Medical Biology - Theses
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ItemStructural studies of the mitochondrial import pathway(University of Melbourne, 2008)
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ItemCD40L expression by dendritic cells : a key to helper-independent killer T cell independent(University of Melbourne, 2008)
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ItemCharacterisation of molecular regulation and intracellular signalling of the Toxoplasma gondii lytic cycle( 2015)Much of the pathogenesis associated with the apicomplexan parasites Toxoplasma and Plasmodium, responsible for toxoplasmosis and malaria respectively, arise due to the destructive lytic cycle of Apicomplexa wherein parasites invade host cells, undergo intracellular replication before actively exiting (egress) the host cell destroying it in the process. In this thesis I attempt to understand some of the regulatory mechanisms controlling key stages of the Toxoplasma gondii lytic cycle specifically focusing on signal transduction pathways, including second messenger signalling, kinase activation and protein phosphorylation. In Chapter 3, I examine the biological function of alpha Soluble NSF Attachment Protein (αSNAP) phosphorylation. Here, I show Ser-6 phosphorylation is required for the correct function of αSNAP in controlling secretory traffic. I demonstrate that during intracellular development conditional expression of a non-phosphorylatable form of αSNAP results in Golgi fragmentation and vesiculation of all downstream secretory organelles. In addition, I show that the vestigial plastid (termed apicoplast) is also affected upon overexpression of αSNAP. This work highlights the importance of phosphorylation control of αSNAP function in Toxoplasma organelle biogenesis and exposes a hereto-unexplored mechanism of regulation of vesicle fusion during secretory pathway trafficking in apicomplexan parasites. In Chapter 4, I investigate the signal transduction pathways controlling Toxoplasma egress and motility. I attempt to understand the signal hierarchy and complexities of the intracellular signalling network by assessing the contribution of activators (Ca2+, cGMP, pH and K+) and protein kinases (PKG, CDPK1, and CDPK3) to egress and motility. As intracellular Ca2+ is required for egress and motility I first assess, in real time, intracellular Ca2+ dynamics by applying genetically encoded biosensor technology, GFP-Calmodulin-M13-6 (GCaMP6). I expand my analysis to include cGMP signal transduction using the recently developed cGMP agonist BIPPO (Appendix A). Importantly, I delineated a signal transduction hierarchy where cGMP signalling precedes Ca2+ release. I further incorporate Ca2+-Dependent Protein Kinases TgCDPK1 and TgCDPK3 into my analysis where I find redundancies between the functions of cGMP kinase Protein Kinase G (TgPKG) and TgCDPK3. This work develops a hierarchical understanding of signal transduction in Toxoplasma egress and motility, identifying key intersections between signalling modules. In my concluding discussion I propose an updated model for intracellular signalling incorporating work presented here with the body of published work. In Chapter 5, I identify a novel contribution of cAMP responsive Protein Kinase A (TgPKA) to invasion. Functional analysis of TgPKA3cat identifies an important role in creation of the intracellular replicative niche during invasion. Conditional knockdown of TgPKA3cat results in a severe invasion defect where after invasion, tachyzoites reactivate and host cell deformation prevents successful parasitism. I show that TgPKA3cat has potential roles in moving junction formation and/or reactivation of Ca2+ signalling and motility. This emphasises the importance of signal transduction through cAMP/PKA during invasion. Taken together this body of work demonstrates the diverse and complex requirement of signal transduction pathways in the Apicomplexan lytic cycle. The data presented here provides novel insights into the identity, hierarchy, and interplay of signal transduction modules and contributes to the understanding of apicomplexan intracellular signalling pathways.
