening and enable cellular immortality. ALT-targeted drugs are being developed and there is a need for a reliable ALT diagnostic. C-Circles, self-primed circular telomeric C-strand DNAs, are the only known specific molecule for ALT and have been detected in the blood of osteosarcoma patients by the quantitative C-Circle Assay (CCA). The aim of this study was to investigate C-Circle secretion by ALT-positive (ALT+) cancer cells and advance the blood based CCA as a clinical diagnostic for ALT. This study provided four novel results (i) ALT+ cancer cells secrete C-Circles in exosomes (ii) validation of the blood-plasma based CCA as a test for ALT (iii) prevalence of ALT in paediatric cancer (including haematological cancer) (iv) red blood cells (RBC) are a major source of the CCA background signal in whole blood from ALT-negative patients. C-Circles were detected in exosomes collected by differential centrifugation from the conditioned media of a panel of 11 ALT+ cancer cell lines, comprising lung adenocarcinoma, glioblastoma, neuroblastoma, osteosarcoma and soft tissue sarcoma cell lines. On average, 2-fold and 4-fold more C-Circles were detected in the exosome-enriched fraction than the fractions of apoptotic bodies and microvesicles, respectively. C-Circles were absent from all extracellular fractions of the matching telomerase-positive cancer cell lines and a human fibroblast strain. The secretion of C-Circles inside exosomes was confirmed by further purification of exosomes by iodixanol density gradient centrifugation and immuno-isolation with anti-CD9, anti-CD63 and anti-CD81 antibodies. Using iodixanol density gradient separation, C-Circles and exosomes peaked in the same density fraction. Exosomes pulled down by the anti-CD81 immuno-isolation had 37% of the total CCA signal. C-Circles in exosome isolates were mostly (60% - 80%) protected from digestion by DNase I, which digested all C-Circles after exosome lysis. On average, ALT+ cancer cells secreted 0.4% of their total intracellular C-Circle content in exosomes per day. Secretion of C-Circles inside exosomes should provide a stable blood-based ALT biomarker, whose levels are independent of cancer cell apoptosis and are protected from blood nucleases. Using blood plasma C-Circle levels of 2-fold that of the healthy control as the threshold for diagnosing an ALT+ cancer, the plasma based CCA had an 86% sensitivity, 100% specificity and 89% concordance, compared with the standard tumour based assay for ALT, performed on 13 matched paediatric cancer specimens. The validated plasma based CCA for ALT determined a 36% prevalence of ALT in a representative population of 152 paediatric cancer patients, including 31% (22/72) in acute lymphoblastic leukemia, 37% (7/19) in acute myeloid leukemia and 40% (2/5) in chronic myeloid leukemia cases. Preliminary evidence was found that the plasma based CCA may be able to follow tumour burden during treatment and in general the prevalence of ALT was higher in the diagnostic specimens (40% (34/85)) than in the specimens taken at the completion of treatment (9% (4/43), P = 0.001). This study confirmed that using blood plasma avoids the CCA background signal present in whole blood from ALT-negative patients and found a substantial amount of this background CCA signal resided in the RBC fraction. In conclusion, this study provided the first evidence that C-Circles were secreted in exosomes by ALT+ cancer cells and that the plasma based CCA could be an accurate diagnostic for ALT, which could provide a minimally invasive diagnostic for detecting ALT activity and following ALT+ disease burden.
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(2022)ThesisBackground: Anterior lumbar interbody fusion (ALIF) remains one of the mainstay surgical approaches in treating painful degenerative disc disease with or without segmental instability in the lower spine. The risk factors and complication profile for ALIF differs significantly from other established fusion techniques. Objectives: The goal of the first part of this thesis is to establish the factors associated with long-term clinical outcome (Chapter 2) and short-term perioperative outcomes (Chapter 3) following ALIF. Chapter 4 focuses on the long-term radiographic evidence for biomaterial alternatives for ALIF implants, namely titanium (Ti)-coated PEEK integrated cages. Methods and Results: From a prospective cohort analysis of 147 patients undergoing ALIF, elderly age (≥64 years old) was associated with an increased rate of subsidence but does not affect clinical outcomes. Obesity was not associated with postoperative complications or follow-up patient-reported outcomes. Failed fusion was significantly higher for smokers, and they were significantly more likely than non-smokers to experience postoperative complications such as pseudoarthrosis. To assess risk factors for perioperative complications and readmissions after ALIF, the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database was analysed. ALIF was associated with prolonged length of stay and higher rate of return to operating theatre compared to posterior lumbar fusion. Obesity and alcohol intake increased the risk of 30-day readmissions. Discharge to non-home destination following ALIF was independently associated with wound complications and venous thromboembolism. Finally, a prospective follow-up study was performed to determine the long-term radiographic outcome following ALIF using Ti-coated PEEK cages with allograft and INFUSE. Effective fusion was achieved at up to 24-month follow-up for various indications including degenerative spine/disc disease, low grade lumbar isthmic spondylolisthesis, spondylotic radiculopathy and discogenic low back pain. Conclusions: Collectively, this thesis highlights the importance of personalising the care of an ALIF surgery patient, through identification and optimization of individual risk factors for short-term and long-term outcomes, as well as through choice of implant biomaterial and design.
