Professor Simpson has a BSc (Hons) and a PhD (Veterinary Science) from the University of Sydney.
Professor Simpson joined the staff at UTS in 1994 and since 2002 has held the position of Professor of Biochemistry in the Department of Medical & Molecular Biosciences and since July 2009 has been Head of that Department.
She has also been Director of the Research Centre for Health Technologies since 2006. Prior to coming to UTS she was a Postdoctoral Fellow at both the University of Sydney and the University of New South Wales.
At UTS Prof. Simpson is involved in teaching final year Science students Biochemistry and Molecular Biology.
Her current research interest is the gene therapy of diabetes mellitus. Specifically, her research is concerned with investigating the possibility of engineering an artificial insulin-producing cell to replace the insulin-secreting beta cells that have been destroyed by the autoimmune process in Type I diabetics.
The principle behind this approach is that non-pancreatic cells can be genetically altered to produce, store and secrete insulin to physiological stimuli such as glucose.
Her laboratory has a Research Fellow, a Research Assistant and Postgraduate students. Since joining UTS she has received research funding from organizations such as the NHMRC, ARC, Juvenile Diabetes Research Foundation, Australian Diabetes Research Trust, Roche Organ Transplant Research Foundation, Rebecca Cooper Foundation, Ramiciotti Research Foundation and commercial sources.
She has collaborations with Dr. Bronwyn O'Brien and Najah Nassif in MMB, Dr. Wayne Hawthorne at Westmead Hospital, Dr. Ian Alexander at Westmead Children's Hospital and Dr. John Feller at the Royal Hospital, Randwick.
International collaborations include Sir Roy Calne and Prof. Andrew Lever, Cambridge University, Prof. Dick White, Nottingham University, Prof. Lee Kok Onn and Dr. Shu Vin Gan, National University of Singapore and a commercial relationship with Austrianova Singapore.
1. Endocrine Society of Australia: 1987 to present.
2. Australian Diabetes Society; 1987 to present.
3. Transplantation Society of Australia and New Zealand: 1989 to present.
4. International Transplantation Society; 1989 to present.
5. Cell Transplant Society, Foundation Member: 1991 to present.
6. Juvenile Diabetes Foundation International: 1991 to present.
7. Juvenile Diabetes Foundation of Australia: 1993 to present.
8. Australian Society of Clinical Biochemists: 1995 to present.
9. International Islet & Pancreas Transplantation Society: 1995 to present.
10. Australasian Gene Therapy Society: 2001 to present, founding member Professor Simpson is also the founding Chair of the UTS Biosafety Committee and Inaugural Treasurer of the Australasian Gene Therapy Society and is a Fellow and Board member of the Australian College of Biomedical Scientists.
Can supervise: YES
Specifically, Prof. Simpson's research is concerned with investigating the possibility of engineering an liver cells to replace the beta cells that have been destroyed by the autoimmune process in Type I diabetics.
The group is approaching this problem from 2 main directions:
1. The use of human liver cell lines that store and secrete insulin to glucose, which could be used to correct diabetes via transplantation of the encapsulated cells into patients, and
2. Direct delivery of the insulin gene and other components directly to the liver of patients to induce them to store mature insulin and secrete it in a physiological manner.
Recently the group has engineered a liver cell line derived that secretes insulin in the physiological range: Melligen cells. Her research with Dr. Binhai Ren and Dr. Bronwyn O'Brien has also led to reversal of diabetes in streptozotocin-diabetic immunocompetant rats and NOD mice by the direct delivery of the insulin gene directly through the portal circulation of the animal.
The animals have maintained normo-glycaemia for over 500 days with normal glucose tolerance and liver function tests. The group is moving to large animal studies.
Prof. Simpson co-ordinates Medical & Diagnostic Biochemistry (91344) and Biochemistry, Genes and Disease (91345), both of which are third year subjects in the B. BIomed. Sci., Med. Sci, B. Biotech and the BSc.
Both of these subjects examine the biochemical and molecular aspects of human disease and examine how the diseases are diagnosed in a clinical biochemistry laboratory and treated by clinicians. She also gives specialist lectures in Molecular Biology II on gene therapy and transgenics.
Gerace, D, Martiniello-Wilks, R, Habib, R & Simpson, AM 2019, 'Luciferase-based reporting of suicide gene activity in murine mesenchymal stem cells.', PloS one, vol. 14, no. 7.View/Download from: UTS OPUS or Publisher's site
Due to their ease of isolation, gene modification and tumor-homing properties, mesenchymal stem cells (MSCs) are an attractive cellular vehicle for the delivery of toxic suicide genes to a variety of cancers in pre-clinical models. In addition, the incorporation of suicide genes in stem cell-derived cell replacement therapies improves their safety profile by permitting graft destruction in the event of unexpected tumorigeneses or unwanted differentiation. Due to the functional requirement of ATP for the Firefly luciferase gene Luc2 to produce light, luciferase-based reporting of cytotoxicity can be engineered into potential cell therapies. Consequently, we nucleofected mammalian expression plasmids containing both the Luc2 and the yeast fusion cytosine deaminase uracil phosphoribosyltransferase (CDUPRT) genes for expression in murine MSCs to assess luciferase as a reporter of suicide gene cytotoxicity, and MSC as vehicles of suicide gene therapy. In vitro bioluminescence imaging (BLI) showed that following the addition of the non-toxic prodrug fluorocytosine (5-FC), CDUPRT-expressing MSCs displayed enhanced cytotoxicity in comparison to Luc2 reporter MSC controls. This study demonstrates the utility of luciferase as a reporter of CDUPRT-mediated cytotoxicity in murine MSC using BLI.
Gerace, D, Martiniello-Wilks, R, Habib, R, Ren, B, Nassif, NT, O'Brien, BA & Simpson, AM 2019, 'Ex Vivo Expansion of Murine MSC Impairs Transcription Factor-Induced Differentiation into Pancreatic β-Cells.', Stem Cells International, vol. 2019.View/Download from: UTS OPUS or Publisher's site
Combinatorial gene and cell therapy as a means of generating surrogate β-cells has been investigated for the treatment of type 1 diabetes (T1D) for a number of years with varying success. One of the limitations of current cell therapies for T1D is the inability to generate sufficient quantities of functional transplantable insulin-producing cells. Due to their impressive immunomodulatory properties, in addition to their ease of expansion and genetic modification ex vivo, mesenchymal stem cells (MSCs) are an attractive alternative source of adult stem cells for regenerative medicine. To overcome the aforementioned limitation of current therapies, we assessed the utility of ex vivo expanded bone marrow-derived murine MSCs for their persistence in immune-competent and immune-deficient animal models and their ability to differentiate into surrogate β-cells. CD45-/Ly6+ murine MSCs were isolated from the bone marrow of nonobese diabetic (NOD) mice and nucleofected to express the bioluminescent protein, Firefly luciferase (Luc2). The persistence of a subcutaneous (s.c.) transplant of Luc2-expressing MSCs was assessed in immune-competent (NOD) (n = 4) and immune-deficient (NOD/Scid) (n = 4) animal models of diabetes. Luc2-expressing MSCs persisted for 2 and 12 weeks, respectively, in NOD and NOD/Scid mice. Ex vivo expanded MSCs were transduced with the HMD lentiviral vector (MOI = 10) to express furin-cleavable human insulin (INS-FUR) and murine NeuroD1 and Pdx1. This was followed by the characterization of pancreatic transdifferentiation via reverse transcriptase polymerase chain reaction (RT-PCR) and static and glucose-stimulated insulin secretion (GSIS). INS-FUR-expressing MSCs were assessed for their ability to reverse diabetes after transplantation into streptozotocin- (STZ-) diabetic NOD/Scid mice (n = 5). Transduced MSCs did not undergo pancreatic transdifferentiation, as determined by RT-PCR analyses, lacked glucose responsiveness, and upon transplantation did no...
Gerace, D, Ren, B, Martiniello-Wilks, R & Simpson, AM 2019, 'High-efficiency lentiviral gene modification of primary murine bone-marrow mesenchymal stem cells', vol. 2029, pp. 197-214.View/Download from: Publisher's site
© Springer Science+Business Media, LLC, part of Springer Nature 2019. Lentiviral vectors are the method of choice for stable gene modification of a variety of cell types. However, the efficiency with which they transduce target cells varies significantly, in particular their typically poor capacity to transduce primary stem cells. Here we describe the isolation and enrichment of murine bone-marrow mesenchymal stem cells (MSCs) via fluorescence-activated cell sorting (FACS); the cloning, production, and concentration of high-titer second generation lentiviral vectors via combined tangential flow filtration (TFF) and ultracentrifugation; and the subsequent high-efficiency gene modification of MSCs into insulin-producing cells via overexpression of the furin-cleavable human insulin (INS-FUR) gene.
Atherosclerosis is the underlying cause of most myocardial infarction (MI) and ischaemic stroke episodes. An early sign of atherosclerosis is hypertrophy of the arterial wall. It is known that increased intima media thickness (IMT) is a non-invasive marker of arterial wall alteration, which can easily be assessed in the carotid arteries by high-resolution B-mode ultrasound. Similarly, the other key element of MI and ischaemic strokes is the N-methyl-D-aspartate (NMDA) receptor which is an ionotropic glutamate receptor that mediates the vast majority of excitatory neurotransmission in the brain. NMDA activation requires the binding of both glutamate and a coagonist like D-serine to its glycine site. A special enzyme, serine racemase (SR), is required for the conversion of L-serine into D-serine, and alterations in SR activities lead to a variety of physiological and pathological conditions ranging from synaptic plasticity to ischemia, MI, and stroke. The amount of D-serine available for the activation of glutamatergic signalling is largely determined by SR and we have developed ways to estimate its levels in human blood samples and correlate it with the IMT. This research based short communication describes our pilot study, which clearly suggests that there is a direct relationship between the SR, D-serine, and IMT. In this article, we will discuss whether the activity of SR can determine the future consequences resulting from vascular pathologies such as MI and stroke.
