Medical Biology - Theses
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ItemFunctional characterisation of Caspase-9 in haematopoiesis( 2012)Caspases are a family of cysteine-aspartic proteases that play essential roles in programmed cell death (apoptosis), programmed necrosis (necroptosis), and inflammation. This work aims to clarify additional reported functions of caspases, and to enhance our understanding of the functional roles of caspases in the blood (haematopoietic system). By genetically dissecting the apoptotic pathway, I show that caspase activation is not required for megakaryocytes to form platelets from their cytoplasm. Rather the opposite is true, apoptotic caspase activation must be restrained for megakaryocytes to survive and produce platelets. In addition, platelets are fully functional without the initiator Caspase-9. Caspase-9-deficient platelets maintain blood clotting (hemostasis), and are capable of facilitating thrombin generation via the exposure of membrane phospholipid phosphatidylserine – supporting the notion that platelet apoptosis and platelet activation are biochemically distinct processes. Herein, I also show that the Bcl-2 regulated caspase cascade is critical for haematopoietic stem cell maintenance. A novel relationship between apoptotic caspase activation and type-1 interferon production – a cytokine known to regulate ‘stem-ness’ – is established. Together, this research refines previously described biological functions for caspases, and provides new insight into the role of caspases in cell death and the physiological consequence of their genetic or pharmacological inhibition.
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ItemDisulfide-rich peptide toxins: structure, function and design of minimal analogues( 2012)Disulfide bonds play important roles in stabilizing the native conformation of peptides and proteins. The cone snail venoms are an abundant source of disulfide-rich peptide toxins, commonly known as conotoxins. Over many years, the cone snails have evolved a number of disulfide scaffolds on which huge and diverse combinatorial libraries of peptides are generated. Many of these peptides are potent and selective modulators of ion channels and receptors. Characterization of new conotoxins could lead to the discovery of novel therapeutics and drug leads. This thesis reports the characterization of a conotoxin from the Conus imperialis that has both a novel cysteine framework and disulfide connectivity pattern and defines a new superfamily. Structural characterization of this conotoxin identifies a novel molecular scaffold that conforms the peptide into a unique helical hairpin fold, highlighting the role of disulfide bonds in generating new structural scaffolds. In characterizing conotoxins with multiple disulfide bonds, in addition to challenges posed synthetically, defining the native disulfide connectivities from a myriad of different possibilities can be a difficult task. From a therapeutic point of view, disulfide bonds can be subject to scrambling and reduction in different environments. Thus there is a need for alternative strategies for replacing disulfide bonds in conotoxins with therapeutic potential. This thesis explores the disulfide connectivity of the previously characterized analgesic µ-conotoxin KIIIA and its disulfide isomers. Interestingly, distinct disulfide isomers of µ-KIIIA are capable of potent blockade of voltage-gated sodium channels (VGSCs). µ-conotoxins such as µ-KIIIA that block the VGSCs serve as potential drug leads for designing novel analgesics, as several neuronal VGSC subtypes have been implicated in the perception of pain. Previous structure-activity studies of μ-KIIIA identified a helical pharmacophore for VGSC blockade. This suggested a route for removing all three disulfide bonds and designing truncated analogues of μ-KIIIA by incorporating the key residues into an α-helical scaffold that could be stabilized by an (i,i+4) constraint. The design, structure and function of minimized helical analogues of μ-KIIIA are described in this thesis.
