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(2014)ThesisThe patient s ability to master everyday living tasks is strongly dependent on the utility of healthy functional hands. Therefore the restoration of hand function after tendon injuries is of utmost importance. Tendon injuries are common, especially lacerations in zone II. In this area flexor tendons follow a complicated anatomy and are difficult to repair. Consequently this thesis focuses on the research of tendon repairs in zone II and aims to improve repair quality and ultimately final functional outcome for the patient. To approach this topic the author of this thesis firstly analysis current concepts of tendon repair research models, secondly investigates common tendon repair techniques and improvements of these techniques and thirdly introduces new techniques for repair of deep flexor tendons in zone II. This thesis consists of ex vivo and in vivo experiments, which all build on another. Main results of these experiments are as follows: In regards to comparability to human tendons, sheep tendons are better tendon surrogates as pig tendons if used in ex vivo laboratory experiments. When focusing on gapping resistance, "locking loop" repair configurations for tendon repairs are not substantially different to "grasping loop" configurations, and only "cross-locks", as used in the Adelaide repair technique, deserve the adjective description "locking". The current gold standard of tendon repairs, the Adelaide repair, produces better repair stability if performed with larger cross locks. The author's interlocking modification of the Adelaide repair can further improve the Adelaide repair's stability. In an ex vivo setting, the author's new tendon repair concept, the knotless 3D barbed suture tendon repair, produces superior repair stability than the Adelaide repair. The turkey tendon model is the first tendon model that replicates human anatomy and tendon sizes and can be used in ex vivo as well as in vivo tendon repair experiments. In an in vivo tendon repair scenario, the use of the knotless 3D barbed suture tendon repair with resorbable barbed sutures produces inferior repair stability compared to the Adelaide repair, but improves functional outcomes. This thesis presents new insights into tendon repair research from a surgical and biomechanical point of view. The use of the novel unknotted barbed suture repair method did show superior results in ex vivo experiments but barb resorption in the in vivo experiments caused high failure rates. Nevertheless, there is a probability that with the development of more stable small barbed suturing materials in the near future it will be possible to further improve deep flexor tendon repairs using this novel repair technique.
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(2017)ThesisMuch is still unknown about sensory and perceptual changes in the cortex associated with complex regional pain syndrome (CRPS). This PhD aimed to investigate cortical integration of tactile sensation of the hand specifically the fingers, and how this might be altered in CRPS. A match-paired cross-sectional design was used in a series of neuroimaging and psychophysical studies on patients with unilateral upper limb CRPS (n=21). Clinical characteristics were described and compared with age, gender and hand dominance matched controls (Chapter 2). Methodological improvements for fine-grained fingertip mapping in the primary somatosensory cortex were piloted (n=1) in two separate experiments (Chapter 3). Single fingertip stimulation versus bilateral simultaneous fingertip stimulation was compared using phase encoded functional magnetic resonance imaging (fMRI). Fine-grained fMRI maps of the fingertips in S1 in CRPS were described. Structural morphometry measures underlying the functional S1 fingertip maps including cortical thickness were analysed with step-wise mixed modelling with a priori hypothesised effects including hand dominance and medication. Patient characteristics including pain- related measures were correlated with morphometry measures (Chapter 4). A new finger illusion experiment was applied for the first time in patients with CRPS (Chapter 5). The pilot found that bilateral tactile stimulus was most suitable for use in CRPS and had superior time efficiency. Disordered S1 functional fingertip maps in CRPS with no distinct pattern were found using this stimulus, when compared to the orderly homogenous map pattern in healthy controls. These functional imaging observations were strengthened by the key finding that increased cortical thickness underlying these maps together with hand dominance predicted group (CRPS versus healthy controls) membership. An abnormal finger illusion response in CRPS compared to controls, also suggests a disruption to normal efficiencies of bimanual hand representation cortically, not previously reported. In conclusion, disruption to cortical integration of tactile sensation in CRPS is suggested from the results. These changes also suggest cortical representation of differences in hand dominance rather than CRPS-sided-differences predicted those with CRPS in this study. Future directions to test these suggested cortically mediated changes in CRPS were explored.
