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Title: RAMAN SPECTROSCOPIC STUDY OF SINGLE RED BLOOD CELLS INFECTED BY THE MALARIA PARASITE PLASMODIUM FALCIPARUM.
Creator: Carter, William, Schulte, Alfons, University of Central Florida
Abstract / Description: Raman micro-spectroscopy provides a non-destructive probe with potential applications as a diagnostic tool for cellular disorders. This study presents micro-Raman spectra of live erythrocytes infected with a malaria parasite and investigates the potential of this probe to monitor molecular changes which occur during differentiation of the parasite inside the cell. At an excitation wavelength of 633 nm the spectral bands are dominated by hemoglobin vibrations yielding information the on...
Show moreRaman micro-spectroscopy provides a non-destructive probe with potential applications as a diagnostic tool for cellular disorders. This study presents micro-Raman spectra of live erythrocytes infected with a malaria parasite and investigates the potential of this probe to monitor molecular changes which occur during differentiation of the parasite inside the cell. At an excitation wavelength of 633 nm the spectral bands are dominated by hemoglobin vibrations yielding information the on structure and spin state of the heme moiety. It also demonstrates the novel use of silica capillaries as a viable method for studying the erythrocytes in an environment that is much closer to their native state, thus opening the possibility of maintaining the cell in vivo for long periods to study the dynamics of the parasite's growth.
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Date Issued: 2007
Identifier: CFE0001780, ucf:47254
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0001780

Title: EVALUATION OF THE EFFICACY OF CHLOROPLAST-DERIVED ANTIGENSAGAINST MALARIA.
Creator: Schreiber, Melissa, Chakrabarti, Debopam, University of Central Florida
Abstract / Description: Malaria is the most prevalent vector-borne parasitic disease worldwide and a major cause of death from infections. There is a great need to develop a low cost vaccine for malaria to control transmission of infection and impact of disease, due to the emergence of anti-malarial resistance. Two leading blood stage malarial vaccine candidates are the apical membrane antigen-1 (AMA-1) and the merozoite surface protein-1 (MSP-1). The aim of this project is to express malarial antigens in tobacco...
Show moreMalaria is the most prevalent vector-borne parasitic disease worldwide and a major cause of death from infections. There is a great need to develop a low cost vaccine for malaria to control transmission of infection and impact of disease, due to the emergence of anti-malarial resistance. Two leading blood stage malarial vaccine candidates are the apical membrane antigen-1 (AMA-1) and the merozoite surface protein-1 (MSP-1). The aim of this project is to express malarial antigens in tobacco plants via plastid transformation and deliver them by subcutaneous or oral gavage of minimally processed transplastomic tissue to evaluate their efficacy to elicit an immune response and protect against malarial infection. Transplastomic lines expressing the malarial antigens fused to the transmucosal carrier Cholera toxin B subunit (CTB-AMA-1) and CTB-MSP-1 were generated. CTB-AMA-1 and CTB-MSP-1 accumulated up to 9.5% and 2% of the total soluble protein, respectively. Chloroplast-derived CTB-AMA-1, CTB-MSP-1, or both antigens were administered to BALB/c mice orally or by subcutaneous injections. The immune response in the experimental animals compared to the control animals was found to be significant. Using an immunofluorescence assay (IFA) and immunoblot, anti-AMA-1 and anti-MSP-1 found in sera of immunized mice recognized the native parasite and the native parasite protein, respectively. Anti-malarial antibodies inhibited parasite invasion into erythrocytes by utilizing an in vitro parasite inhibition assay. Results of these investigations may lead to a cost-effective malarial vaccine, much needed in developing nations.
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Date Issued: 2008
Identifier: CFE0002375, ucf:47807
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0002375

Title: A MATHEMATICAL STUDY OF MALARIA MODELS OF ROSS AND NGWA.
Creator: Plemmons, William, Rollins, David, University of Central Florida
Abstract / Description: Malaria is a vector borne disease that has been plaguing mankind since before recorded history. The disease is carried by three subspecies of mosquitoes Anopheles gambiae, Anopheles arabiensis and Anopheles funestu. These mosquitoes carry one of four type of Plasmodium specifically: P. falciparum, P. vivax, P. malariae or P. ovale. The disease is a killer; the World Health Organization (WHO) estimates that about 40% of the world's total populations live in areas where malaria is an...
Show moreMalaria is a vector borne disease that has been plaguing mankind since before recorded history. The disease is carried by three subspecies of mosquitoes Anopheles gambiae, Anopheles arabiensis and Anopheles funestu. These mosquitoes carry one of four type of Plasmodium specifically: P. falciparum, P. vivax, P. malariae or P. ovale. The disease is a killer; the World Health Organization (WHO) estimates that about 40% of the world's total populations live in areas where malaria is an endemic disease and as global warming occurs, endemic malaria will spread to more areas. The malaria parasite kills a child every 30 seconds. In Africa alone, as many as one million children die annually from malaria before they reach the age of 5. The World Health Organization has an estimate of 100-200 million victims annually. Malaria has many mathematical models and this paper will examine several different models in order to achieve a greater understanding of this disease.
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Date Issued: 2006
Identifier: CFE0001406, ucf:47070
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0001406

