Bibliography: leaves 168-184. The work in this study encompasses oxygen free radical related inflammation in the peripheral lung and in lung cells. Animal and human studies have been used. Methods include cell culture with function studies, protein chemistry, animal and human physiology, and cell and lung structure through histopathology, and various forms of electron microscopy. The work resulting from this thesis has formed an important basis for understanding acute and chronic lung injury.

The transcription factor Pax5 is essential for commitment to the B lineage as the development of these cells is arrested at an early stage in the bone marrow of Pax5 deficient mice. Pax5 deficient pro-B cells display remarkable plasticity and are able to differentiate into other cell lineages both in vitro and in vivo. Several Pax5 target genes have been previously reported but none are able to explain the developmental block observed at the early to late pro-B cell stage. To determine the exact mechanisms by which Pax5 controls B cell development, I have undertaken a cDNA microarray screen with a custom generated B cell-specific cDNA library. By identifying genes that are differentially expressed between Pax5 deficient and wild type pro-B cells, I have identified a number of potential Pax5 target genes. The microarray screen identified lymphoid-restricted membrane protein (Lrmp or Jaw1) as a novel Pax5 activated transcript. Using RT-PCR and a Pax5-estrogen receptor inducible system, I confirmed that Jaw1 is a direct Pax5 target gene whose expression is confined, in resting cells, to the earliest stages of B and T cell development. The biological relevance of Jaw1 for cell fate specification has been tested by transducing bone marrow progenitor cells with murine retroviral vectors and reconstituting haemopoiesis in vivo. I have reported that over-expression of Jaw1 in these cells results in a decrease in the development of B and NK lymphocytes and a marked increase in the development of myeloid cells in the bone marrow of reconstituted mice. This result suggests that Jaw1 may play an important role in the early development of lymphocytes in the bone marrow. Whilst initial analysis of Jaw1 deficient mice has revealed no overt defects in lymphopoiesis, I postulate that Jaw1 is involved in IP3-induced calcium signaling downstream of the pre-BCR and BCR. This hypothesis has resulted from analysis of the function of IRAG, the only known Jaw1 homologue combined with data that Jaw1 is expressed in early B cells in the BM as well as in germinal centers in the spleen. Pax5 mutant mice usually die before weaning and the cause of death is currently unknown, suggesting that Pax5 is expressed in a previously unreported tissue. To investigate this hypothesis I produced a monoclonal antibody to Pax5 and screened for novel expression domains during embryonic development and also in neonate mice. These studies did not detect any new Pax5 expression domains, but did reveal that this antibody cross-reacts with Pax2 and/or Pax8 in the developing kidney and brain. Biochemical analysis of the serum from Pax5 deficient mice also did not reveal the likely cause of death in these animals, beyond the general signs of dehydration and starvation that are likely to be secondary to the underlying defect. / Doctor of Philosophy (PhD)

This is an original study to quantify and grade defect healing in a large animal cranial bone substitute model. The study of various therapies to heal cranial defects requires an appropriate ?critical? animal model. An experimental animal model should be analogous and recognizable as an appropriate challenge to human physiology. In addition, the defect must fail to heal unless treated with the tissue engineering therapy under study. Sheep as a large animal model was chosen because of its ability to tolerate creation of large skull defects analogous to clinical scenario, and its biology of healing as a high order mammal would be closer human beings. There is no agreement on the critical size limits for cranial defects. Various sizes have been termed "critical" in publications utilizing sheep. These ranged from 20-22mm. This study will investigate whether a 20mm defect is adequate. Bilateral circular cranial defects of 10, 20 and 25mm diameters were created in 12 adult sheep. Based on guided tissue engineering principles, defect protection was utilized to prevent in-growth of fibroblasts and other connective tissue cells from the surroundings. As bone tissue regeneration strategies usually involve osteoconduction element, an animal model that considered the defect protection role of osteoconduction would be more appropriate. Repopulation and regeneration of the defect was maximized as an added challenge Bioresorbable polylactic acid co-polymer mesh (MacroPoreTM) and Titanium mesh (TiMeshTM) was used as defect protection. The cranial defects were harvested at 8 and 16 weeks. The end-point analysis included Faxitron X-ray images, DEXA (Dual Energy X-ray Absorptiometry), and histology. The defects were graded to assess their ability to eventually heal. 10mm defects fully healed at 16 weeks. There was new bone formation spanning the entire defect seen on histology. 25mm defects were spanned by thin fibrous tissue only. There was variability in the healing potential of 20mm defect. Based on presence of bone islands within the defect, half of the 20mm defects demonstrated ability to heal while the other half actually had new bone spanning the defects on histology. Critical size cranial defect in sheep for the study of bone graft substitute has to be larger than 25mm diameter. The model is then utilized to study the use of Pro Osteon and AGF compared with the gold standard of autologous bone graft.

