Characterization of the Hsp40 partner proteins of Plasmodium falciparum Hsp70
- Authors: Njunge, James Mwangi
- Date: 2014
- Subjects: Plasmodium falciparum , Heat shock proteins , Malaria -- Chemotherapy , Protein-protein interactions , Erythrocytes -- Biotechnology , Molecular chaperones , Host-parasite relationships , Mitochondria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4117 , http://hdl.handle.net/10962/d1013186
- Description: 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.
- Full Text:
- Date Issued: 2014
- Authors: Njunge, James Mwangi
- Date: 2014
- Subjects: Plasmodium falciparum , Heat shock proteins , Malaria -- Chemotherapy , Protein-protein interactions , Erythrocytes -- Biotechnology , Molecular chaperones , Host-parasite relationships , Mitochondria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4117 , http://hdl.handle.net/10962/d1013186
- Description: 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.
- Full Text:
- Date Issued: 2014
Enabling cumulative knowledge-building through teaching: a legitimation code theory analysis of pedagogic practice in law and political science
- Authors: Clarence, Sherran
- Date: 2014
- Subjects: Law -- Study and teaching (Higher) -- South Africa Political science -- Study and teaching (Higher) -- South Africa Education, Higher -- South Africa College teaching -- South Africa Knowledge, Theory of Social realism
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:1966 , http://hdl.handle.net/10962/d1011763
- Description: Much current research and practice in teaching and learning in higher education tends to overfocus on social aspects of education; on how rather than what students are learning. Much of this research and practice is influenced by constructivism, which has a relativist stance on knowledge, generally arguing, contra positivism, that knowledge is constructed in socio-historical contexts and largely inseparable from those who construct it and from issues of power. This leads to a confusion of knowledge with knowing, and knowledge is thus obscured as an object of study because it is only seen or understood as knowing or as a subject of learning and teaching. This ‘knowledge-blindness’ (Maton 2013a: 4) is problematic in higher education because knowledge and knowing are two separate parts of educational fields, and while they need to be brought together to provide a whole account of these fields, they also need to be analysed and understood separately to avoid blurring necessary boundaries and to avoid confusing knowledge itself with how it can be known. Being able to see and analyse knowledge as an object with its own properties and powers is crucial for both epistemological access and social inclusion and justice, because knowledge and knowledge practices are at the heart of academic disciplines in universities. Social realism offers an alternative to the dilemma brought about by constructivism’s tendency towards knowledge-blindness. Social realism argues that it is possible to see and analyse both actors within social fields of practice as well as knowledge as something that is produced by these actors but also about more than just these actors and their practices; thus knowledge can be understood as emergent from these practices and fields but not reducible to them (Maton & Moore 2010). Social realism, drawing from Roy Bhaskar’s critical realist philosophy (1975, 2008), is intent on looking at the real structures and mechanisms that lie beneath appearances and practices in order to understand the ways in which these practices are shaped, and change over time. Legitimation Code Theory is a realist conceptual framework that has, as its central aim, the uncovering and analysis of organising principles that shape and change intellectual and education fields of production and reproduction of knowledge. In other words, the conceptual tools Legitimation Code Theory offers can enable an analysis of both knowledge and knowers within relational social fields of practice by enabling the analysis of the ways in which these fields, such as academic disciplines, are organised and how knowledge and knowing are understood in educational practice. This study draws on social realism more broadly and Legitimation Code Theory specifically to develop a relatively novel conceptual and explanatory framework within which to analyse and answer its central question regarding how to enable cumulative knowledge building through pedagogic practice. Using qualitative data from two academic disciplines, Law and Political Science, which was analysed using a set of conceptual and analytical tools drawn from Legitimation Code Theory, this study shows that the more nuanced and layered accounts of pedagogy that have been generated are able to provide valuable insights into what lecturers are doing as they teach in terms of helping students to acquire, use and produce disciplinary and ‘powerful’ knowledge (Young 2008b). Further, the study demonstrates that the organising principles underlying academic disciplines have a profound effect on how the role of the knower and the place or purpose of knowledge is understood in pedagogy and this affects how the pedagogy is designed and enacted. This study has argued that if we can research pedagogy rigorously using tools that allow us to see the real mechanisms and principles influencing and shaping it, and if we can reclaim the role of disciplinary knowledge as a central part of the pedagogic relationship between lecturer and students, then we can begin to see how teaching both enables and constrains cumulative learning. Further, we can change pedagogy to better enable cumulative learning and greater epistemological access to disciplinary knowledge and related practices for greater numbers of students. The study concludes by suggesting that the conceptual tools offered by Legitimation Code Theory can provide academic lecturers with a set of tools that can begin to enable them to 'see' and understand their own teaching more clearly, as well as the possible gaps between what they are teaching and what their students are learning. This study argues that a social realist approach to the study of pedagogy such as the one used here can begin not only to enable changes in pedagogy aimed at filling these gaps but also begin to provide a more rigorous theoretical and practical approach to analysing, understanding and enacting pedagogic practice. This, in turn, can lead to more socially just and inclusive student learning and epistemic and social access to the powerful knowledge and ways of knowing in their disciplines.
