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Dissertations / Theses on the topic 'Erythrocytes Malaria Plasmodium falciparum'
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Herricks, Thurston E. "Malaria pathogenesis : deformability limits of malaria infected erythrocytes /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/8622.
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Jones, Matthew L. "Erythrocyte invasion by Plasmodium falciparum." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2009. https://www.mhsl.uab.edu/dt/2009r/jonesm.pdf.
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Cranmer, Susan Louise. "Characterisation of altered monocarboxylate transport in erythrocytes parasitised by Plasmodium falciparum." Thesis, University of Bristol, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358773.
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Pettersson, Fredrik. "Sequestration, virulence and future interventions in Plasmodium falciparum malaria." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-568-2/.
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Vogt, Anna. "Heparan sulfate dependent sequestration during Plasmodium falciparum malaria /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-994-3/.
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Scholander, Carin. "Immunoglobulins in the adhesion of Plasmodium falciparum-infected erythrocytes in malaria /." Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-4235-8/.
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Gardner, Jason Paul. "Surface changes to human erythrocytes on infection by Plasmodium falciparum malaria." Thesis, University of Oxford, 1994. http://ora.ox.ac.uk/objects/uuid:5ecd0f5c-8189-4731-a643-d6cf9463e4e5.
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Of the four Plasmodium species which cause malaria in humans, P. falciparum is responsible for the majority of the morbidity and mortality associated with this disease. The surface expression of parasite-derived proteins in the middle of the asexual cycle coincides with two important modifications of the host erythrocyte. First, a protective immune response is directed against a family of variant antigens, known as P. falciparum Erythrocyte Membrane Protein-1 (PfEMPl). Second, ligands are detected at the surface which mediate the specific cytoadherence of infected erythrocytes to vascular endothelium, such that infected cells are sequestered away from the peripheral circulation in deep vascular beds. The potentially fatal syndrome known as cerebral malaria can ensue when infected cells sequester at high density in the brain. Indirect studies have shown that the antigenic and adhesive phenotypes at the surface are linked to the expression of PfEMPl. However, there is a paucity of biochemical data which relate to PfEMPl, and this problem is addressed in this thesis. This study has confirmed, at the biochemical level, inferences from serology that clonal antigenic variation occurred rapidly. Variation produced a number of novel antigenic and adhesive phenotypes which were associated with unique forms of PfEMPl. Further insights into the mechanism of sequestration were possible because of the finding that single infected erythrocytes had the capacity to bind to at least three putative endothelial cell receptors; CD36, Intercellular Adhesion Molecule-1 (ICAM1), and Thrombospondin (TSP). It was demonstrated for the first time that PfEMPl was responsible for cytoadherence to CD36 and ICAM1, but was probably not involved in adhesion to TSP. Extensive analysis with sequence-specific proteases proved that adhesive interactions with each receptor were separable properties of the surface, and facilitated the proposal of a domain model for PfEMPl. Detailed analysis of the antigenic and adhesive phenotypes of a series of clonally-derived parasites demonstrated that infected cells expressing all variant antigenic types could adhere to CD36 whereas adhesion to ICAM1 was seen in a restricted subset. This may be clinically relevant if, as current data suggests, adhesion of infected cells to ICAM1 is important in the development of cerebral malaria. Identification of all ICAM1 binding phenotypes could lead to the design of novel therapeutic strategies for this life-threatening condition.
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Corrigan, Ruth Alexandra. "Rosetting and the innate immune response to Plasmodium falciparum." Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4041.
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Rosetting is an adhesion property of malaria parasites whereby infected erythrocytes bind to two or more uninfected erythrocytes, forming a so-called rosette. Rosetting of Plasmodium falciparum is associated with disease severity and high parasitaemia in sub-Saharan Africa, although currently the function of rosetting remains unknown. An early IFNg response elicited from the innate immune system is associated with resolution of malaria infection in mice. Published data suggests that optimal IFNg production may require contact between peripheral blood mononuclear cells and P. falciparum infected erythrocytes. The first part of this thesis investigates the hypothesis that rosetting is an immune evasion strategy to hide infected erythrocytes from detection by innate immune cells. Across five laboratory strains of P. falciparum rosetting was not associated with differential IFNg production when parasites were grown in group O blood. Reappraisal of the data with respect to blood group for one strain found that rosetting significantly reduced the IFNg response to parasites grown in group A blood (P=0.022, Wilcoxon signed-rank test), where it is known that rosettes are bigger and stronger. This is consistent with the hypothesis that rosetting is an immune evasion strategy and the first study to find evidence for a function of rosetting. Further work is needed in order to generalise this finding. The cytokine response to P. falciparum varies between people and this variation may be indicative of disease progression. In mice infected with malaria it is also apparent that parasite strain can determine the cytokine response of the host. It is unclear whether P. falciparum strains vary in their ability to induce cytokines. The second part of this thesis investigates variation in cytokine induction between P. falciparum strains. Across four laboratory strains of P. falciparum, IFNg production was significantly dependent on parasite strain (F3,178= 48.49, P<0.001). Production of GM-CSF, IL-1b, IL-6, IL-10 and TNFa significantly correlated with production of IFNg (P<0.001, Pearson correlation) and followed the same strain-dependent pattern. The ratio of pro-inflammatory cytokines to IL-10 was also dependent on parasite strain. These data provide strong evidence for P. falciparum strain-dependent cytokine responses which may be an important determinant of disease outcome. Phagocytosis by splenic macrophages is proposed to be the principle mechanism of parasitaemia control in malaria infection. CD36 mediated phagocytosis may by an important mechanism of non-opsonic parasite clearance. The final part of this thesis investigates the hypothesis that rosetting is an immune evasion strategy of P. falciparum in order to evade phagocytic clearance, in particular that mediated by CD36. Overall the data obtained were inconsistent. Phagocytosis was significantly reduced in rosetting versus non-rosetting parasites in some strains (e.g. R29; P=0.048, paired T test), whereas others showed no effect (e.g. Muz12; P=0.228, paired T test) or increased versus non-rosetting parasites (e.g. HB3, P=0.004, paired T test). The relationship between CD36 binding and phagocytosis was also unclear, and anti-CD36 antibody did not effectively block phagocytosis, suggesting the involvement of alternative mechanisms. Further experiments are needed to clarify these observations. Data presented in this thesis are suggestive that rosetting in non-group O blood may be an immune evasion strategy with regard to IFNg production by innate immune cells, mechanistically linking rosetting with enhanced parasitaemia and disease severity. Furthermore, parasite strain significantly affects cytokine production and may be a determinant of disease outcome. This thesis demonstrates the importance of continued research into the effect of parasite virulence on the immune response, with particular emphasis on rosetting.
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Rowe, Jane Alexandra. "Rosetting of Plasmodium falciparium infected erythrocytes." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260775.
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McCormick, Christopher John. "An investigation of the interactions between Plasmodium falciparum-infected erythrocytes and endothelium." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318757.
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Haeggström, Malin. "Variable surface molecules of the Plasmodium falciparum infected erythrocyte and merozoite /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7357-008-7/.
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Neal, Aaron T. "Identifying genetic determinants of impaired PfEMP1 export in Plasmodium falciparum-infected erythrocytes." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:0cc3f09c-9178-448b-92f8-8f9564398585.
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The virulence of Plasmodium falciparum is largely attributed to the ability of asexual blood-stage parasites to cytoadhere to the microvascular endothelium of the human host. This pathogenic behavior is mediated by the primary parasite virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), an understanding of which is crucial to develop interventions to ameliorate the morbidity and mortality of P. falciparum malaria. The work presented in this thesis describes the application of a phenotype-to-genotype experimental approach to identify novel parasite proteins involved in the trafficking and display of PfEMP1. Guided by the overall hypothesis that the in vitro culture-adapted parasite line 3D7 harbors 1 or more genetic determinants of impaired PfEMP1 trafficking, surface PfEMP1 levels were first measured in 3D7, the presumably trafficking-competent parasite line HB3, and 16 unique progeny from an HB3 x 3D7 genetic cross (chapter 2). These phenotypes were then combined with genome-wide SNP data in QTL analysis to identify genetic polymorphisms potentially responsible for the impaired trafficking in 3D7 (chapter 3). A near-significant QTL containing a single protein-coding gene, the putative kinesin Pf3D7_1245600, was identified, characterized, and investigated in CRISPR-Cas9-driven allele-exchange parasite transfection experiments to establish a causal link between the gene and PfEMP1 trafficking (chapter 4). The parasite transfections were unsuccessful, but the potential role of Pf3D7_1245600 in PfEMP1 trafficking was indirectly assessed through the disruption of microtubules with colchicine (chapter 4), which significantly impacted the surface PfEMP1 levels of HB3 but not 3D7. The findings of this thesis suggest that kinesins and microtubules may play previously unconsidered roles in the regulation, production, or trafficking of PfEMP1.
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Stubberfield, Lisa Marie. "Interactions of Plasmodium falciparum proteins at the membrane skeleton of infected erythrocytes." Monash University, Dept. of Microbiology, 2003. http://arrow.monash.edu.au/hdl/1959.1/9433.
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Claessens, Antoine. "How Plasmodium falciparum malaria parasites bind to human brain endothelial cells." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/4897.
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Cerebral malaria is characterised by an accumulation of infected erythrocytes in the microvasculature of the brain. Plasmodium falciparum infected erythrocytes have been shown to bind to a Human Brain Endothelial Cell line (HBEC-5i) in vitro. This provides a model for the investigation of interactions between P. falcuparum and human brain endothelium. Currently neither the parasite adhesion ligands on infected erythrocytes, nor the host endothelial cell receptors necessary for this interaction have been identified. In this work, the identity of the host receptor on brain endothelial cells was addressed by binding assays of selected and unselected parasites on a wide range of malaria-associated host molecules. The identity of the parasite ligand was investigated by microarray analysis of parasites after selection for cytoadherence to HBEC-5i. The hypothesis being tested was that the gene encoding the parasite cytoadherence ligand would show significant upregulation in selected compared to unselected paarasites. The P. falciparum laboratory strains 3D7, HB3 and IT/FCR3 were selected for binding to HBEC-5i using a panning assay. Compared to unselected parasites, HBEC-5i selected parasites showed a distinct phenotype with reduced platelet-mediated clumping. There was no significant increase in binding of parasites to any of the known endothelial cytoadherence receptors for P. falciparum after selection on HBEC-5i. Binding inhibition assays with various antibodies and soluble receptors did not greatly block the adhesion of parasites to HBEC-5i except for heparin. Altogether, the receptor(s) mediating the interation with HBEC-5i remains unknown. In order to carry out transcriptional analysis of selected and unselected paarasites form all three parasite strains, it was necessary to update the existing microarray chip which is based on the 3D7 genome. This is because each parasite train has a unique repertoire of variant surface antigens (VSAs) including var, rif and stevor genes. Therefore, to fully analysis HB3 and IT genomes. Unique oligonnucleotide probes were then designed for each new sequence and the 3D7-based microarray chip was updated. Transcriptional analysis was then carried out on selected and unselected parasites of all strains. Microarray data clearly indicated that the most highly upregulated genes after selection were group A or group A-like var genes (HB3var3, 3D7_PFDOO2Oc, ITvar7 and ITvar19), showing 11 to over 100 fold upregulation in selected parasites. The rif gene adjacent to the upregulated var gene was also highly expressed. To a lesser extent some exported proteins like RESA-1, PfEMP3 or PHIST family members also showed increased transcription in HBEC-selected parasites (2-3 fold upregulation). Reverse transcriptase-PCR confirmed the upregulation of group A var genes in selected parasites, suggessted that the group A PfEMP1 variants are major candidate ligands for parasite binding to HBEC-5i. These findings are consistent with previous work showing an association between Group A var genes and cerebral malaria.