Ren, B, La, QT, O'Brien, BA, Nassif, NT, Tan, Y, Gerace, D, Martiniello-Wilks, R, Torpy, F, Dane, AP, Alexander, IE & Simpson, AM 2018, 'Partial pancreatic transdifferentiation of primary human hepatocytes in the livers of a humanised mouse model.', The journal of gene medicine, vol. 20, no. 5.View/Download from: UTS OPUS or Publisher's site
Gene therapy is one treatment that may ultimately cure type 1 diabetes. We have previously shown that the introduction of furin-cleavable human insulin (INS-FUR) to the livers in several animal models of diabetes resulted in the reversal of diabetes and partial pancreatic transdifferentiation of liver cells. The present study investigated whether streptozotocin-diabetes could be reversed in FRG mice in which chimeric mouse-human livers can readily be established and, in addition, whether pancreatic transdifferentiation occurred in the engrafted human hepatocytes.Engraftment of human hepatocytes was confirmed by measuring human albumin levels. Following delivery of the empty vector or the INS-FUR vector to diabetic FRG mice, mice were monitored for weight and blood glucose levels. Intraperitoneal glucose tolerance tests (IPGTTs) were performed. Expression levels of pancreatic hormones and transcription factors were determined by a reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry.Diabetes was reversed for a period of 60 days (experimental endpoint) after transduction with INS-FUR. IPGTTs of the insulin-transduced animals were not significantly different from nondiabetic animals. Immunofluorescence microscopy revealed the expression of human albumin and insulin in transduced liver samples. Quantitative RT-PCR showed expression of human and mouse endocrine hormones and β-cell transcription factors, indicating partial pancreatic transdifferentiation of mouse and human hepatocytes. Nonfasting human C-peptide levels were significantly higher than mouse levels, suggesting that transdifferentiated human hepatocytes made a significant contribution to the reversal of diabetes.These data show that human hepatocytes can be induced to undergo partial pancreatic transdifferentiation in vivo, indicating that the technology holds promise for the treatment of type 1 diabetes.
Haddadi, N, Lin, Y, Travis, G, Simpson, AM, Nassif, NT & McGowan, EM 2018, 'PTEN/PTENP1: 'Regulating the regulator of RTK-dependent PI3K/Akt signalling', new targets for cancer therapy.', Molecular cancer, vol. 17, no. 1.View/Download from: UTS OPUS or Publisher's site
Regulation of the PI-3 kinase (PI3K)/Akt signalling pathway is essential for maintaining the integrity of fundamental cellular processes, cell growth, survival, death and metabolism, and dysregulation of this pathway is implicated in the development and progression of cancers. Receptor tyrosine kinases (RTKs) are major upstream regulators of PI3K/Akt signalling. The phosphatase and tensin homologue (PTEN), a well characterised tumour suppressor, is a prime antagonist of PI3K and therefore a negative regulator of this pathway. Loss or inactivation of PTEN, which occurs in many tumour types, leads to overactivation of RTK/PI3K/Akt signalling driving tumourigenesis. Cellular PTEN levels are tightly regulated by a number of transcriptional, post-transcriptional and post-translational regulatory mechanisms. Of particular interest, transcription of the PTEN pseudogene, PTENP1, produces sense and antisense transcripts that exhibit post-transcriptional and transcriptional modulation of PTEN expression respectively. These additional levels of regulatory complexity governing PTEN expression add to the overall intricacies of the regulation of RTK/PI-3 K/Akt signalling. This review will discuss the regulation of oncogenic PI3K signalling by PTEN (the regulator) with a focus on the modulatory effects of the sense and antisense transcripts of PTENP1 on PTEN expression, and will further explore the potential for new therapeutic opportunities in cancer treatment.
Lees, T, Fatima-Shad, K, Simpson, A, Nassif, N, Lin, Y & Lal, S 2018, 'Heart Rate Variability as a Biomarker for Predicting Stroke, Post-stroke Complications and Functionality', Biomarker Insights, vol. 13, pp. 1-13.View/Download from: UTS OPUS or Publisher's site
Background: Heart rate variability (HRV) is a non-invasive measure of the function of the autonomic nervous system, and its dynamic nature may provide a means through which stroke and its associated complications may be predicted, monitored, and managed.
Objective: The objective of this review is to identify and provide a critique on the most recent uses of HRV in stroke diagnosis/management and highlight areas that warrant further research.
Methods: The MEDLINE, CINAHL, and OVID MEDLINE databases were canvassed using a systematic search strategy, for articles investigating the use of HRV in stroke diagnosis and management. Initial paper selections were based on title alone, and final paper inclusion was informed by a full-text critical appraisal.
Results: The systematic search returned 98 records, of which 51 were unique. Following screening, 22 records were included in the final systematic review. The included papers provided some information regarding predicting incident stroke, which largely seems to be best predicted by time- and frequency-domain HRV parameters. Furthermore, post-stroke complications and functionality are similarly predicted by time- and frequency-domain parameters, as well as non-linear parameters in some instances.
Conclusions: Current research provides good evidence that HRV parameters may have utility as a biomarker for stroke and for post-stroke complications and/or functionality. Future research would benefit from the integration of non-linear, and novel parameters, the hybridisation of HRV parameters, and the expansion of the utilisation of predictive regression and hazard modelling.
Gerace, D, Martiniello-Wilks, R, Nassif, NT, Lal, S, Steptoe, R & Simpson, AM 2017, 'CRISPR-targeted genome editing of mesenchymal stem cell-derived therapies for type 1 diabetes: a path to clinical success?', STEM CELL RESEARCH & THERAPY, vol. 8.View/Download from: UTS OPUS or Publisher's site
Haddadi, N, Lin, Y, Simpson, AM, Nassif, NT & McGowan, EM 2017, ''Dicing and Splicing' Sphingosine Kinase and Relevance to Cancer', International Journal of Molecular Sciences, vol. 18, no. 9, pp. 1-25.View/Download from: UTS OPUS or Publisher's site
Sphingosine kinase (SphK) is a lipid enzyme that maintains cellular lipid homeostasis. Two SphK isozymes, SphK1 and SphK2, are expressed from different chromosomes and several variant isoforms are expressed from each of the isozymes, allowing for the multi-faceted biological diversity of SphK activity. Historically, SphK1 is mainly associated with oncogenicity, however in reality, both SphK1 and SphK2 isozymes possess oncogenic properties and are recognized therapeutic targets. The absence of mutations of SphK in various cancer types has led to the theory that cancer cells develop a dependency on SphK signaling (hyper-SphK signaling) or 'non-oncogenic addiction'. Here we discuss additional theories of SphK cellular mislocation and aberrant 'dicing and splicing' as contributors to cancer cell biology and as key determinants of the success or failure of SphK/S1P (sphingosine 1 phosphate) based therapeutics.
Lees, T, Nassif, N, Simpson, A, Shad-Kaneez, F, Martiniello-Wilks, R, Lin, Y, Jones, A, Qu, X & Lal, S 2017, 'Recent advances in molecular biomarkers for diabetes mellitus: a systematic review.', Biomarkers, vol. 22, no. 7, pp. 604-613.View/Download from: UTS OPUS or Publisher's site
CONTEXT: Diabetes is a growing global metabolic epidemic. Current research is focussing on exploring how the biological processes and clinical outcomes of diabetes are related and developing novel biomarkers to measure these relationships, as this can subsequently improve diagnostic, therapeutic and management capacity. OBJECTIVE: The objective of this study is to identify the most recent advances in molecular biomarkers of diabetes and directions that warrant further research. METHODS: Using a systematic search strategy, the MEDLINE, CINAHL and OVID MEDLINE databases were canvassed for articles that investigated molecular biomarkers for diabetes. Initial selections were made based on article title, whilst final inclusion was informed by a critical appraisal of the full text of each article. RESULTS: The systematic search returned 246 records, of which 113 were unique. Following screening, 29 records were included in the final review. Three main research strategies (the development of novel technologies, broad biomarker panels, and targeted approaches) identified a number of potential biomarkers for diabetes including miR-126, C-reactive protein, 2-aminoadipic acid and betatrophin. CONCLUSION: The most promising research avenue identified is the detection and quantification of micro RNA. Further, the utilisation of functionalised electrodes as a means to detect biomarker compounds also warrants attention.
