Medical Biology - Theses
Permanent URI for this collection
Search Results
1 - 10 of 154
-
ItemStructural studies of the mitochondrial import pathway(University of Melbourne, 2008)
-
ItemCD40L expression by dendritic cells : a key to helper-independent killer T cell independent(University of Melbourne, 2008)
-
ItemThe role of BCL2 family proteins in apoptosis regulation during angiogenesis( 2016)Blood vessels are multicellular tubes, lined with endothelial cells (ECs), that form a hierarchical network essential for the distribution of blood, oxygen, nutrients, hormones and immune cells around the body and removal of metabolic waste products from tissues. Angiogenesis, the growth of new vessels from pre-existing ones, is essential to match the size of the blood vessel network to the metabolic demands of growing tissues. During this process, an over-production of vessels results in the formation of a dense vessel plexus that is inefficient for blood flow. From this dense network, excess vessels undergo a process of selective regression termed ‘pruning’ to produce a mature, hierarchical vessel network. EC apoptosis occurs as part of the angiogenic remodelling processes, but its contribution to angiogenic vessel remodelling, be it vessel pruning or some other purpose, has remained unclear. In this thesis I directly investigated the role of EC apoptosis during angiogenesis by analysing mice in which ECs were unable to execute the apoptotic program regulated by BCL2 family proteins. I found that while EC apoptosis improved the efficiency of selective vessel pruning, it was ultimately dispensable for this process. Instead, blood vessels formed in the absence of EC apoptosis contained excessive numbers of ECs resulting in increased diameter of mature capillaries. Having established that the BCL2 family was essential for promoting EC death during angiogenesis, I investigated whether pro-survival members of the family were required for the survival of ECs during angiogenesis. Using the neonatal retina as a model for angiogenesis, I found that while BCL2 was not required for EC survival during angiogenic vessel growth (Chapter 4), MCL1 was required in a dose-dependent manner (Chapter 5). In contrast to normal angiogenesis, BCL2 and MCL1 were both independently required for the growth of abnormal vascular lesions in a murine model of pathological retina angiogenesis (Chapter 6). These studies have conclusively determined the role for EC apoptosis during angiogenic growth and remodelling and provide evidence that targeting distinct BCL2 family pro-survival proteins may be a useful therapeutic approach for targeting pathological angiogenesis.
-
ItemMultiple endocrine neoplasia type 2B: modelling the disease in human cells and avian embryos( 2017)Paediatric cancer initiation is difficult to study because the early stages are often prenatal. Patient cells at the time of detection already have complex genetic (including epigenetic) changes, and the cancer cell population is heterogeneous. One way to model cancer initiation at the organism level is to engineer the specific initiating mutation into the appropriate cell lineage of an experimental animal embryo. For the human cell context, an ideal cell model would start with the normal human cell of origin and create candidate initiating mutations. Multiple Endocrine Neoplasia type 2B (MEN2B) is an autosomal dominant complex oncologic disease of the neural crest (NC) cell lineage, a so-called neurocristopathy. It presents with i) multiple mucosal ganglioneuromas including hyperplasia of the enteric (gut) nervous system leading to gastrointestinal disorders, ii) pheochromocytoma, with sympathoadrenal (SA) hyperplasia and catecholamine disturbance, and iii) medullary thyroid carcinoma with C-cell hyperplasia, elevated calcitonin and calcium metabolism disturbance. In addition, patients have marfanoid facial features. MEN2B results from de novo germline gain-of-function mutations in the gene RET, most often M918T. RET codes for the signalling receptor for the growth factor ligand GDNF, hence in MEN2B cells, RET signalling is divorced from GDNF availability. MEN2B is rare but it is often misdiagnosed especially early in life. This is due to the nature and diversity of the lineages affected; SA and enteric NC-lineage cells and thyroid C-cells, the latter being of foregut endodermal entero-endocrine lineage. We were the first to successfully use CRISPR/Cas9 to mutate the developing chicken embryo in vivo, showing phenotypic abnormality. This included creating a single point mutation by homology directed repair in vivo in NC cells at the avian MEN2B homologous site (M910T). For the human cell context, we combined the CRISPR/Cas9 technology and knowledge of embryo development and cell differentiation to create MEN2B M918T cells using the human embryonic stem cell (hESC) lines H9, HES3 and MEL2. We modified a hESC differentiation protocol to produce enteric NC-like cells, showing in vitro that these cells upregulated key NC and enteric genes. Functionally we also showed higher proliferation and greater axon production in the MEN2B mutant cells: this is consistent with the ganglioneuroma phenotype. In addition, we developed a new differentiation protocol to produce human SA progenitors and medullary chromaffin-like cells, as marked by expression of key catecholamine genes TH and PNMT, and expression of adrenaline and noradrenaline by HPLC. These cells are affected in MEN2B patient pheochromocytoma. We then developed a novel differentiation protocol for thyroid C-cell-like cells from hESCs via Definitive Endodermal Cells. These cells produce Calcitonin and we validated their functionality by ELISA assay and compared the MEN2B clones with the control hESCs. These cells are affected in MEN2B patients resulting in medullary thyroid carcinoma.
