Medicine (St Vincent's) - Theses
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Determining how SRSF2 mutation leads to MDS and CMML and its genetic cooperativities in vivo and in vitro
Myelodysplastic syndromes (MDS) are a group of heterogeneous clonal stem cell malignancies characterised by multilineage cytopenia and dysplasia. MDS mostly occurs in aged populations where there are limited therapeutic options. Compared to MDS, chronic myelomonocytic leukaemia (CMML) presents with a monocytosis feature and has a poor survival of 16 months for high-risk patients. In the past decade, several sequencing studies have defined the complex molecular landscapes of MDS and CMML. SRSF2, a component of RNA splicing machinery, is one of the most frequent mutations in MDS and CMML. To understand the consequences and effects of SRSF2P95H mutation on normal haematopoiesis, several groups, including our lab, have generated in vivo mouse models using various gene targeting strategies and Cre recombinases. These models demonstrated some effects of SRSF2P95H mutation on haematopoiesis, and described certain mis-splicing changes in the Srsf2P95H/+ cells. However, the mechanism and role of SRSF2P95H mutation in promoting and initiating MDS/CMML are still poorly defined. My thesis aimed to address key knowledge gaps by examining the cell of origin, transcriptomic/splicing changes, synthetic lethal genetic interactions, and co-operative interactions of SRSF2P95H/+ mutation in the initiation of MDS/CMML. In the first part of my thesis, I assessed the cell of origin in SRSF2P95H MDS by characterizing a conditional knock-in Srsf2P95H/+ mouse model, using LysM-Cre. After activating Srsf2P95H/+ mutation in myeloid progenitors, I observed no development of MDS even after prolonged ageing (up to 52 weeks) and only mild changes in haematopoiesis. Compared to the stem cell activation model (hScl-CreERT2) that we reported, the results of LysM-Cre demonstrated that a myeloid progenitor is not the cell of origin in SRSF2mut MDS. In the second part of my thesis, I analyzed the transcriptomic and splicing changes of Srsf2P95H/+ cells, using both purified stem and progenitor cell populations as well as Hoxb8 immortalized cell lines. The transcriptome analysis revealed up- and down-regulation of lineage associated genes and up-regulation of MDS associated pathways and the p38 MAPK kinase pathway. The splicing analysis demonstrated skipped exons as the most frequent alternative splicing event. In terms of specific mis-splicing targets, I examined exon inclusion in several reported transcripts and compared the most frequently mis-spliced genes across 12 human SRSF2mut and murine Srsf2mut datasets. Through this analysis, I found that mRNA processing and DNA repair represent the top mis-spliced pathways in Srsf2P95H/+ cells. I also present a pilot study of single cell RNA sequencing of Srsf2P95H/+ stem and primitive progenitor populations, which unveiled a myeloid-biased signature and enhanced myeloid differentiation of the Srsf2P95H/+ stem cells. In the third part of my thesis, I explored the synthetic lethality of Srsf2P95H/+ cells with a pooled CRISPR knock-out screen. I discovered that loss of DNA repair or cell cycle pathways was synthetic lethal with Srsf2P95H/+ mutation. Consistent with this genetic lethality, I demonstrated that Palbociclib, a CDK6 inhibitor, could preferentially target the Srsf2P95H/+ cells. This finding opens up new therapeutic windows beyond known spliceosome inhibitors for SRSF2mut MDS. In the fourth and last part of my thesis, I generated and characterized two multi-genic mutation models: Srsf2P95H/+ Tet2-/- and Srsf2P95H/+ Cbl-/-. In the Srsf2P95H/+ Tet2-/- model, I observed profound myeloid bias, B lymphoid suppression and increased ST-HSC percentages in the stem cell compartment after 52 weeks of activating/deleting mutations in the haematopoietic stem cells. Within the Srsf2P95H/+ Tet2-/- cohort, I also observed development of CMML in both native haematopoiesis and transplantation settings. So far, this is the first model to demonstrate synergistic interactions between Srsf2P95H/+ and Tet2-/- mutation, as well as initiation of CMML in vivo. For the Srsf2P95H/+ Cbl-/- model, I characterized a small cohort of mice due to prolonged breeding difficulties. Nevertheless, I discovered increased myeloid proliferation in the double Srsf2P95H/+ Cbl-/- and Srsf2P95H/+ Cbl+/- mutants. Collectively, the work included in this thesis creates an original contribution to understanding the role of SRSF2P95H mutation in MDS, its synthetic lethal genetic interactions, potential therapeutic targeting of SRSF2P95H cells, and how it co-operates with other recurrent mutations in initiation of CMML.