Lund, ME, Greer, J, Dixit, A, Alvarado, R, McCauley-Winter, P, To, J, Tanaka, A, Hutchinson, AT, Robinson, MW, Simpson, AM, O'Brien, BA, Dalton, JP & Donnelly, S 2016, 'A parasite-derived 68-mer peptide ameliorates autoimmune disease in murine models of Type 1 diabetes and multiple sclerosis.', Scientific Reports, vol. 6, pp. 1-11.View/Download from: UTS OPUS or Publisher's site
Helminth parasites secrete molecules that potently modulate the immune responses of their hosts and, therefore, have potential for the treatment of immune-mediated human diseases. FhHDM-1, a 68-mer peptide secreted by the helminth parasite Fasciola hepatica, ameliorated disease in two different murine models of autoimmunity, type 1 diabetes and relapsing-remitting immune-mediated demyelination. Unexpectedly, FhHDM-1 treatment did not affect the proliferation of auto-antigen specific T cells or their production of cytokines. However, in both conditions, the reduction in clinical symptoms was associated with the absence of immune cell infiltrates in the target organ (islets and the brain tissue). Furthermore, after parenteral administration, the FhHDM-1 peptide interacted with macrophages and reduced their capacity to secrete pro-inflammatory cytokines, such as TNF and IL-6. We propose this inhibition of innate pro-inflammatory immune responses, which are central to the initiation of autoimmunity in both diseases, prevented the trafficking of autoreactive lymphocytes from the periphery to the site of autoimmunity (as opposed to directly modulating their function per se), and thus prevented tissue destruction. The ability of FhHDM-1 to modulate macrophage function, combined with its efficacy in disease prevention in multiple models, suggests that FhHDM-1 has considerable potential as a treatment for autoimmune diseases.
Powell, KL, Stevens, V, Upton, DH, McCracken, SA, Simpson, AM, Cheng, Y, Tasevski, V, Morris, JM & Ashton, AW 2016, 'Role for the thromboxane A2 receptor β-isoform in the pathogenesis of intrauterine growth restriction.', Scientific Reports, vol. 6, pp. 1-15.View/Download from: UTS OPUS or Publisher's site
Intrauterine growth restriction (IUGR) is a pathology of pregnancy that results in failure of the fetus to reach its genetically determined growth potential. In developed nations the most common cause of IUGR is impaired placentation resulting from poor trophoblast function, which reduces blood flow to the fetoplacental unit, promotes hypoxia and enhances production of bioactive lipids (TXA2 and isoprostanes) which act through the thromboxane receptor (TP). TP activation has been implicated as a pathogenic factor in pregnancy complications, including IUGR; however, the role of TP isoforms during pregnancy is poorly defined. We have determined that expression of the human-specific isoform of TP (TPβ) is increased in placentae from IUGR pregnancies, compared to healthy pregnancies. Overexpression of TPα enhanced trophoblast proliferation and syncytialisation. Conversely, TPβ attenuated these functions and inhibited migration. Expression of the TPβ transgene in mice resulted in growth restricted pups and placentae with poor syncytialisation and diminished growth characteristics. Together our data indicate that expression of TPα mediates normal placentation; however, TPβ impairs placentation, and promotes the development of IUGR, and represents an underappreciated pathogenic factor in humans.
Ren, B, Tao, C, Swan, MA, Joachim, N, Martiniello-Wilks, R, Nassif, NT, O'Brien, BA & Simpson, AM 2016, 'Pancreatic Transdifferentiation and Glucose-Regulated Production of Human Insulin in the H4IIE Rat Liver Cell Line', INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 17, no. 4.View/Download from: UTS OPUS or Publisher's site
Lawandi, J, Tao, C, Ren, B, Williams, P, Ling, D, Swan, MA, Nassif, NT, Torpy, FR, O'Brien, BA & Simpson, AM 2015, 'Reversal of diabetes following transplantation of an insulin-secreting human liver cell line: Melligen cells', MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT, vol. 2.View/Download from: UTS OPUS or Publisher's site
McGowan, EM, Simpson, A, McManaman, J, Boonyaratanakornkit, V & Hardikar, AA 2015, 'Hijacking of Endocrine and Metabolic Regulation in Cancer and Diabetes', BioMed Research International, vol. 2015.View/Download from: UTS OPUS or Publisher's site
Gerace, D, Martiniello-Wilks, R & Simpson, AM 2015, 'Diabetes reversal via gene transfer: building on successes in animal models', Research and Reports in Endocrine Disorders, vol. 5, pp. 15-29.View/Download from: UTS OPUS or Publisher's site
Type 1 diabetes (T1D) is caused by the autoimmune destruction of the insulin-producing pancreatic β-cells. People with T1D manage their hyperglycemia using daily insulin injections; however, this does not prevent the development of long-term diabetic complications such as retinopathy, nephropathy, neuropathy, and various macrovascular disorders. Currently, the only "cure" for T1D is pancreas transplantation or islet-cell transplantation; however, this is hampered by the limited number of donors and the requirement for life-long immunosuppression. As a result, the need for alternative therapies is vital. One of the strategies employed to correct T1D is the use of gene transfer to generate the production of an 'artificial' β-cell that is capable of secreting insulin in response to fluctuating glucose concentrations that normally occurs in people without T1D. The treatment of many diseases using cell and gene therapy is generating significant attention in the T1D research community; however, for a cell therapy to enter clinical trials, success and safety must first be shown in an appropriate animal model. Animal models have been used in diabetes research for over a century, have improved our understanding of the pathophysiology of diabetes, and have led to the discovery of useful drugs for the treatment of the disease. Currently, the nonobese diabetic mouse is the animal model of choice for the study of T1D as it most closely reflects disease development in humans. The aim of this review is to evaluate the success of cell and gene therapy to reverse T1D in animal models for future clinical application.
Gerace, D, Martiniello-Wilks, R, O'Brien, BA & Simpson, AM 2015, 'The use of beta-cell transcription factors in engineering artificial beta cells from non-pancreatic tissue', GENE THERAPY, vol. 22, no. 1, pp. 1-8.View/Download from: UTS OPUS or Publisher's site
Lund, ME, O'Brien, B, Hutchinson, AT, Robinson, MW, Simpson, AM, Dalton, JP & Donnelly, SM 2014, 'Secreted proteins from the helminth Fasciola hepatica inhibit the initiation of autoreactive T cell responses and prevent diabetes in the NOD mouse', PLoS One, vol. 9, no. 1, p. e86289.View/Download from: UTS OPUS or Publisher's site
Infections with helminth parasites prevent/attenuate auto-inflammatory disease. Here we show that molecules secreted by a helminth parasite could prevent Type 1 Diabetes (T1D) in nonobese diabetic (NOD) mice. When delivered at 4 weeks of age (coincident with the initiation of autoimmunity), the excretory/secretory products of Fasciola hepatica (FhES) prevented the onset of T1D, with 84% of mice remaining normoglycaemic and insulitis-free at 30 weeks of age. Disease protection was associated with suppression of IFN-? secretion from autoreactive T cells and a switch to the production of a regulatory isotype (from IgG2a to IgG1) of autoantibody. Following FhES injection, peritoneal macrophages converted to a regulatory M2 phenotype, characterised by increased expression levels of Ym1, Arg-1, TGFß and PD-L1. Expression of these M2 genetic markers increased in the pancreatic lymph nodes and the pancreas of FhES-treated mice. In vitro, FhES-stimulated M2 macrophages induced the differentiation of Tregs from splenocytes isolated from naïve NOD mice. Collectively, our data shows that FhES contains immune-modulatory molecules that mediate protection from autoimmune diabetes via the induction and maintenance of a regulatory immune environment.
Nield, BS, Guzowski, R, Nassif, N, Simpson, AM & Martiniello-Wilks, R 2014, 'First use of Re: View: A tool to combine assessment tasks, marking criteria and graduate attributes for biochemistry students', International Journal of Innovation in Science and Mathematics Education, vol. 22, no. 7, pp. 49-64.View/Download from: UTS OPUS
In order to improve clarity of the link between assessment tasks and graduate attributes to students, Re:View was introduced across three undergraduate biochemistry subjects. Re:View is an online assessment tool which provides a direct visual link between graduate attributes and marking criteria. It also provides students with an easy access portal to retrieve their grade and assessor feedback on assessment tasks. Our aim was to improve the second and third year biochemistry student laboratory-based learning experience by developing and clarifying the link between assessment tasks, marking criteria and graduate attributes, using Re:View as the assessment tool. Student opinion showed Re:View was of benefit to align marking criteria with graduate attributes, and provided easy access to feedback which could be used to improve future work. This first use of Re:View, with development of criterion-referenced marking criteria and rubrics, has revolutionised assessment in the three biochemistry subjects under study. With the use of Re:View we have clarified the link between assessment tasks and marking criteria, and enhanced student engagement with laboratory-based assessment tasks, which has improved their written assessment performance.
Ren, B, O'Brien, B, Byrne, M, Ch'ng, E, Gatt, PN, Swan, MA, Nassif, N, Wei, M, Gijsbers, R, Debyser, Z & Simpson, AM 2013, 'Long-term reversal of diabetes in non-obese diabetic mice by liver-directed gene therapy.', The Journal of gene Medicine, vol. 15, no. 1, pp. 28-41.View/Download from: UTS OPUS or Publisher's site
Background Type 1 diabetes (T1D) results from an autoimmune attack against the insulin-producing ß-cells of the pancreas. The present study aimed to reverse T1D by gene therapy. Methods We used a novel surgical technique, which involves isolating the liver from the circulation before the delivery of a lentiviral vector carrying furin-cleavable human insulin (INS-FUR) or empty vector to the livers of diabetic non-obese diabetic mice (NOD). This was compared with the direct injection of the vector into the portal circulation. Mice were monitored for body weight and blood glucose. Intravenous glucose tolerance tests were performed. Expression of insulin and pancreatic transcription factors was determined by the reverse transcriptase-polymerase chain reaction and immunohistochemistry and immunoelectron microscopy was used to localise insulin. Results Using the novel surgical technique, we achieved long-term transduction (42% efficiency) of hepatocytes, restored normoglycaemia for 150 days (experimental endpoint) and re-established normal glucose tolerance. We showed the expression of ß-cell transcription factors, murine insulin, glucagon and somatostatin, and hepatic storage of insulin in granules. The expression of hepatic markers, C/EBP-ß, G6PC, AAT and GLUI was down-regulated in INS-FUR-treated livers. Liver function tests remained normal, with no evidence of intrahepatic inflammation or autoimmune destruction of the insulin-secreting liver tissue. By comparison, direct injection of INS-FUR reduced blood glucose levels, and no pancreatic transdifferentiation or normal glucose tolerance was observed.
