Medicine (RMH) - Theses
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ItemKey clinical and molecular determinants of biological behavior in glioma: examining the role of the Salvador-Warts-Hippo pathway in glioma stem cells( 2011)Analysis of a landmark genetic sequencing project, identifying likely damaging genetic variants in a series of twenty-one glioma stem cells, reveals the Salvador-Wart-Hippo (SWH) pathway to be a key deregulated pathway. A constitutive tumour suppressor pathway, its down-stream effector yes-associated protein, or “YAP”, is implicated in regulating stem cell and progenitor cell self-renewal. We found that YAP is overexpressed in a group of selected glioma stem cells, in contrast to normal brain, and established glioma cell lines. When we compared YAP and constitutive phosphorylated- YAP (p-YAP) expression between the glioma stem cells and their matched bulk tumours, we found p-YAP was consistently more highly expressed in the bulk “parent” tumour, compared to the corresponding stem cell. These findings are consistent with YAP expression being a molecular marker of “stemness”. YAP expression was shown to correlate with an aggressive phenotype within our stem cell series, with respect to adverse survival outcomes and biological behavior. Tumours associated with YAPexpressing stem cells had worse overall survival than their non-YAP-expressing counterparts (median survival 8 months versus 15 months, HR 1.886, 95% CI 0.224 to 0.8413, p value 0.4727). This favorably contrasts with the only other related published translational research to date, showing that lack of YAP expression in gliomas carries a survival advantage. Within a selected cohort of glioma stem cells, YAP-expression correlated with increased proliferative capacity, assessed by LDH assay. We show new and novel evidence that YAP is an oncogene in glioma, and may be a key glioma stem cell marker, further adding to a shift towards re-conceptualizing high-grade glioma as a disease of molecular-genetic aberrations.
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ItemAntidepressant pharmacotherapy in epilepsy: the effects of chronic fluoxetine and citalopram treatments in a rat model of epileptogenesis( 2012)Introduction: In patients with epilepsy there is a high incidence of comorbid psychiatric illnesses, especially mood and anxiety disorders, which have been associated with lower quality of life, impaired function and an elevated risk of suicide. In fact, the occurrence of these illnesses in patients with epilepsy has been reported to be a stronger predictor of quality of life than epilepsy variables such as illness duration or seizure frequency. In addition, there is evidence that the depressed state itself may predispose to seizures and epilepsy. For these reasons, effective management of depressive and anxiety symptoms and syndromes in epilepsy is essential. Selective serotonin reuptake inhibitors (SSRIs) are commonly used to treat depression in epilepsy, therefore it is important to consider their impact on epilepsy. To date, many studies have suggested that SSRI are safe for use in epilepsy, but the majority of these studies administered SSRIs only acutely or for short periods, and investigated effects only on acute seizure endpoints. There is no indication of the effect that chronic SSRI treatment may have on epileptogenesis and the associated neurobiological changes that continue after seizures emerge. This thesis aimed to investigate the effects of chronic SSRI treatment in a rat model of epileptogenesis, as well as investigating common neurobiological substrates of SSRI treatment and epileptogenesis that may also influence the disorder. It was hypothesised that chronic SSRI treatment would slow the rate of kindling epileptogenesis, as well as mitigate effects on common neurobiological substrates. Methods: The amygdala kindling model was used to assess the effects of chronic SSRI treatment (with fluoxetine or citalopram) on epileptogenesis. 9-11 week old male Wistar rats were surgical implanted with a bipolar electrode into the left amygdala for electrical kindling and a subcutaneously implanted osmotic pump filled with fluoxetine (10mg/kg/day, n=19) or vehicle (50% DMSO, n=22) or citalopram (10mg/kg/day, n=26) or vehicle (50% DMSO, n=22), comprising two separate cohorts. All rats were given 30 stimulations and then kindling rate, seizure duration and seizure threshold before and after kindling were monitored. Effects on anxiety- and depressive-like behaviours were also investigated after kindling using two well-validated tests, the elevated plus maze and forced swim test respectively, as well as assessing the corticosterone response to stress and dentate gyrus neurogenesis. Results: The key finding of this study was that rats chronically treated with SSRIs, either fluoxetine or citalopram, demonstrated accelerated rates of kindling epileptogenesis, showing a more rapid progression through the different stages of kindling compared to vehicle treated rats. The increase in seizure duration was also accelerated in the early stages of kindling in both cohorts of SSRI treated rats, however seizure threshold was not significantly different between vehicle and fluoxetine or vehicle and citalopram treated rats, either before or after kindling. This indicates that while epileptogenesis itself progressed at a faster rate during chronic SSRI treatment, accelerating the increase in seizure severity and duration, the local excitability and