Context for improving access to care for children and youth with diabetes in less-resourced countries
There are major deficits in knowledge related to the epidemiology and care of the various types of diabetes in young people in less-resourced countries. Multiple barriers exist at individual, community, health system, national, and international levels that must be overcome to lessen the gap in outcomes between advantaged and disadvantaged regions. This thesis presents 11 published papers by the candidate addressing this gap in knowledge. Type 1 diabetes (T1D) incidence data is presented for three countries with no previous data (Fiji, Bolivia and Azerbaijan), showing differing rates in each country, and in Fiji differing rates in the two main ethnic populations. Novel information on the types of diabetes is presented for Azerbaijan (along with the incidence data aforementioned), Pakistan and Bangladesh. Results in Azerbaijan were similar to those seen in European populations. In Pakistan and Bangladesh, it is common to see atypical forms that clinically present like T1D cases but do not have low C-peptide values or diabetes autoantibodies. Five papers examine costs and access to care. In a survey of 71 countries, availability of nearly all key components of care was greatly reduced in lower-income countries. A study of costs to families in 15 countries demonstrated that the cost of core supplies is prohibitively expensive for many families. A comprehensive review of issues surrounding access to supplies for self-monitoring of blood glucose presents new information on the global market and makes numerous practical recommendations. Progress towards Universal Health Coverage for provision of insulin and blood glucose test strips was evaluated in 44 countries, showing that there was greater progress for insulin than for test strips. A novel framework for describing T1D care levels (Basic, Intermediate and Comprehensive) provides a way of identifying the steps required to improve care in a particular situation, and the data presented from Bolivia shows that Intermediate Care can achieve outcomes similar to those in some highly-resourced countries. The final paper, using robust, novel and replicable methodology, demonstrates the efficacy of traditional evaporative cooling devices used for insulin storage where refrigerators are not available. In conclusion, efforts to improve care for young people with diabetes in less-resourced countries must take into account wide differences in incidence and the types of diabetes that occur between countries. Furthermore, for care to improve, many components of care need to be addressed. The concept of ‘Intermediate Care’ provides an achievable level of care that can result in reasonable outcomes even in poorly resourced health systems.
Detection and monitoring of diabetic polyneuropathy with specialised markers of small and large nerve fibres
Routine nerve conduction studies (NCS) are the current standard test for the evaluation of suspected peripheral neuropathy, but are often normal in patients with diabetic polyneuropathy (DPN). This is because: i. NCS are unable to detect dysfunction of small fibres, which may be affected earlier and are the cause of painful neuropathic symptoms; and ii. Significant large fibre axonal loss +/- demyelination may need to occur before NCS detect this is an abnormality. This research project critically evaluates various newer/novel methods to detect and potentially monitor diabetic neuropathy, and to provide a feasible way that this can be routinely assessed in the neurophysiology laboratory. I chose to specifically analyse the diabetic population as this is by far the most common cause of peripheral neuropathy in the global community, causing significant burden of disease. The various known novel techniques to assess and monitor DPN are evaluated, firstly through that which is available in current literature. These are compared in terms of diagnostic accuracy, as well as availability and cost-effectiveness. Based on the findings from this literature review, various prospective clinical studies are developed to further evaluate the more promising techniques identified. A focus was made towards techniques that can be performed in the neurophysiology laboratory, as routine NCS have, and will remain likely to have a significant role in the assessment of diabetes-related neuropathy. Thus, this clinical visit provides an opportune time to perform these additional tests as a way of providing supplementary information on patient’s peripheral nerve status. Small and large nerve fibres are both affected by diabetes. However, these fibres display very different physiological properties, not just in terms of structure and function, but they are also evaluated by completely different techniques. Small fibres can then be further subdivided into different populations of thinly myelinated A-delta fibres and unmyelinated C-fibres, which again have very different properties and assessment modalities. This thesis is divided into two main sections with research focussed on providing more accurate measurement of the various small fibre populations in the first section, and then improving large fibre testing beyond that of routine NCS in the second section.
Determining the impact of Recql4 mutations on normal homeostasis, tumour development, and functional genetic interactions
Since mutations in the RECQL4 gene were identified as causative of Rothmund-Thomson syndrome (RTS) more than twenty years ago, some inroads have been made in the understanding of this disease and its mutations. It has been discovered that the majority of these mutations are nonsense and frameshift mutations resulting in truncating protein products that delete both the helicase and the C-terminal domain. The deletion of these domains results in a dysfunctional RECQL4 protein and the development of the variable clinical spectrum and the increased predisposition to malignancies, typical of RTS. Several Recql4-mutated mice have been generated as a model for RTS. Although these models have contributed to identify some of the functions of RECQL4, they do not accurately reflect the genetic status of RTS, in which patients generally present with hypomorphic, rather than null alleles. On the other hand, these models have not provided sufficient insight into the specific functions and domains of RECQL4; and the effects of RECQL4 mutations on normal homeostasis, tumour development, and functional genetic interactions. This thesis, through its three related components, aims to address all these gaps by using murine models bearing mutations that inactivate specific functions and domains of RECQL4, and that resemble common mutations seen in humans. In the first component of this thesis, I generated mice carrying an ATP-binding knock-in mutation to assess the physiological requirements and biological functions of the helicase activity, thought to be critical for the overall functions of RECQL4. Through a variety of experiments, I observed that homozygous mice were normal in terms of embryonic development, body weight, haematopoiesis, B and T cell development, and physiological DNA damage repair. Furthermore, to compare the in vivo effects of a helicase-inactive versus truncating mutations, I used conditional deletion models and found that only mice carrying truncating mutations developed bone marrow failure. These findings demonstrated that the ATP-dependent helicase activity of RECQL4 is not essential for its physiological functions, and that truncating mutations are deleterious. For the second component of this thesis, I assessed mice carrying germline truncating RECQL4 mutations to understand the impact of the deletion of the helicase and C-terminal domains on normal homeostasis and tumour development. I found that truncating mutations affected stability and subcellular localisation of RECQL4, which translated to a homozygous embryonic lethality and a haploinsufficient low bone mass phenotype through defects in early osteoblast progenitors. Additionally, I observed that the severity of the defect was related to the degree of the truncation, suggesting that gene dosage is an important determinant of the bone phenotype. However, these mutations were not sufficient to initiate tumorigenesis in mice, even after exposure to irradiation, which pointed to the possibility that mutations in other genes, besides Recql4, might be contributing to this disease. In the third and final component of this thesis, I performed a genome-wide genetic rescue screen to identify genetic interactions with mutant Recql4, an area largely unexplored. Amongst the genes identified, the standout candidate was Klhdc3. In subsequent validation assays, I demonstrated that mutation of Klhdc3 rescued the phenotypes associated with a pathogenic RECQL4 mutation, which is to my knowledge, the first confirmed genetic interaction associated with an improvement of the cellular phenotypes caused by RECQL4 mutations. Collectively, the work presented in this thesis makes an original contribution to knowledge through the finding that the ATP-dependent helicase activity of RECQL4 is dispensable for its physiological functions, the discovery that truncating mutations cause a haploinsufficient low bone mass phenotype and that gene dosage is an unsuspected regulator of bone mass, and the identification of Klhdc3 mutation as being capable of rescuing the proliferation defect caused by a truncating RECQL4 mutation.
