Made available in DSpace on 2011-03-23T21:19:19Z (GMT). No. of bitstreams: 0 / Item created via OAI harvest from source: http://www.bdtd.ufpa.br/tde_oai/oai2.php on 2011-03-23T21:19:19Z (GMT). Item's OAI Record identifier: oai:bdtd.ufpa.br:92 / Malaria is still a serious public health problem, which continues to affect groups of the population living in the Amazon region. The main affected groups live in settlement areas and in artesanal gold mining camps (garimpos). This work aimed to analyze the relationship between the spread of the disease and the migratory process that leads people without any immunity against malaria to the areas of settlement and garimpos. This work also approached issues such as occupational activities, housing, and imported malaria cases from the international frontier with the Guyana. This study focused in the state of Amapa, specifically in the municiples of Ferreira Gomes, Porto Grande, Pedra Branca do Amapari, Serra do Navio, and Oiapoque where, from 1990 to 2003, the highest annual incidences of Plasmodium falciparum have been recorded for the urban sector, settlements, and garimpos. Both the exploratory/descriptive and ecological analysis systems were combined to describe the geographic areas. The data obtained by the Malaria Information System (SISMAL) and Epidemiological Watch System (SIVEP) supported the quantitative analysis and the qualitative approach of the malaria epidemics in the study area. Thematic maps were generated from geographic information system (GIS) data of the population under study and used the ArquiGiz system and the digital cartographic database of the Secretary for the Environment (SEMA). These maps allowed the spatial description, analysis, and discussion of the observed phenomena. The results lead to the conclusion that the occupational activity and housing have a direct relationship with the incidence of malaria. During the period under study, despite the policies used to control malaria, the studied areas remained as high-risk areas, and infection by P. falciparum increased, mainly in the geographic area of Oiapoque, an international frontier, where the imported cases presented the same tendency. / Como a malária ainda se mantém como um grave problema de saúde pública e continua afetando grupos da população que vive na região amazônica, principalmente os que moram em áreas de assentamentos e de exploração de garimpos é que decidiu-se desenvolver estudos voltados para a expansão da endemia e sua vinculação com o processo migratório. Outras questões, como a atividade ocupacional, moradia, e a situação da malária importada pela fronteira internacional, também foram consideradas. O universo alvo do estudo está no estado do Amapá, mais especificamente nos municípios de Ferreira Gomes, Porto Grande, Pedra Branca do Amapari, Serra do Navio e Oiapoque com enfoque no contexto urbano de cada município e nas áreas de assentamentos e garimpos onde, no período de 1990 2003, ocorreu a maior incidência parasitária anual e o maior índice de lâminas positivas, para a infecção pelo Plasmodium falciparum. O estudo exploratório - descritivo vinculado ao estudo ecológico foram utilizados para descrever as áreas geográficas e os dados obtidos pelo Sistema de Informações de Malária (SISMAL) e Sistema de Vigilância Epidemiológica (SIVEP), deram suporte para as análises quantitativas e abordagem qualitativa da situação da malária nas áreas de estudo. Os mapas temáticos foram gerados a partir do georreferenciamento da população estudada utilizando-se a ferramenta ArquiGiz e a base cartográfica digital da Secretaria de Meio Ambiente SEMA , que possibilitaram através de uma visão espacial a discussão, analise e descrição dos fenômenos observados. Mediante os resultados obtidos concluiu-se que a atividade ocupacional e moradia têm relação direta com a incidência da malária, pois no período analisado mesmo com as políticas que foram utilizadas para o controle da endemia as áreas estudadas se mantiveram como de alto risco e a infecção pelo P. falciparum aumentou, principalmente no Oiapoque, fronteira internacional, onde os casos importados apresentaram a mesma tendência.