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1
Yap, Jessica. "Identification of Plasmodium falciparum protein kinase substrates and interacting proteins." Honors in the Major Thesis, University of Central Florida, 2012. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/644.
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Characterization of PfPKA and PfPK5 substrates, as well as the proteins they interact with, will help us to develop innovative therapies targeting binding sites.; Malaria is a devastating disease that results in almost one million deaths annually. Most of the victims are children under the age of five in Sub-Saharan Africa. Malaria parasite strains throughout developing countries are continually building resistance to available drugs. Current therapies such as mefloquine, chloroquine, as well as artemisinin are becoming less effective, and this underscores the urgency for therapeutics directed against novel drug targets. In order to identify new drug targets, the molecular biology of the malaria parasite Plasmodium needs to be elucidated. Plasmodium exhibits a unique cell cycle in which it undergoes multiple rounds of DNA synthesis and mitosis without cytokinesis. Thus, cell cycle regulatory proteins are likely to be promising pathogen-specific drug targets. It is expected that fluctuating activity of key proteins, such as protein kinases, play an essential role in regulating the noncanonical life cycle of Plasmodium. Consequently, malarial kinases are a prime target for therapy. One way to better understand the role of malarial kinases in Plasmodium cell cycle regulation is to identify putative protein kinase substrates and interacting proteins. Two malarial kinases that have been implicated in regulating malaria parasite cell cycle stages were investigated in this study: P. falciparum CDK-like Protein Kinase 5 (PfPK5) and cAMP-Dependent Protein Kinase A (PfPKA). A transgenic P. falciparum line was created for the expression of epitope-tagged PfPK5 for pull-down analysis. Phospho-substrate antibodies were used to identify physiological substrates of both PfPK5 and PfPKA. Immunoblotting with these antibodies identified several potential substrates. Identities of the PfPKA physiological substrates were determined from the global P. falciparum phosphoproteome dataset that has recently been generated in our laboratory.B.S.
Bachelors
Burnett School of Biomedical Sciences
Molecular and Microbiology
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Shonhai, Addmore. "Molecular characterisation of the chaperone properties of Plasmodium falciparum heat shock protein 70." Thesis, Rhodes University, 2007. http://eprints.ru.ac.za/886/.
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Matambo, Tonderayi Sylvester. "Biochemical characterization of plasmodium falciparum heat shock protein 70." Thesis, Rhodes University, 2004. http://eprints.ru.ac.za/73/1/Matambo-MSc.pdf.
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Plamodium falciparum heat shock protein (PfHsp70) is believed to be involved in the cytoprotection of the malaria parasite through its action as a molecular chaperone. Bioinformatic analysis reveal that PfHsp70 consists of the three canonical Hsp70 domains; an ATPase domain of 45 kDa, Substrate binding domain of 15 kDa and a C-terminal domain of 10 kDa. At the C-terminus there is a GGMP repeat motif that is commonly found in Hsp70s of parasitic origins. Plasmodium falciparum genome is 80% A-T rich, making it difficult to recombinantly express its proteins in Escherhia coli (E. coli) as a result of rare codon usage. In this study we carried out experiments to improve expression in E. coli by inserting the PfHsp70 coding region into the pQE30 expression vector. However multiple bands were detected by Western analysis, probably due to the presence of rare codons. The RIG plasmid, which encodes tRNAs for rare codons in particular Arg (AGA/AGG), Ile (AUA) and Gly (GGA) was engineered into the E. coli strain resulting in production of full length PfHsp70. Purification was achieved through Ni^(2+) Chelating sepharose under denaturing conditions. PfHsp70 was found to have a very low basal ATPase activity of 0.262 ± 0.05 nmoles/min/mg of protein. In the presence of reduced and carboxymethylated lactalbumin (RCMLA) a 11-fold increase in ATPase activity was noted whereas in the presence of both RCMLA and Trypanosoma cruzi DnaJ (Tcj2) a 16-fold was achieved. For ATP hydrolysis kcat value of 0.003 min^(-1) was obtained whereas for ADP release a greater k_cat_ value of 0.8 min^(-1) was obtained. These results indicated that rate of ATP hydrolysis maybe the rate-determining step in the ATPase cycle of PfHsp70.
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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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Holton, S. J. "Structural and functional studies of Plasmodium falciparum protein kinase 5 and Cks proteins." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270634.
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McNamara, Caryn. "Characterisation of Human Hsj1a : an HSP40 molecular chaperone similar to Malarial Pfj4." Thesis, Rhodes University, 2007. http://hdl.handle.net/10962/d1007603.
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Protein folding, translocation, oligomeric rearrangement and degradation are vital functions to obtain correctly folded proteins in any cell. The constitutive or stress-induced members of each of the heat shock protein (Hsp) families, namely Hsp70 and Hsp40, make up the Hsp70/Hsp40 chaperone system. The Hsp40 J-domain is important for the Hsp70-Hsp40 interaction and hence function. The type-II Hsp40 proteins, Homo sapiens DnaJ 1a (Hsj1a) and Plasmodium falciparum DnaJ 4 (Pfj4), are structurally similar suggesting possible similar roles during malarial infection. This thesis has focussed on identifying whether Hsj1a and Pfj4 are functionally similar in their interaction with potential partner Hsp70 chaperones. Analysis in silico also showed Pfj4 to have a potential chaperone domain, a region resembling a ubiquitin-interacting motif (UIM) corresponding to UIM1 of HsjIa, and another highly conserved region was noted between residues 232-241. The highly conserved regions within the Hsp40 J-domains, and those amino acids therein, are suggested to be responsible for mediating this Hsp70-Hsp40 partner interaction. The thermosensitive dnaJ cbpA Escherichia coli OD259 mutant strain producing type-I Agrobacterium tumefaciens DnaJ (AgtDnaJ) was used as a model heterologous expression system in this study. AgtDnaJ was able to replace the lack of two E coli Hsp40s in vivo, DnaJ and CbpA, whereas AgtDnaJ(H33Q) was unable to. AgtDnaJ-based chimeras containing the swapped J-domains of similar type-II Hsp40 proteins, namely Hsj1Agt and Pfj4Agt, were also able to replace these in E. coli OD259. Conserved J-domain amino acids were identified and were substituted in these chimeras. Of these mutant proteins, Hsj IAgt(L8A), Hsj1Agt(R24A), Hsj1Agt(H31Q), Pfj4Agt(L 11A) and Pfj4Agt(H34Q) were not able to replace the E. coli Hsp40s, whilst Pfj4Agt(Y8A) and Pfj4Agt(R27A) were only able to partially replace them. This shows the leucine of helix I and the histidine of the loop region are key in the in vivo function of both proteins and that the arginine of helix II is key for Hsj1a. The histidine-tagged Hsj1a protein was also successfully purified from the heterologous system. The in vitro stimulated ATPase activity of human Hsp70 by Hsj1a was found to be approximately 14 nmol Pí[subscript]/min/mg, and yet not stimulated by Pfj4, suggesting a possible species-specific interaction is occurring.
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Botha, Melissa. "Characterisation of the plasmodium falciparum Hsp40 chaperones and their partnerships with Hsp70." Thesis, Rhodes University, 2009. http://hdl.handle.net/10962/d1003997.
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Central to this research, 40 kDa Heat shock proteins (Hsp40s) are known to partner (or cochaperone) 70 kDa Heat shock proteins (Hsp70s), facilitating the selection and transfer of protein substrate to Hsp70 and the stimulation of the protein folding ability of Hsp70. Members of the diverse Hsp70-Hsp40 protein complement of Plasmodium falciparum have been implicated in the cytoprotection of this malaria parasite, and are thought to facilitate the protein folding, assembly and translocation tasks required by the parasite to commandeer the infected human erythrocyte subsequent to invasion. In particular, the parasite has evolved an expanded and specialised 43- member suite of Hsp40 proteins, 19 of which bear an identifiable export motif for secretion into the infected erythrocyte cytoplasm where they potentially interact with human Hsp70. Although type I Hsp40 proteins are representative of typical regulators of Hsp70 activity, only two of these proteins are apparent in the parasite’s Hsp40 complement. These include a characteristic type I Hsp40 termed PfHsp40, and a larger, atypical type I Hsp40 termed Pfj1. Both Hsp40 proteins are predicted to be parasite-resident and are most likely to facilitate the co-chaperone regulation of the highly abundant and stress-inducible Hsp70 homolog, PfHsp70-I. In this work, the co-chaperone functionality of PfHsp40 and Pfj1 was elucidated using in vivo and in vitro assays. Purified recombinant PfHsp40 was shown to stimulate the ATPase activity of PfHsp70-I in in vitro single turnover and steady state ATPase assays, and co-operate with PfHsp70-I in in vitro aggregation suppression assays. In these in vitro assays, heterologous partnerships could be demonstrated between PfHsp70-I and the human Hsp40, Hsj1a, and human Hsp70 and PfHsp40, suggesting a common mode of Hsp70-Hsp40 interaction in the parasite and host organism. The functionality of the signature Hsp40 domain, the Jdomain, of Pfj1 was demonstrated by its ability to replace the equivalent domain of the A. tumefaciens Hsp40, Agt DnaJ, in interactions with the prokaryotic Hsp70, DnaK, in the thermosensitive dnaJ cbpA E. coli OD259 deletion strain. An H33Q mutation introduced into the invariant and crucial HPD tripeptide motif abrogated the functionality of the J-domain in the in vivo complementation system. These findings provide the first evidence for the conservation of the prototypical mode of J-domain based interaction of Hsp40 with Hsp70 in P. falciparum. Immunofluorescence staining revealed the localisation of PfHsp40 to the parasite cytoplasm, and Pfj1 to the parasite cytoplasm and nucleus in cultured intraerythrocytic stage P. falciparum parasites. PfHsp70-I was also shown to localise to the parasite cytoplasm and nucleus in these stages, consistent with the literature. Overall we propose that PfHsp40 and Pfj1 co-localise with and regulate the chaperone activity of PfHsp70-I in P. falciparum. This is the first study to identify and provide evidence for a functional Hsp70-Hsp40 partnership in P. falciparum, and provides a platform for future studies to elucidate the importance of these chaperone partnerships in the establishment and survival of the parasite in the intraerythrocytic-stages of development.