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(2017)ThesisMurine haematopoiesis progresses step-wise through a number of defined stages that includes the yolk sac, aorta-gonad-mesonephros (AGM), foetal liver (FL), placenta and finally the bone marrow. The number of haematopoietic stem cells (HSCs) increases dramatically in the FL during mid-gestation. The cellular and signalling processes that govern HSC expansion and maturation in the FL are not entirely understood. Mesenchymal stem cells (MSC) are known to provide a niche for HSC maintenance in the adult bone marrow. Therefore, we hypothesised that FL MSCs may also play a role in the maturation and expansion of HSCs during embryonic development. Due to the lack of bona fide markers for FL MSCs, we first estimated the number of FL MSCs between E11.5 and E18.5 using colony forming unit- fibroblast (CFU-F) assays. FL CFU-Fs demonstrate serial clonogenicity, long-term self-renewal, and in vitro multipotency and their number progressively increases during FL development. Using two germ layer specific Cre transgenic mouse lines, I discovered that the majority of FL MSCs are derived from MesP1 mesoderm but that at E18.5 but not at E13.5, the FL also contains MSCs derived from Wnt1 neuro-ectoderm. HSC expansion in the FL occurs during E11.5-E16.5. To evaluate the contribution of FL MSCs to HSC expansion, further investigations were focused on the E13.5 time point with E18.5 FL used as a control. MSCs in the E13.5 and E18.5 FL can be significantly enriched by purifying cells expressing PDGFRα. PDGFRα+ cells reside near the peritoneal surface of the hepatic lobes and in the peri-sinusoidal and perivascular regions. Lineage tracing studies coupled with confocal microscopy revealed that PDGFRα+ cells directly contribute to hepatic mesothelial and endothelial cells during FL development and thus to the structural integrity and vasculature of the developing liver. Genome-wide expression analysis revealed that PDGFRα+ cells up- regulate genes and pathways involved in haematopoiesis. E13.5 PDGFRα+ population was also shown to support the ex vivo culture of haematopoietic stem and progenitor cells in a competitive transplantation assay.
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(2019)ThesisSomatic mutations in RNA splicing genes are frequent in many haematological malignancies, but are rarer in acute myeloid leukaemia (AML). However, their roles in leukaemogenesis are poorly described. To gain insights into the functional impact of alternative RNA splicing in leukemia, I investigated the transcriptomes of patients with matching clinical data. As a first step, I developed a bioinformatics pipeline (henceforth SpliceGX) to quantify alternative splicing (AS) and differentially expressed genes (DEGs) within the same samples. I also developed tools to predict the functional impact of AS on the translated proteins. I then applied SpliceGX to interrogate the role AS in disease severity in AML. I then applied SpliceGX in two additional, novel contexts: to study T-cell Acute Lymphoblastic Leukaemia (T-ALL) as well as in T cell development. To investigate alternative RNA splicing in AML, I extracted transcriptomic data from two published cohorts; The Cancer Genome Atlas and Clinseq. I identified 222 AS events that were significantly enriched or depleted in adverse- vs. favourable-risk AML patients with ~ 30% resulting in predicted loss of an annotated protein domain. Most AS events resulted in skipped exons with dysregulation of mTOR and integrin signalling in patients assigned as adverse risk by the European Leukaemia Net (ELN). Motif analysis of alternatively spliced junctions showed usage of non-canonical splice sites. Integrating AS with DEG in adverse- vs. favourable-risk AML further showed that aberrant splicing, even in the absence of known splicing factor mutations, triggers a stress response and the up-regulation of inflammatory genes in AML patients with poor prognosis. Furthermore, using machine learning, I discovered four alternatively spliced transcripts of prognostic significance in AML. Using SpliceGX, I identified characteristic splicing signatures at each waypoint during healthy T cell development, which affected various pathways responsible for cell growth, differentiation, and lineage commitment of thymocytes. In T-ALL, I discovered aberrant splicing of leukaemia driver and splicing factor genes, which indicate that in T-ALL deregulation of cancer genes occurs primarily at the level of splicing, rather than via somatic mutations. In summary, I have developed a bioinformatics pipeline and surveyed RNA splicing in AML and healthy T cell/T-ALL and identified specific events of interest that could be of value as therapeutic targets.