the threshold at which a seizure occurred was not affected by SSRI treatment. In order to investigate potential mechanisms underlying this, neurobiological alterations common to epileptogenesis and SSRI treatment were also investigated. Behavioural analyses found that both fluoxetine and citalopram treatments did not affect anxiety- or depressive-like behaviours, while kindling increased anxiety-like behaviour, but only in the fluoxetine treated cohort. Dentate gyrus neurogenesis was not significantly affected by kindling or drug treatment while stress-induced corticosterone levels were significantly reduced only by fluoxetine treatment. These investigations do not suggest that these alterations are associated with accelerating kindling rate during chronic SSRI treatment, however how these are affected during or immediately after kindling was not investigated. Conclusions: Chronic treatment with fluoxetine and citalopram, at clinically relevant doses, accelerated kindling epileptogenesis in rodents. This highlights the need to investigate the effects of SSRI treatment on epileptogenesis over time in both animal models and people with epilepsy, rather than focusing solely on acute seizure time points. While the investigations in this study do not suggest that alterations in behaviour, neurogenesis or neuroendocrine responses are associated with accelerating kindling rate during chronic SSRI treatment, how these are affected during or immediately after kindling was not investigated. Therefore, future studies should further investigate the mechanisms underlying the effects of SSRIs on epileptogenesis at appropriate time points, such as during kindling epileptogenesis and also in complementary animal models of epilepsy, such as post-status epilepticus and post-traumatic models. It is essential to treat the depressive symptoms that manifest in people with epilepsy; however whether these medications affect the course of epilepsy and how they may do so should become priority areas for future research.
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ItemCharacterisation of the nuclear pore abnormalities in the intestinal zebrafish mutant, flotte lotte (flo)( 2012)The evolution of eukaryotic cells is defined by the compartmentalisation of the genetic material inside the nucleus, segregated from cytoplasm by a nuclear envelope. This barrier is punctuated by approximately 3000 large multi-protein structures known as nuclear pore complexes, which permit the bidirectional transport of protein and RNA molecules between the nucleus and cytoplasm. This study provided the opportunity to investigate the importance of the nuclear pore protein Elys during vertebrate development. Zebrafish mutants generated by ethylnitrosourea (ENU) mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes. Our interest has focused on a panel of ENU generated mutants exhibiting a variety of defects in the formation and differentiation of the intestinal epithelium. One of these mutants, flotte lotte (flo), harbours a premature stop codon in the coding sequence of the nuclear pore component elys (embryonic large molecule derived from yolk sac). Elys is an essential component of the nuclear pore complex, yet surprisingly, its mutation in the flo mutant does not result in a global dysfunction in nuclear pore formation throughout the developing zebrafish embryo. Instead, flo mutants exhibit tissue-specific abnormalities in the development of the intestinal epithelium, liver, pancreas and eye; organs that are highly proliferative from 48hpf. We show that this time-point coincides with the exhaustion of maternally-deposited stocks of elys mRNA from flo embryos. Not surprisingly, we found that the ensuing inability to create new nuclear pore complexes appears to impact most severely on these rapidly proliferating tissues. Using multi-photon microscopy we reconstructed three-dimensional renditions of the endodermal organs in wild-type and flo larvae. Compared to the highly elaborated and polarized intestinal epithelium of wild-type zebrafish, the intestinal epithelium in flo is thin, unfolded and poorly polarised. Moreover, nuclear pore complexes in flo intestinal cells are not embedded in the nuclear envelope but are found in profuse cytoplasmic aggregates. Catastrophic levels of apoptosis accompany the loss of a functional nuclear envelope in intestinal epithelial cells. Thus, flo mutants provide an opportunity to identify signals that commit nuclear pore-deficient cells to an apoptotic fate. We found that the apoptotic response observed in the flo intestine is mediated via a Tp53-independent mechanism. Since Elys function is critical for the integrity of proliferative cells in zebrafish, we investigated whether ELYS is also critical for the proliferation of human cancer cells. We discovered a strong up-regulation of ELYS expression in many cancers when compared to their respective normal tissues. We observed ELYS to be ranked in the top 1% of all up-regulated genes investigated in gene expression studies of colorectal, kidney, liver and breast cancers available in the Oncomine database. The discovery that ELYS is frequently over-expressed in human colorectal cancer suggests that our functional genomics approach to novel cancer gene discovery using zebrafish mutants is valid. Moreover, we propose that targeted approaches to disabling ELYS synthesis or function may activate apoptosis in colorectal cancer cells and provide a useful therapeutic approach in the future.