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-05-26T16:17:01Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoAtividadeAntiplasmodica.pdf: 1151729 bytes, checksum: bbc1a54b6ba8f91eed9599167986532d (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-09-09T12:08:46Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoAtividadeAntiplasmodica.pdf: 1151729 bytes, checksum: bbc1a54b6ba8f91eed9599167986532d (MD5) / Made available in DSpace on 2014-09-09T12:08:47Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoAtividadeAntiplasmodica.pdf: 1151729 bytes, checksum: bbc1a54b6ba8f91eed9599167986532d (MD5) Previous issue date: 2010 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Na busca de novos antimaláricos, duas espécies típicas da região Amazônica e uma fração rica em limonóides foram objeto deste estudo: Carapa guianensis Aubl. (Meliaceae), conhecida popularmente como andiroba, utilizada tradicionalmente como inseticida e no combate da malária. A espécie Piper aduncum L. (Piperaceae), conhecida popularmente como pimenta-de-macaco, usada para tratar doenças inflamatórias e a fração rica em limonóides fracionada do óleo de andiroba. Tanto os óleos brutos como a fração foram submetidos a ensaios in vitro, segundo metodologia descrita por Rieckman e colaboradores (1980) modificada por Carvalho (1990) com os clones do Plasmodium falciparum W₂ e Dd₂. Estes ensaios demonstraram que os óleos apresentaram atividade antiplasmódica, sendo que na concentração de 0,82ng/mL e 8,2μg/mL do óleo de andiroba a inibição do clone W₂ foi de 100% e do Dd₂ de 71% após 72h de exposição, respectivamente. Para a fração na concentração de 3,1μg/mL o clone W₂ foi de 100% e do Dd₂ a de 82% após 72h de exposição. O óleo de pimenta-de-macaco teve na concentração de 1,30ng/mL para o clone W₂ a inibição de 100% e para o Dd₂ a de 77%, após 72h de exposição, para a concentração de 10,3μg/mL. Os resultados com o óleo de pimenta-de-macaco, na concentração de 1,30ng/mL a inibição foi de 100% para clone W₂ e para o clone Dd₂, na concentração de 10,3μg/mL, a inibição foi de 77% após 72h de exposição. / In search of new antimalarial drugs, two typical species of the Amazon region and a fraction rich limonoids were the object of this study: Carapa guianensis Aubl. (Meliaceae), known popularly as andiroba traditionally used as an insecticide and fighting malaria, the species Piper aduncum L. (Piperaceae), known popularly as the pimento-de-macaco, used to treat inflammatory diseases and the fraction rich limonoids obtained from Carapa guianensis. Crude oil and fraction were tested in vitro using methods described by Rieckman and col. (1980) modified by Carvalho (1990) with Plasmodium falciparum clones W₂ and Dd₂. These studies showed that the oils had antiplasmodial activity, with a concentration of 0.82ng/mL and 8.2mg/mL andiroba oil showed an inhibition he W₂ clone was 100% and Dd₂ to 71% (IC₅₀ 9.4 μg/ml) after 72h of exposure respectively. For the fraction at a concentration of 3.1mg/mL, clone W₂, was 100% and Dd₂ to 82% (IC₅₀ 0.4 μg/ml), after 72h of exposure. The pimento-de-macaco oil overalls had a concentration of 1.30ng/mL for the W₂ clone inhibition of 100% and the Dd₂ to 77% after 72h of exposure to a concentration of 10.3mg/mL. The results with the chili oil overalls at a concentration of 1.30ng/ml the inhibition was 100% in clone W₂ and Dd₂ clone at a concentration of 10.3mg/mL, inhibition was 77% after 72h of exposure.

