2012 Summer Researchers Participants-HHMI Undergraduate Program - Carnegie Mellon University

2012 HHMI Summer Researchers Participants

Victor Bass

Victor Bass
Mentor: Jon Minden

Analyzing the Effects of L-Asparaginase on Two Leukemia Cell Line Proteomes using 2D DIGE

L-asparaginase (LA) is the main chemotherapeutic drug used to treat childhood acute lymphoblastic leukemia (ALL). It works by converting asparagine, an auxotrophic marker for certain types of leukemia cells, to aspartic acid. Even though it is very effective against ALL, LA is rarely used against other types of leukemia such as acute myeloid leukemia (AML). Some cell lines of AML have been shown to be sensitive to LA, while some others are resistant. This project aims to identify possible LA-resistance mechanisms in AML cells, which could then be used to increase the effectiveness of the drug for AML treatment. Our approach uses 2-Dimensional Difference Gel Electrophoresis (2D DIGE) to detect candidate proteins. In DIGE, two cell extracts are tagged with two different fluorescent dyes and then electrophoresed on the same 2D gel. Difference proteins are detected by fluorescence imaging and can be excised and identified by mass spectrometry. DIGE can be combined with our Structured Illumination Gel Imager to find extremely low abundance difference proteins. Using DIGE, we compared the proteomes of two AML cell lines, one sensitive to LA and one resistant, before and after treatment with LA. Even though both of our cell lines are myeloid leukemia cell lines, we found very large differences in their proteomes. This suggests that the difference in drug sensitivity may be due to differences in metabolism between the cell lines. Additionally, no protein expression changes were observed in the resistant line after drug treatment. Several difference proteins were found in the sensitive line and were excised, but we did not have time to carry out mass spectrometry. This work was done in collaboration with Dr. Jenny Jun and Professor Mark Minden of the Ontario Cancer Institute at the University of Toronto.

Alexander Boscia

Alexander Boscia
Mentor: Stephanie Tristram-Nagle

Secondary Structure Determination of Lentivirus Lytic Peptide-2 Variants in HIV-1 and Tcell Membrane Mimics Using Circular Dichroism Spectroscopy

The envelope glycoprotein (Env) of HIV-1 consists of two subunits: gp120 and gp41. These subunits play a major role in the fusion of HIV-1 with Tcell membranes in vivo. The lentiviral lytic peptide-2 (LLP-2) of gp41 has been of particular interest in the Nagle/Tristram-Nagle Laboratory.  Four different membrane mimics of HIV and Tcell (HIV, HIV-neg, HIV-chol, Tcell) as well as five LLP-2 variants (WT, MX2, CracWT+pal, CracWT-pal, CracMX2+pal) were used. The membrane mimics were designed to assess the importance of charge and cholesterol in the membrane mimics; the peptide mutants were designed to assess the influence of charge and the presence of a CRAC, a cholesterol-binding peptide motif, on the membrane mimics. During the school year, I aided in data collection at the Cornell High Energy Synchrotron Source (CHESS). X-ray scattering was used to characterize the effect of the peptide variants on the membrane mimics. I assisted in the analysis of the wide-angle X-ray scattering (WAXS) data using a theory developed in the Nagle/Tristram Nagle lab to calculate SXray, an order parameter, for each membrane. SXray is a measure of the in-plane chain ordering of a lipid bilayer (the greater the value, the more ordered the membrane). This summer I focused on structural characterization of LLP-2 itself using circular dichroism (CD) spectroscopy. CD refers to the fact that light may be circularly polarized clockwise or counterclockwise. Certain molecules absorb clockwise and counterclockwise circularly polarized light differently, resulting in absorption curves that, when analyzed can estimate the proportions of a protein’s secondary structure. Analysis of our CD data through Dichroweb revealed that LLP-2 is largely helical.