Simpson, AM, Ren, B, O'Brien, BA & Nassif, NT 2013, 'Response to the letter to the editor by M. Elsner et al: "Comment on Binhai Ren et al (2013;15:28-41). Long term reversal of diabetes in non-obese diabetic mice by liver-directed gene therapy"', JOURNAL OF GENE MEDICINE, vol. 15, no. 8-9, pp. 309-310.View/Download from: UTS OPUS or Publisher's site
Gerace, D, Ren, B, Hawthorne, W, Byrne, M, Phillips, P, O'Brien, B, Nassif, N, Alexander, I & Simpson, AM 2013, 'Pancreatic transdifferentiation in porcine liver following lentiviral delivery of human furin-cleavable insulin', Transplantation Proceedings, vol. 45, no. 5, pp. 1869-1874.View/Download from: UTS OPUS or Publisher's site
Type I diabetes mellitus (TID) results from the autoimmune destruction of the insulin-producing pancreatic ß-cells. Gene therapy is one strategy being actively explored to cure TID by affording non-ß-cells the ability to secrete insulin in response to physiologic stimuli. In previous studies, we used a novel surgical technique to express furin-cleavable human insulin (INS-FUR) in the livers of streptozotocin (STZ)-diabetic Wistar rats and nonobese diabetic (NOD) mice with the use of the HMD lentiviral vector. Normoglycemia was observed for 500 and 150 days, respectively (experimental end points). Additionally, some endocrine transdifferentiation of the liver, with storage of insulin in granules, and expression of some ß-cell transcription factors (eg, Pdx1, Neurod1, Neurog3, Nkx2-2, Pax4) and pancreatic hormones in both studies. The aim of this study was to determine if this novel approach could induce liver to pancreatic transdifferentiation to reverse diabetes in pancreatectomized Westran pigs. Nine pigs were used in the study, however only one pig maintained normal fasting blood glucose levels for the period from 10 to 44 days (experimental end point). This animal was given 2.8 × 10(9) transducing units/kg of the lentiviral vector expressing INS-FUR. A normal intravenous glucose tolerance test was achieved at 30 days. Reverse-transcription polymerase chain reaction analysis of the liver tissue revealed expression of several ß-cell transcription factors, including the key factors, Pdx-1 and Neurod1, pancreatic hormones, glucagon, and somatostatin; however, endogenous pig insulin was not expressed.
Tohidi-Esfahani, D, Graham, L, Hannan, G, Simpson, AM & Hill, R 2011, 'An Ecdysone Receptor From The Pentatomomorphan, Nezara Viridula, Shows Similar Affinities For Moulting Hormones Makisterone A And 20-Hydroxyecdysone', Insect Biochemistry and Molecular Biology, vol. 41, no. 2, pp. 77-89.View/Download from: UTS OPUS or Publisher's site
It has been suggested that Pentatomomorpha utilise the C(28) ecdysteroid, makisterone A (MakA), as the major moulting hormone rather than the more common C(27) hormone, 20-hydroxyecdsyone (20E). The present study is the first to examine this postulate at
Tohidi-Esfahani, D, Lawrence, M, Graham, L, Hannan, G, Simpson, AM & Hill, R 2011, 'Isoforms Of The Heteropteran Nezara Viridula Ecdysone Receptor: Protein Characterisation, Rh5992 Insecticide Binding And Homology Modelling', Pest Management Science, vol. 67, no. 11, pp. 1457-1467.View/Download from: UTS OPUS or Publisher's site
Abstract: BACKGROUND: Certain bisacylhydrazine compounds such as tebufenozide (RH5992) have been shown to act as order-specific insecticides. Their compatibility with predatory Heteroptera, which are used as biological control agents, has also been demonstrated. However, the molecular mode of action of these ecdysone agonists has not been explored in a heteropteran, much less one that is a significant agricultural pest, such as Nezara viridula. RESULTS: Alternatively spliced ligand-binding regions of the N. viridula ecdysone receptor were expressed, purified and characterised by 2D gel analysis, mass spectrometry, homology modelling and competitive binding of a bisacylhydrazine insecticidal compound (RH5992) and various ecdysteroids. Ligand binding by the two splice isoforms was indistinguishable, and relative affinities were found to occur in the order muristerone A > ponasterone A > 20-hydroxyecdysone > inokosterone > RH5992 > alpha-ecdysone. CONCLUSION: The predicted difference in amino acid sequence between the ligand-binding domains of the N. viridula ecdysone receptor splice variants was verified by mass spectrometry. Both splice variant isoforms exhibit a greater affinity for the bisacylhydrazine insecticide RH5992 than do the other hemipteran ecdysone receptors characterised to date. Their affinities for a range of ecdysteroids also distinguish them from the ecdysone receptors of other Hemiptera characterised thus far. Homology models of both N. viridula receptor isoforms provide further insight into the bisacylhydrazine- and ecdysteroid-binding properties of these receptors, including their similar affinity for 20-hydroxyecdysone and the postulated pentatomomorphan moulting hormonemakisterone A.
Lutherborrow, M, Appavoo, M, Simpson, AM & Tuch, BE 2009, 'Gene expression profiling of HUH7-ins Lack of a granulogenic function for Chromogranin A', Islets, vol. 1, no. 1, pp. 60-72.View/Download from: UTS OPUS or Publisher's site
Previously, the insulin producing liver cell line HUH7-ins has been shown to synthesize, store and secrete insulin in response to glucose via secretory granules. The current study characterized the gene expression profile of HUH7-ins with the aim to identify changes possibly involved in the formation of granules. Additionally, experiments were conducted to determine the influence of chromograninA (egA) on secretory granule biogenesis (5GB) in HUH7-ins. Expression of 165 genes were significantly changed in HUH7-ins, though interestingly the majority of secretory granule associated genes, such as the chromogranins were unchanged. egA was over-expressed in glucose unresponsive HUH7-ins cells to test whether egA played a role in 5GB and would restore the regulated secretory phenotype. Over expression affected neither the storage nor regulated secretion of insulin. These data suggest that 5GB may by regulated at a post-transcriptional level with no evidence to indicate that egA regulates SGB in the cell line HUH7-ins.
Lutherborrow, MA, Appavoo, M, Simpson, AM & Tuch, BE 2009, 'Gene expression profiling of HUH7-ins: lack of a granulogenic function for chromogranin A.', Islets, vol. 1, no. 1, pp. 62-74.View/Download from: Publisher's site
Previously, the insulin producing liver cell line HUH7-ins has been shown to synthesize, store and secrete insulin in response to glucose via secretory granules. The current study characterized the gene expression profile of HUH7-ins with the aim to identify changes possibly involved in the formation of granules. Additionally, experiments were conducted to determine the influence of chromogranin A (CgA) on secretory granule biogenesis (SGB) in HUH7-ins. Expression of 165 genes were significantly changed in HUH7-ins,though interestingly the majority of secretory granule associated genes, such as the chromogranins were unchanged. CgA was over-expressed in glucose unresponsive HUH7-ins cells to test whether CgA played a role in SGB and would restore the regulated secretory phenotype. Over-expression affected neither the storage nor regulated secretion of insulin. These data suggest that SGB may by regulated at a post-transcriptional level with no evidence to indicate that CgA regulates SGB in the cell line HUH7-ins.
Feller, JM, Simpson, AM, Nelson, M, Swan, MA, O'Connell, PJ, Hawthorne, WJ, Tao, CZ & O'Brien, B 2008, 'Growth-promoting effect of Rh(D) antibody on human pancreatic islet cells', Journal of Clinical Endocrinology and Metabolism, vol. 93, no. 9, pp. 3560-3567.View/Download from: UTS OPUS or Publisher's site
Context/Objective: Hyperinsulinism with islet cell hyperplasia is a frequent complication, of unknown cause, in hemolytic disease of the newborn, occurring in Rh(D)-positive infants of Rh-isoimmunized Rh(D)-negative mothers, but not in infants with other hemolytic disorders. We investigated the possibility that trans-placentally acquired anti-D Ig is the cause of both conditions. Design: Monolayer cultures of human islet cells were exposed to sera from Rh-isoimmunized mothers and newborns, where jaundice, hyperinsulinism, and hypoglycemia in the infant had ensued. Parallel cultures with anti-D, specific anti-D monoclonal antibodies, normal human Ig (15 Î¼g/ml), and serum controls were also undertaken. Islet cell proliferation was determined by [ 3H]thymidine incorporation. Insulin storage and chronic and acute insulin secretion to glucose were analyzed by RIA. Rh(D) surface antigen expression was determined on islet cells by flow cytometric analysis. Results: Islet cell proliferation and insulin secretion were significantly greater in coculture with test sera (P < 0.01; n = 8) and with anti-D (P < 0.001; n = 8), compared with either controls or Ig. After 8 d of growth, the static incubation experiment showed a 3.5-fold response to glucose stimulus in all sera. Rh(D) antigen expression was detected on the islet cell surface by flow cytometry, and islet cell morphology was normal. Colocalization of the proliferation marker Ki67 with insulin by immunofluorescent staining further indicated that Rh(D) antibody promoted islet growth. Conclusions: The anti-Rh(D) islet cell proliferative effect generates neonatal hyperinsulinism in Rh isoimmunization. Anti-Rh(D) may have application for islet cell proliferation in diabetes mellitus treatment for Rh(D)-positive subjects. Further analysis is required. Copyright © 2008 by The Endocrine Society.
