Alzheimer's disease (AD) is the most common form of dementia, a disease that gradually destroys brain cells and leads to progressive decline in mental function. The presence of high densities of neuritic plaques composed of Abeta in the cerebral cortices is a criterion for the post-mortem diagnosis of AD. The view that Abeta deposition drives the pathogenesis of AD (amyloid hypothesis) has received support from a wide range of molecular, genetic, and animal studies. This hypothesis has been the focus of therapeutic intervention leading to the development of anti-Abeta immunotherapy as a potential treatment. There is a great deal of evidence that supports the capacity of immunization against Abeta to reduce amyloid pathology and restore memory function in transgenic mouse models of amyloidogenesis. However, as a result of anti-Abeta immunotherapy, many investigators have reported increased severity of cerebral amyloid angiopathy (CAA) and increased incidences of microhemorrhage. The mechanism/s responsible for the redistribution of Abeta to the vasculature is unclear. We examine two possible mechanisms that may influence the severity of CAA following immunization; the rate of Abeta clearance with deglycosylated antibodies via a dose response study and anti-Abeta antibody epitope specificity. Dose response results with a deglycosylated antibody showed that lower doses resulted in greater clearance of amyloid and significant improvements in cognition, suggesting that clearance mechanisms become saturated with high doses of antibody. Treatment with antibodies directed against different epitopes of Abeta implied that the degree of parenchymal Abeta clearance determines the extent of vascular Abeta accumulation; epitope specificity is not critical in directing the vascular accumulation. Passive anti-Abeta immunization can prevent Abeta deposition in APP transgenic mice. We investigated amyloid accumulation after immunization was terminated, and discovered that after treatment, amyloid began to accumulate as a factor of time and gradually built up but never reached the Abeta levels in control APP mice. These data suggest that delayed deposition of amyloid leads to long term delays in AD associated pathology. These data strongly support the use of prophylactic immunotherapy treatments, and it appears that existing amyloid deposits will require interventions that actively clear amyloid as the only means to efficiently reduce brain Abeta in AD.

published_or_final_version / abstract / Orthopaedics and Traumatology / Master / Master of Philosophy

No description available.

Movement is central for life, and all animals depend on accurate regulation of movement for purposeful behavior. There is great diversity of movements, ranging between simple and vital breathing movements to minute and subtle movements of the face used to communicate emotions. Consequently, motor neurons, which are the only route of central nervous system output, are essential for all motor behaviors. To control the many motor behaviors expressed by an animal, motor neurons are exposed to a large number and variety of modulating synaptic inputs and have evolved into subtypes with specific functions. In this thesis, motor neuron subtypes and the synaptic input to motor neurons from Renshaw cells and Ia afferents have been studied. Novel molecular markers that identify subtypes of motor neurons are described. Three markers, Chodl, Calca and ERRβ, have been used to study the degeneration of subtypes of motor neurons in a mouse model of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Another marker, 5-ht1d, has been used to record the electrophysiological character of gamma motor neurons. In mice that lack 5-ht1d, motor neurons develop with reduced proprioceptive input. Remarkably, these mice had fewer foot faults than control animals when challenged to cross a narrow beam suggesting that the amplitude of monosynaptic proprioceptive input to motor neurons is not essential for motor coordination. In a final set of experiments, genetic removal of vesicular transport of neurotransmitter from Renshaw cells suggest that Renshaw cells are not integral for motor circuit function or motor behaviors. However, they are involved in the development of motor circuits in the spinal cord. Together, this thesis provides novel molecular tools for studies of motor neuron subtypes and novel data regarding the development and function of spinal motor circuits.

Multiple checkpoints control the survival and activation of auto-reactive B cells. The discovery of the TNF family cytokine BAFF has been crucial to understanding peripheral B cell tolerance mechanisms. Homeostatic levels of BAFF are tightly regulated to maintain tolerance in the periphery. Chronically increased levels of BAFF lead to the survival of autoreactive B cells. Autoimmune patients display elevated serum BAFF levels. BAFF Tg mice model this situation with systemically high levels of BAFF and the subsequent development of two separate but related autoimmune syndromes; systemic lupus erythematosus (SLE) and Sj??gren???s syndrome (SS). The work conducted in this thesis further investigates the defects in tolerance down-stream of self-reactive B cell survival, which may contribute to autoimmune disease development in BAFF Tg mice. Expansion of the Marginal zone (MZ) B cell population correlates with the pathogenesis of several models of autoimmune disease. BAFF Tg mice are unique in that they not only display an increased splenic MZ B cell population, but also MZ B cells are found in the salivary glands of mice developing SS. The examination of genes differentially regulated between MZ and Follicular (Fo) B cells led to the investigation of sphingosine-1-phosphate receptor biology. The expression of S1P receptors was shown to be required for the positioning of MZ B cells in the spleen. Chronic BAFF stimulation alters the retention of MZ B cells through the alteration of S1P receptors and decreased integrin activation. The alteration of S1P receptors and increased ligand sensitivity leads to the accumulation of MZ B cells in the inflamed salivary glands of BAFF Tg mice. This works provides a potential mechanism for the tissue specificity seen in systemic autoimmune disease. The provision of T cell help to auto-reactive B cells is thought to underlie the development of SLE. BAFF Tg mice deficient in T cells surprisingly developed an SLE-like disease indistinguishable from that of BAFF Tg mice. Autoimmunity in BAFF Tg mice did however require signals through the toll-like receptor (TLR)-associated signalling adaptor, MyD88, which controlled the production of pathogenic autoantibodies. Therefore, autoimmunity in BAFF Tg mice results from altered B cell tolerance, which requires TLR signalling and is independent of T cell help. It is likely that autoimmune patients with elevated levels of BAFF show a similar basis for disease.

