Medicine & Health

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Now showing 1 - 10 of 30
  • (2021) Hanssen, Kimberley
    Thesis
    Multidrug resistance is a leading contributor to treatment failure in cancer patients. As elevated levels of the antioxidant glutathione (GSH) and increased expression of drug efflux pumps may both contribute to chemotherapy resistance, depleting GSH and blocking drug efflux in cancer cells may improve response to treatment. Multidrug resistance protein 1 (MRP1) is a membrane transporter that is frequently overexpressed in cancer cells. By actively effluxing a wide range of chemotherapeutic agents, MRP1 can protect the cancer cell from chemotherapy. GSH is also transported by MRP1 as a low-affinity endogenous substrate. Some small molecules (MRP1 'modulators'), upon binding to MRP1, greatly enhance this innate GSH transport whilst simultaneously blocking the efflux of chemotherapeutics. As this stimulated GSH efflux can deprive the cell of GSH, in parallel with the blocked chemotherapy efflux enhancing intracellular chemotherapy concentrations, MRP1 modulators might be used to exploit the high expression of MRP1 in cancer cells to improve treatment response. MRP1 was found to be frequently expressed in patient tumours of two difficult to treat cancer types: non-small cell lung (NSCLC) and ovarian cancer. As this MRP1 expression might be leveraged with an MRP1 modulator, two modulators were tested on high MRP1-expressing NSCLC and ovarian cancer cell lines. As single agents, the modulators reduced MRP1 drug transport by >85% and improved the efficacy of MRP1-substrate chemotherapeutics (2 5-fold). In combination with the GSH synthesis inhibitor buthionine sulfoximine, complete GSH depletion, diminished clonogenic capacity, enhanced radiosensitivity, and extended chemosensitivity were achievable selectively in high MRP1-expressing cancer cells. As MRP1 expression in NSCLC was also associated with NRF2 activation, a key driver of treatment resistance in NSCLC, the modulator and buthionine sulfoximine combination was tested and found to be effective against cell lines representative of this highly resistant subset of NSCLC. Given the therapeutic potential for MRP1 modulators, their MRP1 binding site and mechanism of action as GSH transport modulators was investigated to provide a basis for future structure-guided design of more potent and selective modulators. Overall, these findings support that the high expression of MRP1 in cancers can be exploited with MRP1 modulators to chemosensitise and radiosensitise NSCLC and ovarian cancers.

  • (2020) Chen, Kerrie-Anne
    Thesis
    This thesis examines the clinical translation of two novel therapeutic drugs in rare paediatric neurological disease via expanded access programs (EAP). Patients with rare diseases commonly encounter barriers to care and lack of disease-modifying treatments. EAPs help address this need by providing access to therapeutic drugs before commercial availability, however, there is usually limited knowledge regarding the safety and full impact of the drug. Two drugs were recently made available via an EAP for children in New South Wales: cannabidiol for paediatric drug-resistant epilepsy (DRE) and nusinersen for spinal muscular atrophy (SMA). In a time when there was no evidence regarding the use of cannabidiol in paediatric DRE, an EAP was established due to unprecedented patient demand. Our study analysed the safety, adverse effects and preliminary efficacy of cannabidiol in patients with DRE. Cannabidiol was well tolerated, with sedation commonly reported (37.5%). Elevated transaminases were seen in 5% and demonstrated the necessity of medical monitoring. Although many patients reported improved overall health, indirect measures of seizure control did not show improvement, thus signifying the need for larger, randomised-controlled trials. In children with SMA type 1 (SMA1) treated with nusinersen, respiratory, bulbar and nutritional outcomes were analysed, as previous studies had demonstrated increased survival and motor function but had not assessed the broader impact on the burden of disease. Our study demonstrated substantial ongoing comorbidities due to respiratory and bulbar weakness, with the need for ongoing nocturnal noninvasive ventilation, gastrostomy feeding and recurrent acute hospitalisations. Greater burden of disease was seen more in patients with two SMN2 copies. Most children showed improvement in motor outcome assessments, a stark change in the natural history of SMA. These findings show the impact of nusinersen in modifying the phenotype of children with SMA1, yet ongoing significant morbidities and requirement for multidisciplinary supportive medical care. In conclusion, these studies provide additional real-world clinical data on the implementation and extent of efficacy of two novel drugs for rare neurological diseases. EAPs serves to address an unmet clinical need and facilitate the translation of research into practice to advance future health practice and research in rare diseases.