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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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Clitheroe, Crystal-Leigh. "In-silico analysis of Plasmodium falciparum Hop protein and its interactions with Hsp70 and Hsp90." Thesis, Rhodes University, 2013. http://hdl.handle.net/10962/d1003819.
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A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.
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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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Holland, Zoe. "Plasmodium falciparum protein kinase CK2." Thesis, University of Glasgow, 2008. http://theses.gla.ac.uk/606/.
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Malaria, caused by infection with intracellular protozoan parasites of the genus Plasmodium, is responsible for 300 to 600 million clinical cases annually (Snow et al., 2005), resulting in the deaths of up to three million people every year (Breman, 2001, Breman et al., 2004). There is a clear need for further research aimed at identifying novel drug targets (Ridley, 2002). Reversible phosphorylation of proteins is a major regulatory mechanism in most cellular processes, and protein kinases are considered promising drug targets, comprising as much as 30% of all protein targets under investigation (Cohen, 2002). The divergences between human and plasmodial protein kinases suggest that specific inhibition of the latter is an achievable goal (Doerig, 2004, Doerig and Meijer, 2007). This study investigates protein kinase CK2 of Plasmodium falciparum, seeking to establish by reverse genetics and biochemical approaches whether it represents a possible antimalarial drug target. Protein-kinase CK2, formerly known as Casein Kinase II, is a dual-specificity (Serine/Threonine and Tyrosine) protein kinase ubiquitously expressed in eukaryotes. It has over 300 cellular substrates catalogued to date (Meggio and Pinna, 2003). Consistent with its multiple substrates, the enzyme plays a crucial role in many cellular processes, and is essential to viability in yeast and slime mould (Padmanabha et al., 1990, Kikkawa et al., 1992). The human CK2 holoenzyme consists of two catalytic a or a’ subunits and two regulatory b subunits, and recent evidence indicates that the latter interact with several protein kinases in addition to CK2a (reviewed in (Bibby and Litchfield, 2005)), pointing to a likely role in the integration of numerous signalling pathways. A putative CK2a orthologue and two predicted CK2b subunits were identified in the P. falciparum genome (Ward et al., 2004, Anamika et al., 2005). Here we present the biochemical characterisation of the PfCK2a orthologue and both PfCK2b orthologues, and demonstrate by using a reverse genetics approach that each of the three subunits is essential for completion of the erythrocytic asexual cycle of the parasite, thereby validating the enzyme as a possible drug target. Recombinant PfCK2a possesses protein kinase activity, exhibits similar substrate and co-substrate preferences to those of CK2a subunits from other organisms, and interacts with both of the PfCK2b subunits in vitro. PfCK2a is amenable to inhibitor screening, and we report differential susceptibility between the human and P. falciparum CK2a enzymes to a small molecule inhibitor. Taken together, the data indicate that PfCK2a is an attractive, validated target for antimalarial chemotherapeutic intervention.
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Hillebrand, Arne Thomas. "Funktionelle Analyse kleiner, nichtkodierender RNAs in den Organellen von Plasmodium falciparum und Charakterisierung neuer RNA-Bindeproteine in Apicomplexa." Doctoral thesis, Humboldt-Universität zu Berlin, 2019. http://dx.doi.org/10.18452/20433.
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Die Infektionskrankheit Malaria wird von einzelligen Parasiten der Gattung Plasmodium verursacht und stellt vor allem im südlichen Afrika eine große Herausforderung dar. Die Zellen der Parasiten enthalten zwei endosymbiontische Organellen, den Apicoplast und das Mitochondrium. Beide Organellen besitzen ein reduziertes Genom. Die Struktur des mitochondrialen Genoms ist ungewöhnlich. Mit nur 6 kb gehört es zu den kleinsten beschriebenen Genomen und enthält neben drei proteinkodierende Gene auch 34 kleine rRNA-Gene. Um das Genom zu exprimieren wird eine Vielzahl von kernkodierten Faktoren benötigt. Die Regulation der Expression, die Prozessierung der polycistronischen Primärtranskripte und die Regulation des RNA-Metabolismus des Mitochondriums ist jedoch weitestgehend unbekannt. In dieser Arbeit konnten kurze RNAs in den Mitochondrien von P. falciparum mittels Hochdurchsatzsequenzierung identifiziert werden. Solche RNA-Akkumulationen an Transkriptenden werden in den Organellen höherer Pflanzen durch PPR-Proteine (Pentatricopeptide repeat) verursacht. Um zu untersuchen, ob in P. falciparum PPR-ähnliche, helikale Proteine vorhanden sind, wurde genomweit nach Proteinen mit repetitiven, helikalen Elementen gesucht. Dabei konnte eine vorher unbekannte Proteinfamilie identifiziert werden, die aufgrund ihrer 37 Aminosäure langen Motive Heptatricopeptide repeat Proteine (HPR) genannt wurde. In P. berghei konnte für 7 HPR-Proteine eine mitochondriale Lokalisation betätigt werden. Außerdem zeigten Deletionsversuche, dass die meisten HPR-Proteine in den Blutstadien essentiell sind. In vitro RNA-Bindestudien konnte für ein rekombinantes HPR-Protein eine unspezifische Interaktion mit mitochondrialen Transkripten nachgewiesen werden, während keine Bindung an DNA erfolgt. Eine breite Suche in verschiedenen phylogenetischen Gruppen zeigte, dass HPR-Proteine in verschiedensten eurkaryotischen Taxa vorhanden sind, mithin früh in der Evolution der eukaryotischen Zelle entstanden sind.Malaria is caused by a single celled parasite of the genus Plasmodium. Especially in Sub-Saharan Africa, -this disease is a huge challenge for the health system. The cells of the parasites contain two organelles of endosymbiotic origin, the apicoplast and the mitochondrion. Both organelles still contain a reduced genome. For the expression of the genome, the organelles depends on a large set of nuclear encoded proteins. The mitochondrial genome has a unique structure. With only 6 kb it is one of the smallest genomes discovered to date and it contains only three protein coding genes along with 34 small ribosomal genes. The regulation of expression, the processing of the polycistronic primary transcript and the regulation of the RNA metabolism in the mitochondria of Plasmodium remains largely unknown. Through high-throughput sequencing of cellular RNA, we discovered a population of small RNAs originating in the mitochondria of P. falciparum. Similar RNA accumulations can be detected in the organelles of higher plants and are caused by helical-hairpin repeat proteins like PPR proteins (pentatricopeptide repeat). To search for plant-like RNA binding proteins similar to PPR proteins we scanned the nuclear genome of P. falciparum for helical-hairpin repeat proteins. We found a novel protein family with repetitive helical elements of 37 amino acid length we termed heptatricopeptide repeat (HPR) proteins. In the rodent Malaria parasite P. berghei, the mitochondrial localization for 7 HPR-Proteins was verified. In knockout studies, we also showed that almost all HPR proteins are essential for blood stages of P. berghei. In RNA-binding assays, one recombinant HPR protein showed unspecific interaction with mitochondrial transcripts but not with DNA. By broadening the search, we discovered that HPR proteins are found in multiple eukaryotic taxa.
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Mpangase, Phelelani Thokozani. "Integrating protein annotations for the in silico prioritization of putative drug target proteins in malaria." Diss., University of Pretoria, 2012. http://hdl.handle.net/2263/24715.