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ItemSHARPIN and RIPK1 are key regulators of cell death and inflammation in vivo( 2015)Therapeutic tumour necrosis factor (TNF) inhibition has been remarkably successful in the treatment of inflammatory and autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. Receptor interacting serine/threonine protein kinase 1 (RIPK1) and SHANK-associated RH domain interacting protein (SHARPIN) are proteins that regulate TNF and other immune signalling pathways. In mice, RIPK1 deficiency causes perinatal lethality whilst SHARPIN deficiency due to the inactivating chronic proliferative dermatitis gene mutation results in multi-organ disease including severe dermatitis. In both cases the underlying mechanism for the pathology has been unknown. TNF is best known for upregulating pro-survival and inflammatory transcriptional pathways although cell death in the form of caspase-8-dependent apoptosis or Receptor interacting serine/threonine protein kinase 3- (RIPK3) and Mixed lineage kinase domain-like- (MLKL) dependent programmed necrosis can also result. Whilst apoptosis is generally viewed as being immunologically inert and non-inflammatory, programmed necrosis, known as necroptosis, results in the release of cellular contents into the extracellular matrix and this can drive inflammation. In vitro, SHARPIN and RIPK1 regulate the transcriptional arm of TNF signalling and RIPK1 is also required for TNF-induced necroptosis. Both SHARPIN and RIPK1 also regulate transcriptional and cell death signalling from multiple other immune receptors. We therefore hypothesised that deregulated cell death was causative for the phenotypes caused by RIPK1 and SHARPIN deficiency. This work shows that SHARPIN deficient keratinocytes and dermal fibroblasts are sensitive to TNF-induced cell death. The SHARPIN deficient skin phenotype was completely prevented by deletion of the gene encoding TNF or its death receptor Tumour necrosis factor receptor 1 (TNFR1). SHARPIN deficient mice with caspase-8 heterozygosity and RIPK3 deficiency were almost fully protected from multi-organ inflammation and dermatitis, whilst unexpectedly, combined SHARPIN, RIPK3 and caspase-8 deficiency resulted in lethality. We also show that RIPK1 deficient mice on a C57BL/6 background die perinatally due to TNFR1-independent RIPK3- and MLKL-dependent systemic inflammatory disease, providing strong evidence that RIPK1 is both dispensable for, but required to inhibit, necroptosis. Mice doubly deficient for RIPK1 and RIPK3 or RIPK1 and MLKL were overtly normal at birth but after three to five days became runted and appeared to succumb to an intestinal phenotype marked by excessive apoptosis. Caspase-8 deficiency did not prevent the RIPK1 deficient perinatal lethality but prevented the intestinal phenotype. Finally, analogous to the deleterious effects of caspase-8 and RIPK3 in SHARPIN deficient mice, mice triply deficient for RIPK1, RIPK3 and caspase-8 were viable well into adulthood, and fertile, but eventually developed severe lymphoproliferative disease. These data show that in mice, RIPK1 or SHARPIN deficiency results in widespread inflammation and pathology due to unrestrained caspase-8-dependent apoptosis and RIPK3- and MLKL-dependent necroptosis, in a tissue specific manner. The results highlight that excessive apoptosis can be inflammatory, and that TNF can cause inflammation indirectly by causing excessive cell death and not only by upregulating inflammatory cytokines. RIPK1 activates necroptosis and has been successfully therapeutically targeted in pre-clinical trials. This research shows that RIPK1 also inhibits necroptosis in some tissue types, indicating a protective function for RIPK1 that must be preserved when therapeutically targeting it. These findings may have implications for the treatment of human disease states including stroke, heart attack and inflammatory bowel disease thought to involve excessive and pathological cell death.
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ItemTowards the synthesis of rocaglamide congeners as potential inhibitor of the eukaryotic initiation factor eIF4A( 2015)Silvestrol is one of the naturally occurring cyclopenta[b]benzofurans which exhibits cytotoxic and antiproliferative activities via the inhibition of the eukaryotic initiation factor eIF 4A. However, current synthetic challenges limit further biological studies these compounds. In this work, the use of phase transfer catalysts improved the outcome of the classical Algar-Flynn-Oyamada reaction to furnish a range of 3-hydroxyflavone precursors, thus enabling access to simplified silvestrol analogues. Biochemical evaluation of these congeners showed that the C8-methoxy substitution was important for the translation inhibitory effect. We also reported our investigation on the Pauson-Khand reaction to affect the carbocylization of enynes containing substituted alkenes as an alternative approach to homologous tricyclic scaffolds.