Investigation of actions of Parathyroid hormone-related protein (PTHrP) independent of PTH1R
Parathyroid hormone-related protein (PTHrP; gene name PTHLH/Pthlh) acts as a paracrine/autocrine factor to produce a range of effects, including contributing to skeletal development and determining trabecular bone mass. By activating the receptor (PTH1R) that PTHrP shares with parathyroid hormone (PTH), it transduces cascades coupled with the stimulation of cyclic adenosine monophosphate (cAMP) protein kinase A (PKA) and CREB responses. However, in addition to its amino-terminal region that mediates similar actions to PTH, PTHrP has structural features that allow it to exert different activities independent of PTH1R. Relatively little is known about the signalling pathways that guide these actions; however, the novel specific functional regions that exist in PTHrP allow us to ask how PTHrP acts independently and uses non-canonical pathways. The primary aim of my thesis is to unveil the PTHrP functions that act through non-canonical pathways associated with regions of the molecule other than the PTH1R binding region. Recently, it has been demonstrated that PTHrP overexpression drives dormant human MCF7 breast cancer cells in mice to colonise the bone marrow and induces osteolytic damages. However, I found that although MCF7 cells activate cAMP in response to prostaglandin E2 (PGE2) and calcitonin, the cells do not respond to PTHrP or PTH. This suggests that PTH1R in those cells is not functionally linked to adenylyl cyclase. My analysis suggests that MCF7 cells express PTH1R mRNA and proteins; however, such PTH1R does not bind sufficient exogenous PTHrP to activate cAMP. RNA-Seq analysis identified many genes induced by PTHrP overexpression in MCF7 cells, indicating responsiveness to PTHrP, although a shift in cell differentiation because of clonal drift cannot be ruled out. Several potential alternative signalling pathways were identified, notably those related to calcium signalling. These data suggest that the effect of PTHrP overexpression on MCF7 cell dormancy may occur through PTH1R-independent actions of PTHrP. PTHrP is also essential for bone formation, as indicated by mouse models with genetic depletion of PTHrP, both globally and within the osteoblast lineage. It has been established that Dmp1Cre.Pthlhf/f mice have significantly lower levels of PTHrP in osteocytes resulting in reduced bone mass and strength. This differs strikingly from Dmp1Cre.Pth1rf/f mice with a deficiency of PTH1R in osteocytes which exhibit high bone mass. This implies that PTHrP signalling in osteocytes that promotes bone formation occurs through a non-PTH1R-mediated pathway. To identify alternative signalling pathways of PTHrP, RNA-Seq was conducted on OCY454 osteocytes overexpressing secreted PTHrP and mutant forms of PTHrP lacking both the nuclear localisation sequence and C-terminus, the nuclear localisation sequence, or lacking the signalling peptide required for secretion. In these cells, the expression of PTHrP-activated genes is significantly higher when the PTHrP NLS and C-terminus domains are absent. This shows that the NLS and/or the C-terminus of PTHrP may have a negative impact on genes regulated through PTH1R. However, unlike deletion of the NLS and C-terminus, deletion of NLS alone did not change the number of genes regulated by PTHrP nor the magnitude change. This difference indicates that the C-terminus, but not the NLS, may limit gene expression in response to PTH/PTHrP signalling in OCY454 cells. Notably, there was no change in cAMP response, CREB responsive gene alterations, or CREB phosphorylation in response to exogenous PTHrP in cells overexpressing the C-terminus, nor was there any difference in the effects of exogenously supplied C-terminus PTHrP compared to full-length PTHrP. This suggests the inhibitory action of the PTHrP C-terminus is intracrine, with regulation taking place beyond CREB activation. I next performed studies to determine whether the PTHrP C-terminus reduces PTH1R-mediated signalling using qPCR on UMR106.01 overexpressing the C-terminus, focusing on the marked down-regulation of osteocalcin (protein name: BGLAP, bone gamma carboxyglutamate protein; gene name: Bglap1/2) expression, as an example, targeted by PTH/PTHrP signalling. The induction of Bglap1/2 expression by PTH or PTHrP was significantly lower in osteoblastic-like osteosarcoma UMR106.01 cells overexpressing the PTHrP C-terminus. Subsequent analysis suggests this C-terminus gene suppression effect is cytosolic but is not dependent on modifying cAMP/PKA signalling. My preliminary data suggest that modification of Wnt signalling pathway may be required for this C-terminus effect. The RNAseq data from OCY454 PTHrPFL cells revealed that osteocyte-derived PTHrP might regulate mineralisation due to significant regulation of mineralisation genes in the gene profile, compared to vector control cells. Since bones from 12-week-old Dmp1Cre.Pthlhf/f mice were previously