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ItemTargets of antibodies to the surface of Plasmodium falciparum-infected erythrocytes and protective immunity to human malaria( 2012)Effective clinical immunity that protects against symptomatic malaria in humans develops gradually after repeated exposure to Plasmodium falciparum. Naturally acquired antibodies targeting antigens expressed on the surface of infected erythrocytes (IE) represent an important component of protective immunity against malaria. During intra-erythrocytic development, P. falciparum dramatically remodels the host erythrocyte membrane through the export of novel parasite proteins. Among these are antigens expressed on the IE surface, known as variant surface antigens (VSA), that include PfEMP1, RIFIN, STEVOR, SURFIN proteins and possibly others such as PfMC2TM and modified host band 3. These antigens are highly polymorphic and some are known to undergo clonal antigenic variation for immune evasion. Numerous studies have reported that individuals living in malaria endemic regions were capable of agglutinating P. falciparum-IEs suggesting the recognition of VSAs expressed on the IE surface. Longitudinal studies further suggested that anti-VSA antibodies were associated with protection from P. falciparum malaria. Previous studies have only been able to measure the acquired antibody response towards all VSAs expressed on the IE surface, due to a lack of tools to dissect the antibody responses to individual VSAs. Although studies have also evaluated antibodies to recombinant proteins, it has been difficult to directly quantify the contribution of each native VSA to the overall antibody response to the IE surface. The aims of this thesis were to quantify the significance of VSAs as targets of naturally acquired antibodies, with a particular focus on P. falciparum erythrocyte membrane protein 1 (PfEMP1) and evaluate the importance of naturally acquired antibodies to PfEMP1 and other antigens that afford clinical protection from symptomatic P. falciparum malaria. Novel approaches using transgenic P. falciparum with inhibited PfEMP1 expression have enabled the quantification of PfEMP1 relative to other VSAs as a target of acquired antibodies. This was achieved by the transfection of parasites with a construct that encodes a var gene promoter without a downstream var gene (vpkd; presented in Chapter 3) thus resulting in a PfEMP1-deficient line and the transfection of parasites with a construct that has a deletion of the pfsbp1 gene required for PfEMP1 trafficking (SBP1KO; presented in Chapter 4). These approaches were then applied to human studies in Kenya and Papua New Guinea (PNG) and comparisons between parental and PfEMP1-deficient transgenic parasites allowed for the determination of antibodies specific to PfEMP1. The functional significance of naturally acquired antibodies was determined using assays that specifically measure antibody-mediated phagocytosis of IEs by undifferentiated monocytes. In addition, the clinical importance of PfEMP1-specific antibody responses was further investigated in a longitudinal cohort study with PNG school children. Characterisation of the transgenic vpkd parasites demonstrated reduced var gene transcription by Northern blot analyses and the absence of PfEMP1 proteins by Western blot analyses of IE membrane extracts, thus suggesting that PfEMP1 expression was inhibited in these parasites. In addition, characterisation of the SBP1KO parasites confirmed the absence of PfSBP1 protein in Western blot analyses and immunofluorescence microscopy of pigmented trophozoite IEs. However, other VSAs such as RIFIN and STEVOR, and other IE membrane proteins such as PfEMP3 were still expressed by the transgenic vpkd and SBP1KO parasites. Furthermore, transmission electron microscopy of pigmented trophozoite IEs confirmed the presence of knob structures on the IE surface of the vpkd parasites, similar to parental parasites. These findings suggest that despite the inhibition of PfEMP1, other IE membrane proteins and knob assembly occurred normally in the transgenic vpkd and SBP1KO parasites. Among malaria-exposed children and adults from Kenya and PNG, IgG binding to the surface of erythrocytes infected with the transgenic vpkd parasites was substantially reduced compared to parental. This suggests that majority of the acquired antibody response to the IE surface was predominantly directed towards PfEMP1, while