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(2007)Journal ArticleBiallelic promoter methylation and transcriptional silencing of the MLH1 gene occurs in the majority of sporadic colorectal cancers exhibiting microsatellite instability due to defective DNA mismatch repair. Long-range epigenetic silencing of contiguous genes has been found on chromosome 2q14 in colorectal cancer. We hypothesized that epigenetic silencing of MLH1 could occur on a regional scale affecting additional genes within 3p22, rather than as a focal event. We studied the levels of CpG island methylation and expression of multiple contiguous genes across a 4 Mb segment of 3p22 including MLH1 in microsatellite-unstable and -stable cancers, and their paired normal colonic mucosa. We found concordant CpG island hypermethylation, H3-K9 dimethylation and transcriptional silencing of MLH1 and multiple flanking genes spanning up to 2.4 Mb in microsatellite-unstable colorectal cancers. This region was interspersed with unmethylated genes, which were also transcriptionally repressed. Expression of both methylated and unmethylated genes was reactivated by methyltransferase and histone deacetylase inhibitors in a microsatellite-unstable colorectal carcinoma cell line. Two genes at the telomeric end of the region were also hypermethylated in microsatellite-stable cancers, adenomas, and at low levels in normal colonic mucosa from older individuals. Thus, the cluster of genes flanking MLH1 that was specifically methylated in the microsatellite-unstable group of cancers extended across 1.1 Mb. Our results show that coordinate epigenetic silencing extends across a large chromosomal region encompassing MLH1 in microsatellite-unstable colorectal cancers. Simultaneous epigenetic silencing of this cluster of 3p22 genes may contribute to the development or progression of this type of cancer.
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(2013)Journal ArticleActivated pancreatic stellate cells (PSCs) are responsible for the fibrotic matrix of chronic pancreatitis and pancreatic cancer. In vitro protocols examining PSC biology have usually involved PSCs cultured on plastic, a non-physiological surface. However, PSCs cultured on physiological matrices e.g. MatrigelTM (normal basement membrane) and collagen (fibrotic pancreas), may have distinctly different behaviours compared to cells cultured on plastic. Therefore, we aimed to i) compare PSC gene expression after culture on plastic, MatrigelTM and collagen I; ii) validate the gene array data for transgelin, the most highly dysregulated gene in PSCs grown on activating versus non-activating matrices, at mRNA and protein levels; iii) examine the role of transgelin in PSC function; and iv) assess transgelin expression in human chronic pancreatitis sections. Culture of PSCs on different matrices significantly affected their gene expression pattern. 146, 619 and 432 genes respectively were differentially expressed (p < 0.001) in PSCs cultured on collagen I vs MatrigelTM, MatrigelTM vs plastic and collagen I vs plastic. The highest fold change (12.5 fold upregulation) in gene expression in cells on collagen I vs MatrigelTM, was observed for transgelin (an actin stress fibre associated protein). Transgelin was significantly increased in activated PSCs versus quiescent PSCs. Silencing transgelin expression decreased PSC proliferation and also reduced platelet derived growth factor (PDGF)-induced PSC migration. Notably, transgelin was highly expressed in chronic pancreatitis in stromal areas and peri-acinar spaces but was absent in acinar cells. These findings suggest that transgelin is a potentially useful target protein to modulate PSC function so as to ameliorate pancreatic fibrosis.