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Maphumulo, Philile Nompumelelo. "Characterisation of a plasmodium falciparum type II Hsp40 chaperone exported to the cytosol of infected erythrocytes." Thesis, Rhodes University, 2013. http://hdl.handle.net/10962/d1015681.
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Heat Shock 40 kDa proteins (Hsp40s) partner with heat shock 70 kDa proteins (Hsp70s) in facilitating, among other chaperone activities; correct protein transport, productive protein folding and assembly within the cells; under both normal and stressful conditions. Hsp40 proteins regulate the ATPase activity of Hsp70 through interaction with the J-domain. Plasmodium falciparum Hsp70s (PfHsp70s) do not contain a Plasmodium export element (PEXEL) sequence although PfHsp70-1 and PfHsp70-3 have been located outside of the parasitophorous vacuole. Studies reveal that a type I P. falciparum (PfHsp40) chaperone (PF14_0359) stimulates the rate of ATP hydrolysis of the cytosolic PfHsp70 (PfHsp70-1) and that of human Hsp70A1A. PFE0055c is a PEXEL-bearing type II Hsp40 that is exported into the cytosol of P. falciparum-infected erythrocytes; where it potentially interacts with human Hsp70. Studies reveal that PFE0055c associates with structures found in the erythrocyte cytosol termed “J-dots” which are believed to be involved in trafficking parasite-encoded proteins through the erythrocyte cytosol. If P. falciparum exports PFE0055c into the host cytosol, it may be proposed that it interacts with human Hsp70, making it a possible drug target. The effect of PFE0055c on the ATPase activity of human Hsp70A1A has not been previously characterised. Central to this study was bioinformatic analysis and biochemical characterisation PFE0055c using an in vitro (ATPase assay) approach. Structural domains that classify PFE0055c as a type II Hsp40 were identified with similarity to two other exported type II PfHsp40s. Plasmids encoding the hexahistidine-tagged versions of PFE0055c and human Hsp70A1A were used for the expression and purification of these proteins from Escherichia coli. Purification was achieved using nickel affinity chromatography. The urea-denaturing method was used to obtain the purified PFE0055c whilst human Hsp70A1A was purified using the native method. PFE0055c could stimulate the ATPase activity of alfalfa Hsp70, although such was not the case for human Hsp70A1A in vitro.
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Myrie, Latoya T. "Optimization of the conditions necessary to show binding of the Plasmodium yoelii RHOP-3 rhoptry protein to mouse erythrocytes." Cleveland, Ohio : Cleveland State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=csu1213361087.
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Thesis (M.S.)--Cleveland State University, 2008.Abstract. Title from PDF t.p. (viewed on July 29, 2008). Includes bibliographical references (p. 66-73). Available online via the OhioLINK ETD Center. Also available in print.
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Emsri, Pongponrat Sornchai Looareesuwan. "Ultrastructural studies of Plasmodium falciparum in human organs : the interactions between the parasitized erythrocytes and the host cells /." Abstract, 2000. http://mulinet3.li.mahidol.ac.th/thesis/2543/43E_Emsri-P.pdf.
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Curra, Chiara. "Protein trafficking and host cell remodeling in malaria parasite infection." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20219.
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Pour assurer ses besoins de croissance, multiplication, et survie, Plasmodium modifie sa cellule hôte, l'érythrocyte, après l'invasion. Le parasite met en place ainsi un système d'échanges (import/export) avec sa cellule hôte et le milieu extérieur. Nous avons identifié dans la base de données de Plasmodium berghei, le parasite de rongeurs, une famille de gènes, sep, correspondant à la famille etramp chez Plasmodium falciparum. Cette famille de gènes code pour des petites protéines exportées, et conservées dans tout le genre Plasmodium. Les protéines SEP (13?16 kDa) contiennent en N-terminal un peptide signal prédit, un domaine hydrophobe interne, et elles diffèrent au niveau des régions C-terminal et 3' UTR. Toutefois, les protéines SEP sont exprimées à différents moments du cycle de Plasmodium. Durant le cycle érythrocytaire, PbSEP1 et PbSEP3 sont exprimées à partir du stade trophozoïte, et la même quantité de protéine est détectée au stade schizonte et gamétocyte, pendant que PbSEP3 est hautement détectée dans les trophozoïtes mûrs et les gamétocytes. Chez le moustique, PbSEP1 et PbSEP3 sont détectées seulement chez les ookinètes, alors que PbSEP2 est très abondante dans les ookinètes, oocystes, et sporozoïtes des glandes salivaires. Les protéines SEP ont également des localisations différentes. Dans l'érythrocyte, PbSEP1 est localisée dans la membrane de la vacuole parasitophore, alors que PbSEP2 et PbSEP3 sont exportées au-delà de cette vacuole, et sont ainsi localisées dans la cellule hôte, en association avec des structures vésiculaires. Dans cette étude, nous avons identifié les signaux d'adressage des protéines SEP dans la vacuole parasitophore et dans la cellule hôte, chez Plasmodium berghei. L'autre partie du travail, effectuée à l'Université de Montpellier II, a consisté à étudier la localisation de deux protéines du squelette sous- membranaire de l'érythrocyte, la dématine, et l'adducine, durant le développement intra-érythrocytaire de Plasmodium falciparum. Le but de cette étude étant d'identifier un mécanisme potentiel d'internalisation des composants du squelette sous-membranaire de l'érythrocyte dans le parasite. Des études d'immuno-localisation ont montré que la dématine et l'adducine sont internalisées à partir du stade trophozoïte, et sont localisées probablement à la vacuole parasitophore (membrane et/ou lumière). Cette internalisation a été confirmée par des études de fractionnement cellulaire et d'accessibilité à la protéinase K, montrant que la dématine est totalement internalisée, alors l'adducine ne l'est que partiellement, suggérant une localisation de la protéine à la périphérie du parasitePlasmodium endurance depends on the ability of the parasite to reorganize the cytosol of the erythrocyte, a terminally differentiated cell, and remodel its skeleton membrane immediately after invasion. In this way the parasite can organize the import/export of the molecules necessary to its survival. The comprehension of cellular trafficking mechanisms which occur during Plasmodium infection is a very important step and fundamental contribute to understand the biology of the malaria parasite.We identified in database of the rodent malaria parasite Plasmodium berghei the gene family sep, corresponding to etramp in P. falciparum, encoding small exported proteins conserved in the genus Plasmodium. SEP proteins (13?16 kDa) contain a predicted signal peptide at the NH2-terminus, an internal hydrophobic region while they differ in their C-terminal region; the genes share the upstream regulative region while differ in the 3' UTR. Despite this, we showed that SEPs have a different timing of expression and a different localization: in the erythrocytic cycle PbSEP1 and PbSEP3 start to be expressed at trophozoite and the same amount of protein is detected also in schizonts and gametocytes, while PbSEP2 is highly detected in mature trophozoites and even more in gametocytes. In mosquitoes stages PbSEP1 and PbSEP3 are expressed only in ookinetes, while PbSEP2 is very abundant in ookinetes, oocysts and in sporozoites of the salivary glands. SEPs also have a different localization in the iRBC: PbSEP1 is targeted to the membrane of the parasitophorous vacuole, while PbSEP2 and 3 are exported beyond the parasite membrane and translocated to the host cell compartment in association with vesicle-like structures. In this study we identified the specific signals necessary for the correct timing of expression and to direct SEP proteins to the vacuolar membrane and to the host cell compartments.The second part of the work was carried out in Montpellier II University and aims to identify the localization of two RBC membrane skeleton components, dematin and adducin, during Plasmodium falciparum infection. Our purpose is to recognize a possible mechanism of internalization of host cytoskeleton components to the parasite compartments. In fact, IFA experiments carried on iRBCs showed that dematin and adducin start to be internalized at trophozoite stage and localize at the periphery of the parasite, most probably at the parasitophoruos vacuole (PV) membrane/lumen. Dematin and adducin internalization during Plasmodium infection is also demonstrated by subcellular fractionation and proteinase K assay: while dematin is fully internalized, adducin is partially protected and suggesting a localization of the protein at the periphery of the parasite where it can be exposed to PK degradation
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Beattie, Pauline. "Isolation of a TNF-inducing factor from erythrocytic cultures of Plasmodium falciparum." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.268199.
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Bond, P. M. "Diversity in Plasmodium falciparum with particular reference to the infected erythrocyte." Thesis, University of Liverpool, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.234840.
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Njunge, James Mwangi. "Characterization of the Hsp40 partner proteins of Plasmodium falciparum Hsp70." Thesis, Rhodes University, 2014. http://hdl.handle.net/10962/d1013186.