other VSAs appear to play a minor role in relation to immunity. These key findings were confirmed with two genetically different parasite lines, 3D7 and E8B. Furthermore, using sera from children, adults and pregnant women available from Kenya or PNG, IgG binding to the surface of erythrocytes infected with the transgenic SBP1KO parasites was also markedly reduced compared to parental, suggesting that antibodies primarily targeted major antigens expressed on the IE surface that are dependent on PfSBP1 for trafficking. Currently, only PfEMP1 is known to be trafficked by PfSBP1 and this study has demonstrated that other VSAs such as RIFIN and STEVOR proteins remain expressed by the SBP1KO parasites. Comparing the antibody responses between PNG adults and children demonstrated that IgG binding to the vpkd and SBP1KO parasites was substantially reduced in both groups, suggesting that both adults and children had a great proportion of PfEMP1-specific antibodies. Evaluating the effect of trypsin treatment of IEs on antibody binding showed that most serum samples targeted trypsin sensitive epitopes expressed on the IE surface, consistent with PfEMP1 being the major target of antibodies. However, some samples appeared to target trypsin resistant epitopes on the IE surface of the vpkd and SBP1KO parasites. This study provides major new evidence that PfEMP1 is the dominant target of naturally acquired antibodies to the IE surface. In assays that specifically measure antibody-mediated phagocytosis by undifferentiated monocytes, the level of opsonic phagocytosis activity was greatly reduced in the transgenic vpkd parasites compared to parental. These results suggest that PfEMP1-specific antibodies are essential to promote IE opsonisation for phagocytosis by monocytes, an important mechanism in parasite clearance. Thus, these finding provide further evidence that PfEMP1 represents the major target of functional antibodies. Some measurable level of opsonic phagocytosis activity was still detected with the transgenic vpkd parasites although the level of IgG binding to these parasites were extremely low, suggesting that antibodies to non-PfEMP1 antigens may also function to opsonize IEs for phagocytic clearance. The clinical importance of antibodies to PfEMP1 and other VSAs was further evaluated in a longitudinal study conducted with school children from Madang, PNG. Antibodies to the 3D7 parental and 3D7-PfEMP1 (reflected in the difference between IgG binding to 3D7 parental versus 3D7vpkd) were associated with a significantly reduced risk of symptomatic P. falciparum malaria whereas antibodies to 3D7vpkd (reflecting antibodies to non- PfEMP1 antigens) were not associated with protective immunity. Children with antibodies to other isolates such as E8B and XIE-ICAM also had a reduction in malaria risk, however these associations were not statistically significant. It should be noted that there was insufficient statistical power in the current study to detect differences in small effect sizes and weak associations. The protective association with 3D7 observed in the current study of PNG school children complements a longitudinal study conducted with children in Chonyi, Kilifi (J. Chan and K. Howell et al 2012, J Clin Invest, in press) whereby antibodies to 3D7 parental and 3D7-PfEMP1 were associated with protection but antibodies to 3D7vpkd were not. Therefore, these findings indicate that PfEMP1 represents a major target of naturally acquired antibodies that are associated with protective immunity. However, these studies do not exclude an important role for other VSAs as targets of protective antibodies and further studies are essential to understand their significance as antibody targets and their association with protection from malaria. The results presented in this thesis provide major new evidence that among the VSA families present on the surface of P. falciparum-IEs, PfEMP1 represents the dominant target of naturally acquired human antibodies and antibodies to PfEMP1 contribute to protective immunity against malaria. Novel approaches using PfEMP1-deficient transgenic parasites performed in this study offered a unique insight to determine the relative contribution of PfEMP1 and other VSAs to the overall antibody response to the IE surface. Therefore, the work presented in this thesis enhances the understanding of humoral immunity to malaria and will aid the development of vaccines against malaria.