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(2019)ThesisPancreatic cancer (PC) is a lethal malignancy characterised by chemoresistance and metastatic spread. This is largely due to the presence of dense stromal fibrosis which distorts tumour vasculature. This limits nutrient, oxygen and chemotherapy perfusion into pancreatic tumours and promotes oxidative stress. The craftsmen of fibrosis are cancer-associated pancreatic stellate cells (CA-PSCs) who drive disease progression, chemoresistance and metastasis. To survive in the tumour microenvironment, CA-PSCs and PC cells undergo significant metabolic changes. A key alteration is increased expression of nutrient transporters to promote nutrient uptake. Solute carrier transporters have gained significant attention as a novel class of therapeutic targets, particularly solute carrier transporter 7A11 (SLC7A11) and solute carrier transporter 7A5 (SLC7A5), which are upregulated in PC cells and CA-PSCs. This study aimed: (1) to assess SLC7A11 inhibition in cancer-associated pancreatic stellate cells in vitro and in a clinically relevant orthotopic mouse model of PC; and (2) to investigate the function of SLC7A5 and SLC3A2 in cancer-associated pancreatic stellate cells in vitro. This study demonstrated that inhibiting SLC7A11 in CA-PSCs reduced proliferation. This limited cystine uptake, glutathione synthesis and anti-oxidant capacity of CA-PSCs and sensitised to oxidative stress. In a co-culture setting, inhibiting SLC7A11 in both PC cells and CA-PSCs significantly reduced colony number. We evaluated the therapeutic efficacy of two different SLC7A11 inhibitors, sulfasalazine and erastin. Sulfasalazine proved ineffective at reducing orthotopic pancreatic tumour growth, indicating it cannot be repurposed as PC therapy. Excitingly, erastin showed significant anti-proliferative effects in both PC cells and CA-PSCs. In CA-PSCs, the anti-proliferative effects were mediated by the induction of both ferroptosis and apoptosis. Lastly, we observed inhibition of SLC7A5 in CA-PSCs reduced proliferation by inducing senescence and limiting leucine uptake. In summary, this thesis demonstrated inhibition of SLC7A11/SLC7A5 has the potential to target PC cells and CA-PSCs, representing a dual targeting approach. Our data supports the concept of personalised medicine in which a patient will be treated based on their tumour’s genetic/metabolic profile, overcoming the current ‘one-drug-fits-all’ approach. Importantly, our findings could be translated to other hard to treat solid tumours.
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(2019)ThesisChiari malformation type I is a disorder in which the cerebellar tonsils herniate through the foramen magnum. A majority of patients with this condition also develop a fluid filled cavity (syrinx) within the spinal cord. Syrinxes are associated with both sensory and motor disturbances, with extreme cases leading to autonomic dysfunction. The mechanisms that lead to the accumulation of fluid remain unknown. There is no universally accepted treatment and current surgical treatments have variable, but often unsatisfactory, outcomes and side effects are common. There is a need to understand cerebrospinal fluid (CSF) circulation in Chiari malformation to identify the mechanism(s) that cause syrinx formation. This would enable a mechanistically based treatment to be developed. This thesis presents six interrelated studies investigating CSF circulation and the influence of tonsillar herniation on CSF dynamics, to better understand potential mechanisms for syrinx formation. Methods used include novel real-time magnetic resonance imaging (MRI) of CSF and blood flow, morphological assessments of anatomical MRI, subject-specific computational models of the spinal subarachnoid space, and idealised models of the perivascular space. The subject-specific models showed that in Chiari patients the peak systolic CSF pressure was increased and occurred earlier in the cardiac cycle, compared with controls. The perivascular space model suggested that these subarachnoid pressure characteristics may cause increased flow into the cord that is favourable for syrinx formation in Chiari patients. Increased overcrowding below the foramen magnum in syrinx free subjects led to an earlier systolic pulse, whereas in syrinx subjects it caused a delay. This difference in behaviour may be related to syrinx patients having a smaller midsagittal cross-sectional area and could explain why not all patients develop a syrinx. Real-time imaging of CSF flow in controls were inconsistent with the currently accepted mechanism for respiratory CSF circulation, suggesting instead that respiratory CSF flow is primarily dependent on the balance of the thoracic and lumbar spinal pressures. Real-time imaging in Chiari patients found coughing and straining produced high velocity cranial flow at the foramen magnum, which may be a marker for Chiari associated headache, but whether this influences syrinx formation requires further investigation.
