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Full Text, Thesis (Masters), Master of Science (MSc), School of Environment and Sc, Science, Environment, Engineering and Technology, Ras-GTPase activating protein SH3 domain binding proteins (G3BP) are a small family of RNA-binding proteins that have been implicated in regulating gene expression when a cell undergoes transformation or in response to external stimuli. Changes in the cellular expression of G3BPs are correlated to several cancers including head and neck, thyroid, colon, pancreatic and breast cancer. G3BPs are components of stress granules (SGs) and can regulate their formation. SGs are specialised ribonucleoprotein (RNP) particles that respond to cellular stresses, including chemotherapy, to triage mRNA with selected transcripts being degraded, stored or translated. The net result to the cell is a change in the gene expression which confers a survival response to the cell. This can include the upregulation of genes needed to survive external stresses and this can include the development of drug resistance in cells. Many viruses, including Chikungunya, target SG and dismantle the granules so that the cell cannot respond to the viral infection. Non-structural protein3 (nsP3), from the Chikungunya virus, has been shown to translocate G3BP from SG, and the inability of the cell to form SG is required for the virus to complete an infectious cycle. Interestingly, the formation of SG is correlated to drug-resistance and blocking their formation has been shown to reestablish the efficacy of the chemotherapeutic, bortezomib. The present study explored the disruption of SG formation, caused by the interaction of nsP3 with G3BP, and as a result restored cytotoxicity of bortezomib and improved the selectivity of the bortezomib for cancer cell lines when compared to HEK cells. In addition, the interaction of nsP3 with G3BP also disrupted the activity of G3BP and led to the significantly downregulation of G3BP target genes NDUFV1 and IFITM3. These findings represent a tool for future study into strategies to block G3BP activities and disrupt SG formation in anti-cancer therapeutics.

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Natural Sciences, Science, Environment, Engineering and Technology, This thesis examined exploitation of cell death to combat cancer from two angles: investigating cell death signalling pathways to find new targets for treatment, and using the novel anti-austerity approach to combat the tolerance of cancer cells to nutrient deprivation. Cancers often display high levels of genetic diversity, even within cancers of a particular organ. These genetic differences often make treatments which work with a particular cancer ineffective on another - even from the same origin, and lead to differences in outcomes to selective pressures such as nutrient deprivation. Current treatments are aimed at killing rapidly proliferating cells and often have severe side effects. Their efficacy is highly dependent on early diagnosis and many cancers are resistant to chemotherapeutic drugs. In addition, cancer cells on the inside of tumours have the ability to survive under adverse conditions such as nutrient deprivation due to intermittent blood supply, Therefore, alternative treatment strategies must be explored. Defects in cell death play a large contributing factor to cancer progression and tumour growth and the evasion of cell death by cancers presents a major problem in its treatment Conventional cancer treatment relies on the activation of cell death in fast growing cancer cells while attempting to leave normal cells undamaged. Therefore, selective triggering of cell death in a cancer cell would be extremely useful. In order to achieve this goal a thorough understanding of the cell death response of a particular cell type to a particular stimulus must be known. Moreover, any novel or unusual cell death signalling pathways would provide more opportunities and targets for attempting to trigger selective cell death in cancers.

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Biomolecular and Physical Sciences, Science, Environment, Engineering and Technology, Colorectal cancer (CRC) progresses from precursor lesions or polyps via two main pathways – the traditional and serrated pathway. The traditional pathway is the conventional type, while the serrated neoplastic pathway has been recently identified and is not well characterised. The aim of this study was to evaluate molecular factors associated with the serrated pathway and study its progression from a serrated polyp to colorectal cancer using patient samples and mouse models. The first part of the study was to evaluate CIMP status of all the serrated polyp subtypes and its association with functionally important genes such as MLH1, p16 and IGFBP7 in patient samples. The mouse models were then utilized to assess the contribution of BRAF V600E mutation along with the cell cycle regulators and tumour suppressor genes, p16Ink4a and p19Arf in the initiation and progression of CRC. The CIMP study was a prospective study in which the real-time based MethyLight assay was employed to evaluate CIMP and methylation status of p16, IGFBP7 and MLH1 in 154 serrated polyps and 63 adenomas. Samples were subjected to bisulfite modification and methylation levels were assessed using the Weisenberger et al panel of methylation markers (IGF2, SOCS1, NEUROG1, RUNX3 and CACNA1G) with ALU as the reference gene.

Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder resulting in a myriad of abnormalities, including progressive neurodegeneration and cancer predisposition. At the cellular level, A-T is a disease of chronic oxidative stress (OS) causing damage to proteins, lipids, and DNA. OS is contributed to by pro-oxidative transition metals such as iron that catalyze the conversion of weakly reactive oxygen species to highly reactive hydroxyl radicals. Iron-associated OS has been linked to neurodegeneration in Alzheimer's and Parkinson's diseases and development of lymphoid tumors (which afflict ~30 of A-T patients). To investigate iron regulation in A-T, iron indexes, regulatory genes, and OS markers were studied in livers of wild-type and Ataxia telangiectasia mutated (Atm) null mice on control or high-iron diets. Atm-/- mice had increased serum iron, hepatic iron, and ferritin and significantly higher Hepcidin compared with wild-type mice. When challenged with the high-iron diet, Bmp6 and Hfe expression was significantly increased. Atm-/- mice had increased protein tyrosine nitration and significantly higher Heme Oxygenase (decycling) 1 levels that were substantially increased by a high-iron diet. Ferroportin gene expression was significantly increased; however, protein levels were unchanged. We demonstrate that Atm-/- mice have a propensity to accumulate iron that is associated with a significant increase in hepatic OS. The iron-induced increase in hepcidin peptide in turn suppresses ferroportin protein levels, thus nullifying the upregulation of mRNA expression in response to increased OS. Our results suggest that increased iron status may contribute to the chronic OS seen in A-T patients and development of disease pathology.© 2011 the American Physiological Society.

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), Griffith University. School of Biomolecular and Physical Sciences, Science, Environment, Engineering and Technology, Zellweger syndrome (ZS) represents the most severe form of the spectrum of autosomal recessive peroxisomal disorders. These disorders result from mutations in PEX genes, which encode proteins (peroxins) that are required for peroxisome biogenesis. The inability to form functional peroxisomes in ZS leads to the loss of peroxisomal functions such as very long chain fatty acid oxidation and synthesis of bile acids and plasmalogens. Clinically, this results in a complex array of symptoms of which the neuropathogenesis is most pronounced. The mechanism behind the neuropathogenesis (and other clinical symptoms) is not yet understood. Over the last two decades, several murine models that feature most ZS symptoms have been generated to aid in understanding the pathogenesis of this debilitating disease. However, the utility of some of these mouse mutants has been limited by the neonatal lethality of pups. Recent work in this laboratory resulted in the generation of a brain specific Pex13 mutant – Pex13 encodes a peroxisomal membrane protein essential for peroxisome biogenesis. This Pex13 brain mutant was generated by nestin-driven, Cre recombinase-mediated, tissue specific disruption of Pex13. Pex13 brain mutants feature many biochemical characteristics of a milder form of ZS and exhibits postnatal survival, making them suitable for studying postnatal neuropathogenesis in ZS. The goal of this thesis was to characterise macroscopic and neurological changes of the Pex13 brain mutant, and ultimately to gain a better understanding of the neuropathogenesis of ZS. An average excision level of Pex13 of 70-90% was found to result in a pronounced mutant phenotype in all Pex13 brain mutants. This macroscopic phenotype was characterised by severe growth retardation, early death, and prominent abnormalities in motor control. A higher level of Pex13 excision did not result in earlier death or a more pronounced phenotype. However, a correlation was identified between disease sev

