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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.
Show less - Date Issued
- 2007
- Identifier
- CFE0001780, ucf:47254
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001780
- Title
- UNDERSTANDING THE ROLE OF PLASMODIUM FALCIPARUM VAMP8 SNARE HOMOLOGUE.
- Creator
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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.
Show less - Date Issued
- 2013
- Identifier
- CFH0004525, ucf:45157
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004525
- Title
- VESICLE TARGETING IN PLASMODIUM FALCIPARUM: THE IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF PLASMODIUM FALCIPARUM FAMILY OF SNARE PROTEINS.
- Creator
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Ayong, Lawrence, Chakrabarti, Debopam, University of Central Florida
- Abstract / Description
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Proteins of the SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) super-family have been characterized as playing an essential role in vesicle targeting and fusion in all eukaryotes. The intracellular malaria parasite Plasmodium falciparum exhibits an unusual endomembrane system that is characterized by an unstacked Golgi apparatus, a developmentally induced apical complex, and various organellar structures of parasite origin in the infected host cells. How malaria...
Show moreProteins of the SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) super-family have been characterized as playing an essential role in vesicle targeting and fusion in all eukaryotes. The intracellular malaria parasite Plasmodium falciparum exhibits an unusual endomembrane system that is characterized by an unstacked Golgi apparatus, a developmentally induced apical complex, and various organellar structures of parasite origin in the infected host cells. How malaria parasites target nuclear-encoded proteins to these novel compartments is a central question in Plasmodium cell biology. Ultrastructural studies elsewhere have implicated the participation of specialized vesicular elements in transport of virulence proteins, including various cytoadherance and host cell remodeling factors, into the infected erythrocyte cytoplasm. However, little is known about the machineries that define the directionality of vesicle trafficking in malaria parasites. We hypothesized that the P. falciparum SNARE proteins would exhibit novel features required for vesicle targeting to the parasite-specific compartments. We then identified for the first time and confirmed the expression of eighteen SNARE genes in P. falciparum. Members of the PfSNAREs exhibit atypical structural features (Ayong et al., 2007, Molecular & Biochemical Parasitology, 152(2), 113-122). Among the atypical PfSNAREs, PfSec22 contains an unusual insertion of the Plasmodium export element (PEXEL) within its profilin-like longin domain, preceded by an N-terminal hydrophobic segment. Localization analyses suggest that PfSec22 is predominantly a vesicle-associated SNARE of the ER/Golgi interface, but which associates partially with mobile extraparasitic vesicles in P. falciparum-infected erythrocytes at trophozoite stages. We showed that PfSec22 export into host cells occurs via a two-step model that involves extraparasitic vesicle budding from the parasite plasma membrane and fusion with the parasitophorous vacuolar membrane. Export of PfSec22 was independent of its membrane-insertion suggesting that this protein might cross the vacuolar space as a single-pass type IV membrane protein. We demonstrated that the atypical longin domain dictates the steady-state localization of PfSec22, regulating its ER/Golgi trafficking and export into host cells. Our study provides the first experimental evidence for SNARE protein export in P. falciparum, and suggests a role of PfSec22 in vesicle trafficking within the infected host cell (Ayong et al, Eukaryotic Cell, Epub Jul 17, 2009). Next, to define the physiological function of the PfSec22 protein in Plasmodium parasites, we investigated its cognate partners. Using purified recombinant proteins we showed that PfSec22 forms direct binding interactions with six other PfSNAREs in vitro. These included the PfSyn5, PfBet1, PfGS27, PfSyn6, PfSyn16 and PfSyn18 PfSNAREs. By generating GFP-expressing parasites, we successfully localized the SNARE proteins PfSyn5, PfBet1 and PfGS27 to the parasite cis-Golgi compartment. We confirmed the association of PfSec22 with PfSyn5, PfBet1 and PfGS27 in vivo by immunoprecipitation analyses. Our data indicate a conserved ER-to-Golgi SNARE assembly in P. falciparum, and suggest that the malaria Sec22 protein might form novel SNARE complexes required for vesicle traffic within P. falciparum-infected erythrocytes
Show less - Date Issued
- 2009
- Identifier
- CFE0002852, ucf:48053
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0002852
- Title
- IDENTIFICATION OF NOVEL ANTIMALARIALS FROM MARINE NATURAL PRODUCTS FOR LEAD DISCOVERY.
- Creator
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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.
Show less - 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
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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.
Show less - Date Issued
- 2007
- Identifier
- CFE0001879, ucf:47407
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0001879
- Title
- ELUCIDATING THE MOLECULAR PATHWAY OF ATYPICAL PLASMODIUM FALCIPARUM CDK-RELATED KINASES THROUGH SUBSTRATE CHARACTERIZATION.
- Creator
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Segarra, Daniel, Chakrabarti, Debopam, University of Central Florida
- Abstract / Description
-
Plasmodium falciparum, the organism responsible for the most prevalent and most virulent cases of malaria in humans, poses a major burden to the developing world. The parasite is increasingly developing resistance to traditional therapies, such as chloroquine, so the need to determine novel drug targets is more prevalent than ever. One such method involves targeting proteins unique to the malarial proteome that do not have homologues in humans. An especially promising group of targets are...
Show morePlasmodium falciparum, the organism responsible for the most prevalent and most virulent cases of malaria in humans, poses a major burden to the developing world. The parasite is increasingly developing resistance to traditional therapies, such as chloroquine, so the need to determine novel drug targets is more prevalent than ever. One such method involves targeting proteins unique to the malarial proteome that do not have homologues in humans. An especially promising group of targets are protein kinases, which are involved in many different biochemical pathways within the cell. Eukaryotic cell cycle progression is moderated by a family of protein kinases known as the cyclin-dependent kinases (CDKs). These kinases depend on the binding of a cognate regulatory unit (cyclin) in order to enter its activated state. Once activated, these cyclins then mediate phosphorylation events that are crucial to cell cycle advancement . Cyclin Dependent Kinases (CDKs) are common to most eukaryotes and are responsible for regulating the cell cycle of growth and proliferation. Proteins have been previously identified in Plasmodium that have sequence homology to traditional CDK and have a potential function to be classified as "CDK-like" kinases. Three kinases that fit this description are Plasmodium falciparum Kinase 5, 6, and mrk, or MO15- Related Kinase. These kinases are expected to have roles in both malarial growth and regulation of the cell cycle. Bacterial constructs were generated to express and purify recombinant forms of these kinases and potential substrates. Once the potential interactors were isolated, in vitro protein kinase assays were used to validate the interactions to the kinases as substrates. In summary, the study has identified substrates that are directly phosphorylated by PfPK6, and demonstrated that the identified proteins are not directly phosphorylated by PfPK5 and Pfmrk.
Show less - Date Issued
- 2015
- Identifier
- CFH0004861, ucf:45486
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004861
- Title
- IDENTIFICATION OF PLASMODIUM FALCIPARUM PROTEIN KINASE SUBSTRATES AND INTERACTING PROTEINS.
- Creator
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Yap, Jessica, Chakrabarti, Debopam, University of Central Florida
- Abstract / Description
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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.
Show less - Date Issued
- 2012
- Identifier
- CFH0004157, ucf:44829
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004157
- Title
- IDENTIFICATION OF POTENTIAL LEAD ANTIMALARIAL COMPOUNDS FROM MARINE MICROBIAL EXTRACTS.
- Creator
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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.
Show less - Date Issued
- 2013
- Identifier
- CFH0004332, ucf:45035
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004332