O'Brien, B, Archer, NS, Simpson, AM, Torpy, FR & Nassif, N 2008, 'Association of SLC11A1 promoter polymorphisms with the incidence of autoimmune and inflammatory diseases: A meta-analysis', Journal Of Autoimmunity, vol. 31, no. 1, pp. 42-51.View/Download from: UTS OPUS or Publisher's site
Solute carrier family 11 member a1 (SLC11A1) exerts pleiotropic effects on macrophage function. Expression of SLC11A1 is regulated by a (GT)(n) microsatellite promoter repeat polymorphism of which nine alleles have been described. Enhanced activation of
Simpson, AM 2008, 'Gene Therapy Reverse Diabetes', Australasian Science, vol. April, pp. 23-25.
Simpson, AM & O'Brien, B 2008, 'Diabetes therapy by lentiviral hepatic insulin gene expression without transofrmation of liver. Reply to Elsner M, Jorns A, Lenzen S (letter)', Diabetologia, vol. 51, pp. 696-696.
Lawandi, J, O'Brien, B & Simpson, AM 2007, 'An Insulin Secreting Liver Cell Line, Tao, Is Resistant To The Cytotoxic Effects Of Pro-inflammatory Cytokines Via Nf-kb-dependent Pathways', Journal Of Gene Medicine, vol. 9, no. 6, pp. 527-527.
Mccann, L, Haywood, M, Ren, B, Simpson, AM, Guilhaus, M, Wasinger, V, Raftery, MJ & Davey, RA 2007, 'Identification Of Vascular Surface Proteins By In Vivo Biotinylation: A Method Sufficiently Sensitive To Detect Changes In Rat Liver 2 Weeks After Partial Hepatectomy', Journal Of Proteome Research, vol. 6, no. 8, pp. 3108-3113.View/Download from: UTS OPUS or Publisher's site
We have developed a methodology to selectively isolate and identify proteins associated with the luminal surface of blood vessels using in vivo biotinylation, streptavidin-affinity chromatography, and SDS-PAGE/LC-MS/MS. This had sufficient sensitivity to
Ren, B, O'Brien, B, Swan, MA, Koina, ME, Nassif, N, Wei, MQ & Simpson, AM 2007, 'Long-term Correction Of Diabetes In Rats After Lentiviral Hepatic Insulin Gene Therapy', Diabetologia, vol. 50, no. 9, pp. 1910-1920.View/Download from: UTS OPUS or Publisher's site
Aims/hypothesis Type 1 diabetes results from the autoimmune destruction of pancreatic beta cells. Exogenous insulin therapy cannot achieve precise physiological control of blood glucose concentrations, and debilitating complications develop. Lentiviral v
Ren, B, O'Brien, B, Swan, MA & Simpson, AM 2005, 'In vivo delivery of the human insulin gene results in long-term reversal of streptozotocin-induced type 1 diabetes in rats', Journal Of Gene Medicine, vol. 7, no. 8, pp. 1124-1124.
Khoury, P, Tuch, BE & Simpson, AM 2004, 'Dynamics Of Insulin Release From Transplanted Genetically Engineered Liver Cells', Immunology And Cell Biology, vol. 82, no. 2, pp. 1-1.
Simpson, AM, Tao, CZ, Elgundi, Z, Castro, M & Swan, MA 2004, 'Glucose Responsive Insulin Secretion From The Engineered Human Liver Cell Line Huh7ins Is Regulated By Atp-sensitve Potassium Channels (k-atp)', Immunology And Cell Biology, vol. 82, no. 2, pp. 1-1.
Liu, GJ, Simpson, AM, Swan, MA, Tao, C, Tuch, BE, Crawford, RM, Jovanovic, A & Martin, DK 2003, 'ATP-sensitive potassium channels induced in liver cells after transfection with insulin cDNA and the GLUT 2 transporter regulate glucose-stimulated insulin secretion.', The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, vol. 17, no. 12, pp. 1682-1684.
As part of our research into the liver-directed gene therapy of Type I diabetes, we have engineered a human hepatoma cell line (HEPG2ins/g cells) to store and secrete insulin to a glucose stimulus. The aim of the present study was to determine whether HEPG2ins/g cells respond to glucose via signaling pathways that depend on ATP-sensitive potassium channels (KATP). Using patch-clamp electrophysiology with symmetrical KCl solutions, the single-channel conductance of KATP was 61pS. KATP was inhibited by ATP (1 mM) or cAMP (50 microM) applied to the cytosolic side of the membrane. Single KATP channels and macroscopic whole-cell currents were inhibited by glucose (20 mM) and glibenclamide (20 microM) and were activated by diazoxide (150 microM). Immunoprecipitation and Western blot analysis confirmed the presence of Kir6.2 KATP channel subunit protein in HEPG2ins/g and HEPG2ins cells. Using radioimmunoassay techniques, we report that exposure of the cells to tolbutamide (100 microM) resulted in an increase in insulin secretion from 0.3 +/- 0.05 to 1.8 +/- 0.2 pmol insulin/10(6) cells and glibenclamide (20 microM) from 0.4 +/- 0.06 to 2.1 +/- 0.3 (n=4), similar to what is seen on glucose (20 mM) stimulation. Diazoxide (150 microM) completely inhibited glucose-stimulated insulin release. Glucose 20 mM and glibenclamide 100 microM increased intracellular Ca2+ level in the HEPG2ins/g cells. However, glucose 20 mM did not stimulate a rise in intracellular Ca2+ in the un-transfected parent cell-line HEPG2. We used confocal microscopy to confirm that glucose (20 mM) stimulated the release of insulin from the fluorescently labeled secretion granules in the cells. Furthermore, glibenclamide (20 microM) also stimulated the release of insulin from fluorescently labeled secretion granules, and diazoxide (150 microM) blocked that stimulated release of insulin. Our results suggest that HEPG2ins/g cells respond to glucose via signaling pathways that depend on KATP, similar to a norm...
Liu, GJ, Simpson, AM, Swan, MA, Tao, CZ, Tuch, BE, Crawford, RM, Jovanovic, A & Martin, DK 2003, 'ATP-sensitive potassium channels induced in liver cells after transfection with insulin cDNA and the GLUT2 transporter regulate glucose-stimulated insulin secretion', Faseb Journal, vol. 17, no. 10, pp. 1-21.View/Download from: UTS OPUS or Publisher's site
Lutherborrow, M, Simpson, AM & Tuch, BE 2003, 'Microarray Analysis Of A Beta Cell Surrogate: The Insulin Producing Liver Cell Line Hep G2ins/g', Journal Of Gene Medicine, vol. 5, no. 5, pp. 1-2.
Simpson, AM, Elgundi, Z, Tao, CZ, Swan, MA & Winch, D 2003, 'Insulin Trafficking In A Glucose-responsive Human Liver Cell Line-huh7-egfpins', Journal Of Gene Medicine, vol. 5, no. 5, pp. 1-1.
Tuch, BE, Szymanska, B, Yao, M, Tabiin, MT, Gross, DJ, Holman, S, Swan, MA, Humphrey, R, Marshall, GM & Simpson, AM 2003, 'Function of a genetically modified human liver cell line that stores, processes and secretes insulin', Gene Therapy, vol. 10, no. 6, pp. 490-503.View/Download from: UTS OPUS or Publisher's site
Bai, L, Tuch, BE, Hering, B & Simpson, AM 2002, 'Fetal pig β cells are resistant to the toxic effects of human cytokines', Transplantation, vol. 73, no. 5, pp. 714-722.View/Download from: Publisher's site
Background. The cytokine tumour necrosis factor-α (TNF-α) is thought to be responsible for primary non-function of islets when transplanted. This, and two other cytokines, interleukin-1β (IL-1β) and interferon -γ (IFN-γ) are also implicated in the autoimmune destruction of β cells. It is unknown if the fetal pig β cell, which is being transplanted as a treatment for type 1 diabetes, is affected by these cytokines. Methods. We compared the effects of the cytokines on the function and viability of adult and fetal pig β cells. The cells were cultured for up to 3 days in the presence of 2000 pg/ml of human IL-1β, 1000 U/ml of TNF-α, and 1000 U/ml of IFN-γ, as well as 1000 U/ml of porcine IFN-γ Cumulative insulin levels, insulin content, metabolic activity, and viability of these cells were examined. Additionally, nitric oxide production and the activity of antioxidant enzymes in these cells were also determined. Results. TNF-α and the combination of the three human cytokines caused a transient increase in cumulative insulin levels. TNF-α alone enhanced insulin content on day 3. There was no effect of these human cytokines on mitochondrial function and viability. In contrast, porcine IFN-γ inhibited fetal pig β cell function and also caused their death. Adult pig islets are sensitive to the toxic effects of human TNF-α, IL-1β, the combination of the three cytokines, and porcine IFN-γ. The activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase were significantly higher in fetal pig β cells than in adult islets, implying that this may be the reason for the lack of adverse effects of the cytokines on the fetal β cell. Conclusion. Fetal pig β cells are resistant to the toxic effect of the human cytokines, TNF-α and IL-1β, in vitro. This resistance suggests that fetal, but not adult β cells, when transplanted into humans with type 1 diabetes may be protected from primary nonfunction and will be partially protected from autoimmune des...
