2011 Summer Researchers Participants-HHMI Undergraduate Program - Carnegie Mellon University

2011 HHMI Summer Researchers Participants

Medini Annavajhala

Medini Annavajhala
Mentor: Terrence Collins

Sade Atoyebi

Olufolasade (Sade) Atoyebi
Mentor: Newell Washburn

Improvement of the Anti-inflammatory Protein, Anti-Tumor Necrosis Factor (TNF)-Alpha, through Conjugation with Hyaluronic Acid

Modulation and control of the inflammatory response has begun to be explored through the formation of synthetic conjugates between biocompatible polymers and proteins involved in inflammation. Sun et al. have recently discovered that attaching the anti-inflammatory TNF- cytokine to polysaccharides substantially decreases the KD of the protein, indicating that polymer-protein conjugates improve binding affinity. Though important binding qualities have been revealed, binding to large antibodies have life-threatening side effects.  In this project, the binding of a synthetic anti-TNF α to hyaluronic acid (HA) will be studied to see if the increased binding affinity is consistent. HA has been studied for its ability to form a viscous aqueous solution for drug delivery. The amino acid sequence in the active domain of the anti-TNF α was chosen as the region to synthesize, which has been found to be more biocompatible.  It is theorized that the anti-TNF α sequence bound to the HA will experience a decreased diffusion coefficient and maintain its activity at the wound site longer. Synthesis of this material will be accomplished by functionalizing HA with a thiol moiety. The synthetic anti-TNF α will be prepared through solid phase peptide synthesis. The peptide will be covalently attached to a bifunctional, medium molecular weight PEG linker by the amine of the peptide. The vinyl group of the PEG will covalently attach the peptide to the HA polymer through a Michael-type addition reaction. A Forte Bio Octet system will be used to explore kon, koff, KD. The binding of unmodified TNF- α to anti-TNF αwill be used as a standard binding control. It is predicted that the binding affinity will increase, the dissociation constant will decrease, the diffusion of the polymer-protein TNF- α pair from the injury site will decrease, and the activity of cytokine will remain the same when bound to HA. If successful, this will provide a method for reducing inflammation at the site of injury.

Yookyung Bae

Yookyung Bae
Mentor:Catalina Achim

Redox Driven Translocation Mechanism of Copper in Peptide Nucleic Acid (PNA)

Peptide Nucleic Acid (PNA) has shown vast advantages in the research field due to its neutral and flexible backbone, which lends it greater specificity for binding to DNA than DNA itself. PNA will be modified with 8-hydroxyquinoline (Hq) and Bipyridine (Bpy) ligands and the binding of copper to these two ligand sites will be analyzed by UV-Vis titration. The structural and the thermodynamic properties of the ligand-modified PNA strands will be studied by applying melting curve analysis to explain the dissocation characteristics and CD spectroscopy to study the chirality of the PNA strands. These studies and UV-Vis titrations will provide the stoichiometry and stability constants of the complexes formed between copper and the ligand-modified PNAs. By understanding how copper binds to these ligand sites, this information can be used for a nanosystem that can act as molecular switch which can have various applications in nanotechnology and medicine. For example, the PNA can serve as template for molecular electronics circuits or as “glue” in the construction of synthetic, multiprotein systems.

Lisa Brubaker

Lisa Brubaker
Mentor: Veronica Hinman

Orthologous Protein Expression in Strongylocentrotus purpuratus and Pateria miniata

