Medical Biology - Theses
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ItemPlasmodium chabaudi adami: vaccine antigens and antigenic variation( 2003)There is an abundance of information available on the molecular mechanisms of antigenic variation in Plasmodium falciparum. The variant antigen PfEMP1, which mediates antigenic variation as well as cytoadherence and rosetting, has been extensively characterised. Genes coding for the antigen belong to the gene family var, and several var genes have been cloned and characterised. The rodent malaria parasite P. chabaudi is a widely studied in vivo model for P. falciparum. The P. c. chabaudi AS parasite strain has been shown to exhibit antigenic variation and the variant antigen has been detected by surface fluorescence. As with P. falciparum, there is a link between antigenic variation and cytoadherence, however genes coding for the variant antigen in P. chabaudi have not been cloned to date. Therefore, potentially useful in vivo experiments on antigenic variation are restricted. In this thesis it is shown for the first time that the P. c. adami DS parasite strain also exhibits antigenic variation. Chapter 3 describes efforts to locate genes coding for variant antigens in P. c. adami DS. The main strategy involved a genome survey, by sequencing and analysing randomly selected clones from a P. c. adami DS genomic library. DNA sequences were compared to Plasmodium spp. sequence databases to look for similarity to var genes or other genes encoding variant antigens. Of the 297 clones analysed none had significant sequence similarity to genes coding for variant antigens. However, in a small proportion of sequences some similarity to var genes was noted. Several genes of potential interest were identified, most importantly the gene coding for the vaccine candidate rhoptry associated protein 1 (RAP1), which was subsequently cloned and characterised. Further attempts to locate var gene homologues in P. c. adami involved amplification of P. c. adami genomic DNA using degenerate oligonucleotide primers corresponding to conserved regions of var genes. This strategy proved to be unsuccessful, most likely due to lack of sequence similarity between P. falciparum and P. c. adami genes. In several vaccination studies with the apical membrane antigen 1 (AMA1) of P. c. adami DS, mice were significantly protected against homologous parasite challenge. However, some mice developed late, low-level breakthrough parasitaemias. In Chapter 4, the characterisation of two such breakthrough parasitaemias is described. The ama1 genes of the breakthrough parasites were found to be identical to the ama1 gene of the parental parasites. Similarly, no alteration in AMA1 expression was observed. However, the breakthrough parasites were found to be more resistant than the parental parasites to the effects of passive immunisation with rabbit antisera to AMA1, RAP1 and possibly also MSP119. P. chabaudi infections in mice have been previously shown to consist of a primary parasitaemia followed by a short period of subpatency, and a recrudescent parasitaemia. In surface immunofluorescence studi Chapter 4 describes similar surface immunofluorescence assays carried out with P. c. adami infected erythrocytes, and quantitation of fluorescence by flow cytometry. As with P. c. chabaudi, the recrudescent parasites were found to be antigenically distinct from the primary parasitaemia, indicating that antigenic variation had taken place. Because breakthrough parasites from the AMA1 vaccination trial were similar to recrudescences in peak and duration, we hypothesised that breakthrough parasitaemias, like recrudescent parasitaemias, occur as a result of antigenic variation. In Chapter 4 it was shown by surface immunofluorescence and flow cytometry using hyperimmune sera raised against different parasite populations, that breakthrough parasites express antigens on the surface of late trophozoite- and schizont infected erythrocytes that differ from those expressed by the parental and recrudescent parasites. These results support the hypothesis that switching of the variant antigen on the infected erythrocyte surface enables parasites to evade protective antibody responses directed against merozoite antigens. Chapter 5 describes the cloning and characterisation of P. c. adami RAP1 which was identified in the process of the genomic survey described in Chapter 3, as well as P. berghei RAP1. Both rodent parasite orthologues of RAP1 were found to have 30% sequence similarity to P. falciparum RAP1, and 6 of 8 cysteines were conserved in the rodent parasite orthologues. However the three polypeptides vary significantly in size. P. c. adami RAP1 and P. berghei RAP1 consist of 691 aa and 604 aa respectively, whereas P. falciparum RAP1 consists of 783 aa residues. These size differences reflect very different N-terminal sequences prior to the first cysteine, whereas the cysteine-rich C-terminal regions are more conserved. Both P. falciparum RAP1 and P. c. adami RAP1 contain N-terminal repeats, however they bear no sequence similarity to each other. P. berghei RAP1 lacks N-terminal sequence repeats that are characteristic of P. falciparum and P. c. adami RAP1. The large cysteine-rich C-terminal region P. c. adami RAP1 (PcRAP1 C3) was expressed in E. coli as a hexa-his fusion protein. Rabbit antiserum to recombinant PcRAP1 C3 was used to characterise the expression and sub-cellular localisation of the RAP1 antigen. P. c. adami RAP1 was found to have a Mr of approximately 80,000 and was shown by immunofluorescence to localise to the merozoite rhoptries. Passive immunisation of mice with rabbit anti-RAP1 serum was shown to protect against fulminant parasitaemia and mortality. In a mouse vaccination trial using the recombinant PcRAP1 C3 polypeptide partial protection was conferred against homologous parasite challenge.