Bai, L, Tuch, BE, Hering, BJ & Simpson, AM 2002, 'Fetal pig beta cells are resistant to the toxic effects of human cytokines', Transplantation, vol. 73, no. N/A, pp. 714-722.View/Download from: UTS OPUS or Publisher's site
Humphrey, R, Smith, MS, Kwok, J, Tuch, BE, Si, Z & Simpson, AM 2001, 'In Vitro Dedifferentiation of Fetal Porcine Pancreatic Tissue Prior to transplantation as islet-Like Cell Clusters', Cells Tissues Organs, vol. 168, no. 3, pp. 158-169.View/Download from: UTS OPUS
Liu, GJ, Simpson, AM & Martin, DK 2001, 'Glucose Activates K-atp Signaling Pathways In The Insulin Secreting Liver Cell Lin Hepg2ins/g', Molecular Biology Of The Cell, vol. 12, no. 0, pp. 1-1.
Tabiin, MT, Tuch, BE, Bai, L, Han, X & Simpson, AM 2001, 'Susceptibility of Insulin-Secreting Hepatocytes to the Toxicity of Pro-Inflammatory Cytokines', Journal of Autoimmunity, vol. 17, no. 3, pp. 229-242.View/Download from: UTS OPUS or Publisher's site
The liver has been suggested as a suitable target organ for reversing type I diabetes by gene therapy. Whilst gene delivery systems to the hepatocyte have yet to be optimized in vivo, whether insulin-secreting hepatocytes are resistant to the autoimmune process that kills pancreatic -cells has never been addressed. One of the mechanisms by which -cells are killed in type I diabetes is by the release of the cytokines interleukin-1 (IL-1), tumour necrosis factor- (TNF-) and interferon- (IFN-) by immune cells. To test the effect of the cytokines on insulin-secreting hepatocytes in vitro we exposed the betacyte, also called the HEP G2ins/g cell which possesses cytokine receptors and can synthesize, store and secrete insulin in a regulated fashion to a glucose stimulus, to the above mentioned cytokines for 14 days. Viability of the HEP G2ins/g cells was similar to that of other liver cell lines/primary cells which were more resistant to the cytokines than the -cell line NIT-1. The cytokines had no adverse effect for the first six days on insulin secretion, content and mRNA levels of the HEP G2ins/g cells and insulin secretion in response to 1-h exposure to 20 mM glucose was enhanced 14-fold. Our results indicate that genetically engineered hepatocytes and primary liver cells are more resistant than pancreatic -cells to the adverse effects of cytokines offering hope that insulin secreting hepatocytes in vivo made by gene therapy are less likely to be destroyed by cytokines released during autoimmune destruction.
Vo, L, Tuch, BE, Wright, DC, Keogh, GW, Simpson, AM, Roberts, S, Yao, M, Tabiin, MT, Valencia, SK & Scott, H 2001, 'Lowering of Blood Glucose to Nondiabetic Levels in a Hyperglycemic Pig By Allografting of Fetal Pig Isletlike Cell Clusters', Transplantation, vol. 71, no. 11, pp. 1671-1677.View/Download from: UTS OPUS or Publisher's site
Tasevski, V, Benn, D, King, M, Luttrell, B & Simpson, AM 2000, 'Mitogenic Effect of Lithium in FRTL-5 Cells can be Reversed by Blocking De Novo Cholesterol Synthesis and Subsequent Signal Transduction', Thyroid, vol. 10, no. 4, pp. 305-311.View/Download from: Publisher's site
Lithium therapy is the therapeutic mainstay for bipolar disorder and has been associated in the thyroid with euthymic goiter, hyper and hypothyroidism as well as thyroid autoimmune disease. The FRTL-5 cell line is a well known model of thyroid cell physiology, where lithium has been shown to increase 3H-thymidine uptake at concentrations of 2 mM. This mitogenic effect was not associated with adenylate cyclase as measured by cyclic adenosine monophosphate (cAMP) production. The de novo synthesis of cholesterol is an important signal transduction pathway in FRTL-5 cells, where newly synthesized Rho GTPase is geranylgeranylated, enabling membrane localization of the G-protein and subsequent G1 to S-phase transition, resulting from extracellular stimulation. Here we confirm lithium mitogenicity at therapeutically relevant concentrations (1 mM) and demonstrate a lithium-associated accumulation of FRTL-5 cells in S-phase of the cell cycle. These effects could be abolished by Pravastatin, a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), the rate-limiting enzyme in the formation of intermediates (de novo cholesterol synthesis) required for G-protein prenylation. Pravastatin, similar to lithium, showed no effect on cAMP production either under basal or thyroid stimulating hormone (TSH)-stimulated conditions indicating that de novo cholesterol synthesis is not involved with adenylate cyclase. The inhibitory effect of pravastatin could be overcome by reinitiating de novo cholesterol synthesis. This was achieved by the addition of the cell permeable, first metabolite (mevalonate) after HMG-CoA, which allowed the cycle to continue, leading eventually to protein prenylation, despite the presence of Pravastatin. These novel findings demonstrate lithium involvement in de novo cholesterol synthesis and G-protein prenylation, an important signal transduction pathway in FRTL-5 cells.
Tuch, BE, Yao, M, Tabiin, MT, Simpson, AM, Gross, DJ, Holman, S, Humphrey, R & Szymanska, B 2000, 'Reversal Of Diabetes By Transplantation Of Insulin-producing Human Liver Cells', Diabetologia, vol. 43, no. 0, pp. 1-1.
Simpson, AM, Szymanska, B, Tuch, BE & Marshall, GM 1999, 'Secretion And Storage Of Insulin From A Human Hepatoma Cell Line (huh7-ins)', Transplantation Proceedings, vol. 31, no. 1-2, pp. 812-812.
Tuch, BE, Wright, DJ, Martin, T, Keogh, GW, Deol, H, Simpson, AM, Roach, W & Pinto, A 1999, 'Differentiation Of Fetal Pig Endocrine Cells After Allografting Into The Thymus Gland', Transplantation, vol. 67, no. 8, pp. 1184-1187.View/Download from: Publisher's site
Background. The thymus of large animals, such as the pig, is thought to be an appropriate site for transplanting adult islets, which contain numerous beta cells, for the purpose of reversing diabetes, Whether fetal islet-like cell clusters (ICCs), which
Tuch, BE, Wright, DJ, Martin, T, Keogh, GW, Deol, H, Simpson, AM, Roach, W & Pinto, A 1999, 'Fetal Pig Endocrine Cells Develop When Allografted Into The Thymus Gland', Transplantation Proceedings, vol. 31, no. 1-2, pp. 670-670.
Tasevski, V, Benn, D, Peters, G, Luttrell, B & Simpson, AM 1998, 'The Fischer Rat Thyroid Cell Line Frtl-5 Exhibits A Nondiploid Karyotype', Thyroid, vol. 8, no. 7, pp. 623-626.View/Download from: Publisher's site
The FRTL-5 cell line is a stable thyroid cell line derived from the thyroid gland of the Fischer rat under defined culture conditions, which has been widely adopted as a model system for the study of thyroid cell function and for bioassay. While characte
Tuch, BE, Tabiin, MT, Casamento, F, Simpson, AM & Marshall, GM 1998, 'Transplantation Of Genetically Engineered Insulin-producing Hepatocytes Into Immunoincompetent Mice', Transplantation Proceedings, vol. 30, no. 2, pp. 473-473.
Simpson, AM, Andrews, S, Hill, R, Hannan, G & Tuch, BE 1997, 'Inducible Insulin Expression In A Human Hepatoma Cell Line', Diabetologia, vol. 40, no. 0, pp. 1501-1501.
Simpson, AM, Marshall, GM, Tuch, BE, Maxwell, L, Szymanska, B, Tu, J, Beynon, S, Swan, MA & Camacho, M 1997, 'Gene Therapy Of Diabetes: Glucose-stimulated Insulin Secretion In A Human Hepatoma Cell Line (hep G2inslg)', Gene Therapy, vol. 4, no. 11, pp. 1202-1215.View/Download from: Publisher's site
In order to design a feasible somatic cell gene delivery system for the treatment of type I diabetes, a suitable cell type needs to be determined. We have previously shown that the stable transfection of the full-length insulin cDNA into the human liver
Tuch, BE, Beynon, S, Tabiin, MT, Sassoon, R, Goodman, RJ & Simpson, AM 1997, 'Effect Of Beta-cell Toxins On Genetically Engineered Insulin-secreting Cells', Journal Of Autoimmunity, vol. 10, no. 3, pp. 239-244.View/Download from: Publisher's site
The betacyte is a genetically engineered insulin-secreting liver cell line that is glucose responsive. Whether this cell. is affected by specific beta-cell toxins is unknown. To explore this possibility we exposed these cells and those from the NIT-1 bet
Marshall, GM, Cheung, B, Stacey, K, Camacho, M, Simpson, AM, Kwan, E, Smith, ST, Haber, M & Morris, M 1995, 'Increased Retinoic Acid Receptor-gamma Expression Suppresses The Malignant Phenotype And Alters The Differentiation Potential Of Human Neuroblastoma-cells', Oncogene, vol. 11, no. 3, pp. 485-491.