-
ItemRegulation of BH3-only proteins by IKK, downstream of beta-common receptor signalling( 2017)Apoptosis is a tightly regulated process and is responsible for maintaining tissue homeostasis, controlling cell numbers and the elimination of damaged or unwanted cells. Intrinsic apoptosis, also known as the mitochondrial apoptotic pathway, is activated by a variety of cellular stresses including growth factor deprivation (serum or cytokine), DNA damaging agents, toxins, hypoxia or viral infections. Activation of the mitochondrial pathway by a cellular stress results in the up-regulation of pro-apoptotic BH3-only proteins, which in turn bind to neutralise Bcl-2 anti-apoptotic proteins and activate downstream apoptotic effectors. The BH3-only proteins Puma and Bim are known potent inducers of cell death and are able to bind all Bcl-2 anti apoptotic family members. Our lab and other groups have shown that Puma is subject to posttranslational regulation by phosphorylation at a conserved residue, Serine-10, following a period of cytokine deprivation (IL-3) of factor dependent myeloid cells (FDMs), and subsequent re-stimulation with that cytokine (Fricker, O’Prey, Tolkovsky, & Ryan, 2010; Sandow et al., 2012). Serine-10 phosphorylation targetted Puma for ubiquitination and proteasomal degradation. Mutation of Serine-10 to an Alanine abolished Puma phosphorylation. Surprisingly, the kinase found to be responsible for this phosphorylation was IKK1, a member of IκB kinase complex IKK, a crucial member of NF-κB activation, downstream of TNF. This study suggests the existence of a previously unrecognised link between beta common receptor cytokine signalling, IKK activation and the regulation of BH3-only proteins. In this thesis, we explored whether other BH3-only proteins can be regulated by IKK, following beta common receptor cytokine stimulation (IL-3 or GM-CSF). We found Bim protein levels were up-regulated after a period of cytokine deprivation and rapidly degraded, following re-stimulation with either IL-3 or GM-CSF. Strikingly, we also observed the phosphorylation of IκBα, following this time course. Bim protein degradation was abolished in the presence of an IKK inhibitor, delayed by the deletion of IKK1 and to a lesser extent, the deletion of IKK2. Further to that, stimulation of WT MEFs or FDMs with TNF, a well-known activator of IKKs, resulted in Bim size shift and degradation. Through comparative sequence alignment we observed that Serine-10 is conserved in Bim. However, mass spectrometry analysis did not reveal the phosphorylation of this Serine as a result IL-3 cytokine deprivation and re-stimulation. Overall, mass spectrometry analysis revealed the phosphorylation of Bim sites which have been previously reported. To study the physiological role of IKK in the haematopoietic compartment, we used the leukaemia model MLL-AF9, which is associated with poor prognosis in patients. Previous studies have shown that IKK suppression delays the progression of myeloid leukaemia. Our results showed that mice intravenously injected with primary leukaemic bone marrow cells deficient of IKK1 did not succumb to leukaemic disease as early as their WT littermates. These results provide evidence for a link between members of the intrinsic and extrinsic pathways of apoptosis and may provide new therapeutic avenues for acute myeloid leukaemia.