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ItemRegulation of neural connectivity by the Epha4 receptor tyrosine kinase( 2001-10)Interactions between the Eph family of receptor tyrosine kinases, and their ligands, the ephrins, are required for the normal development and maintenance of many patterns of connectivity within the nervous system. Eph receptors and ephrins are expressed widely throughout both the developing and mature nervous system where they function as important regulators of cell migration and axon guidance. The studies presented in this thesis examine the role of one particular member of the Eph receptor family, EphA4, in regulating mechanisms that underlie the development and maintenance of certain neural connections within the nervous system. This thesis demonstrates that the EphA4 receptor is expressed within specific regions of the developing and mature nervous system, some of which are associated with the control of locomotor activity. Consistent with these observations are the locomotor defects exhibited by animals with a targeted disruption of the EphA4 gene. These animals exhibit abnormal bilateral limb movements and have severe disruptions of a number of major axonal pathways. One of these disrupted axonal pathways, the corticospinal tract (CST), is a particularly important mediator of locomotor activity. This thesis reveals that EphA4 is expressed on the axons that comprise the CST. It demonstrates that although EphA4 is not required for the initial development of the CST, repulsive interactions between EphA4-bearing CST axons and ephrinB3, a ligand for EphA4 that is expressed at the midline of the spinal cord, appear to prevent CST axons from aberrantly recrossing the spinal midline during development.
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ItemBiochemical basis of B cell dysfunction in Lyn kinase deficient mice( 2003-11)B lymphocytes constitutes an important arm of the immune system, and their response to antigen is largely dependent upon signal transduction through the B cell receptor (BCR). Such a potent receptor, however, needs to be further balanced by positive and negative regulators to prevent harmful effects that may arise from inappropriate stimulation. Src family protein tyrosine kinase Lyn is involved in both positive and negative regulation, since the both gain-of-function Lyn and loss-of-function Lyn mutations caused autoimmunity in mice. The exact signalling pathway(s) regulated by Lyn in B cells, however, are still not clear. Work presented in this thesis attempts to elucidate the biochemical mechanisms that underline the double-edged nature of Lyn in BCR signalling. (For complete abstract open document)
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ItemB cell selection in the germinal centre( 2002-10)Central to the humoral immune response is the production of memory B cells and the secretion of highly specific antibody, by high affinity antibody forming cells (AFCs). Both these cell types are produced in germinal centres (GC) during primary responses but have several different properties. AFCs arise early in the reaction, are highly selected for affinity and specifically migrate to the bone marrow (BM) where they persist for many months. Memory cells are produced later, are not so stringently selected and recirculate throughout the lymphoid system, but also persist for many months. Therefore, there is a selective difference in the outcome of the GC reaction. (For complete abstract open document)
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ItemThe control of normal and leukaemic human cells by the colony stimulating factors( 1986)To examine the control of human haemopoietic cells by the haemopoietic hormones (the Colony Stimulating Factors, CSF’s) both purified factors and purified cell populations are required. Studies were initially performed to characterize the action of purified murine and purified and partially-purified human CSF’s on human marrow cells. Of the purified murine CSF’s, G-CSF was active on human bone marrow cells. The murine molecule Eosinophil Differentiation Factor (EDF) was shown to an Eosinophil-CSF with activity on murine and human cells. Recombinant human GM-CSF (rHGM-CSF) was shown to possess all the biological activities of the partially purified native molecule CSF-α. Both the glycosylated and non-glycosylated preparations of rHGM-CSF showed equivalent biological activity in vitro. Subsequent studies were performed to purify and characterize a population of normal committed-granulocyte progenitor cells and the action of CSF’s on these cells was examined. Normal human promyelocytes and myelocytes were obtained using the monoclonal antibody WEM-G11 and the fluorescence activated cell sorter. These cells demonstrated transient, CSF-stimulated proliferation in vitro and generated neutrophilic clones of less than 40 cells in size (clusters). These cells were stimulated to proliferate by human CSF-α, CSF-β and murine G-CSF. Clone transfer experiments documented the ability of clones initiated by one CSF (α or β) to proliferate when transferred to cultures stimulated by the other CSF. The cross-species activity of human CSF-β and murine G-CSF, and the ability of CSF-β to compete with radio-iodinated murine G-CSF for binding-sites on murine (and human) cells suggested that CSF-β was the human equivalent of murine G-CSF. A comparative study of leukaemic promyelocytes demonstrated that fractionated promyelocytes-myelocytes from patients with chronic myeloid leukaemia also showed transient clonal proliferation in vitro and responded to CSF-α, CSF-β and murine G-CSF. Promyelocytes from the blood of these patients generated clones of only two cells in CSF-unstimulated cultures. This behaviour was mimicked when normal promyelocytes-myelocytes were pulse-stimulated by CSF for 45 min. Leukaemic cells from patients with acute myeloid leukaemia also demonstrated CSF-stimulated proliferation in vitro and were responsive to CSF-α, CSF-β, rHGM-CSF and murine G-CSF. There was however considerable heterogeneity in the CSF-responsiveness of these cells. Differentiation-induction by CSF in leukaemic cells was examined using the human leukaemic cell line HL60. When stimulated by CSF, these cells showed increased expression of myeloid surface antigens (as monitored by three monoclonal antibodies) and decreased numbers of clonogenic cells. In some experiments the number of clonogenic cells was reduced to zero. In an attempt to establish a sensitive micro-assay for CSF, two target cell populations were examined. Normal promyelocytes-myelocytes displayed CSF-stimulated proliferation in a micro-assay system but this was not associated with a heightened sensitivity to CSF. The survival of mature human neutrophils and eosinophils in vitro was however shown to be enhanced by CSF’s in a “lineage-specific” manner and this assay was between 10^2-10^3 times more sensitive to CSF than agar cultures. These studies demonstrated that the Colony Stimulating Factors enhanced survival, stimulated proliferation and stimulated differentiation-commitment of normal and leukaemic human cells.