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(2011)Journal ArticleObjective Three decades into the HIV epidemic and with the advancement of HIV treatments, condom and non-condom-based anal intercourse among gay men in resource-rich countries needs to be re-assessed. Methods The proportions of men engaging in a range of anal intercourse practices were estimated from the ongoing cross-sectional Gay Community Periodic Surveys in six states in Australia from 2007 to 2009. Comparisons were made between HIV-negative men, HIV-positive men with an undetectable viral load and those with a detectable viral load. Results Condoms play a key role in gay men's anal intercourse practices: 33.8% of HIV-negative men, 25.1% of HIV-positive men with an undetectable viral load and 22.5% of those with a detectable viral load reported consistent condom use with all male partners in the 6 months before the survey. Among HIV-negative men, the second largest group were men who had unprotected anal intercourse (UAI) only in the context of HIV-negative seroconcordant regular relationships. Among HIV-positive men, the second largest group was men who had UAI in casual encounters preceded by HIV status disclosure to some, but not all, casual partners. Conclusions A minority, yet sizeable proportion, of men consistently engaged in a number of UAI practices in specific contexts, suggesting they have adopted deliberate HIV risk-reduction strategies. While it is important that HIV behavioural prevention continues to reinforce condom use, it needs to address both the challenges and opportunities of the substantial uptake of non-condom-based risk-reduction strategies.
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(2007)Journal ArticleThe metabolic response to two different forms of high intensity intermittent cycle exercise was investigated in young females. Subjects (8 trained and 8 untrained) performed two bouts of high intensity intermittent exercise: short sprint (SS) (8 s sprint, 12 s recovery) and long sprint (LS) (24 s sprint, 36 s recovery) for 20 min on two separate occasions. Both workload and oxygen uptake were greater in the trained subjects but were not significantly different for SS and LS. Plasma glycerol concentrations increased during exercise but peaked earlier for the trained women. Lactate concentrations rose over the 20 min and were higher for the trained women. Catecholamine concentration was also higher postexercise when compared to pre-exercise for both groups. Both SS and LS produced similar metabolic response although both lactate and catecholamines were higher after the 24 s sprint. In conclusion, these results show that high intensity intermittent exercise resulted in significant elevations in catecholamines that appear to be related to increased venous glycerol concentrations. Trained compared to the untrained women tended to show increased plasma glycerol concentrations, earlier during high intensity exercise. Keywords: intermittent exercise, fat oxidation, RER, catecholamines
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(2008)Journal ArticleAbstract OBJECTIVE: to determine the effects of a 15-week high intensity intermittent exercise (HIIE) program on subcutaneous and trunk fat and insulin resistance of young women. DESIGN AND PROCEDURES: subjects were randomly assigned to one of three groups: HIIE (n = 15), steady state exercise (SSE; n=15), or control (CONT; n=15). HIIE and SSE groups underwent a 15-week exercise intervention. SUBJECTS: forty-five women with a mean BMI of 23.2 + 2.0 kg/m2 and age of 20.2 + 2.0 years. RESULTS: both exercise groups demonstrated a significant improvement, P < 0.05, in cardiovascular fitness. However, only the HIIE group had a significant reduction in total body mass (TBM), fat mass (FM), trunk fat, and fasting plasma insulin levels. There was significant fat loss, P < 0.05, in legs compared to arms in the HIIE group only. Lean compared to overweight women lost less fat after HIIE. Decreases in leptin concentrations were negatively correlated with increases in (r = -.57, P < 0.05) and positively correlated with decreases in TBM (r = .47; P < 0.0001). There was no significant change in adiponectin levels after training. CONCLUSIONS: HIIE three times per week for 15 weeks compared to the same frequency of SSE exercise was associated with significant reductions in total body fat, subcutaneous leg and trunk fat, and insulin resistance in young women. Keywords: intermittent sprinting, body fat, insulin
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(2000)Journal ArticleThe route of estrogen replacement therapy has a major impact on the growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis. Estrogen administration by the oral, but not the transdermal, route reduces IGF-I and increases GH levels in postmenopausal women. This perturbation of the GH-IGF-I axis occurs with different forms of estrogen treatment, indicating that the dissociation of the somatotropic axis and concomitant increase in GH-binding protein levels are intrinsic effects of the oral route of estrogen administration. In clinical studies, oral estrogen reduced postprandial lipid oxidation, compared with transdermal estrogen. Oral estrogen was also associated with a reduction in lean body mass and an increase in fat mass, compared with transdermal estrogen. In contrast, the route of estrogen therapy had no impact on carbohydrate metabolism or the estrogen-induced increase in bone mineral density. The findings of route-dependent changes in body composition add a new dimension to health considerations concerning estrogen therapy in postmenopausal women and may have significant implications for estrogen replacement therapy in young hypogonadal females.
