Medicine & Health

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  • (2018) De Rosa, Nicholas
    Thesis
    Introduction: Skin disease is common in immunosuppressed solid organ transplant recipients (SOTRs) and skin cancer, in particular, is a major cause of morbidity and mortality in these patients. This study aims to determine the rates and risk factors for skin cancer in Australian heart and lung transplant recipients (HLTRs). It also aims to examine the spectrum of skin diseases encountered in HLTRs and their effect on quality of life (QOL). Methods: Ninety-four participants were recruited from the Dermatology Outpatient Clinic at St. Vincent’s Hospital Sydney between March and December 2016. Retrospective skin cancer diagnoses were obtained from medical records and participants were also examined prospectively for malignant and non-malignant skin disease. A questionnaire and the Dermatology Life Quality Index were administered to all participants. The probabilities of developing non-melanoma skin cancer (NMSC), squamous cell carcinoma (SCC), and basal cell carcinoma (BCC) were estimated using Kaplan-Meier analysis. The association of risk factors with skin cancer development were examined using the Cox proportional hazards model. The association of examined variables with DLQI score were assessed using univariate and multivariate logistic regression analysis. Results: Retrospectively, there were 801 post-transplant skin cancers in 61% of participants. At 5 and 10 years post-transplantation the probabilities were 41% and 67% for developing NMSC, 27% and 53% for developing BCC, and 33% and 62% for developing SCC, respectively. A prospective dermatological diagnosis was made in 87% of the participants over the study period. Actinic keratosis was the most common diagnosis, affecting 53% of participants, followed by skin cancer in 44% and warts in 15% of participants. Other non-malignant skin diseases were less common. Risk factors significantly associated with skin cancer included older age at transplantation, history of pre-transplant skin cancer, and history of ≥5 post-transplant skin cancers. Fitzpatrick skin type 3-6 was associated with a decreased risk of NMSC. Skin disease had a negative effect on the QOL of a minority of HLTRs. The use of tacrolimus was associated with better QOL scores on multivariate analysis. Conclusion: Australian HLTRs have high rates of skin cancer that exceed the rates reported for other SOTRs.

  • (2015) Beniamen, Daniella
    Thesis
    The alpha (1A)-adrenergic receptors (α1A-ARs) play a pivotal role in regulating cardiac contractility. However direct evidence for their action on myocardial microvasculature is not known. Myocardial angiogenesis has generated great interest for its potential as a therapeutic alternative in the treatment of ischaemic disorders. Current pro-angiogenic therapies aim to instigate de novo formation of microvessels with the potential of reversing myocardial injury due to ischaemia, although the underlying mechanistic role is yet to be elucidated. To examine this we investigated the angiogenic role of the α1A-AR in mice with cardiac-specific transgenic (TG) α1A-AR overexpression (66-fold). At baseline, α1A-AR transgenic adult mice displayed enhanced angiogenesis within the myocardium compared to their non-transgenic littermates (NTLs) through up-regulated VEGF-A mRNA expression in isolated cardiomyocytes (CMs), and increased myocardial capillary density. This study investigates the mechanistic role implicating α1A-ARs expressed specifically on CMs in stimulating angiogenesis through VEGF-A and thereby enhancing myocardial perfusion. At 24 hours and 7-days post-myocardial infarction (MI) performed by occlusion of the left anterior descending artery (LAD), infarct size and CM apoptosis was markedly reduced, whereas CM proliferation was increased in α1A-AR TG mice, indicating a delay in left ventricular remodelling and increase in CM viability. Capillary density also increased in the infarct, border and remote zones of α1A-AR TG mice subjected to MI for 7 days compared to NTLs, suggesting a cross-talk between CMs and endothelial cells (ECs) promotes neo-angiogenesis that favourably enhances myocardial perfusion to areas under ischaemic stress. Utilizing an In vitro model, human umbilical vein endothelial cells (HUVECs) were treated with cultured CM conditioned medium from either α1A-AR TG mice or NTLs with or without the α1A-AR agonist A61603. We reported an A61603-dependent increase in HUVEC tubule formation on matrigel and increased VEGF-A mRNA expression in α1A-AR CMs receiving 25nM A61603 treatment compared to NTLs. Thus increased vessel formation suggests that the paracrine mechanism of action between CMs and ECs is initiated by VEGF-A up-regulation due to α1A-AR stimulation. Our data reveal the α1A-AR to be capable of activating EC migration, sprouting and maturation within the cardiac microenvironment, such that is necessary for enhanced myocardial perfusion.