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Human malaria is an economically important disease caused by single-celled parasites of the Plasmodium genus whose biology displays great evolutionary adaptation to both its mammalian host and transmitting vectors. This thesis details the 70 kDa heat shock protein (Hsp70) and J protein chaperone complements in malaria parasites affecting humans, primates and rodents. Heat shock proteins comprise a family of evolutionary conserved and structurally related proteins that play a crucial role in maintaining the structural integrity of proteins during normal and stress conditions. They are considered future therapeutic targets in various cellular systems including Plasmodium falciparum. J proteins (Hsp40) canonically partner with Hsp70s during protein synthesis and folding, trafficking or targeting of proteins for degradation. However, in P. falciparum, these classes of proteins have also been implicated in aiding the active transport of parasite proteins to the erythrocyte cytosol following erythrocyte entry by the parasite. This host-parasite “cross-talk” results in tremendous modifications of the infected erythrocyte, imparting properties that allow it to adhere to the endothelium, preventing splenic clearance. The genome of P. falciparum encodes six Hsp70 homologues and a large number of J proteins that localize to the various intracellular compartments or are exported to the infected erythrocyte cytosol. Understanding the Hsp70-J protein interactions and/or partnerships is an essential step for drug target validation and illumination of parasite biology. A review of these chaperone complements across the Plasmodium species shows that P. falciparum possesses an expanded Hsp70-J protein complement compared to the rodent and primate infecting species. It further highlights how unique the P. falciparum chaperone complement is compared to the other Plasmodium species included in the analysis. In silico analysis showed that the genome of P. falciparum encodes approximately 49 J proteins, 19 of which contain a PEXEL motif that has been implicated in routing proteins to the infected erythrocyte. Most of these PEXEL containing J proteins are unique with no homologues in the human system and are considered as attractive drug targets. Very few of the predicted J proteins in P. falciparum have been experimentally characterized. To this end, cell biological and biochemical approaches were employed to characterize PFB0595w and PFD0462w (Pfj1) J proteins. The uniqueness of Pfj1 and the controversy in literature regarding its localization formed the basis for the experimental work. This is the first study showing that Pfj1 localizes to the mitochondrion in the intraerythrocytic stage of development of P. falciparum and has further proposed PfHsp70-3 as a potential Hsp70 partner. Indeed, attempts to heterologously express and purify Pfj1 for its characterization are described. It is also the first study that details the successful expression and purification of PfHsp70-3. Further, research findings have described for the first time the expression and localization of PFB0595w in the intraerythrocytic stages of P. falciparum development. Based on the cytosolic localization of both PFB0595w and PfHsp70-1, a chaperone – cochaperone partnership was proposed that formed the basis for the in vitro experiments. PFB0595w was shown for the first time to stimulate the ATPase activity of PfHsp70-1 pointing to a functional interaction. Preliminary surface plasmon spectroscopy analysis has revealed a potential interaction between PFB0595w and PfHsp70-1 but highlights the need for further related experiments to support the findings. Gel filtration analysis showed that PFB0595w exists as a dimer thereby confirming in silico predictions. Based on these observations, we conclude that PFB0595w may regulate the chaperone activity of PfHsp70-1 in the cytosol while Pfj1 may play a co-chaperoning role for PfHsp70-3 in the mitochondrion. Overall, this data is expected to increase the knowledge of the Hsp70-J protein partnerships in the erythrocytic stage of P. falciparum development, thereby enhancing the understanding of parasite biology.
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Reinhardt, Christina Kimberly. "Funktionelle Analysen der Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1)- Domäne DBL3 gamma." [S.l. : s.n.], 2006.
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Bera, Sagorika. "Analysis of Antibody-Induced Plasmodium falciparum Sporozoites Through Scanning Electron Microscopy." Scholar Commons, 2017. http://scholarcommons.usf.edu/etd/6679.
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Malaria is a devastating disease that continues to affect millions of people worldwide every year. Specifically, Plasmodium falciparum is the most common human malaria parasite, particularly in sub-Saharan Africa. P. falciparum causes the most malignant and debilitating symptoms with the highest mortality and complication rates. Even with the worldwide efforts of many researchers and organizations, the road to discovering a vaccine has been difficult and challenging. Due do to the improvements in in vitro liver stage assays as well as rodent models of mammalian malaria, pre-erythrocytic stages of malaria have become a more accessible target for experimental studies. These vaccine candidates target Plasmodium sporozoites in the liver and liver stages to prevent development to the blood-stage forms, which is responsible for the debilitating symptoms of the disease. Scanning electron microscopy has been used for decades to provide insight on the morphology and topography of specimens, which cannot be seen through a light microscope. The purpose of this study was to analyze the morphology of sporozoites with some target antibodies. Sporozoites have previously shown uncharacterized appearances and development in an immunofluorescent stain at different concentrations of particular antibodies. With this further understanding on the morphological impact few of the target antibodies have on sporozoites through scanning electron microscopy, further grasp can be acquired.
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Miliu, Alexandra Victoria. "Role of the phosphatases over the erythrocytic cycle of the malaria parasite Plasmodium falciparum." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT029/document.
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Plasmodium falciparum, l'agent étiologique du paludisme, est un parasite intracellulaire obligatoire du phylum des Apicomplexa, responsable de 445 000 décès par an. Le développement de Plasmodium dans les globules rouges (GRs) humains correspond à la phase symptomatique de la maladie. Il commence par la pénétration active de la cellule hôte par la forme invasive nommée mérozoïte, suivie par la multiplication du parasite dans un processus appelé schizogonie pour former 16 à 32 nouveaux mérozoïtes qui sont alors libérés des GRs (étape de sortie) et peuvent alors initier un nouveau cycle. Au cours de son développement intra-érythrocytaire de 48h, ce parasite utilise la phosphorylation réversible de protéines pour réguler les étapes d‘invasion, de schizogonie et de sortie du GR, mais nos connaissances actuelles sur la contribution des phosphatases parasitaires dans ces mécanismes demeurent très incomplètes.L'objectif de ma thèse était d’identifier et de caractériser des phosphatases potentiellement impliquées dans la sortie ou l'invasion des GRs par P. falciparum. J'ai centré mon travail sur 4 d'entre elles, à savoir PP1, PP4, PP7 et Shelph2, sur la base de leur profil d'expression transcriptionnelle tardive au cours du cycle intra-érythrocytaire, qui correspond à ces deux évènements cellulaires. La première partie de cette étude est consacrée à la caractérisation fonctionnelle de Shelph2, une phosphatase d'origine bactérienne. Par génétique inverse utilisant la stratégie CRISPR-Cas9, nous avons étiqueté le gène au locus endogène et montré que Shelph2 est stockée dans des vésicules apicales des mérozoïtes en formation. Nous avons également démontré que cette phoshpatase n’est pas essentielle pour le développement intra-érytrocytaire du parasite dans les GRs car la délétion du gène n'affecte pas les étapes d'invasion, de multiplication des parasites ou de leur sortie des GRs, ce qui suggère la possibilité d’une redondance fonctionnelle avec d'autres phosphatases parasitaires.Dans la deuxième partie de ce travail, nous avons cherché à décrire les rôles de PP1, PP4 et PP7. Les gènes codant pour ces enzymes étant décrits comme probablement essentiels, nous avons mis en place au laboratoire une stratégie de knock-down conditionnel (ribozyme glmS), avec l’idée de déstabiliser l’ARNm après auto-clivage du ribozyme lors de l’addition d‘un métabolite, ici la glucosamine. Nous avons introduit avec succès la séquence glmS en 3 'des gènes d’intérêt pour PP4 et PP7, mais nous n’avons pas observé de déplétion protéique significative lors de l’addition de glucosamine, empêchant d’utiliser ces lignées pour étudier les fonctions de PP4 et PP7. Cependant, ces lignées parasitaires modifiées ont été utilisées pour analyser la localisation subcellulaire de ces phosphatases. Comme alternative au ribozyme, nous avons utilisé une approche de knock-out inductible (iKO) basée sur une recombinase Cre dimérisable (système DiCre) qui excise des fragments d'ADN situés entre deux sites loxP. Nous avons établi deux lignées de parasites, iKO-PP7 qui n'a pas encore été caractérisée et la souche iKO-PP1. En utilisant les parasites iKO-PP1, nous avons montré que PP1 était principalement une phosphatase cytosolique majoritairement exprimée au stade schizontes. De plus, l'excision inductible du gène PP1 à deux moments différents du cycle érythrocytaire de P. falciparum nous a permis de révéler que PP1 joue deux rôles essentiels, l'un pendant la schizogonie et l'autre au moment de la sortie du parasite. A notre connaissance, ce travail représente la première description d'une phosphatase parasitaire requise pour ces étapes du développement asexué de P. falciparumPlasmodium falciparum, the etiologic agent of malaria, is an obligate intracellular parasite of the Apicomplexa phylum that is responsible for 445000 deaths annually. Plasmodium development in human red blood cells (RBCs) corresponds to the symptomatic phase of the disease. It starts by the active penetration of the host cell by the invasive form named merozoite, followed by the parasite multiplication in a process called schizogony to form 16-32 new merozoites that are released from the RBC (egress step) and start a new cycle. During its 48h intra-erythrocytic development, this parasite uses reversible protein phosphorylation to regulate invasion, schizogony as well as egress, but our current knowledge on the contribution of parasite phosphatases in these cellular events is still very poor. The objective of my thesis was to identify and functionally characterize phosphatases potentially involved in egress or invasion during P. falciparum RBC cycle. I focused my work on 4 of them, namely PP1, PP4, PP7 and Shelph2, on the basis of their late transcriptional expression profile during the intra-erythrocytic cycle, as this profile matches the timing of these two essential events. The first part of this study is dedicated to the functional characterization of Shelph2, a phosphatase of bacterial origin. By reverse genetics using CRISPR-Cas9 strategy, we endogenously tagged the gene, and showed that Shelph2 is stored in apical vesicles in the developing merozoites. We also demonstrated that it is dispensable for parasite RBC development, as the deletion of the gene did not affect invasion, parasite multiplication nor egress, suggesting possible functional redundancy with other parasite phosphatases.In the second part of this work, we aimed to describe the roles of PP1, PP4 and PP7. As they were described as likely essential, we set up in the laboratory a conditional knock-down strategy named the glmS ribozyme, with the idea of destabilizing the mRNA following self-cleavage of the ribozyme upon metabolite addition, here glucosamine. We successfully introduced the glmS sequence in 3’ of the genes of interest for PP4 and PP7 but we did not observe any significant protein depletion upon glucosamine addition, thus preventing us to use these lines to study PP4 and PP7 functions. Yet, these engineered parasite lines were used to analyze the subcellular localization of these phosphatases. As an alternative to the ribozyme, we used an inducible knock-out (iKO) approach based on a dimerizable Cre recombinase (DiCre system) that excises DNA fragments located between two loxP sites. We established two parasite lines, the iKO-PP7 that has not been further characterized and the iKO-PP1 strain. Using the iKO-PP1 parasites, we showed that PP1 is predominantly a cytosolic phosphatase mostly expressed during schizogony. Furthermore, the inducible excision of PP1 gene at two different time points of P. falciparum RBC cycle permitted us to reveal that PP1 plays two essential roles, one during schizogony and the other one at the time of parasite egress. This is to our knowledge the first description of a parasite phosphatase required for these developmental steps
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Alghamdi, Sultan Ahmed. "Genetic determinants of selectivity of erythrocyte invasion in the human malaria parasite Plasmodium falciparum." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6605/.