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ItemBiochemical characterisation of factors stimulating bone marrow colony growth in vitro( 1970)A general study of factors stimulating formation of colonies of granulocytic and monocytic cells from mouse bone marrow cells grown in agar cultures has been made. Factors from mouse serum, urine and tissue extracts, human serum and urine and mouse embryo cell “conditioned medium” were examined. (For full abstract open document)
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ItemStudies on the cellular basis of the immunological defect following thymectomy in the mouse( 1969)Answers have been sought for two key questions relevant to the proposed mode of action of the thymus as a “central” lymphoid organ. Does the peripheral lymphocyte population contain a large number of thymus-derived cells and do these cells respond to antigens by producing a progeny of antibody-forming cells? The number of small lymphocytes emerging from a thoracic duct fistula in young adult neonatally-thymectomized CBA mice was approximately 2% of that in intact mice of the same age and body weight. Multiple injections of chromosomally-marked thymus cells increased the size of the lymphocyte pool and the majority of thoracic duct cells, stimulated into division, carried the chromosome marker of the thymus cell donor. Thymectomy in adult life was not followed by a significant decrease in the output of thoracic duct lymphocytes until many months after the operation. Whole body x-irradiation resulted in a dramatic reduction in the number of cells drained from a thoracic duct fistula and, in mice protected from the lethal effects of haemopoietic failure, the reestablishment of the lymphocyte population was dependent upon the presence of the thymus. The data supports an increasing bulk of indirect evidence which, when taken in toto, strongly suggests that a large proportion of recirculating lymphocytes are thymus -derived cells or their descendants. The number of sheep erythrocyte antigen-reactive cells (ARC) in the thoracic duct lymphocyte population of neonatally-thymectomized mice was markedly reduced when compared with the number in the population from normal mice. The bone marrow did not contain ARC but was a potent source of ARC precursors. Neonatally-thymectomized mice did not lack precursor cells in the bone marrow but apparently lacked the thymus influence necessary for the differentiation of these precursors into ARC. Further studies on the thymus-dependent development of ARC hinted at the possibility that the entity known as "an ARC" required the presence of both thymus- and bone marrow-derived cells to express itself in terms of haemolysin production. Neonatally-thymectomized CBA mice failed to respond in normal fashion to a primary injection of sheep erythrocytes (SRBC) and the peak number of haemolysin plaque-forming cells (PFC) in the spleen was reduced by a factor of 1 log 10. The PFC response was increased by injections of either thymus or thoracic duct lymphocytes from CBA, (CBA x C57BL)F 1 hybrid, and C57BL donor mice. In thymectomized mice reconstituted with semiallogeneic and allogeneic cellular inocula, the PFC carried the immunogenetic characteristics of the host and not those of the inoculated cells. Hence, thymus and thoracic duct cells were not reconstitutive simply by virtue of the ability to transform into PFC. Thymus and thoracic duct cell inocula contained "reactor cells" which responded to SRBC antigens in irradiated mice by undergoing a burst of mitosis. Thoracic duct lymphocytes, unlike thymus and bone marrow cells, were able to produce PFC when injected together with SRBC into irradiated mice. However, the PFC response in irradiated recipients of thoracic duct lymphocytes was increased substantially by a simultaneous injection of bone marrow cells. Combinations of cells from semiallogeneic mice did not interact upon transfer to irradiated recipients but clear evidence of synergism in PFC production was apparent in adult-thymectomized irradiated mice protected with CBA bone marrow cells and injected two weeks later with (CBA x C57BL)F 1 thoracic duct cells. In this case, the vast majority of PFC were derived from the bone marrow inoculum. The results suggest that the bone marrow contains only PFC precursors, the thymus only "reactor cells", but that the thoracic duct lymph contains both cell types. It seems that the normal 19S haemolysin response to SRBC in the CBA mouse requires the collaboration of bone marrow-derived PFC precursors and thymus-derived "reactor cells". The neonatally-thymectomized mouse contains adequate numbers of PFC precursors but, after challenge with SRBC, few are recruited into 19S haemolysin production because of the severe deficiency in thymus-derived ''reactor cells".