reported to have impaired bone material strength; I next sought to understand how osteocyte-derived PTHrP modifies mineralisation in vivo. I used high-resolution synchrotron-based Fourier Transform Infrared (FTIR) microspectroscopy to determine the bone composition of 12-week-old Dmp1Cre.Pthlhf/f mice and Dmp1Cre controls. Male Dmp1Cre.Pthlhf/f mice showed significantly higher amide I: II ratio (i.e. less compacted collagen) and lower mineral: matrix ratio compared with controls. This suggests a requirement of PTHrP for both collagen organisation and mineral formation. Surprisingly, preliminary in vitro results showed that both PTHrP knocked-down cells and overexpression cells had a low mineralised deposit phenotype. My preliminary data suggest that PTHrP is likely to contribute to bone strength via proper mineral deposition and compaction of collagen, and by modifying osteocyte differentiation. In conclusion, my thesis describes three major findings. Firstly, although PTHrP promotes breast tumour MCF7 cells aggressively growing in bone, this is not dependent on PTH1R/cAMP/PKA activation. Secondly, in osteocytes, which express PTHR1, the PTHrP C-terminus appears to inhibit Bglap1 and Bglap 2 transcription induced by PTH/PTHrP, and this occurs through intracellular pathways independent of PTH1R/cAMP/PKA. Moreover, PTHrP is essential for normal bone composition. It is likely that PTHrP contributes to the promotion of mineralisation, thereby contributing to bone strength via collagen compaction, and by modifying osteocyte differentiation. Deciphering these actions above and their crosstalk with non-PTH1R-mediated processes will increase our understanding of how PTHrP manipulates breast cancer metastasis and bone composition.
Understanding the effects of Radiotherapy and Surgical injury on Local immunity and Lymphatic Architecture and Function
This thesis aims to provide insight into the pathophysiological mechanisms behind two important clinical problems encountered in patients surviving oncological treatments: 1) Infections in previously radiated sites and 2) Lymphoedema that follows surgical injury to lymphatics or radiation of nodal basins (RTx). Both infection and lymphoedema have significant impact on patient morbidity and quality of life (QOL). Lymphoedema, in particular, has a significant “psychological and social health cost”, and is associated with significant costs in providing long term conservative management to prevent symptom progression and maintenance of QOL outcomes for patients. Therefore, socioeconomic implications of these diseases certainly warrants further investigation of the disease pathophysiology and potential avenues for prevention and treatment. This thesis provides a review of knowledge regarding normal structure, function and development of the lymphatic system, lymphangiogenesis and lymphatic remodelling and how these processes relate to lymphoedema. In addition, the processes of antigen capture and transfer to lymph nodes for presentation to lymphocytes will also be addressed as a background to understanding infections in radiated sites. Subsequently, methods to investigate lymphatic morphology utilising a murine Prox1-eGFP model and confocal imaging were detailed, as was an automated segmentation and analysis method to allow high throughput analysis of lymphatic vessel morphology following sugical or RTx injury in detail. Functional techniques utilised to assess antigen presentation and fluid transport to draining lymph nodes (dLNS)have also been described in detail. We have demonstrated in this thesis that RTx significantly reduces CCL21 expression by LECs and has subsequent effects on antigen presentation from RTx tissue. These results have implications for the increased rates of infection noted after RTx. Moreover, RTx significantly impairs early wound lymphangiogenesis in vivo. While surgical injury alone induces remodelling of both lymphatic capillaries and deep collecting lymphatic vessels (CLVs) distal to sites of surgical, combined injury with RTx and Surgery markedly exacerbates these remodelling responses. By using longer time points, we have also explored possible mechanisms behind chronic lymphoedema, and have demonstrated that RTx continues to impair wound lymphatic regeneration in combination with severe surgical injury. Furthermore, the distal limb appears to demonstrate evidence of progressive remodelling at late time points following lymphatic injury. As with earlier time points, combined injury with RTx and Surgery, appears to promote exacerbated secondary lymphangiogenesis in the distal limb at later time points. Potential molecular mechanisms behind these differences between the groups have then been subsequently explored through RNA sequencing and multiple PCR analyses of total tissue mRNA. The broader implications of the findings of this thesis to clinical lymphoedema and RTx are then addressed, as well as potential future directions for research to build on the findings of this thesis.