Full textAbstract:
The aim of this study was to investigate the genetic basis of selectivity in invasion of the red blood cells by the human malaria parasite Plasmodium falciparum. Multiple invasions of a single host red blood cell by more than one merozoite, which can be described or assessed in terms of the selectivity index (SI), has been reported to be related to the severity of malaria disease. In this study, selectivity index, defined as the ratio of the number of multiply-infected red cells observed to that expected from random invasion, as modelled by a Poisson distribution was determined for certain clones of P.falciparum. SI was measured under static and shaking culturing conditions for P. falciparum clones 3D7 and HB3 and 18 progeny clones derived from a genetic cross between these two parasite clones. P. falciparum clone 3D7 was found to have a significantly lower SI than HB3 under both static and shaking culture conditions. There was no relationship between SI and days in continuous culture for clone 3D7 under shaking and static conditions; the phenotype therefore appears to be stable over time. The genetic basis of the difference in selectivity index between P. falciparum clones 3D7 and HB3 was investigated in progeny clones from a cross between these two clones, to ascertain the inheritance pattern of the phenotype. Under static conditions, ten progeny clones had a selectivity index lower than either parent, one progeny clone had higher selectivity index than both parent, and six progeny clones had selectivity index intermediate between the parents . Under shaking conditions, fifteen progeny clones were observed to have a selectivity index lower than either parent. These observations suggest the involvement of more than one parasite gene in selectivity index. A Quantitative Trait Locus (QTL) analysis was performed in order to identify genomic regions influencing SI in the progeny clones. The highest LOD score of 5.06 was obtained for a QTL on chromosome 13 for SI measured in parasites cultured under shaking conditions. This QTL denoted, PF_SI_1, extends for approximately 100kb on chromosome 13 and contains 19 open reading frames. This finding indicates the presence of a gene or genes on chromosome 13 that influence the parasite’s selection of erythrocytes for invasion.
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Fröhlich, Benjamin [Verfasser], and Motomu [Akademischer Betreuer] Tanaka. "Haemoglobinopathies and the Protection Against Severe Malaria: Probing Cytoadhesion and Mechanics of Plasmodium falciparum Infected Erythrocytes / Benjamin Fröhlich ; Betreuer: Motomu Tanaka." Heidelberg : Universitätsbibliothek Heidelberg, 2019. http://d-nb.info/1188412574/34.
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Wright, Katherine Elizabeth. "Structure of the essential malaria invasion protein RH5 in complex with its erythrocyte receptor and inhibitory antibodies." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:5da6ecfc-8768-42cd-ad04-373b24fa659d.
Full textAbstract:
Invasion of host erythrocytes is an essential stage in the life cycle of Plasmodium parasites and in development of the pathology of malaria. The stages of erythrocyte invasion, including initial contact, apical reorientation, junction formation, and active invagination, are directed by the coordinated release of specialised apical organelles and their parasite protein contents. Among these proteins, and central to invasion by all species, are two parasite protein families, the reticulocyte-binding protein homologue (RH) and the erythrocyte-binding like (EBL) proteins, that mediate host-parasite interactions. RH5 from Plasmodium falciparum (PfRH5) is the only member of either family demonstrated to be necessary for erythrocyte invasion in all tested strains, through its interaction with the erythrocyte surface protein basigin. Indeed, antibodies targeting either PfRH5 or basigin can block parasite invasion with high efficiency in vitro, making PfRH5 an excellent candidate for a vaccine to protect against the most deadly form of malaria. Here I present crystal structures of PfRH5 in complex with basigin and with two distinct inhibitory antibodies. This is the first structure of any RH protein, revealing a novel fold in which two three-helical bundles come together to form a kite-like architecture. The two immunoglobulin domains of basigin and the inhibitory antibodies bind to one tip of the kite. These findings provide the first structural insights into erythrocyte binding by the Plasmodium RH protein family and identify novel inhibitory epitopes to guide the design of a new generation of vaccines against the blood-stage parasite.
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Ferrer, Savall Jordi. "Individual-based modeling of Plasmodium falciparum erythrocyte infection in in vitro cultures." Doctoral thesis, Universitat Politècnica de Catalunya, 2010. http://hdl.handle.net/10803/6597.
Full textAbstract:
La malària és encara avui en dia una malaltia que causa aproximadament un milió de morts a l'any a tot el món. La seva eradicació suposa un gran repte per a la humanitat i per a la comunitat científica, en particular. El cultiu in vitro del paràsit és essencial per al desenvolupament de nous medicaments. Els mètodes de cultiu actuals es basen en l'heurística i requereixen millores.En aquesta tesi es presenta una aproximació teòrica al procés d'infecció a eritròcits en cultius in vitro amb Plasmodium falciparum, un dels protozous paràsits causants de la malària. El treball està centrat en la construcció i avaluació de models d'una complexitat adequada per tractar els problemes específics detectats pels experts en l'àmbit, i inclou també la formulació d'algorismes de simulació i el disseny de protocols experimentals.
Aquest tipus de treball requereix de la col·laboració multidisciplinària. La visió dels experts en malària es complementa amb la modelització i simulació, que permet la comprovació dels supòsits preestablerts, la comprensió de fenòmens observats i la millora dels mètodes de cultiu actuals. Així doncs, cal establir i desenvolupar eines que permetin crear, analitzar i compartir models amb grups que estudien la malària des d'altres perspectives. En aquesta tesi, s'ha optat per la modelització basada en l'individu (IbM) i orientada a la reproducció de múltiples patrons (PoM). El model s'ha formulat seguint l'ODD, un protocol estàndard en el camp de l'ecologia teòrica, que s'ha adaptat a la representació de comunitats microbianes.
Els models basats en l'individu (IbMs) defineixen un conjunt de normes que regeixen el comportament de cada cèl·lula i les seves interaccions amb les altres cèl·lules i amb el seu entorn immediat. A partir d'aquestes regles, i tenint en compte una certa diversitat dins de la població i un cert grau d'aleatorietat en els processos individuals, els IbMs mostren explícitament el comportament emergent del sistema en conjunt. Complementàriament, s'han aplicat conceptes propis de la termodinàmica per tal d'entendre
l'aparició de patrons macroscòpics a partir de l'estructura de la població (per exemple de la distribució de les fases d'infecció entre els glòbuls vermells infectats).
Aquesta recerca ha comportat la la creació i aplicació del model i simulador INDISIM-RBC, que ha demostrat ser una bona eina per millorar la comprensió dels cultius estudiats. Es tracta d'un model mecanicista, basat en l'individu, que reprodueix quantitativament els patrons observats en cultius reals a diferents nivells de descripció, i que en prediu el comportament sota determinades condicions.
Hem demostrat que INDISIM-RBC pot ser emprat per a estudiar en detall alguns aspectes del cultiu del paràsit causant de la malària que calia aclarir. Permet realitzar experiments virtuals i així impulsar noves línies de recerca i explorar noves tècniques de cultiu. En particular, INDISIM-RBC s'ha utilitzat per millorar els protocols experimentals actuals del cultius estàtics, definint la geometria òptima de l'hematòcrit i els protocols de subcultiu més adequats per als cultius continus.
El treball realitzat en malària s'ha comparat amb la investigació duta a terme pel grup de recerca em relació amb d'altres comunitats microbianes. D'aquesta manera, podem estudiar les propietats emergents dels sistemes microbians en general en relació als efectes de la individualitat de la cèl·lula, la diversitat de les poblacions, l'heterogeneïtat en el medi, o el caràcter local de les interaccions, entre d'altres. Aquesta visió general proporciona eines conceptuals que poden ser emprades per refinar l'anàlisi dels processos d'infecció sota estudi.
Malaria is still a major burden that causes approximately one million deaths annually worldwide. Its eradication supposes a great challenge to the humanity and to the scientific community, in particular. In vitro cultivation of the parasite is essential for the development of new drugs. Current culture methods are based on heuristics and demand for specific improvements.
The present thesis is a theoretical approach to in vitro cultivation of the protozoan parasite Plasmodium falciparum infecting human red blood cells. It mainly focuses on the process of building a model of appropriate complexity to deal with the specific demands above mentioned, but it also includes the formulation and implementation of algorithms, and the design and execution of experimental trials.
This kind of work requires multidisciplinary collaboration: the insight of the experts in malaria research is complemented with modeling and simulation, which allows for checking settled assumptions, increasing the understanding on the system and improving the current culturing methods.
The use of tools for building, analyzing and sharing models is an imperative to this end. In this thesis, Pattern-oriented Modeling (PoM) has been adopted as the most appropriate way for raising of models and the ODD protocol (Objectives, Design Concepts and Details) has been proposed as the standard tool for communicating them.
Individual-based Modeling (IbM) has been used to tackle malaria culture systems. IbMs define a set of rules governing each cell, its interactions with others and with its immediate surroundings. From this set of rules, and taking into account diversity within the population and a certain degree of randomness in the individual processes, IbMs explicitly show the emerging behavior of the system as a whole. Methods from statistical thermodynamics have been applied to understand the emergence of macroscopic patterns from the population structure (e.g. distribution of infection stages among infected red blood cells).
The research resulted in the development of the model and simulator INDISIM-RBC, which has proved to be a good tool to improve understanding of the cultures under study. It is a mechanistically rich individual-based model and it quantitatively reproduces and predicts several patterns observed in real cultures at different levels of description.
We demonstrated that INDISIM-RBC can be used to study in detail several aspects of malaria cultivation that remained unclear, as well as to perform virtual experiments. Consequently, it can be used to open novel lines of research and to examine potential experimental techniques. INDISIM-RBC has also been used to improve the current experimental culturing protocols in static cultivation by obtaining the optimal geometry of the hematocrit layer and subcultivation periods in the continuous cultures.
This study on malaria has been compared to the research carried out by the group regarding other microbial communities. Thereby studying general emerging properties of microbial systems in general, with regard to the effect of cell individuality, heterogeneity and diversity, the local nature of interactions; and biological and spatial complexity. In doing so, the acquired holistic view has been used to develop tools that allow for a better characterization and study of the infection process, in particular.
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Downing, Sarita Louise. "Characterization of Plasmodium falciparum merozoite apical membrane antigen-1 protein changes prior to erythrocyte invasion." Diss., University of Pretoria, 2016. http://hdl.handle.net/2263/61680.
Full textAbstract:
Malaria is a global pandemic that affects millions of people each year. It is a parasitic
infection caused by the Plasmodium family, with Plasmodium falciparum being the most
virulent strain. Malaria is transmitted to humans by the female Anopheles mosquito. The
parasite undergoes two different cycles of its life cycle within the human host: the liver
and intraerythrocytic life cycle. The latter consists of an asexual and sexual cycle. The
intraerythrocytic cycle is perhaps the most important stage of the parasite's life cycle as it
promotes the spread of the disease within and between hosts. The focus of this
investigation was aimed at the invasion process of the merozoites into the erythrocytes.