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ItemWeight loss after reoperations for laparoscopic adjustable gastric banding( 2012)Background: Reoperation after laparoscopic adjustable gastric banding occurs with relative frequency and this is often cited as a major weakness of the procedure. There are currently no guidelines for the best reoperative procedure in these cases. Few studies report weight loss after reoperation, especially after band salvage procedures, despite being a critical factor in deciding which reoperative procedure to select. This study aims to assess the impact of major reoperations after laparoscopic adjustable gastric banding on weight loss at 12 and 24 months and on patient satisfaction with the procedure. Methods: An analysis of New York University Langone Medical Centre’s prospectively collected bariatric database was conducted. Adult patients who underwent laparoscopic adjustable gastric banding from 1 January 2001 to 30 June 2009 were identified. Patients who required major reoperation were then studied. Major reoperation included band repositioning, replacement, hiatal hernia repair and band removal. Basic demographic data, weight outcomes and surgical details were recorded. Weights at 12 and 24 months after reoperation were recorded and compared with initial weight and weight at reoperation. Patient satisfaction with the procedure was assessed by a qualitative telephone survey. Results: Of the 4680 patients retrieved, 4652 met inclusion criteria. There were 664 patients who underwent one or more major reoperations. There were no procedure-related deaths in the reoperation group. The 30-day patient complication rate for all reoperations was 1.8%. Patients undergoing 1 reoperation represented the largest subgroup (507 patients). For this subgroup, initial weight and Body Mass Index (BMI) were 122.7±23.3kg and 44.0±6.7kg/m2. At reoperation, weight, BMI and percentage excess weight loss (%EWL) were 91.4±22.7kg, 32.8±7.0kg/m2 and 51±24%. Twelve months after reoperation, weight, BMI and %EWL were 92.7±21.5kg, 33.3±6.8kg/m2 and 49±23% and at 24 months were 91.8±21.0kg, 33.2±6.7kg/m2 and 49±24%. Weight loss was sustained both at 12 months and 24 months after reoperation and did not differ from those without reoperation at the same length of time after primary banding. Patient satisfaction with the band was not affected by reoperation. Conclusion: Reoperation after laparoscopic adjustable gastric banding can be achieved with minimal morbidity. Weight loss is sustained 12 and 24 months after reoperation for laparoscopic adjustable gastric banding.