Catherine Byrd

Catherine Byrd
Mentor: Chien Ho

Role of Woolly Mammoth Central Cavity Hemoglobin Residues in Oxygen Delivery

Human adult hemoglobin (Hb A) increases its oxygen-binding affinity at low temperatures. During hypothermic treatments, the hemoglobin in red blood cells cannot effectively deliver oxygen to the tissues due to low body temperatures. A preliminary biochemical-biophysical study found that the oxygen affinity of woolly mammoth hemoglobin (Hb WM) is much less dependent on temperature, thus allowing the hemoglobin to function in the icy conditions of the Arctic. Therefore, study of Hb WM could provide new insights in designing hemoglobin-based oxygen carriers (HBOCs) for treating patients under therapeutic hypothermia. Amino acid sequence comparisons among the hemoglobins from Asian elephant, woolly mammoth, and Hb A suggest that the mammoth-specific /101Gln residue, located in the central cavity of the Hb molecule, could be responsible for the reduced temperature effect in rHb WM by creating additional proton-linked chloride binding sites. Mutations that change the charges in the central cavity should affect the oxygen-binding affinity. Using site-specific mutagenesis, we have constructed several mutants of rHb WM with a /101 and a α5 substitution. Oxygen-binding properties of these mutants are measured under various experimental conditions of temperature and allosteric effectors (Cl- and IHP). 1H-NMR spectra in such conditions are collected to detect changes in the tertiary or quaternary structures of the rHbs. The results have shown that both chloride and IHP play a role in lessening the temperature effect on oxygen affinity in rHb WM and its mutants.

Alexander Chen

Alexander Chen
Mentor: Alison Barth

Paige Davison

Paige Davison
Mentor: Brooke McCartney

Susan Hannes

Susan Hannes
Mentor: Newell Washburn

Testing Cytokine-Neutralizing Gels in a Mouse Model of Inflammatory Bowel Disease: Applications to Colitis-Associated Colon Cancer

Colon Carcinoma, more commonly referred to as colorectal cancer, is a leading cause of cancer-related deaths within the United States. Though there is no single cause, it has been observed that patients affected by chronic inflammatory disorders have an increased incidence of cancer development at lesions of inflammation within the mucoso-layer of the colon and rectum. Consequently, the development and treatment of inflammatory conditions such as inflammatory bowel disease (IBD), a chronic immunoinflammatory response leading to colitis-associated colon cancer (CACC), has become a focal point in current related research. The association between chronic inflammation in IBD and increased risk of cancer has previously been explored with respect to abnormal expression of chronic inflammation-associated human tumor antigen, MUC1 (Beatty et.al). In addition to abnormal MUC1 expression, high levels and dysregulation of pro-inflammatory cytokine expression, tumor necrosis factor-α (TNF-α) during chronic inflammation, results. It has been theorized that minimizing inflammation in IBD colons, can alter the tumor-promoting microenvironments of chronic inflammation, potentially reducing the progression of IBD and development of colon cancer (Beatty, et.al). To explore the effect of reducing chronic inflammation, the use of an anti-inflammatory conjugate gel will be administered in vivo to a MUC1+/IL-10-/- mouse model, generated by crossing IL-10-/- mice with MUC1 transgenic mice, which results in the development of Human MUC1-positive IBD. To mimic the characteristic chronic inflammatory tumor-promoting environment of Human IBD, mice will be administered dextran sodium sulfate (DSS) to induce colitis, and subsequently be treated with a direct application of an anti-inflammatory suppository to the colon, developed through conjugation of TNF-α antibody to weakly mucoadhesive polysaccharide, hyaluronic acid (HA). Previously tested in vitro on a burn model, it was found that modulating the activities of a cytokine at the apex of the signaling cascade (TNF-α), was effective in reducing necrosis, resulting in the stabilization and neutralization of free TNF antigen, and also reducing the diffusion rate of the antigen within the system (Washburn, et.al). Consequently, motivation developed to test a theory hinging on these results: when administered in vivo to an area of inflammation, potential exists for stabilization and neutralization of this antigen to reduce levels of chronic inflammation, by disrupting its ability to activate a chain of cellular mediators in the signal transduction pathway leading to pathogenesis of IBD. This will allow a conclusion to be made with respect to the effectiveness of the conjugate at stabilizing TNF-α in IBD, which contributes to CACC.