Title: Proteomic Analysis Delineates the Signaling Networks of Plasmodium falciparum.
Creator: Pease, Brittany, Chakrabarti, Debopam, Khaled, Annette, Jewett, Mollie, Chakrabarti, Ratna, Cole, Alexander, University of Central Florida
Abstract / Description: Malaria is a life-threatening disease caused by Plasmodium parasites that are spread through the bites of infected mosquito vectors. It is a worldwide pandemic that threatens 3.4 billion people annually. Currently, there are only a few validated Plasmodium drug targets, while drug resistance continues to rise. This marks the urgency for the development of novel parasite-specific therapeutics. Plasmodium falciparum diverges from the paradigm of the eukaryotic cell cycle by undergoing multiple...
Show moreMalaria is a life-threatening disease caused by Plasmodium parasites that are spread through the bites of infected mosquito vectors. It is a worldwide pandemic that threatens 3.4 billion people annually. Currently, there are only a few validated Plasmodium drug targets, while drug resistance continues to rise. This marks the urgency for the development of novel parasite-specific therapeutics. Plasmodium falciparum diverges from the paradigm of the eukaryotic cell cycle by undergoing multiple rounds of DNA replication and nuclear division without cytokinesis. A better understanding of the molecular switches that coordinate the progression of the parasite through the intraerythrocytic developmental stages will be of fundamental importance for the design of rational intervention strategies. To achieve this goal, we performed an isobaric tag-based approach for a system-wide quantitative analysis of protein expression and site-specific phosphorylation events of the Plasmodium asexual developmental cycle in the red blood cells. This study identified 2,767 proteins, 1,337 phosphoproteins, and 6,293 phosphorylation sites. Approximately 34% of identified proteins and 75% of phosphorylation sites exhibit changes in abundance as the intraerythrocytic cycle progresses. Because the links between Plasmodium protein kinases as key cell cycle regulators to cellular events are largely unknown, it is of importance to define their cognate physiological substrates. To test the hypothesis that genetic screening would be a useful approach for discovery of candidate substrates of a protein kinase, we used the orphan kinase PfPK7 as a model. Our comparison of the phosphoproteome profiles between the wild-type 3D7 and PfPK7- parasites identified 146 proteins with 239 phosphorylation sites exhibiting decreased phosphorylation in the absence of PfPK7 at the developmental stages where nuclear division and merozoite formation occur. Further analysis of the decreased phosphorylated events revealed three motifs that are enriched among phosphorylated sites in proteins that are down regulated. In vitro kinase assays were done to validate the potential substrates of PfPK7 and to elucidate the signaling events that are regulated by PfPK7. In parallel to our experimental analysis, we used a computational approach for substrate prediction from our phosphoproteome dataset. This analysis identified 43 distinct phosphorylation motifs and a range of proline-directed potential MAPK/CDK substrates. To identify substrates/ interactors of Plasmodium CDK-like kinases, we also used HA-tagged CDK-like kinases, PfPK6 and Pfmrk lines. Co-immunoprecipitation of the HA-tagged PfPK6 and Pfmrk baits, followed by mass spectrometric analyses, identified the components of the protein interaction complexes of these kinases. Our analyses of HA-PfPK6 and HA-Pfmrk immunoprecipitates identified 15 and 21 proteins in the interaction complex, respectively. The ability of recombinant PfPK6 and Pfmrk to interact and/or utilize any of the proteins identified in the interaction complex as substrates was verified through in vitro kinase assays and pull-down analysis. This study is the most comprehensive definition of the constitutive and regulated expression of the Plasmodium proteome during the intraerythrocytic developmental cycle, and offered an insight into the dynamics of phosphorylation during the asexual cycle progression [1]. In summary, this study has 1) defined the constitutive and regulated expression of the Plasmodium proteome during its asexual life cycle, 2) demonstrated that fluctuation and reversible phosphorylation is important for the regulation of P. falciparum's unique cell cycle, 3) provided the foundation for quantitative phosphoproteomic analysis of kinase negative mutants to understand their function, 4) provided a major step towards defining kinase-substrate pairs operative within parasite's signaling networks, and 5) generated a preliminary interactome for PfPK6.
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Date Issued: 2015
Identifier: CFE0005863, ucf:50898
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005863

Title: CHARACTERIZATION OF PROTEIN PRENYLTRANSFERASESAND PROTEIN PRENYLATION INPLASMODIUM FALCIPARUM.
Creator: DaSilva, Thiago Gaspar, Dobopam Chakrabarti, Dr, University of Central Florida
Abstract / Description: 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...
Show moreMalaria 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.
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Date Issued: 2004
Identifier: CFE0000100, ucf:46182
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0000100

