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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: 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: ELUCIDATING THE MOLECULAR PATHWAY OF ATYPICAL PLASMODIUM FALCIPARUM CDK-RELATED KINASES THROUGH SUBSTRATE CHARACTERIZATION.
Creator: 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.
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Date Issued: 2015
Identifier: CFH0004861, ucf:45486
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH0004861

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 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: 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: 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: 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: VESICLE TARGETING IN PLASMODIUM FALCIPARUM: THE IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF PLASMODIUM FALCIPARUM FAMILY OF SNARE PROTEINS.
Creator: Ayong, Lawrence, Chakrabarti, Debopam, University of Central Florida
Abstract / Description: 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
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Date Issued: 2009
Identifier: CFE0002852, ucf:48053
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0002852

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: 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: 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: MICRO-SPECTROSCOPY OF BIO-ASSEMBLIES AT THE SINGLE CELL LEVEL.
Creator: Kera, Jeslin, Chakrabarti, Debopam, Schulte, Alfons, University of Central Florida
Abstract / Description: In this thesis, we investigate biological molecules on a micron scale in the ultraviolet spectral region through the non-destructive confocal absorption microscopy. The setup involves a combination of confocal microscope with a UV light excitation beam to measure the optical absorption spectra with spatial resolution of 1.4 ?m in the lateral and 3.6 ?m in the axial direction. Confocal absorption microscopy has the benefits of requiring no labels and only low light intensity for excitation...
Show moreIn this thesis, we investigate biological molecules on a micron scale in the ultraviolet spectral region through the non-destructive confocal absorption microscopy. The setup involves a combination of confocal microscope with a UV light excitation beam to measure the optical absorption spectra with spatial resolution of 1.4 ?m in the lateral and 3.6 ?m in the axial direction. Confocal absorption microscopy has the benefits of requiring no labels and only low light intensity for excitation while providing a strong signal from the contrast generated by the attenuation of propagating light due to absorption. This enables spatially resolved measurements of single live cells and bio-molecules with less than 10^9 molecules in the probe volume. Employing a multichannel detection system, the absorption spectrum of hemoglobin in a single red blood cell is measured on the timescale of seconds. We also extend the spectral range from the visible range to the experimentally more challenging ultra-violet region where characteristic absorption bands of bio-molecules are observed. Exploiting the ultra-violet range, amino acids, nucleic acids solutions, and plant cells are investigated. We measure the spatially resolved absorption spectra at the nucleus of an onion cell and cytoplasm to probe DNA base-pair absorption. Small variations in our micro-absorption data are seen around 260 nm, possibly due to the abundance of DNA in the nucleus. This thesis contributes to the goal of spectroscopic identification of spatial heterogeneities at the single cell level and the label-free detection of proteins and nucleic acids.
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Date Issued: 2017
Identifier: CFH2000356, ucf:45905
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFH2000356

Title: A Solid Phase Assay for Topoisomerase I interfacial Poisons and Catalytic Inhibitors.
Creator: Cyril Sagayaraj, Vidusha, Muller, Mark, Zhao, Jihe, Chakrabarti, Debopam, University of Central Florida
Abstract / Description: We report a mechanism based screening technique to rapidly identify eukaryotic topoisomerase I targeting agents. The method is based on genetic tagging of topoisomerase I to immobilize the enzyme on a solid surface in a microtiter well format. DNA is added to the wells and retained DNA is detected by Picogreen fluorescence. Compounds that result in an increase in Picogreen staining represent potential topoisomerase interfacial poisons while those that reduce fluorescence report catalytic...
Show moreWe report a mechanism based screening technique to rapidly identify eukaryotic topoisomerase I targeting agents. The method is based on genetic tagging of topoisomerase I to immobilize the enzyme on a solid surface in a microtiter well format. DNA is added to the wells and retained DNA is detected by Picogreen fluorescence. Compounds that result in an increase in Picogreen staining represent potential topoisomerase interfacial poisons while those that reduce fluorescence report catalytic inhibitors; therefore, the solid phase assay represents a 'bimodal' readout that reveals mechanisms of action. The method has been demonstrated to work with known interfacial poisons and catalytic inhibitors. In addition to specific topoisomerase targeting drugs, the method also weakly detects other relevant anticancer agents, such as potent DNA alkylating and intercalating compounds; therefore, topoisomerase I HTS represents an excellent tool for searching and identifying novel genotoxic agents. This method is rapid, robust, economical and scalable for large library screens.
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Date Issued: 2011
Identifier: CFE0004473, ucf:49304
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004473