  • (2021) Krysta-Matter, Adriana
    Thesis
    Background: Oocyte quality is the rate-limiting factor to the success of in-vitro fertilisation (IVF). Growth differentiating factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are key oocyte-secreted growth factors that act on the surrounding cumulus cells (CC) to regulate their functions and oocyte quality. Reliable quantification of these factors and the relationship between their levels and oocyte quality have not been demonstrated to date. Measuring GDF9 and BMP15 from CC discarded during IVF treatment could be useful for studying the role of these factors in female reproductive potential. Aim: To develop specific ELISAs for GDF9 and BMP15 quantification in discarded CC from ICSI patients and correlate their levels to patient characteristics and reproductive outcomes. Method: GDF9 and BMP15 ELISAs were developed and applied to extracts from discarded CC. Optimised extraction conditions allowed for a simultaneous extraction of protein and DNA (used as a measure of cell count for protein normalisation). A pooled extract from human granulosa cells was used as a standard, as non-parallelism was observed between CC extract and recombinant GDF9 and BMP15. Results: The ELISAs developed were specific for GDF9 and BMP15. Maternal age and polycystic ovaries/polycystic ovary syndrome were factors for change in BMP15 on pooled CC from individual patients in the first study (n=120) but not in the second (n=81). Embryo developmental trajectory was not associated with their levels. GDF9 and BMP15 measured on CC from individual oocytes (n=181) varied considerably between oocytes within and between patients, and GDF9 levels were higher in older patients. GDF9 and BMP15 were not associated with embryo developmental outcomes but their ratio was associated with blastocyst quality. Correlation between GDF9 and BMP15 on individual oocytes was remarkably strong (p<0.0001). Conclusion: The current study showed that levels of CC-bound human GDF9 and BMP15 were not linked with embryo developmental outcomes using existing assays. Studying the relative dosage of these proteins may provide a useful diagnostic. Strong correlation between GDF9 and BMP15 suggested that native GDF9 and BMP15 form a heterodimer in-vivo. Further research into the native forms of human GDF9 and BMP15, and their levels is needed to gain a better understanding of their physiological roles. Development of new generation assays able to measure different forms of the two proteins will be required

  • (2020) Chen, Jingwei
    Thesis
    TERT gene rearrangement with transcriptional super-enhancers leads to TERT over-expression and neuroblastoma. No targeted therapy has been tested in patients. Here I show that the BET bromodomain protein BRD4 was required for TERT over-expression in TERT-rearranged neuroblastoma cells. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. Carfilzomib and OTX015 synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT over-expression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT rearranged neuroblastoma cell lines or patient-derived xenograft tumor cells, OTX015 and carfilzomib synergistically and considerably blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression and improved mouse survival. OTX015 and carfilzomib combination therapy is therefore ready for translation into the first clinical trial of targeted therapy against TERT rearranged neuroblastoma.