-
ItemThe identification of small molecules that target the insulin or Type1 insulin-like growth factor receptor ectodomain( 2017)The insulin receptor (IR) and Type 1 insulin-like growth factor receptor (IGF-1R) are homodimeric α2β2 receptor tyrosine kinases. While both IR and IGF-1R possess high affinity for their cognate ligands (insulin, and IGF-I and IGF-II, respectively), they can also bind and be activated by the reciprocal ligands. Activation of either receptor may effect downstream PI3K-AKT and RAS-RAF- MAPK signalling, with varying outcomes on cellular glucose metabolism, differentiation and proliferation. Aberrant signalling leads to a number of clinical manifestations, including diabetes and cancer, making both receptors attractive pharmaceutical targets. The primary extracellular ligand-binding site of these receptors is a tandem element comprising the first leucine-rich repeat domain (L1) of one receptor α-chain in association with the C-terminal region (αCT) of the alternate receptor α-chain. Disruption of the L1 and αCT association abrogates ligand binding and may have additional effect on basal receptor activation. I have obtained two crystal structures that describe the respective ways in which IR αCT and IGF-1R αCT engage the IR L1 domain, as well as a third structure that reveals that the high-affinity IR agonist peptide S519C16 shares the same disposition as IR αCT upon the IR L1 domain surface. Together, these three structures imply that S519C16 derives its agonistic functionality from its displacement of αCT away from receptor L1 domain and justify the search for and development of small-molecule therapeutics that may modulate the activity of IR/IGF-1R through a similar mechanism. Both biochemical and computational methods have been employed to identify such molecules. Of 114,000 compounds screened in a high-throughput in vitro assay, 22 compounds were found to disrupt the association between a truncated IR or IGF-1R L1-containing construct and S519C16 with IC50 < 10 μM. Nine of these compounds were also found to antagonise ligand-dependent IR activation, consistent with the expectation that the compounds disrupt the interaction occurring between IR L1 and IR αCT in holo-IR. Additional chemical scaffolds capable of antagonising the interaction between truncated IR and S519C16 have also been identified using integrated structural, biochemical and computational methods. It is anticipated that the identified small-molecule antagonists of the interaction between S519C16 and IR may be developed into therapeutic compounds capable of modulating IR and/or IGF-1R activity in vivo.
-
ItemCharacterization of cyclic nucleotide-mediated signal transduction pathways in Toxoplasma gondii( 2017)T. gondii is amongst the most common human pathogens, chronically infecting more than 30% of the human population. T. gondii has a complex life cycle, often spanning multiple hosts. T. gondii parasites invade target host cells to establish infection. Residing inside host cells, parasites are immotile and active replicate. After multiple rounds of replication, T. gondii activates their motility and egress from host cells. Invasion and egress relies on parasites motility, which is strictly controlled by a number of second messenger-mediated signalling transduction pathways. The signalling pathways that controls parasite motility have become a focus of investigations in the past two decades due to its potential as a therapeutic target. Previous studies showed that activation of Ca2+-mediated signalling pathways and cGMP-mediated signalling pathways drives parasites microneme secretion and motility. Conversely, cAMP-mediated signalling pathways may work as suppressors on parasites motility. In Chapter 3, I evaluated the function of a putative guanylyl cyclase in T. gondii asexual growth. TgGC accumulates at apical tip of the parasites and alters its localization during intracellular growth and extracellular motility. TgGC is critical for T. gondii growth and it plays an important role in parasites microneme secretion, invasion and egress, highly suggestive of its function in cGMP generation. We showed that cGMP-mediated signalling pathways localize upstream of cytosolic Ca2+ rise and could be occurring by activation of phosphatidylinositol-specific phospholipase C. In Chapter 4, I investigated the regulation of cAMP signalling. I identified the cAMP-dependent protein kinase A regulatory domain TgPKAr that regulates activity of catalytic domain TgPKAc1, which has been suggested as a negative regulator of motility and Ca2+ signalling. TgPKAr and TgPKAc1 localize at the parasite periphery, which occurs likely through palmitoylation and myristoylation on the N-terminal TgPKAr sequence. Overall, my work highlights the pivotal role signal transduction pathways play in T. gondii parasites and sheds light on the complex hierarchy and interplay between second messengers-mediated signalling cascades that regulate parasites motility and infectivity.