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ItemThe role of the transcription/repair factor TFIIH in the intestinal dysplasia of the zebrafish mutant sycorax( 2012)The gene expression profiles of various cancer types show significant similarities to those of their developing tissue of origin, with a number of genes that are expressed in the developing intestine but not the adult intestine being re-expressed in colorectal cancer (CRC) cells. This study utilised a zebrafish intestinal mutant, sycorax, to further the current understanding of the genetics underlying vertebrate intestinal development and development and progression of CRC. The sycorax zebrafish mutant displays a dysplastic intestinal epithelium, with multilayering and disorganisation, reminiscent of a pre-neoplastic lesion. The mutation underlying the sycorax phenotype is a premature stop codon in the general transcription 2 H, polypeptide 4 (gtf2h4) gene, which is predicted to generate a truncated Gtf2h4 protein. Gtf2h4 is one of the components of the multisubunit complex, general transcription factor IIH (TFIIH), which is integral to a number of cellular processes. TFIIH is one of the general transcripton factors involved in transcription initation of all protein coding genes via RNA polymerase II. It also fine tunes the expression of a small proportion of genes by integrating signals from gene specific activators and repressors. TFIIH is involved in the regulation of transcription of rRNA by RNA polymerase I, in addition to being a crucial component of the DNA repair pathway known as nucleotide excision repair. Finally, TFIIH plays a role in the regulation of cell cycle progression. Northern blot analysis and metabolic labeling of newly synthesized rRNA in wild type and sycorax embryos demonstrated an increase in rRNA transcription in sycorax embryos. sycorax embryos were also shown to have reduced DNA repair capabilities following UV irradiation. Increased levels of proliferation in the intestine, as demonstrated by EdU incorporation, are hypothesised to contribute to the multilayering of the sycorax intestinal epithelium. Gene expression analysis of wild type and sycorax intestines identified a number of differentially expressed genes, many of which are involved in the regulation of cell proliferation. Upregulated genes in the sycorax intestine include c-myc and c-myb, which have previously been shown to be directly transcriptionally regulated by TFIIH, and are proposed to contribute to the increased levels of proliferation observed in the sycorax intestine. Furthermore, knockdown of c-myb in sycorax embryos rescues the intestinal dysplasia. These results demonstrate that sycorax embryos display abnormalities in all the processes in which TFIIH plays a role; therefore, the sycorax mutant provides a novel opportunity to further study the function of TFIIH in an entire vertebrate organism. Strikingly, as the sycorax intestinal phenotype is reminiscent of a pre-neoplastic lesion, and the oncogenes c-myc and c-myb are upregulated in the sycorax intestine, this study has identified a possible role for altered TFIIH function in CRC.
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ItemThe association of antiepileptic drugs (AEDs) with bone disease, balance function, falls and fractures: studies of the risk( 2012)Aims: Patients with epilepsy taking long-term anti-epileptic drug (AED) therapy have been reported to have an elevated risk of fractures, falls, balance impairment and bone disease. In the work for this thesis, four studies were undertaken which were designed to investigate the associations of AED therapy with the prevalence of falls and fractures in patients taking AEDs compared to non-AED users; to assess the level of patient awareness regarding AED-related bone health, falls and fracture risk; to examine potential associations of AED therapy, both recent and long-term, on bone measures; and to evaluate any association of chronic therapy on balance function. Included in the evaluation were risk factors including age, AED polytherapy, therapy length, gender and types of AED (enzyme-inducing vs. non-enzyme inducing). Methods: Data on falls, fracture history and associated risks were collected in 150 epilepsy outpatients taking AEDs and 506 controls, and analysed cross-sectionally by univariate and multivariate methods to assess the prevalence of falls and fractures, and associated factors. Baseline and follow-up bone assessments (separated by at least two years) were performed in two cohorts, including: i) 54 twin and sibling pairs discordant for chronic therapy, and ii) 57 newly-diagnosed epilepsy patients recently started on therapy and 54 controls, using dual energy X-ray absorptiometry, and peripheral quantitative