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Health Challenge Past Interns: 2009

Natural Sciences

Abdallah Bukari, 2010, Chemical Engineering

Independent Study of Nanoparticles for the Delivery of TB Drugs

Princeton University, Princeton NJ

Robert Prud'homme

"The purpose of this internship was to research with the Prudíhomme group a novel drug delivery system for the administration of tuberculosis therapeutics. This delivery system, which can be incorporated into current therapeutic regimens, will quickly and efficiently kill off a large percentage of the infection in a short time, reducing the duration of therapy and frequency of dosing needed. The improved performance of this regimen is derived from the design of the NP system.  Usually, there is little control over the release and biodistribution of combination therapy drugs in circulation.  A NP system which co-encapsulates active drugs and is efficiently taken up into macrophages ensures controlled release of both drugs at the infection site and thus more efficient killing of the bacteria. We utilized a Flash Nano-Precipitation (FNP), a continuous process developed by the Prudíhomme group for the encapsulation of highly lipophilic active pharmaceutical ingredients in polymer protected NPs, to co-encapsulate a currently marketed anti-tubercular drug (rifampicin) and recently discovered peptidoglycan-inhibiting drugs (SQ641, SQ922, SQ997). Active targeting techniques, such as receptor-specific ligands, were incorporated into the NP design to provide control over biodistribution of NPs. NPs were tested for stability and size distribution over time. Additionally, we tried methods to concentrate suspended NPs in solution." (See presentation.)

Erick Kazarian, 2012, Molecular Biology

Perioperative Pain Protocol and Infection Diagnostics

Lankenau Institute for Medical Research, Pennsylvania

"My internship position was responsible for assisting Dr. Deirmengian in what turned out to be multiple research opportunities. The two primary focuses were to gather all past and present patient information for a long term study to assess the success of pain management on revision hip and knee replacement surgeries. The Second research proposal was to gather information regarding the success of current ways to detect infection in pre-surgical patients, and compare the current statistics to the new system that Dr. Deirmengian has discovered. My primary responsibility in the second proposal was to gather the current information and present it in a way that could be compared against the statistics that the newer system produces." (See presentation.)

Zoe Li, 2012, Molecular Biology

Internship: Histone Modifications in Plasmodium Falciparum

Organization : Princeton University, Princeton, NJ

Adviser: Manuel Llinas

"Plasmodium falciparum causes malaria in over 300 million people each year yet little is understood about the parasiteís transcriptional regulation. In the parasiteís asexual stage, the parasite undergoes a 48-hour cycle of red blood cell (RBC) invasion, which causes the clinical symptoms of malaria. Only a handful of transcription factors have been identified in P. falciparum so epigenetic factors may play a significant role in transcriptional regulation.  Chromatin modification, predominantly via methylation or acetylation marks, is one form of epigenetic mark and has been shown to be involved in transcriptional regulation in many organisms. The marks are classically found on nucleosomes, which are comprised of DNA and histones, the proteins around which DNA are bound.  As an example, certain histone post-translational modifications (PTMs) such as lysine trimethylations and acetylations have been found to be strongly associated with silent or active genes. Through a mass spectrometry-based approach, we have identified a novel histone PTM, H3H39me1."

"My work centers on using chromatin immunoprecipitation against this modified histone and the DNA that it binds to allow us to map where this modification occurs genome-wide.  Ultimately, the goal is to correlate the presence of this mark with active or silent genes, thus further establishing a correlation between chromatin state and gene expression in P. falciparum." (See presentation.)

Julie Sung, 2011, Molecular Biology

Internship: Investigating Slow-Growth Inducing Fosmids of B. Pseuodomallei

Organization: Genomic Institute of Singapore, Singapore

Advisers: Jason Kreisberg and Patrick Tan

"The bacteria, Burkholderia pseuodmallei, which causes the disease melioidosis, is a disease that is endemic to Thailand and Northern Australia.  The bacteria live in the soil, are highly resistant to antibiotics, and cause respiratory infection.  If not treated properly, the fatality rate is 50%.  Melioidosis has been a neglected disease but has potential as a bioterrorism weapon."
"Certain fosmids made from the DNA of B. pseudomallei that when inserted into E. coli bacteria, cause slow growth in the E. coli bacteria.  In order to test if the fosmids were causing the slow growth, the E. coli were cured of the fosmid to see if they returned to wild-type growth.  Electrocompetent E. coli bacteria were also transformed and tested to see if they acquired the slow growth phenotype.  As a control, E. coli were also grown while maintaining the presence of the fosmid.  Even though most of the E. coli were able to be cured and transformed, the slow growth phenotype was difficult to maintain.  This could be due to the E. coli overcoming the cause of slow growth over time."
"One fosmid was able to be maintained, and transposon mutagenesis was performed on this fosmid to see if the wild type rate of growth could be recovered.  While this did not occur, two of the transposed fosmids grew more slowly than the original 15B23 strain.  The 15B23 fosmid is currently being sequenced and perhaps then a gene or genes can be identified that are responsible for the slow growth." (See presentation.)