Associations between Hepatitis B virus mutations and infection kinetics, host responses and outcomes
The hepatitis B virus (HBV) has infected more than 2 billion people worldwide and is associated with a significant global burden of disease due to its main associated complications of cirrhosis and hepatocellular carcinoma (HCC). The risk of developing cirrhosis or HCC is highly variable, with some people who have quiescent disease throughout their lifetime, whilst others develop aggressive disease associated with high mortality and morbidity rates. HBV is prone to mutations during viral replication and, as a result, it acquires multiple mutations due to pressures either from exogenous vaccines or therapeutic agents or from the host’s immune system. This propensity to mutate has resulted in the formation of genotypes and viral variants. Genotypes of HBV have a sequence variation of 8% or more and they have a specific geographical distribution. The most common viral variants of HBV are the basal core promoter (BCP) and precore (PC) mutations. BCP diminishes the production of the e antigen (HBeAg) whilst PC abolishes it completely. The role of the HBeAg is not completely understood. It is not required for infectivity or any major steps of the viral life cycle. It is thought to be an immune-modulator although the mechanisms of immune modulation are yet to be elucidated. There is a robust body of epidemiological evidence that links viral variants and genotypes to distinct clinical phenotypes. However, the actual host-virus mechanistic interactions are unknown. The HBV, in itself, is not generally cytopathic. It is the immune response to the virus that is thought to be responsible for the inflammatory response and subsequent scarring and liver fibrosis. Currently, there is no definitive cure for chronic hepatitis B (CHB) infection. Antiviral therapy suppresses viral replication but does not eliminate the virus. Due to its close interaction with the immune response, an understanding of the host-virus immuno-pathogenesis is needed in order to explore novel immune-based therapies. The first aim of this thesis was to understand the characteristics of CHB in Australia. Using a population sample from a large tertiary Victorian hospital, a detailed study of the virological characteristics including genotypes, and BCP and PC mutations was performed. We focused on the association between virological factors including genotypes and mutations and clinical phenotypes. Genotypes A, B, C and D were the most prevalent, with a clear preservation of the relationship between genotype and country of origin of the patient. Genotypes B and C were the most common, a reflection of the large migrant population from South East Asia. HBeAg-negative CHB was common and was strongly associated with the presence of the PC variant. A large proportion of patients with HBeAg-negative CHB had an active hepatitis and they were in the immune escape phase. BCP and PC mutations were highly prevalent and were independently associated with the presence of advanced fibrosis, regardless of age, sex or genotype. These findings suggest that there may be a role for sequencing for these mutations in clinical practice and use them as biomarkers. Based on this clinical observation, we also hypothesized that HBeAg was modulating the immune response to enable chronicity in early stages of disease but then becomes a liability in later phases. This formed the basis of studies of the immune response with a specific focus on HBeAg. The second aim of this thesis was to explore some of the key immune pathways that modulate HBV infection. The hypothesis was that HBeAg modifies innate host immunity to establish chronic infection in the early phases of disease. Much of the literature in HBV has focused on the adaptive immune response but we were interested in the early phases of the natural history, where HBeAg is almost universally present and its absence is associated with reduced infectivity. The role of HBeAg in the early stages of HBV infection was studied using an immunocompetent mouse model transfected with HBeAg- negative strains of HBV. Pertinent findings included a sudden, early drop in viraemia in HBeAg- negative mutants compared to wild type (WT) HBV. A corresponding transaminitis strongly suggested that in absence of HBeAg, there was a more robust innate immune and inflammatory response to the virus. Complementation studies confirmed that the effect was due to HBeAg and no other viral components. Using gene-modified animals and selective antibody depletion, a series of studies were performed to examine individual arms of the innate immune response. We showed that the Type 1 interferon (IFN-I) response was much stronger in absence of HBeAg. Correspondingly, transcriptomic analysis revealed the downregulation of multiple IFN-I pathways and proteome/ phosphor-proteome studies showed changes in the expression of interferon regulating genes associated with HBeAg. Collectively our data showed that HBeAg allows the virus to evade the innate immune response by antagonising IFN-I in the initial stages of infection. The emergence of HBeAg- negative mutants in the later stages of the natural history of CHB suggests that HBeAg presents a liability to the virus. In our mouse model, following a significant drop in viraemia in the initial phases of infection, there was a recrudescence in viral load in the later stages of disease in HBeAg deficient mutants. We hypothesized that in the later, adaptive immune response, HBeAg is an immune target and there is a strong selective pressure that favours HBeAg- deficient viral variants. A series of experiments using gene-targeted animals was performed to examine the roles of various mediators of the adaptive immune system. These studies showed that the absence of HBeAg was associated with reduced function of CD4+ T cells and a reduction in TNF and IFN- gamma activity. HBeAg deficiency was also associated with an escape from the antibody response. In conclusion, HBeAg is an important antigen in the modulation of the immune response in HBV. Sequencing for HBeAg negative variants may have a role in clinical practice and further longitudinal, prospective studies are warranted. Further dissection of the proteomic and transcriptomic signatures associated with HBeAg negative mutants and their correlation with clinical samples would be the next steps to elucidate the intricate role of HBeAg in modulating disease behaviour and outcomes.
