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(2007)ThesisIntroduction: Around a quarter of patients with spinal cord injury develop post traumatic syringomyelia (PTS), causing progressive neurological deficits. Current surgical treatment is unsatisfactory. Endogenous stem cell therapy, aiming at replacing lost tissue and repairing damaged ones by endogenous progenitors, may offer hope. Investigation into the reaction of endogenous progenitors in PTS may extend our knowledge about stem cell biology and help to develop a new treatment option for PTS. Endogenous stem cells were found to differentiate into astrocytes. Reactive astrocytes and gliosis are shown to have an important role in spinal cord injury, such as protecting neurons, limiting inflammation and regulating local environment to suit progenitors. We hypothesize that reactive astrocytes may play an important protective and potential therapeutic roles in PTS. The aim of this thesis is to study proliferation, differentition and location of endogenous progenitors and their roles in PTS. Materials and methods: Excitotoxic injury model of PTS was performed in adult Wistar rats. Proliferating cells were marked by either exogenous mitotic marker bromodeoxyuridine or endogenous mitotic marker Ki67.lmmunofluorescence techniques targeting mitotic markers were used to trace the proliferating cells. Immunofluorescent double staining techniques were used to phenotype the proliferating cells. Results: A large number of endogenous progenitors appear in PTS from 24 hours to at least 8 weeks post injury (PI). They proliferate much faster in PTS than in the control animals. Although less endogenous progenitors are observed after 4 weeks PI, their number is still much higher than that in the control animals. Immediately after injury, progenitors exist mainly in the white matter, but the majority of them shift their position closer to the lesion within 2 days. In the chronic stage, the majority of stem cells are located in and around the lesion site. Endogenous progenitors differentiate into astrocytes but not oligodendrocytes or neurons within 8 weeks. Astrocytes respond to injury by upgrading GFAP (1 day PI), becoming hypertrophic (7 days PI) and forming glial scar (2 weeks PI) in PTS. The development of a glial scar corresponds with the stage of cyst stability or reduction in size. Conclusions: Endogenous progenitors exist in PTS and they respond to injury by proliferating and shifting their position towards the lesion. These studies are important in understanding the endogenous stem cell response to PTS and lay the groundwork for future studies examining stem cell therapy for the condition. Endogenous progenitors in the PTS model differentiate into astrocytes, which help to form the glial scar lining the syrinx. Reactive gliosis may play an important role to seclude the injury site from healthy tissue, prevent a cascading wave of uncontrolled tissue damage and restrict the syrinx enlargement.
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(2007)ThesisDiabetes can be considered to be one of the main health epidemics of the 21st century. Studies conducted by the World Health Organisation (WHO) indicate that the number of people with diabetes in the year 2000 was 171 million and this is projected to increase to 366 million by 2030 (Wild et al. 2004). The increasing incidence of both Type 1 and Type 2 diabetes is due to population growth, aging, urbanisation, obesity and physical inactivity. The current treatment by insulin injections for individuals with Type 1 diabetes fails to overcome the long term microvascular and macrovascular complications associated with the disease. A major challenge in the treatment of diabetes is to provide patients with an insulin source that is capable of regulating blood glucose levels (BGL) on a minute to minute basis. Advances in medical research have enabled the investigation of a variety of potential alternative therapies that may provide Type 1 diabetic patients with a more superior control of BGL and consequently minimise complications. The utilisation of pancreases obtained from fetal pigs offers potential therapeutic value in the treatment of Type 1 diabetes. Islet-like cell clusters (ICCs) are obtained from such tissue following partial mechanical and enzymatic digestive procedures. ICCs are primarily composed of immature duct cells which, when transplanted, will mainly differentiate into insulin producing β cells. Such cells are able to normalise BGL in immunodeficient diabetic recipients and in immunocompetent recipients when anti-rejection drugs are administered. This study investigates