The Plasmodium merozoite utilises a cascade of proteins during the erythrocyte invasion
process, which is a swift action that takes place in approximately 30 seconds. A number
of surface proteins are expressed during merozoite development and are distributed along
the merozoite surfaces to assist with attachment and invasion, the most crucial being
MSP-1, AMA-1 and RON-2. MSP-1 and AMA-1 are vital targets for the development of
malaria vaccines.
AMA-1 is the central target protein of this investigation as it plays an essential role in the
invasion process. AMA-1 commits the merozoite to invade the erythrocyte, as it assists
the RON proteins in the formation of an irreversible tight-junction with the membrane of
the erythrocyte. Antibodies, specific to AMA-1, bind to the protein, which prevents the
formation of the tight junction and inhibits the invasion of the merozoite into the
erythrocyte, therefore preventing the spread of the disease.
However, before invasion, AMA-1 undergoes a number of proteolytic processes. It is
synthesized as an 83 kDa (AMA-183) precursor protein in the apical organelle of the
merozoite. This is then cleaved at the N-terminus to give rise to a 66 kDa (AMA-166)
fragment, which is secreted onto the surface of the merozoite. The AMA-166 fragment is
then cleaved into either a 48 kDa (AMA-148) or 44 kDa (AMA-144) fragment. One of
these three fragments is then used by the merozoite for erythrocyte invasion.
The aim of this investigation was to isolate and characterise each of the fragments of the
Plasmodium falciparum AMA-1 (PfAMA-1) protein using the 3D7 lab strain of P. falciparum and to visualise the merozoite-erythrocyte invasion process, to possibly
identify which of the AMA-1 fragments are involved in the invasion process. In order to
achieve this large clusters of merozoites from sorbitol-synchronised cultures were
isolated. Schizonts were isolated from culture by magnetic separation and incubated with
E64 to prevent the release of merozoites. Merozoites that were required for the isolation
of PfAMA-1 were harvested from the schizonts by saponin lysis, then homogenised,
separated by SDS-PAGE and digested for LC-MS/MS analysis. Merozoites that were
required for the visualisation procedures were not incubated with E64, to allow natural
egression from the erythrocyte.
The transmission electron microscopy results produced clear images of the merozoiteerythrocyte
invasion process and the positioning of PfAMA-1 on the merozoite, before
and after schizont rupture, was visualised from results obtained from confocal
microscopy. Then PfAMA-1 was identified in isolated merozoite samples by LC-MS/MS
analysis. However, due to its low abundance, isolation of high enough concentrations of
PfAMA-1 to characterise its different fragments was not achieved.
Further investigation into the development of the culturing and isolating methods could
help in future projects aimed at isolating higher concentrations of merozoite proteins
from synchronised cultures with a lower merozoite egression window period, in order to
accomplish detailed analysis on invading proteins for the future development of
treatments against malaria.Dissertation (MSc)--University of Pretoria, 2016.
Pharmacology
MSc
Unrestricted
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Musasia, Fauzia Khagai [Verfasser], and Michael [Akademischer Betreuer] Lanzer. "Antibody mediated clearance of ring-infected erythrocytes as a mechanism of protective immunity against Plasmodium falciparum malaria / Fauzia Khagai Musasia ; Betreuer: Michael Lanzer." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1206347864/34.
Full text
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Baron, Kim L. "Enzymatic and chemical modifications of erythrocyte surface antigens to identify Plasmodium falciparum merozoite binding sites." Diss., University of Pretoria, 2014. http://hdl.handle.net/2263/46043.
Full textAbstract:
Malaria is a disease caused by the protozoan parasite Plasmodium where the species that causes the most severe form of malaria in humans is known as Plasmodium falciparum. At least 40% of the global population is at risk of contracting malaria with 627 000 people dying as a result of this disease in 2012. Approximately 90% of all malaria deaths occur in sub-Saharan Africa, where approximately every 30 seconds a young child dies, making malaria the leading cause of death in children under the age of five years old.
The malaria parasite has a complex life cycle utilising both invertebrate and vertebrate hosts across sexual and asexual stages. The erythrocyte invasion stage of the life cycle in the human whereby the invasive merozoite form of the parasite enters the erythrocyte is a central and essential step, and it is during this stage that the clinical symptoms of malaria manifest themselves. Merozoites invade erythrocytes utilising multiple, highly specific receptor-ligand interactions in a series of co-ordinated events. The aim of this study was to better understand the interactions occurring between the merozoite and erythrocyte during invasion by using modern, cutting-edge proteomic techniques. This was done in the hope of laying the foundation for the discovery of new key therapeutic targets for antimalarial drug and vaccine-based strategies, as there is currently no commercially available antimalarial vaccine and no drug to which the parasite has not at least started showing resistance.
In this study healthy human erythrocytes were treated separately with different protein-altering enzymes and chemicals being trypsin, the potent oxidant sodium periodate (NaIO4), the amine cross-linker tris(2-chloroethyl)amine hydrochloride (TCEA) and the thiol cross-linker 1,11-bis(maleimido)triethylene glycol (BM(PEG)3). The resulting erythrocyte protein alterations were visualised as protein band differences on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), where treated and untreated control erythrocyte ghost protein fingerprints were visually compared to one another. The protein bands showing differences between treated and control samples were in-gel digested using trypsin then sequenced by liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified using proteomics-based software. In this way, the erythrocyte proteins altered by each enzyme/chemical treatment were identified.
Malaria invasion assays were performed where each treatment group of erythrocytes as well as the control erythrocytes were incubated separately with schizont stage malaria parasites for the duration of one complete life cycle. Using fluorescent staining and flow cytometry, the invasion inhibition efficiency for each treatment group was evaluated. By utilising these methods, the identification and the relative importance of the erythrocyte proteins involved in the invasion process were determined. Protein fingerprints of control and treated erythrocyte ghosts were visualised and optimised on SDS PAGE where induced protein band differences were successfully identified by LC-MS/MS. It was found that each treatment altered erythrocyte proteins with changes found in Band 3, actin, phosphoglycerate kinase 1, spectrin alpha, spectrin beta, ankyrin, haemoglobin, Bands 4.1 and 4.2, glycophorin A and stomatin. The invasion assays revealed that TCEA inhibited invasion to the greatest extent as compared to the other treatments, followed by BM(PEG)3 and trypsin. Sodium periodate-treated erythrocytes could not be assessed using the invasion assay due to auto-haemolysis. Band 3, glycophorin A, Band 4.1 and stomatin appear to be of higher relative importance in the invasion process as compared to the other altered erythrocyte proteins. These results confirmed the known roles of spectrin alpha, spectrin beta, glycophorin A, Band 3 and Band 4.1 in invasion, and suggested that ankyrin, Band 4.2 and stomatin may also be involved.
This study highlighted the potential that modern, cutting-edge proteomic techniques provide when applied to previous comparative studies found in older literature, as previously unidentified proteins that can be involved in invasion were revealed.
These results can be used as a foundation in future studies in order to identify new key targets for the development of new antimalarial drug- and vaccine-based strategies, with the hope of preventing the suffering of the millions of malaria-inflicted people worldwide, and ultimately eradicating this deadly disease.Dissertation (MSc)--University of Pretoria, 2014.
tm2015
Pharmacology
MSc
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Chauvet, Margaux. "Étude de la modulation, par l'hémoglobine S, de la présentation des antigènes plasmodiaux à la surface du globule rouge infecté par Plasmodium falciparum, et de la réponse immunitaire contre le paludisme Impact of hemoglobin S trait on cell surface antibody recognition of Plasmodium falciparum infected erythrocytes in pregnancy-associated malaria." Thesis, Sorbonne Paris Cité, 2019. http://www.theses.fr/2019USPCB037.