Title: Discovery and characterization of antimalarial compounds with novel cellular mechanisms of action.
Creator: Roberts, Bracken, Chakrabarti, Debopam, Jewett, Travis, Self, William, University of Central Florida
Abstract / Description: Malaria kills over 500,000 people each year and over a third of the global population is at risk of infection. Though the human race has been fighting the malaria war for over 4,000 years and we have made great strides in eliminating malaria from many countries, we are treading on the edge of what could be another malaria epidemic primarily due to widespread drug resistance. There are documented cases of resistance for every known antimalarial in use today, including Artemisinins. It is...
Show moreMalaria kills over 500,000 people each year and over a third of the global population is at risk of infection. Though the human race has been fighting the malaria war for over 4,000 years and we have made great strides in eliminating malaria from many countries, we are treading on the edge of what could be another malaria epidemic primarily due to widespread drug resistance. There are documented cases of resistance for every known antimalarial in use today, including Artemisinins. It is critical that we open a new window of discovery in development of next generation antimalarials that circumvent current resistance paradigms. These compounds must attack new targets, have different speeds of action, and ideally possess powerful transmission blocking potential if they are to be successful antimalarial candidates. Screening endeavors historically focused on either synthetic or natural product libraries. Recent efforts have focused on combining privilege elements of natural products into synthetically tractable compounds to create hybrid libraries. To discover novel antimalarial pharmacophores, we have screened natural products derived from marine biodiversity as well as natural product-inspired synthetic libraries. Our phenotypic screening of 3,164 marine natural products from the Harbor Branch Oceanographic Institute, 56 high density combinatorial natural product based libraries from the Torrey Pines Institute for Molecular Studies, alkaloid, terpene, and macrocyclic libraries from Memorial Sloan Kettering Cancer Center, and 594 natural productinspired compounds from Asinex have identified several new selective antiplasmodial hit chemotypes. iv In this study, we have focused on compounds that exhibit cellular actions differing from current antimalarials. Two of the scaffolds, UCF 201 and 501, a spirocyclic chromane and a nitroquinoline, respectively, act early in the development cycle and block invasion. The alkaloid derived compound M03 blocks egress. UCF 501 cures malaria in the rodent model and significantly inhibits stage V gametocytogenesis. Given that discovery of transmission blocking agents are a priority in the malaria elimination strategies, this result is significant. This work is of high impact as it addresses a critical need in the field- next generation antimalarial scaffolds for malaria therapy and elimination campaign.
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Date Issued: 2017
Identifier: CFE0006785, ucf:51815
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0006785

Title: Identification of Novel Antimalarial Scaffolds From Marine Natural Products.
Creator: Roberts, Bracken, Chakrabarti, Debopam, Jewett, Travis, Self, William, University of Central Florida
Abstract / Description: Malaria, the disease caused by Plasmodium sp., claims the lives of over 1 million people every year, with Plasmodium falciparum causing the highest morbidity. Rapidly acquiring drug resistance is threatening to exhaust our antimalarial drug arsenal and already requires the utilization of combination drug therapy in most cases. The global need for novel antimalarial chemical scaffolds has never been greater.Screening of natural product libraries is known to have higher hit rates than synthetic...
Show moreMalaria, the disease caused by Plasmodium sp., claims the lives of over 1 million people every year, with Plasmodium falciparum causing the highest morbidity. Rapidly acquiring drug resistance is threatening to exhaust our antimalarial drug arsenal and already requires the utilization of combination drug therapy in most cases. The global need for novel antimalarial chemical scaffolds has never been greater.Screening of natural product libraries is known to have higher hit rates than synthetic chemical libraries. This elevated hit rate is somewhat attributed to the greater biodiversity available in natural products. Marine life is the most biodiverse system on the planet, containing 34 of the 36 known phyla of life, and is expected to be a rich source of novel chemotypes. In collaboration with the Harbor Branch Oceanographic Institute in Ft. Pierce we have screened a library of over 2,800 marine macroorganism peak fractions against Plasmodium falciparum using the SYBR green I fluorescence-based assay. In this screening process we have identified six compounds from five novel chemical scaffolds all of which have low micromolar to submicromolar IC50 values and excellent selectivity indices. Additionally, one of these chemical scaffolds, the bis(indolyl)imidazole, was selected for further in vitro pharmacological and structure-activity relationship (SAR) profiling, key steps in the challenging process of identifying a new antimalarial drug lead compound.
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Date Issued: 2012
Identifier: CFE0004792, ucf:49748
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0004792