Title: UNDERSTANDING THE ROLE OF PLASMODIUM FALCIPARUM VAMP8 SNARE HOMOLOGUE.
Creator: Camacho Ferreira, Katherine, Chakrabarti, Debopam, University of Central Florida
Abstract / Description: Malaria is one of the worlds most deadly infectious diseases and results in almost a million deaths each year, largely in children under the age of five in Sub-Saharan Africa. Outside Africa, malaria is responsible for a large number of cases in the Amazon rainforest of Brazil, Middle East, and in some areas of Asia . According to the World Health Organization, there was an estimated 655, 000 deaths from malaria in 2012. Malaria is caused by a eukaryotic Apicomplexan parasite, Plasmodium,...
Show moreMalaria is one of the worlds most deadly infectious diseases and results in almost a million deaths each year, largely in children under the age of five in Sub-Saharan Africa. Outside Africa, malaria is responsible for a large number of cases in the Amazon rainforest of Brazil, Middle East, and in some areas of Asia . According to the World Health Organization, there was an estimated 655, 000 deaths from malaria in 2012. Malaria is caused by a eukaryotic Apicomplexan parasite, Plasmodium, which has three distinct life cycles occurring in the midgut of the female Anopheles mosquito, the liver of the human host, and human erythrocytes. When the parasite infects the erythrocyte, some induced cell host modifications are made in order to accommodate growth. During its intra-erythrocytic life cycle, the malaria parasite traffics numerous proteins to a set of unique destinations within its own plasma membrane including the digestive vacuole, the apicoplast, rhoptries, and micronemes. Vesicular transport is an essential process in eukaryotic cells. This coordinated process is responsible for moving thousands of proteins between compartments within the cell. Essential to the targeting and fusion of protein transport vesicles in eukaryotes are SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), a family of fusogenic proteins that are localized to distinct intracellular compartments . Studies performed in our laboratory have identified 18 proteins putatively belonging to the PfSNARE family . To date the exact role of PfSNAREs in the unique trafficking pathways of malaria is undetermined. Of particular interest to our study is PfVAMP8. In model eukaryotic organisms, VAMP8 containing vesicles deliver cargo to lysosomes and are involved in endocytosis. The food vacuole of the parasite is very similar to that of lysosomes and is essential to parasite survival. The study aims to identify the organelle(s) to which PfVAMP8 is localized and characterize membrane-association properties of this parasite's R-SNARE protein. We believe that PfVAMP8 would localize to unique compartments in the parasite protein network flow. An in depth understanding of its mechanisms and localizations could be a key in developing novel anti-malarials. This study aims to identify the organelle(s) to which PfVAMP8 are localized, determine the trafficking determinants of this protein and determine this proteins' expression and membrane association during the intra-erythrocytic stages of Plasmodium falciparum. Our immunofluorescence studies with known biological markers reveals that, PfVAMP8 passes through the endoplasmic reticulum, Golgi, and localizes to the food vacuole during trophozoite and schizont stage. Further characterization of the membrane association properties of the protein in this study reveals that PfVAMP8 is a soluble integral membrane protein with amphipathic characteristics.
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Date Issued: 2013
Identifier: CFH0004525, ucf:45157
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004525

Title: 3-AMINO-2-PIPERIDINEQUINOLINE A NOVEL NATURAL PRODUCT-INSPIRED SYNTHETIC COMPOUND WITH ANTIMALARIAL ACTIVITY.
Creator: Valor, Cristhian, Chakrabarti, Debopam, University of Central Florida
Abstract / Description: Malaria afflicts about 500 million people worldwide thus causing significant global economic toll. The drugs available to treat the disease are rapidly losing their efficacy because of widespread prevalence of drug resistant parasites. Thus there is an urgent need to discover novel malaria therapeutics. This research is focused on to study the properties of a novel natural-like synthetic scaffold and analyze its selectivity, and cellular mechanism of action in Plasmodium falciparum. We have...
Show moreMalaria afflicts about 500 million people worldwide thus causing significant global economic toll. The drugs available to treat the disease are rapidly losing their efficacy because of widespread prevalence of drug resistant parasites. Thus there is an urgent need to discover novel malaria therapeutics. This research is focused on to study the properties of a novel natural-like synthetic scaffold and analyze its selectivity, and cellular mechanism of action in Plasmodium falciparum. We have identified a novel compound, 3-amino-2-piperidinequinoline (APQ), which we termed UCF401. APQ demonstrated IC50 at submicromolar concentrations against Plasmodium falciparum using the SYBR Green-I fluorescence assay measuring cellular proliferation. This compound also demonstrated low cytotoxicity against the NIH3T3 and HEPG2 cells using MTS assays, showing an IC50 of 174 uM and 125 uM respectively, suggesting of excellent selectivity. We evaluated the compliance of APQ with Lipinski's parameters and determined the in vitro physicochemical profiles of the compound. Our results show that APQ is a Lipinski parameter compliant and has good physicochemical properties. The cellular mechanism of action of APQ was characterized through the assessment of the effects of the compound at different stages of the parasite's intraerythrocytic life cycle. This assay was done by treating a synchronized cell line with the compound at 5X the IC50 value and then imaging the cells at 12-hour intervals. We found that APQ arrests parasite development at the trophozoite stage. In addition we determined that APQ is parasitocidal after a 96 h exposure. These results demonstrate that APQ can be considered as a validated hit and/or early lead.
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Date Issued: 2014
Identifier: CFH0004593, ucf:45233
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004593