Le paludisme à P. falciparum constitue le problème de santé publique principal sur l'archipel des Comores. Le présent travail propose une synthèse des informations relatives à son épidémiologie, associée à une application des méthodes récentes d'épidémiologie moléculaire et de génétique des populations plasmodiales à la situation comorienne. Celle-ci apparaît contrastée. Ainsi, Grande Comore, Mohéli et Anjouan sont des îles marquées par une morbidité palustre importante, et leur population plasmodiale est globalement homogène. La morbidité est en revanche beaucoup plus faible à Mayotte, où les cas sont pour moitié d'origine importée depuis les îles voisines, et pour moitié autochtones et concentrés dans le foyer résiduel et génétiquement isolé de Bandraboua selon une distribution microépidémique. Les niveaux de chimiorésistance, globalement élevés, sont également contrastés, du fait de pressions médicamenteuses différentes. En complément des stratégies actuelles de lutte antipaludique, ces informations conduisent donc à recommander : (1) la nécessité d'une surveillance de la chimiorésistance séparée pour chaque île, (2) le principe d'une élimination du paludisme sur les 4 îles à la fois et non pas sur une seule, et (3) un renforcement de la prévention des épidémies sur la commune de Bandraboua. Mais l'arrivée des ACT, la large distribution des moustiquaires imprégnées et le récent traitement de masse sur Mohéli devraient modifier à court terme cette situation épidémiologique. Ils devraient également relancer la pertinence d'une surveillance de la chimiorésistance excentrée à Marseille, où la représentativité génétique des parasites importés est pour l'instant limitée.

Immunity to asexual blood-stage of Plasmodium falciparum malaria is believed to be associated with protective antibodies of certain immunoglobulin classes and subclasses. This thesis addressed the importance of antibodies in relation to malaria infection and their effective interactions with Fc gamma receptor IIa (FcyRIIa) polymorphisms. Our data indicate that the frequency of FcyRIIa-R/R131 genotype was statistically significantly higher in Sudanese patients with severe malaria, while the FcyRIIa-H/H131 genotype showed a significant association with mild malaria. The levels of IgG1 and IgG3 subclass antibodies were statistically higher in the mild malaria patients. The Fulani ethnic group in West Africa has been shown to be relatively resistant to malaria. We investigated the possible impact of FcyRIIa polymorphisms in the Fulani and non-Fulani in Mali and Sudan, and analysed their malaria-reactive IgG subclass profiles. The FcyRIIa-H/H131 genotype and H131-allele were found to be prevalent in the Fulani while R131-allele was prevalent in non-Fulani. The Fulani had higher serum levels of IgG1-3, with higher proportion of IgG2 than the non-Fulani. Most clinico-epidemiology studies have been in areas with holo- and hyper-malaria endemicity. We have analysed antibody responses to a panel of six blood-stage antigens in relation to clinical malaria outcome in mesoendemic Sudan. Our results revealed a linear association with anti-AMA-1 IgG1 antibodies and reduced risk of severe malaria while a non-linear relationship with IgG3 antibodies was observed for MSP-2, MSP-3 and GLURP. In the combined final model, the highest levels of IgG1 subclass antibodies to AMA-1, GLURP-R0, and the highest levels of IgG3 subclass antibodies reactive to 3D7 MSP-2 were independently associated with a reduced risk of clinical malaria. Taken together, these data suggest a possible association between FcyRIIa-R/H131 and anti-malarial antibody responses in the clinical outcome of malaria.

Malaria is caused by a protozoan parasite belonging to the genus Plasmodia. Plasmodium falciparum is responsible for the fatal form of human malaria. Spread of drug resistant parasites warrants for sound biological understanding of the parasite at both cellular and biochemical level. Heat shock proteins are highly conserved group of proteins required for correct folding, transport, and degradation of substrate proteins in vivo. Hsp60 is found in eubacteria, mitochondria, and chloroplasts, where in cooperation with Hsp10, it participates in protein folding. Keeping in mind the central importance of chaperones in biological processes, our lab has been interested in examining roles of heat shock proteins in malarial parasite during its asexual growth in human erythrocytes. During its life cycle, the parasite continually shuttles between a cold-blooded insect vector with the body temperature of 27°C and a warm-blooded human host with the body temperature of 37°C and parasite experiences episodes of heat shock periodically. Therefore malaria parasite serves as good model to study heat shock protein functions. Like all biological systems, the malaria parasite