computed tomography. Balance examinations were performed for 26 AED-discordant twin and sibling pairs, with re-assessment at least one year later. The annual rate of change of bone and balance measures were calculated. Cross-sectional and longitudinal data were analyzed by univariate and multivariate analyses. Results: Compared to controls, epilepsy outpatients had a higher risk of fractures at vertebrae (p = 0.037), clavicle (p = 0.019) and ankle sites (p = 0.048), and multiple fracture episodes (p = 0.008). Female users had more non-seizure falls (31% vs. 17%, p = 0.027) and multiple falls (18% vs. 5%, p = 0.028) compared to female non-users. Less than 30% of patients knew of the association of AED use with increased risk for fractures, reduced bone mineral density (BMD) or falls. Prolonged AED therapy (> 20 years) significantly predicted an increased rate of bone loss for forearm BMD (-0.53%/yr; p = 0.040) and whole-body BMD (-0.37%/yr; p = 0.043). AED therapy was a significant predictor of increased bone loss at whole-body BMC (-0.26%/year; p = 0.041). Use of enzyme-inducing AEDs was associated with increased bone loss at the total hip (-1.65%/yr; p = 0.013) and whole-body BMD (-1.41%/yr; p = 0.019). In the initial years of AED therapy carbamazepine monotherapy (an enzyme-inducer) was associated with increased bone loss at the total hip (p = 0.034) and inter-trochanteric regions (p = 0.035). Postural sway deterioration was greater in AED users than non-users, with anterior-posterior (A-P) tilting and a concurrent distraction task (p = 0.016), and medial-lateral tilting without distraction (p = 0.027). In females (but not in the small male subsample), sway deterioration was greater in users with the A-P tilting platform and a concurrent distraction task (p = 0.035). Lower limb muscle strength and gait did not deteriorate over time. Conclusions: AED users demonstrated a higher risk of fractures, particularly with prolonged therapy. Female users were at increased risk of falls than female non-users. Greater BMD loss was associated with prolonged therapy and enzyme-inducing AEDs. Carbamazepine use in early therapy predicted increased hip region bone loss. Postural stability deteriorated over time in AED users compared to non-users. Awareness amongst epilepsy patients of AED-related effects was low. Findings suggests an association between chronic therapy and progressive underlying pathophysiology, adversely affecting bone measures and balance, both risk factors for fractures. Patients may benefit from taking NEIAEDs, rather than EIAEDs, to reduce associated long-term risks of bone fragility. These risks need consideration when managing patients taking AEDs.
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ItemInsights into mechanisms and effects of omega-3 polyunsaturated fatty acid supplementation in human atrial fibrillation( 2012)Atrial fibrillation (AF) is the most common cardiac arrhythmia in humans, causing significant morbidity, mortality and health care expenditure. Anti-arrhythmic drugs are the cornerstone of AF management but are limited in their efficacy, side effects and potential for pro-arrhythmia. A concordance of in vivo and in vitro animal experimental studies have demonstrated that omega-3 polyunsaturated fatty acids (ω-3 PUFAs) in fish oils may have anti-arrhythmic and anti-fibrillatory effects. However, definitive demonstration of the efficacy of fish oils in human AF has remained elusive. This thesis systematically evaluates the mechanisms and effects of ω-3 PUFAs in human AF incorporating the vast body of pre-clinical information from animal experimental paradigms, information on ω-3 PUFA dosing, duration of supplementation, kinetics of membrane incorporation and the importance of form of administration. The thesis characterises the effects of ω-3 PUFAs on human atrial electrical and mechanical function and examines the efficacy of ω-3 PUFAs on clinical AF endpoints. Chapter 1 describes the current state of knowledge about AF mechanisms in the absence and presence of structural heart disease, mechanisms of action and efficacy of contemporary anti-arrhythmic and anti-remodeling drugs and the pre-clinical and clinical evidence for ω-3 PUFAs in cardiac arrhythmias. Chapter 2 assesses the use of AF inducibility as a metric for future experimental chapters and its appropriateness for use as a clinical endpoint after electrical isolation of the pulmonary veins for AF. This study demonstrates that in the absence of structural heart disease or clinical AF, inducible and sustained AF occurs at a similar frequency to that noted in patients with a history of AF. It highlights the criticality of the induction protocol, inducibility definitions and number of inductions on rates of