Electrical stimulation of retinal neurons is known to elicit visual sensations. When applied to the retina in a spatial pattern, electrical stimulation may be capable of providing rudimentary patterned vision that may be of benefit to sufferers of degenerative retinal disorders. No such device has yet been devised to provide for chronic study of the psychophysical perceptions elicited from a prosthesis for retinal stimulation. In this study, steps towards achieving this goal have been successfully carried out. Foregoing research was reviewed such that appropriate stimulation parameters were incorporated in the design of a 100 stimulation channel, complimentary metal oxide semiconductor (CMOS) integrated circuit, small enough in size so as to be capable of being implanted within the ocular anatomy or surrounding orbit. The device, and its associated external hardware and software were designed, modeled, fabricated, and interfaced with stimulating electrodes in acute testing in a highorder mammal (Ovis aries) so as to assess the capabilities of the device to elicit cortical potentials as a direct result of stimulation of the neural retina. Testing was performed under conditions similar to those anticipated in chronic in-situ configurations wherein radio-frequency telemetry was used to deliver power and configuration parameters to the device thus avoiding the passage of wires through tissue in order to communicate to the implant circuit. The results of the testing indicate that the circuit is indeed capable of eliciting physiological responses in the animal and evidence is present that these responses could be elicited in patterned form. Further work undertaken includes the development of surgical methods for implantation, and application of the prosthesis circuit in functional electronic stimulation.

The focus of this dissertation was leptin and the leptin receptor, and the role of these genes (OB and OB-R) in the development of obesity and type 2 diabetes in humans and Psammomys obesus, a polygenic rodent model of obesity and type 2 diabetes. Studies in humans showed that circulating leptin concentrations were positively associated with adiposity, and independently associated with circulating insulin and triglyceride concentrations. Analysis of two leptin receptor sequence polymorphisms in a Caucasian Australian population and a population of Nauruan males, with very high prevalence rates of obesity, showed no associations between sequence variation within the OB-R gene and obesity- or diabetes-related phenotypic measures. In addition, these two OB-R polymorphisms were not associated with longitudinal changes in body mass or composition in either of the populations examined. A unique analysis of the effects of multiple gene defects in the Nauruan population, demonstrated that the presence of sequence alterations in both the OB and OB-R genes were associated with insulin resistance. Psammomys obesus is regarded as an excellent rodent model in which to study the development of obesity and type 2 diabetes in humans. Examination of circulating leptin concentrations in Psammomys revealed that, as in humans, leptin concentrations were associated with adiposity, and independently associated with circulating insulin concentrations. This animal model was utilised to examine expression of OB-R, and the regulation of expression of this gene after dietary manipulation. OB-R is known to have several isoforms, and in particular, OB-RA and OB-RB gene expression were examined. OB-RB is the main signalling isoform of the leptin receptors. It has a long intracellular domain and has previously been shown to play an important role in energy balance and body weight regulation in rodents and humans. OB-RA is a much shorter isoform of OB-R, and although it lacks the long intracellular domain necessary to activate the JAK/STAT pathway, OB-RA is also capable of signalling, although to a lesser degree than OB-RB. OB-RA is found to be expressed almost ubiquitously throughout the body, and this isoform may be involved in transport of leptin into the cell, although its role remains unclear. OB-RA and OB-RB were both found to be expressed in a large number of tissues in Psammomys obesus. Interestingly, obese Psammomys were found to have lower levels of expression of OB-RA and OB-RB in the hypothalamus, compared to lean animals. This finding raises the possibility that decreased leptin signalling in the brain of obese, hyperleptinemic Psammomys obesus may contribute to the leptin resistance previously described in this animal model. However, the primary defect is unclear, as alternatively, increased circulating leptin concentrations may lead to down-regulation of leptin receptors. The effect of fasting on leptin concentrations and gene expression of OB-RA and OB-RB was also examined. A 24-hour fast resulted in no change in body weight, but a reduction in circulating leptin concentrations, and an increase in hypothalamic OB-RB gene expression in lean Psammomys. In obese animals, fasting again did not alter body weight, but resulted in an increase in both circulating leptin concentrations and hypothalamic OB-RB gene expression. In the liver, fasting resulted in a large increase in OB-RA gene expression in both lean and obese animals. These results highlighted the fact that regulation of leptin receptor gene expression in polygenic models of obesity and type 2 diabetes is complex, and not solely under the control of circulating leptin concentrations. Sucrose-feeding is an established method of inducing obesity and type 2 diabetes in rodents, and this experimental paradigm was utilised to examine the effects of longer term perturbations of energy balance on the leptin signalling pathway in Psammomys obesus. Addition of a 5% sucrose solution to the diet of lean and obese Psammomys resulted in increased body weight in both groups of animals, however only obese Psammomys showed increased fat mass and the development of type 2 diabetes. The changes in body mass and composition with sucrose-feeding were accompanied by decreased circulating leptin concentrations in both groups of animals, as well as a range of changes in leptin receptor gene expression. Sucrose-feeding increased hypothalamic OB-RB gene expression in obese Psammomys only, while in the liver there was evidence of a reduction in OB-RA and OB-RB gene expression in both lean and obese animals. The direct effects of sucrose on the leptin signalling pathway are unclear, however it is possible to speculate that the effect of sucrose to decrease leptin concentrations may have been involved in the exacerbation of obesity and the development of type 2 diabetes in obese Psammomys, From these studies, it appears that sequence variation in the OB and OB-R genes is unlikely to be a major factor in the etiology of obesity in human populations. The ability to examine regulation of expression of these genes in Psammomys obesus, however, has demonstrated that the effects of nutritional modifications on leptin receptor gene expression need closer attention. The role of the OB and OB-R genes in metabolism and the development of type 2 diabetes also warrants further examination, with particular attention on the differential effects of dietary modifications on leptin receptor gene expression across a range of tissues.