The embryo of the sea urchin Strongylocentrotus purpuratus is organized very similarly to that of the sea star Asterina miniata. A striking difference between these embryos, however, is the embryo of the sea urchin contains a region of additional cells (primary mesenchyme cells or PMCs) that specifies the embryonic skeleton. Tbrain (Tbr) and Hesc are two transcription factors (TFs) which play crucial roles in PMC specification in sea urchins but have different functions in sea stars. It was previously determined that the RNA encoding Tbr is located in the PMCs in sea urchins. In sea stars, on the other hand, Tbr RNA is located primarily in the vegetal pole (the region corresponding to the endomesoderm) but is also sparsely scattered throughout the embryo. Hesc RNA is cleared from PMCs in sea urchins but is ubiquitous in sea star embryos. Our goal is to determine why these orthologous proteins play different roles in each organism and if these differences in function are caused by amino acid level mutations. In order to elucidate these dissimilarities we need to confirm the level of translation of Tbr and Hesc RNA and when (at what developmental stage) and where in the embryo these proteins are nuclear (active). This knowledge will help us understand whether or not the Tbr and Hesc proteins are expressed in the same locations as the Tbr and Hesc RNA (respectively); and whether or not each protein functions as predicted. Western blotting will be used to determine the amount of Tbr and Hesc protein present in the embryos of each organism. Immunofluorescence will be used to determine the distribution of the Tbr and Hesc orthologs. From these analyses we hope to determine the temporal and spatial activity of Tbr and HesC. This information will immediately enhance our knowledge about these proteins and will be crucial in guiding future experiments (such as chromatin immunoprecipitation (ChIP)). This summer we also hope to optimize a ChIP sequencing protocol for echinoderm embryos. The western blots and immunofluorescence assays will enable us to validate the antibodies used for this protocol. ChIP-sequencing will allow us to isolate cis regulatory modules (CRMs) for the target genes of Tbr and Hesc. We will then be able to compare target genes between the Tbr and Hesc orthologs. This information will also enable us to determine species-specific interactions and compare binding site preferences for both Tbr and Hesc orthologs. In summary, we hope to gain a deeper understanding of two orthologous proteins (Tbr and Hesc) in sea urchins and star fish in order to compare the evolution of the developmental mechanisms of these two organisms.

Catherine Bryd

Catherine Byrd
Mentor: Chien Ho

Roles of Amino Acid Residues in Woolly Mammoth Hemoglobin on the Temperature Effect of Oxygen Binding

A biochemical-biophysical study of the recombinant hemoglobins (Hbs) of woolly mammoth (rHb WM) and Asian elephant (rHb AE) as compared to normal human hemoglobin (Hb A) will be carried out to investigate the molecular basis of environmental adaptation of Hb. Preliminary studies show that the O2-binding affinity for rHb WM is less sensitive to temperature change than that of rHb AE. This means that in colder environments, Hb WM’s oxygen affinity stays relatively constant whereas, other Hbs’ affinity increases. The mammoth-specific β/δ101Gln residue, which is located in the central cavity of the Hb molecule, could create additional H+-linked Cl binding sites in rHb WM, and may be responsible for its lower temperature effect. In this study, mutants with a β/δ101 substitution (β/δ101Gln→Glu, Lys, and Gly) will be made, and the O2-binding affinity (P50) and cooperativity (Hill coefficient) of these mutants will be measured under various experimental conditions to investigate whether the β101Gln residue is critical for a stronger response to the allosteric effectors (phosphate, IHP, and Cl). This study will gain insights into the role of the β101Gln residue in the function of rHb WM.

Yoonjin Choi

Yoonjin Choi
Mentor: Linda Peteanu

Joe DeFazio

Joseph DeFazio
Mentor: Neraj Gandhi

 