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ItemStatistical analyses of high-throughput sequencing data to study chromatin structure and organisation( 2015)Massively parallel sequencing technology is a powerful experimental tool for molecular biology research. The most obvious application is RNA-seq, where cellular RNA is sequenced to quantify the expression of each gene in the genome. Another application is that of ChIP-seq, which is widely used to identify the genomic binding sites of transcription factors or other proteins. The Hi-C and ChIA-PET methods aim to examine pairwise interactions between genomic loci, such as those between enhancers and genes. Together, these techniques can be used to to study the structure and organisation of the genome, and how they relate to genome function, i.e., as mechanisms of gene regulation. This thesis describes novel statistical and computational approaches for the analysis of data from these sequencing experiments. In particular, this work focuses on the detection of differential features between conditions, e.g., changes in the binding profile for ChIP-seq or in the interaction intensities for Hi-C. Such changes are easier to detect using established methods like those in the edgeR package. Molecular changes are also more likely to be relevant, as they can be associated with the biological differences between conditions. For ChIP-seq data, the aim of the analysis is to identify regions of differential binding between conditions. This is done in a de novo manner that does not depend on pre-specified regions of interest. Instead, empirically defined peaks or sliding windows must be used. Peak calling must be performed independently of the tests for differential binding, in order to maintain type I error control for the latter. Similarly, the false discovery rate may be misinterpreted when multiple overlapping windows are present for each binding site. Some strategies are proposed here to maintain error control in both analyses. The impact of normalization on differential binding analyses is also discussed. Composition and efficiency biases may be present between libraries. However, methods for scaling normalization will only be able to remove one of these biases. The assumptions of some of these methods are examined in a mathematical framework, along with the effect of each method on the analysis. For complex trended biases, a method for non-linear normalization is proposed that is more robust than other approaches at low counts. For Hi-C data, an analysis pipeline is described that spans from read alignment to detection of differential interactions. Several strategies for the alignment of chimeric reads are assessed. The effect of different parametrizations during counting and filtering are examined. The need for non-linear normalization and modelling of biological variability is also demonstrated on real data. A method is proposed to avoid misinterpretation of the false discovery rate, and to combine results from analyses at different spatial resolutions. A similar pipeline is described to detect specific protein-mediated interactions from ChIA-PET data. The inadequacy of an existing method is explored with respect to the non-randomness of ligation. A more robust analysis is described that exploits hetero-linker data to define the null hypothesis. This new approach also accounts for overdispersion between biological replicates, though some care is required in handling low counts. Some work is also done pertaining to general analyses of sequencing data. The statistical concept of independent filtering for count data is explored, and the use of the average abundance as an independent filter statistic is proposed. The effect of zero counts on the residual degrees of freedom in generalized linear models is studied, and a refinement to existing methods is proposed to avoid loss of error control during testing. Intersection-union tests are also examined to integrate analyses from multiple datasets, where several new procedures are proposed to increase power relative to conventional methods. In summary, this thesis describes a number of novel methods for the analysis of ChIP-seq, Hi-C and ChIA-PET data, as well as that of sequencing data in general.
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ItemStructural transitions during cell death: bak activation and oligomerisation( 2015)Apoptotic stimuli activate and oligomerise the pro-apoptotic proteins Bak and Bax resulting in mitochondrial outer membrane permeabilisation and subsequent cell death. This thesis investigates structural transitions occurring to Bak during apoptosis. I present crystal structures of a Bak core/latch dimer and demonstrate the dissociation of the core and latch domains upon Bak activation. I provide the first high-resolution details for the core domain dimer, a subunit upon which the larger Bak oligomer builds. Cellular assays, guided by the presented crystal structures, confirm the physiological relevance of these key events in the intrinsic apoptotic pathway (Chapter 2). I also describe the first crystal structures of Bak in complex with the BH3-domain of Bim (Chapter 3). These studies complement previous work performed on Bax and support an analogous mechanism of activation and oligomerisation. Certain detergents have been reported to activate Bak in vitro. Here I demonstrate that some detergents can oligomerise Bak and/or promote hetero-complexes between Bak and the pro-survival protein Mcl-1. I describe the production of homo-oligomeric and hetero-oligomeric complexes of Bak, which may be amenable to structural studies (Chapter 4). The literature on apoptosis assumes that mouse and human Bak are analogous in structure and therefore function. Here I report structural differences between Bak homologs from these two species (Chapter 5). These differences exist at the site of ligand binding, yet it remains unclear whether they result in functional variations. These data may aid in the development of novel agonists and antagonists of Bak, and could prove fundamental to designing murine-based pre-clinical trials. The BH3-only protein from the Schistosoma mansoni worm has been identified as a potential direct activator of Bak and Bax. This direct activator BH3-only protein is unique as it only binds to one of the pro-survival proteins (Mcl-1). Here I describe the crystal structure of the Schistosoma BH3-domain (sBH3) in complex with Bax. Liposome release assays demonstrate that sBH3 can directly activate Bak and Bax and induce the formation of membrane permeabilising oligomers (Chapter 6). These studies support current models for the activation and oligomerisation of Bax. Defining the structural characteristics of the intrinsic apoptotic pathway provides novel opportunities for drug design. Organic agonists of Bak may prove useful for the treatment of cancers, while antagonists could provide therapy for diseases characterised by excessive cell death.
