Health Challenge Past Interns: 2008
Fatu S. Conteh, 2010, Chemistry
Finding the Biochemical Target(s) of Artemisinin
Manuel Llinas & Dr. Joel Freundlich
Llinas lab, Princeton University
:It's a fact: malaria kills over a million people a year and stands in the way of economic development for many countries where the disease is endemic. Past efforts to eradicate this deadly disease with drug treatments like quinine, chloriquine, and other antimalarials have proven futile as the malaria parasite (Plasmodium Falciparum) has developed resistance to these drugs. However, artemisinin, with no known stable resistance, has been ganarring momentous support from both the non-profit and for-profit sectors as the first line drug treatment for uncomplicated and multi-drug resistant P. Falciparum malaria."
"Currently, according to the World Health Organization, since 2001, a total of 56 countries have adopted one of the WHO recommended artemisinin-based combination therapies, several as first-line treatment and a few as second-line. With this increasing use of Artemisinin comes the threat of the malaria parasite developing resistance to the drug. There is a growing concern in the field of malaria therapy that if or when the parasite develops resistance to artemisinin, like it has done with other antimalarials, no one will know how to combat the resistance because the mechanism of action of artemisinin is uncertain.
"There are two strong competing hypothesis as to how the drug actually kills the parasite. One hypothesis, the multiple targets hypothesis, contends that artemisinin kills the parasite by reacting with the iron in hemin to produce oxygen radicals. The other hypothesis argues that artemisinin acts via a specific protein. My research this past summer was focused on using novel protocols in proteomics to find the protein target(s) of artemisnin, with hopes of disproving one of these two hypothesis. The results I obtained from my protein gels at the end were inconclusive and further experiments are needed to clarify my data."
William Dixon, 2009, Molecular Biology
Characterization of the FlpA Protein and Its Role in Flagellar Placement and Pathogenesis of Pseudomonas aeruginosa
"Over the summer I worked with the bacteria Pseudomonas aeruginosa in the Gitai lab in Princeton, NJ. P. aeruginosa is a gram-negative bacteria that infects immune-challenged humans as well as many model organisms such as C. elegans. It is most dangerous to humans with cystic fibrosis and severe burn victims. To infect, P. aeruginosa uses flagella, a propeller-like tail, and type IV pili, a shorter structure that functions somewhat like a grappling hook. After initial infection, P. aeruginosa forms biofilms for better growth and increased antibiotic resistance. If the flagella or pili are mislocalized, meaning they are not placed at one of the poles of the bacteria, it has been shown the bacterium has reduced infection strength."
"The previous student in the lab where I worked, Natsai Nyakudarika, ran a screen for bacteria with transposon-insertion mutations that affected flagellar function. She looked for bacteria defective in swimming motility, followed by checking these mutants for mislocalization of the protein flhF, a protein responsible for localization of the flagella. She discovered a gene she named flagellar localizing protein A (flpA) that had two mutations responsible for irregular flagellar placement. My goal was to ascertain the role of flpA in flagellar localization."
"In the course of my research, I discovered that multiple genes may be responsible for the defect in flagellar placement. My current focus is to identify which genes direct flagellar placement, how they direct flagellar placement, and what their importance in virulence is. I plan on using the C. elegans organism to determine the virulence levels of mutant P. aeruginosa." (See presentation.)
James Marvel, 2009, Ecology and Evolutionary Biology
Max Planck Institute Field Assistantship
Stephen Pacala, Martin Wikelski
Radolfzell am Bodensee, Germany
"The money I received through this grant was used to cover the costs of conducting senior thesis research this past summer in Germany. I collaborated with Bryson Voirin, a graduate student at the Max Planck Institute for Ornithology who was conducting a pilot project for what is to be his Ph.D. We shared similar goals in what we wanted to get out of this project, so I had significant liberty in shaping how I wanted the experimental design to look. We also worked closely with Martin Wikelski, former Princeton professor and current director of the Max Planck Institute for Ornithology, in addition to collaborating with researchers at local universities."
