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The effects of ceramide synthases and protein kinase c epsilon on glucose homeostasis and lipid metabolism(2015) Diakanastasis, BarbaraThesisInsulin resistance contributes strongly to Type 2 Diabetes, which is a global epidemic. A strong link exists between dietary lipid excess and the development of insulin resistance. Two key bioactive lipid metabolites implicated in the development of insulin resistance are the sphingolipid ceramide and diacylglycerol, an activator of protein kinase c epsilon (PKCe). The mechanisms linking the actions of these metabolites to insulin resistance are unclear. The aims of this thesis were to (i) assess the effect of ceramide synthase (CerS) overexpression on key elements of skeletal muscle sphingolipid metabolism and insulin action and (ii) examine the effect of PKCe deletion solely in adipose tissue on glucose and lipid homeostasis. CerS isoforms were overexpressed in lipid-treated L6 skeletal myotubes. This did not cause compensatory changes in the mRNA expression of sphingolipid metabolism proteins. The effect of CerS overexpression on flux through ceramide synthesis pathways was assessed. Increased flux via the salvage pathway was seen after CerS overexpression. In addition, GLUT4 translocation to the plasma membrane, a key aspect of skeletal muscle insulin action, was increased or unaltered after CerS overexpression. Glucose tolerance was tested in fat-fed mice with PKCe deletion only in adipose tissue. Improved glucose tolerance was seen in these mice during short and long term fat feeding. This was linked to a greater decrease in plasma fatty acids and without compensatory rises in insulin secretion, suggesting this deletion enhanced whole body insulin sensitivity. This was confirmed by euglycaemic-hyperinsulinaemic clamp. Improved hepatic insulin sensitivity, potentially via decreased plasma fatty acids, was shown to drive this improvement. PKCe deletion in adipose tissue was also linked to smaller adipocyte size, an indicator of improved insulin sensitivity. Gene expression analysis showed that these protective effects were not via altered mRNA expression of lipid metabolism or inflammatory genes. Increased protein expression of lipid esterification enzymes however, was observed. This research has highlighted a novel protective role for ceramide during skeletal muscle insulin resistance upon specific CerS isoform overexpression. We have also shown for the first time that (i) exclusive PKCe deletion in adipose tissue improves glucose and lipid homeostasis during insulin resistance (ii) hepatic insulin sensitivity is modulated indirectly by PKCe.
(2000) Bova, RonaldoThesis
(2012) Stokes, Rebecca AnneThesisIslet transplantation is a promising therapy for β-cell replacement in people with type 1 diabetes. However many constraints exist including low donor availability and loss of up to 70% of β-cells during the islet isolation process and the immediate post transplant period. Much of this islet loss is due to hypoxia. This project investigated the role of transcription factors Aryl-hydrocarbon-receptor-nuclear-translocator (ARNT) and Hypoxia-Inducible-Factor-1α (HIF-1α) in islet transplantation, to study the mechanism of β-cell loss in the immediate post transplant period and consequently aim to improve outcomes. Hypoxia-Inducible-Factor-1α (HIF-1α) is upregulated in response to hypoxia, which is experienced after transplantation of islets. ARNT and HIF-1α are transcription factors which regulated β-cell function. HIF-1α is oxygen sensitive and binds with its partner ARNT to regulate transcription, including genes important for transplant outcomes. This thesis hypothesized that the increase in HIF-1α post transplantation was a compensatory response to protect β-cells during transplantation. β-ARNT and β-HIF-1α mice have specific β-cell deletion of ARNT and HIF-1α respectively, and are effective models for investigating the role of ARNT and HIF-1α in β-cell dysfunction post transplantation. Both β-ARNT and β-HIF-1α donor transplants had impaired glucose tolerance and function post transplantation. Hence, ARNT and HIF-1α are required in β-cell survival, stress response and for β-cell function in normal glucose tolerance. Desferrioxamine (DFO), an iron chelator, increases HIF-1α and improved β-cell function and survival in transplanted human islets and achieved improved transplant outcomes. Iron chelation with DFO markedly improved transplant success in a HIF-1α-dependent manner thus demonstrating the mechanism of action. Human islets treated with DFO decreased apoptosis and showed improved glucose control, as well as increased β-cell mass, glucose oxidation, endothelial preservation and beneficial β-cell genes post transplantation. Higher ATP concentrations were evident with both short and long term hypoxia in DFO treated islets. This thesis showed that HIF-1α and ARNT are important for β-cell function and survival. DFO improved outcomes of human islet transplants and the mechanism of benefit required HIF-1α. Thus, HIF-1α is a protective factor and is required for successful islet transplant outcomes. These results provide a potential therapy for successful human islet transplant outcome.