  • (2021) Prentice, Bernadette
    Thesis
    Cystic Fibrosis (CF) is frequently complicated by Cystic Fibrosis-related diabetes (CFRD). CFRD has a direct impact on the morbidity of patients with CF and leads to early mortality. However, it is not yet known at what age pre-diabetic “early glucose abnormalities” begin or whether they are clinically important. The first study of the thesis was to determine whether children with CF less than ten years of age have glucose abnormalities and if present to examine the clinical importance of glucose abnormalities in this cohort that do not have diabetes. I was able to demonstrate that in children with CF, a high intermediate glucose peak on a 5-point Oral Glucose Tolerance Test (OGTT) is negatively correlated with both lung function and nutritional status. The poor clinical status of these children was not predicted by the two-hour level, the current gold standard diagnostic criterion for CFRD. This study also demonstrated that Continuous Glucose Monitoring (CGM) detected a higher frequency of glucose abnormities than the OGTT. The second part of the thesis evaluated the association between early indicators of airways disease (inflammation) and CGM detected glucose abnormalities in CF. I was able to show that children with pre-diabetic glucose abnormalities on CGM have a greater degree of pulmonary inflammation and were more likely to have a past history of Pseudomonas aeruginosa infection. In the third study, a longitudinal evaluation of these participants showed for the first time that for most children with CF who exhibit early glucose abnormalities, they will have glucose abnormalities that fluctuate. However, some children will exhibit persistently high glucose levels. The chapters of this thesis demonstrate for the first time the importance of pre-diabetic CGM detected glucose abnormalities in such young children with CF. These findings suggest that current screening recommendations will miss important, clinically significant glucose abnormalities that may impact on early lung disease in children with CF.

  • (2020) Coffey, Michael
    Thesis
    Gastrointestinal disease in cystic fibrosis (CF) begins In Utero and continues throughout life. Intestinal dysbiosis and inflammation have been observed in children with CF, however, the mechanisms linking them, and clinical implications are poorly understood. The intestinal tract as a therapeutic target is relatively underexplored. The overarching hypothesis is the intestinal milieu (bacteria, viruses and host-expressed proteins) is altered in children with CF compared to healthy controls (HC), and these changes have implications for intestinal inflammation and clinical outcomes. This thesis presents a series of observational studies characterising alterations in the intestinal milieu in children with CF, with a focus on identifying mechanisms for inflammation, disease, influences on clinical outcomes and potential therapeutic strategies. This thesis presents a novel research program commenced to address the limitations of current microbiome research and explore the gut-lung and gut-brain axes. This thesis demonstrates that in children with CF: (i) the intestinal milieu is structurally and functionally altered compared to HC, (ii) pro-inflammatory microbiota and bacteriophage lysins are increased in abundance, (iii) Ruminococcaceae may be beneficial for growth and lung function, (iv) upstream regulators for intestinal inflammation include interferon gamma, tumour necrosis factor (TNF) 5 and transglutaminase 2, and (v) xanthine dehydrogenase/oxidase (generates reactive oxygen species) negatively correlate with growth measures. Several potential strategies warranting further investigation for intestinal disease in children with CF include: (i) probiotics, (ii) short-chain fatty acid sources, (iii) antioxidants (e.g. glutathione), (iv) lactotransferrin, (v) TNF inhibitors, and (vi) amino acids (e.g. phenylalanine). A systematic review of probiotics for people with CF revealed low-quality evidence suggestive that probiotics may be beneficial to the health of children and adults with CF, however they are associated with a small number of adverse events. A novel probiotic randomised controlled trial was commenced and its design addresses several key gaps in our understanding of probiotics for children with CF. This thesis contributes to our understanding of the clinical relevance of the structural and functional alterations in the intestinal milieu of children with CF. It provides future directions for both observational and interventional research studies in children with CF.