Title: IDENTIFICATION OF NOVEL ANTIMALARIALS FROM MARINE NATURAL PRODUCTS FOR LEAD DISCOVERY.
Creator: Alvarado, Stephenie, Chakrabarti, Debopam, University of Central Florida
Abstract / Description: An estimated 500 million cases of malaria occur each year. The increasing prevalence of drug resistant strains of Plasmodium in most malaria endemic areas has significantly reduced the efficacy of current antimalarial drugs for prophylaxis and treatment of this disease. Therefore, discovery of new, inexpensive, and effective drugs are urgently needed to combat this disease. Marine biodiversity is an enormous source of novel chemical entities and has been barely investigated for antimalarial...
Show moreAn estimated 500 million cases of malaria occur each year. The increasing prevalence of drug resistant strains of Plasmodium in most malaria endemic areas has significantly reduced the efficacy of current antimalarial drugs for prophylaxis and treatment of this disease. Therefore, discovery of new, inexpensive, and effective drugs are urgently needed to combat this disease. Marine biodiversity is an enormous source of novel chemical entities and has been barely investigated for antimalarial drug discovery. In an effort to discover novel therapeutics for malaria, we studied the antimalarial activities of a unique marine-derived peak fraction library provided by Harbor Branch Oceanographic Institute (HBOI). Within this unique library, we have screened 2,830 marine natural product (MNP) peak fractions through a medium throughput screening effort utilizing the SYBR Green-I fluorescence based assay, and have identified 253 fractions that exhibit antimalarial activity. From those inhibiting fractions we have identified twenty species of marine organisms that inhibit Plasmodium falciparum growth, from which thirty-five fractions were selected for further study. Among those thirty-five, eighty-three percent were also found to inhibit the chloroquine resistant strain of P. falciparum, Dd2. The most potent inhibitors were then screened for their cytotoxic properties using the MTT cell viability assay. Among the samples that exhibited potent inhibition of P. falciparum growth were fractions derived from a sponge of the genus Spongosorites sp.. This genus of sponge has been reported to contain the nortopsentin and topsentin class of bis-indole imidazole alkaloids. Nortopsentin A inhibited the parasite growth at the trophozoite stage with an IC50 value of 1.6 µM. This is the first report of antimalarial activity for this class of compound.
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Date Issued: 2010
Identifier: CFE0003472, ucf:48932
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0003472

Title: A MEMBER OF THE NOVEL FIKK FAMILY OF PLASMODIUM FALCIPARUM PUTATIVE PROTEIN KINASES EXHIBITS DIACYLGLYCEROL KINASE ACTIVITY AND IS EXPORTED TO THE HOST ERYTHROCYTE.
Creator: Curtis, David, Chakrabarti, Debopam, University of Central Florida
Abstract / Description: Plasmodium falciparum is one of four species known to cause malaria in humans and is the species that is associated with the most virulent form of the disease. Malaria causes nearly two million deaths each year, many of these occurring among children in under-developed countries of the world. One reason for this is the prevalence of drug resistant strains of malaria that mitigate the efficacy of existing drugs. Hence, the identification of a new generation of pharmacological agents for...
Show morePlasmodium falciparum is one of four species known to cause malaria in humans and is the species that is associated with the most virulent form of the disease. Malaria causes nearly two million deaths each year, many of these occurring among children in under-developed countries of the world. One reason for this is the prevalence of drug resistant strains of malaria that mitigate the efficacy of existing drugs. Hence, the identification of a new generation of pharmacological agents for malaria is extremely urgent. The recent identification of a group of novel protein kinases within the Plasmodium falciparum genome has provided researchers with a basis for what many hope to be new potential drug targets for malaria. Identified within the Plasmodium genome and a few select apicomplexans, these novel proteins have been predicted to be protein kinases based solely on certain sequence features shared with other eukaryotic protein kinases (ePKs). However, to date, no significant studies to determine the function of these novel kinases have been performed. Termed FIKKs, these proteins all possess a non-conserved N-terminal sequence that contains a Plasmodium export element (Pexel) which may target the proteins for export from the parasite and a conserved C-terminal catalytic domain containing a FIKK sequence common to all twenty members of this family. We analyzed the localization of one of the FIKK proteins, FIKK11, encoded by the PF11_0510 locus, during intraerythrocyte differentiation of P. falciparum by Western blot analysis and indirect immunofluorescence assay. Western blot analysis demonstrated that FIKK 11 is expressed within the parasite at all stages of its erythrocytic life cycle with its highest expression occurring during the schizont stage. Immunofluorescence assays showed that this protein is exported from the Plasmodium parasite into the host erythrocyte cytosol which is consistent with studies on other Plasmodium proteins that also have the Pexel motif. To determine the enzymatic activity of FIKK11, we overexpressed the recombinant protein in E. coli and then purified it. However, no protein kinase activity was detected using several commonly used protein kinase substrates including histone H1, myelin basic protein, or dephosphorylated casein. We also did not detect any kinase activity of the native enzyme using pull-down assays of the Plasmodium falciparum cell extract against those same substrates. In addition, kinase substrate peptide array analysis of FIKK11 showed no evidence of protein kinase activity either for FIKK11. Interestingly, however, we were able to detect some kinase activity using the recombinant protein alone with no substrate. The lack of the glycine triad within subdomain I of these FIKK kinases as compared with most traditional eukaryotic protein kinases may explain why we were unable to find any interactions between FIKK11 and other commonly protein kinase substrates. Of interest was the observation that the protein reproducibly exhibited what appeared to be an autophosphorylation activity when using the standard protein kinase assay. Further analyses, however, showed that FIKK11 actually possesses diacylglycerol kinase activity utilizing 1-Stearoyl-2-arachidonoyl-sn-glycerol as a substrate. This is the first evidence of diacylglycerol kinase activity in Plasmodium falciparum. Because FIKK11 is exported into the host cell and is localized on the erythrocyte membrane, its enzymatic activity may potentially have relevance in the pathophysiology of the disease.
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Date Issued: 2007
Identifier: CFE0001879, ucf:47407
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0001879