Brian Hayashi

Brian Hayashi
Mentor: Gordon Rule

 

Sarah Horner

Sarah Horner
Mentor: Mark Macbeth

The Effect of Cell Signaling Molecules on RNA Modification

This summer I researched the effect of inositol hexakisphosphate (IP6) and its precursors on the function of adenosine deaminases that act on tRNAAla at position 37 (ADAT1) in Saccharomyces cerevisiae (S. cerevisiae). IP6 is a compound that is synthesized naturally in S. cerevisiae through a series of four reactions. The pathway proceeds with the cleavage of PIP2 (phosphatidylinosiol 4,5-bisphosphate) by phospholipase C to produce IP3. IP3 is in turn phosphorylated to IP4 (inositol 1,4,5,6-tetrakisphosphate) and IP5 (inositol 1,3,4,5,6-pentakisphosphate) using Ipk2. Then, IP5 is phosphorylated by Ipk1 into IP6. IP6 is required for the activity of ADAT1, which converts adenosine to inosine at position 37 of tRNAAla. Inosine is “read” as guanosine thereby changing the overall sequence of tRNAAla. This modified tRNAala product of the reaction between IP6 and ADAT1 is thought to preserve translational fidleity. Four knockout strains were obtained that were devoid of either phospholipase C, Ipk2, Ipk1, or ADAT1. Once the mutants were grown, an extract of tRNA was assayed using RT-PCR to determine if there was editing of tRNAAla at position 37. The IP6 intermediates could not restore the editing function of ADAT1. Further studies will be conducted to determine the effect of cell stress on editing.

Maya Khandker

Maya Khandker
Mentor: Anna Fisher

Justine Kim

Justine Kim
Mentor: Bryan Tillman

Vivien Kuo

Vivien Kuo
Mentor: Joel McManus

Ling-Ling Lee

Ling-Ling Lee
Mentor: Bruce Armitage

Angela Lo

Angela Lo
Mentor: Chien Ho

Temperature Dependence of Autoxidation with Hemoglobin

Extracellular hemoglobin that have been chemically changed or genetically expressed in microbial host systems have been developed for the purpose of oxygen-carrying therapeutics. Clinical studies have shown that these types of hemoglobin most likely deliver sufficient amounts of oxygen to the tissues. However, there are still concerns regarding the autoxidation, or spontaneous oxidation, of these hemoglobin types and their redox reactions with other tissue oxidants that may prevent these recombinant hemoglobin from becoming clinically useful.  Research has shown that the L29F mutant inhibits while the L28F mutant has no effect on autoxidation. And with regards to the hemoglobin structure, previous NMR studies have also shown that the distal histidyl hydrogen bond in the β subunit is weaker than that in the α subunit. In addition, these hydrogen bonds are more mobile at lower pH and/or higher temperature. This project will test whether hemoglobin autoxidation is affected by the strength of the hydrogen bond between the distal histidyl residue and the oxygen ligand.  Since hydrogen bond strength is pH and/or temperature dependent, studies will be conducted to see if the autoxidation of αL29F and αL29W mutants act as a function of temperature and will compare the autoxidation rates to that of wild type hemoglobin under the same experimental conditions.  Experiments will include the use of visible spectrophotometry under various temperatures.  With such limited amounts of safe donated blood, recombinant hemoglobin research is crucial for the development and application of suitable hemoglobin-based oxygen carriers (HBOCs) and in hemoglobin therapeutics in a blood substitute system.