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ItemThe independent and overlapping roles of chromatin regulators MOZ, QKF, and BMI1( 2012)Chromatin plays an integral role in regulating stem cells and embryonic development. Two complexes that play an important role in regulating chromatin in stem cells and development are the MOZ/QKF complex, and the BMI1-PRC1 complex. The MOZ/QKF complex is associated with a transcriptionally active state, whereas the BMI1-PRC1 complex is a transcriptional repressor. Curiously, the MOZ/QKF and BMI1-PRC1 complexes have previously been shown to have very similar functions, despite having opposing transcriptional activities. In this study, I aim to broaden our understanding of the role of MYST-family histone acetyltransferases MOZ (MYST3, KAT6A), and QKF (Querkopf, MORF, KAT6B), in stem cells and embryonic development. In this thesis, I show that high Qkf levels are a marker for self-renewing and multipotent neural stem cells (NSCs). A novel relationship is established in this study, whereby in the adult forebrain NSC niche, Qkf is most strongly expressed in the rare NSC population, and decreases progressively as commitment and differentiation proceeds in the neural lineage. Consistently, in a Bmi1 deficient state where the self-renewal of NSCs is severely compromised, there was an age-related decline in the highest Qkf expressing fraction. During early embryonic development, MOZ and BMI1 are required for the specification of the anterior-posterior segment identity via the regulation of Hox genes. My work shows that MOZ and BMI1 play opposing roles in specification of the axial skeleton. Both were required to determine the correct anterior expression boundaries of Hox genes. In contrast to playing opposing roles in the specification of body segment identity, MOZ and BMI1 played a synergistic role in regulating the self-renewal and proliferation of haematopoietic stem cells. Furthermore, I show that MOZ is a positive regulator of the lymphocyte lineage. Utilising primary mouse embryonic fibroblasts, I establish that MOZ, in a similar manner to BMI1, is a negative regulator of cellular senescence. Together, the data presented in this thesis show that MOZ, QKF, and BMI1 play important and interconnected roles in regulating stem cell self-renewal, multipotency, and cellular senescence.
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ItemThe role of polycomb group genes in cancer( 2012)This thesis examines the role of a specific class of epigenetic modifiers, the polycomb group (PcG) proteins, in the context of cancer development. Polycomb group proteins form distinct transcriptional repressor complexes – polycomb repressive complex 1 (PRC1) and 2 (PRC2) – that coordinate changes in chromatin structure via post-translational modification of histone protein tails. PcG genes are aberrantly expressed in various types of cancers, but the underlying molecular mechanisms by which this drives malignancy remain poorly understood. Indeed recent findings demonstrate that PRC2 possesses dual roles in the development of cancer that depend on tumour cell type. A deeper understanding of the mechanism by which PcG proteins contribute to cancer is required to facilitate the design of potential therapeutic agents. To further investigate the role of PcG genes in cancer, two distinct mouse models of cancer were employed in this thesis – a haematological cancer model using the Eμ-myc transgenic mice, a model of B-cell lymphoma, and an epithelial cancer model of intestinal neoplasia using the APCMin mice. Eμ-myc mice carrying heterozygous loss-of-function alleles in PRC2 components Suz12, Ezh2, or Eed, exhibited accelerated lymphoma development, while heterozygosity of Bmi1, a central component of PRC1, delayed disease onset. This finding was further confirmed in transplantation experiments using Eμ-myc foetal liver cells that expressed shRNAs targeting Suz12 or Ezh2. This novel finding established that PRC2 behaves as a tumour suppressor in B-cell lymphoma while PRC1 acts as an oncogene. Furthermore, it supports previous observations that PRC2 and PRC1 have opposing roles in the haematopoeitic stem cell compartment by regulating distinct target genes. Analyses of pre-neoplastic Eμ-myc mice carrying a loss-of-function allele of Suz12 showed accumulation of the B-lineage cells in the bone marrow, spleen and peripheral blood, without obvious defects in apoptosis or proliferation. The number of clonogenic B-lymphoid progenitors in the bone marrow was increased in Suz12-deficient mice. This demonstrates a role for PRC2 in regulating B-lymphopoiesis, and suggests that partial loss of PRC2 function enhances self-renewal of B-lineage precursors, an observation supported by gene expression analysis of lymphomas which revealed that stem-cell related gene signatures were consistently represented in Suz12-deficient lymphomas. This novel finding was extended to investigate the development of intestinal neoplasia in APCMin mice. Unexpectedly, heterozygous loss of Suz12 or Bmi1 did not influence the overall survival kinetics in APCMin mice, and these mice exhibited similar phenotypic features and developed similar number of intestinal polyps at the moribund stage. This suggests that the intestinal system is more robust when faced with partial loss of PRC1 or PRC2. This is in direct contrast to the haematological system, which is extremely sensitive to dosage differences of PRC1 and PRC2. Collectively, this study has established that PRC2 normally suppresses Myc-driven lymphomagenesis, likely by restricting the self-renewal of B cell progenitors. The influence of polycomb repressors in cancer development is highly cell type-specific and context-dependent.