Title: Multi-target high-throughput screening assays for antimicrobial drug discovery.
Creator: Grube, Christopher, Roy, Herve, Chakrabarti, Debopam, Moore, Sean, Koculi, Eda, University of Central Florida
Abstract / Description: The rise of antibiotic resistant microbes (bacteria, fungi, and parasites), combined with the current void of new drugs entering the clinical setting, has created an urgent need for the discovery of new antimicrobials. High-throughput screening (HTS) assays represent a fast and cost-efficient method for identifying new therapeutic compounds and have been the longstanding gold standard for drug discovery. The focus of this dissertation is on the development and implementation of novel...
Show moreThe rise of antibiotic resistant microbes (bacteria, fungi, and parasites), combined with the current void of new drugs entering the clinical setting, has created an urgent need for the discovery of new antimicrobials. High-throughput screening (HTS) assays represent a fast and cost-efficient method for identifying new therapeutic compounds and have been the longstanding gold standard for drug discovery. The focus of this dissertation is on the development and implementation of novel methodologies to increase the throughput of target-based HTS by designing assays that allow multiple drug targets to be probed simultaneously. During my graduate studies, I developed three distinct HTS assays. In each of these assays, drug targets were incorporated into synthetic pathways obeying various reaction topologies (e.g., cyclical, parallel, or linear). Each of these reaction topologies conferred specific advantages and limitations to the individual assays. The first assay reconstitutes the bacterial tRNA-dependent pathway for lipid aminoacylation. This two-step pathway combines a tRNA aminoacylation step catalyzed by an aminoacyl-tRNA synthetase (aaRS), and a transferase step, which transfers the amino acid born by the tRNA onto membrane lipids. aaRSs are essential enzymes in all domains of life and represent longstanding drug targets in pathogenic species. The transferase reaction in the pathway is also an appealing drug target since it impacts the cellular permeability of antibiotics. Inhibitors of this reaction could dramatically increase the efficacy of existing therapeutics. The second assay I developed also targets aaRSs, but utilizes a parallel topology that permits the probing of the synthetic and editing activities of up to four aaRSs simultaneously. The third assay utilizes a linear topology that reconstitutes the entire purine salvage pathway from Plasmodium falciparum. Because parasites are unable to synthesize purines de novo, this pathway represents an appealing target for novel antimalarials. Pilot screens using this assay revealed inhibitors for multiple enzymes in the pathway, validating the design of the system. This body of work aims to shift the current paradigm of single-target systems that have historically dominated the HTS field, toward multi-target designs that can be used to more efficiently screen compound libraries against essential pathways in pathogenic microbes.
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Date Issued: 2019
Identifier: CFE0007642, ucf:52469
Format: Document (PDF)
PURL: http://purl.flvc.org/ucf/fd/CFE0007642

malaria (8)	+ -
Plasmodium (5)	+ -
falciparum (4)	+ -
Malaria (3)	+ -
Plasmodium falciparum (3)	+ -

phosphoproteomics (3)	+ -
plasmodium (3)	+ -
Antimalarial (2)	+ -
PfPK5 (2)	+ -
PfPK6 (2)	+ -
antimalarial (2)	+ -
drug discovery (2)	+ -
kinase (2)	+ -
APQ (1)	+ -
Absorption and Transmission (1)	+ -
Amino Acids (1)	+ -
Aminoacyl-tRNA synthetase (1)	+ -
Anti cancer compounds (1)	+ -
Antimalarial drug discovery (1)	+ -
Confocal Microscope (1)	+ -
Cristhian Valor (1)	+ -
DNA binding proteins (1)	+ -
Dd2 (1)	+ -
Diacylglycerol (1)	+ -
Diacylglycerol Kinase (1)	+ -
Drug Discovery (1)	+ -
Drug discovery (1)	+ -
Drug resistance (1)	+ -
Eukaryotic topoisomerase I (1)	+ -
FIKK (1)	+ -

ETD Collection (9)	+ -
Honors Collection (9)	+ -

Chakrabarti, Debopam (18)	+ -
University of Central Florida (18)	+ -
Jewett, Travis (2)	+ -
Roberts, Bracken (2)	+ -
Self, William (2)	+ -

Alvarado, Stephenie (1)	+ -
Ayong, Lawrence (1)	+ -
Balde, Zarina Marie G (1)	+ -
Camacho Ferreira, Katherine (1)	+ -
Carbonell, Abigail (1)	+ -
Chakrabarti, Ratna (1)	+ -
Cole, Alexander (1)	+ -
Cummins, Andi J (1)	+ -
Curtis, David (1)	+ -
Cyril Sagayaraj, Vidusha (1)	+ -
Grube, Christopher (1)	+ -
Jewett, Mollie (1)	+ -
Kera, Jeslin (1)	+ -
Khaled, Annette (1)	+ -
Koculi, Eda (1)	+ -
Moore, Sean (1)	+ -
Muller, Mark (1)	+ -
Pease, Brittany (1)	+ -
Roy, Herve (1)	+ -
Schreiber, Melissa (1)	+ -
Schulte, Alfons (1)	+ -
Segarra, Daniel (1)	+ -
Sullenberger, Catherine (1)	+ -
Valor, Cristhian (1)	+ -
Yap, Jessica (1)	+ -

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