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Current anti-malarial methods have been effective in reducing the number of malarial cases. However, these methods do not completely block the transmission of the parasite. Research has shown that repeated use of the current anti-malarial drugs, which include artemisinin-based drug combinations, might be toxic to humans. There have also been reports of an emergence of artemisinin-resistant parasites. Finding anti-malarial drugs through the drug discovery process takes a long time and failure results in a great financial loss. The failure of drug discovery projects can be partly attributed to the improper selection of drug targets. There is thus a need for an eff ective way of identifying and validating new potential malaria drug targets for entry into the drug discovery process. The availability of the genome sequences for the Plasmodium parasite, human host and the Anopheles mosquito vector has facilitated post-genomic studies on malaria. Proper utilizationof this data, in combination with computational biology and bioinformatics techniques, could aid in the in silico prioritization of drug targets. This study was aimed at extensively annotating the protein sequences from the Plasmodium parasites, H. sapiens and A. gambiae with data from di fferent online databases in order to create a resource for the prioritization of drug targets in malaria. Essentiality, assay feasibility, resistance, toxicity, structural information and druggability were the main target selection criteria which were used to collect data for protein annotations. The data was used to populate the Discovery resource (http://malport. bi.up.ac.za/) for the in silico prioritization of potential drug targets. A new version of the Discovery system, Discovery 2.0 (http://discovery.bi.up.ac.za/), has been developed using Java. The system contains new and automatically updated data as well as improved functionalities. The new data in Discovery 2.0 includes UniProt accessions, gene ontology annotations from the UniProt-GOA project, pathways from Reactome and Malaria Parasite Metabolic Pathways databases, protein-protein interactions data from. IntAct as well as druggability data from the DrugEBIlity resource hosted by ChEMBL. Users can access the data by searching with a protein identi er, UniProt accession, protein name or through the advanced search which lets users filter protein sequences based on different protein properties. The results are organized in a tabbed environment, with each tab displaying different protein annotation data. A sample investigation using a previously proposed malarial target, S-adenosyl-Lhomocysteine hydrolase, was carried out to demonstrate the diff erent categories of data available in Discovery 2.0 as well as to test if the available data is su fficient for assessment and prioritization of drug targets. The study showed that using the annotation data in Discovery 2.0, a protein can be assessed, in a species comparative manner, on the potential of being a drug target based on the selection criteria mentioned here. However, supporting data from literature is also needed to further validate the findings.Dissertation (MSc)--University of Pretoria, 2012.
Biochemistry
unrestricted
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Fenton, Brian Forbes Neil. "Studies on polymorphic proteins of Plasmodium falciparum." Thesis, University of Edinburgh, 1987. http://hdl.handle.net/1842/14835.
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Haußig, Joana. "Genetic characterization of Plasmodium berghei apicoplast proteins." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://dx.doi.org/10.18452/16808.
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Malaria wird durch den einzelligen Parasiten Plasmodium verursacht. Hierbei handelt es sich um einen obligat intrazellulären, eukaryotischen Erreger, der zum Phylum der Apicomplexa gehört. Apicomplexa zeichnen sich durch das einzigartige Vorhandensein eines ungewöhnlichen Plastids, genannt Apicoplast, aus. Die Exklusivität dieser Organelle und ihre metabolische Notwendigkeit für das Parasitenwachstum haben sie als attraktives pharmakologisches Ziel bestätigt. In dieser Arbeit wurden, unter Anwendung des Nagetier-Malariaerregers Plasmodium berghei, zwei verschiedene Aspekte von Apicoplast Proteinfunktionen untersucht. Zum Ersten wurde ein bislang unbeschriebenes Plasmodium Apicoplast Protein, Plasmodium-specific Apicoplast protein important for Liver Merozoite formation (PALM), charakterisiert. Drei voneinander unabhängige palm— Parasitenlinien, wurden durch zielgerichtete Gendeletion generiert. Die PALM Knockout-Mutanten entwickelten sich während eines Großteils des Lebenszyklus normal, jedoch war die Abgabe von Merozoiten in den Blutstrom und die Fähigkeit eine Blutstadien-Infektion zu etablieren signifikant beeinträchtigt. Experimentelle Immunisierung von Mäusen mit palm— Sporozoiten bewirkte einen starken und langanhaltenden Schutz gegen Reinfektion mit Malaria. Diese Ergebnisse lassen darauf schließen, dass Parasiten mit einem Arrest in den finalen Schritten der Bildung von Leberstadien-Merozoiten einen Vorteil gegenüber genetisch attenuierten Parasiten der ersten Generation haben, die in der frühen Leberstadienentwicklung arretiert sind. Zum Zweiten wurden die sechs Nucleus-kodierten Komponenten der [Fe-S] Cluster Biosynthese im Apicoplast systematisch durch experimentelle Genetik analysiert. Insgesamt zeigen meine Studien, dass bisher unbekannte Ziele im Plasmodium Apicoplast für Interventionsstrategien gegen Malaria geeignet sind.Malaria is caused by Plasmodium, an obligate intracellular eukaryotic pathogen that belongs to the phylum Apicomplexa. Apicomplexan parasites harbor an unusual plastid organelle, termed apicoplast. Because this unique organelle is indispensable for parasite growth it is a validated and attractive drug target. Using the rodent malaria parasite Plasmodium berghei, two different aspects of apicoplast protein functions were analyzed in this study. Firstly, a previously uncharacterized Plasmodium apicoplast protein, Plasmodium-specific Apicoplast protein important for Liver Merozoite formation (PALM), was investigated. Three independent palm— knockout parasite lines were generated by targeted gene deletion. While the resulting knockout mutants developed normally for most of the life cycle, merozoite release into the blood stream and the ability to establish an infection was severely impaired. Experimental immunization of mice with palm— sporozoites elicited unprecedented potent and long-lasting protection against malaria re-infection. The results indicate that a tailor-made arrest in the final steps of hepatic merozoite formation could be an improvement over first-generation early liver-stage genetically arrested parasites (GAPs). Secondly, the six nuclear-encoded components of the apicoplast [Fe-S] cluster biosynthesis pathway were systematically targeted by experimental genetics. Together, my studies show that the Plasmodium apicoplast harbors previously unrecognized targets for anti-malaria intervention strategies.
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Cockburn, Ingrid Louise. "Modulation of Plasmodium falciparum chaperones PfHsp70-1 and PfHsp70-x by small molecules." Thesis, Rhodes University, 2013. http://hdl.handle.net/10962/d1001747.
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The heat shock proteins of ~ 70 kDa (Hsp70s) are a conserved group of molecular chaperones important in maintaining the protein homeostasis in cells, carrying out functions including refolding of misfolded or unfolded proteins. Hsp70s function in conjunction with a number of other proteins including Hsp40 cochaperones. Central to the regulation Hsp70 activity is the Hsp70 ATPase cycle, involving ATP hydrolysis by Hsp70, and stimulation of this ATP hydrolysis by Hsp40. PfHsp70-1, the major cytosolic Hsp70 in the malaria parasite, Plasmodium falciparum, and PfHsp70-x, a novel malarial Hsp70 recently found to be exported to the host cell cytosol during the erythrocytic stages of the P. falciparum lifecycle, are both thought to play important roles in the malaria parasite’s survival and virulence, and thus represent novel antimalarial targets. Modulation of the function of these proteins by small molecules could thus lead to the development of antimalarials with novel targets and mechanisms. In the present study, malarial Hsp70s (PfHsp70-1 and PfHsp70-x), human Hsp70 (HSPA1A), malarial Hsp40 (PfHsp40) and human Hsp40 (Hsj1a) were recombinantly produced in Escherichia coli. In a characterisation of the chaperone activity of recombinant PfHsp70-x, the protein was found to have a basal ATPase activity (15.7 nmol ATP/min/mg protein) comparable to that previously described for PfHsp70-1, and an aggregation suppression activity significantly higher than that of PfHsp70-1. In vitro assays were used to screen five compounds of interest (lapachol, bromo-β-lapachona and malonganenones A, B and C) belonging to two compound classes (1,4 naphthoquinones and prenylated alkaloids) for modulatory effects on PfHsp70-1, PfHsp70-x and HsHsp70. A wide range of effects by compounds on the chaperone activities of Hsp70s was observed, including differential effects by compounds on different Hsp70s despite high conservation (≥ 70 % sequence identity) between the Hsp70s. The five compounds were shown to interact with all three Hsp70s in in vitro binding studies. Differential modulation by compounds was observed between the Hsj1a-stimulated ATPase activities of different Hsp70s, suggestive of not only a high degree of specificity of compounds to chaperone systems, but also distinct interactions between different Hsp70s and Hjs1a. The effects of compounds on the survival of P. falciparum parasites as well as mammalian cells was assessed. Bromo-β-lapachona was found to have broad effects across all systems, modulating the chaperone activities of all three Hsp70s, and showing significant toxicity toward both P. falciparum parasites and mammalian cells in culture. Malonganenone A was found to modulate only the malarial Hsp70s, not human Hsp70, showing significant toxicity toward malarial parasites (IC₅₀ ~ 0.8 μM), and comparatively low toxicity toward mammalian cells, representing therefore a novel starting point for a new class of antimalarials potentially targeting a new antimalarial drug target, Hsp70.
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Daniyan, Michael Oluwatoyin. "The plasmodium falciparum exported Hsp40 co-chaperone, PFA0660w." Thesis, Rhodes University, 2014. http://hdl.handle.net/10962/d1011780.