- Full Text:
- Date Issued: 2014
- Authors: Clarence, Sherran
- Date: 2014
- Subjects: Law -- Study and teaching (Higher) -- South Africa Political science -- Study and teaching (Higher) -- South Africa Education, Higher -- South Africa College teaching -- South Africa Knowledge, Theory of Social realism
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:1966 , http://hdl.handle.net/10962/d1011763
- Description: Much current research and practice in teaching and learning in higher education tends to overfocus on social aspects of education; on how rather than what students are learning. Much of this research and practice is influenced by constructivism, which has a relativist stance on knowledge, generally arguing, contra positivism, that knowledge is constructed in socio-historical contexts and largely inseparable from those who construct it and from issues of power. This leads to a confusion of knowledge with knowing, and knowledge is thus obscured as an object of study because it is only seen or understood as knowing or as a subject of learning and teaching. This ‘knowledge-blindness’ (Maton 2013a: 4) is problematic in higher education because knowledge and knowing are two separate parts of educational fields, and while they need to be brought together to provide a whole account of these fields, they also need to be analysed and understood separately to avoid blurring necessary boundaries and to avoid confusing knowledge itself with how it can be known. Being able to see and analyse knowledge as an object with its own properties and powers is crucial for both epistemological access and social inclusion and justice, because knowledge and knowledge practices are at the heart of academic disciplines in universities. Social realism offers an alternative to the dilemma brought about by constructivism’s tendency towards knowledge-blindness. Social realism argues that it is possible to see and analyse both actors within social fields of practice as well as knowledge as something that is produced by these actors but also about more than just these actors and their practices; thus knowledge can be understood as emergent from these practices and fields but not reducible to them (Maton & Moore 2010). Social realism, drawing from Roy Bhaskar’s critical realist philosophy (1975, 2008), is intent on looking at the real structures and mechanisms that lie beneath appearances and practices in order to understand the ways in which these practices are shaped, and change over time. Legitimation Code Theory is a realist conceptual framework that has, as its central aim, the uncovering and analysis of organising principles that shape and change intellectual and education fields of production and reproduction of knowledge. In other words, the conceptual tools Legitimation Code Theory offers can enable an analysis of both knowledge and knowers within relational social fields of practice by enabling the analysis of the ways in which these fields, such as academic disciplines, are organised and how knowledge and knowing are understood in educational practice. This study draws on social realism more broadly and Legitimation Code Theory specifically to develop a relatively novel conceptual and explanatory framework within which to analyse and answer its central question regarding how to enable cumulative knowledge building through pedagogic practice. Using qualitative data from two academic disciplines, Law and Political Science, which was analysed using a set of conceptual and analytical tools drawn from Legitimation Code Theory, this study shows that the more nuanced and layered accounts of pedagogy that have been generated are able to provide valuable insights into what lecturers are doing as they teach in terms of helping students to acquire, use and produce disciplinary and ‘powerful’ knowledge (Young 2008b). Further, the study demonstrates that the organising principles underlying academic disciplines have a profound effect on how the role of the knower and the place or purpose of knowledge is understood in pedagogy and this affects how the pedagogy is designed and enacted. This study has argued that if we can research pedagogy rigorously using tools that allow us to see the real mechanisms and principles influencing and shaping it, and if we can reclaim the role of disciplinary knowledge as a central part of the pedagogic relationship between lecturer and students, then we can begin to see how teaching both enables and constrains cumulative learning. Further, we can change pedagogy to better enable cumulative learning and greater epistemological access to disciplinary knowledge and related practices for greater numbers of students. The study concludes by suggesting that the conceptual tools offered by Legitimation Code Theory can provide academic lecturers with a set of tools that can begin to enable them to 'see' and understand their own teaching more clearly, as well as the possible gaps between what they are teaching and what their students are learning. This study argues that a social realist approach to the study of pedagogy such as the one used here can begin not only to enable changes in pedagogy aimed at filling these gaps but also begin to provide a more rigorous theoretical and practical approach to analysing, understanding and enacting pedagogic practice. This, in turn, can lead to more socially just and inclusive student learning and epistemic and social access to the powerful knowledge and ways of knowing in their disciplines.
- Full Text:
- Date Issued: 2014
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