Nick DelRose

Nicolas DelRose
Mentor: Tina Lee

Allyson Dill

Allyson Dill
Mentor: Mark Macbeth

Structural Analysis of an Adenosine Deaminase that Acts on Double-Stranded RNA

Adenosine deaminases that act on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in double-stranded RNA substrates. Inosine is interpreted by the cell’s translational machinery as guanosine; thus ADAR activity effectively causes an A to G point mutation. Through post-transcriptional modification, ADARs indirectly diversify eukaryotic genomes. ADARs are required for viability in mammals, particularly in neuronal function - editing activity has been shown to target mRNAs related to neuronal activity such as those that encode the AMPA GluR ion channel B subunit and the serotonin 2C receptor. ADAR activity has also been implicated in the antiviral response and RNA interference. I am studying human ADAR 1 (hADAR1), which includes a C-terminal catalytic domain, three double-stranded RNA binding motifs (dsRBMs), and in the full-length isoform, two N-terminal Z-DNA binding motifs. The structural mechanisms for hADAR1 catalysis and substrate specificity are not well understood. As such, my goal is to solve the three-dimensional structure of hADAR1 using X-ray crystallography, and to study the structural features of enzyme-substrate interactions with in vitro assays. I have cloned several constructs of hADAR1 into the galactose-inducible pJEL vector (AMPR, URA3) for overexpression in S. cerevisiae strain BCY123. Each construct contains an N-terminal His-10 tag followed by a TEV protease cleavage site, and the purification procedure utilizes nickel affinity chromatography, treatment with TEV protease, cation exchange chromatography, and gel filtration chromatography. The specific experimental goals I will be working towards this summer include crystallization of hADAR1’s catalytic domain, cocrystallization of hADAR1-R721 (includes the catalytic domain and one dsRBM) with a dsRNA substrate containing a transition state analog, activity assays of hADAR1 truncations on a well-known substrate (the R/G site of the GluR-B pre-mRNA), a footprinting assay to map out the binding sites of each hADAR1 domain on the pre-mRNA surrounding the R/G site, and an assay for inositol hexakisphosphate (IP6) requirement for activity by hADAR1.

Elias Fatsi

Elias Fatsi
Mentor: Ellen Peterson

Katherine Forsyth

Katherine Forsyth
Mentor: Alex Evilevitch

Osmotic Pressure Measurement of Bacteriophage Lambda

The subject of this project is to quantitatively determine the pressure inside an overpackaged bacteriophage lambda (phage λ). While our lab has previously determined the genome pressure inside wild type phage λ and its shorter genome mutants, the experiment has never been conducted on an overpackaged phage. Viral DNA is extremely tightly packaged inside the viral capsid, and due to the negative charge and stiffness of the DNA, it exerts very high pressure (tens of atmospheres) from the neighboring strands, which facilitates the ejection of the viral DNA into the host cell. It is hypothesized that in overpackaged phage λ, containing 110% of the wild type (100%) genome length of approximately 17,000 base pairs, the pressure inside the capsid is greatly increased due to increased DNA-DNA repulsion. Whether the increase correlates linearly or exponentially with the amount of genome length added is unknown. To address the aim of the project, osmotic pressure suppression of DNA ejection from phage will be used in order to measure the internal DNA pressure. Polyethylene glycol (PEG) is used as an osmolite in the presence of both the 110% phage lambda and the protein lambda receptor (lamB), which will induce the ejection of DNA. The amount of ejected DNA will be measured using Ultraviolet/Visible (UV/Vis) Spectrometry. As the concentration of PEG increases, the amount of DNA ejected will decrease, and eventually an equilibrium will be reached with no DNA ejected, when external osmotic pressure will be matching the pressure inside the capsid. The results wil be directly compared to the genome pressures for 78, 94 and 100% DNA length in lambda. This work will help to give insights into the mechanisms of viral DNA ejection into the host cell.

Naomi Gunawardena

Naomi Gunawardena
Mentor: Aaron Mitchell

Candida albicans Plasma Membrane Proteins that Govern Cell Wall Integrity

Candida albicans is a major fungal pathogen of humans. Its ability to adapt to its external surroundings allows it to transform from commensal to pathogenic growth within its human host. The C. albicans plasma membrane has many significant functions in the cell, providing a protective barrier around the cell and acting as a dynamic interactive surface with its environment. The goal of this project was to screen a plasma membrane mutant library for altered sensitivity to a variety of stresses in order to identify genes of interest as prospective targets for antifungal therapies. Previously, an insertion mutant clone library was used to create homozygous C. albicans insertion mutants for 97 genes, many of which had multiple isolates. I tested these mutant strains for growth by spot assays on YPD (control) and YPD + sodium chloride (osmotic stress), YPD + hydrogen peroxide (oxidative stress), YPD + Caspofungin (cell wall stress) and YPD + Congo red (cell wall stress). 30 different strains displayed sensitivity to Caspofungin and Congo red, implicating possible roles in cell wall integrity. Many of the genes associated with phenotype alterations were previously uncharacterized open reading frames, so these results already help to annotate the C. albicans genome. Of the previously-characterized genes, most had not been associated with cell wall integrity. This group includes three glucose transporter genes and two choline transporter genes. My future directions include complementation of each gene of interest, and more detailed studies to elucidate the mechanistic relationships between protein function and biological function.