AF inducibility and persistence. Chapter 3 and 4 are the first randomised human studies of their kind to examine the effects of long-term ω-3 PUFA supplementation (>30 days) on human atrial electrophysiology in the absence of confounders such as structural heart disease or a history of clinical AF or flutter (Chapter 3) and on pulmonary vein and left atrial electrophysiology in the presence of paroxysmal AF (Chapter 4). Both studies showed that long-term supplementation results in chronic incorporation of ω-3 PUFA in plasma phospholipids. This results in significant prolongation of right, left atrial and pulmonary venous refractoriness. Long-term oral ω-3 PUFAs had no effect on atrial or pulmonary venous conduction. Most importantly, both studies showed a significant reduction in vulnerability toward and persistence of AF, an effect attributed to refractory period prolongation. Together, these studies provide mechanistic insights into the anti-fibrillatory effects of long-term oral ω-3 PUFA exposure. Chapter 5 examines the effects of long-term ω-3 PUFA supplementation on human atrial mechanical function after reversion of persistent atrial arrhythmias to sinus rhythm. Reversion of persistent atrial arrhythmias to sinus rhythm is associated with transient depression of left atrial mechanical function, a phenomenon known as atrial mechanical stunning. Stunning is implicated in the heightened risk of thromboembolic complications such as stroke, failure of improvement in cardiac output and exercise tolerance, and increased risk of recurrence atrial arrhythmias. The main finding was that patients randomised to fish oils, compared to controls, had markedly reduced incidence of atrial mechanical stunning. This study provides insights into how fish oils can attenuate adverse atrial remodeling in response to persistent atrial arrhythmias. Chapter 6 is a randomised clinical trial examining the efficacy of long-term fish oils in the prevention of persistent AF recurrence post electrical cardioversion in a high risk population. Patients were randomised to control or fish oil groups; the latter was commenced >1 month prior to cardioversion and continued till return of AF or maximum of 1 year. The main finding was that fish oil patients had higher rates of pharmacological reversion, and a significantly lower risk of persistent AF recurrence compared to controls at 1 year. These effects were seen in the presence or absence of concurrent anti-arrhythmic drugs. This study shows the critically of long-duration, high dose supplementation to allow sufficient time for maximal myocardial incorporation and for the all expected electrophysiologic, anti-remodeling and anti-inflammatory mechanisms of ω-3 PUFA to take effect before assessment of clinical endpoints, an approach rarely used in previous clinical studies. Chapter 7 is a prospective randomised study examining if long-term ω-3 PUFA supplementation (6 or 12 months) reduces burden of paroxysmal atrial tachycardia/fibrillation (AT/AF) in a high risk population of elderly patients with sinus node dysfunction, implanted dual chamber pacemakers and a history of the same arrhythmias. The main findings were that whilst fish oils did not suppress AT/AF burden per se, they significantly attenuated temporal progression of AT/AF burden over time that was seen in controls over 18 months of follow up. There was no demonstrable effect on arrhythmia triggers, but significantly shorter episodes of AF were seen suggesting that the predominant effect of fish oils are on atrial substrate. This study demonstrates that long-term ω-3 PUFA supplementation reduces paroxysmal atrial arrhythmia burden likely mediated by effects on atrial structural remodeling. Chapter 8 is the first human study of its kind examining the effects of acute, intravenously delivered, high dose ω-3 PUFAs on human atrial electrophysiology. The main findings were that when given intravenously, fish oils are predominantly present as free ω-3 PUFAs, with little or no membrane incorporation. Free ω-3 PUFAs predominantly caused atrial conduction slowing with minimal effects on atrial refractoriness which was in contrast to the previous observations that incorporated ω-3 PUFAs had no effect on atrial conduction, but prolonged atrial refractoriness. Moreover, free ω-3 PUFA reduced AF inducibility and persistence, organised inducible AF into atrial flutter and significantly increased the inducibility of atrial flutter in patients with no clinical history of this arrhythmia. This study provides novel insights into the complex effects of incorporated versus free ω-3 PUFAs on atrial electrophysiology and provides evidence for a future clinical study examining the efficacy of high dose IV fish oil on acute AF termination.