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(2010)Recorded/Rendered Creative WorkABSTRACT Pancreatic stellate cells (PSCs) produce the stromal reaction of pancreatic cancer (PC) and their interaction with cancer cells facilitates cancer progression. This study investigated the role of human PSCs (hPSCs) in the metastatic process and tumor angiogenesis using an in vivo (orthotopic model) and in vitro (cultured PSC and PC cells) approach. A gender mismatch study [injection of male hPSCs + female PC cells into the pancreas of female mice] was conducted to determine whether hPSCs accompany cancer cells to metastatic sites. Metastatic nodules were examined by fluorescent in situ hybridization for the presence of the y chromosome. Angiogenesis was assessed by i) immunostaining tumors for CD31, an endothelial cell marker; and ii) in vitro quantifying human microvascular endothelial cell (HMEC-1) tube formation upon exposure to conditioned media from hPSCs. Transendothelial migration was assessed by examining the movement of fluorescently labeled hPSCs through an endothelial cell monolayer. Human PSCs i) were found in multiple metastatic sites in each mouse injected with male hPSCs + female PC cells; ii) increased CD31 expression in primary tumors from mice injected with MiaPaCa-2 and hPSCs and stimulated tube formation by HMEC-1 in vitro; iii) exhibited transendothelial migration which was stimulated by cancer cells. Human PSCs accompany cancer cells to metastatic sites, stimulate angiogenesis and have the capacity to intravasate/extravasate to and from blood vessels.
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(2015)ThesisParkinson’s disease (PD) is a neurodegenerative disorder that manifests as a result of degeneration of the nigrostriatal system. While L-Dopa still remains the most effective symptomatic treatment, its benefits are limited as it is associated with the development of L-Dopa-induced dyskinesias (LIDs). Furthermore, there is currently no treatment that has proved effective in halting the nigrostriatal degeneration, thus alternative therapeutic targets are needed. To identify such targets, we first established the MPTP mouse model of PD to allow for the investigation of both pathological and behavioural outcomes. In contrast to previous studies, we demonstrated no significant effect of MPTP on motor function at 1 week and 3 months post-MPTP, despite stereological quantification showing a decrease in dopamine cells with increasing MPTP dose, suggesting the MPTP model is more appropriate for identifying effects on cell survival. A screen of novel therapeutic agents and genetic modifications in mice in the MPTP model revealed the growth factor activin A and the kainate receptor subunits GluK1 and GluK3 as the most promising neuroprotective targets. We showed activin A increased survival of dopamine neurons at 1 and 8 weeks following MPTP. Interestingly, animals receiving activin A did not have a corresponding increase in dopamine levels and striatal terminal protection. This nigral protective effect of activin A was replicated in the 6-OHDA model of PD. Animals receiving activin A also had lower numbers of astrocytes and microglia following MPTP and LPS, suggesting that activin A’s neuroprotective effects may be driven by its anti-inflammatory properties. In addition, activin A was shown to increase the efficacy of low doses of L-Dopa in 6-OHDA-lesioned mice, suggesting activin A may act as an L-DOPA-sparing agent. We also showed that blocking GluK1 or GluK3, through the use of selective antagonists or knockout animals, increased survival of dopaminergic neurons following MPTP, however there was no corresponding increase in dopamine levels and striatal terminal protection. Our results indicate that activin A, GluK1 and GluK3 are potential neuroprotective targets for PD.