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ItemThe role of HBO1 during embryonic development( 2012)This thesis presents the first in vivo characterisation of HBO1 (histone acetyltransferase bound to ORC1; MYST2/KAT7) function at the molecular and cellular level. Specifically, HBO1 function was investigated during (a) early embryonic development, by analysing Hbo1 null mutant mice and (b) in brain development during late gestation, by analysing conditional transgenic mice displaying HBO1 deficiency in the central nervous system. HBO1 is essential for H3K14 acetylation (H3K14ac) and the activation of key developmental regulatory genes in both mouse models. In the first study, this process is crucial for the patterning of rapidly differentiating embryonic tissues such as the mesenchyme, vasculature, heart and neural tube during post-gastrulation development. In the second study, HBO1 mediated H3K14ac is indispensible for the expression of genes required for neuronal lineage commitment and differentiation. During embryonic brain development, HBO1 is critical for dentate gyrus patterning, neuronal migration, cortical lamination and layer specific gene expression. Unexpectedly, in contrast to numerous earlier reports implicating HBO1 in DNA replication, this thesis demonstrates that HBO1 is not essential for DNA replication. Together, in vivo and in vitro data presented in this thesis illustrate the dependency on HBO1 for H3K14ac, transcriptional activation and cellular differentiation.
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ItemFunctional studies of dendritic cells in type 1 diabetes: non-obese diabetic (NOD) mouse model( 2012)Dendritic cells (DC) are highly efficient antigen presenting cells and are important in regulating immune defense and tolerance. Both conventional DC (cDC) and plasmacytoid DC (pDC) are found in lymphoid tissues, such as the thymus and spleen. cDC can be further segregated into two populations: the CD8α+Sirpα- (CD8+) and CD8α-Sirpα+ (CD8-) cDC. Thymic cDC play an important role in the negative selection of auto-reactive T cells and the induction of naturally occurring CD4+CD25+Foxp3+ regulatory T cells (nTregs). The splenic cDC, particularly the CD8+ cDC, can cross-present auto-antigen in steady state and delete auto-reactive T cells in peripheral tissues. In addition, pDC have also been shown to induce or regulate immune tolerance in various autoimmune disease models via cross talk with regulatory T cells, producing anti-inflammatory cytokines, including TGF-β, IL-10 or typtophan-catabolising enzyme indoleamine 2,3-dioxygenase (IDO) which can inhibit the activity of T cells. Human type 1 diabetes (T1D) mellitus is an autoimmune disease that results from the autoimmune destruction of β-cells in the pancreas. The non-obese diabetes (NOD) mouse is an important animal model for T1D and shares many pathological features with human T1D. Both genetic and environmental factors are believed to play an important role in T1D development. However, the mechanisms for disease initiatory and the loss of immune tolerance remain unclear. In addition, infectious pathogens such as Lymphocytic Choriomeningitis virus (LCMV), mycobacterial reagents (e.g. BCG, CFA) or CpG oligonucleotide can delay or prevent the onset of diabetes in NOD mice. Again, the mechanisms for these effects remain elusive. Until recently, no thorough studies have been performed on the phenotype and function of steady state DC subsets in autoimmune T1D mouse models. This has prompted us to investigate whether altered functions of DC subsets contribute to the break of immune tolerance in autoimmune mouse models. In this study, characteristics of steady state DC subsets in the lymphoid organs of NOD mice were determined and compared with that of the diabetes resistant control mice NOR, Idd11 congenic