microencapsulation as an immunoprotective strategy that has the potential to remove the need for immunosuppression when such cells are transplanted. A review of the literature related to current medical research in the field of diabetes is presented in Chapter 1. In order to achieve the aims of the study, an understanding of how fetal pig ICCs behave when placed within a barium alginate microcapsule both in vitro and in vivo is essential and this data is presented in Chapter 3. This chapter demonstrates that ICCs will survive and differentiate in their typical manner when enclosed within microcapsules and transplanted. Such encapsulated cells will function to normalise BGL when transplanted into diabetic immunodeficent mice for at least 25 weeks and the animals exhibit increased bodyweight. Microcapsules retrieved at this time point were observed to be intact with no breakages or evidence of cellular overgrowth. Transplantation of encapsulated insulin-producing cells into immunocompetent mice are described in Chapter 4. Allotransplantation of a microencapsulated mouse insulin-producing cell line into these diabetic mice also exhibited graft function, resulting in normal BGL in recipients. Large animal experiments are described in Chapter 5. Allotransplantation of microencapsulated fetal pig ICCs into diabetic pig recipients displayed evidence of transient graft function in terms of lower BGL and reduced exogenous insulin requirements. The xenotransplantion of encapsulated fetal pIg ICCs into diabetic immunocompetent mice described in Chapter 4 proved to be more challenging. The transplantation of such cells in this environment did not yield particularly positive results. BGL remained elevated in these recipients and the animals lost bodyweight post transplantation. This area of research warrants further investigation as it is likely that further measures such as transient immunosuppression in combination with microencapsulation will allow fetal pig ICCs to function in a xenograft setting.
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(2007)ThesisBladder cancer (BlCa) is the second most common genitourinary cancer, affecting both men and women. Most (70%) cases present at the superficial stage; 20% of these recur with muscle-invasive disease. Major genetic alterations associated with BlCa include: loss/gain in expression or mutations in Retinoblastoma (RB) gene, human epidermal growth factor receptors (HERs), H-ras, p53 and FGFR3. Only p53 mutations are well correlated with invasive BlCa; other changes show variable correlations with disease status. To understand the progression of BlCa, a model of nine human BlCa cell sublines derived from a single parent but differing in in vivo characteristics, has been developed previously. These cells represent a heterogenous population from a single tumour and a model of different stages of BlCa progression, from non-tumourigenic to invasive. Two sublines were selected for further investigation: C3 (non-tumourigenic) and B8 (invasive). These were transfected with green (C3-GSP-2) and red fluorescent reporters (B8-RSP-gck) respectively to investigate the effects of their co-injection in vivo, specifically, promotion of C3 tumour growth by B8 cells. Surprisingly, B8 tumour growth was inhibited by C3 cells in vivo at different cell numbers and proportions of cells injected. Microarray analysis of C3 and B8 cells revealed differential expression of 1367 genes with dramatic differences in the transforming growth factor-ß and integrin-mediated pathways. Gene expression of BMP2, INHBB, FST, NOG, ID4 and TGF- ß1, in the TGF- ß pathway was further analysed with qRT-PCR in all nine sublines. Expression of BMP2 was significantly related to tumourigenic potential (p=0.0238, Mann-Whitney) and INHBB to invasive ability (p=0.0476, Mann-Whitney). The BlCa model did not include a metastatic component. To broaden the model, cell lines were established from an invaded lymph-node (B8-RSP-LN) and a bone-metastasis (B8-RSP-BN) after subcutaneous and intra-cardiac injection of B8-RSP-gck cells. No significant differences were observed in the migratory capability and anchorage-independent colony formation of these metastatic cells compared with B8 cells. Evaluation of expression of the panel of TGF-beta genes (BMP2, INHBB, FST, NOG, ID4 and TGF- ß1) and metastasis-related genes (MMP9, MMP2 and KAI1) indicated that expression of BMP2, FST, ID4 and MMP9 was decreased or lost in the metastatic sublines.
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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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