  • (2018) Kadam, Pooja
    Thesis
    Introduction Telangiectatic matting (TM) is a morphological description referring to vessels with a small diameter of less than 0.2 mm that can appear sporadically or in well-defined patches (hence the term ‘matting’) primarily on the lower limbs. The aetiology underlying TM remains uncertain however angiogenic and inflammatory mechanisms are believed to play a pathogenic role. The aim of this study was to investigate the pathogenesis of TM and identify possible risk factors. Methods This study had two parts. The clinical records of consecutive patients were retrospectively analysed to identify risk factors for TM. In the second part, the haemostatic and coagulation profile of 12 patients with TM were analysed and compared with 12 controls using standard coagulation tests, platelet function and a global assay of coagulation namely rotational thromboelastometry (ROTEM). Results In 352 consecutive patients presenting to a phlebology practice, 25 patients had TM (7.1%). All 25 patients were female with the median age of 45 (27-57) years. A comprehensive medical history was taken. Amongst 27 possible risk factors assessed, statistically significant associations included recurrent epistaxis, easy bruising, hypersensitivity (eczema, hives, hay fever, rhinitis), previous treatment with sclerotherapy or vascular laser for lower limb veins and a family history of telangiectasias. The haemostatic and coagulation profile of patients with TM did not differ significantly from those without TM. Conclusion TM is associated with both hypersensitivity and a bleeding tendency. Our study revealed no significant increase in the incidence of haemostatic abnormalities in patients with TM compared with the control group. Given the significant association with hypersensitivity disorders we believe underlying mast cell hyperreactivity may contribute to both hypersensitivity and a bleeding tendency and predispose patients to TM. Further research studying mast cell activity is warranted to establish a definite link.

  • (2018) Kozan, Pinar
    Thesis
    Background: High dietary acid load relates to increased risk of type 2 diabetes in epidemiological studies. We aimed to investigate whether buffering a high acid load meal with an alkalising treatment changes post meal glucose metabolism. Methods: Non-diabetic participants (n=32) were randomized to receive either NaHCO3 1680mg or placebo, followed by a high acid load meal in a double-blind placebo-controlled crossover (1-4 weeks apart) study. Thirty (20 males) participants completed the study. Venous blood pH, serum bicarbonate, blood glucose, serum insulin, C-peptide, non-esterified fatty acid (NEFA), and plasma glucagon-like peptide-1 (GLP-1) concentrations were measured at baseline (fasting) and at 15-30min intervals for 3h post meal. Results: The treatment was well tolerated. Venous blood pH declined in the first 15min post meal with the placebo (p=0.001), but not with NaHCO3 (p=0.86) and remained lower with the placebo for 3h (pinteraction=0.04). The iAUC of pH was significantly higher following the NaHCO3 treatment versus the placebo (p=0.02). However, postprandial glucose, insulin, C-peptide, NEFA and GLP-1 were not different between treatments (pinteraction≥0.07). Conclusions: An alkalising medication administered pre-meal has no acute effect on glycaemia and insulin response in healthy individuals. Long-term interventions in at-risk populations are necessary to investigate the effect of sustained alkalisation on glucose metabolism.