Title: IDENTIFICATION AND CHARACTERIZATION OF INTERACTORS OF PLASMODIUM FALCIPARUM PFPK6, AN ATYPICAL PROTEIN KINASE.
Creator: Cummins, Andi J, Chakrabarti, Debopam, University of Central Florida
Abstract / Description: Plasmodium falciparum, the organism that causes the most prevalent and most virulent cases of malaria in humans, poses a major health burden on the developing world, especially in the tropical regions of Sub-Saharan Africa, Southeast Asia, and Latin America. The burden of the disease is intensified by the fact that the parasite has developed widespread resistance to all current antimalarial therapies, such as chloroquine. This drug resistance underscores the need to develop novel therapeutics...
Show morePlasmodium falciparum, the organism that causes the most prevalent and most virulent cases of malaria in humans, poses a major health burden on the developing world, especially in the tropical regions of Sub-Saharan Africa, Southeast Asia, and Latin America. The burden of the disease is intensified by the fact that the parasite has developed widespread resistance to all current antimalarial therapies, such as chloroquine. This drug resistance underscores the need to develop novel therapeutics that target the parasite, but show low toxicity in the human host. Protein kinases, because of their integral roles in cell signaling networks, are considered to be attractive drug targets. Cyclin dependent kinases, or CDKs, and Mitogen-Activated Protein kinases, or MAPKs, are common to eukaryotes and regulate cellular processes of growth and proliferation. Plasmodium falciparum Protein Kinase 6, or PfPK6, is an atypical protein kinase that shows similarities to both MAPKs and CDKs. PfPK6 is expected to have an important role in the intraerythrocytic cell cycle progression and growth in the malaria organism, as it has been found to be essential in the parasite. In order to better understand the function of PfPK6 within Plasmodium, we have identified serveral potential substrates and interactors of the kinase using co-immunoprecipitation with an HA epitope-tagged cell line of PfPK6, as well as phosphoproteomic analysis. These methods resulted identification of 15 novel protein interactors, with 4 being studied for further investigation, and 45 putative substrates after strict peptide filtering, five of which are used in this study. In order to verify putative substrates and interactors, both in vitro and in vivo methods were used. In vitro kinase assays using GST-PfPK6 with 5 recombinant substrates confirmed direct phosphorylation of two novel substrates: MAL7P1.38, a regulator of chromosome condensation, and PF10_0047, a putative RNA binding protein. After attempts to generate bacterial constructs of several putative interactors and a global failure of a usable amount of protein to express under IPTG induction conditions, an alternative form of expression using a cell free Transcription and Translation reaction (TNT) with Wheat Germ Extract was used to generate radiolabeled PF11_0154, PFF0625w, and PF11_0305. Pull down analysis using GST-PfPK6 showed the kinase�s ability to �pull� the interactors out of solution, confirming the interactions defined by the initial epitope tagged Co-Immunoprecipitation. Additionally, for in vivo analysis, parasites were transfected with RFP- PFF_0695w, an uncharacterized Plasmodium protein, in order to cellular localization of this interactors. Immunofluorescence assays of transfected lines showed punctate forms of PFF_0695w in the host erythrocyte in the late trophozoite and schizont stages of the parasite development, suggesting this interactor is a previously undiscovered protein in the Plasmodium secretome. The research presented here is an initial step to defining the interactome of PfPK6.
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Date Issued: 2016
Identifier: CFH2000041, ucf:45517
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH2000041

Title: IDENTIFICATION OF PHYSIOLOGICAL SUBSTRATES OF PLASMODIUM FALCIPARUM PFPK5, A CDK-LIKE KINASE.
Creator: Sullenberger, Catherine, Chakrabarti, Debopam, University of Central Florida
Abstract / Description: Malaria is one of the most devastating infectious diseases causing 1-3 million fatalities a year. The majority of these cases occur amongst children in developing countries. Malarial strains in these areas are exhibiting increasing resistance to canonical treatments proving the importance of new drug targets for anti-malarials. Identification of new drug targets is dependent upon a better understanding of the molecular biology of the parasitic agent of malaria, Plasmodium. The regulation of...
Show moreMalaria is one of the most devastating infectious diseases causing 1-3 million fatalities a year. The majority of these cases occur amongst children in developing countries. Malarial strains in these areas are exhibiting increasing resistance to canonical treatments proving the importance of new drug targets for anti-malarials. Identification of new drug targets is dependent upon a better understanding of the molecular biology of the parasitic agent of malaria, Plasmodium. The regulation of Plasmodium's complex life cycle is still not well understood. Elucidation of signaling pathways involved in Plasmodium cell cycle regulation will provide insights into how the parasite thrives in human cells. A subset of kinases, referred to as cyclin-dependent kinases (CDKs), are crucial regulators of eukaryotic cell cycle progression. In silico studies show high homology between mammalian CDK's and a group of CDK-like Plasmodium kinases including PfPK5 (Plasmodium falciparum protein kinase 5). Plasmodium homologues to CDK regulators, cyclins, have also been identified. Understanding the role of PfPK5 in cell cycle regulation would require analysis of subcellular localization and cell cycle-dependent expression. Immunofluorescence assays demonstrate that PfPK5 is localized in the nucleus. PfPK5's expression profile, as determined by western blotting, shows highest expression in the schizont stage, the stage when the atypical multiple nucleated form of the parasite is observed. Possible PfPK5 interacting partners were detected by performing an anti-PfPK5 immunoprecipitation assay. Additionally, a hemagglutinin (HA)-tagged PfPK5 construct was made to increase the sensitivity of immunoprecipitation assay and identification of PfPK5 interacting partners. The characterization of PfPK5 and its interacting partners may prove useful in identification of novel drug targets in the future.
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Date Issued: 2011
Identifier: CFH0003848, ucf:44701
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0003848