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Biomolecular and Physical Sciences, Science, Environment, Engineering and Technology, The Ataxia telangiectasia (A-T) syndrome is a multisystem disorder exhibiting a complex array of clinical features for which there is no effective therapy. The earliest and most debilitating aspect of this disease is progressive ataxia resulting from neurodegeneration of the cerebellum. Several lines of evidence suggest a potential function for ATM in the maintenance of cellular homeostasis, and disruptions in functional ATM appear to result in alterations cholesterol homeostasis. This association may provide some insight into the neurodegenerative aspect of this disease. Cholesterol homeostasis is particularly significant in the central nervous system (CNS) with several neurodegenerative disorders involving disturbances in cholesterol homeostasis. The goal of this thesis was to attempt to establish a potential relationship between ATM and cholesterol homeostasis. As well it was hoped the establishment of a mechanism for such a relationship may help contribute to the establishment of therapies for this debilitating aspect of A-T...

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Biomolecular and Physical Sciences, Science, Environment, Engineering and Technology, Zellweger syndrome (ZS) is the most severe form of a spectrum of disorders resulting from mutations in PEX genes, genes that encode proteins necessary for peroxisome biogenesis. Loss of functional peroxiosmes leads to disruption of multiple metabolic pathways involving the peroxisome, including very long chain fatty acid oxidation and plasmalogen and bile acid synthesis. ZS patients exhibit a range of clinical abnormalities, including facial dysmorphism, cataracts, hypotonia, seizures, psychomotor retardation, and hearing impairment. In terms of tissue pathology, there are also wide ranging effects, including neuronal migration defects, hepatomegaly, retinopathy, and renal cysts. Pex13 encodes a peroxisomal membrane protein that is essential for peroxisome biogenesis. Previous work in this laboratory resulted in the generation of a Pex13-null mouse model for the purpose of investigating the pathogenesis of Zellweger syndrome. The work in the first part of this thesis extends these studies and describes the generation and initial characterisation of tissue-specific Pex13 mouse models. These tissue-specific models are expected be useful tools for analysis of the impact of localised, brain- and liver-specific elimination of peroxisomes on the pathogenesis of ZS. In addition, in the second part of the thesis, a separate and novel hypothesis is addressed as an explanation for the molecular pathogenesis of ZS, through investigating the relationship between reduced peroxisome abundance and microtubule-mediated peroxisome trafficking. Pex13 brain mutant mice were generated by mating the previously generated Pex13-floxed mice with mice expressing Cre recombinase under the control of the neuron-specific rat nestin promoter. Pex13 brain mutant mice displayed growth retardation beginning at day 5 postnatal, with gradual deterioration until death at approximately day 22 postnatal. Other clinical features included contracted postures, under-developed lower body mass, abnormal and

Full Text, Thesis (PhD Doctorate), Doctor of Philosophy (PhD), School of Biomolecular and Biomedical Sciences, Science, Environment, Engineering and Technology, Ataxia-telangiectasia (A-T) is a debilitating disease caused by the functional loss of the ataxia-telangiectasia mutated (ATM) protein. A-T is characterised by immunodeficiency, reduced growth, insulin resistance and a predisposition to cancer. However, the hallmark symptom of ataxia, which presents within the first years of life, is caused by degeneration of cerebellar Purkinje and granule cells. At the cellular level, the loss of ATM leads to increased oxidative stress. The major function of ATM in normal cells is to activate DNA repair pathways in response to double strand breaks. However, the mechanisms leading to neurodegeneration of cerebellar Purkinje cells are still being elucidated. The focus of this study was to examine how oxidative stress contributes to defective cell signalling and the A-T phenotype. Examination of the insulin and epidermal (EGF) receptor signalling pathways by Western blotting indicated abnormal signalling in fibroblasts from A-T patients. Reduced expression of the membrane receptors and some effector proteins were a likely cause of the observed signalling defects. Fluorescence studies on transferrin receptor recycling identified delayed intracellular trafficking as a novel characteristic of the cellular A-T phenotype, and a contributing factor to altered signalling...