mice (C57BL/6 x NOD intercross mice which have similar genetic background as NOD mice, but have a difference diabetes incidence level), as well as C57BL/6 mice. In addition, the frequency and the number of nTregs in NOD mice were also analyzed. A reduced percentage of splenic CD8+ cDC and an increased number of pDC in the thymus and spleen were found in NOD mice. Data showed that a CD8+ cDC deficiency, which is associated with diabetes onset in NOD mice, maps to the Idd11 locus. Further analysis of alterations in DC function and their potential contribution to the impairment of immune tolerance in NOD mice was carried out. A novel auto-antigen cross-presentation assay using islet-specific glucose-6-phosphate catalytic subunit-related protein (IGRP) was established to test the cross-presentation capacity of NOD DC. The results demonstrated a comparable cross-presentation ability between NOD and NOR cDC on a per cell basis. However, a lower capacity to cross-present cell associated auto-antigen in the spleen was found in NOD mice. Moreover, an elegant protocol, in which cells with the ability to cross-present can be deleted by horse cytochrome c (cyt c) treatment, was employed for the detection of cross-presenting cells. Results showed that the cells in NOD mice were insensitive to cyt c treatment, implying that the reduced percentage of the CD8+ cDC in NOD spleen was most likely due to the absence of the cDC subset with cross-presentation potential. These observations suggested that the reduced number of cross-presenting CD8+ cDC in NOD mice might result in a deficiency in maintaining peripheral immune tolerance with an accumulation of auto-reactive T cells in the peripheral, which could lead to the development of diabetes in NOD mice. pDC is a subset of DC that can protect the host from viral infection through the production of large amounts of type-1 interferon, including IFN-α, IFN-β etc., to inhibit the replication of viruses. The results from this study showed that pDC in NOD mice produced higher levels of IFN-α and IL-3 upon TLR ligand stimulation. Subsequent analysis showed that high dose of IFN-α could inhibit the proliferation of pathogenic CD8+ T cells. In addition, IL-3 could promote the development of CD103+ cDC with the ability to cross-present auto-antigens for the deletion of auto-reactive T cells in the peripheral tissues. These observations may help to explain the findings, which showed that infection by microbial agents could delay or prevent the onset of diabetes in NOD mice. Moreover, this study also revealed a reduction in the number of T regulatory cells in pancreatic lymph nodes, but not in the thymus and spleen of NOD mice. However, the ratio of Foxp3+ Tregs to CD4+ T cells was lower in thymus, spleen as well as in pancreatic lymph nodes of NOD mice. These findings suggests that the reduced ratio of Tregs to CD4+ T cells, rather than reducing absolute number of Tregs may result in less efficient control of auto-reactive T cells, and these may contribute to the development of diabetes in NOD mice. Overall, this study thoroughly examined and compared the phenotype, the number and the function of individual resident DC subsets in the thymus and spleen of NOD, NOR and Idd11 congenic mice. This study provides novel evidence for the importance of cross-presenting CD8+ cDC in the maintenance of peripheral immune tolerance. In addition, the distinct capacities of NOD pDC to produce IL-3 and much higher level of IFN-α in response to TLR activation were first demonstrated in this study. The effects of these two cytokines on T cells and DC function further suggested a potential mechanism for pDC in the prevention of T1D upon microbial stimulation. Further investigation of DC biology in T1D patients will be crucial for developing an effective approach for T1D immunotherapy.