Susan Hannes

Susan Hannes
Mentor: Newell Washburn

Improvement of the Anti-inflammatory Protein, Anti-Tumor Necrosis Factor (TNF)-Alpha, through Conjugation with Hyaluronic Acid

Modulation and control of the inflammatory response has begun to be explored through the formation of synthetic conjugates between biocompatible polymers and proteins involved in inflammation. Sun et al. have recently discovered that attaching the anti-inflammatory TNF- cytokine to polysaccharides substantially decreases the KD of the protein, indicating that polymer-protein conjugates improve binding affinity. Though important binding qualities have been revealed, binding to large antibodies have life-threatening side effects.  In this project, the binding of a synthetic anti-TNF α to hyaluronic acid (HA) will be studied to see if the increased binding affinity is consistent. HA has been studied for its ability to form a viscous aqueous solution for drug delivery. The amino acid sequence in the active domain of the anti-TNF α was chosen as the region to synthesize, which has been found to be more biocompatible.  It is theorized that the anti-TNF α sequence bound to the HA will experience a decreased diffusion coefficient and maintain its activity at the wound site longer. Synthesis of this material will be accomplished by functionalizing HA with a thiol moiety. The synthetic anti-TNF α will be prepared through solid phase peptide synthesis. The peptide will be covalently attached to a bifunctional, medium molecular weight PEG linker by the amine of the peptide. The vinyl group of the PEG will covalently attach the peptide to the HA polymer through a Michael-type addition reaction. A Forte Bio Octet system will be used to explore kon, koff, KD. The binding of unmodified TNF- α to anti-TNF αwill be used as a standard binding control. It is predicted that the binding affinity will increase, the dissociation constant will decrease, the diffusion of the polymer-protein TNF- α pair from the injury site will decrease, and the activity of cytokine will remain the same when bound to HA. If successful, this will provide a method for reducing inflammation at the site of injury.

Sherry He

Siping (Sherry) He
Mentor: A. Javier Lopez

Xuexia Jiang

Xuexia Jiang
Mentor: Robert Murphy

Terri Jones

Terrell (Terri) Jones
Mentor: Markus Dittrich

Diane Koeller

Diane Koeller
Mentor: Mark Macbeth

Allyson Koyen

Allyson Koyen
Mentor: Brooke McCartney

Dara Krute

Dara Krute
Mentor: Steve Garoff

 

 Benjamin Kuo

Benjamin Kuo
Mentor: John Woolford

Investigating Protein Interactions of Has1 Involved in 60S Ribosome Assembly

Ribosome biogenesis is a highly regulated, step-wise pathway, which consists of ~200 assembly factors that help fold and process rRNA, as well as bind ~80 ribosomal proteins to rRNA. Has1, a DEAD-box protein, is shown to play a role in the 60S pathway, more specifically, in the 27SA3 to 27SBS processing step. Hence, it is of interest to determine the protein targets and cofactors of Has1 to further understand its role in the 60S ribosome assembly pathway. Since Has1 functions in the steps mentioned above, we hypothesize that Has1 will have protein interactions with some of the proteins also involved in those steps. Hence, a two-hybrid assay is used to screen for potential protein interactions with Has1. Out of the 36 proteins, which include assembly factors and ribosomal proteins, 8 of them were two hybrid positive with Has1. Then, mutants of Has1 were used to see which domain of Has1 was required for those interactions to remain two-hybrid positive. Moreover, TAP purifications using high salt wash was also performed in an attempt to identify salt stable complexes of Has1. The 8 two hybrid positive interactions are potential real interactions involved in 60S ribosome assembly, where an orthogonal approach will be used to identify those real interactions.