  • (2017) Cao, Yiming
    Thesis
    Liver transplantation is a highly efficacious intervention for end stage liver failure. Unfortunately, there is considerable waiting time for suitable donor organs leading to significant post-operative morbidity and mortality from marginal donors and recipients, as well as through attrition on the waiting list. The introduction of donation after circulatory death (DCD) organs has to some extent relieved the stress on the waiting list, but unfortunately, these organs are associated with worse outcomes than the more traditional form of donation – donation after brain death. As such, interventions to improve these organ qualities are pivotal in facilitating maximal benefit to be derived from these valuable donations. The current thesis seeks to explore such measures from an experimental and clinical perspective. The former is with regards to the design of a clinically applicable large animal model of donation after circulatory death, followed by ex situ perfusion of the isolated liver. The latter is concerned with donor practices, especially life support withdrawal practices and administration of ante-mortem heparin, that can be modified to benefit the recipient without impacting on the end of the life care of the donor. Our experimental results demonstrated the feasibility of maintaining the hepatocellular function for up to 4 hours using ex situ perfusion of livers retrieved under a clinical DCD protocol. Potential mechanisms are discussed, but it is clear that ex situ perfusion is able to mitigate a portion of the damage incurred by the liver during the preceding warm ischaemia. Furthermore, our clinical results indicate that further minimisation of the warm ischaemic time by withdrawing donor life support in the operating theatre as opposed to intensive care unit can confer significant benefit upon the recipient, with additional benefits afforded by administration of ante-mortem heparin. In conclusion, we have been able to demonstrate that warm ischaemia is a critical factor in determining subsequent recipient outcomes, and minimisation of this time period even within the acceptable threshold of 30 min can have significant benefits. Once warm ischaemia has occurred, it is possible to reverse some of the damage. This can be achieved by normothermic ex situ perfusion, a technique that should be subject to more rigorous investigation in a clinical or clinically applicable animal setting in the future.

  • (2016) Rasouli, Mina
    Thesis
    Recent efforts in cancer therapy investigations have been focused on developing a rational strategy to specifically target cancer cells to reduce toxic side effects. Uncontrolled cell division (mitosis) is a hallmark of cancer cells. Classical anti-mitotic drugs such as Taxanes and Vinca alkaloids work by disrupting microtubule dynamics, which results in a block in cell division and induction of mitotic arrest. This prolonged mitotic arrest can trigger cell death, which is often curative in some cancer patients. However, despite the success of these chemotherapies, many patients have innate or develop acquired resistance. Furthermore, higher doses of these drugs cause toxicities, which limit the efficacy of these compounds. To overcome these limitations research has focused on finding novel drug combinations that enhance the effects of mitotic chemotherapies. Recently, blocking mitotic exit has emerged as a promising strategy for killing cancer cells and enhancing mitotic chemotherapy response. In order to find novel regulators of mitotic exit, we analysed published genome wide RNA interference screens and online-databases to identify novel genes whose knockdown increased mitotic index and raised the sensitivity of cells to Taxol. In this thesis, we explored two of these potential new target genes, STK16 (serine/threonine kinase 16) and Protein phosphatase 2 A (PP2A). The effects of targeting STK16 and PP2A (using specific siRNA or chemical inhibitors) were examined in combinations with Taxol. HeLa cells stably expressing histone H2B fused to red fluorescent protein (H2B-mCherry) were treated with combination of drugs and monitored by live cell imaging systems. Phenotypes of the cells were tracked during the treatment to measure duration of mitotic arrest and percentage of dead cells. Knockdown of STK16 failed to affect mitotic progression or the duration of mitotic arrest in combination with Taxol. In contrast, siRNA knockdown of the B55 subunit of PP2A and inhibition of PP2A with the chemical inhibitor (okadaic acid) both extended duration of mitotic arrest and significantly increased the percentage of mitotic death in HeLa cells. By rescuing mitotic arrest, we also showed that there was link between duration of mitotic arrest and death in HeLa cells. However, these findings were not replicated in two other Taxol resistant cell lines. These cell lines showed significant increases in mitotic length in response to PP2A inhibition; however, this failed to translate into increased sensitivity to Taxol. Taken together these results indicate that blocking mitotic exit may be a potential target for increasing the efficiency of anti-mitotic drug therapy in some cells such as HeLa cell line but it could not be generalized in all cells.