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Plasmodium falciparum is the pathogen that is responsible for the most virulent, severe and dangerous form of human malaria infection, accounting for nearly a million deaths every year. To survive and develop in the unusual environment of the red blood cells, the parasite causes structural remodelling of the host cell and biochemical changes through the export of virulence factors. Among the exportome are the molecular chaperones of the heat shock protein family, of which Hsp40s and Hsp70s are prominent. PF A0660w, a type II P. falciparum Hsp40, has been shown to be exported in complex with PfHsp70-x into the infected erythrocyte, suggesting possible functional interactions. However, the chaperone properties of PF A0660w and its interactions with proteins of parasite and human origin are yet to be investigated. Using a codon optimised coding region, PF A0660w was successfully expressed in E. coli M 15 [pREP4] cells. However, the expressed protein was largely deposited as insoluble pellet, and analysis of the pellets revealed a high percentage of PF A0660w, characteristic of inclusion body formation. PF A0660w was purified from inclusion bodies using additive enhanced solubilisation and refolding buffers followed by nickel affinity chromatography. SDS-PAGE and western analysis revealed that the purified protein was of high purity. Size exclusion chromatography showed that the protein existed as a monomer in solution and the secondary structure analysis using Fourier transformed infrared spectroscopy (FTIR) confirmed the success of the refolding approach. Its monomeric state suggests that PF A0660w may be functionally different from other Hsp40 that form dimers and that for PF A0660w, dimer formation may not be needed to maintain the stability of the protein in solution, but may occur in response to functional necessities during its interaction with partner Hsp70. PFA0660w was able to significantly stimulate the ATPase activity ofPfl-Isp70-x but not Pfl-Isp70-1 or human Hsp70 (HsHsp70), suggesting a specific functional interaction. Also, PF A0660w produced a dose dependent suppression of rhodanese aggregation and cooperated with Pfl-Isp70-1, PfHsp70-x and HsHsp70 to cause enhanced aggregation suppression. Its ability to independently suppress aggregation may help to maintain substrates in an unfolded conformation for eventual transfer to partner Hsp70s during refolding processes. Also, the in vivo characterisation using a PF A0660w peptide specific antibody confirmed that PF A0660w was exported into the cytosol of infected erythrocytes. Its lack of induction upon heat shock suggests that PF A0660w may not be involved in the response of the parasite to heat stress. Overall, this study has provided the first heterologous over-expression, purification and biochemical evidence for the possible functional role of PF A0660w, and has thereby provided the needed background for further exploration of this protein as a potential target for drug discovery.
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Engel, Jessica Alexandra. "Investigating Plasmodium falciparum Histone Deacetylase 1 Complex Proteins." Thesis, Griffith University, 2017. http://hdl.handle.net/10072/367801.
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Malaria causes substantial morbidity and mortality worldwide. Although there has been a considerable decline in global malaria incidence and mortality rates since 2000, it is estimated that more than 400,000 deaths occurred in 2015 as a result of this parasitic disease. The lack of a broadly effective licensed vaccine and the threat of malaria parasite resistance to current drugs means there is an urgent need for the development of new therapies with novel parasite targets. With a renewed call for global malaria eradication, novel therapeutic strategies are crucial to continue progress achieved over the last decade in combating malaria and to achieving a malaria-free world.
Targeting epigenetic mechanisms within Plasmodium parasites represents a promising therapeutic approach for malaria. Histone deacetylase (HDAC) enzymes, the focus of this thesis, act in conjunction with histone acetyltransferases (HATs) to reversibly acetylate histone and non-histone proteins. Some HDACs, in particular class I and II HDACs, are already validated drug targets for cancer therapy and are showing promise as antimalarial drug targets. However, besides their classical role in regulating gene expression, knowledge of the roles that HDACs play in the Plasmodium parasites, is limited. Like higher eukaryotic HDACs, P. falciparum HDACs (PfHDACs) are believed to localise in multi-protein complexes with accessory proteins and to regulate lysine acetylation of both histones and non-histone proteins.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
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DaSilva, Thiago Gaspar. "CHARACTERIZATION OF PROTEIN PRENYLTRANSFERASES AND PROTEIN PRENYLATION IN PLASMODIUM FALCIPARUM." Master's thesis, University of Central Florida, 2004. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4401.
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Malaria kills at least one million people each year, mostly children - a death every 30 seconds. Almost one half of the world population is at risk from malaria. Antimalarial drugs are the only means for the treatment of about 500 million annual global malaria cases. Because of prevalent drug-resistance it is extremely urgent to identify new drug targets. Many proteins involved in eukaryotic signal transduction and cell cycle progression undergo post-translational lipid modification by a prenyl group. Protein prenyltransferases, which catalyze the post-translational prenyl modification, have been established as a target for anticancer therapy. Research done in our laboratory has demonstrated recently that prenyl modification of proteins could be a novel target for the development of antimalarial drugs.The goal of this study is to understand the molecular mechanism of protein prenylation in Plasmodium. The key to use of prenyltransferase inhibitors for the pharmacological intervention is a thorough understanding of the in vivo prenylation pathways in the malaria parasite. Knowledge of the physiological functions of the cellular protein substrates of malarial prenyltransferases is an important first step in the elucidation of the mechanism of antimalarial action of inhibitors of protein prenylation. The research described in this thesis revealed the evidence for the existence of farnesylated and geranylgeranylated malaria parasite proteins. The study shows that the dynamics of protein prenylation changes with the intraerythrocytic development cycle of the parasite. We detected that prenylated proteins in the 50 kDa range were mostly farnesylated and that the proteins in the 22-25 kDa range were mostly geranylgeranylated. The prenylation of P. falciparum proteins is inhibited by prenyltransferase inhibitors. We have also demonstrated unique features of protein prenylation in P. falciparum compared to the human host such as farnesylation of proteins are sensitive to inhibition by geranylgeranyltransferase inhibitors.. In-silico search of the malarial genome sequence identified potential protein prenyltransferase substrates. One of these substrates is a SNARE protein Ykt6 homologue. The malarial Ykt6 was recombinantly expressed and subjected to an in-vitro prenylation assay. We showed that the recombinant Ykt6 was indeed a substrate for the malarial prenyltransferase.M.S.
Department of Molecular Biology and Microbiology
Health and Public Affairs
Molecular Biology and Microbiology
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Parish, Lindsay A. "Protein trafficking and 4.1R relocalization in Plasmodium falciparum-infected erythrocytes." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2009. https://www.mhsl.uab.edu/dt/2009p/parish.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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Rao, Aditya. "Tarfetpf: A Plasmodium faciparum protein localization predictor." Thesis, University of Skövde, School of Humanities and Informatics, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-24.
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Rao, Aditya. "TargetPf: A Plasmodium falciparum protein localization predictor." Thesis, University of Skövde, School of Humanities and Informatics, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-914.
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Background: In P. falciparum a similarity between the transit peptides of apicoplast and mitochondrial proteins in the context of net positive charge has previously been observed in few proteins. Existing P. falciparum protein localization prediction tools were leveraged in this study to study this similarity in larger sets of these proteins.
Results: The online public-domain malarial repository PlasmoDB was utilized as the source of apicoplast and mitochondrial protein sequences for the similarity study of the two types of transit peptides. It was found that
many of the 551 apicoplast-targeted proteins (NEAT proteins) of PlasmoDB may have been wrongly annotated to localize to the apicoplast, as some of these proteins lacked annotations for signal peptides, while others also had annotations for localization to the mitochondrion (NEMT proteins). Also around 50 NEAT proteins could contain signal anchors instead of signal peptides in their N-termini, something that could have an impact on the current theory that explains localization to the apicoplast [1].
The P. falciparum localization prediction tools were then used to study the similarity in net positive charge between the transit peptides of NEAT and NEMT proteins. It was found that NEAT protein prediction tools like PlasmoAP and PATS could be made to recognize NEMT proteins as NEAT proteins, while the NEMT predicting tool PlasMit could be made to recognize a significant number of NEAT proteins as NEMT. Based on these results a conjecture was proposed that a single technique may be sufficient to predict both apicoplast and mitochondrial transit peptides. An implementation in PERL called TargetPf was implemented to test this conjecture (using PlasmoAP rules), and it reported a total of 408 NEAT
proteins and 1504 NEMT proteins. This number of predicted NEMT proteins (1504) was significantly higher than the annotated 258 NEMT proteins of plasmoDB, but more in line with the 1200 predictions of the tool PlasMit.
Conclusions: Some possible ambiguities in the PlasmoDB annotations related to NEAT protein localization were identified in this study. It was also found that existing P. falciparum localization prediction tools can be made to detect transit peptides for which they have not been trained or built for.
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Günther, Svenja. "Lipoic acid protein ligases in Plasmodium spp. /." Connect to e-thesis. Move to record for print version, 2008. http://theses.gla.ac.uk/71/.
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Thesis (Ph.D.) - University of Glasgow, 2008.Ph.D. thesis submitted to the Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.
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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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Günther, Svenja. "Lipoic acid protein ligases in Plasmodium spp." Thesis, University of Glasgow, 2008. http://theses.gla.ac.uk/71/.