expresses several chaperones including proteins of the Hsp40, Hsp60, Hsp70, Hsp90 and Hsp100 families. Towards this we have systematically characterized different families of stress proteins Hsp40, Hsp60, Hsp70, Hsp90 as well as Hsp100. In addition to cloning their genes we have studied their expression, localization, abundance, complexes and their biological roles. Earlier studies from our lab showed PfHsp90 is essential for parasite growth and survival in human erythrocytes. Our present study attempts to study heat shock protein 60 of the malarial parasite (PfHsp60). In this connection we have been successful to clone and express PfHsp60 gene from Plasmodium falciparum in E. coli and to raise antibodies specific to PfHsp60. We have examined its expression and import in the mitochondrion of malarial parasite during its asexual growth in human erythrocytes. Analysis of the total parasite lysates resolved by two-dimensional gel electrophoresis followed by western blotting using specific antibodies showed PfHsp60 exhibits an isoelectric point corresponding to its signal uncleaved precursor (pI - 6.2). Mass spectrometric analysis of the spot corresponding to precursor PfHsp60 confirmed the presence of signal peptide region. Co-immunoprecipitation analysis of total parasite lysates with antibodies specific to PfHsp60 showed precursor PfHsp60 to be associated with PfHsp70 and PfHsp90. Co-immunoprecipitation from the mitochondrial and cytoplasmic fraction confirmed the position of mature PfHsp60. Indirect immunofluorescence analysis also showed presence of a pool of PfHsp60 in the cytoplasm of the parasite, in addition to its expected localization in the mitochondrion. Treatment of parasite infected erythrocytes with an inhibitor of Hsp90 disrupted its association with cytoplasmic chaperones and targeted precursor Pfhsp60 for intracellular degradation. On the other hand treatment with the mitochondrial import inhibitor further inhibited the import of precursor PfHsp60 into the mitochondrion and stabilized its interaction with cytosolic chaperones. Previous reports have shown that there are four fold accumulations of PfHsp60 transcripts in heat shocked parasites. However, the expression of PfHsp60 was not induced upon heat shock in the blood stages of P.falciparum. Biochemical data indicate that the mitochondrion is not the source of ATP in the parasite. Furthermore the genome does not seem to encode the critical subunits of Fo-F1 ATP synthase. Yet, the active mitochondrial electron transport chain serves for regeneration of ubiquinone required for pyrimidine biosynthesis. The active electron transport chain is critical for parasite survival. Recent study with the lab-grown 3D7 strain of malaria parasite concluded that mitochondria are not required for energy conversion. Transcriptome analysis of the parasite derived directly from blood samples of infected patients showed that genes encoding the proteins of mitochondrial biogenesis, oxidative phosphorylation, respiration and highlighted the mean expression level for PfHsp60 is dramatically up regulated in parasites. Gene up regulation doesn’t always translate to increase in protein function or metabolic up regulation. When we analyzed the total parasite lysates of field isolates resolved by two-dimensional gel electrophoresis also showed presence of the precursor form of Pfhsp60 in the cytoplasm of the parasite. Overall, our observations indicated accumulation of precursor PfHsp60 in the parasite cytoplasm suggesting an inefficient mitochondrial protein import in the malarial parasite. The defect in mitochondrial protein import is possibly reflective of the compromised energy state of the parasite mitochondrion. This fits with the model that has been reported in mutant strains of yeast, Saccharomyces cerevisiae lacking functional F o-F1-ATPase. These strains were found to grow very poorly under anaerobic conditions and are known to accumulate Hsp60 protein in the cytoplasm mainly its precursor form. Under optimal growth conditions most eukaryotes maintain close co-ordination between gene expression, translation and translocation efficiently. As a result, mitochondrial precursor proteins are usually not found to accumulate in the cytoplasm. To our knowledge this the first report suggesting an inefficient co-ordination in the synthesis and translocation of precursor PfHsp60 and possibly other proteins during asexual growth of malarial parasite in human erythrocytes under optimal growth conditions. Finally, expression of the PfHsp60 gene in E.coli resulted in its association with bacterial GroEL subunits co-fractionating with a size of 920 kDa, corresponding to the tetra decameric form. The observation indicated possible existence of a hybrid chaperonin complex consisting of subunits from ectopically expressed PfHsp60 and endogenous GroEL.