Jessika Louissaint

Jessika Louissaint
Mentor: Veronica Hinman

Characterization of Function & the Expression of the Hedgehog (HH) Gene during Neurogenesis in a Non-Chordate Deuterostome Patiria miniata

The phyla Echinodermata (Echinoderms) belong to the Superphyla Deuterostomes, under the umbrella of the Bilateria.  Deuterostome embryos are classified by radial cleavage during their eight-cell stage and the development of their anal region at the blastopore, the first opening into the archenteron, or the embryo’s primitive gut. Echinoderms consist of marine animals that develop via bilateral symmetry, typically being five-point radial symmetry. The Hinman lab group focuses on sea stars Patiria miniata (formerly Asterina miniata) and sea urchins Strongylocentrotus purpuratus to understand how roles of various genes in embryonic development have evolved. My project seeks to understand the function of the hedgehog gene (hh) in echinoderms. In chordates, which are organisms containing a notochord and hollow dorsal nerve cord (and which including humans, mice, and chickens), an ortholog of hh contributes to various roles in signaling pathways, specifically the specification of the dorsal ventral neural tube along the dorsal ventral axis. Although hh is not known to have a role in patterning neurons in the ectoderm of non-chordates, we have shown it to be expressed in the ectoderm of the mouth in sea stars; thus, our goal is to investigate the functionality of hh in patterning echinoderm neurons, specifically genes known to be expressed in the mouth of the P. miniata. The first step is to characterize mouth genes of the species through creation of a figure plate of whole mount in situ hybridizations of genes thought to be involved mouth (upper and bottom lips) and gut activity/development, including gsc, hbd, hex, soxc, and hh. To further characterize hh activity throughout echinoderms, we will perform knockdowns of hh to examine the effects of differing concentrations of the gene on the neuronal development and the vitality of the embryos.

Mridula Nadamuni

Mridula Nadamuni
Mentor: Nathan Urban

High-Throughput Method for Behavioral Phenotyping

The need for efficient, high-throughput models of mouse behavioral phenotyping has been identified since 2003 [Paylor]. Analyzing behavior in mouse models currently requires recording video and hand-scoring to detect changes in a wide range of behaviors such as digging, grooming, drinking, walking, etc. This process is time-intensive, subject to variability, and fails to capture important components of mouse behavior such as vocalizations. To address this, we propose a new method of audio-based analysis of behavior. We aim to develop a method for analyzing large volumes of audio signals from mouse cages using recognition software that scans for specific behavioral fingerprints. Automating this process will allow us to capture data over significantly longer periods of time, improving the reliability of the results. I will begin by collecting large volumes of data, audio and some video, from multiple mice over several days.

I will work with commercially available software to recognize audio signals corresponding to typical behaviors and vocalizations, especially in the ultrasonic range. I will then test the efficacy of this method by hand-scoring a subset of the data and comparing the results against the automated analysis. An additional component of this project is to train mice to use touchscreens and monitor their interactions with devices like the iPod. The work builds on previous efforts in the Crawley Lab, at the National Institutes of Mental Health (NIMH). Interactions with touch screens may serve to provide additional information on normative mouse behavior. I will attempt to develop an app for the iPod for the purpose of training mice to use touch screens. If this is successful, the results of this project may be used to inform behavioral assessments in mouse models of autism, addiction and other neurological conditions. Ultimately, a high-throughput screening system will result in a better understanding of behavioral patterns in mouse models of disease.

  1. Paylor, Richard. High Throughput Screening Strategies. Department of Molecular and Human Genetics, Division of Neuroscience Baylor College of Medicine, Houston, TX. 2003.
Steven Nguyen

Steven Nguyen
Mentor: Subha R. Das

Synthesis of S-Adenosyl Methionine Analogues via Click Chemistry

The chemically reactive methyl group on S-adenosyl methionine (SAM) is critically involved in methylation during key biological processes such as transcription and signal transduction. Chemical analogues of SAM have shown interesting antiviral effects through inhibition of viral-specific, SAM-dependent processes. In this project we propose to use principles of click chemistry to obtain analogues of SAM through the high efficiency click reaction between an azide and alkyne. The idea is to install the ‘clickable’ groups (terminal propargyl group or azido group) onto adenosine and an amino acid (ideally on the side chain). With prepared ‘clickable’ staring material, the modified adenosine and amino acids will be reacted in the presence of copper(I) to form  triazole linked analogues of SAM. Following purification and characterization, samples will be evaluated for antiviral effects through the NIH-NIAID screening service.