"The idea behind the project was to use immuno-compromised chickens as sentinel test animals to assess the spatio-temporal abundances of pathogens in a particular environment. This research was originally going to be conducted in California through UC Davis, but it was moved at the last minute to Germany, where a more accessible facility that would better suit our needs was already in place at the Max Planck Institute. The first goal then, was to determine which environments we would be investigating, as our original plan to disperse chickens all along the western United States coastline to examine latitudinal and altitudinal gradients. In the area of Germany where we were working, the landscape was dominated by vast stretches of farmland with isolated patches of forest scattered throughout. Thus, we decided it would be most interesting and practical to see if we could quantify the degree to which anthropogenic habitat fragmentation affected pathogen dispersal."
"Once the sites were selected, we began to approach the logistical aspects of both compromising the birds’ immune systems, and ways to keep them alive and fully exposed to the environment for five days. There are several established methods for immuno-compromising chickens, and after a thorough literature review and discussions with chicken pathologists, we decided the most effective means would be a bursectomy. This is a procedure in which the bursa of fabricius, an organ responsible for immune responses in birds, is removed from the chickens. We traveled to a medical school in Hungary to learn this obscure procedure, as the next closest scientist who knew how to perform the operation lived in Japan. This minimally invasive procedure was performed on day-old chicks, when the tissue was softest, facilitating the organ’s removal."
"After learning how to bursectomize chickens, we then focused our attention on how to raise them and keep them alive outside when they were in such a fragile state. At the institute we established a clean room with filtered air and individual cages where we raised the chickens after they had been bursectomized. We then began to experiment with placing the chickens outside, as no one has ever experimented with taking bursectomized chickens out of a laboratory setting. We established that they were viable, and proceeded to design cages suitable to keep the chickens alive for the time they spent in the preselected environments. The greatest obstacle was heating, as chicks need to be kept at 80 – 90˚F during their first weeks. We collaborated with engineers and local technicians before arriving at the brilliantly simple solution of using a candle. We tried out dozens of candles before finding graveyard candles that burned for 5-days straight."
"Once all of this had been set in place, we were ready to put the chickens out in the environment. We conducted two trials, each involving 81 chickens, divided into 27 cages. We placed these cages in previously established transects of forest, farmland, and swamp. After five days in the field, the chickens were collected and blood samples were taken. I am unfortunately still waiting on the data, but the blood samples just arrived in the United States this week. They will be sent to a lab in UC Davis where they will be analyzed for pathogen load and acute phase proteins. This data will hopefully allow us to understand the spatio-temporal distribution of diseases in a patchy environment, in addition to a quantitative assessment of the stress placed upon each chicken’s immune system. If the data proves informative, a significant aspect of my thesis will be devoted to analyzing how the methodologies we developed may be employed in directing public health policies in different areas. For example, my collaborator already has plans to employ these methods in the tropics, to assess variability of pathogen virulence." (See presentation.)
Agatha O. Offorjebe, 2009, Ecology and Evolutionary Biology
Urban Malaria in Sub-Saharan Africa
Professor Simon Levin
"Based on the most recent Nigeria Demographic Health Survey (DHS) in 2003, bed net usage is much lower in urban areas than in rural areas. This trend is the opposite of what is found in most other malaria endemic African nations. The objective of this research project was to discover why this was the case and how this trend could be reversed. (See presentation.)
Richmond Adusei Owusu, 2009, Chemistry
The search for a new tuberculosis drug: targeting the Mycobacterium tuberculosis malate synthase
Dr. Joel Freundlich/ Prof. Erik Sorensen
"My research started with the synthesis and chemical analysis of various analogs of phenyldiketoacids (PKBAs). Upon successful synthesis and characterization, I submit the compounds to collaborators at the Texas A&M University for testing of the inhibitory activity of the compounds in malate synthase enzyme and M. tuberculosis whole-cell assays. Compounds showing high inhibition of the malate synthase in both assays will be further tested in animal models. The goal is to make appropriate modifications to the parent compound in order to increase its inhibitory activity against the enzyme. The finding if a highly potent analog will ultimately lead to the filing for patent rights to the compound and an ultimate development of the compound into a new drug for tuberculosis. This research should be useful in helping solve the current problem of increasing tuberculosis that the world faces. In a broad scientific perspective, the research will also contribute enormously to the understanding of disease-causing microorganism, their mechanism of persistence and adaptations that ultimately help these pathogens develop resistance to medications. Such an understanding will help combat microorganism resistance to antibiotics and pave a way for a breakthrough against pathogenic diseases."