Jonathan Turner, 2010, Molecular Biology

Preliminary Characterization of a Putative Flagellar Localization Complex in Pseudomonas Aeruginosa

Princeton University, Princeton, NJ

Zemer Gitai

"Studies of bacterial protein localization give insight into the fundamentals of intracellular structure and provide opportunities for developing novel antimicrobial therapeutics. Studies of polar flagellar localization in the opportunistic pathogen Pseudomonas aeruginosa have shown that a polar-localized protein, FlhF, is responsible for localizing the flagellum to the pole. Motility screens of a P. aeruginosa transposon mutant library isolated three proteins necessary for polar localization of FlhF and, thus, the flagellum." 

"These proteins, PA0406, PA2982, and PA2983, are homologous to the components of the conserved TonB energy transduction complex, suggest- ing a flagellar localization complex with functional homology to the TonB complex. Current work is focused on demonstrating complex formation by co-precipitation of the component proteins, and on establishing a system for direct observation of the dynamics of related proteins, including FlhF, during the process of Pseudomonas pathogenesis in the amoeba Dictyostelium discoideum and human tissue culture." (See presentation.)

Emma Yates, 2011, Chemistry

Emma Yates

Direct Approach to the Functionalized Propellane Core of Pleuromutilin, Towards Possible New Antibiotics Against Tuberculosis

Princeton University, Princeton, NJ

Erik Sorensen

"Pleuromutilin is a natural product with antibiotic activity against tuberculosis, but that has never been approved for human oral administration because of poor pharmacokinetic properties.  We have been synthesizing the first pleuromutilin derivatives that include changes to the architectually novel propellane
core of the molecule, in hopes of creating analogs with improved activity and metabolic stability sufficient to allow them to be explored from a clinical perspective. This summer I worked on optimizing our route towards our key intermediate compound and developed a novel in situ silyation protocol which appears to be generally applicable in the stoichiometric Nozaki-Hiyama-Kishi reaction.  Though an adaption of this protocol we are now able to gain access to enantiopure material, and work continues in our laboratory towards synthesizing and testing racemic and enantiopure pleuromutilin derivatives." (See presentation.)

Neal Yuan, 2010, Molecular Biology

Elucidating the Significance of ApiAP2 Transcription Factors in the Malaria Parasite Plasmodium Falciparum

Princeton University, Princeton, NJ

Manuel Llinas

"Despite its widespread impact, Plasmodium falciparum, the causative agent of the most virulent form of malaria, is a parasite whose biology is one of the least understood among infectious agents. The parasite exhibits a highly adapted lifestyle that involves a 48-hour intraerythrocytic development cycle in humans during which the parasite undergoes significant morphological transformations. Such a complex developmental process necessitates a sophisticated network of genetic regulation that has yet to be fully understood.  Even so, the promoters and transcription factors necessary for transcriptional regulation have for the most part remained elusive. "
"Previous research has identified for the first time a putative family of P. falciparum transcription factors, the Apicomplexan AP2s (ApiAP2s). My project has employed a multifaceted approach to help further elucidate their role in gene regulation. In order to clarify the in vivo activity of ApiAP2s, I am studying the stage specific importance of ApiAP2 proteins by gene knockdown as well as a suspected ApiAP2 DNA-binding motif using luciferase expression assays.  These experiments will build on in vitro and computational work with the goal of elucidating in vivo some of the first minimal transcriptional networks in P. falciparum.  As an additional project, I have used protein binding microarrays to characterize the in vitro DNA-binding preferences of a previously uncharacterized ApiAP2, uniquely found in the malaria parasite Plasmodium vivax.  Studying the regulatory targets of this ApiAP2 may help reveal genes that are specific to this parasites pathogenicity.  Together, these experiments will help further articulate the importance of ApiAP2 transcription factors, while also opening many new avenues for further inquiry."

Alex Zozula, 2010, Chemistry

Investigating the Mechanism of Naphthalene 1,2-Dioxygenase in Whole Cells

Princeton University, Princeton, NJ

John Groves

"The hydroxylation of unactivated hydrocarbons is one of the most difficult chemical reactions known. Yet despite its complexity, many organisms are able to use hydrocarbons as their sole energy source, and an wide array of enzymes have evolved to facilitate these reactions. Naphthalene 1,2-dioxygenase (NDO), a Rieske protein homologue that catalyzes the stereoselective oxidation of naphthalene to (1R,2S)-1,2-dihydronaphthalene-1,2-diol is one such enzyme. The cis-1,2-dihydroxylation of olefins and arenes carried out by NDO is of immense synthetic interest; NDO has the ability to catalyze the creation of two chiral centers in a reaction that proceeds almost to completion."
"Despite all the work on NDO in recent years, the mechanisms for itsdi- and mono-oxygenations have not been elucidated. In vitro work had suggested that mono-oxygenation proceeds through a radical pathway, indicative of the formation of a high-valent Fe(V)-oxo-hydroxo species before substrate attack. I have demonstrated that the same mechanism is at work in vivo using an even wider range of diagnostic substrates than used in vitro. As further evidence of the formation of a Fe(V)- oxo-hydroxo species, I performed studies using 18O labeled water and showed that there is non-trivial exchange into the iron center based on product analysis; oxo-hydroxo tautomerization, expected for a high- valent Fe(V) species, would result in this solvent exchange. I have also synthesized a range of selectively deuterated materials to serve as intramolecular probes of NDO's kinetic isotope effect. This represents one of the first instances of a KIE being determined solely by product analysis for an in vivo system. (See presentation.)