Lateral hip pain (LHP), characterised by non-specific symptoms in the region of the greater trochanter, is a condition frequently encountered by physiotherapists and other health professionals. However, the pathogenesis of LHP is not well understood. Although pathology of the gluteal tendons and their associated bursae have long been implicated in the cause of this problem, trochanteric bursitis has emerged as the primary clinical diagnosis. In order to determine a differential diagnosis, clinicians are reliant on information collated from the patient history and physical examination, yet the validity of many of the tests used to diagnose LHP has not been established. Abnormalities of the gluteal bursae may give rise to LHP and therefore to ensure precision of clinical assessment and treatment techniques, knowledge of bursal morphology is essential. However, a review of the literature revealed that there are no complete morphological accounts of all the bursae in this area. Therefore, the main purposes of this study were (a) to determine the morphology of the bursae associated with the greater trochanter and (b) to examine the physiotherapy and radiological diagnoses of LHP, and the validity of selected tests used in the diagnosis of LHP. In the anatomical study, the bursae deep to each of the layered gluteal tendons were examined in 21 embalmed human hips (9 male, 12 female; mean age 79 years, SD 9.4 years) using macro-dissection and histological techniques. Morphological associations, size, positions and histological characteristics of the bursae were recorded. A total of 121 bursae were identified in ten different locations, with an average of six bursae per hip. Variation was evident, but it was typical that at least two bursae were found deep to gluteus maximus (GMax) and the fascia lata, and gluteus medius (GMed). In approximately two-thirds of specimens a single bursa was situated deep to the tendon of gluteus minimus (GMin). All of these bursae demonstrated a synovial lining, which was predominantly areolar in type. This study revealed that numerous bursae are intimately associated with the greater trochanter, and provides new morphological detail which is of significance when considering clinical and biomechanical models of LHP. A clinical study was undertaken whereby 40 consecutive patients (37 female, 3 male; mean age 54.4 years, SD 9.5 years) with unilateral LHP were recruited prospectively. Each eligible participant underwent a standardised physiotherapy assessment followed by a magnetic resonance (MR) imaging study of the pelvis and both hips. The MR images were analysed in random order by three radiologists blinded to clinical findings and symptomatic side, and the intra-and inter-observer reliability for image analysis was examined using the kappa statistic. To determine the validity of selected clinical tests as evaluated against MR imaging, sensitivity, specificity, and positive and negative likelihood ratios (LRs) were calculated, and the chi-squared test was used to determine association. As demonstrated by MR imaging, GMed tendon pathology, bursitis, osteoarthritis (OA) and gluteal muscle atrophy are all associated with the report of LHP. Interestingly, these various pathologies were identified in asymptomatic as well as symptomatic limbs. However, while bursitis was equally prevalent in symptomatic and asymptomatic hips, GMed tendon pathology and OA were observed more frequently on the symptomatic side. Furthermore, muscle atrophy which predominantly affected GMin, was specific to symptomatic hips. Large variation was evident in the strength of agreement between radiologists and there was little agreement between physiotherapy and radiological diagnoses of pathology. Physiotherapists frequently diagnosed trochanteric bursitis as a cause of LHP and while palpation was identified as the most provocative test for reproducing patients complaint of LHP, it was not shown to be a valid technique. Instead, the outcomes pertaining to the validity of the clinical tests indicate that attention should be focused towards the assessment and treatment of gluteal tendon pathology. The two tests that appeared to be most useful for diagnosing gluteal tendon pathology were pain reproduction with passive hip abduction and resisted testing of GMed and GMin. While these findings demonstrate that various pathologies are associated with the report of LHP, they also highlight some problems associated with the use of MR imaging as a reference standard. Before further clinical validation studies of LHP are undertaken in larger populations, it is recommended that verification of MR imaging outcomes are performed against surgical and histological findings.