Human neuroblastoma (NB) tumor cell lines treated in vitro with the retinoid, all-trans-retinoic acid (aRA), form neurites and undergo growth arrest, Retinoids exert their diverse morphologic effects through a signalling pathway which involves the nuclea
Simpson, AM, Tuch, BE, Swan, MA, Tu, J & Marshall, GM 1995, 'Erratum: Functional expression of the human insulin gene in a human hepatoma cell line (HEP G2) (Gene Therapy 1995; 2: 223-231)', Gene Therapy, vol. 2, no. 10, p. 789.
Simpson, AM, Tuch, BE, Swan, MA, Tu, J & Marshall, GM 1995, 'Functional expression of the human insulin gene in a human hepatoma cell line (HEP G2)', Gene Therapy, vol. 2, no. 3, pp. 223-231.
Simpson, AM, Iland, H, Clarke, R & Tuch, BE 1993, 'Transformation of pituitary and fibroblast cell lines using human insulin c-DNA and a dexamethasone-inducible promoter', Transplantation Proceedings, vol. 25, pp. 2915-2916.
Simpson, AM, Tuch, BE & Vincent, PC 1991, 'Characterization of endocrine-rich monolayers of human fetal pancreas that display reduced immunogenicity', Diabetes, vol. 40, pp. 800-809.View/Download from: Publisher's site
Tuch, BE, Simpson, AM & Campbell, IA 1991, 'Role of tumor necrosis factor-alpha and interferon-gamma as growth factors to the human fetal beta-cell', Journal of Clinical Endocrinology and Metabolism, vol. 73, pp. 1044-1050.View/Download from: Publisher's site
Simpson, AM, Tuch, BE & Vincent, PC 1990, 'Monolayers of human and porcine fetal pancreas display reduced immunogenicity', Transplantation Proceedings, vol. 22, pp. 2169-2170.
Simpson, AM & White, IG 1988, 'Measurement and manipulation of cytoplasmic free calcium of ram and boar spermatozoa using quin 2', Cell calcium, vol. 9, no. 1, pp. 45-56.View/Download from: Publisher's site
The highly selective fluorescent Ca2+ indicator `quin 2 has been loaded into ram and boar spermatozoa as the acetoxymethyl ester, `quin 2/AM, which is hydrolysed and trapped in the cytoplasm. Loadings of several mM were not toxic to spermatozoa as judged by motility. Fluorescence measurements (mean + S.E.M.) indicated a normal cytoptasmic free-calcium concentration, [Ca2+]i, of 193nM f 0.2 (n= 10) for ejaculated ram sperm, 175nM 5 3.9 (n= 10) for cauda epididymal boar sperm and 105nM `_ 10 (n= 10) for the caput sperm. After cold shock ejaculated mm and cauda epididymal boar sperm did not retain quin 2, due presumably to structural damage. However, cold shocked caput boar sperm could be readily loaded with quin 2 and had a [Ca2+]i similar to control sperm.
Simpson, AM & White, IG 1987, 'Interrelationships between motility, c-AMP, respiration and calcium uptake of ram and boar sperm', Animal Reproduction Science, vol. 15, no. 3-4, pp. 189-207.View/Download from: UTS OPUS or Publisher's site
Uptake of radioactive calcium by washed ejaculated ram and epididymal boar spermatozoa was inhibited by theophylline and dibutyryl cyclic AMP (diBc-AMP) which increases cyclic AMP (c-AMP) levels in cells, by the uncouplers 2,4-dinitrophenol (2,4-DNP) and carbonylcyanide m-chlorophenylhydrazone (CCP), by sodium azide, rotenone and antimycin A and by ruthenium red and La3+. Nicotine, eserine and ouabain stimulated the calcium uptake of caput boar sperm and decamethonium inhibited it. Inhibition of calcium uptake was accompanied by a decrease in oxygen uptake in the case of the metabolic inhibitors (rotenone, antimycin A and sodium azide) and substances that interfere with cell calcium transport (ruthenium red and La3+). Respiration was increased in the presence of 2,4-DNP and CCP, and theophylline and diBc-AMP. The phosphodiesterase inhibitors (caffeine and theophylline), diBc-AMP, 2,4-DNP, bicarbonate ion and ouabain increased the motility of caput boar sperm. Most other substances depressed motility. The ionophore A23187 severely inhibited the motility of ram and boar sperm with an accompanying increase in their calcium and oxygen uptake which was largely unaffected by addition of inhibitors, activators and surfactants. This may be explained by the operation of competing mitochondrial and plasma membrane pumps. However, a selective increase in the permeability of the sperm plasma membrane on addition of the polyene antibiotic filipin released calcium accumulated in the presence of A23187. This provides further evidence for the lack of mitochondrial involvement in the influx of calcium produced by the ionophore.
Simpson, AM, Swan, MA & White, IG 1987, 'Calcium uptake, respiration, and ultrastructure of sperm exposed to ionophore A23187', Archives of Andrology, vol. 19, no. 1, pp. 5-18.
Calcium accumulation by ram sperm in the presence of 3.0 μM ionophore A23187 increased greatly when the pH of the medium was raised from 7.5 to 8.5. The increase in calcium uptake was remarkable between pH 7.5 and 8.0 and was paralleled by increased oxygen consumption. There was extensive vesiculation of the plasma membrane and membranes of the acrosome and postnuclear cap of ram sperm incubated with the ionophore and calcium at pH 8.0. The mitochondria of the midpiece contained pale and expanded cristae similar to ATP-deprived mitochondria in the 'condensed' configuration. Such changes were not observed in ram sperm incubated under similar conditions at pH 7.0. The ionophore-induced calcium and oxygen uptake of cauda and caput epididymal boar sperm also increased with increased pH, but the effect commenced at a lower pH. Controls cauda epididymal boar sperm (i.e. without ionophore) had higher oxygen uptake, glucose breakdown, and lactic acid production than caput sperm. The influence of pH on the operation of the membrane calcium pumps between ram boar sperm indicates species differences.
Simpson, AM, Swan, MA & White, IG 1987, 'Susceptibility of epididymal boar sperm to cold shock and protective action of phosphatidylcholine', Gamete Research, vol. 17, pp. 355-373.View/Download from: Publisher's site
Simpson, AM & White, IG 1986, 'Effect of cold shock and cooling rate on calcium uptake of ram spermatozoa', Animal Reproduction Science, vol. 12, no. 2, pp. 131-143.View/Download from: UTS OPUS or Publisher's site
Rapid cooling (cold shock) of washed ejaculated ram sperm irreversibly reduced motility and respiration and greatly increased uptake of 4sCa2+. The effect was greater as the temperature of cooling was reduced from 15°C to 0°C, and a substantial increase in sperm calcium levels was even observed after slow cooling to temperatures below 10°C. The rise in calcium uptake on freezing sperm to -79°C was not as great as that on cold shocking sperm to 0°C. Inactivation of sperm by mild heat (50°C) had no significant effect on calcium uptake but subsequent cold shock increased the sperm calcium. Reverse immobilization of sperm by low concentrations of formaldehyde significantly reduced calcium uptake on cold shock. Addition of detergents to sperm immediately reduced motility, respiration and calcium uptake of control and cold-shocked sperm to zero.
Gerace, D, Martiniello-Wilks, R & Simpson, AM 2016, 'Viral-Mediated Gene Therapy for the Generation of Artificial Insulin-Producing Cells as a Therapeutic Treatment for Type 1 Diabetes Mellitus' in Hardikar, AA (ed), Pancreatic Islet Biology, Springer, Germany, pp. 241-257.View/Download from: UTS OPUS or Publisher's site
Over the past decade, several approaches have been employed to develop cell and gene therapy strategies that generate artificial insulin-producing cells (IPCs) for potential therapeutic applications in the treatment of type 1 diabetes mellitus (T1D) . The genetic engineering of functional IPCs necessitates a broad understanding of the pancreatic developmental process and the β cell transcription factors that govern mature β cell differentiation and function. To successfully obtain functional IPCs, the type of vectors utilised for gene transfer and the selection of a suitable target cell for subsequent differentiation into IPCs is of fundamental importance. Techniques for manufacturing IPCs include the dedifferentiation and directed transdifferentiation of autologous or allogeneic cells ex vivo followed by transplantation and the in vivo transdifferentiation of target tissue via viral gene transfer. Ultimately, the goal is to construct IPCs that have the capacity to process, store and secrete insulin in response to fluctuating blood glucose levels, whilst avoiding the administration of immunosuppressants and recurrent autoimmune destruction, thereby indefinitely restoring normoglycaemia.