Studies investigating the clinical impact and immunological host response to viral eradication in hepatitis C infection
Australia now has improved access to HCV treatment for individuals with all stages of liver disease. There is limited data however, documenting any long-term clinical benefit of viral eradication in those with early stage liver disease. We report on the long-term outcomes of a well-characterised cohort of CHC subjects with predominantly early stage liver disease, whom paired liver fibrosis assessments were performed more than 10 years apart. We show in a real-world setting, that both early and curative HCV treatment halts fibrosis progression. Our data supports the early treatment of all people with CHC regardless of liver fibrosis stage, to prevent long-term liver sequelae. In subjects with acute / subacute HCV infection, we identify predictors of innate immunological response in peripheral blood mononuclear cells, to differentiate spontaneous clearers to those who progress to chronic infection. Our data strongly implicates TLR4 signaling in the persistence of HCV infection. Those who developed chronic infection had higher TLR4 expression on peripheral monocytes and NK cells and increased IFN-gamma response to TLR4 stimulation. We also observed increased TLR7 responsiveness in this group. We confirm the previously noted associations of IFNL4 genotype and plasma IP-10. Our data presents TLR4 as a potential biomarker for predicting clearance. This would clinically translate to individuals presenting with acute / subacute HCV infection, being able to defer drug therapy and its associated morbidity and cost. In subjects with chronic HCV infection, we demonstrate a clear effect of direct antiviral agent (DAA) mediated viral suppression therapy on patterns of TLR signaling in subjects with chronic HCV-1. We show that peripheral monocytic TLR2, TLR4 and TLR7 signaling is down-regulated early on in treatment, with a strong trend to higher baseline TLR signaling being associated with viral clearance with the DAAs. We are the first study to demonstrate a relationship between TLR signaling activity and IFN-free therapy for HCV. We hypothesise that the HCV virus directly stimulates the TLR pathways to induce an antiviral effect, with higher TLR signaling evident among those who respond to DAA therapy. This was accompanied by a reduction in PBMC ISG expression, NK activation markers and plasma levels of inflammatory cytokines / chemokines, with restoration of the innate immune response. Despite excellent treatment options, the mechanisms responsible for viral eradication in HCV have remained poorly defined. Previous data suggest a link between innate immunity and HCV pathogenesis, as well as spontaneous viral clearance. The data suggested that antiviral therapy alone was not sufficient to clear virus and supported a key role for innate immunity contributing to viral clearance. Our results strongly implicate TLR signaling / expression in HCV viral clearance. We believe TLR agonists must be considered as a potential HCV vaccine candidate and further research in this area is required.
Activation of islet inflammation by cytokine signalling in pancreatic beta cells: understanding the role of protein tyrosine phosphatases
Type 1 diabetes is characterised by the autoimmune destruction of insulin producing beta-cells in the islets of Langerhans of the pancreas. Immune cells release pro-inflammatory cytokines such as interferon-g (IFN-g), tumour necrosis factor-a (TNF-a) and interleukin- 1b (IL-1b) into the islet microenvironment which activate phosphorylation cascades and gene expression in beta-cells that increase their susceptibility to autoimmune attack and destruction. Protein tyrosine phosphatases (PTPs) regulate phosphorylation based signalling pathways and have previously been shown to negatively regulate IFN-g induced cell death of beta-cells in vitro. We previously showed that during immune infiltration to the islet PTPs, including PTPN1 and PTPN6, are rendered catalytically inactive through oxidation resulting in loss of signal regulation. The overall aim of this thesis is to observe if antioxidant treatment can reduce autoimmune development in the NOD/Lt mouse through reduction of oxidised PTPs and dissect the role of PTPN1 and PTPN6 in the regulation of cytotoxic signalling events in the NIT-1 beta-cell line and isolated NODPI islets in vitro. Chapter 3 studies the effect of the mitochondrial targeted antioxidant mito-TEMPO on insulitis and diabetes development in the NOD/Lt mouse. Delivery of mito-TEMPO through drinking water reduced levels of oxidised PTPs in the pancreas of NOD/Lt mice but had no effect on the development of insulitis, activity or number of CD8+ and CD4+ T- cells in the periphery in NOD/Lt mice or diabetes development in a diabetes transfer model. Chapter 4 describes the regulation of cytokine signalling in NIT-1 cells and isolated NODPI islets by PTPN1. Inhibition of PTPN1 activity reduced IFN-g, TNF-a and IL-1b induced death of NIT-1 cells in vitro. Activation of the IFN-g, TNF-a and IL-1b signalling pathways and downstream transcription of pro-inflammatory gene signatures associated with autoimmune diabetes were also reduced with PTPN1 inactivation. Furthermore, PTPN1 inhibition reduced IFN-g induced MHC-I expression on the surface of NODPI beta-cells and reduced the ability of autoreactive NOD8.3 CD8+ T-cells to destroy isolated NOD/Lt islets. These studies showed that PTPN1 is a positive regulator of cytotoxic signalling in NIT-1 cells and NODPI islets and promotes immune cell mediated death, suggesting it may be a potential therapeutic target for type 1 diabetes. Chapters 5&6 study the role of PTPN6 in cytokine signalling regulation in NIT-1 cells. PTPN6 inhibition was found to enhance TNF-a induced NIT-1 cell death independent of IFN-g and IL-1b in vitro. TNF-a induced JNK signalling was enhanced with PTPN6 inhibition which resulted in reduced anti-apoptotic BCL-2 protein expression and enhanced caspase-3 cleavage. Pan-caspase inhibition prevented TNF-a induced cell death suggesting that cells were dying through apoptosis. TNF-a induced cell death was also prevented with RIPK1 inhibition which prevented enhanced caspase-8 cleavage in PTPN6 deficient cells. Collectively these studies showed that PTPN6 negatively regulates TNF-a induced intrinsic and extrinsic apoptosis of beta-cells in vitro. Overall, the data indicate that PTPs play a nonredundant role in the regulation of cytotoxic signalling in NIT-1 cells and NODPI islets. This finding is consistent with results in other disease pathologies. The results provide a mechanistic insight into how PTPN1 and PTPN6 have opposite roles in regulating cytokine signalling, highlighting how PTPN1 antagonism and PTPN6 agonism may prove beneficial in reducing beta-cell death in vitro. Whether these results are directly translatable into in vivo models of autoimmune diabetes remains undetermined. The use of PTPN1 inhibitors or PTPN6 agonists currently under development would allow direct translation of these results.