  • (2021) Barlow, Ellen
    Thesis
    Aims: This vulvar cancer thesis had four major aims. To: (i) analyse incidence and mortality trends in Australian women over the years from 1982 to 2011, (ii) investigate the independent prognostic significance of HPV, p16 and p53 status, (iii) determine the incidence of, and risk factors for morbidity following groin node dissection, and (iv) explore the pattern of local recurrences and determine their relationship with the extent of the histopathological margin. Methods: Four studies were performed. (i) Australian population-based vulvar cancer data were analysed for changes in age-standardized incidence and mortality rates. Subsequently, data collected over a 29-year period from the database of a single-institution were analysed in three studies. (ii) immunohistochemistry was used to determine p53 and p16 status, and HPV status was determined by PCR detection of HPV DNA in 119 patients, (iii) clinical and histopathological data for 333 patients (525 groins) treated with groin node dissection were retrospectively analysed for post-operative morbidity, and (iv) data on 345 patients treated primarily with surgery were retrospectively analysed for risk factors associated with local recurrence. Results: (i) vulvar cancer incidence was significantly increasing in women under 60 years and mortality decreasing in women over 60 years, (ii) p16, p53 and HPV DNA status were not independent prognostic factors, (iii) the number of lymph nodes resected was the only factor significantly associated with all complications, and (iv) primary site recurrences were increased in patients with histopathological margins < 8mm. Treatment of patients with sub-optimal margins decreased the risk of recurrence. Conclusions: (i) Vulvar cancer incidence has increased by more than 80% in women younger than 60 years in Australia, consistent with increased exposure to the human papillomavirus in cohorts of females born after 1950, (ii) vulvar cancer treatment decisions should continue to be based on clinical indicators rather than on p16 or p53 status, (iii) a more extensive lymph node dissection is a significant risk factor for all post-operative groin complications, and (iv) Guidelines should continue to recommend a surgical margin of 1 cm.

  • (2022) Forest, Chelsea
    Thesis
    In the last 40 years there have been many strides taken towards better and more selective cancer treatment using nanoparticles. Nanoparticles can have inherent passive accumulation in tumour cells, known as the enhanced permeability and retention effect (EPR) which makes them a strong therapy candidate; however this effect is not as well defined or effective as once thought. There is a large variance of efficacy between different patients due to the heterogeneity of tumours, therefore a more targeted nanoparticle systems needs to be designed to increase selectivity and efficacy. This thesis describes the design, synthesis, and characterisation of 20 novel ellipsoidal polymersomes decorated with peptide ligands for selective targeting of medulloblastoma, a childhood brain cancer. These ligands were FSRPAFL 1 a medulloblastoma cell targeting peptide and T7 26 a transferrin targeting peptide designed to aid in crossing the blood brain barrier (BBB). A new synthetic method was designed to attach the peptide ligands post self-assembly, so the peptides were attached to the hydrophilic corona rather than the hydrophobic membrane of the polymersomes. Analysis of these polymersomes showed more ligand available for binding but this did not translate to increased cell association due to an over saturation of ligand. The ratio and density of the targeting peptide 1 and BBB peptide 26 was altered on the surface of the polymersomes and it was found that the polymersomes with 100% T7 ligand showed rapid and high cell association with two different subtypes of SHH medulloblastoma (DAOY and UW228) as well as high association with brain endothelial cells that make up the BBB (HBEC-5i) making it a promising candidate as a drug delivery system for SSH medulloblastoma. Finally, linearly conjugated dual peptides made up of both targeting peptide 1 and T7 peptide 26 sequence, were synthesised and attached to the polymersome hydrophilic corona and analysed against the non-conjugated dual-functionalised peptide polymersomes. There was no significant difference between the two ligand conjugation method analysed but further research should be conducted to confirm this. The work described in this Thesis has shed light on the multitude of nuances that make up the composition of mono and dual functionalised peptide nanoparticle systems and how these can influence biological function. Future work will allow for a better understanding of fundamental questions about targeted nanoparticles therapies and how ligand characteristics directly impact biological function, selectivity and efficacy.