  • (2013) Chopra, Samarth
    Thesis
    Aims and Objective: Monotherapy for localised prostate cancer with radical prostatectomy, external beam radiotherapy or brachytherapy produce similar oncological outcomes. Hence there is increasing need to evaluate and communicate the quality of life outcomes of these treatments. The aim of our prospective study was to determine how the various types of radical treatment at a tertiary referral centre affected the health related quality of life (HRQOL) domains of urinary, sexual, bowel, and hormonal features relative to men managed with active surveillance. Materials and Methods: We measured HRQOL outcomes from baseline through to 24 months after treatment using the EPIC questionnaire (Expanded Prostate Cancer Index Composite) in a cohort of 851 patients first treated for stage T1 or T2 prostate cancer from May 2007 to February 2011. Patients elected one of five management options: (1) Robotic prostatectomy (RALP) further subdivided by surgeon experience (1a) RALP <200 and (1b) RALP 200+ (2) Radical retro pubic prostatectomy (ORP); (3) High dose rate brachytherapy (HDR); (4) Low dose rate brachytherapy (LDR) and (5) active surveillance (AS). Demographic and treatment variables were recorded at baseline. We used multivariate linear regression analyses with generalised estimating equations (GEE) to estimate adjusted mean differences in outcome scores between treatments. Results: A total of 770 (96%) men with complete data were included in the analysis. All baseline clinical and demographic characteristics showed significant heterogeneity within treatment types (P<0.001). Questionnaire completion rates ranged from 88% to 100% for any given treatment/month. There was a significant change in sexual quality of life in each group (P<0.005) from baseline to follow-up except in the AS group. Men in the RALP 200+ showed a significant trend towards return to baseline urinary and sexual function. Men in the HDR or LDR brachytherapy groups had worse bowel function and urinary irritation/obstructive scores after treatment but these scores improved between 12-24months. Conclusion: Treatment for localised prostate cancer has significant and persistent adverse effects on quality of life. However, men managed by active surveillance maintained a reasonably stable quality of life scores across all EPIC domains. In the absence of randomized clinical trials, observational studies such as ours can help portray a realistic expectation in terms of changes in quality of life for men treated for localised prostate cancer.

  • (2012) Agapiou, David
    Thesis
    A previous study published by us identified twelve differentially expressed microRNAs between AML FAB M1 and M5, from these five candidate microRNAs (microRNA-130a, -135b, -146a, -181a and -181b) were selected for further study based on their predicted targeting for inhibition of nine transcription factor mRNAs involved in myeloid differentiation and development (Lutherborrow et al., 2011). In this study through the use of a luciferase reporter assay six of the predicted gene targets (FOS, HOXA10, MAFB, IRF8, KLF4 and MCL-1) identified were as bona fide targets of four of the five candidate microRNAs. The next aim was to assess the effect of over-expressing the microRNAs in AML cell line models (HL-60 and NB4); optimisation of three transfection methods was undertaken. The lipid based transfection reagent lipofectamine 2000 delivered oligonucleotides into HL-60 and NB4 cells at transfection efficiencies of 10% and 40% respectively, but was unable to successfully introduce plasmid to levels required for functional studies. Electroporation was found to be highly variable and toxic resulting in high levels of cell death when introducing oligonucleotides or plasmids. Nucleofection, a more specific method of electroporation, was optimised and was able to successfully transfect a GFP plasmid into the HL-60 cell line at efficiencies of 21-46%, but was unable to transfect oligonucleotides. In preparation for future studies based on the transfection optimisation results, inducible microRNA expression vectors for the candidate microRNAs were successfully cloned. The third part of my work was to examine changes in the candidate microRNA expression levels during normal primary macrophage differentiation to determine if published changes seen in cell line experiments occur in primary cells. Interestingly microRNA-146a was identified as being significantly increased during monocyte to macrophage differentiation in contrast to the published results. Since NF-κβ signalling is known to be important in malignancies, an experiment was performed examining the effect of the candidate microRNAs on NF-κβ signalling using a NF-κβ specific luciferase reporter. Of the candidate microRNAs, only microRNA-146a over-expression resulted in a statistically significantly decreased of luciferase activity (by 60%) and therefore NF-κβ signalling. In summary this work has identified six microRNA:mRNA interactions involved in the myeloid development and it can be hypothesised that these interactions could be involved in the formation of the myeloid differentiation blocks seen in AML. Transfection optimisation has elucidated that stable plasmid over-expression is the best method for microRNA alteration in AML cell lines. Additionally this study has provided a foundation for future projects to further examine the roles of these microRNAs in the pathogenesis of AML.