Iron is one of the essential elements involved in various fundamental biological activities. However, excess iron may bypass the negative feedback regulatory systems, leading to the formation of iron overload. The increase of iron deposition generates cellular toxicity and subsequently damages vital organs. Primary and secondary iron overload are affecting patients worldwide. Iron overload cardiomyopathy is the primary cause of cardiac dysfunction and cardiovascular mortality in β-thalassaemia major patients. Current effective therapy includes chelation treatment with conventional and new iron chelators, while potential new therapies are currently under development. The pathophysiology of iron overload cardiomyopathy remains unclear. Controversial findings on the mechanism of excessive iron entry into cardiomyocytes exist. Using novel real-time approach to trace iron entry into HL-1 cardiomyocytes, the only beating cardiac cell line with mature cardiac phenotype available currently, we visualized the patterns of iron entry following ferric iron incubation with and without ascorbate. Iron entry could be partly blocked by pretreatment with L-type calcium channel blockers but not T-type calcium channel blocker. Such blockage effect by L-type calcium channel blockers occurred in ferric iron overload. This finding suggested a role of L-type calcium channels for ferric iron uptake into cardiomyocytes under iron overload condition. For the pathophysiology of iron cardiac toxicity, we assessed the iron overload induced apoptosis using both in vitro and in vivo approaches. The results demonstrated that iron-overloaded mouse HL-1 atrial cardiomyocytes and human embryonic stem cell derived ventricular cardiomyocytes underwent apoptosis via the mitochondria-mediated caspase-3 dependent pathway. Supportive data was found in iron-overloaded mouse myocardium by an increase in DNA fragmentation. However, despite the blockage of iron entry, L-type calcium channel blockers did not significantly prevent iron induced apoptosis in vitro. The mechanism of cardiac contractile dysfunction caused by iron overload on cardiomyopathy has not yet been fully characterized. Given the central role of titin, the giant myofilament protein, as the main determinant in myocardial passive tension, stiffness, diastolic and systolic cardiac function, as well as myocardial twisting and untwisting motion, we investigated its expression in iron-overloaded cardiomyocytes in vitro and in vivo. Our results indicated that significant degradation of cardiomyocytes titin was induced by iron overload. This was associated with the cleavage at the elastic domain. Its potential upstream protease, calpain, was further identified to be activated under iron overload. The specific role of titin proteolysis in iron-overloaded cardiomyocytes merited further investigation. The findings in this project provided new insights to the pathophysiology of iron overload cardiomyopathy, in terms of the route for iron entry, iron induced cardiac apoptosis, and titin proteolysis. Novel therapeutic approaches for prevention and treatment of iron overload cardiomyopathy can focus on inhibiting excessive iron uptake, as well as by targeting pathways involved in cardiac apoptosis and titin proteolysis. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize / published_or_final_version / abstract / toc / Microbiology / Doctoral / Doctor of Philosophy

No description available.