Malaria is a life-threatening vector borne infectious disease caused by protozoan parasites of the genus Plasmodium. More than 100 species of Plasmodium can infect numerous animal species such as reptiles, birds and various mammals. However, human malaria is caused by four Plasmodium species -Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae, and occasionally by the simian malaria parasite, Plasmodium knowlesi. Of these, P. falciparum and P. vivax are the major causative agents and P. falciparum is the most virulent. About 300-500 million malaria infections occur every year leading to over 1-2 million deaths, of which 75% occur in African children of less than 5 years infected with P. falciparum. In spite of major global efforts to eliminate this disease over the past few decades, it continues to persist as a major affliction of human-kind imposing serious health and economic burden, especially to the poor countries. In India, the present scenario is about 2 million malaria positive cases every year, with almost 50% being caused by P. falciparum. Although remarkable attempts have been made over the years to develop vaccines against sexual and asexual stages of malaria parasite, an effective vaccine is still not in sight and remains as a distant goal. Hence, highly potent, less toxic and affordable antimalarial drugs remain as a first line therapy for malaria. Unfortunately, these parasites have been evolving against every known antimalarial drug and many of these drugs have lost their potency due to rapid emergence and spread of drug resistant strains. With development of resistance against frontline antimalarials such as chloroquine and antifolates, artemisinin and its derivatives seem to be the only effective antimalarials. However, recent reports on the possible emergence of artemisinin resistant strains, have led to the implementation of artemisinin-based combination therapies as a strategy to prevent drug resistance. Also, this continuous emergence of drug resistance has necessitated the development of new antimalarial drugs to combat this disease. While, Anopheles mosquitoes transmit parasites that infect humans, monkeys and rodents, Culex and Aedes mosquitoes predominate in the natural transmission to birds, and vectors of reptilian parasites are largely unknown. Of the approximately 400 species of Anopheles throughout the world, about 60 are malaria vectors under natural conditions, and 30 of which are of major importance. Ironically, the strategies implemented for controlling Anopheles, have also been hampered by insecticide resistance and other practical difficulties that exist in the scope of their applicability. In the past few years several milestones have been achieved in parasite genome, transcriptome and proteome studies, which could be exploited for the development of new drugs and drug targets. One such promising target includes the metabolic pathways of the malaria parasite which differ significantly from its human host. This thesis entitled “Unique Features of the Heme-Biosynthetic Pathway in Human Malaria Parasite, Plasmodium falciparum” unravels the unique biochemical features of heme-biosynthetic enzymes of P. falciparum, which have the potential for being drug targets. This pathway was first identified in this laboratory over 15 years ago. In the present study, five of the 7 enzymes of this pathway have been cloned, expressed, properties studied and sites of localization identified. With the knowledge on the first two enzymes coming from earlier studies, it is now possible to depict the unique hybrid pathway for heme biosynthesis in P. falciparum with full experimental validation.

Malaria is a life threatening parasite disease caused and transmitted by infected female anopheles mosquito. However, the parasite, Plasmodium falciparum, has become resistant to most anti malarial drugs, such as chloroquine, which contributes to fever and anaemia because of its ability to digest the haemoglobin in the red blood cells. The aims of this project were to establish whether “Bac to Bac” Baculoviral Expression System is suitable for expression of pfmdr 1 gene and for purification of the pgh 1 protein. The pfmdr 1 gene encodes an ABC transporter protein, pgh 1, fixed in the cell membrane of the Plasmodium falciparuum gut, which assist in elimination of drug compounds. Furthermore, “Bac to Bac” Baculoviral Expression System uses vectors with histidine tags to clone the pfmdr 1 gene and subsequently transform these into DH10Bac cells to produce the recombinant bacmid DNA. Since pfmdr 1 gene is an AT-rich sequence, PCR was optimized, by lowering the annealing and extension temperature to 47Co and 66Co respectively. The results show that “Bac to Bac” Baculoviral Expression System can be used to express the pfmdr 1 gene, though further experiments has to be performed.