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Protozoan parasites of the genus Plasmodium are the causative agent of malaria. The four human pathogenic species infect more than 500 million people each year, causing the death of at least 1 million people. The most severe form of human malaria is caused by P. falciparum, which is responsible for 90% of the malaria deaths. A major problem in the treatment of this disease is resistance of the parasites against most of the existing chemotherapies. Therefore, there is an urgent need to identify, validate and assess potential new drug targets. The prerequisite of a potential drug target is that it should not be of significance for the human host or it should be sufficiently different from the human counterpart, so that parasite-specific inhibition is feasible. Lipoic acid metabolism in Plasmodium differs from that of mammals in some ways and therefore it might be a promising target for the development of new antimalarials. This study investigated the importance of lipoic acid ligation in P. falciparum using reverse genetic approaches, to assess whether this pathway has potential for drug design. In addition, a spectrophotometric assay system was developed that allowed the biochemical characterisation of lipoic acid ligases and can be adapted to high-throughput screening approaches of inhibitors for these enzymes. Lipoic acid, also known as 6,8-thioctic acid, is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADH) and the glycine cleavage system (GCV). The KADH include the pyruvate dehydrogenase (PDH), branched chain alpha-keto acid dehydrogenase (BCDH) and alpha-ketoglutarate dehydrogenase (KGDH), which are an integral part for any cell's metabolism. In Plasmodium spp. the lipoic acid dependent enzyme complexes are found in the apicoplast, a plastid related organelle, and in the mitochondrion and thus two organelle specific lipoylation pathways are present in these parasites. Biosynthesis of the cofactor occurs in the apicoplast. Octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) catalyses the attachment of octanoyl-acyl carrier protein (octanoyl-ACP) to the PDH and lipoic acid synthase (LipA) then catalyses the insertion of two sulfurs into the octanoyl-chain to form lipoamide. In the mitochondrion, scavenged lipoic acid is ligated to the enzyme complexes by the action of lipoic acid protein ligase A (LplA1), in an ATP-dependent reaction. However, a second lipoate protein ligase A (LplA2) was identified in the genome of P. falciparum, but its subcellular localisation could not be predicted using the available prediction programs. To further analyse its localisation, parasites were generated expressing full length LplA2 in frame with green fluorescent protein (GFP). In addition, immunofluorescence analyses on wild-type parasites using LplA2 specific antibodies were performed. These studies showed that LplA2 is dually targeted to the apicoplast as well as to the mitochondrion, raising the question about potential redundancy between the ligases present in the parasites. To further analyse this possibility, knock-out studies of lplA1 and lplA2 were performed in the human and rodent malaria parasites P. falciparum and P. berghei, respectively. Knock-out studies showed that LplA1 and LplA2 are non-redundant and strongly suggested that LplA1 is crucial for intraerythrocytic development, whereas LplA2 is essential for sexual development in the mosquito. According to these results it appears that (1) a key regulator of lipoic acid metabolism in Plasmodium spp. is stage specific expression of the relevant proteins and (2) both ligases are potential drug targets as knock-out of lplA1 appeared impossible in the blood stages and knock-out of lplA2 resulted in the interruption of parasite sexual development in the mosquito, and thus transmission of the parasites would be blocked if LplA2 was inhibited. To further analyse the biochemical properties of P. falciparum LplA1 and LplA2, a spectrophotometric assay system was developed, which is also suitable for the development of a high-throughput assay system. The spectrophotometric assay monitors the first part of the LplA reaction - the activation of lipoic acid by ATP. The released pyrophosphate is converted to phosphate which is detected by acidic ammonium molybdate. Using the Escherichia coli LplA protein as a positive control, kinetic parameters for the bacterial protein were determined that are in reasonable agreement with the published data. The results validate the assay and suggest that it might be suitable for inhibitor screening in the future.
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Green, Judith Louise. "Genes encoding rhoptry proteins of the malaria parasite plasmodium." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.300303.
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Hliscs, Marion. "Functional Characterization of Actin Sequestering Proteins in Plasmodium berghei." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16452.
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Plasmodien spp. sind obligat intrazellulär lebende Parasiten, welche einen evolutionär konservierten aktinabhängigen molekularen Motor für die Fortbewegung und den Wirtszellein- und -austritt nutzen. In dieser Arbeit werden die Aktinregulatoren Adenylyl- Zyklase- assoziierte Protein (C-CAP), Profilin sowie die Aktin depolymerizierenden Faktoren 1 und 2 (ADF1, ADF2) in Plasmodium berghei charakterisiert. Die Geninaktivierung von C-CAP besitzt keinen Einfluss auf die Entwicklung von pathogenen Blutstadien. C-cap(-) Ookineten bewegen sich jedoch deutlich langsamer, sind aber in der Lage den invertebraten Wirt zu infizieren. Defekte treten während der extrazellulären Replikationsphase im Mosquito auf und führen zu Abbruch des Lebenszykluses. Die erfolgreiche Komplementierung der Defekte mit dem orthologen Gen aus Cryptosporidium parvum CpC-CAP bestätigt die funktionale Redundanz zwischen beiden Proteinen. Profilin, als ein weiteres G-Aktin bindendes Protein, ist hingegen nicht in der Lage die Defekte des c-cap(-) Parasiten auszugleichen. Mittels transgener Parasiten welche ein C-CAPmCherry Fusionsprotein exprimieren, wird das C-CAP Protein im Zytoplasma lokalisiert. Erstmals wird mit dieser Arbeit ein G-Aktin bindendes Protein, C-CAP beschrieben, welches eine essentielle Funktion während der Oozystenreifung in Plasmodium berghei besitzt. Die Transkription der Aktinregulatoren Profilin, ADF1 und ADF2 wird in Sporozoiten drastisch herunterreguliert und Profilin kann als Protein nicht mehr nachgewiesen werden. Um die Funktion von C-CAP und Profilin zu überprüfen, wurden beide Proteine spezifisch in Sporozoiten überexprimiert. Diese Parasiten sind nicht in der Lage die Speicheldrüsen des Wirtes zu besiedeln, was zum Abbruch des Lebenszykluses führt. Anhand dieser Ergebnisse entwickele ich ein „minimalistisches“ Model zur Beschreibung der Aktinregulation in Sporozoiten in welchem das ADF1 als regulatorisches Protein im Mittelpunkt steht.Plasmodium spp. are obligate intracellular parasites, which employ an conserved actin-dependent molecular motor machinery that facilitates their motility, host cell invasion and egress. In this work I report implications of the actin-regulators adenylyl cyclase-associated protein (C-CAP), profilin and actin depolymerization factor 1 and 2 (ADF1, ADF2) in distinct and previously unanticipated cellular processes during the life cycle of in the rodent malarial parasite Plasmodium berghei. Fluorescent tagging of the endogenous C-CAP genetic locus with mCherry revealed cytosolic distribution of the protein. Gene deletion demonstrates that the G-actin binding protein C-CAP is entirely dispensable for the pathogenic blood stages. Ookinetes show reduced motility, but are competent infecting the mosquito host. Defects emerging in the extracellular replication phase, leading to attenuation of oocyst maturation. Successful trans-species complementation with the C. parvum C-CAP ortholog, rescues the c-cap(-) phenotype and proves functional redundancy. The actin regulator profilin fails to rescue the defects of c-cap(-) parasites, despite sharing its actin sequestering activity with C-CAP. Taken together, C-CAP is the first G-actin sequestering protein of Plasmodium species that is not required for motility but performs essential functions during oocyst maturation. Characterization of the actin regulators profilin, ADF1 and ADF2 revealed dramatic transcriptional down-regulation and the absence of the profilin protein in sporozoites. To test whether G-actin binding proteins interfere with sporozoite functions, I ectopically overexpressed of profilin and C-CAP stage-specifically in sporozoites. This conducted to abolishment of salivary gland invasion and lifecycle arrest. Based on these unexpected findings and the available literature data, I developed a “minimalistic model” for actin regulation in sporozoites that predicts ADF1 as the main actin-turnover regulating factor.
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Longhurst, Hilary Jane. "Studies on malarial histones." Thesis, Open University, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262662.
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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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Ramakrishnan, Chandra. "Analysis of CTRP, a Plasmodium ookinete surface protein." Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.585532.
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The Plasmodium surface protein CTRP mediates ookinete motility, adhesion to the mosquito midgut and the basal lamina and invasion. It is part of the TRAP protein family members of which play similar roles in other stages of Plasmodium and in all apicomplexan species examined to date. Uniquely, CTRP contains multiple copies ofvon Willebrand factor A-related (A) domains and thrombospondin type 1 repeat-like (TS) domains. CTRPko ookinetes are non-motile, cannot adhere to the midgut and do not develop into oocysts demonstrating that CTRP is essential. To understand the roles of A and TS domains, endogenous CTRP was replaced with mutants lacking either all the A domains or all the TS domains. The impact of domain deletions on oocyst number was studied. Ookinetes carrying mutant CTRP lacking TS domains produced similar numbers of oocysts to wildtype, whereas CTRP lacking A domains did not produce oocysts. Thus the A domains are essential for the formation of oocysts, whereas TS domains are non-essential. . 3sS-methionine labelled ookinetes were used for co-immunoprecipitation with CTRP to identify binding partners of CTRP. Three putative interaction partners of 110, 100 and 60 kDa molecular weight were isolated. Further work is needed to obtain these co- precipitates in sufficient amounts for unequivocal characterisation by mass spectrometry. To evaluate CTRP as a transmission-blocking vaccine target, antibodies were raised against the third TS domain of CTRP. They recognised CTRP in Western blots and immunofluorescence assays. In transmission-blocking assays however, they failed to reduce transmission. The TS domains of CTRP are clearly not an appropriate target for transmission-blocking vaccines, however further work is required to determine whether A domains can be targeted effectively. Future studies on CTRP and related proteins may elucidate the structural and essential functional requirements of proteins involved in invasion, and provide new ration ales for vaccine and possibly drug design.
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Walker, Alison Dalgity. "Protein variation in the malaria parasite Plasmodium falciparum." Thesis, University of Edinburgh, 1986. http://hdl.handle.net/1842/13171.
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Awah, Nancy. "Malarial anaemia : the potential involvement of Plasmodium falciparum rhoptry proteins." Licentiate thesis, Stockholms universitet, Wenner-Grens institut, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-8460.