Vivien Kuo

Vivien Kuo
Mentor: Jon Minden

Characterizing a pH Sensitive Fluorescent Protein, pHluorin, as a Reporter for Autophagy

Apoptosis is a programmed cell-death, and autophagy is a similar process in which cells devour their own organelles during times of starvation. Both processes are essential to the embryonic development of multicellular organisms. The interplay and mechanisms between the two processes, however, is unclear. In order to study these processes, a pH sensitive fluorescent molecule called pHluorin will be used to mark the Atg8a protein, a protein that is normally cytoplasmic but accumulates in autophagic vesicles upon initiation of the autophagic pathway. This should allow observers to see puncta (the pHluorin aggregates) in both acidified and non-acidified conditions under a fluorescent microscope. The purpose is to characterize pHluorin as an autophagy reporter. The main experiments performed involved using a drug or starving cells to attempt to induce autophagy. Rapamycin, a drug involved in the TOR pathway, which has been shown in previous literature that it increases the autophagic flux. Placing cells in media containing fewer or no nutrients is also another common way to increase flux. The cell line used is S2 cells, which are macrophage derivatives from D. Melanogaster. The results are that Rapamycin has little to no effect on S2 cells, regardless of concentration, and that starvation in HBSS solution does not display a significant autophagic phenotype. The results from starvation in HBSS solution are inconsistent – some display no puncta at all, some starved cells display significantly more puncta than control cells, but some control cells have also displayed as many puncta as starved cells. The results are inconclusive because there is no consistent phenotype related to autophagy in starved cells. Further experiments should be done on a different cell line that has been shown to express autophagy more dramatically. Or, the study should be done in vivo in the fly fat body, as previous literature has used this organ to study autophagy.

Jiayue Li

Jiayue (Amy) Li
Mentor: Christopher Langmead

Wendy Li

Wendy Li
Mentor: Chien Ho

Characterization of Immune Cells under Different Conditions of Cell Labeling with Magnetic Resonance Imaging Contrast Agents

Currently, the labeling and tracking of cells in vivo by magnetic resonance imaging (MRI) has great potential for diagnosing and determining treatment for many diseases, such as graft rejection. By tracking the accumulation of labeled immune cells (particularly macrophages and T-cells) at the site of organ rejection, we have shown that cardiac rejection can be monitored through MRI. In order for cardiac transplant rejection to be monitored successfully, however, the effects of cell-labeling and drug treatments on immune cells in vivo and in vitro must first be understood. This project will first examine through flow cytometry whether labeling of immune cells with iron-oxide particles containing a terminal carboxyl group and attached to a cationic amine group (IOPC-NH2 particle, an MRI contrast agent) will affect cell-cell interactions or change the expression of cell surface markers. This project will also determine by flow cytometry and ELISA whether two immunosuppressive drugs, clodronate and cyclosporine A, affect the efficiency of labeling cells with IOPC-NH2 or affect the expression of cell surface markers and cell production by labeled cells.

Margaret Libonati

Margaret Libonati
Mentor: Veronica Hinman

Identification and Analysis of the Sea Star Gene gcm cis-regulatory Module

The expression of pigment cells in sea urchins is an example of the evolution of new cell types. A possible event that could lead to novel cell types is a change in cis-regulation of genes involved in expression of specific cell types. The cis-regulatory module (CRM) is a segment of the gene with transcription factor (TF) binding sites, which makes it essential for the regulation and expression of the gene. The purpose of this project is to identify and analyze the CRM of the sea star gene, gcm. Gcm is responsible for the activation of expression of pigment synthesis genes such as pks and fmo. Gcm­-expressing cells originate from the sea star ectoderm, but gcm is expressed in the sea urchin mesoderm. Because the sea star mesoderm lacks pigment cells, embryonic pigment cell expression may have evolved in sea urchins. We propose that a change in the cis-regulation of the gcm gene enables gcm expression in the sea urchin mesoderm and resulted in a novel cell type. By comparing the previously identified sea urchin gcm CRM (Ransick et al., 2006) with that of the sea star, it will be possible to determine how a change in cis-regulation of gcm results in gcm and pigment synthesis gene expression in sea urchin in addition to a gain of expression in the mesoderm. The first goal of this project is to isolate the region of the sea star gcm gene containing a CRM. The major techniques used in this portion of the project are PCR, restriction enzyme digestion, ligation and transformation using electroporation. Regions possibly containing the CRM will be analyzed using a program called ClusterBuster in order to find putative transcription factor binding sites. Cloned regions of sea star gcm possibly containing CRMs will be injected into the sea star embryo. Secondly, specific expression within the ectoderm will be analyzed using in situ hybridization in order to further the understanding of the evolutionary differences between the gcm of sea urchins and sea stars. In addition, this project will include the injection of the known sea urchin CRM into sea star embryos. If new patterns of expression are observed in the sea star embryos, this may indicate that specific transcription factors are necessary for the regulation of gcm expression in sea urchins, but not in sea star regulation of gcm expression. This will lead to more research on the TFs involved in gcm regulation and expression and will expand the gene regulatory network.