This thesis describes studies directed towards understanding structure-function relationships of triosephosphate isomerase (TIM), from a protozoan parasite Plasmodium falciparum and a thermophilic archaea Methanocaldococcus jannaschii. Triosephosphate isomerase, a ubiquitous glycolytic enzyme, has been the subject of biochemical, enzymatic and structural studies for the last five decades. Studies on TIM have been central to the development of mechanistic enzymology. The present study investigates the role of specific residues in the structure and function of Plasmodium falciparum triosephosphate isomerase (PfTIM). The structure and stability of a tetrameric triosephosphate isomerase from Methanocaldococcus jannaschii (MjTIM) is also presented. Chapter 1 provides a general introduction to the glycolytic enzyme triosephosphate isomerase, conservation of TIM sequences, its fold and three dimensional organization. The isomerisation reaction interconverting dihydroxyacetone phosphate and glyceraldehyde 3phosphate catalyzed by triosephosphate isomerase is an example of a highly stereospecific proton transfer process (Hall & Knowles, 1975; Rieder & Rose, 1959). This chapter briefly reviews mechanistic features and discusses the role of active site residues and the functional flexible loop 6. Triosephosphate isomerase adopts the widely occurring ( β/ α)8 barrel fold and mostly occurs as a dimer (Banner et al., 1975). Protein engineering studies, related to folding, stability and design of monomeric TIM are also addressed. A brief introduction to thermophilic TIMs and higher oligomeric TIMs is given. The role of this enzyme in disease states like hemolytic anemia and neuromuscular dysfunction is surveyed. The production of methylglyoxal, a toxic metabolite, as a byproduct of the TIM reaction is also considered. Many proteins utilize segmental motions to catalyze a specific reaction. The omega loop (loop 6) of triosephosphate isomerase is important for preventing the ene-diol intermediate from forming the cytotoxic byproduct, methylglyoxal. The active site loop-6 of triosephosphate isomerase moves about 7Ǻ on ligand binding. It exhibits a hinged lid motion alternating between two well defined, “open” and “closed”, conformations (Joseph et al., 1990). Though the movement of loop 6 is not ligand gated, in crystals the ligand bound forms invariably reveal a closed loop conformation. Plasmodium falciparum TIM is an exception which predominantly exhibits “open” loop conformations, even in the ligand bound state (Parthasarathy et al., 2002). Phe 96 is a key residue that is involved in contacts between the flexible loop-6 and the protein body in PfTIM. Notably, in all TIM sequences determined thus far, with the exception of plasmodial sequences, this residue is Ser 96. In Chapter 2 the mutants F96S, F96H and F96W are reported. The crystal structures of the mutant enzymes with or without bound ligand are described. In all the ligand free cases, loop-6 adopts an “open” conformation. Kinetic parameters for all the mutants establish that residue 96 does not play an essential role in modulating the loop conformation but may be important for ligand binding. Structural analysis of the mutants along with WT enzyme reveals the presence of a water network which can modulate ligand binding. Subunit interfaces of oligomeric proteins provide an opportunity to understand protein- protein interactions. Chapter 3 describes biochemical and biophysical studies on two separate dimer-interface destabilizing mutants C13E and W11F/W168F/Y74W of PfTIM. The intention was to generate a stable monomer by disrupting the interaction hubs. C13 is a part of a large hydrophobic patch (Maithal et al., 2002a) at the dimer interface. Introduction of a negative charge at position 13 destabilizes the interface and reduces activity. Y74 is a part of an aromatic cluster of the interface (Maithal et al., 2002b). The Y74W triple mutant was designed to disrupt the aromatic cluster by introducing additional atoms. Tryptophan is also a fluorophore, allowing studies of the dimer disruption by fluorescence, after mutating the two inherent tryptophan residues, W11 and W168 to phenylalanine. The mutants showed reduced activity and were more sensitive than the wild type enzyme to chemical denaturants as well as thermal denaturation. Evidenced for monomer formation is presented. These studies together with previous work reveal that the interface is important for both activity and stability. In order to develop