  • (2011) Porta Cubas, Ana
    Thesis
    Breast cancer is extremely heterogeneous, to the extent that some consider it to be composed of distinct diseases. Over the last 10 years, research has driven a new classification of Breast cancers, based on the expression of different molecular markers. This newer system is able to divide Breast cancers into at least 5 different subgroups. One subgroup, the ‘Basal breast cancers’ is of particular concern due to its younger age at diagnosis, increased aggressiveness, shorter survival and lack of targeted therapies. Studies have found that cell lines representing this subtype are sensitive to the multikinase inhibitor ‘Dasatinib’. This suggests that kinase/s targeted by Dasatinib may be overexpressed or deregulated in Basal breast cancers, and may thus represent novel therapeutic targets. To investigate this hypothesis, mRNA and protein expression of some of the known Dasatinib kinase targets was compared between cell lines representing Basal breast cancer and the less aggressive Luminal breast cancer. One candidate kinase, Lyn, was significantly overexpressed at the mRNA and protein level in Basal versus Luminal breast cancer lines, and exhibited increased activity in the Basal subgroup. To investigate the mechanisms regulating Lyn activation, cell lines were stimulated with candidate growth factors. HGF stimulated Lyn activity in one cell line suggesting that Lyn forms part of the Met signalling pathway. However, Lyn knock down did not affect the activity or total levels of signalling proteins comprising the Met pathway, nor did it affect HGF-induced cell scattering. Interestingly, Lyn depletion did affect the morphology of cells upon starvation, with cells becoming more dissociated and fibroblastic. Lastly, the translational relevance of these findings was tested by investigating Lyn expression in a Breast cancer patient cohort by immunohistochemical staining. Lyn was found to be significantly overexpressed in the Basal subgroup of patients and strongly associated with it. In addition, Lyn expression was associated with poor prognosis. Thus, while the functional role of Lyn in Basal breast cancer requires further characterisation, it represents a novel biomarker of the Basal breast cancer subgroup and may represent a therapeutic target in Basal breast cancer cells.

  • (2010) Rao, Geetha
    Thesis
    The interactions between the nervous and immune systems have been explored over the last thirty years. Neuropeptide Y (NPY) and its receptors (Y1, Y2, Y4, Y5 and y6) have been closely studied in this area. NPY is a stress hormone expressed both in the nervous and immune systems. Furthermore, receptors such as Y1 are also expressed in immune tissue. Previous work from our lab using mice that lack Y1 (Y1-/-) has revealed that Y1-/- mice have significantly reduced mature B cell numbers in the periphery. Otherwise, very little is understood regarding the NPY system and B cells. Therefore, the purpose of this thesis was to investigate the role of the NPY system on B cell function and development. Initially, B cells were investigated in mice deficient in NPY (NPY-/-), PYY (a close relative of NPY) and mice deficient in both NPY and PYY (NPY-/-/PYY-/-). Profiling of B cell compartments showed some differences, where NPY-/- and NPY-/-/PYY-/- mice had a significantly reduced number of transitional type 2 B cells due to a decrease in CD23 expression. These mice also had impaired post-immune IgG3 production. However, these findings were later attributed to the genetic background of the mice, rather than the loss of NPY. B cells were then assessed in Y1-/- mice. Similar to previous findings, Y1-/- mice had reduced B cell numbers in peripheral lymphoid organs, however, the reason for this remains unclear. Analysis of B cells from mice lacking Y1 in B cells only and from mice in a reciprocal bone marrow chimera experiment could not conclusively show whether reduced B cell numbers were due to direct or indirect Y1 receptor signaling on B cells. When analysing Y1-/- B cell proliferation in vitro, a significantly higher response to stimulation via toll-like receptor (TLR) 9 was observed when compared to controls, indicating a role for the Y1 receptor in a TLR9 mediated response. Finally, an antibody reacting specifically to NPY was generated from a C57BL/6 mouse. In conclusion, despite some experimental difficulties encountered, this thesis provides further evidence for neuro-immune crosstalk, with specific reference to B cells.