Doctor of Philosophy(PhD) / Melanotransferrin or melanoma tumour antigen p97 (MTf) is a transferrin homologue that is found predominantly bound to the cell membrane via a glycosylphosphatidylinositol anchor. The molecule is a member of the transferrin super-family that binds iron through a single high affinity iron(III)-binding site. Melanotransferrin was originally identified at high levels in melanoma cells and other tumours, but at lower levels in normal tissues. Since its discovery, the function of MTf has remained intriguing, particularly regarding its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways e.g., DNA and haem synthesis, it is important to understand the function of melanotransferrin in the transport of this vital nutrient. Melanotransferrin has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis, cell migration and eosinophil differentiation. Despite these previous findings, the exact biological and molecular function(s) of MTf remain elusive. Therefore, it was important to investigate the function of this molecule in order to clarify its role in biology. To define the roles of MTf, six models were developed during this investigation. These included: the first MTf knockout (MTf -/-) mouse; down-regulation of MTf expression by post-transcriptional gene silencing (PTGS) in SK-Mel-28 and SK-Mel-2 melanoma cells; hyper-expression of MTf expression in SK-N-MC neuroepithelioma cells and LMTK- fibroblasts cells; and a MTf transgenic mouse (MTf Tg) with MTf hyperexpression. The MTf -/- mouse was generated through targeted disruption of the MTf gene. These animals were viable, fertile and developed normally, with no morphological or histological abnormalities. Assessment of Fe indices, tissue Fe levels, haematology and serum chemistry parameters demonstrated no differences between MTf -/- and wild-type (MTf +/+) littermates, suggesting MTf was not essential for Fe metabolism. However, microarray analysis showed differential expression of molecules involved in proliferation such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein d (Apod) in MTf -/- mice compared with MTf +/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to down-regulate MTf mRNA and protein levels by >90% and >80%, respectively. This resulted in inhibition of cellular proliferation and migration. As found in MTf -/- mice, melanoma cells with suppressed MTf expression demonstrated up-regulation of MEF2A and TCF4 in comparison with parental cells. Furthermore, injection of melanoma cells with decreased MTf expression into nude mice resulted in a marked reduction of tumour initiation and growth. This strongly suggested a role for MTf in proliferation and tumourigenesis. To further understand the function of MTf, a whole-genome microarray analysis was utilised to examine the gene expression profile of five models of modulated MTf expression. These included two stably transfected MTf hyper-expression models (i.e., SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type with downregulated MTf expression (i.e., SK-Mel-28 melanoma). These findings were then compared with alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the microarray data were also assessed in another model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and upregulation, three genes were identified as commonly modulated by MTf. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and Tcf4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. Hence, this study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation. To further understand the function of MTf, transgenic mice bearing the MTf gene under the control of the human ubiquitin-c promoter were generated and characterised. In MTf Tg mice, MTf mRNA and protein levels were hyper-expressed in a variety of tissues compared with control mice. Similar to the MTf -/- mice, these animals exhibited no gross morphological, histological, nor Fe status changes when compared with wild-type littermates. The MTf Tg mice were also born in accordance with classical Mendelian ratios. However, haematological data suggested that hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. In conclusion, the investigations described within this thesis clearly demonstrate no essential role for MTf in Fe metabolism both in vitro and in vivo. In addition, this study generates novel in vitro and in vivo models for further investigating MTf function. Significantly, the work presented has identified novel role(s) for MTf in cell proliferation, migration and melanoma tumourigenesis.

[Truncated abstract] One of the barriers to understanding and preventing proliferative diabetic retinopathy in humans has been the lack of an appropriate animal model. Historically dog, rat and mouse models of diabetic retinopathy have been studied but none of these exhibit the later changes of proliferative diabetic retinopathy. Animals can be rendered diabetic by surgical pancreatectomy or the use of chemicals such as allozan or streptozotocin or by feeding of a high galactose diet. Alternatively, spontaneous rodent models of diabetes have been examined such as the BB rat, KK mouse or NOD mouse. However, in each case the retinal vascular changes observed are those of early nonproliferative diabetic retinopathy comprising at most saccular microaneurysms, increased thickness of the capillary basement membrane, acellular capillaries and pericyte ghosts. … Fluorecein angiography of this transgenic line clearly demonstrates the presence of leaky new vessels, by the appearance of leakage spots scattered throughout the retina from 1 month of age. These mice constitute a valuable model of diabetic retinopathy. Neovascularization in this animal model is induced by VEGF as in human diabetic retinopathy. The source of VEGF in human diabetic retinopathy is the ischemic inner retina. In this transgenic model the source of VEGF are the photoreceptor cells, which are situated just underneath the inner retina. The neovascularization is not dependent on a particular developmental stage and there is no spontaneous regression of new vessels. Thus any results generated in this model are highly relevant to human diabetic retinopathy.

Thesis (Ph. D.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains viii, 143 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph.D.)--McMaster University, 2003. / Advisor: W. J. Muller. Includes bibliographical references (leaves 259-282). Also available via World Wide Web.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 133-150) Also available in print.