a model for understanding the relationship between protein stabilization and oligomeric status, characterization of the TIM from Methanocaldococcus jannaschii (MjTIM) has been undertaken. Chapter 4 describes the purification and characterization of MjTIM. The MjTIM gene was cloned and expressed in pTrc99A and protein was isolated from AA200 E. coli cells. Hyperexpressed protein was purified to homogeneity and relevant kinetic parameters have been determined. The tetrameric nature of MjTIM is established by gel filtration studies. Circular dichroism (CD) studies establish the stability of the overall fold, even at temperatures as high as 95ºC. A surprising loss of enzyme activity upon prolonged incubation at high temperature was observed. ESI-MS studies establish that oxidation of thiol groups of the protein may be responsible for the thermal inactivation. Chapter 5 describes the molecular structure of MjTIM, determined in collaboration with Prof. MRN Murthy’s group at the Indian Institute of Science (Gayathri et al., 2007). The crystal structure of the recombinant triosephosphate isomerase (TIM) from the archaeabacteria Methanocaldococcus jannaschii has been determined at a resolution of 2.3 Å. MjTIM is tetrameric, as suggested by solution studies and from the crystal structure, as in the case of two other structurally characterised archaeal TIMs. The archaeabacterial TIMs are shorter compared to the dimeric TIMs, with the insertions in the dimeric TIMs occurring in the vicinity of the putative tetramer interface, resulting in a hindrance to tetramerization in the dimeric TIMs. The charge distribution on the surface of archaeal TIMs also facilitates tetramerization. Analysis of the barrel interactions in TIMs suggests that these interactions are unlikely to account for the thermal stability of archaeal TIMs. A feature of the unliganded structure of MjTIM is the complete absence of electron density for the loop 6 residues. The disorder of the loop may be ascribed to a missing salt bridge between residues at the N- and C- terminal ends of the loop in MjTIM. Chapter 6 is a follow up of an interesting observation made by Vogel and Chmielewski (1994), who noticed that subtilisin cleaved rabbit muscle triosephosphate isomerase religated spontaneously upon addition of organic solvents. Further extension of this nicking and religation process with PfTIM emphasizes the importance of tertiary interactions in contributing to the stability of the (β/α)8 barrel folds (Ray et al., 1999). This chapter establishes that subtilisin nicking and religation is also facile in thermophilic MjTIM. Fragments generated by subtilisin nicking were identified using MALDI mass spectrometry at early and late stages of the cleavage for both the dimeric PfTIM and tetrameric MjTIM. This chapter also describes the comparative thermal and denaturant stability of both the enzymes. The accessibility of the Cys residues of MjTIM has been probed by examining the rates of labeling of thiol groups by iodoacetamide. The differential labeling of Cys residues has been demonstrated by mass spectrometry. Chapter 7 summarizes the main results and conclusions of the studies described in this thesis.

<p>Malaria is a life threatening parasite disease caused and transmitted by infected female anopheles mosquito. However, the parasite, Plasmodium falciparum, has become resistant to most anti malarial drugs, such as chloroquine, which contributes to fever and anaemia because of its ability to digest the haemoglobin in the red blood cells. The aims of this project were to establish whether “Bac to Bac” Baculoviral Expression System is suitable for expression of pfmdr 1 gene and for purification of the pgh 1 protein. The pfmdr 1 gene encodes an ABC transporter protein, pgh 1, fixed in the cell membrane of the Plasmodium falciparuum gut, which assist in elimination of drug compounds. Furthermore, “Bac to Bac” Baculoviral Expression System uses vectors with histidine tags to clone the pfmdr 1 gene and subsequently transform these into DH10Bac cells to produce the recombinant bacmid DNA. Since pfmdr 1 gene is an AT-rich sequence, PCR was optimized, by lowering the annealing and extension temperature to 47Co and 66Co respectively. The results show that “Bac to Bac” Baculoviral Expression System can be used to express the pfmdr 1 gene, though further experiments has to be performed.</p>

Thesis (M.S.P.H.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 114 pages. Includes bibliographical references.