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ItemCostimulatory requirements of helpindependent anti-influenza CTL( 2012)This thesis focused on the role of costimulatory molecules, CD28 and CD40L, on the induction and expansion of help-independent CD8+ T cell response during primary influenza virus infection. It has been conventionally believed that the help provided by CD4+ T cells is mediated by CD40-CD40L interactions, which results in the licensing of DCs. However, during some infections such as influenza virus infection, CD8+ T cell responses can be mounted without the requirements of CD4+ T cell help. Although there have been numerous studies on the role of costimulation in CD8+ T cell immunity, many were done in the presence of CD4+ T cells. Hence it is difficult to distinguish which T cell subset (CD4+ or CD8+ T cell), or both, the costimulation has had an impact on. This thesis has dissected the role of CD28 and CD40L costimulation on the activation and expansion of help-independent CD8+ T cells during primary influenza infection. This thesis demonstrated that, unlike CD4+ T cell help, the generation of influenza-specific CD8+ T cells was dependent on the direct costimulation of CD28 and CD40L. The impaired generation of influenza-specific CD8+ T cells in the absence of CD28 costimulation, and CD40L costimulation to a lesser extent, correlated with virus titre in the lungs. In addition, this thesis also demonstrated that CD28 costimulation was required in the early activation of “helpless” influenza-specific CD8+ T cells. In contrast, the CD40L costimulation was required at the late phase of the primary response, suggesting the role of CD40L costimulation in the expansion of “helpless” CD8+ T cells, after the initial activation by CD28 costimulation. In vitro studies revealed that CD80/86 and CD40L on DCs were important for the priming of “helpless” CD8+ T cells. Correlating with in vivo studies, CD28-CD80/86 costimulation was required for the early activation of CD8+ T cells, while CD40- CD40L costimulation was required at the late phase for the expansion of CD8+ T cells. CD28-CD80/86 costimulation had also been demonstrated to promote the survival of CD8+ T cells.
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ItemPlasmodium falciparum merozoite invasion mechanisms and inhibitors of invasion( 2012)Malaria threatens 40% of the global population resulting in approximately 225 million cases of disease and 800 000 deaths per year. Recently marked improvements in the implementation of control measures and increased use of artemisinin combination therapies (ACTs) have contributed to a reduction in malaria mortality and morbidity (World Health Organization, Global Malaria Programme, 2010). Furthermore the licensing and roll-out of the first malaria vaccine, RTS,S, is hoped to occur by 2015 (White, 2011; Agnandji et al., 2011). However, a sustained reduction in malaria burden and eradication of malaria seems unlikely with current control strategies alone. The largest malaria burden is caused by infection with P. falciparum parasites. All symptomatic illness occurs during asexual replication in the blood that is initiated when the merozoite form of the parasite invades red blood cells (RBCs). A limited understanding of merozoite invasion and immune mechanisms inhibiting invasion has hampered rational vaccine and drug development targeting this stage of the parasite life cycle. This thesis is based on the study of merozoite invasion of the RBC with a particular focus on the role of merozoite surface proteins in invasion and mechanisms of inhibition targeting these proteins. Part of the difficulty in studying P. falciparum merozoite invasion is due to the lack of efficient techniques to isolate merozoites that maintain invasive capacity. Chapter 3 describes the development of methods to isolate merozoites that maintain viability. Importantly, the method only requires basic laboratory equipment, therefore is accessible to both resource rich and developing laboratories. Highly synchronized cultures were treated with a cysteine protease inhibitor to block schizont rupture and then merozoites isolated via membrane filtration (Boyle et al., 2010a). Approximately 15% of isolated merozoites maintained viability and were able to successfully invade when incubated with RBCs. This allowed for the development of methods to fix merozoites during invasion for microscopy and invasion inhibition assays. Invasion of isolated merozoites was independent of serum components and the invasive half-life of merozoites was approximately 8 minutes. Merozoite isolation and invasion assays are now being used by a number of research groups and are a powerful technique to study merozoite invasion mechanisms (Riglar et al., 2011) and inhibitors of invasion. In Chapter 4, microscopy of invading merozoites is used to investigate the shedding of merozoite surface antigens during invasion. The initial steps of merozoite invasion are hypothesized to be mediated by merozoite surface proteins that contact with the RBC via weak receptor-ligand interactions. During invasion it is thought that merozoite surface