The contribution of the pulmonary circulation and cardiac function to exercise intolerance in people with diabetes
Diabetes mellitus (DM), and in particular type 2 DM (T2DM), is approaching epidemic proportions globally. Relative to people without diabetes, exercise performance is reduced by approximately 20% in T2DM subjects universally, whereas impairment in type 1 DM (T1DM) is less consistently reported. Risk of cardiovascular disease (CVD) is significantly elevated in both T1DM and T2DM, and thus emphasis is placed on cardiovascular constraints to exercise capacity, including subclinical cardiac dysfunction and the entity of diabetic cardiomyopathy (DMC). A clear bidirectional association between heart failure and T2DM exists, with numerous pathophysiological mechanisms identified in animal models. Longitudinal data suggests a low prevalence of heart failure in T1DM, on the other hand, which insinuates the pathological mechanisms thought to underpin DMC may be exclusive of chronic hyperglycemia which unifies the diagnosis of T1DM and T2DM. The pulmonary microvasculature is targeted in diabetes, meanwhile, and may be an under-appreciated contributor to exercise limitation. It is thought that the lungs are protected from deleterious effects of subclinical microvascular disease by virtue of their substantial size, although the ability to assess pulmonary microvascular function during exercise has been limited. This thesis assesses exercise performance (VO2peak) in people with T1DM and T2DM, evaluates the contribution of cardiac function and the pulmonary circulation using comprehensive traditional and novel echocardiographic analyses, and investigates the effect of phosphodiesterase type 5 (PDE5) inhibitor Sildenafil on exercise capacity. VO2peak is demonstrated to be impaired in only the T2DM cohort of the study group, and independently associated with sedentary lifestyle and reduced left ventricular end diastolic volume. Comprehensive resting and exercise echocardiographic measurements of biventricular systolic function and LV diastolic function, in addition to speckle-tracking global longitudinal strain and LV twist mechanics, are normal. The novel echocardiographic assessment of pulmonary microvascular function by measuring the pulmonary transit of agitated contrast (PTAC) identifies less PTAC (low-PTAC) in DM subjects, and associations with reduced RV function, higher pulmonary artery pressures, and lower exercise capacity. Finally, Sildenafil improves a number of central hemodynamic parameters but does not improve VO2peak. Results of this thesis support the argument that in the presence of impaired exercise performance and longstanding DM, subclinical cardiac dysfunction and DMC should not be universally anticipated. Diagnosis of pulmonary microvascular dysfunction using PTAC is feasible and readily accessible, and pulmonary microvascular disease is associated with reduced exercise capacity. Nonetheless, quantification of pulmonary microvascular disease remains imperfect, and the pulmonary vasodilator Sildenafil is ineffective in improving exercise performance presumably due to the mismatch between the pathophysiology thought to underpin diabetic lung disease and the more proximal smooth muscle vascular target of Sildenafil.