Title: IDENTIFICATION OF PLASMODIUM FALCIPARUM PROTEIN KINASE SUBSTRATES AND INTERACTING PROTEINS.
Creator: Yap, Jessica, Chakrabarti, Debopam, University of Central Florida
Abstract / Description: 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...
Show moreMalaria 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. Characterization of PfPKA and PfPK5 substrates, as well as the proteins they interact with, will help us to develop innovative therapies targeting binding sites.
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Date Issued: 2012
Identifier: CFH0004157, ucf:44829
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004157

Title: HIGH PRESSURE AND MICRO-SPECTROSCOPIC STUDIES OF SINGLE LIVING ERYTHROCYTES AND THE INTRAERYTHROCYTIC MULTPLICATION CYCLE OF PLASMODIUM FALCIPARUM.
Creator: ARORA, SILKI, SCHULTE, ALFONS, University of Central Florida
Abstract / Description: A novel experimental approach for micro-absorption spectroscopy and high-pressure microscopy of single cells is developed and applied to the investigation of morphological, volume, and spectroscopic changes in healthy red blood cells (RBCs) and erythrocytes infected with the malaria parasite Plasmodium falciparum. Through real-time optical imaging of individual erythrocytes (size ~ 7 microns) we determine the change in volume over the pressure range from 0.1 to 210 MPa. The lateral diameter...
Show moreA novel experimental approach for micro-absorption spectroscopy and high-pressure microscopy of single cells is developed and applied to the investigation of morphological, volume, and spectroscopic changes in healthy red blood cells (RBCs) and erythrocytes infected with the malaria parasite Plasmodium falciparum. Through real-time optical imaging of individual erythrocytes (size ~ 7 microns) we determine the change in volume over the pressure range from 0.1 to 210 MPa. The lateral diameter of healthy RBCs decreases reversibly with pressure with an approximate slope of 0.015 microns / MPa. In infected cells, clear differences in the deformability and between the compression and decompression curves are observed. The results are discussed with respect to the elasticity of the phospholipid membrane and the spectrin molecular network. Employing micro-absorption spectroscopy with spatial resolution of 1.4 microns in the lateral and 3.6 microns in the axial direction the visible absorption spectrum of hemoglobin in a single red blood cell is measured under physiological conditions. The spectra of cells infected with the malaria parasite show changes in peak positions and relative intensities in the Soret and the alpha- and beta-bands. These indicate hemoglobin degradation that can be correlated with the stages of the parasite multiplication cycle and can be used as a potential diagnostic marker. The research is further extended towards the understanding of pressure effects on the ligand binding kinetics to heme proteins. For a well characterized reaction at ambient pressure, CO binding to myoglobin in solution, we investigate the transient absorption following laser flash photolysis over eight decades in time at variable pressure and temperature. The data demonstrate that pressure significantly affects the amplitudes (not just the rates) of the component processes. The amplitude of the geminate process increases with pressure corresponding to a smaller escape fraction of ligands into the solvent and a smaller inner barrier.
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Date Issued: 2011
Identifier: CFE0004039, ucf:49157
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004039

Title: Mobile Phone Short Message Service (SMS) to Improve Malaria Pharmacoadherence in Zambia.
Creator: Steury, Elinda, Sole, Mary Lou, Bushy, Angeline, Conner, Norma, Haiduven, Donna, University of Central Florida
Abstract / Description: ABSTRACTMalaria significantly contributes to morbidity and mortality rates in Zambia. The currently accepted malaria treatment is artemisinin-based combination therapy (ACT); it is more than 97% effective when the regimen is strictly adhered to. However, the mean ACT adherence rate in sub-Saharan Africa is only approximately 38-48%. Poor pharmacoadherence remains a significant barrier to malaria control and elimination.The purpose of this study was to determine if adherence rates to a six...
Show moreABSTRACTMalaria significantly contributes to morbidity and mortality rates in Zambia. The currently accepted malaria treatment is artemisinin-based combination therapy (ACT); it is more than 97% effective when the regimen is strictly adhered to. However, the mean ACT adherence rate in sub-Saharan Africa is only approximately 38-48%. Poor pharmacoadherence remains a significant barrier to malaria control and elimination.The purpose of this study was to determine if adherence rates to a six-dose ACT antimalarial treatment differ between patients in Zambia who received short message service (SMS) reminders and those who did not. An experimental, randomized, controlled trial was conducted to collect data from a sample of 96 adult patients with malaria who presented to Fisenge Clinic in the Copperbelt Province of Zambia. Participants were randomly assigned to a control or intervention group. The intervention group received SMS messages to remind them to take their medication according to the regimen. An electronic pillbox was used to measure pharmacoadherence for both groups, and patients were classified as probably adherent or probably non-adherent.Data were analyzed using Chi-square for association between the SMS intervention and pharmacoadherence, and logistic regression used for predictors of adherence. No significant association was found between SMS reminders and pharmacoadherence among malaria patients being treated with ACT when evaluated with respect to those who received the SMS reminders and those who did not (?2=0.19, df=1, p=0.67). Binary logistic regression indicated that there were no variables associated with adherence (p(>)0.05).Findings from this study contribute to the research regarding the use of mobile phones to promote adherence. This is the first study of its kind using SMS directly to the patient for ACT adherence in sub-Saharan Africa known to the author. It is possible that the use of the electronic pillbox and/or the novelty of participating in a research study contributed to higher levels of adherence than previously found in this geographical area. While data suggested that there was no association between SMS and adherence, further research is needed to explore the value of this intervention.
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Date Issued: 2014
Identifier: CFE0005421, ucf:50430
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0005421