proteins are cleaved and then shed from the merozoite to allow invasion to occur. This has been most clearly demonstrated for MSP1, with compounds that inhibit MSP1 cleavage and/or shedding also inhibiting invasion (Blackman et al., 1994; Singh et al., 2006; Woehlbier et al., 2010; Fleck et al., 2003; Blackman and Holder, 1992). Contrary to the current paradigm, it was found that merozoite surface proteins MSP2 and MSP4 were not shed from the merozoite surface during invasion and were instead carried into the RBC without apparent cleavage. Post invasion, MSP2 was rapidly degraded within a few minutes, whereas MSP4 was maintained for a number of hours. Interestingly, during invasion some MSP2 antibodies were found to be internalized into the RBC. Internalized antibodies were maintained for approximately 24 hours post invasion. This work establishes that there is differential cleavage and shedding of merozoite surface proteins during invasion and suggests that some merozoite surface proteins may have roles outside initial contact events. Chapter 5 investigates the mechanisms by which antibodies inhibit merozoite invasion in the presence of physiological relevant concentrations of complement-active serum. This work was possible due to capacity of isolated merozoites to invade in both the absence of serum and high serum concentrations. While the importance of IgG in mediating parasite clearance is well established (Sabchareon et al., 1991; McGregor, 1964b), the mechanisms of antibody function remain poorly understood. Naturally acquired antibodies from malaria-exposed individuals, as well as antibodies from vaccinated rabbits and humans had complement-dependent inhibition activity targeting merozoite invasion. The complement component C1q was required and appeared to be sufficient for complement-dependent inhibition. MSP1 and MSP2 were identified as targets of complement-dependent antibody mediated inhibition. Antibody mediated complement-dependent inhibition of invasion is a novel mechanism targeting merozoites that may be important in understanding protective immunity and for evaluating candidate merozoite vaccines. Finally, Chapter 6 explores the interaction of heparin with merozoite surface proteins and the potential of heparin-like-molecules (HLMs) as the basis for novel drug development. Heparin is a known inhibitor of merozoite invasion, and appears to act by inhibiting early contact events. Utilizing a heparin-binding assay with native merozoite proteins, heparin was shown to bind the processed fragment of MSP1, known as MSP1- 42. A panel of novel HLMs were screened for growth/invasion inhibition activity and a number of highly inhibitory compounds were identified. This work will be a basis for further studies to identify novel invasion inhibitors that may be used as the basis for drug development. The development of a method to isolate viable merozoites has allowed this thesis to explore a number of aspects of merozoite invasion mechanisms and inhibition of invasion. As well as increasing our understanding of P. falciparum merozoite biology and immunity to malaria, it is hoped that this work will contribute to the development of tools to combat malaria disease.
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ItemUnderstanding transcriptional regulation of terminal T cell differentiation: a focus on Blimp1( 2012)The transcription factor Blimp1 is a critical regulator of the terminal differentiation of effector CD8+ T cells during acute viral infections. Blimp1-deficient CD8+ T cells failed to develop into short-lived effector cells. Blimp1 is also indispensable for the differentiation of activated Treg cells and is required for IL-10 production by Treg cells. However, it was not known what genes were regulated by Blimp1 in those two populations and how they were regulated. Here I present data showing that Blimp1 globally regulates genes involved in migration, effector function and transcription in T cells that promote the cell fate associated with terminal differentiation. Although Blimp1 is required for the downregulation of transcriptional signatures associated with naïve and memory T cells including Tcf7 and Id3, its expression is equally crucial for gene upregulation in vivo, such as Gzmb in CD8+ T cells and Il10 in Treg cells. Using Il10 as a model gene, I show data supporting the role of Blimp1 together with IRF4 in mediating open chromatin for Il10 transcription. Numerous cytokines have been shown to upregulation Blimp1 expression in vitro but their in vivo roles and effects on Blimp1 were not addressed. In particular, IL-2 has been described as an important inducer of Blimp1 in vitro and IL-2 signalling is essential for CTL differentiation. I show here that IL-2 is not mandatory for Blimp1 expression in vitro or in vivo, and Blimp1 could be redundantly upregulated by the pro-inflammatory cytokine IL-12. However, IL-2 is required in an environment where inflammation is limited and induces both Blimp1 and T-bet in effector CTLs. T-bet and Blimp1 function independently in parallel transcriptional pathways that mediate similar aspects of effector CTL differentiation.