Peter Shengyang Wu, 2009, Chemistry
Summer Research in Virology and Systems Biology
Princeton University Department of Molecular Biology and Chemistry
"Current antiviral drugs work through one of the five following strategies 1) by blocking viral attachment to the host cell, 2) by disrupting replication of viral DNA/RNA, 3) by halting transcription of viral DNA or translation of viral mRNA, 4) or by preventing viral release from the host cell. However no antiviral drug yet takes advantage of the fact that viruses are almost completely dependent on host cell enzymes to produce key precursors for their growth, such as lipids for viral envelopes or amino acids for viral proteins."
"Drugs that inhibit specific human metabolic enzymes, such as statin drugs and methotrexate, have long been used as effective therapies for high cholesterol and leukemia respectively. Their success demonstrates that regardless of the cause of an illness, many diseases can be effectively managed at the level of metabolism. We assert that viral infections too may be viewed and treated, in part, as metabolic disorders. Inhibiting host-cell enzymes critical to virus replication is a promising new way to approach antiviral drug treatment. The difficulty however is to determine which specific enzymes in the set of over three-thousand known reactions in the human metabolism are critical to virus replication. However, not all the metabolic fluxes can be quantified through direct measurement, and those that cannot must be estimated in other ways. Flux balance analysis (FBA) is a computational technique used to predict the optimal capabilities of large-scale networks at steady state. It has been used, for example, to accurately predict the growth rate of a E. coli culture and the viability of E. coli gene knockouts.
"Flux balance analysis on the entire human metabolism is a new, unexplored, area. In 2006, Duarte et al. presented a global reconstruction of the human metabolism based on information from genomic data and published literature. To date it is the most comprehensive data set of its kind.
In my summer work, I apply FBA to the human model in an attempt to quantify the effect of human cytomegalovirus (HCMV) infection on host cell metabolic fluxes. Ichoose to study HCMV because of the large amount of transcriptional and metabolomic data available to our group that can be used to verify predictions made by the flux balance model. However the method described may be applied to any virus given an accurate description of its chemical composition."
"I use FBA to answer the following questions: Given a set of experimentally observed fluxes into and out of a host cell, what is the maximum number virus particles that can be produced? What single and double-knockouts of the host-cell enzymes will halt viral replication? What specific reactions are upregulated during viral replication compared to a mock infected cell? Enzymes catalyzing reactions that are both critical and upregulated comprise a promising set of antiviral drug targets. Thanks, huge thanks, to the Grand Challenges in Health for funding my summer work!" (See presentation.)
James Yan, 2009, Chemistry
Studying Siderophore-Mediated Bacterial Iron Acquisition
"Iron is essential to life. All living organisms, including microbes, require iron to live and their ability to acquire iron is extremely important. My internship was focused on researching a mechanism of iron acquisition utilized by bacteria and fungi. Bacteria and fungi can produce molecules called siderophores when they are low on iron. They send the siderophores out into the environment, where they bind to iron. The microbes can then take up the iron-bound siderophores as a source of iron. Siderophores are essential for bacteria living in low iron environments, such as when they are infecting people. The ability to acquire iron is essential for the survival and virulence of the bacteria, and this mechanism is used by such bacteria as the ones that cause pneumonia and tuberculosis."
"My research studied the mechanism of how one specific siderophore, acinetoferrin, acquired iron from a human iron transport protein, transferrin. Transferrin is the primary iron transport molecule in humans, and could provide a source of iron for infecting bacteria. Discovering the mechanism of siderophore iron acquisition could provide insight in combating bacterial infections and provide a novel way of dealing with antibiotic-resistant strains."
"Our main approach to studying the siderophore was studying the interaction between transferrin and an analogue of the siderophore. Our hypothesis is that the siderophore displaces a synergistic anion in the protein iron-binding site, then chelates the iron and escapes the binding site by undergoing a conformational change. Our analogue in theory should be able to displace the anion, but lacks the necessary residues to chelate the iron completely. If we can see the analogue complexed with the iron in the protein binding site, then it supports our hypothesis that the first step for iron acquisition is displacement of the anion. We tested this by attempting to iron-load transferrin with the analogue and analyzing the protein with gel electrophoresis and UV-vis spectroscopy. Additionally, we are attempting to use mass spectrometry to find the product of a photochemical reaction of the analogue when it is complexed with iron. Future work also involves studying another analogue." (See presentation.)