-
ItemA CRISPR/Cas9-based investigation of inflammasomes in infectious disease and autoinflammation( 2017)Inflammasomes are a family of innate immune signalling platforms that are activated in response to tissue damage or infection. Inflammasome stimulation results in activation of the inflammatory protease caspase-1, which induces a lytic cell death program known as pyroptosis, and maturation and release of the pro-inflammatory cytokines Interleukin-1β (IL-1β) and IL-18. The potent inflammatory cascade triggered through activation of the inflammasomes is protective against many bacterial pathogens that either invade host cells or produce toxins that deregulate key homeostatic mechanisms within innate immune cells such as monocytes and macrophages. De-regulation of inflammasome signalling, such as gain-of-function mutations in inflammasome components, can result in autoinflammatory pathology. In order to investigate the function and regulation of inflammasomes, Clustered, Regularly Interspersed, Short, Palindromic Repeats (CRISPR)/Cas9 gene editing technology has been utilised to delete various inflammasome components from human myeloid cell lines or from mice. The alternative inflammatory caspases, caspase-11 in mice and caspases-4 and -5 in humans are activated directly by cytoplasmic lipopolysaccharide (LPS), a key component of the cell wall of gram-negative bacteria. These caspases are able to induce pyroptosis independently of caspase-1, but are only able to trigger IL-1β and IL-18 release in a caspase-1-dependent manner. In this thesis, the roles of caspase-4 and caspase-5 in the response to cytoplasmic lipopolysaccharide (LPS) and invasive gram-negative bacteria have been investigated in a human monocytic cell line. While both caspases responded to infection with live gram-negative bacteria, free LPS that was transfected into the cytoplasm activated only caspase-4. This suggests that caspases-4 and -5 may be activated by distinct stimuli or through different mechanisms. This work also interrogates the role of the inflammasome-forming receptor pyrin, in both autoinflammatory disease and the anti-bacterial immune response. A serine to arginine mutation in pyrin at amino acid position 242 results in a newly described autoinflammatory condition known as Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND). A monocytic cell line expressing the S242R mutant of pyrin has been created and it was demonstrated that this mutation results in spontaneous inflammasome activity. Under homeostatic conditions, serine 242 is phosphorylated and interacts with the 14-3-3 family of adapter proteins to keep pyrin inactive. Deletion of specific 14-3-3 isoforms also resulted in spontaneous production of mature IL-1β. Finally, the expression of pyrin in various myeloid compartments and its role in in vivo models of bacterial infection have been investigated using a pyrin-deficient mouse line. Two isoforms of pyrin were detected that were differentially expressed among myeloid populations. Additionally, no role for the pyrin inflammasome was observed in a Dextran Sodium Sulfate (DSS)-induced colitis model, or Citrobacter rodentium, Salmonella Typhimurium or Mycobacterium tuberculosis infection models.
-
ItemBiochemical and biophysical investigations into key malaria parasite proteins( 2017)Plasmodium falciparum, the most pestilential of the malaria parasite species, is responsible for ~450,000 direct deaths annually. Clinical disease is a consequence of the blood stage of the parasite’s lifecycle involving a plethora of host-parasite interactions. Key to these interactions are the P. falciparum reticulocyte binding-like homologue (PfRh) proteins responsible for binding erythrocyte receptors and gaining entry to host cells. For example, PfRh4 binds to human complement receptor-1 (CR1) on erythrocytes for sialic-acid-independent invasion. Another protein important for invasion is the PfRh5-interacting protein (PfRipr), an essential member of the PfRh5-associated invasion complex (PAIN-complex) along with CyRPA, the cysteine-rich protective antigen. Loss of function of PfRipr in P. falciparum parasites prevents erythrocyte entry and ablates Ca2+-influx into the erythrocyte; essential events during invasion. This study aimed to biochemically and structurally investigate truncated recombinant versions of PfRh4 and PfRipr. Homology modelling suggested that PfRh4 is rich in alpha-helical secondary structure. The sequence of PfRipr suggested the presence of ten epidermal growth factor-like (EGF) modules, two towards the N-terminus and eight in the C-terminal domain. In this project, monoclonal antibodies made against recombinant PfRh4 were shown, via indirect immunofluorescent assays, to localize to the apical tip of merozoites. Monoclonal antibody 5H12, raised against PfRh4, reduces parasite invasion of erythrocytes by ~75% in growth-inhibition assays with neuraminidase pre-treated erythrocytes. Attempts to produce a stable truncated recombinant PfRh4 protein for structural studies were unsuccessful. An ELISA-based assay using ten alanine-scan mutants suggested the CR1-binding site lies outside of amino acids 283 – 341 of PfRh4. PfRipr truncations, defined by the boundaries of EGF-like repeats predicted based on sequence homology, were produced recombinantly in Escherichia coli and Pichia pastoris. These proteins had a circular dichroism signature suggestive of β-strand-containing proteins with disordered regions. EGF-containing PfRipr truncations did not bind recombinant PfRh5 according to ELISA and size-exclusion chromatography assays. EGFs 1-2, 5-7 and 7-10 of PfRipr did not bind CyRPA via size-exclusion chromatography or NMR. Crystallisation trials performed on EGF modules failed to yield crystals suitable for data collection. A 15N isotopically-labelled sample of EGF5-7 gave good quality HSQC NMR spectra. A 15N isotopically-labelled sample of EGF5-7 gave good quality HSQC NMR spectra. A suite of three-dimensional NMR spectra collected on a 13C,15N-EGF5-7 sample, at three different temperatures, allowed for >86% of backbone assignments. T1/T2 relaxation analysis and heteronuclear NOE data were suggestive of an elongated, rigid protein undergoing intermolecular self-association. Further evidence for EGF5-7 being an elongated protein was provided via SAXS analysis. Chemical shifts facilitated prediction of secondary structure in EGF 5-7 consistent with an EGF-like fold. Melting studies performed on EGF5-7 showed no evidence of denaturation over the temperature range 20˚C - 95˚C indicating a thermally-stable protein. The addition of Ca2+ to the 15N-EGF5-7 sample caused chemical shift perturbations consistent with high-affinity binding. The discovery of inhibitory monoclonal antibodies recognising a conformational epitope on EGF7 provided evidence of the functional importance of this region within PfRipr. The work described in this thesis provides methods for the industrially-scalable production and biophysical investigations of P. pastoris or E. coli-produced disulfide-rich P. falciparum antigens of interest to vaccinologists.