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ItemMechanistic and structure-function characterisation of the epigenetic regulator Smchd1( 2015)Epigenetic regulation of gene expression is fundamental in controlling biological processes in multicellular organisms. Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic repressor that plays critical roles in X chromosome inactivation, genomic imprinting and monoallelic expression of clustered protocadherin genes. In addition, SMCHD1 is crucial for suppressing the D4Z4 repeat implicated in pathogenesis of facioscapulohumeral muscular dystrophy (FSHD). However, the exact molecular mechanism by which Smchd1 regulates gene expression is unclear. Likewise, little is known about the structure and function of Smchd1 protein apart from that it contains two predicted domains, an N-terminal GHKL-ATPase domain and a C-terminal Smc hinge domain. To understand how Smchd1 mediates epigenetic regulation at the molecular level, I determined genome-wide Smchd1 binding sites in male murine neural stem cells by performing chromatin immunoprecipitation coupled with next-generation sequencing. Together with profiling gene expression and epigenetic marks in wild type and Smchd1-deficient cells, I found that Smchd1 binding at several of its known target genes is correlated with differential gene expression, concomitant with changes in epigenetic modifications. Unexpectedly, a significant proportion of Smchd1 occupancy overlaps with that of CCCTC-binding factor (Ctcf) at distal cis-regulatory elements, indicative of a functional relationship between Smchd1 and Ctcf. Indeed, I demonstrated that Smchd1 and Ctcf could evoke opposing effects on the expression of many protocadherin genes. As Ctcf is implicated in mediating chromatin interactions, these results indicate that Smchd1 may impart epigenetic regulation via physical association with chromatin and maintaining a repressive chromatin state that antagonises Ctcf facilitated chromatin interactions. In order to gain further mechanistic insights into how Smchd1 functions, I conducted structural-functional characterisation of the Smchd1 protein. I determined the domain boundaries and generated recombinant Smc hinge domain with its flanking coiled-coil and recombinant protein corresponding to the N-terminal region of Smchd1 encompassing the putative GHKL-ATPase domain. By performing small-angle X-ray scattering (SAXS) analysis, structural characteristics of those two domains were revealed for the first time. By utilizing a suite of biochemical and biophysical assays, I was able to show that the hinge domain could directly bind to nucleic acids in vitro. Furthermore, I found that a single-amino acid substitution within the hinge domain, equivalent to a modification implicated in FSHD pathogenesis, displayed significantly compromised binding activities. These results support the notion that the Smchd1-chromatin interaction via the hinge domain is critical for its role in epigenetic regulation. In addition, I demonstrated that a putative gain-of-function mutation of Smchd1, could potentially induce a conformational change and acquisition of ATP-binding activity of the N-terminal region of Smchd1. Preliminary results also suggest that this mutation could enhance Smchd1 function in the cellular context, potentially resulting from the altered structural-functional properties. Together, work from this thesis provides unprecedented molecular explanations for Smchd1’s action in epigenetic regulation, which are supported by structural-functional characteristics of the Smchd1 protein uncovered by this study. These results not only offer valuable insights into Smchd1 function, but also may inform future development of much needed therapy for FSHD disease where SMCHD1 is critically involved.
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ItemManipulation of apoptotic signaling promotes cell-mediated immunity and death of infected cells during chronic viral infection( 2015)Overwhelming chronic viral infections produced by human immunodeficiency virus (HIV), Hepatitis B and C viruses (HBV and HCV respectively) subvert immunity to enable their persistence. These viruses maintain high levels of viraemia and antigaemia that in part drives immunodeficiency during infection. Cytotoxic CD8+ T cells are critical effectors of the immune system that promote the death of infected cells. However, CD8+ T cells gradually loss the ability to secrete effector cytokines, proliferate, kill infected cells and dramatically decline in number as chronic viral infections persist, a phenomena commonly termed ‘exhaustion’. A number of groups have tried to reverse the ‘exhausted’ T cell phenotype with cytokine therapy or antagonism of inhibitory receptors, however the proteins driving the loss of effector CD8+ T cells responding to the infection are largely unknown. Two programmed death pathways, intrinsic and extrinsic apoptosis, are critical for immune homeostasis before, during and after short-lived acute infections. Bim, a pro-apoptotic protein of the intrinsic apoptotic pathway plays a prominent role during all stages of immune homeostasis. Considering it’s indispensable immunoregulatory role, I wanted to examine if Bim regulated CD8+ T cell attrition during chronic viral infection. To test this I used the well characterised lymphocytic choriomeningitis virus (LCMV) murine model of chronic viral infection. I infected Bim knock out mice and mice with cell specific deletion of Bim to show a significant increase in the number of