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Malaria remains a challenging health problem in malaria endemic regions. Infection with malaria invariably leads to anaemia. The groups at risk of developing malarial anaemia include children below the age of five years and pregnant women, especially primigravidae. Several factors have been suggested to be responsible for its aetiology, including increased destruction of infected and normal red blood cells together with bone marrow suppression. However, until recently, the molecular mechanisms involved have remained elusive. The aim of the work presented herein was to investigate the mechanisms responsible for the destruction of normal red blood cells in anaemia, and more specifically to define the role of the ring surface protein (RSP/RAP) -2 and other members of the low molecular weight rhoptry associated protein (RAP) complex, RAP-1 and -3. In the first study we showed that antibodies to the RAP complex could mediate the destruction of RSP-2 tagged erythroid cells by phagocytosis or by complement activation and then lysis. In addition, antibodies to RAP-1 and RAP-2 could induce the death of RSP-2/RAP-2 tagged erythroblasts. We further investigated the frequency and functionality of naturally occurring RSP-2/RAP-2 antibodies in the sera of anaemic and non-anaemic Cameroonian children. We found that all sera investigated contained RSP-2/RAP-2 reactive antibodies by both immunoflorescence and flow cytometry. The anaemic group of children had significantly higher levels of antibodies of the IgG isotype than the non-anaemic individuals, while the levels of IgM were similar in both groups. With respect to IgG subclasses, low levels of IgG1 and -3 antibodies were detected. Higher levels of IgG3 were seen in the non-anaemic individuals as compared to anaemic subjects. With regards to antibody functionality, the non-anaemic individuals recognised a greater proportion of RSP-2/RAP-2 tagged erythrocytes and activated complement to a greater extent than the anaemic individuals. From our findings, we can conclude that antibodies to the RAP complex are potentially involved in erythroid cell destruction during malaria which may result in anaemia, and that high levels of such antibodies may be detrimental to the host.
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Ochola, Lynette Isabella. "The role of Rab5 proteins in endocytosis in Plasmodium falciparum." Thesis, University of Nottingham, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.428280.
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Clarke, Amy Marigot. "Functional, biochemical and structural analyses of two plasmodium membrane proteins." Thesis, University of St Andrews, 2013. http://hdl.handle.net/10023/3603.
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Protozoan parasites of the genus Plasmodium are the causative agent of malaria. The most severe form of human malaria is caused by P. falciparum, responsible for approximately three quarters of a million deaths each year. One major problem in the treatment of malaria is resistance to existing chemotherapies. Consequently, there is an urgent need to identify and validate novel drug targets. A possible recently identified drug target is the PfNitA protein of P. falciparum which contains orthologues in other Plasmodium species but is absent from humans. The gene is annotated as a putative formate-nitrite transporter and orthologues are found in a range of prokaryotes as well as the lower eukaryotes algae and fungi. To determine the biological function of the protein, pfnita was expressed in Escherichia coli strains lacking the endogenous formate and nitrite transporters. In order to analyse the essentiality of the gene a reverse genetics approach was taken and the data discussed. Results indicate that the PfNitA protein is located in the plasma membrane and digestive vacuole of intraerythrocytic parasites suggesting a role in the uptake or excretion of metabolites. A second complexity with regard to treatment is the lack of a vaccine. A problem in crating a vaccine is antigenic variation. The PIR family of proteins contain a so-called hypervariable domain that has led to the suggestion that the family may play a role in antigenic variation. The objective of the work carried out in this thesis was to investigate the topology and structure of the PcCir2 protein of Plasmodium chabaudi, using E. coli as the expression host. The topology of Cir2 has been examined by means of reporter fusions and overexpression/purification studies undertaken towards crystallisation. As the PcCir2 amino acid sequence does not show significant homology to other proteins, structural data may provide insights into potential functional or binding domains.
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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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Korbmacher, François. "Towards functional assignment of Plasmodium membrane transport proteins: an experimental genetics study on four diverse proteins." Doctoral thesis, Humboldt-Universität zu Berlin, 2021. http://dx.doi.org/10.18452/23029.
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Etliche Membran Transport Proteine (MTP) sind essentiell in den Plasmodium Blutstadien, und geraten zunehmend in den Fokus der Wirkstoffentwicklung. Die physiologischen Rollen der Transporter sind jedoch oft ungeklärt. In dieser Arbeit wurden mittels experimenteller Genetik funktionelle Charakteristika der MTPs untersucht. Am Maus Parasiten Plasmodium berghei und der Plasmodium falciparum Blutstadien-Kultur wurden vier MTPs ausgewählt: ein konservierter Folat Transporter (FT2), sowie eine P. falciparum-spezifisches P-Typ ATPase und zwei essentielle MTPs (CRT und ATP4). Diese Auswahl verkörpert ein breites Spektrum an MTP Kandidaten und reflektieren zudem das Potenzial und die Grenzen funktioneller Analysen von Plasmodium MTPs mittels reverser Genetik. Für den Folat Transporter 2 (FT2) wurde eine Kombination von transgenen Strategien auf P. berghei angewandt. Durch ein endogenes tag von FT2 wurde die Lokalisierung im Apicoplast, sowie dessen Expression über fast den kompletten Zyklus hinweg gezeigt. Nach der Deletion von FT2, wiesen die Parasiten einen Defekt während der Sporulation auf. Demzufolge bilden sich nur nicht infektiöse Sporozoiten, was letztendlich zur Unterbrechung des Lebenszyklus der Parasiten führt. Eine Aminophospholipid P-Typ ATPase, wurde mittels CRISPR/Cas9 in P. falciparum genetisch deletiert und die Mutante analysiert. Im Gegensatz zu den meisten vitalen P-Typ ATPasen erweist sich das Gen in den asexuellen Blutstadien als entbehrlich. Des Weiteren bilden die MTPs ATP4 und CRT einen einflussreichen Faktor bei Malaria-Therapien. Eine umfassende Analyse von räumlichen und zeitlichen Expressionsmustern von transgenen Parasiten mit mCherry-getaggten Proteinen zeigt ein Expression der beiden MTPs über die Blutstadien hinaus, was auf zusätzliche Funktionen in den jeweiligen Stadien verweist. Diese Studie trägt, basierend auf Lokalisation, Expression und funktioneller Deletion, zur funktionellen Entschlüsselung der vier untersuchten MTPs bei.Many membrane transport proteins (MTP) are essential for Plasmodium infection and gain importance as candidate drug targets in malaria therapy, whereas the physiological functions often remain enigmatic. In this thesis, we applied experimental genetics to determine key characteristics of four Plasmodium MTPs. We employed the murine malaria model parasite Plasmodium berghei and in vitro blood cultures of Plasmodium falciparum. We selected one conserved MTP called FT2, which was previously shown to transport folate, a P-type ATPase that is specific for P. falciparum as well as two essential MTPs, CRT and ATP4. These targets exemplify the range of druggable candidates and illustrate the potential and limitations of reverse genetics to decipher their physiological roles. A combination of transgenic and knockout strategies was applied to the P. berghei folate transporter 2 (FT2). We show that endogenously tagged FT2 localises to the apicoplast membranes, and is broadly expressed throughout the parasite’s life cycle. Analysis of FT2-deficient parasites revealed a severe sporulation defect in the vector; the vast majority of ft2– oocysts form large intracellular vesicles which displace the cytoplasm. Very few sporozoites are generated and these are non-infectious to the mammalian host, resulting in a complete arrest of Plasmodium transmission. A candidate aminophospholipid P-type ATPase, was assessed by a CRISPR/Cas9-mediated gene disruption. Compared to many vital P-type ATPases this gene is dispensable for asexual blood replication. Two MTPs, ATP4 and CRT are prime targets for antimalarial therapies. A comprehensive spatio-temporal expression analysis of transgenic parasites expressing mCherry-tagged proteins revealed expression beyond blood infection, indicative of functions in additional parasite stages. The findings of this study contribute towards a better understanding of the roles of the four MTPs based on localisation, expression and functional deletion.
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Joseph, Diego F., Jose A. Nakamoto, Ruiz Oscar Andree Garcia, Katherin Peñaranda, Ana Elena Sanchez-Castro, Pablo Soriano Castillo, and Pohl Milón. "DNA aptamers for the recognition of HMGB1 from Plasmodium falciparum." Public Library of Science, 2019. http://hdl.handle.net/10757/655484.
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Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-mediated detection. However, the expression of commonly used biomarkers varies geographically and the sensibility of immunodetection can be affected by batch-to-batch differences or limited thermal stability. In this study we aimed to overcome these limitations by identifying a potential biomarker and by developing molecular sensors based on aptamer technology. Using gene expression databases, ribosome profiling analysis, and structural modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium falciparum is highly expressed, structurally stable, and present along all blood-stages of P. falciparum infection. To develop biosensors, we used in vitro evolution techniques to produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations suggested three predominant aptamer motifs. Representatives of the aptamer families were tested for binding parameters to the HMG-box domain using microscale thermophoresis and rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude between nano- and micromolar ranges while the aptamer-HMG-box interaction occurred in a few seconds. The specificity of aptamer binding to the HMG-box of P. falciparum compared to its human homolog depended on pH conditions. Altogether, our study proposes HMGB1 as a candidate biomarker and a set of sensing aptamers that can be further developed into rapid diagnostic tests for P. falciparum detection.Grand Challenges Canada
Revisión por pares
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Gurung, Pratima. "Deciphering the role of G-quadruplexes and their interacting proteins in Plasmodium falciparum." Thesis, Montpellier, 2020. http://www.theses.fr/2020MONTT010.