Full textAbstract:
Le paludisme est une maladie tropicale résultant de l'infection par le parasite Plasmodium falciparum transmis par piqûre de moustique. Les symptômes du paludisme résultent du développement de P. falciparum dans les globules rouges (GR). Depuis des siècles, le paludisme a exercé des pressions sur le génome humain, sélectionnant des mutations conférant une protection contre les formes sévères de la maladie. C'est le cas de la mutation du gène de l'hémoglobine (HbA), composant principal des GRs. La forme mutée du gène produit une hémoglobine anormale (HbS). Contrairement à la drépanocytose (HbSS), le portage hétérozygote (trait drépanocytaire) de cette mutation (HbAS) est asymptomatique. Les porteurs sains HbAS développent moins de symptômes graves du paludisme. Aujourd'hui, les mécanismes responsables de cette protection restent partiellement élucidés. Lors de son développement, P. falciparum modifie la membrane et le cytosquelette du GR afin d'exposer des protéines parasitaires à la surface de l'hématie. Parmi ces protéines, l'adhésine majeure parasitaire "P. falciparum erythrocyte membrane protein 1" (PfEMP1), se lie aux récepteurs endothéliaux, entraînant la cytoadhérence et la séquestration des GRs infectés (iGRs). Cette cytoadhérence permet d'éviter le passage et la clairance splénique des iGRs. Des études ont montré que les iGRs HbAS cytoadhèreraient moins, en association avec une présentation anormale de PfEMP1. Cette thèse porte sur l'étude des mécanismes de résistance du trait drépanocytaire contre le paludisme à P. falciparum. Le premier projet de cette thèse porte sur le phosphoprotéome des membranes de GRs HbAA et HbAS infectés par P. falciparum. Les protéines parasitaires exposées à la surface du GR interagissent avec des protéines érythrocytaires impliquées dans l'ancrage de la membrane du GR au cytosquelette. Il s'agit des protéines du complexe Ankyrine-R et du complexe jonctionnel. Le stress oxydant généré par le trait drépanocytaire et par l'invasion parasitaire perturbe l'équilibre kinase/phosphatase dans la cellule, pouvant entraîner des modulations de la phosphorylation des protéines, interférer dans les interactions protéiques et par conséquent dans la présentation des antigènes parasitaires. Des extraits membranaires de GRs HbAA et HbAS infectés ont été produits et analysés en spectrométrie de masse et en Western-Blot. Cette étude a montré que le trait drépanocytaire modulait la phosphorylation des protéines érythrocytaires de la membrane du iGR (transporteurs membranaires et protéines du cytosquelette majoritairement), mais aussi celle de protéines parasitaires. Le deuxième projet porte sur la réponse anticorps anti-VAR2CSA dans le cadre du paludisme gestationnel selon le portage de l'HbS. Le paludisme gestationnel est une des formes sévères du paludisme, due à la cytoadhérence des iGRs dans le placenta. Cette cytoadhérence résulte de l'interaction d'un PfEMP1 particulier, VAR2CSA, à la chondroïtine sulfate A des syncytiotrophoblastes. 159 plasmas de femmes HbAA et HbAS Béninoises, collectés à l'accouchement, ont été utilisés pour mesurer leur capacité de reconnaissance de VAR2CSA à la surface de GRs HbAA et HbAS infectés. La reconnaissance immune des iGRs HbAS par les plasmas provenant des mères HbAS est significativement plus faible que celle des iGRs HbAA par les plasmas des mères HbAA. Par ailleurs, d'autres maladies génétiques affectant le GR peuvent influencer la réponse en anticorps spécifiques aux GRs parasités. Les co-portages du déficit en G6PD et de l'alpha-thalassémie avec l'HbS ont ainsi été évalués pour ce groupe d'étude. Respectivement, 26,7% et 51,7% des femmes étaient porteuses du déficit G6PD ou de l'alpha-thalassémie. Ces données soulignent l'importance de considérer simultanément les différents désordres érythrocytaires existant parmi la population considérée, pour étudier les mécanismes protecteurs conférés par le portage d'HbS contre le paludismeMalaria is a tropical disease resulting from infection by the parasite Plasmodium falciparum transmitted by mosquito bite. The symptoms of malaria are caused by the development of P. falciparum in red blood cells (RBCs). For centuries, malaria has put pressure on the human genome, having selected mutations conferring protection against severe forms of the disease. This is the case of the mutation of the hemoglobin gene (HbA), the principal constituent of RBCs. The mutated form of the gene produces abnormal hemoglobin (HbS). In contrast to sickle cell disease (HbSS), the heterozygous carriage (sickle cell trait) of this mutation (HbAS) is asymptomatic. Healthy HbAS carriers are protected from severe symptoms of malaria. Today, the mechanisms responsible for this protection remain partially understood. During its intra-erythrocytic development, P. falciparum modifies the RBC membrane and cytoskeleton to expose parasite proteins at the surface of the erythrocyte. Among these proteins, the major parasitic adhesin, "P. falciparum erythrocyte membrane protein 1" (PfEMP1), binds to endothelial receptors, resulting in cytoadherence and sequestration of infected RBCs. This cytoadherence permits the infected RBCs to avoid splenic clearance. Studies have shown that infected HbAS RBCs have a reduced cytoadherence, in association with an abnormal PfEMP1 display. This PhD project attempts to decipher the mechanisms of resistance conferred by the sickle cell trait against P. falciparum malaria. The first part of this project considers the phosphoproteome of the infected HbAA and HbAS red cell membranes. Parasitic proteins exposed on the surface of RBCs interact with erythrocyte proteins involved in the anchorage of the cytoskeleton to the erythrocyte membrane. These human proteins belong to the Ankyrin-R and the junctional complexes. The oxidative stress generated by sickle cell trait, and by parasite invasion, disrupts the kinase / phosphatase balance, leading to modulation of protein phosphorylation. As protein interactions could be regulated by their state of phosphorylation, this modulation may interfere in parasite antigens' display. Thus, protein membrane extracts of infected HbAA and HbAS RBCs were produced and analyzed by mass spectrometry and Western-Blot. This study showed that the sickle cell trait modulated the phosphorylation of erythrocyte proteins of the infected RBCs (membrane transporters and cytoskeletal proteins mainly), but also that of parasite proteins. The second part of the project deals with the anti-VAR2CSA antibody response in the context of pregnancy-associated malaria according to the heterozygous carriage of hemoglobin S. Placental malaria is one of the severe forms of malaria, resulting from the cytoadherence of infected RBCs in the placenta. This cytoadherence results from the interaction of a particular PfEMP1, VAR2CSA, with chondroitin sulfate A expressed on syncytiotrophoblasts. 159 plasma samples of HbAA and HbAS Beninese women, collected at delivery, were used to measure their ability to recognize VAR2CSA on the surface of infected HbAA and HbAS RBCs. Immune recognition of infected HbAS RBCs by plasma from HbAS mothers is significantly lower than the immune recognition of infected HbAA RBCs by HbAA mothers' plasma. In addition, other genetic diseases affecting RBCs may influence the antibody response to parasitized red blood cells. Co-carriage of G6PD deficiency and alpha-thalassemia with HbS were assessed for this study group. G6PD deficiency and alpha-thalassemia were present in, respectively, 26.7% and 51.7% of the women. These data underline the importance of simultaneously considering the different erythrocyte disorders present at high prevalence among the population considered, in order to study the protective mechanisms conferred by the carriage of HbS against malaria
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Moles, Meler Ernest. "Development of polyvalent erythrocyte- and parasitized erythrocyte-targeted nanovectors as novel site-specific drug delivery approaches for Plasmodium falciparum malaria chemotherapy." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/360333.
Full textAbstract:
Bearing in mind the absence of an effective preventive vaccine against malaria (WHO, 2015) and its severe clinical manifestations that are principally associated with red blood cell (RBC) destruction and parasitized-RBC (pRBC) cytoadherence to host cells causing in turn nearly half a million deaths every year, this disease represents nowadays a major threat to life and consequently its control and ultimate global eradication must be undertaken without preconceptions.
Additionally, the basic rationale followed by most of the currently marketed antimalarial approaches is based on the administration of single/multiple drugs on their own, strategy that promotes the emergence of drug-resistant parasites owing to the limitation in delivering drug payloads into the pRBC high enough to kill the intracellular parasite while at the same time minimizing the risk of causing toxic side effects to the patient. Such dichotomy has been successfully addressed through the preparation of RBC- and pRBC-targeted drug delivery systems in the form of antibody-vectorized liposomes (iLPs), among other types of nanovectors, improving in this way the activity of antimalarials (Chandra, 1991; Owais, 1995; Urbán, 2011).
Nevertheless, the aforesaid iLP models have been poorly characterized with a minimal knowledge regarding: their mechanism of interaction with the target cell, release kinetics of encapsulated material or antibody coupling yield; and their recognized antigens have not been reported or are still unknown. Besides, the improvement in drug activity they have provided has been rather modest when evaluated against the human-infecting Plasmodium falciparum species. The main scope of this PhD thesis has been in this regard the characterization and development of more effective immunoliposomal nanovectors against P. falciparum with special attention given to the obtainment of chemotherapeutic approaches displaying multiple mechanisms of action.
Antibody coupling yields of >40% were obtained by means of their derivatization with the SATA crosslinker and the incorporation of maleimide-containing, PEGylated phospholipids into LPs, which results in the highly stable thioether linkage. SATA/antibody molar ratios of up to 10× provided adequate antigen recognition and minimal iLP aggregation. Furthermore, the pH- driven active encapsulation of chloroquine (CQ) and primaquine antimalarials into DSPC-based LPs together with their conjugation with a monoclonal antibody specific for the glycophorin A antigen of RBCs (GPA-iLPs) enabled these iLPs (i) to completely recognize and become retained into both RBCs/pRBCs, as well as (ii) a total and stable drug encapsulation along with its effective intracellular release under parasite culture conditions. The improved antimalarial efficacy of CQ-loaded, GPA-iLPs was demonstrated in vivo in P. falciparum-infected, humanized mice through the reduction of their parasite densities to undetectable levels (<0.01% parasitemia) and following a 4 × 0.5 mg CQ/kg dosage schedule. Free CQ at a dosage 3.5 times higher was at least 40-fold less effective.
Moreover, lumefantrine-laden iLPs targeted against rosette-forming variants of PfEMP1 exhibited a great potential for severe malaria therapeutics by means of mechanically disrupting already generated rosettes while at the same time eliminating those parasites forming them. An increased activity of lumefantrine in reducing the number of rosette-forming pRBCs was obtained when delivered by homologous PfEMP1-targeted-iLPs with a ~5.5-fold decreased IC50 compared to either free drug or non-targeted LPs. The rosette-disrupting activity of anti- PfEMP1 antibodies was importantly preserved after their conjugation to LPs. Finally, based on a recent study in Toxoplasma gondii (Nagamune, 2008), the biosynthesis of ABA by P. falciparum was explored as a new target route for the design of antimalarials. However, ABA could not be found in late form-pRBC extracts even though the sensitivity for its detection had been extraordinarily improved (LOD of 0.03 ng/ml).
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Curtis, David Floyd. "A Member of the Novel FIKK Family of Plasmodium falciparum Putative Protein Kinases Exhibits Diacylglycerol Kinase Activity and Is Exported to the Host Erythrocyte." Master's thesis, University of Central Florida, 2007. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2363.
Full textAbstract:
Plasmodium falciparum is one of four species known to cause malaria in humans and is the species that is associated with the most virulent form of the disease. Malaria causes nearly two million deaths each year, many of these occurring among children in under-developed countries of the world. One reason for this is the prevalence of drug resistant strains of malaria that mitigate the efficacy of existing drugs. Hence, the identification of a new generation of pharmacological agents for malaria is extremely urgent. The recent identification of a group of novel protein kinases within the Plasmodium falciparum genome has provided researchers with a basis for what many hope to be new potential drug targets for malaria. Identified within the Plasmodium genome and a few select apicomplexans, these novel proteins have been predicted to be protein kinases based solely on certain sequence features shared with other eukaryotic protein kinases (ePKs). However, to date, no significant studies to determine the function of these novel kinases have been performed. Termed FIKKs, these proteins all possess a non-conserved N-terminal sequence that contains a Plasmodium export element (Pexel) which may target the proteins for export from the parasite and a conserved C-terminal catalytic domain containing a FIKK sequence common to all twenty members of this family. We analyzed the localization of one of the FIKK proteins, FIKK11, encoded by the PF11_0510 locus, during intraerythrocyte differentiation of P. falciparum by Western blot analysis and indirect immunofluorescence assay. Western blot analysis demonstrated that FIKK 11 is expressed within the parasite at all stages of its erythrocytic life cycle with its highest expression occurring during the schizont stage. Immunofluorescence assays showed that this protein is exported from the Plasmodium parasite into the host erythrocyte cytosol which is consistent with studies on other Plasmodium proteins that also have the Pexel motif. To determine the enzymatic activity of FIKK11, we overexpressed the recombinant protein in E. coli and then purified it. However, no protein kinase activity was detected using several commonly used protein kinase substrates including histone H1, myelin basic protein, or dephosphorylated casein. We also did not detect any kinase activity of the native enzyme using pull-down assays of the Plasmodium falciparum cell extract against those same substrates. In addition, kinase substrate peptide array analysis of FIKK11 showed no evidence of protein kinase activity either for FIKK11. Interestingly, however, we were able to detect some kinase activity using the recombinant protein alone with no substrate. The lack of the glycine triad within subdomain I of these FIKK kinases as compared with most traditional eukaryotic protein kinases may explain why we were unable to find any interactions between FIKK11 and other commonly protein kinase substrates. Of interest was the observation that the protein reproducibly exhibited what appeared to be an autophosphorylation activity when using the standard protein kinase assay. Further analyses, however, showed that FIKK11 actually possesses diacylglycerol kinase activity utilizing 1-Stearoyl-2-arachidonoyl-sn-glycerol as a substrate. This is the first evidence of diacylglycerol kinase activity in Plasmodium falciparum. Because FIKK11 is exported into the host cell and is localized on the erythrocyte membrane, its enzymatic activity may potentially have relevance in the pathophysiology of the disease.M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular and Microbiology MS
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Lansche, Christine [Verfasser], and Michael [Akademischer Betreuer] Lanzer. "Protection against severe malaria by hemoglobin S and C: A quantitative understanding of the cytoadhesion behavior of Plasmodium falciparum infected erythrocytes / Christine Lansche ; Betreuer: Michael Lanzer." Heidelberg : Universitätsbibliothek Heidelberg, 2018. http://d-nb.info/1177251809/34.