Xenotransplantation of adult human olfactory stem cells into mice with early-onset sensorineural hearing loss(2011) Pandit, SonaliThesisThe main goals of this thesis are: • To investigate the survival of adult human olfactory stem cells xenotransplanted into a mouse model of early-onset hearing loss (A/J mice). • To evaluate the ability of the stem cells to rescue hearing in early-onset progressive sensorineural hearing loss. The main results of this thesis are: • Xenotransplanted adult human olfactory stem cells survived for 4 weeks after the surgery (end point of the study). • The stem cells were mainly found in perilymphatic compartments of the cochlea (scala vestibuli and scala tympani) while only a few stem cells were present in scala media. • The stem cells did not integrate into cochlear tissues. • Post-surgery hearing threshold levels in stem cell-transplanted mice were found to be significantly lower than threshold levels of sham-injected mice (P < .05) for both click and pure tone stimuli. In addition, the threshold shift (difference between pre and post-surgery hearing thresholds) was significantly less in the stem cell transplanted animals than the sham-injected animals for click stimulus. Overall, adult human olfactory stem cell transplantation can help preserve hearing during early-onset sensorineural hearing loss.This improvement in hearing could be due to paracrine effect of adult human olfactory stem cells.
(2006) Patel, Nirmal PrafulThesisA practical consideration in the development of cellular therapy technology for the inner ear is the development of an in vitro model for assessing the optimal conditions for successful application of cells. The first part of this thesis describes the adaptation of the cochleovestibular structure harvested from P1 mouse pups for analysis of factors critical for the optimal implantation of stem cells in the inner ear. Results of these studies establish that the c17.2 neural stem cell line can be introduced into the cochleovestibular structure in vitro. Using this model, c17.2 cells demonstrated survival predominantly within the vestibule and basal spiral ganglion regions. Furthermore, the addition of the ototoxin, cisplatin and the neurotrophin, Brain Derived Neurotrophic Growth Factor (BDNF) enhanced the survival and migration/dispersion of c17.2 cells within the cochleovestibular explant. The second part of this thesis examines the hypothesis that olfactory neurosphere (ONS) and progenitor cells harvested from the olfactory epithelium represent a viable source of graft material for potential therapeutic applications in the inner ear. Olfactory epithelium represents a unique source of pluripotent cells that may serve as either homografts or autografts. The feasibility of ONSs to survive and integrate into a mammalian cochlea in vivo was assessed. The ONSs were isolated as a crude fraction from the olfactory epithelium of P1 to P3 day old swiss webster mouse pups, ubiquitously expressing the Green Fluorescent Protein (GFP) marker. The ONSs were microinjected into the cochleae of adult CD1 male mice. Four weeks following their implantation, ONS cells expressing the GFP marker and stained by Nestin were identified in all areas of the cochlea and vestibule, including the spiral ganglion. Robust survival and growth of the implanted ONS and ONS derived cells in the cochlea also included the development of “tumor-like” clusters, a phenomenon not observed in control animals implanted with c17.2 neural stem cells. Collectively, the results of this thesis illustrate the potential of olfactory neurosphere and progenitor cells to survive in the inner ear and expose a potential harmful effect of their transplantation.
(2013) Ye, Si Yuan SunnyThesisFor breast cancer patients, cancer recurrence and secondary metastasis still remain serious clinical problems that are inadequately prevented or managed. Part of the underlying cause may be due to the incomplete eradication of the rare sub-population of self-renewing, highly tumourigenic cancer stem cells (CSC) found within breast cancer tumours. Hence, breast CSCs need to be identified, isolated at high purity, and studied to find therapeutic targets. The transcriptional regulator protein Id1 is known to mediate self-renewal and differentiation processes in normal stem cells, and has also been implicated in breast cancer cell proliferation and invasion. Thus, the purpose of this thesis was to investigate whether Id1 marks the sub-population of rare cancer stem cells in triple-negative breast cancer (TNBC), an aggressive type of breast cancer with poor prognosis and high risk of relapse. A p53-null mouse model of mammary carcinoma that was previously developed in our lab was used for these studies. To understand the characteristics of the mouse model, p53-null tumours were analysed through array CGH and gene expression profiling which revealed the tumours to represent the claudin-low molecular subtype of TNBC. Strikingly, copy number amplification of Met was found in almost all tumours, and shown to be functionally important for cell proliferation in certain tumours. To functionally characterise the tumour-forming capacity of Id1+ cells, p53-null tumour cells were firstly transduced with an Id1GFP reporter lentivirus to label Id1+ cells as GFP+. Two rounds of limiting dilution tumour formation assays were then performed using FACS-purified populations of Id1GFP+, Id1GFP- and unsorted cells. Id1GFP+ enriched for a tumourigenic subset of cells in one p53-null tumour clone, but showed mixed results or no effect in two other tumour clones. Interestingly, Id1 expression did not correlate with the CD29+/CD24+ phenotype, which identified tumourigenic cells in other reported studies of p53-null mouse mammary carcinoma. The findings presented in this thesis demonstrate that Id1 marks a sub-set of prospective CSCs in certain cases of claudin-low breast cancer. Further investigation is required to determine the specific biological contexts of breast cancer under which Id1 is, or is not, a useful CSC marker.