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Le paludisme continue d'être l'une des principales causes de morbidité et de mortalité dans les pays en développement. Le développement de la résistance aux médicaments antipaludiques disponibles et la rareté des vaccins efficaces ont rendu nécessaire la recherche urgente de nouvelles cibles antipaludiques. La forme grave du paludisme est causée par Plasmodium falciparum, qui présente un cycle de vie complexe impliquant diverses formes morphologiquement et fonctionnellement distinctes chez deux hôtes différents - l'humain et le moustique anophèle. Afin de se développer dans l'environnement de deux hôtes distincts, ces parasites utilisent différents mécanismes pour réguler leur expression génétique étroitement coordonnée. Ce projet de thèse se concentre sur l'exploration de cette régulation, qui est médiée par des structures secondaires d'ADN riches en guanine, principalement des G-quadruplexes. Ces structures se trouvent dans une large gamme d'organismes et sont impliquées dans la régulation des gènes tels que la transcription, la réplication de l'ADN et la maintenance télomérique. Récemment, on a découvert qu'elles sont également impliquées dans le processus de virulence pour échapper à la réponse immunitaire de l'hôte dans de nombreux pathogènes tels que les bactéries, les protozoaires et les virus. Chez Plasmodium, les motifs formant le quadruplex-G sont enrichis dans les régions télomériques et sous-télomériques, où les gènes de virulence sont présents. L'existence de G4 dans le génome de ces parasites riches en AT indique leur rôle dans le mécanisme de régulation des gènes et de variation antigénique. Cependant, il y a un manque de preuves expérimentales pour appuyer cette hypothèse. Le but de ce projet est de fournir la première étude complète de l'interactome G4 afin de comprendre le rôle des mécanismes de régulation médiés par le G4 dans la biologie du Plasmodium. En utilisant une combinaison d'approches non biaisées (levure hybride et test d'ADN pull-down), nous avons identifié ~152 protéines interagissant potentiellement avec les motifs G4 Plasmodium falciparum. Il a été démontré que les orthologues de certaines de ces protéines interagissent avec les G4, ce qui renforce nos résultats. De plus, pour comprendre comment ces candidats contribuent aux processus de régulation médiés par les G4, nous avons sélectionné et caractérisé deux protéines (GBP2 et DNAJ) pour effectuer des études fonctionnelles après validation de leurs propriétés de liaison. Il est démontré que ces protéines jouent un rôle important dans la biologie du Plasmodium. Dans cette étude, nous avons découvert que la GBP2 est une protéine dispensable qui interagit avec la G4 sélectionnée. Même si la délétion du gène n'est pas mortelle pour les parasites, elle affecte toujours l'expression des gènes var. Alors que l'ADNJ putatif est une protéine essentielle et que sa délétion entraîne l'arrêt des parasites aux derniers stades du cycle érythrocytaire. Collectivement, cette étude fait la lumière sur ce mécanisme de régulation basé sur la structure de l'ADN, encore peu étudié, et fournit la première étude systématique de l'interactome G4. Etant donné leur rôle essentiel dans le développement des parasites, une caractérisation plus poussée des candidats obtenus permettra probablement de générer de nouvelles cibles pour les antipaludiques qui contribueront à long terme à l'éradication de la maladieMalaria continues to be one of the major causes of morbidity and mortality in the developing countries. The development of the resistance against the available antimalarial drugs and scarcity of effective vaccines have demanded the urgent need of finding new antimalarial targets. The severe form of malaria is caused by Plasmodium falciparum, which manifests a complex life cycle involving various morphologically and functionally distinct forms within two different hosts - human and Anopheles mosquitoes. In order to thrive in two distinctive host’s environment, these parasites employ different mechanisms to regulate their tightly coordinated gene expression. This thesis project is focused on exploring the regulation, which is mediated by guanine-rich DNA secondary structures, predominantly G-quadruplexes. These structures are found in wide range of organisms and are involved in gene regulation such as transcription, DNA replication and telomeric maintenance. Recently, they are also found to be involved in the process of virulence to evade the host’s immune response in numerous pathogens such as bacteria, protozoa and viruses. In Plasmodium, the G-quadruplex forming motifs are found to be enriched in the telomeric and sub-telomeric regions, where the virulence genes are present. The G4 existence in these AT biased genome points towards their role in the mechanism of gene regulation and antigenic variation. However, there is a lack of experimental evidence to support this hypothesis. The aim of this project is to provide the first comprehensive survey of the G4-interactome in order to understand the role of G4-mediated regulatory mechanisms in Plasmodium biology. Using a combination of unbiased approaches (Yeast one-hybrid and DNA pull-down assay), we have identified ~152 putative G4 interacting proteins in Plasmodium falciparum. The orthologs of some of these proteins were shown to interact with G4s, thus strengthening our results. Furthermore, to understand how these candidates contribute to G4 mediated regulatory processes, we have selected and characterized two proteins (GBP2 and DNAJ) to perform functional studies following validation of their binding properties. These proteins are shown to play an important role in Plasmodium biology. In this study, we have found that the GBP2 is a dispensable protein that interacts with the selected G4. Even though the deletion of the gene is not lethal to the parasites, it still affects the expression of var genes. Whereas putative DNAJ is an essential protein and its deletion results into the arrest of the parasites at the late stages of erythrocytic cycle. Collectively, this study sheds light on this understudied DNA structure based regulatory mechanism and provide the first systematic survey of the G4 interactome. Given their essential role in parasite development further characterization of obtained candidates will likely generate new targets for antimalarial drugs that will in the long term contribute to the eradication of the disease
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LaCrue, Alexis Nichole. "Characterization of thesporozoite and eythrocytic stages (SES) protein." Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/4648.
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Thesis (Ph. D.)--University of Missouri-Columbia, 2007.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2007" Includes bibliographical references.
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Hinds, Louise. "Identification and characterisation of a moved plasmodium falciparum protein." Thesis, University College London (University of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.497504.
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George, Miriam Thankam. "Immunological Characterization Of Duffy Binding Protein Of Plasmodium vivax." Scholar Commons, 2015. http://scholarcommons.usf.edu/etd/5689.
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Plasmodium vivax Duffy binding protein (DBP) is an essential ligand for reticulocyte invasion making it a premier asexual blood stage vaccine candidate. However, strain-specific immunity due to DBPII allelic variation may complicate vaccine efficacy, suggesting that an effective DBPII vaccine needs to target immune responses to conserved epitopes that are potential targets of strain-transcending neutralizing immunity. Anti DBPII monoclonal antibodies, which were previously characterized by COS7 cell binding assay as inhibitory and non-inhibitory to DBPII-erythrocyte binding, were mapped to DBPII gene fragment libraries using phage display. Inhibitory mAb 3C9 binds to a conserved conformation-dependent epitope in subdomain 3 while non-inhibitory mAb 3D10 binds to a linear epitope in subdomain 1 of DBPII.
More definitive epitope mapping of mAb 3D10 was achieved using a random peptide library displayed on phage. Since DBP region II is mostly made up of alpha-helices, we used a randomized helical scaffold library, the Affibody library, displayed on phage, to determine epitope of conformation-dependent antibodies.
The immunogenicity of the identified epitopes was evaluated in mice and the immune sera evaluated for binding to DBPII by ELISA and inhibition of DBPII-erythrocyte binding by the COS7 cell assay. Immune serum from the mAb3C9 epitope blocked DBPII-erythrocyte, suggesting this epitope could be a good subunit vaccine target.
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Das, Neves Guevara Diaz S. A. "Protein interactions in the Plasmodium falciparum merozoite motor complex." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1418839/.
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The actomyosin motor that drives both motility and host cell invasion in the human malaria parasite Plasmodium falciparum is thought to be coupled through aldolase to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins including apical membrane antigen (AMA1) and members of the erythrocyte binding antigen (EBL) and reticulocyte binding homologue (Rh) protein families have been implicated in host cell binding and moving junction formation. We show that P. falciparum aldolase binds to P. falciparum actin. Using a kinetic assay, we show that TRAP family members also bind to aldolase. A direct binding method confirmed that merozoite TRAP (MTRAP) and TRAP bind aldolase and indicated that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the CTD abolished binding to aldolase. MTRAP CTD was phosphorylated by calcium dependent protein kinase 1 (CDPK1) and protein kinase A, kinases that are known to phosphorylate other motor proteins, and this modification increased the affinity of binding ten-fold. Similarly AMA1, EBA and Rh protein families also bound to aldolase, with the affinity also increased by CTD phosphorylation. Therefore other proteins involved in host cell recognition and invasion, in addition to members of the TRAP family, may be connected to the motor through aldolase. If this is the case their affinity for aldolase may also be important in addition to their host ligand specificity in determining the use of, and efficiency of alternate invasion pathways. These interactions also contributed to stimulate actin polymerization and enhance aldolase enzymatic activity, increasing the enzyme affinity for it’s substrate, and potentially to the compartmentalization of the glycolytic pathway and consequent ATP availability for motor complex function.
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Meredith, Sandra Allison. "The characterization of adaptor protein homologues in Plasmodium falciparum." Doctoral thesis, University of Cape Town, 2009. http://hdl.handle.net/11427/3291.
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Includes abstract.Includes bibliographical references (leaves 148-171).