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Mauritz, Jakob Martin Andreas. "Homeostasis and volume regulation in the Plasmodium falciparum infected red blood cell." Thesis, University of Cambridge, 2011. https://www.repository.cam.ac.uk/handle/1810/240497.
Full textAbstract:
The thesis reports on the application of advanced microanalytical techniques to answer a fundamental open question on the homeostasis of Plasmodium falciparum infected red blood cells, namely how infected cells retain their integrity for the duration of the parasite asexual reproduction cycle. The volume and shape changes of infected cells were measured and characterized at femtolitre resolution throughout the intraerythrocytic cycle using confocal microscopy. Fluorescence lifetime imaging and electron probe X-ray microanalysis were applied for the quantification of intracellular haemoglobin and electrolyte concentrations. The cytomechanical properties of uninfected and infected red cells were studied using a novel optical stretcher device, which enabled individual cells to be trapped and manipulated optomechanically in microfluidic channels. Combined, these methods offered a unique insight into the homeostatic and rheological behaviour of malaria-infected red cells. The results were analysed by comparison with predictions from a detailed physiological model of the homeostasis and volume regulation of infected cells, providing broad support to the view that excess haemoglobin consumptions by the parasite was necessary for the integrity of infected cells (the colloidosmotic hypothesis). The dissertation is introduced with an overview of malaria, red blood cells homeostasis and the changes induced by Plasmodium falciparum infection. In the following, this description is extended to an in-depth theoretical analysis of the infected red blood cell homeostasis, from which the need to characterise certain parameters arises. The subsequent chapters address sequentially the assessment of the haemoglobin and electrolyte concentration, cell shape and volume changes and ultimately alterations in cell elasticity. The experimental part is complemented with a comparison of the resulting data to the predictions from the theoretical analysis and an outlook on future work.
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Venter, Tarryn Lee. "Characterisation of the pre-invasion glycophosphatidylinositol-anchored surface proteins of Plasmodium falciparum merozoites." Diss., University of Pretoria, 2017. http://hdl.handle.net/2263/63040.
Full textAbstract:
Plasmodium falciparum is a protozoan parasite responsible for causing the most severe form of
malaria in humans. This species is responsible for over 90% of malaria mortalities which occur
predominantly in Africa. An increase in drug resistant parasites in recent years is threatening the
progress made against malaria and thus new antimalarial drugs and vaccines are needed to
combat this disease.
During the intraerythrocytic phase, merozoites egress from mature schizonts to invade new
uninfected erythrocytes. Glycophosphatidylinositol (GPI) -anchored proteins cover most of the
exterior surface of the merozoite prior to invasion, while other GPI-anchored proteins are released
onto the merozoite surface through apical organelle secretions. These proteins are involved in
interactions with erythrocytes and are thought to be vital to erythrocyte invasion. GPI-anchored
proteins have also been implicated as a cause of pathogenic symptoms and activation of immune
components. These proteins are then released or cleaved to enable merozoite entry into the
erythrocyte. Several enzymes are thought to be involved in their cleavage including the serine
proteases subtilisin-like proteases (SUB) 1 and 2, and phosphatidylinositol-phospholipase C (PIPLC);
GPI-anchored proteins are also generally sensitive to phospholipase A2 (PLA2). Cleaved
proteins are released into the host blood system, while uncleaved proteins are carried into the
erythrocyte during invasion.
Merozoites have a limited period in which they retain invasive capacity. A previous lack of
available techniques that are specifically adapted to merozoite analysis has resulted in an
incomplete understanding of invasion and GPI-anchored protein involvement in invasion. This
study aimed to determine how GPI-anchored proteins on the merozoite surface are altered in the
invasive phase, and explore the possibility of using merozoite GPI-anchored proteins as potential
drug targets to block erythrocyte invasion. Optimised methods of in vitro parasite culturing which produce highly synchronised merozoites
was essential to this study. Parasite culturing techniques were optimised by utilising low
haematocrit cultures with frequent culture splitting and optimised synchronisation. The
“Malarwheel” is a tool that was developed for this research to provide a means for scheduling
sorbitol treatments and MACs isolations. This tool and optimised culturing methods enabled large
volumes of highly synchronised invasive merozoites to be harvested. Four compounds (vanadate,
edelfosine, dioctyl sodium sulfosuccinate (DSS), and gentamicin) suspected to interfere with GPIanchored
cleavage or processes were screened on intraerythrocytic stages and merozoites. Antimalarial and anti-invasive properties of these compounds were screened by modified malaria
SYBR Green I-based fluorescence (MSF) assay and merozoite invasion assays (MIA)
respectively. DSS and gentamicin showed limited potential as antimalarials or as anti-invasive
agents. Vanadate and edelfosine both showed antimalarial and anti-invasive activity, while
edelfosine was the most potent anti-invasive agent at physiological concentrations. The merozoite GPI-anchored proteome was analysed by sodium dodecyl sulphatepolyacrylamide
gel electrophoresis (SDS-PAGE) followed by complete gel lane analyses
conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on soluble and
pelleted merozoite proteins in samples from either invasive or non-invasive merozoites. Thirteen
known or predicted GPI-anchored proteins were identified in samples. Several changes were
identified in merozoite GPI-anchored proteins between the invasive phase and after its
completion, and minor differences were observed following treatment with edelfosine. Edelfosine
showed partial inhibition of erythrocyte invasion, however, the primary cause of inhibition cannot
be directly related to interferences with GPI-anchored proteins. These results suggest that GPIanchored
proteins are controlled by various complex processes, and are cleaved or processed
by diverse mechanisms during the invasive phase. These mechanisms may be controlled by
multiple signals which effect proteins or groups of proteins in specific ways. These signals may
be influenced by “checkpoints” during invasion processes including the time period after egress
from schizonts, and possibly the recognition of erythrocyte targets. These methods and results provide a foundation for future research to enable culturing of P.
falciparum parasites specifically for merozoite research, and to identify merozoite proteins active
during the invasive phase. These results confirm and challenge previous ideas reported in
literature on the GPI-anchored processes of merozoites and further characterise less studied GPIanchored
proteins. The results suggest that the processes controlling GPI-anchored proteins may
be more complex than previously thought. These results form a basis to further identify and
characterise GPI-anchored proteins in the aim to develop antimalarial medications and vaccines
that target merozoites and their GPI-anchored processes.Dissertation (MSc)--University of Pretoria, 2017.
Pharmacology
MSc
Unrestricted
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Duvalsaint, Marvin Duvalsaint. "The Effects of Phytohormones and Isoprenoids in Dihydroartemisinin-induced Dormancy in the Erythrocytic Stages of Plasmodium falciparum." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6495.
Full textAbstract:
Our ability to control malaria has been challenged by increasing antimalarial resistance. Plasmodium falciparum undergoes dormancy in the blood stages which is hypothesized to be a means by which they are able to survive under drug pressure. This helps select for resistant parasites which grow following removal of drug. The mechanisms behind dormancy and the subsequent recrudescence are not fully understood but translating knowledge from related organisms which undergo a similar phenomenon might shed some light. Higher plants utilize dormancy during the early development stages to survive under unfavorable conditions, increasing fitness of the seedling and ensuring viability when this is released and it develops into a mature plant. Abscisic acid (ABA) and gibberellic acid (GA) antagonistically regulate this in response to environmental cues. We have found that both can be supplemented to dihydroartemesinin-induced dormant parasites to stimulate early recovery. Fluridone, an ABA inhibitor that releases dormancy in plants, was found to prolong it and cause a delay in recrudescence. These effects were observed in artemisinin sensitive and resistant strains.
The apicoplast is required for recovery and supplementation of essential isoprenoid, isopentyl pyrophosphate (IPP), in apicoplast deficient parasites is sufficient enough to compensate for the lack of the organelle in antibiotic treated parasites. IPP plays an important role in development and metabolism of blood stage parasites as a key component of numerous secondary metabolites and protein activity by prenylation of isoprenoids. Its role in dormancy has not been explored prior to this study.
Carotenoids are long-chained ABA precursors consisting of two molecules of geranylgeranyl pyrophosphate (GGPP). Several carotenoids as well as enzymes in that pathway have been identified in the blood stages of P. falciparum. The Apicomplexan parasite, Toxoplasma gondii synthesizes ABA, where it plays a role in signaling and development. To date ABA has not been detected in P. falciparum due to limitations in methods previously utilized. We have found that parasites with fosmidomycin inhibition of isoprenoids can be rescued with GGPP supplementation which we planned to use to further elucidate the carotenoid biosynthetic pathway.
We hypothesized that Plasmodium has retained the ability to biosynthesize ABA and aimed to confirm this. We developed a novel method to label GGPP with 13C on three of its isoprene units. This would be used to metabolically label isoprenoid inhibited P. falciparum for incorporation through the carotenoid pathway for detection of 13C-ABA.
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Blanc, Manuel. "Biochemical, biophysical, and structural studies of a protein complex implicated in the erythrocyte interaction with the malaria parasite Plasmodium falciparum." Thesis, Keele University, 2015. http://eprints.keele.ac.uk/588/.