Simpson, AM, Swan, MA, Liu, GJ, Tao, CZ, O'Brien, B, Ch'ng, E, Castro, LM, Ting, HJ, Elgundi, Z, An, T, Lutherborrow, M, Torpy, FR, Martin, DK, Tuch, BE & Nicholson, GM 2013, 'Insulin trafficking in a glucose responsive engineered human liver cell line is regulated by the interaction of ATP-sensitive potassium channels and voltage- gated calcium channels' in Molina, FM (ed), Gene Therapy - Tools and Potential Applications, In-Tech, Rijeka, Croatia, pp. 703-726.View/Download from: UTS OPUS or Publisher's site
Type I diabetes is caused by the autoimmune destruction of pancreatic beta (â) cells . Current treatment requires multiple daily injections of insulin to control blood glucose levels. Tight glucose control lowers, but does not eliminate, the onset of diabetic complications, which greatly reduce the quality and longevity of life for patients. Transplantation of pancreatic tissue as a treatment is restricted by the scarcity of donors and the requirement for lifelong immunosuppression to preserve the graft, which carries adverse side-effects. This is of particular concern as Type 1 diabetes predominantly affects children. Lack of glucose control could be overcome by genetically engineering "an artificial â-cell" that is capable of synthesising, storing and secreting insulin in response to metabolic signals. The donor cell type must be readily accessible and capable of being engineered to synthesise, process, store and secrete insulin under physiological conditions.
Suurbach, JH, Tan, M, Simpson, AM, Kench, J, Gupta, R & Martiniello-Wilks, R 2017, 'PRECLINICAL EVALUATION OF A BMSC DELIVERED GENE-DIRECTED ENZYME PRO-DRUG THERAPY (GDEPT) LOCAL BYSTANDER EFFECT IN IMMUNE INTACT MOUSE MODELS OF PRIMARY AND METASTATIC PROSTATE CANCER', JOURNAL OF GENE MEDICINE, Joint 10th Annual Scientific Meeting of the Australian-Gene-and-Cell-Therapy-Society (AGCTS) and Australasian-Society-for-Stem-Cell-Research (ASSCR), WILEY, Univ Technol Sydney, Sydney, AUSTRALIA.View/Download from: UTS OPUS
Ren, B, La, QT, O'Brien, BA, Nassif, NT, Tan, Y, Gerace, D, Martiniello-Wilks, R, Torpy, F, Dane, AP, Alexander, IE & Simpson, AM 2017, 'EXPRESSION OF HUMAN PANCREATIC TRANSCRIPTION FACTORS IN THE LIVERS OF FRG MICE', JOURNAL OF GENE MEDICINE, Joint 10th Annual Scientific Meeting of the Australian-Gene-and-Cell-Therapy-Society (AGCTS) and Australasian-Society-for-Stem-Cell-Research (ASSCR), WILEY, Univ Technol Sydney, Sydney, AUSTRALIA.View/Download from: UTS OPUS
Gerace, D, Martiniello-Wilks, R, Nassif, NT, Ren, B & Simpson, AM 2017, 'Ex Vivo Expanded Murine Mesenchymal Stem Cells as Targets for the Generation of a Cell Replacement Therapy for Type 1 Diabetes', DIABETES, 77th Scientific Sessions of the American-Diabetes-Association, AMER DIABETES ASSOC, San Diego, CA, pp. A83-A83.View/Download from: UTS OPUS
Lawandi, J, Tao, C, Ren, B, Williams, P, Ling, D, Swan, MA, Nassif, NT, Torpy, FR, O'Brien, BA & Simpson, AM 2015, 'MELLIGEN CELSS: AN INSULIN-SECRETING HUMAN LIVER CELL LINE WHICH IS RESISTANT TO CYTOKINE-INDUCED IMMUNE ATTACK', JOURNAL OF GENE MEDICINE, 9th Biennial Meeting of the Australasian-Gene-and-Cell-Therapy-Society (AGCTS), WILEY-BLACKWELL, Univ Melbourne, Univ Coll, Parkville, AUSTRALIA, pp. 189-189.View/Download from: UTS OPUS
Simpson, A, Ren, B, O'Brien, BA, Alexander, IE, Nassif, NT, Tan, Y & Martiniello-Wilks, R 2015, 'GENE THERAPY FOR DIABETES: REVERSAL OF DIABETES IN THE HUMANISED FRG MOUSE MODEL.', TRANSPLANTATION, Joint Congress of the International-Pancreas-and-Islet-Transplantation-Association, International-Xenotransplantation-Association and Cell-Transplant-Society, LIPPINCOTT WILLIAMS & WILKINS, Melbourne, AUSTRALIA, pp. S67-S67.View/Download from: UTS OPUS
Gerace, D, Martiniello-Wilks, R & Simpson, AM 2015, 'BIOLUMINESCENT IMAGING OF MESENCHYMAL STEM CELL ENGRAFTMENT IN IMMUNE COMPETENT AND IMMUNE DEFICIENT ANIMAL MODELS OF TYPE 1 DIABETES', JOURNAL OF GENE MEDICINE, 9th Biennial Meeting of the Australasian-Gene-and-Cell-Therapy-Society (AGCTS), WILEY-BLACKWELL, Univ Melbourne, Univ Coll, Parkville, AUSTRALIA, pp. 201-201.
Gerace, D, Martiniello-Wilks, R & Simpson, AM 2015, 'Persistence of Luciferase Expressing Bone Marrow-Derived Mesenchymal Stem Cells (BMSCs) in Non-Obese Diabetic (NOD) and NOD/Scid Mice', MOLECULAR THERAPY, 18th Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT), NATURE PUBLISHING GROUP, New Orleans, LA, pp. S101-S101.View/Download from: Publisher's site
Ren, B, O'Brien, BA, Alexander, IE, Nassif, NT, Tan, Y, Martiniello-Wilks, R & Simpson, AM 2015, 'Pancreatic Transdiffereniation of Human Hepatocytes in the Livers of a Humanized Mouse Model', MOLECULAR THERAPY, 18th Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT), NATURE PUBLISHING GROUP, New Orleans, LA, pp. S110-S110.View/Download from: UTS OPUS or Publisher's site
Simpson, A, Ren, B, O'Brien, BA, Alexander, IE, Nassif, NT, Tan, Y & Martiniello-Wilks, R 2015, 'Gene therapy for diabetes: reversal of diabetes in the humanised FRG mouse model', XENOTRANSPLANTATION, IPITA/IXA/CTS Joint Congress, WILEY-BLACKWELL, Melbourne, AUSTRALIA, pp. S41-S42.View/Download from: UTS OPUS
Nield, BS, Martiniello-Wilks, R, Guzowski, R, Simpson, A & Nassif, N 2013, 'First use of Re:view – a tool to combine assessment tasks, marking criteria and graduate attributes', Australian Conference on Science and Mathematics Education, Canberra, ACT.View/Download from: UTS OPUS
Gerace, D, Ren, B, Hawthorne, WJ, Byrne, MR, Phillips, P, O'Brien, BA, Nassif, N, Alexander, IE & Simpson, AM 2013, 'REVERSAL OF DIABETES IN A PORCINE MODEL FOLLOWING LIVER-DIRECTED GENE THERAPY', JOURNAL OF GENE MEDICINE, 8th Meeting of the Australasian-Gene-Therapy-Society, WILEY-BLACKWELL, Univ Technol, Sydney, AUSTRALIA, pp. 326-326.View/Download from: UTS OPUS
Simpson, AM, Tao, C, Lawandi, J, Swan, MA & O'Brien, BA 2008, 'Physiological insulin secretion from a novel human insulin-secreting liver cell line: Melligen cells', DIABETES, 67th Annual Meeting of the American-Diabetes-Association, AMER DIABETES ASSOC, San Francicso, CA, pp. A88-A88.
Biady, J, O'Brien, B & Simpson, AM 2007, 'An insulin secreting liver cell line, TAO, is resistant to the cytotoxic effects of pro-inflammatory cytokines via NF-KB-dependent pathways', Journal of Gene Medicine, Wiley, Canberra, pp. 527-527.
Biady, J, Tao, CZ, O'Brien, B & Simpson, AM 2005, 'Susceptibility of an insulin-secreting liver cell line to the toxic effects of cytokines involved in the autoimmune destruction of pancreatic beta cells', Journal of Medicine, Wiley, Melbourne, pp. 1124-1124.
Ch'ng, E, Tao, CZ, Martin, DK, Lutherborrow, M, Tuch, BE & Simpson, AM 2005, 'Glucose-stimulated insulin secretion from an engineered human liver cell line is regulated by the l-type calcium channel', Journal Of Gene Medicine, 4th Meeting of the Australasian-Gene-Therapy-Society, Melbourne, Australia, pp. 1131-1131.
Lawandi, J, Tao, CZ, O'Brien, B & Simpson, AM 2005, 'Susceptibility of an insulin-secreting liver cell line to the toxic effects of cytokines involved in the autoimmune destruction of pancreatic beta cells', Journal Of Gene Medicine, 4th Meeting of the Australasian-Gene-Therapy-Society, Unknown, Melbourne, Australia, pp. 1124-1124.
Tuch, BE, Tabiin, MT, Simpson, AM & Marshall, GM 1997, 'Reversal of diabetes with genetically engineered insulin producing hepatocytes', Experimental and Clinical Endocrinology and Diabetes.
Tuch, BE, De Silva, C, Keogh, GW, Simpson, AM & Smith, MSR 1995, 'Survival of allografted fetal pig pancreatic islet-like cell clusters [ICCS]', Transplantation Proceedings, p. 3375.
Tuch, BE, Beretov, J, Mackie, JD, Beynon, S, Simpson, AM & Rolph, M 1994, 'Preventing the rejection of grafted human fetal pancreas', Transplantation Proceedings, p. 704.
Vine, M, Lees, T, Nassif, N, Simpson, A & Lal, S 2017, 'Investigating the associations between ADHD symptomology and chronic illness: cardiovascular disease and diabetes mellitus'.View/Download from: UTS OPUS