Plasmodium falciparum is becoming increasingly more resistant to regular antimalarial drugs, making it necessary to identify novel drug candidates and drug targets. Components of the endocytic and secretory pathway in asexual stage parasites are attractive targets because they play a fundamental role in the normal processes of parasite metabolism. Adaptor protein complexes are components of protein coats that associate with transport vesicles of the endocytic and secretory pathways in mammalian cells. Homologues of several adaptor protein subunits are encoded by the parasite genome. The presence of these genes suggests that the parasite experiences clathrin-mediated transport processes. This study reports the cloning and characterization of selected malarial homologues of these adaptor proteins, namely three medium (μ) chain adaptin homologues and two sigma (σ) chains.
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Khalid, Muhammad. "Partial Characterization Of Plasmodium Falciparum Protein Kinase ABCk2 (PfABCk2)." Scholar Commons, 2018. https://scholarcommons.usf.edu/etd/7315.
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Malaria is a major threat to the public health worldwide as it is affecting populations in tropical and subtropical areas globally. Among those populations are around 40% of pregnant women and children who are susceptible to this disease. Plasmodium falciparum is the most lethal agent that causes malaria in human. Currently, there is drug resistance against antimalarial drugs in parasite against treatment of malaria infections, it is essential to search for new drug targets in order to find cure and alleviate suffering of human population.
There are approximately 100 protein kinases in P. falciparum that are involved in phosphorylation of asexual blood stage. Hence, the phosphorylation plays an important part in the development of different stages of malarial parasites. Due to their significance in the parasite life cycle, one of the protein kinase of P. falciparum belongs to the ABC-1 family of proteins. PfABCK2 can be a therapeutic target due to its higher expression during the late schizont stage of blood stage form.
The bioinformatic analysis and preliminary results of PfABCK2 showed the heterologous expression of this protein. Hence, the gene of PfABCk2 was ligated into pET21a+ vector with His-tag at C-terminus and transformed into BL-21 (DE3) competent cells that were verified through Miniprep and DNA sequencing. Furthermore, this gene construct is utilized to heterologous express this protein with IPTG and afterwards purified the recombinant protein kinase using nickel affinity chromatography as shown on 10% SDS-PAGE with the expected 36 kDa protein band. Therefore, the aim of this study is to partially characterize PfABCK2 protein kinase utilizing molecular cloning, heterologous express and protein kinase activity assay.
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Higham, Christopher W. "A study of lactate dehydrogenase from Plasmodium falciparum." Thesis, University of Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299529.
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Bernabeu, Aznar Maria. "Functional analysis of variant proteins in Plasmodium vivax: Implications in pathology." Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/121241.
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P. vivax is the most widespread malaria, it accounts for 25-40% of the global disease burden and it is estimated that causes from 132 to 391 million clinical cases each year. It is amply accepted that P. vivax do not cytoadhere to the inner capillaries having an obligate passage through the spleen. Our group advanced a hypothesis postulating that this parasite induces a reorganization of the spleen and the formation of a barrier of fibroblastic origin where infected reticulocytes specifically cytoadhere through VIR proteins. The VIR multigene subtelomeric family encodes for 346 genes and is divided into 12 different subfamilies. Despite they were originally located at the surface of infected reticulocytes; motif analysis suggested that they could present different subcellular localizations and exert different functions. In the absence of a long term P. vivax in vitro culture, we generated P. falciparum transgenic lines expressing VIR proteins from subfamily A (vir17), C (vir14) and D (vir10). Immunofluorescence assays revealed that they presented different subcellular localizations when expressed in P. falciparum. While VIR14 and VIR10 were exported to the membrane of the infected erythrocyte, VIR17 remained inside the parasite and was located surrounding the membrane in nascent merozoites. Later, a new clustering approach redefined the VIR repertoire and confirmed that members of subfamily A could no longer be considered VIR proteins, as well as members of subfamily D and H. Cytoadhesion assays revealed that VIR14, expressed in the surface of P. falciparum infected erythrocytes , mediated cytoadherence to CHO cells expressing endothelial receptors CD36, ICAM-1, VCAM and E- Selectine. However, only significant cytoadhesion levels to ICAM-1 were maintained under flow conditions. In addition, the transgenic line expressing VIR14 presented high adhesion to spleen fibroblasts. Similar adhesion levels were reported in some isolates of P. vivax infected reticulocytes. Finally, adhesion-inhibition experiments, using specific polyclonal antibodies against conserved motifs of VIR proteins, revealed that these proteins mediated adhesion to spleen fibroblasts whereas ICAM-1 was not the main receptor involved in this adhesion. Collectively, the data presented here suggest that VIR proteins mediate cytoadhesion in P. vivax and could have and important role in avoiding parasites spleen clearance.Plasmodium vivax és el paràsit causant de la malària humana amb distribució geogràfica més àmplia i s’estima que cada any produeix de 132 a 391 milions de casos clínics. Sempre s’ha cregut que P. vivax no citoadhereix als capil•lars interns dels òrgans i que per aquest motiu té un pas obligat per la melsa, però encara es desconeix com el paràsit és capaç d’evitar l’eliminació per part d’aquest òrgan. El nostre grup va postular que el paràsit indueix la reorganització de la melsa, mitjançant la formació d’una barrera d’origen fibrocític a la que el paràsit s’adhereix mitjançant les proteïnes VIR. La família multigènica VIR va ser descoberta pel nostre grup i presenta 346 gens que es divideixen en 12 subfamílies diferents. Els darrers anàlisis bioinformàtics van demostrar que podrien tenir localitzacions sub-cel•lulars diferents i exercir diferents funcions. Degut a la impossibilitat de cultivar aquest paràsit, en aquesta tesi s’han generat línies transgèniques de P. falciparum expressant gens vir de la subfamília A (vir17), C (vir14) i D (vir10). Els assajos d’immunofluorescència han revelat que aquestes proteïnes, quan s’expressen en P. falciparum presenten localitzacions sub-cel•lulars diferents. No obstant, l’aplicació posterior d’un nou algoritme va determinar que els membres de les subfamílies A i D ja no poden ser considerades VIR, juntament amb la subfamília H. En paral•lel, es van realitzar assajos de citoadhesió que van revelar que el VIR14, que s’expressa a la superfície de l’eritròcit, presenta adhesió a cèl•lules CHO que expressaven diferents receptors endotelials humans. No obstant, quan els assajos van ser realitzats en condicions de flux, només es va mantenir l’adhesió a ICAM-1. A més a més, assajos posteriors van demostrar que la línia transgènica que expressa el VIR14 presenta una gran adhesió a fibroblasts de melsa. Per extrapolar aquests resultats, vam realitzar assajos utilitzant soques de camp de P. vivax que van mostrar nivells d’adhesió variables a fibroblasts de melsa i van suggerir que les proteïnes VIR poden estar implicades en aquesta adhesió. En resum, les dades presentades en aquesta tesi suggereixen que les proteïnes VIR són almenys en part, les responsables de la citoadhesió del paràsit evitant així l’eliminació per part de la melsa.
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Dhanasarnsombut, Kelwalin. "Unstructured proteins of the malaria parasite Plasmodium falciparum as vaccine candidates." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8034.
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Malaria vaccine research has been battling with persistent challenges, including polymorphisms of vaccine antigens, difficulties with production processes, and limited immune protection against the disease. Intrinsically unstructured proteins (IUPs) are a fairly newly classified group of proteins that have no stable 3D structure and are generally heat-resistant. They usually contain low complexity regions and repetitive sequences, both of which are distinct characteristics of the malaria proteome. Surprisingly, some of the vaccine candidates that have been extensively studied were later reported to have unstructured regions, some of which serve as targets of protective immunity. In keeping with their interesting immunological profiles and their unique properties, which are exceptionally beneficial for vaccine production, malarial IUP antigens may be good vaccine candidates. This PhD project has the following aims:- 1) to develop a synthetic unstructured protein antigen based on the Block 2 region of MSP-1, named the MSP-1 hybrid 2) to characterize a novel vaccine antigen derived from the MSP-3.3 protein, namely an IUP region of PF10_0347 gene product, for its potential as a vaccine candidate 3) to develop a second-generation vaccine by combining the MSP-1 hybrid, with two allelic variants of MSP-2, to overcome antigenic polymorphism and strain-specific immune responses 4) to validate protocols for IUP identification from proteins extracted from the malaria parasite. This study showed that 1) MSP-1 hybrid production was scalable, yielding high protein yields with comparable immunological properties to small-scale production. MSP-1 hybrid was shown to be compatible with different adjuvants, and elicited specific antibodies covering the whole range of Block 2 allelic diversities. 2) A novel antigen, MSP-3.3C, an IUP based on the 3’ region of the PF10_0347 gene, was cloned, expressed and purified. Anti-MSP3.3C antibodies showed very strong parasite growth inhibitory effects in vitro. 3) The MSP-multihybrid antigen was expressed using simple techniques, but only at low levels. It contains epitopes from all three parasite antigen components, and is recognized by specific naturally acquired antibodies. 4) an unconventional 2D gel technique was tested as a method of malaria parasite IUP identification. Plans for further validation of this technique were discussed.
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Love, Timothy. "Prenylated Proteins in Plasmodium falciparum and their Role in FTI Response." Honors in the Major Thesis, University of Central Florida, 2004. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/715.
Full textAbstract:
This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
Burnett School of Biomedical Sciences
Molecular Biology and Microbiology
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Stafford, William Herbert Lee. "Analysis of the merozoite surface protein-1 complex and protein secretion in plasmodium falciparum." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338464.
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