Molecular Investigation of the Fanconi Anemia DNA repair pathway
Chemotherapeutic drugs often kill cancer cells by inducing toxic DNA interstrand crosslinks (ICLs) that inhibit DNA replication or transcription. Cells from patients with the genetic disorder Fanconi Anaemia (FA) are extremely sensitive to such DNA crosslinking agents. A critical step in the repair of ICLs is the biochemical modification of FANCI:FANCD2 protein by monoubiquitination. This monoubiquitination reaction is defective in 95% of FA patients, however the exact function of this process remains unclear. This thesis aims to better investigate how FANCI:FANCD2 monoubiquitination is regulated. To determine how monoubiquitinated proteins regulate the FA DNA repair pathway, several targets of monoubiquitination including FANCI:FANCD2, PCNA trimer and nucleosomes ubiquitinated by BRCA1:BARD1 were purified using Avi-ubiquitin. This thesis presents a minimal approach to purify challenging DNA repair proteins, which could potentially be applied to elucidate signalling mechanisms of other ubiquitination-mediated DNA repair pathways. Biochemical reconstitution of the FA pathway using recombinant human proteins revealed that FANCI:FANCD2 monoubiquitination is dependent on DNA and low ionic strength buffers. In addition, electrophoretic mobility shift assays revealed that monoubiquitination locks FANCI:FANCD2 on double-stranded DNA (dsDNA). Affinity pulldown of monoubiquitinated FANCI:FANCD2 confirmed that the complex remained bound to dsDNA, reflecting the monoubiquitination’s role in stabilising the FANCI:FANCD2 complex. The increased affinity of monoubiquitinated proteins on dsDNA suggests a conformational change on FANCI:FANCD2 after monoubiquitination. Using a newly developed Avi-tag-ubiquitin construct, the first successful purification of dually-monoubiquitinated FANCI:FANCD2 complex bound to DNA was reported. Strikingly, single-particle electron microscopy analysis revealed that monoubiquitinated FANCI:FANCD2 forms oligomers on dsDNA. This unexpected discovery hints that the function of monoubiquitination is to lock FANCI:FANCD2 oligomers on chromatin to initiate DNA repair. FANCI phosphorylation was previously reported to play a key role in stimulating FANCI:FANCD2 monoubiquitination. Mass spectrometry and western blot using phospho-specific antibodies revealed that ATR-mediated FANCI phosphorylation at specific serine-glutamate (SQ) clusters regulate both ubiquitination and deubiquitination of FANCI:FANCD2 complex. These results indicate a mechanistic view where FANCI phosphorylation is required for ubiquitination-mediated DNA repair which gives insights into the biology of FA pathway and potential chemosensitiser development. Collectively, the results presented in this thesis elucidate the role of monoubiquitination in locking FANCI:FANCD2 on DNA to form oligomers to initiate DNA repair. This work uncovers a new role of monoubiquitinated FANCI:FANCD2 during interstrand crosslink repair and provides new potential chemotherapeutic targets.
Novel aspects of commonly encountered pituitary adenomas in clinical practice
Clinically relevant pituitary adenomas are common in the general population occurring in 1 per 865-1470 people. Prolactinomas and non-functioning pituitary adenomas account for more than 80% of pituitary adenoma types. My thesis is composed of several studies, undertaken at a single centre specialising in the multidisciplinary care of pituitary diseases, investigating clinical issues pertinent to the management of patients with the above adenomas. The findings of these studies contribute to the understanding of outcomes in the local practice, as well as being an Australian contribution to the international experience of managing these adenomas. Firstly, I examine the issue of valvular heart disease in prolactinoma patients treated with cabergoline therapy. By studying a local cohort and by a systematic review I found that the prevalence of cabergoline-associated valvular heart disease (CAV) in prolactinoma patients to be extremely low, with just three confirmed cases amongst 1800 patients described in the literature. I have shown evidence that a simple annual cardiovascular examination is a suitable screening tool for this rare condition and made recommendations on when to consider a diagnostic echocardiogram. Secondly, I examine two clinical issues affecting patients with surgically treated non-functioning pituitary macroadenomas (NFPMAs): the issues of regrowth and recurrence, and of hormonal outcomes. In the largest Australian cohort of cases with NFPMAs to be described, it was found that residual disease is a common finding post-surgery. In cases with residual disease, regrowth occurred in 40% of these cases compared to only 12.5% in cases without residual disease, at a median of 3.6 years of follow-up. Not surprisingly, larger baseline adenoma size was a predictor of regrowth and recurrence. In multivariate analysis, the presence of residual disease and younger age (under 41 years) at presentation were independent risk factors for regrowth and recurrences. Based on this risk factor of younger age, I have recommended that these cases have lifelong radiological follow-up. Results of hormonal outcomes in cases with NFPMAs treated with surgery showed the novel finding of gender differences in hormonal outcomes, with males having a higher prevalence of multiple (more than two) hormone deficiencies (MHD) at presentation than females, and after surgery. In particular, pre-menopausal females had very few hormone deficiencies post-operatively and demonstrated a propensity to recover function. These gender subgroup differences are the first to be noted in the literature and have important clinical implications particularly for pre-menopausal females where fertility preservation is an issue. I also found that pre-menopausal females had smaller adenomas than males suggesting that they may present earlier in the natural history of the disease. In multivariate analysis, larger adenoma size remained a significant factor associated with post-operative MHD. The final part of this thesis is an exploratory study of intrasellar pressure (ISP) and how it relates to adenoma size and hormonal outcomes in cases with NFPMAs undergoing surgery. I did not find that ISP was correlated to adenoma size, but results did suggest that raised ISP may play a role in more frequent hormone deficiencies at presentation. In this study adenoma size was again noted to be showing a trend towards influencing hormonal outcomes. The findings from this study will require a larger cohort in order to better quantify how ISP and adenoma size interact to contribute to the pathophysiology of hormone deficiencies. In these three studies of NFPMAs adenoma size has emerged as an important factor influencing surgical and hormonal outcomes; smaller adenomas are more likely to be fully resected and less likely to result in hormone deficiencies. This thesis lays the foundations for advocating for the consideration of early surgical intervention of NFPMAs (when adenomas are smaller and earlier in the natural history of the disease), in order to optimise surgical and hormone outcomes. The above should now be an important clinical consideration in the multidisciplinary care of these patients.