antigen-specific CD8+ T cells in the absence of Bim. Furthermore, equivalent rescue of effector cell numbers were seen with the loss of Bak and Bax, the critical gatekeepers of intrinsic apoptosis. Therefore Bim appears to be the sole intrinsic apoptotic protagonist. Cells rescued from apoptosis retained their ability to kill and to secrete cytokine, despite high expression of the inhibitory receptor PD-1. In spite of increased numbers of functional antigen-specific cells, no mice cleared chronic LCMV infection. However, the data is the first to identify Bim as the sole therapeutic target necessary to inhibit intrinsic apoptotic signaling and rescue functional effector cells during chronic viral infection. Having uncovered a critical effector protein controlling CD8+ T cell fate, I then examined the utility of promoting Fas/FasL mediated apoptotic death to promote viral clearance during chronic LCMV and HBV infection. FasLΔs/Δs mice possess a mutation in the extracellular cleavage domain of FasL. I found FasLΔs/Δs mice demonstrated enhanced FasL expression during chronic LCMV infection, that led to rapid control of a semi-acute strain of LCMV and a consistent trend toward accelerated control of HBV viraemia, within our murine model of chronic HBV infection. In addition, I found lpr-/- mice, which lack Fas receptor expression, fail to control HBV viraemia, thereby uncovering a previously unappreciated role for Fas signaling in the control of HBV infection. Lastly, I tested the ability of clinically relevant cancer therapeutics to sensitize infected cells to death within the LCMV and HBV chronic infection models. I found a strong synergism between the FasLΔs/Δs mice and the SMAC mimetic compound Birinapant in promoting HBV control and clearance of viraemia. Manipulation of cellular death pathways with cancer therapeutics represents a novel reimagining of this class of drugs and has important implications for future therapies against infectious diseases. In summary, to combat chronic viral infections I have promoted effector cell survival through Bim antagonism, enhanced cell-mediated cytolysis through FasL/Fas signaling within infected cells and further potentiated FasL/Fas sensitivity with SMAC mimetic treatment. Together, the data described herein has uncovered three avenues for the development of novel therapeutic interventions against chronic viral infection.
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ItemMerozoite antigens of Plasmodium falciparum elicit strain transcending opsonising immunity( 2015)Despite progress towards reducing the global burden, malaria continues to cause approximately 200 million cases and 600,000 deaths annually (World Health Organization, 2014). Although several malaria vaccines are currently in clinical trials, no advanced vaccine candidate has yet demonstrated sufficient efficacy to be a stand-alone vaccine against the highly variant Plasmodium falciparum parasite. Development of effective vaccine strategies requires knowledge of the essential mechanisms for protective immunity and robust assays to serve as correlates of protective immunity. However, exactly which antibody functions are necessary to control parasitemia and clinical symptoms during natural infection remains unclear. The merozoite represents an attractive vaccine target, as antibodies to numerous merozoite antigens have been associated with protective immunity in human cohort studies. This thesis aimed to investigate the importance of merozoite opsonising antibodies for immunity to malaria. Opsonising antibodies, and the Fc Receptor-mediated functions these antibodies elicit, have been poorly studied in malaria partly due to limitations of in vitro assays. Therefore, in this thesis a merozoite phagocytosis assay was developed and validated (Chapter 3), and robust and reproducible phagocytosis responses from THP-1 cells were observed. This assay was then used to measure merozoite opsonisation in a longitudinal study of semi-children from Papua New Guinea (PNG), and phagocytosis responses were demonstrated to correlate with protection from clinical disease and high-density parasitemia (Chapter 4). Due to the highly diverse nature of P. falciparum merozoites, it was important to assess whether merozoite opsonisation involved strain-specific or strain-transcending specificities (Chapter 5). Highly consistent opsonisation and associations with immunity were observed across a panel of common laboratory strains and PNG parasites adapted to growth in vitro. Through use of transgenic parasite lines, the absence of MSP3, MSP6, MSPDBL1 or MSP1-19 was not observed to impact the overall level of merozoite phagocytosis. By depleting antibody reactivity to 3D7 merozoites, opsonisation of merozoites from PNG strains also declined, suggestive of conserved antigenic targets across parasite strains. The findings in this thesis have demonstrated the importance of opsonising antibodies and their associated phagocytic responses for protective immunity to malaria. Robust, reproducible and well-validated assays are a priority to aid pre-clinical and clinical malaria vaccine development. The consistent responses and protective associations provide strong support for merozoite opsonisation as a robust correlate of protective immunity in malaria endemic populations. As the majority of merozoite opsonising antibodies were strain-transcending, uncovering these conserved domains within merozoite surface antigens may yield important novel vaccine candidates with which to tackle this deadly disease.