Title: CHARACTERIZATION OF NOVEL ANTIMALARIALS FROM COMPOUNDS INSPIRED BY NATURAL PRODUCTS USING PRINCIPAL COMPONENT ANALYSIS (PCA).
Creator: Balde, Zarina Marie G, Chakrabarti, Debopam, University of Central Florida
Abstract / Description: Malaria is caused by a protozoan parasite, Plasmodium falciparum, which is responsible for over 500,000 deaths per year worldwide. Although malaria medicines are working well in many parts of the world, antimalarial drug resistance has emerged as one of the greatest challenges facing malaria control today. Since the malaria parasites are once again developing widespread resistance to antimalarial drugs, this can cause the spread of malaria to new areas and the re-emergence of malaria in areas...
Show moreMalaria is caused by a protozoan parasite, Plasmodium falciparum, which is responsible for over 500,000 deaths per year worldwide. Although malaria medicines are working well in many parts of the world, antimalarial drug resistance has emerged as one of the greatest challenges facing malaria control today. Since the malaria parasites are once again developing widespread resistance to antimalarial drugs, this can cause the spread of malaria to new areas and the re-emergence of malaria in areas where it had already been eradicated. Therefore, the discovery and characterization of novel antimalarials is extremely urgent. A previous drug screen in Dr. Chakrabarti's lab identified several natural products (NPs) with antiplasmodial activities. The focus of this study is to characterize the hit compounds using Principal Component Analysis (PCA) to determine structural uniqueness compared to known antimalarial drugs. This study will compare multiple libraries of different compounds, such as known drugs, kinase inhibitors, macrocycles, and top antimalarial hits discovered in our lab. Prioritizing the hit compounds by their chemical uniqueness will lessen the probability of future drug resistance. This is an important step in drug discovery as this will allow us to increase the interpretability of the datasets by creating new uncorrelated variables that will successively maximize variance. Characterization of the Natural Product inspired compounds will enable us to discover potent, selective, and novel antiplasmodial scaffolds that are unique in the 3-dimensional chemical space and will provide critical information that will serve as advanced starting points for the antimalarial drug discovery pipeline.
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Date Issued: 2018
Identifier: CFH2000405, ucf:45893
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH2000405

Title: IDENTIFICATION OF POTENTIAL LEAD ANTIMALARIAL COMPOUNDS FROM MARINE MICROBIAL EXTRACTS.
Creator: Carbonell, Abigail, Chakrabarti, Debopam, University of Central Florida
Abstract / Description: Malaria, caused by the parasite Plasmodium falciparum, has a long history as a global health threat. The vector-borne disease causes millions of deaths yearly, especially in developing countries with tropical climates that facilitate transmission. Compounding the problem is the emergence of drug-resistant strains due to overuse of outdated treatments. New compounds with antiplasmodial activity are needed to be developed as effective drugs against malaria. The hypothesis for this project is...
Show moreMalaria, caused by the parasite Plasmodium falciparum, has a long history as a global health threat. The vector-borne disease causes millions of deaths yearly, especially in developing countries with tropical climates that facilitate transmission. Compounding the problem is the emergence of drug-resistant strains due to overuse of outdated treatments. New compounds with antiplasmodial activity are needed to be developed as effective drugs against malaria. The hypothesis for this project is that marine microorganisms have a high likelihood of yielding novel antiplasmodial chemotypes because of their high diversity, which has not yet been explored for antimalarial development. In this project, microbes harvested and fermented by the Harbor Branch Oceanographic Institute in Fort Pierce, Florida were explored as sources for antiplasmodial natural products. Using a SYBR Green I fluorescence-based assay, 1,000 microbial extracts were screened for inhibition of the multidrug-resistant Plasmodium falciparum strain Dd2. Dose-response analysis was performed on 46 fractions from isolates whose extracts demonstrated [greater-than or equal to] 70% inhibition of Dd2 at 1 [micro]g/mL. To evaluate cytotoxicity, the MTS cell viability assay was used to calculate IC50 of extracts from active isolates in NIH/3T3 embryonic mouse fibroblasts. Several extracts demonstrated low IC50 in Dd2 and high IC50 in 3T3, suggesting that they contain potential lead antimalarial compounds. Extracts with high selectivity indices (potent plasmodial inhibition with low mammalian toxicity) have been prioritized for dereplication, with the goal of identifying novel active components that can be developed as antimalarial drugs.
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Date Issued: 2013
Identifier: CFH0004332, ucf:45035
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004332