This study is an investigation of the epistemological beliefs and study habits of students undertaking first-year courses in Biological Chemistry and Biochemistry. In particular, we were interested in the relationship between students’ epistemological beliefs about learning and knowledge, approaches to learning, and achievement. The study adopted a mixed-methods approach in which quantitative and qualitative data have provided complementary insights into the beliefs and approaches adopted by these students. Our findings indicate that most students tend to adopt beliefs that knowledge and learning involves the accumulation of information and the capacity to reproduce on demand in examinations. Approaches to learning reflect these beliefs and are dominated by rote learning and preference for assessment by examination. Few students adopt strategies that emphasise the relationship of concepts to those already learnt or to applications relevant to biological science. Implications of this study for reform of university teaching practices as well as secondary practices are discussed.

In foundation biochemistry and biological chemistry courses, a major problem area that has been identified is students’ lack of understanding of pH, acids, bases, and buffers and their inability to apply their knowledge in solving acid/base problems. The aim of this study was to explore students’ conceptions of pH and their ability to solve problems associated with the behavior of biological acids to understand the source of student difficulties. The responses given by most students are characteristic of an atomistic approach in which they pay no attention to the structure of the problem and concentrate only on juggling the elements together until they get a solution. Many students reported difficulty in understanding what the question was asking and were unable to interpret a simple graph showing the pH activity profile of an enzyme. The most startling finding was the lack of basic understanding of logarithms and the inability of all except one student to perform a simple calculation on logs without a calculator. This deficiency in high school mathematical skills severely hampered their understanding of pH. This study has highlighted a widespread deficiency in basic mathematical skills among first year undergraduates and a fragmented understanding of acids and bases. Implications for the way in which the concepts of pH and buffers are taught are discussed.

Fibrogenic stresses promote progression of renal tubulointerstitial fibrosis, disparately affecting survival, proliferation and trans-differentiation of intrinsic renal cell populations through ill-defined biomolecular pathways. We investigated the effect of fibrogenic stresses on the activation of cell-specific mitogen-activated protein kinase (MAPK) in renal fibroblast, epithelial and endothelial cell populations. The relative outcomes (cell death, proliferation, trans-differentiation) associated with activation or inhibition of extracellular-regulated protein kinase (ERK) or stress activated/c-Jun N terminal kinase (JNK) were analysed in each renal cell population after challenge with oxidative stress (1 mmol/L H2O2), transforming growth factor-beta1 (TGF-beta1, 10 ng/mL) or tumour necrosis factor-alpha (TNF-alpha, 50 ng/mL) over 0-20 h. Apoptosis increased significantly in all cell types after oxidative stress (P < 0.05). In fibroblasts, oxidative stress caused the activation of ERK (pERK) but not JNK (pJNK). Inhibition of ERK by PD98059 supported its role in a fibroblast death pathway. In epithelial and endothelial cells, oxidative stress-induced apoptosis was preceded by early induction of pERK, but its inhibition did not support a pro-apoptotic role. Early ERK activity may be conducive to their survival or promote the trans-differentiation of epithelial cells. In epithelial and endothelial cells, oxidative stress induced pJNK acutely. Pretreatment with SP600125 (JNK inhibitor) verified its pro-apoptotic activity only in epithelial cells. Transforming growth factor-beta1 did not significantly alter mitosis or apoptosis in any of the cell types, nor did it alter MAPK activity. Tumor necrosis factor-alpha caused increased apoptosis with no associated change in MAPK activity. Our results demonstrate renal cell-specific differences in the activation of ERK and JNK following fibrotic insult, which may be useful for targeting excessive fibroblast proliferation in ch