-
ItemInvestigating the role of NK cells in allogeneic haematopoietic stem cell transplantation( 2017)Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is used to treat a range of haematological malignancies. However, the use of allo-HSCT is limited due to significant toxicity related to conditioning intensity, opportunistic infection, graft failure, and graftversus host disease (GVHD). Standard conditioning regimen including myeloablative total body irradiation (TBI) is considered to be the trigger for pro-inflammatory cytokine release and further induce acute GVHD (aGVHD). To decrease the risk of aGVHD, a reduced intensity conditioning (RIC) regimen was introduced to clinical practice because of its lower toxicity. However, the insufficient suppression of host immune system caused by RIC might lead to higher chances of graft failure. In this case, to identify the risk factors to graft rejection after RIC regimen was crucial. And by targeting the major contributor to graft failure post-transplantation, RIC can be utilised to prevent aGVHD while high donor engraftment can still be achieved. Recipient natural killer (NK) cells were found to be significantly more radio-resistant than other cytotoxic lymphocytes following TBI and thus could be a considerable contributor to acute allograft rejection. MHC-mismatched transplantation allo-BMT mouse models were utilised in this project to determine the extent by which radio-resistant recipient NKs are involved in causing acute allograft rejection. By using wild-type (WT) and the NK cell deficient (Bcl2fl/fl Ncr1-iCre) mice as recipients, donor cells were rapidly rejected in WT recipients with RIC-allo-BMT, whereas Bcl2fl/fl Ncr1-iCre recipients that were irradiated with the RIC 2×400 rad TBI achieved long-term engraftment and lower aGVHD clinical scores. This positive outcome with reduced TBI dose was attributed to lesser donor T cell expansion, lower pro-inflammatory cytokine levels and higher myeloid cell reconstitution in the NK cell deficient recipients, compared to recipients that had undergone transplantation with myeloablative TBI (2×600 rad). These findings clearly indicate that recipient radio-resistant NK cells are the main cause of graft failure and can be targeted to lower conditioning intensity and promote engraftment. Besides the Bcl2fl/fl Ncr1-iCre mouse models, other NK cell deficient or aberrant models were also studied in order to understand further how NK cells can regulate aGVHD and donor engraftment. Using an alternative approach with greater clinical relevance, WT mice administered with the BCL2-inhibitor S63845 and/or the MCL1-inhibitor ABT-199 exhibited similar outcomes as the BCL2- and MCL1-deficient mouse models. Inhibitor treatment prior to allogeneic transplantation with RIC regimen reduced the number of host residual NK cells. And in this case, mice that had the inhibitor treatment and RIC-allo-BMT had reduced risk of graft failure and could also be free from aGVHD. Another key finding of this study, which has significant potential for clinical translation was validation of the therapeutic graft-versus-leukaemia (GVL) effect in the RIC-allo-BMT models. Either genetically modified NK cell deficient mouse RIC-allo-HSCT models or inhibitor treatment mouse RIC-allo-HSCT models were proven to have intact GVL effect. Therefore, the less sufficient anti-neoplastic efficacy of RIC regimen could be compensated by the intact GVL effect. And most importantly, the overall mortality related to leukaemia relapse of the NK cell deficient RIC-allo-BMT models was significantly lower than the WT RIC-allo-BMT models.