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ItemRegulation of receptors in TGF-β signaling and its impact in diseases( 2012)Appropriate cellular signaling is essential to control cell proliferation, differentiation and cell death. Aberrant signaling can have devastating consequences and lead to disease states including cancer. The transforming growth factor-β (TGF-β) signaling pathway is a prominent signaling pathway that has been tightly regulated in normal cells while its deregulation strongly correlates with the progression of human cancers. The regulation of the TGF-β signaling pathway involves a variety of physiological regulators. Many of these molecules act to alter the activity of Smad proteins. In contrast, the number of molecules known to affect TGF-β signaling pathway at the receptor level is relatively low and there are no known direct modulators for the TGF-β type II receptor (TβRII). Here we identify SPSB1 (a SPRY domain-containing SOCS box protein) as a novel regulator of the TGF-β signaling pathway. SPSB1 negatively regulates TGF-β signaling pathway through its interaction with both endogenous and over-expressed TβRII (and not TβRI) via its SPRY domain. As such, TβRII and SPSB1 colocalize on the cell membrane. SPSB1 maintains TβRII at a low level by enhancing the ubiquitination levels and degradation rates of TβRII through its SOCS box. More importantly, silencing SPSB1 by siRNA results in enhanced TGF-β signaling and migration and invasion of tumor cells. Thus SPSB1 acts as a new regulatory component of the TGF-β signaling pathway that targets TβRII for degradation and provides fine control of its signaling. Transformation by oncogene Ras overcomes TGF-β mediated growth inhibition in epithelial cells. However, it cooperates with each other to mediate epithelial to mesenchymal transition (EMT). The mechanism how these two pathways interact with each other is controversial. Here we report that Ras interacts with the SPRY domain of the TGF-β type II receptor (TβRII) targeting molecule SPSB1 to enhance TGF-β signaling. Oncogenic Ras interacts and colocalizes with the TβRII negative regulator SPSB1 on the cell membrane, consequently promoting SPSB1 protein degradation via enhanced mono- and di-ubiquitination. Reduced SPSB1 levels result in the stablization of TβRII, in turn the increase of receptor levels significantly enhace Smad2/3 phosphorylation and signaling. Importantly, forced expression of SPSB1 in Ras transformed cells suppresses TGF-β signaling and its mediated migration and invasion. Lastly, this study tested a strategy for non-invasive intracellular delivery of the SPSB1 protein into living cells by fusing a membrane penetrating sequence (MTS) to its N-terminal (MTS-SPSB1). One of the original SOCS-box containing proteins, SOCS3 has been developed into an experimental therapeutics treating inflammation in mouse model by fusing MTS with SOCS3, making it cell penetratable. I generated the MTS-SPSB1 protein by using the insect cell expression system. My results reveal that the MTS fusion does not affect the function of the SPSB1 protein, however, it results in the anchoring of the recombinant MTS-SPSB1 protein on the outer cell surface. As a result, the internalization of the recombinant MTS-SPSB1 protein in cells is likely mediated by the endocytic pathways. This is the first report showing the MTS fusion can result in the anchoring of the cargo protein on the outer cell surface.
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ItemBiliary tract injury( 2012)Aim - The aim of the thesis was to examine the causes, mechanisms, recognition and treatment of biliary injury. The hypothesis was that the management principles for biliary injury were similar regardless of the cause. Methods - Biliary injuries secondary to operative damage (iatrogenic), following transplantation or as a result of blunt or penetrating (traumatic) trauma were examined. The following databases were analyzed; A prospective database of biliary injuries in Victoria from 1997 - 1999, a database of iatrogenic biliary injuries from the Royal Infirmary of Edinburgh from 1984 - 2003, a prospective database for all liver transplants performed by the Scottish Liver Transplant Unit (SLTU) until September 2001 and the prospectively gathered trauma registry at The Royal Melbourne Hospital from 1999 - 2011. Retrospective case note review was performed for further data collection. Patient data was entered onto a Microsoft Access database and statistical analysis performed with SSPS versions using Cox regression for multivariate analysis, the Mann Whitney U test for independent variables and the Log rank test when appropriate. Not all data sets were of sufficient size to allow statistical analysis. Management of biliary injury included non-operative, percutaneous, endoscopic and surgical options. Results - Iatrogenic injuries were recorded in 33 patients from the Victorian audit and 123 patients from the Royal Infirmary of Edinburgh. Fifty five (14.6%) of 379 consecutive orthotopic liver transplants at the SLTU