(2010) Walters, Stacey NicoleThesisUnderstanding the factors that control T cell responses is a major focus of immunology. Despite this the factors that control T cell development, homeostasis and function are still not completely understood. To date the major function of the TNF-family cytokine BAFF is that it is an essential B cell survival and activation factor. The observation that T cells express the BAFF receptors BR3 and TACI suggests a role for BAFF on T cells. This is supported in vitro; exogenous BAFF co-stimulating both human and mouse T cell activation, and in vivo; BAFF Transgenic mice (Tg) have enhanced DTH responses. Conversely blockade of BR3 impairs the development of antigen specific T cell responses. Given the background data on BAFF and T cells, the hypothesis of this thesis was that BAFF would play a potentially powerful pro-inflammatory role in regulating T cell dependent immune responses. To directly assess the affect of BAFF upon T cells the T cell dependent model of allograft rejection was utilised. Contrary to expectations BAFF-Tg mice appeared to have impaired T cell immunity, as indicated by their acceptance of islet allografts. However, further investigation revealed that effector T cell function was intact and the allograft acceptance in BAFF-Tg mice was due to an increased number of CD4+ Foxp3+ regulatory T cells (Tregs). Indeed, depletion of Tregs in BAFF-Tg mice restored normal allo-reactivity demonstrating the expanded Tregs were responsible for maintaining allograft tolerance. The mechanism by which BAFF was expanding Tregs was shown to be independent of the T cell itself and directly related to the presence of B cells. An IL10 B cell regulatory mechanism was then investigated and it was found that although excessive BAFF could expand Tregs in the absence of IL10 they were non functional. In conclusion, the findings presented in this thesis demonstrate that BAFF can play a dichotomous anti-inflammatory role in T cell biology by promoting the expansion of Treg cells that allow for allograft acceptance.
Investigation of the sarcoma fusion gene landscape through targeted sequencing of archived patient samples(2021) Wooi, DansonThesisFusion genes are known to be key diagnostic, prognostic and therapeutic targets in blood cancers. However in sarcoma, the fusion gene landscape has yet to be fully elucidated due to the heterogeneity of fusion gene partners and rarity of the samples. Current fusion gene diagnostics are only able to detect presence of the fusion and are unable to identify novel fusion gene partners or splice isoforms. Therefore to expand on the knowledge of fusion genes in sarcoma, my study aims to sequence RNA from archived sarcoma patient samples by targeting a panel of 255 genes known to be involved in fusion. Through targeted sequencing, accurate fusion gene detection can be achieved along with information on novel fusion gene partners or splice junctions. Finally gene expression analysis was performed to gain insight into the transcriptome of sarcoma and identify the relationship of fusion genes to clinical prognostic markers. In this study, RNA was sourced from formalin fixed paraffin embedded (FFPE) samples, which is often degraded and chemically modified. However based on quality assessment, fusion gene detection could still be carried out in libraries as small as 1 million reads. Fusion genes were found to be identified in 44% (43/98) of patient samples with 38 of these fusions consistent with those reported in literature. Novel fusion isoforms were also found to contain different breakpoints but retain the same protein domains as the recurrent fusion isoform. Through gene expression analysis of fusion positive and negative Ewing’s sarcoma, it was found that 52% of differentially expressed protein coding isoforms contained fewer regulatory elements compared to the principal isoform of the gene. This suggests that presence of the fusion in Ewing’s sarcoma may be related to eased expression of certain genes. To identify any other links fusion genes may have to gene expression, levels of prognostic immune markers were compared to fusion type. While fusion type did not appear to be associated with expression of these immune genes, varying expression levels between samples may potentially act as a predictor for immunotherapy success.