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(2018)ThesisLow back pain is the leading cause of disability worldwide. Attentional bias has been hypothesised to underlie the development of persistent low back pain, however this has not been investigated using reliable outcome measures and with appropriate consideration for confounding. Researchers have recently begun to use eyetracking technology to assess attentional bias in painful conditions, however the reliability of eye tracking to assess the different stages of attentional bias (early, late, overall) is unclear. The primary research questions were: (1) What is the reliability of eye tracking to assess attentional bias? (2) Is there evidence that people with acute low back pain have an attentional bias to threat words? (3) Does attentional bias cause persistent low back pain? A systematic review and a test-retest reliability study were conducted to investigate the reliability of eye tracking to assess attentional bias. Reliable eye-tracking attentional bias outcomes were compared in people with and without acute low back pain. The causal relationship between attentional bias of people with acute low back pain and persistent low back pain (persistent pain, disability and pain intensity at three and six months) was determined. Directed acyclic graphs were used to identify confounding variables that may bias the causal role of attentional bias in the development of persistent low back pain. The results of the systematic review were inconclusive. The results of the test-retest reliability study indicated that eye tracking outcome measures that assess late and overall stages of attentional bias are reliable for investigating attentional bias. There was no evidence that people with acute low back pain have an attentional bias to threat words. An attentional bias away from emotionally threatening words was identified a causal factor in the development of low back pain related disability, but not in the development of persistent pain or pain intensity related to low back pain. Reliable eye tracking outcome measures that assess the early stage of attentional bias are not available and require further investigation. Future research should include attentional bias in causal models that seek to explore why some people develop persistent low back pain.
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(2018)ThesisToday, there is still a great clinical need for heart regenerative therapies. Future therapies should be directed by lessons learnt from vessel developmental biology. The coronary vasculature derives its constituents from different origins during its formation. Endothelial and periendothelial cell heterogeneity is partly due to these diverse developmental origins and may acquire specialised functions depending on their origin. This thesis describes a Pdgfra+ coronary vascular progenitor population and its temporal-spatial relationship to the mesoderm origin, and contribution to mature vascular networks during heart development. Pdgfra+ cardiac CFU-F isolated from the developing heart at different time points displayed different stem cell characteristics, cell morphology and location within the heart. Some Pdgfra+ progenitors derived from the mesoderm. Other origins such as the neural crest contributed to aorta formation during late embryonic stages. A Pdgfra-Nestin hierarchy exists during Pdgfra+ progenitor population differentiation into endothelial cells and pericytes. Pdgfra+ progenitors transit through Nestin expressing cells before differentiating into endothelial cells and pericytes. Nestin+ cell populations are more differentiated than Nestin- populations and can only form endothelial cell tubes without proper pericyte coverage in vivo. Nestin- populations form endothelial tubes with proper pericyte coverage. Pdgfra+ progenitor commitment to coronary vasculature formation occurs during early embryonic time points at E7.5. Maturing vascular networks present in the E18.5 heart derive from the Pdgfra+ progenitors marked at E7.5. In comparison, Pdgfra+ progenitors present within the heart at E8.5 – E14.5 do not contribute significantly to coronary vasculature within the E18.5 heart. The work conducted for this thesis indicates that only Pdgfra+ progenitor populations from the early embryonic E7.5 heart contribute significantly to coronary vasculature formation during heart development. The Pdgfra+ progenitors differentiate into endothelial cells and pericytes by first transiting through Nestin expressing cells.