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(2022)ThesisThe two leading causes of failure of orthopaedic implants are aseptic loosening and periprosthetic joint infection. Since the numbers of primary and revision joint replacement surgeries are increasing, strategies to mitigate these failure modes have become increasingly important. However, most recent work has focused on the design of coatings to prevent infection or to enhance bone mineralisation. However, long-term success of the implants is contingent on addressing both of these issues. Consequently, the present work focussed on multifunctional orthopaedic coatings that inhibit microbial cells while still promoting osseointegration. Nanoceria has considerable potential to be used in biomedical applications owing to its unique bio-responsive redox switching and its capacity to be doped with different therapeutic ions of varying functionalities. Therefore, the effect of different cations incorporated in ceria on cellular behaviour in vitro as well as the anti-bacterial performance were investigated. The two main foci were: (1) characterisation of the bioceramic materials and (2) biological response to undoped and doped ceria ceramics in vitro using bacteria colonies forming unit (CFU) and cytotoxicity Ceria (CeO2) thin films (~820 nm thickness) doped with 0-9 mol% Ga or Mn were fabricated by spin coating on 3D-printed Ti6Al4V followed by heat treatment at 650°C for 2 h, and these were characterised by transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM) (microstructure), 3D laser scanning confocal microscopy (topography), glancing angle X-ray diffraction (GAXRD) (structure and mineralogy), and X-ray photoelectron spectroscopy (XPS) (surface chemistry). In vitro testing was conducted, including inhibition of bacterial growth, simulated body fluid (SBF) testing, and cell attachment and proliferation studies. The data are interpreted in terms of the following: (1) The roles of the sol-gel precursor viscosity, which affected pore filling and surface coverage, (2) Lattice contraction, which contradicted the XPS data, (3) Intervalence charge transfer, which increased the Ce3+ concentration but was a minor effect, (4) Substitutional solid solubility, which is consistent with Hume-Rothery’s rules and the GAXRD data, (5) Redox charge compensation, where the defect equilibria highlight the key role of this mechanism, which decreased the Ce3+ concentration and provided the majority effect, (6) Electronegativity, which plays a small, if any, role in affecting the ion valences but is important in initiating intervalence charge transfer, (7) Multivalence charge transfer, which combined the electron exchanges between film matrix, dopants, and Ti substrate. The most significant outcome was that the bioactivity of ceria derives directly from the Ce3+ concentration, which itself results from solid solubility (substitutional and interstitial) and charge compensation and redox. This challenges the common assumption of the dominance of oxygen vacancies in the performance of ceria. The antibacterial activity was dependent on the type, amount, and valence of the dopant, where opposite trends were observed for gram-positive S. aureus and gram-negative E. coli bacteria. All of the doped samples resulted in enhanced cell proliferation, although this was greatest at the lowest dopant concentration. Surface hydroxyapatite formation on the samples was achieved by soaking in SBF at 2 weeks and 1 month.
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(2022)ThesisLess 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.
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(2021)ThesisUse of the best available evidence is fundamental to provide high-value care for people with low back pain. However, the global burden of low back pain continues to rise, partly attributable to low-value and mismanaged healthcare. The findings from this thesis have provided new data on the quality, mechanisms and reporting of research evidence that informs the care provided to people with low back pain. Chapter Two provided evidence that muscle relaxant medicines, considered first-line pharmacological care by the American College of Physicians guideline, do not provide clinically important improvements in pain and function and are associated with increased risk of experiencing an adverse event in adults with low back pain. Chapter Three and Four highlighted the need and provided recommendations for improved methodological quality and reporting of randomised controlled trials investigating physiotherapy treatments in low back pain. Chapter Five showed that the policies of 10 leading pain journals do not support transparent and open communication of pain research. These findings led to an update in the policies of a leading pain journal. Chapter Six identified how patient education, recommended first-line non-pharmacological care for low back pain, can be adapted to produce clinically important improvements in function. Chapter Seven found suboptimal reporting and methodological limitations in studies conducting mediation analysis in health research. This finding provided evidence for the need to develop a specific reporting guideline for studies conducting mediation analyses. Chapters Eight and Nine describe the development of a guideline for reporting mediation analyses, including a checklist of reporting items derived from an international Delphi exercise. This program of work provides new evidence to improve the evidence-based management of low back pain relating to quality, mechanisms and reporting. The implications for future research and clinical practice are discussed. Through the conscious and judicious use of the best available evidence, we can improve the care, and ultimately the lives of people with low back pain.