Marianne Pan

Marianne Pan
Mentor: Partha Biswas

Ashley Reeder

Ashley Reeder
Mentor: Brooke McCartney

Darlene Reid

Darlene Reid
Mentor: Catalina Achim

Charge Transfer Study of Peptide Nucleic Acids linked to Cytochrome c

Metallo-proteins and –enzymes can transfer electrons and catalyze chemical reactions in living organisms in a highly correlated way.  Organization of such proteins on the nanometer scale could enable scientists to create sensitive and specific biosensors or research tools.  Peptide nucleic acid (PNA), a synthetic analogue of DNA that has a pseudopeptide backbone, is a candidate for a “universal” and tunable linker of such proteins. Cytochrome c is an electron transfer protein typically found in the mitochondrion of eukaryotic cells. As a biological molecule that can be oxidized or reduced, Cytochrome c is ideal to use as a redox reporter in the study of charge transfer in PNA by cyclic voltammetry.  We hypothesize that a PNA strand less than seven nucleotides long will be able to transfer charge between a Au electrode and Cytochrome c.  PNA with pyridine or carboxylate end group are used to attach Cytochrome c. The PNAs are synthesized, purified and characterized by manual peptide synthesis, HPLC and MALDI- ToF, respectively. UV-Vis titrations of Cytochrome c with PNA provide insight on how the Cytochrome binds to the PNA. Electrochemistry is used to measure the rate constant for charge transfer from Cytochrome c to the Au through the PNA.

 John Sadeghi

John Sadeghi
Mentor: Stephen Meriney

Rachel Sewell

Rachel Sewell
Mentor: Eric Ahrens

19F MRI Assessment of Systemic Inflammation Following TBI and Combined TBI and Hemorrhagic Shock in Mice

Traumatic brain injury (TBI) is one of the leading causes of mortality and morbidity in the United States, and has gained attention due to the conflicts in Iraq and Afghanistan. It is now understood that mortality is effectively doubled following a secondary insult. This research proposal plans to take advantage of novel perfluorocarbon-based MRI cell tracking agents, developed by Dr. Eric Ahrens, to examine systemic inflammatory involvement following experimental TBI and an imposed secondary insult of hypotension.  With this contrast agent it is possible to label macrophages in situ and noninvasively detect the trafficking and accumulation of these labeled immune cells in vivo.  This is due to the 19F MRI signal, not present naturally, providing a unique marker for tracking these cells. Specifically, this study will quantify macrophage accumulation in a mouse model of TBI plus hemorrhagic shock (HS).  Three groups of mice will be used for this study: sham, controlled cortical impact (CCI at a velocity of 5m/sec and a depth of 1mm), and CCI + HS. Mice undergoing HS will sustain a shock period of 35 min with a mean arterial blood pressure (MABP) of 25-27 mmHg, followed by resuscitation until MABP is ≥70 mmHg. The mice will then be injected with V-sense (VS580H) 48 hours post injury. Mice will be imaged in 3D with 1H MRI for anatomical reference and 19F MRI to localize the fluorine-labeled macrophages. A fluorine reference standard will be imaged alongside the mice for quantification. Voxel Tracker is a program that will be used to quantify areas of 19F signal accumulation. Organs (brain, heart, liver, spleen, lung, etc.) will be excised and subjected to 19F NMR spectroscopy with a TFA reference standard. The V-sense signal will be integrated for each organ and compared to the in vivo MRI analysis.

Anthony Spadaro

Anthony Spadaro
Mentor: David Geller

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Sophia Wu
Mentor: Newell Washburn

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Angela Yi
Mentor: Chuck Ettensohn

William Yoo

William Yoo
Mentor: Adam Linstedt