Andrew McCoy

Andrew McCoy
Mentor: Peter Berget

Thrombin Biosensors and Characterization of the Fluorogen-Activating Protein (FAP) Platform

Proteases are proteins that cleave peptide bonds in other proteins and play a significant role in normal and pathological physiology. Thrombin is a serine protease involved at multiple steps in the blood coagulation pathway including activation of Factors V, VIII and XI, cleavage of fibrinogen and of Protease Activated Receptors (PARs). New technologies that are specific and can measure protease activity directly are needed for research and clinical studies. Using a fluorogen activating protein (FAP) platform, thrombin biosensors were designed with cleavage sites from PAR-1 and with various linker lengths separating the fluorogen activating component from the signal blocking component of the biosensor. These constructs were expressed in Escherichia coli (E.coli) and the cleavage activity of the purified biosensors was assayed in vitro using fluorometry and SDS-PAGE analysis. The biosensors with PAR-1 sequences were cleaved with thrombin and fluorogen was activated following cleavage.  A control sequence was not cleaved. Kcat/Km and fluorometry results indicate that the length of the cleavage sequence and the length of the linker impacted the cleavage of the biosensor. Further studies of structure, specificity and functionality will characterize the biosensors and allow for the development of general design specifications.

Darlene Reid

Darlene Reid
Mentor: Catalina Achim

Anna Romanova

Anna Romanova
Mentor: Nathan Urban

Oliver Ruiz

Oliver Ruiz
Mentor: Edda Thiels

Effect of Repeated Exposure to Nicotine on Reward-predictive Cue-evoked Activation of Extracellular Signal-regulated Kinase in the Nucleus Accumbens

Environmental stimuli that are reward-paired increase operant behaviors that serve to obtain the reward. For instance, a tone that is repeatedly paired with food increases lever-pressing for food in rats. This type of conditioned stimulus (CS)-potentiation of reward seeking can be detrimental when it contributes to uncontrollable reward seeking, as in drug addiction. We found that a food-paired CS evokes an increase in activation of extracellular signal-regulated kinase (ERK) in the nucleus accumbens (NAc) of rats, and that inhibition of ERK activation in the NAc abolishes CS-mediated enhancement of reward seeking. These observations suggest that NAc ERK activation is critical for the ability of a CS to potentiate reward seeking. Recently, we found that repeated exposure to amphetamine after daily tone-food conditioning enhances the magnitude of the CS-evoked NAc ERK signal and augments CS-potentiated reward seeking. Here, I aimed to determine whether nicotine treatment causes a similar enhancement of CS-evoked NAc ERK activation. Rats were given daily tone-food conditioning immediately followed by injection of either nicotine or vehicle solution. Control rats received tone presentations without food followed by drug injections. Forty-eight hours after the last training session and injection, rats were exposed to the tone without food.  Immediately after this test, brains were removed and the NAc excised and probed for total and active ERK using Western blot analysis. Analysis of food cup approach during training and the test revealed that nicotine treatment did not affect acquisition of the discriminative conditioned response. Biochemical analyses to determine the effect of nicotine on basal and CS-evoked NAc ERK activation are currently underway. The biochemical results will reveal whether nicotine impacts NAc ERK regulation by CSs in a similar or different manner from that of amphetamine. The results will provide insights into mechanisms through which these addictive drugs can alter reward seeking.

Maneesha Sakhuja

Maneesha Sakhuja
Mentor: Manojkumar Puthenveedu

Katherine Siewart

Katherine Siewert
Mentor: Dannie Durand

Uncovering Domain Shuffling in the MAGI Gene Family