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(2021)ThesisThe following thesis examines the implementation of objective methods to measure patient physical capabilities within the spine surgery setting. With a broad aim to introduce accurate and robust assessment tools that are not influenced by subjective patient factors and experiences our methods have been devised after a wide literature search to examine the current state-of-play. Following this literature review two separate arms have been conducted, one in the clinical setting on a large scale using affordable and accurate devices while a second arms aim’s to examine range-of-motion as a potential outcome measure. The latter arm within the scope of the thesis is limited to a validation study with 2 separate emerging technologies; an inertial based sensor system and a digital goniometer. Together these two arms pave way for clinicians not just in the spine surgery setting to employ similar assessment methods in aims to achieve safe, affordable, remote and objective measures of patient outcome. Briefly we have been able to validate the digital goniometer for clinical use, while our accelerometer data is promising for use in the large volume outpatient setting.
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(2021)ThesisPeripheral neuropathy is a common and debilitating complication of diabetes and chronic kidney disease (CKD). The pathophysiological mechanisms contributing to peripheral neuropathy in these conditions remain unclear. In this thesis, axonal excitability studies and nerve ultrasonography were utilised to assess peripheral nerve structure and function in human subjects with either diabetes, CKD, or both to investigate the mechanisms underlying peripheral nerve dysfunction in each condition. To commence, it was essential that an instrument to assess the severity of neuropathy in patients CKD with and without type 2 diabetes (T2DM) was formally validated. Having validated The Total Neuropathy Score in Chapter 1, axonal excitability studies were then utilised to determine the relative contributions of T2DM and CKD underlying nerve dysfunction in diabetic kidney disease in Chapter 2. It was established that CKD, and not diabetes, underlies axonal pathophysiology in patients with diabetic kidney disease. Studies were then conducted in autoimmune diabetes. In type 1 diabetes (T1DM), despite good glycaemic control as measured by HbA1c, development of peripheral neuropathy frequently occurs. In search of an explanation, the association between acute glucose control and nerve structure and function was explored in Chapter 3. Greater acute glucose variability and longer time spent in hyperglycaemia were associated with worse nerve excitability measures, altered corneal nerve morphology, and a higher number of corneal micro-neuromas. In Chapter 4, the mechanisms underlying axonal dysfunction in a recently recognised form of autoimmune diabetes known as latent autoimmune diabetes in adults (LADA) were then investigated. The basis of nerve dysfunction in LADA was different to T1DM and T2DM, and LADA patients exhibited more severe changes in nerve excitability and ultrasound measures. Investigations were then undertaken in T2DM. There is a strong association between the metabolic syndrome (MetS) and the development of peripheral neuropathy. To explain this relationship, the effect of the metabolic syndrome (MetS) in T2DM was examined in Chapter 5. A reduction in the function of the Na+/K+ pump was found to explain the more severe changes in nerve structure and function in T2DM patients with MetS compared to patients with T2DM alone. Finally, in exploration of potential neuroprotective options for peripheral neuropathy, the effect of anti-diabetic medication on nerve function was then investigated in Chapter 6. Exenatide treatment was associated with better nerve function in cross-sectional and prospective studies. Prominent abnormalities remained in patients receiving SGLT2 inhibitor or DPP-4 inhibitor therapy.
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(2021)ThesisNephrotoxicity limits the use of calcineurin inhibitors (CNI) in transplantation. Currently, CNI dosage is adjusted using serum creatinine and plasma CNI levels to protect the graft from toxic damage from overmedication or rejection from undermedication. Reduced long-term survival of transplanted kidneys indicates the need for a suitable biomarker of CNI toxicity. In a rodent model, we identified a panel of five urinary biomarkers of CNI nephrotoxicity comprising KIM-1, calbindin, clusterin, EGF and E cadherin. KIM-1, calbindin, and clusterin were differentially increased in CNI toxicity compared to controls among nine known biomarkers of kidney damage. EGF and E cadherin were identified by quantitative proteomics applied to pooled CNI toxic rodent urine samples. Kidney tissue mRNA expression confirmed increased KIM-1, calbindin, clusterin and EGF. These and other known damage biomarkers were then measured in a pilot study in human urine samples from consecutive kidney transplant recipients over one year. Serum creatinine did not change with plasma CNI levels highlighting that reliance on serum creatinine to identify CNI toxicity places the graft at risk of toxic damage. In contrast, multiple urinary biomarkers of kidney injury were increased despite apparently acceptable target plasma tacrolimus levels highlighting a need for greater awareness of potential CNI toxicity. The rodent model also showed that metformin ameliorated CNI toxicity. Combining CNI with metformin prevented CNI-associated reduction in inulin GFR and urinary biomarker increases. In exploring the mechanisms of metformin, we observed that CNIs reduced renal PGC 1a (a major regulator of mitochondrial homeostasis) at both mRNA and protein levels. We hypothesised that metformin increased PGC 1a by increasing AMPK. PGC 1a protein and mRNA increased when metformin was added to CNI treatment. Tissue AMPK levels correlated positively with PGC 1a in the CNI and CNI-metformin combination treatment groups indicating that PGC 1a may be mechanistically involved in the metformin therapeutic pathway in ameliorating CNI toxicity. Overall, these studies suggest strategies for both detecting and preventing toxicity and for monitoring clinical protection of human kidneys against CNI-associated injury.
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(2022)ThesisThe 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.