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(2018)ThesisThe mammary gland is a unique model to study regulation of stem cell homeostasis and differentiation due to its postnatal development. Embedded within the bilayered mammary epithelium are populations of mammary stem cells (MaSCs), which give rise to inner luminal and outer basal/myoepithelial cells. Developmental transcription factors (TFs) control stem cell self-renewal and differentiation. TFs driving luminal differentiation are well studied, however the regulators of MaSC maintenance and myoepithelial differentiation of basal cells are poorly understood. The helix-loop-helix (HLH) TF Inhibitor of Differentiation 4 (ID4) is exclusive to basal cells and is essential for appropriate mammary gland development. ID proteins lack DNA binding domains, and function by binding to and inhibiting basic HLH (bHLH) TFs. The overarching aims of this thesis were to i) isolate and characterise mammary epithelial cells expressing ID4, and ii) integrate multi-omic approaches to define the cis-regulatory mechanism through which ID4 controls cell fate. I found that selecting for cells with high ID4 expression enriched for basal cells with stem cell characteristics. Moreover, basal cells with low ID4 expression exhibited a differentiated myoepithelial phenotype. ID4 was required to inhibit expression of genes involved in muscle contraction, and to activate genes involved in cell growth, metabolism and cell cycle, suggesting that ID4 blocks myoepithelial specialisation and maintains proliferation of MaSCs. ID4 was found to interact with ubiquitously expressed bHLH TFs, E2A, HEB, and ITF-2 (E-proteins) using an unbiased proteomics approach. E-proteins co-regulated a large subset of ID4 target genes. This was unexpected, due to the typical role of ID proteins in antagonising E-protein activity. Motif enrichment revealed E-protein binding motifs in the promoters of genes co-regulated by both ID4 and E-proteins. This raises the possibility of a novel mechanism for ID4 in acting as a chromatin-bound co-factor for E-proteins, rather than simply sequestering E-proteins in the nucleoplasm. Chromatin Immunoprecipitation followed by DNA-Sequencing (ChIP-Seq) experiments will directly test this hypothesis. I discovered that ID4 dictates the MaSC fate by inhibiting myoepithelial differentiation, potentially by cooperating with E-proteins at the level of chromatin. Lessons learnt from the mammary gland may have broader implications in the regulation of adult stem cells by HLH TFs.
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(2018)ThesisEstrogen receptor positive (ER+) breast cancer constitutes 70% of all breast cancers and anti-ER therapies such as aromatase inhibitors and tamoxifen represent the main therapeutic strategies in the treatment of this disease. Unfortunately, up to 30% of all primary ER+ tumours will ultimately develop endocrine-resistance and progress on ER-targeted therapies resulting in disease-related morbidity. As a result, there is an urgent medical need for novel therapeutic strategies capable of managing endocrine-resistant breast cancer. Androgen receptor (AR) is expressed in up to 90% of ER+ breast cancers. AR functions as a tumour suppressor in primary ER+ breast cancer and high AR positivity is strongly associated with a favourable patient outcome in the ER+ setting. However, the role of AR in endocrine-resistant breast tumours is highly controversial with data supporting both oncogenic and tumour suppressive functions reported in the literature. Here I have used different modulatory approaches on in vitro and in vivo preclinical models to dissect the functions of AR and determine the best approach to target AR in endocrine-resistant breast cancer. I use an siRNA-mediated approach to knock down AR in cell line models and discover that the basal expression of AR contributes to endocrine-resistance and that loss of AR restores classical ER signalling and reverses endocrine-resistance. However, inhibiting the transcriptional activity of AR with enzalutamide does not recapitulate this effect, suggesting that it is the non-canonical activity of AR which contributes to endocrine-resistance. In contrast, I show that activation of AR by either 5-α dihydrotestosterone (DHT) or selective AR modulator enobosarm in vitro and in patient derived (PDX) models of endocrine-resistance results in significant growth suppression. Mechanistically, this growth-inhibitory effect of AR activation is associated with downregulation of ER signalling. Moreover, I identify AR-regulated genes from the global gene expression of an ER+AR+ endocrine-resistant PDX model treated with DHT and establish a highly prognostic AR gene signature based on primary ER+ patients in the METABRIC dataset. This suggests that activity of AR is tumour-suppressive independent of endocrine-sensitivity. In summary, I demonstrate that activation, not antagonism, is the optimal AR-targeted therapeutic strategy in the management of endocrine-resistant breast cancer.