Title: Infectious Disease Risks in Developing Countries: A Non-Market Valuation Exercise.
Creator: Samajpati, Shreejata, Gerking, Shelby, Dickie, Mark, Caputo, Michael, Roy, Joyashree, University of Central Florida
Abstract / Description: This dissertation focuses on the non-market valuation of health-risks of malaria, an infectious disease that imposes a substantive public health burden across the globe, hitting particularly hard the tropical developing nations of Africa and Asia. The United Nations Millennium Development Goals include malaria control as a priority and large investments are underway to promote effective prevention and treatment. Despite such concerted supply-side efforts, malaria-related mortality and...
Show moreThis dissertation focuses on the non-market valuation of health-risks of malaria, an infectious disease that imposes a substantive public health burden across the globe, hitting particularly hard the tropical developing nations of Africa and Asia. The United Nations Millennium Development Goals include malaria control as a priority and large investments are underway to promote effective prevention and treatment. Despite such concerted supply-side efforts, malaria-related mortality and morbidity still abound due to a complex interface of factors like climate-change, poverty, inadequate control behavior, infection and prevention externalities, parasite resistance etc. This research project digs into the demand-side of the health problem, considers the "externality" dimension to prevention, and primarily asks the question: how do individuals in developing countries view competing disease-control (prevention) measures, viz. a publicly-administered community-level malaria control measure as against private preventive choices. A theoretical model is developed to help explore the public-private interplay of health risks of malaria. The malaria-endemic regions of Kolkata (India) and its rural fringes comprise the site for an empirical investigation. A field survey (Malaria Risk and Prevention Survey, October-December, 2011) incorporating a mix of stated and revealed preference techniques of health valuation is implemented. Risk-perceptions of respondents are elicited using a measurable visual-aid and individuals' perceived valuations of health-risk reductions, randomly offered with the public and private health treatments, are empirically ascertained. Using a Likelihood Ratio Test on the structural risk parameters, it is seen that individuals' valuations of health risk reductions are the same across the private and public treatments. The comparative valuation exercise, thus, corroborates the externality dimension to malaria control, calling for greater public action to combat malaria. The viability of such a scaled-up public malaria program, in the context of Kolkata, is discussed by comparing the public treatment willingness to pay estimates with the annual estimated costs that the Kolkata Municipal Corporation, the civic body in the city of Kolkata, maintains on account of vector control. Results from the comparative valuation exercises also support the idea that private prevention is generally responsive to prevention costs, indicating the importance of price incentives to induce greater prevention. The issues of health valuation and price sensitivity are further explored across various split-samples differentiated on the basis of socio-economic attributes, disease exposure, actual prevention efforts and perceived malaria risks of survey respondents. Such auxiliary exercises help analyze the valuation question in greater depth, and generate policy insights into the potential factors that shape private prevention behavior.
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Date Issued: 2012
Identifier: CFE0004594, ucf:49195
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004594

malaria (11)	+ -
Malaria (5)	+ -
Plasmodium (3)	+ -
falciparum (3)	+ -
phosphoproteomics (3)	+ -

plasmodium (3)	+ -
Plasmodium falciparum (2)	+ -
antimalarial (2)	+ -
drug discovery (2)	+ -
kinase (2)	+ -
APQ (1)	+ -
Africa (1)	+ -
Antimalarial drug discovery (1)	+ -
Contingent Valuation Method (1)	+ -
Cristhian Valor (1)	+ -
Dd2 (1)	+ -
Developing Countries (1)	+ -
Diacylglycerol (1)	+ -
Diacylglycerol Kinase (1)	+ -
Economic Epidemiology (1)	+ -
Erythrocytes (1)	+ -
Externalities (1)	+ -
FIKK (1)	+ -
Falciparum (1)	+ -
Heme protein dynamics (1)	+ -
High Pressure (1)	+ -
Immunology (1)	+ -
India (1)	+ -
Infectious Diseases (1)	+ -
Kolkata (1)	+ -

ETD Collection (10)	+ -
Honors Collection (7)	+ -

University of Central Florida (17)	+ -
Chakrabarti, Debopam (11)	+ -
ARORA, SILKI (1)	+ -
Alvarado, Stephenie (1)	+ -
Balde, Zarina Marie G (1)	+ -

Bushy, Angeline (1)	+ -
Camacho Ferreira, Katherine (1)	+ -
Caputo, Michael (1)	+ -
Carbonell, Abigail (1)	+ -
Carter, William (1)	+ -
Chakrabarti, Ratna (1)	+ -
Cole, Alexander (1)	+ -
Conner, Norma (1)	+ -
Cummins, Andi J (1)	+ -
Curtis, David (1)	+ -
DaSilva, Thiago Gaspar (1)	+ -
Dickie, Mark (1)	+ -
Dobopam Chakrabarti, Dr (1)	+ -
Gerking, Shelby (1)	+ -
Haiduven, Donna (1)	+ -
Jewett, Mollie (1)	+ -
Khaled, Annette (1)	+ -
Pease, Brittany (1)	+ -
Plemmons, William (1)	+ -
Rollins, David (1)	+ -
Roy, Joyashree (1)	+ -
SCHULTE, ALFONS (1)	+ -
Samajpati, Shreejata (1)	+ -
Schreiber, Melissa (1)	+ -
Schulte, Alfons (1)	+ -

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