had biliary complications. Thirty three patients (0.1%) of 26,014 blunt and penetrating trauma patients had injuries to the biliary tree and gallbladder. Of the 123 iatrogenic injuries from the Royal Infirmary of Edinburgh, 55 (44.7%) had an attempted repair prior to referral, 59 (47.9%) were repaired after referral and 9 (7.3%) were managed without surgery. For the 59 patients repaired after referral a successful repair was possible in 22 (88%) of 25 patients repaired within the first two weeks compared with 20 (91%) of 22 repaired after 6 weeks (p=0.615). Nine patients were considered for hepatic resection. Five patients developed hepatic failure and were considered for liver transplantation with only two reaching transplantation. Of the 55 grafts from the SLTU with biliary complications, 28 biliary leaks occurred with 17 anastomotic leaks successfully treated non-operatively. Of the thirty anastomotic strictures, six (38%) of the 16 early anastomotic strictures required surgery for complete resolution, compared with 12 (86%) of the 14 late anastomotic strictures (p=0.0106). Of the blunt and penetrating biliary injuries there were 10 gallbladder and 23 biliary tree injuries. Fourteen patients had injuries to the intra-hepatic biliary tree and nine to the extra-hepatic biliary tree. Delay in the recognition of biliary injury following iatrogenic injury continues to be prevalent, with the delay often associated with sepsis, jaundice and peritonitis. Injury following liver transplantation is complicated by the association of hepatic arterial thrombosis and immunosuppression, whilst traumatic injury is frequently associated with intra-abdominal organ injury. The timing of repair and utilization of temporizing measures such as biliary drainage depends upon the associated injuries and presence of sepsis or jaundice. For all types of biliary injury, surgical reconstruction with Roux-en-Y hepaticojejunostomy remains the gold standard for repair. Successful long lasting repair is possible in the majority when managed by a specialist hepatobiliary team. Conclusion - The management of biliary injuries is multi-factorial and requires tailoring according to patient variables. However, common management pathways exist regardless of the cause.
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ItemThe effect of patient factors and clinician choices on management of colorectal cancer and other malignancies( 2011)Cancer is one of the leading causes of morbidity and mortality world-wide, and colorectal cancer is one of the most common malignancies in the developed world. Being able to predict the most appropriate strategies for diagnosis, treatment and monitoring for any malignancy, both in the adjuvant and metastatic disease settings, is crucial as more management options come into play. In particular, the choices surrounding chemotherapy dosing can depend on many factors, and increasing interest is developing regarding optimization and individualization of treatment strategies for cancer based on these factors. Modern oncology is currently focused on biomarker-based research and translation to care. Although this thesis does not incorporate any pre-clinical biomarker-based research, patient variables (such as age and comorbidities) can be regarded as a type of clinical ‘biomarker’ – for example, age is a very strong prognostic factor for a number of malignancies, perhaps even more important than particular laboratory-based biomarkers in many circumstances. This research will focus on key aspects of patient care, from surgery to chemotherapy, radiation therapy and disease monitoring, which may be potentially regarded as ‘biomarkers’ - stratifying patients into those who may benefit the most, and least, from various treatment modalities and strategies. This body of work focuses primarily on colorectal cancer. The thesis provides a comprehensive ‘snapshot’ of current management strategies in Australia for colorectal cancer – from diagnosis through to surgical and oncological management – and each paper compares the findings with what is currently regarded as ‘gold-standard’ practice. It is well known that patients on clinical trials are mostly younger and fitter than those seen in routine practice, and treating physicians cannot always apply the findings from randomized controlled studies to the individual cancer patient. It is useful to understand in parallel with the evidence gained from clinical trials, its applicability and modifications employed in routine practice and as such, this research has been largely conducted using a prospectively collected comprehensive cancer database; together with surveys of Australian oncologists and review of available literature. Many issues requiring treatment decisions for colorectal cancer are also applicable to many malignancies, and the thesis also includes some papers which relate to cancer management in general – in particular, the impact of patient age and comorbidity on treatment decisions, and the effect of liver dysfunction on chemotherapy choices for cancer patients.