Full textAbstract:
This thesis describes biochemical and biophysical studies of two protein domains that are believed to be involved in the interaction between the merozoites of Plasmodium falciparum and human red blood cells. The parasite protein fragment derives from the erythrocyte binding antigen 181 (EBA-181) invasion protein, and the human protein fragment comes from the 4.1R erythrocyte skeletal protein. The initial goal of the PhD project was to derive structural information on the nature of this complex, with a perspective towards generating new therapeutic approaches. Extensive biochemical and biophysical characterisation of the complex was carried out and is described in detail in Chapter 3 of the thesis: the results confirm the interaction, add insights to the stability of the complex and suggest the presence of significant disorder in both the individual proteins and the complex. Structural studies were carried out using small-angle neutron and X-ray scattering, used in conjunction with selective deuteration. These studies, which are described in Chapter 4, provide low resolution images of the individual proteins and of the complex; these have been compared to structure predictions using bioinformatics. In Chapter 5, solution state NMR studies were carried out, principally on the EBA-181 protein, but with preliminary results from titration work designed to further probe the nature of the interaction between the two proteins. Chapter 6 concludes the thesis with a summary of the work placed in context of the host-pathogen interaction, and proposes directions for future work.
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Roth, Lisa Katharina [Verfasser], and Iris [Akademischer Betreuer] Bruchhaus. "The effects on the malaria parasite Plasmodium falciparum (WELCH, 1897) in response to an interaction of parasitized erythrocytes with various human endothelial receptors / Lisa Katharina Roth ; Betreuer: Iris Bruchhaus." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2019. http://d-nb.info/1189817519/34.
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Henry, Benoît. "Splénomégalie, déformabilité des globules rouges circulants et héritabilité de la diversité phénotypique chez des sujets exposés à Plasmodium falciparum." Thesis, Sorbonne Paris Cité, 2019. https://theses.md.univ-paris-diderot.fr/HENRY_Benoit_complete_depot_2.pdf.
Full textAbstract:
En Afrique sub saharienne, les membres du groupe ethnique Peulh ont une réponse phénotypique singulière à l’infection par Plasmodium falciparum, comportant une prévalence plus élevée de la splénomégalie et de l’anémie, ainsi qu’une charge parasitaire circulante plus basse et qu’une réponse immune humorale anti parasitaire plus intense. Ce phénotype considéré comme « protecteur » est proche d’une forme rare et chronique d’infection palustre, la splénomégalie palustre hyper-réactive. Les déterminants de cette présentation phénotypique particulière sont mal connus. Une origine génétique est suspectée. Nous avons émis l’hypothèse que des spécificités des hématies (parasitées ou non) jouaient un rôle dans la genèse de ce phénotype.Nous avons étudié la réponse phénotypique à l’infection palustre au sein d’une population multi-ethnique (Bariba, Gando, Otamari, Peulh) de près de 800 individus (193 familles) vivant dans le nord-ouest du Bénin, en zone d’hyper endémie palustre. Huit passages transversaux entre juin 2015 et décembre 2017 ont été réalisés. Nous avons confirmé, dans le groupe Peulh, une sur-prévalence de la splénomégalie. Les IgM plasmatiques totales, mesurées à 2 reprises, étaient aussi plus élevées chez les Peulhs. L’évolution temporelle de la prévalence de l’infection palustre, de la fièvre et de l’anémie différait selon les ethnies de façon inconstante.Nous avons ensuite analysé le phénotype érythrocytaire au sein de cette même population lors d’un passage transversal fin 2017. A cette date, la splénomégalie était significativement plus prévalente chez les Peulhs, mais la tendance à l’anémie et à une moindre charge parasitaire, bien que présente, n’était pas significative. Il existait une sur-prévalence non significative d’infection palustre dans le groupe Peulh. Les hématies circulantes, étudiées par microsphiltration et ektacytométrie, étaient plus déformables dans le groupe Peulh. L’analyse uni puis multivariée des facteurs associés aux valeurs de déformabilité a montré que les déterminants en étaient l’ethnie et la présence de marqueurs d’infection palustre (test diagnostique rapide ou PCR) ; la déformabilité élevée des hématies n’étant observée que chez les sujets Peulhs infectés. Au sein d’un sous-groupe de 120 sujets, l’infection des hématies in vitro par P. falciparum n’a pas montré de différence inter ethnique en termes de déformabilité des hématies infectées ou de croissance parasitaire. En revanche, il existait une corrélation positive, plus marquée chez les Peulhs, entre déformabilité des hématies circulantes et croissance plasmodiale in vitro. L’héritabilité de la déformabilité érythrocytaire (valeurs de microsphiltration) était très importante chez les Peulhs et les sujets infectés. Le groupe Peulh possédait également une plus grande proportion de cellules B mémoires IgM positives en circulation.Ces données confirment l’existence d’une réponse phénotypique particulière de l’ethnie Peulh à l’infection palustre, sujette à de fortes variations temporelles. La déformabilité élevée des hématies circulantes chez les Peulhs, trait hautement héritable associé à une corrélation entre déformabilité des hématies circulantes et croissance in vitro du parasite, pourrait être expliquée, dans ce groupe, soit par une réponse hématopoïétique exacerbée à l’infection, soit par une déformabilité basale plus importante dans un sous-groupe de sujets Peulhs, favorisant l’infection, soit enfin par une filtration splénique des hématies exacerbée par l’infection palustre. Ces éléments suggèrent que chez les sujets exposés au paludisme, des spécificités érythrocytaires ou spléniques pourraient intervenir en amont de la réaction immune adaptative, et ouvrent la voie à l’identification des déterminants génétiques de ce nouveau traitIn subsaharan Africa, the Fulani people display a specific phenotypic response to the infection with Plasmodium falciparum, defined by the over-prevalence of splenomegaly and anemia, less frequent or lower parasitemia), and a stronger anti plasmodial immune response. This “protective” phenotype is reminiscent of hyper-reactive malarial splenomegaly, a rare and chronic form of malarial infection. Determinants of this specific phenotype remain elusive, but a genetic basis is suspected. We hypothesized that specificities of erythrocytes (parasitized or not) would play a role in the emergence of this phenotype.We have studied the phenotypic response to malarial infection in a 800 subjects (193 families) multi-ethnic cohort (Bariba, Gando, Otamari, Fulani) living in sympatry in Northern Benin, a malarial hyperendemic region. Eight cross-sectional studies were performed between June, 2015 and December, 2017. We confirmed, among Fulani, a greater prevalence of splenomegaly. Total plasma IgM were also higher among Fulani at 2 time points. Analysis of temporal trends of Plasmodium infection markers, fever and anemia showed inconstant inter-ethnic differences.We then evaluated erythrocyte phenotype in the same cohort, during a cross-sectional study in December, 2017. At this time, prevalence of splenomegaly was significantly higher among Fulani, but the trend towards anemia and lower parasitic load, although present, was unsignificant. We found a higher, non-significant, prevalence of malarial infection among Fulani. Deformability of circulating erythrocytes, measured through ektacytometry and microsphiltration, was higher among Fulani. Uni- then multivariate analysis of factors associated with erythrocyte deformability showed that the major determinants of this trait were ethnicity and markers of plasmodial infection (rapid diagnostic test or PCR); increase in deformability being almost exclusively observed among infected Fulani subjects. In a subgroup of 120 subjects, in vitro infection of erythrocytes with P. falciparum did not show inter-ethnic differences regarding erythrocyte deformability or parasite growth. However, a positive correlation was observed between circulating erythrocytes deformability and parasite growth. This was more pronounced in Fulani. Heritability of erythrocyte deformability (through microsphiltration) was very high in Fulani and in infected subjects. Fulani also displayed a higher proportion of circulating IgM-positive memory B cells.These data confirm the reality of a peculiar phenotypic response to malarial infection among Fulani; this phenotype is nevertheless subject to marked temporal variations. The enhanced deformability of circulating erythrocytes in Fulani, its strong heritability, and the correlation between circulating erythrocytes deformability and parasite growth after in vitro infection could be explained by three non-mutually exclusive hypotheses: and enhanced erythropoietic response to malarial infection in Fulani; an increased circulating erythrocytes deformability in a subgroup of Fulani, which would favor infection; or by an enhanced splenic filtration of erythrocytes by the spleen in reaction to infection in Fulani. These elements suggest that in malaria-exposed subjects, erythrocytes or spleen-related specificities could act upstream of antimalarial immune response. This also paves the way to the identification of genes involved in this novel trait
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Beaudry, Jeanette T. "Effect of hemoglobins S and C on the in vivo expression and immune recognition of Plasmodium falciparum erythrocyte membrane protein 1 variants in Malian children." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:21f27887-e7e8-4480-a8e4-c7072f3b392c.
Full textAbstract:
The enormous mortality burden exerted by P. falciparum malaria has evolutionarily selected for red blood cell (RBC) polymorphisms which confer protection against the severe manifestations of this disease. Although the epidemiological protection by these polymorphisms has been well-established for the past half-century, the mechanisms underlying this protection are still being uncovered. Recent studies implicate impaired cytoadherence to microvascular endothelial cells (MVECs) due to reduced surface levels and altered display of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) as a mechanism of protection against severe malaria by sickle hemoglobin (Hb) S and HbC. Consequently, in this thesis, I have described three separate, but related investigations into whether hemoglobins S and C influence a parasite’s cytoadherence binding phenotype (Chapter 3), the PfEMP1 variants that parasites express in vivo (Chapter 4), and the IgG recognition of PfEMP1 domains in Malian children (Chapter 5). We found that parasites from HbAS children show statistically insignificant increased binding to MVECs and that parasites did not express a restricted subset of var genes in HbAS and HbAC children. Compared to HbAA and HbAC children, HbAS children demonstrated a slower rate of acquisition of IgG responses to a repertoire of PfEMP1 domains. These findings suggest that, although hemoglobin type influences the binding phenotype of P. falciparum isolates and the acquisition of PfEMP1-specific IgG responses, other factors more likely determine the expressed var gene repertoire within parasites than hemoglobin type.
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Diehl, Mathias [Verfasser], and Michael [Akademischer Betreuer] Lanzer. "Using new genetic tools to elucidate the importance of exported proteins in intra-erythrocytic survival of the malaria parasite Plasmodium falciparum / Mathias Diehl ; Betreuer: Michael Lanzer." Heidelberg : Universitätsbibliothek Heidelberg, 2020. http://d-nb.info/1218432330/34.
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Lenstra, Reijer. "Biologie moleculaire du parasite de la malaria, plasmodium falciparum : etude des knobs et recherches de genes a potentiel codant pour une proteine riche en histidine associee aux knobs." Paris 7, 1988. http://www.theses.fr/1988PA077103.
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Kriek, Neline. "Protein transport in Plasmodium falciparum infected erythrocytes." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270202.
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Treutiger, Carl Johan. "Host cell adhesion of Plasmodium falciparum-infected erythrocytes /." Stockholm, 1998. http://diss.kib.ki.se/1998/91-628-2952-1.
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