  • (2021) Seneviratne, Janith
    Thesis
    Peripheral neuroblastic tumors (PNTs) represent a spectrum of tumors derived from the neural crest and account for 10% of paediatric malignancies. PNTs comprise four histological subtypes, including neuroblastoma (NB). Half of high-risk NB patients relapse after chemotherapy, and relapsed patients seldom respond to therapy afterward. This study aimed to identify chemoresistance mechanisms and effective combination therapies against chemoresistant NB. In the first results chapter, intratumoural heterogeneity in PNT subtypes were first investigated using single cell RNA sequencing (scRNA-seq) on 7 primary PNTs spanning all histological subtypes. This revealed extensive transcriptome heterogeneity among PNTs and identified malignant neuroblasts differentiating through a novel “transitional” state. This state resembled known chemoresistant cell phenotypes and predicted poor NB patient prognoses. In the second results chapter, drug resistance mechanisms in NB were explored in vitro using longitudinal IMR-32 cisplatin-resistant cell line models. Following scRNA-seq, resistant cells undergoing cytoskeletal remodelling were identified and then characterised in vitro. Additionally, tumour samples from 5 high-risk NB patients at diagnosis and post-chemotherapy were analysed with scRNA-seq, where an enrichment of oncogenic cell states was observed. In the third results chapter, potential combination therapies were identified using a combinatorial drug screen against the chemoresistant SK-N-BE(2)-C cell line. The highest synergy manifested between two histone methyltransferase inhibitors, GSK343 and SGC0946. Mechanistic studies revealed the induction of ER stress programs by the combination treatment. The administration of this combination therapy in vivo reduced NB tumour growth. In the fourth results chapter, an integrative approach was used to identify mitochondrial drug targets in NB, whereby the mitochondrial translocase SLC25A5 was prioritized. Targeting SLC25A5 with the selective inhibitor, PENAO, reduced cell viability in NB cell lines, however TP53 mutations conferred chemoresistance. The histone deacetylase inhibitor SAHA combined with PENAO overcame mutant TP53 mediated chemoresistance to synergistically reduce NB cell and tumour growth. Collectively, this thesis has identified transcriptional cell states that drive chemoresistance and combination therapies that overcome chemoresistant NB, thereby contributing novel therapeutic targets and treatment avenues for NB patients.

  • (2021) Avnet, Hagai
    Thesis
    Fetal cardiac function analysis may provide crucial pathophysiological insights into fetal hemodynamic status, clarify cardiovascular adaptation to perinatal complications and improve clinical diagnosis and management of complicated pregnancies. A broad range of ultrasound techniques aim to predict cardiac dysfunction before there are clinical signs of fetal distress to allow early intervention. This thesis aimed to assess Pulsed-Wave Myocardial Performing Index (PW-MPI), Tissue Doppler Imaging MPI (TDI-MPI) and Tricuspid and Mitral Annular Plane Systolic Excursion (TAPSE and MAPSE) for evaluating fetal cardiac function. This was performed in several aspects: TDI has been proposed to be robust for detecting subclinical cardiac dysfunction over conventional Doppler. We found (79 cases) similar achievability and reproducibility, yet a poor correlation between TDI- and PW-MPI. Most probably due to considerably variable TDI waveform quality. This raised concerns about the technique for TDI-MPI. Online international study aiming to evaluate TDI-MPI demarcation and reliability. We compared annotations of 4 international experts (120 images) and found very high intra-observer repeatability, lower inter-observer reproducibility and variability in landmark definition, questioning the reliability of TDI-MPI. Evaluation of STIC M-mode for measuring TAPSE and MAPSE (102 cases). We found high achievability, high intra-observer repeatability for both sides and higher inter-observer reproducibility for TAPSE compared to MAPSE. We concluded that STIC allows easy and accessible evaluation of systolic function, that is reliable and repeatable, more so for TAPSE than MAPSE. Multimodality, global evaluation, which included a comparison of all modalities in 79 normal pregnancies, and in 30 scans from pregnancies complicated with IUGR or monochorionic twins. Achievability rates were high for all modalities. No single method was superior for both sides, TAPSE was more suited to the right heart and automated PWD-MPI for the left. These reflect each modality's specific qualities that accommodate best to the different structure and function of each heart side. This provides a useful first step for developing a clinical multimodality global scoring system that integrates the most favourable imaging technique for each side of the heart to optimally quantify the global heart function. This thesis also provides a firm basis for further research in this field.