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(2018)ThesisCurrent trends in regenerative medicine for tissue repair focus on generating tissue-specific stem cells. However, given the complexity of most tissues, the ideal stem cell would be one that could undergo multilineage context-dependent differentiation to bring about holistic repair of the injured tissue. This thesis describes application of a vector- and transcription factor-free method to reprogram human somatic cells into induced Multipotent Stem (iMS) cells utilizing the combination of 5-Azacytidine and recombinant human Platelet Derived Growth Factor-AB. I optimized xenofree conditions for this Demethylation Cytokine-induced (DCi) reprogramming technique that yielded autologous iMS cells at high efficiency from human adipocytes harvested from subjects aged 18-80 years. Human iMS cells display in vitro colony forming and serial re-plating ability, multilineage differentiation capacity and maintain a stable karyotype over several months. They express MSC markers but not markers of the blood lineage. iMS cells can be expanded long-term in medium containing autologous/allogeneic human serum. They have a transcriptional profile distinct to adipocytes or tissue-derived mesenchymal stem cells. IPA analysis revealed activation of genes associated with embryonic stem cells, EMT, PDGF signaling and downstream JAK/STAT, PI3K/AKT/mTOR pathways in iMS cells compared to adipocytes. Although iMS cells expressed pluripotency factors (OCT4, Nanog, SOX2 and SSEA4) they lacked spontaneous teratogenicity characteristic of pluripotent cells. When transplanted into injured intervertebral disc of NOD/SCID mice, human iMS cells were retained at transplant site for the duration of assessment (1 year) with no evidence of malignant transformation. iMS cells displayed in vivo plasticity and directly contributed to formation of new blood vessels, bone, cartilage and smooth muscle at the site of injury. To assess the specificity of cell plasticity, human iMS cells were also injected into cardiotoxin injured tibialis anterior muscle of SCID/beige mice. Donor iMS cells contributed to hCD56 expressing muscle satellite cells and hSpectrin expressing myofibres without heterotopic transformation or aberrant differentiation. Together these findings demonstrate the feasibility and utility of DCi reprogramming for generation of safe, therapeutically relevant autologous iMS cells, and provide a solid foundation to evaluate their tissue regenerative potential in controlled clinical trials.
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(2018)ThesisThe National Bowel Cancer Screening Program (NBCSP) in Australia will be fully rolled out by 2020, offering free biennial screening with immunochemical Faecal Occult Blood Testing (iFOBT) to people aged 50-74 years, but current participation rates are low at 40%. Section 1 of this thesis describes the development, calibration and validation of a microsimulation model, Policy1-Bowel, which simulates colorectal cancer (CRC) natural history and screening in Australia. I demonstrate the model’s cross-validity and external validity by comparing the estimates with other microsimulation model outcomes and with long-term CRC incidence and mortality outcomes in large randomised-controlled trials that offered guaiac FOBT or sigmoidoscopy screening. Section 2 evaluates the effectiveness and cost-effectiveness of the current NBCSP in Australia. 2-yearly iFOBT screening at 50-74 years (i.e. the fully rolled-out NBCSP) is estimated to reduce CRC incidence and mortality by 23-33% and 36-52%, respectively, at 40-60% participation. The program is highly cost-effective due to the cancer treatment costs averted. More than 59,000 and 83,800 deaths were predicted to be prevented by the NBCSP in 2015-2040 at participation rates of 40% and 60%. It is predicted that annual expenditure on colorectal cancer control will be reduced within a decade of full rollout of the program. Section 3 examines alternate CRC screening approaches, comparing 2-yearly iFOBT screening with screening approaches that use iFOBT, colonoscopy, sigmoidoscopy, computed tomographic colonography, faecal DNA and plasma DNA at different screening intervals in people aged 50-75 years. 2-yearly iFOBT was found to be the most cost-effective, and one of the most effective approaches. I also examine the impact of extending the NBCSP target age to include people in their forties and/or eighties. The current target age range has the most favourable benefit-to-harm balance and is the most cost-effective; extending screening to 45 years could be cost-effective, but would substantially increase colonoscopy demand and be associated with a less favourable benefits-to-harms trade-off. The findings of the thesis suggest that fully implemented NBCSP is currently the optimal approach for CRC screening in Australia. Increasing participation in the program is a critical with potential to save tens of thousands of lives over the next two decades.