2013 Summer Researchers Participants-HHMI Undergraduate Program - Carnegie Mellon University

2013 HHMI Summer Researchers Participants

Jeremy Applebaum

Jeremy Applebaum
Mentor: Marcel Bruchez

Characterization of Fluorogen-Induced Dimerization Modules for Heterodimerization

The ErbB family of receptor tyrosine kinases is involved in many vital cellular processes.  Upon the binding of specific ligands, the receptors form hetero- or homodimers, initiating a signaling cascade. Yet, the progression and pathogenesis of various types of cancers is linked to the over-expression of certain receptors in the ErbB family, specifically ErbB2. Selective dimerization of the ErbB receptors to inhibit the formation of a heterodimer with ErbB2 may prove to be a new method of cancer treatment. Flurogen-activating proteins (FAPs) act as dimerization inducers by allowing for the selectivity of the dimerization components. With their high affinity for certain fluorogenic dyes, FAPs can also promote considerable fluorescence upon binding. FAPs have the significant feature of inducing dimerization only in the presence of the fluorogenic dye. The FAP system can be extended by testing varying fluorogen and FAP pairings for fluorescence capabilities. Ultimately, we seek to have an understanding of compatibilities amongst various FAP and fluorogenic dye pairs. This will allow us to successfully use covalently linked fluorogens to dimerize two FAPs and the fused receptor of interest. A single-chain variable fragment library will be tested for fluorescence amongst five fluorogenic dyes. The development of the FAP system will allow for the chemical induced dimerization of the ErbB receptor family, along with producing a fluorescent signal that will indicate the dimerization state. Such advancements would facilitate the study of receptor activation and the resultant signaling pathways.

Raghunandan Avula

Raghunandan Avula
Mentor: Jon Minden


Leela Chockalingam
Mentor: Kacey Marra

Yidan Cong

Yidan Cong
Mentor: Dannith Ly

Dagney Cooke

Dagney Cooke
Mentor: Jon Minden

Aditya Das

Aditya Das
Mentor: Alex Evilevitch

Matthew Early

Matthew Early
Mentor: Chuck Ettensohn

Brian Hayashi

Brian Hayashi
Mentor: Gordon Rule

Memphis Hill

Memphis Hill
Mentor: Aaron Mitchell

The Role of p53 in Cellular Transformation by the Large T-antigen of Simian Virus 40 and JCV Polyomaviruses

In healthy cells, transformation and tumorigenesis are prevented by the activities of p53 and the Rb protein family (pRb). The large T-antigen (TAg) of Simian Virus 40 (SV40) binds to and inhibits regulatory functions of pRb, resulting in uncontrolled proliferation of the cell. This should signal p53 to induce transcription of genes involved in cell cycle arrest and apoptosis. However, TAg binds the DNA-binding domain of p53, inhibiting activation of p53 target genes. Inhibition of pRb and p53 are required for transformation. Oncoproteins of viruses from many other families inhibit p53 by targeting it for degradation, but TAg binding stabilizes p53. Cells expressing TAg accumulate high levels of p53 protein, implying a possible p53 gain-of-function. We hypothesize that this high level of p53 may enhance the transforming ability of TAg from two different polyomaviruses, SV40 and JCV. Mouse embryonic fibroblasts (MEFs) expressing either SV40 or JCV TAg were used to test the role of p53 in cell transformation. The transformation capability of MEFs expressing p53 was compared to that of p53 knockout (p53KO) MEFs using soft agar assays, which measure a cell’s ability to grow and form a colony without adhering to a surface, also known as anchorage independence. Our results showed less colony formation with the p53 knockout MEFs, suggesting that the TAg-p53 complex is required for higher efficiency of cell transformation. This will be further explored by re-introducing p53 to the KO cells and performing soft agar assays.

Hiromi Holt

Hiromi Holt
Mentor: Aryn Gittis


Allison Huang
Mentor: Dannith Ly

Filip Istvanic

Filip Istvanic
Mentor: Richard Steinman

Effects of Cyclin Dependent Kinase Inhibition on Fibrosis

The cyclin dependent kinase (cdk) inhibitor roscovitine is currently in clinical cancer trials. This drug prevents activation of the cell cycle by inhibiting cdks 1, 2, 5, 7, and 9. Cdks are protein kinases involved in both cell cycle progression and gene expression. Our lab recently discovered that roscovitine selectively lowers matrix protein expression (including collagen and fibronectin) in fibroblasts. Excess matrix proteins cause fibrosis and scar tissue. If the antifibrotic effect of roscovitine results from its cdk-inhibiting activity, this observation would implicate cdks for the first time in scar formation. We hypothesize that inhibition of cdks 1, 2, 5, or 9 will reduce matrix protein levels in cultured cell lines. We confirmed that roscovitine lowered collagen production by fibroblasts, but were unable to demonstrate that roscovitine suppression of matrix proteins occurred through accelerated protein degradation. Inhibition of cdk 2 alone, cdk 9 alone, or cdks 1, 2, and 5 (i.e. via butyrolactone) did not suppress collagen levels to the same extent as roscovitine did. However, a combination of cdk9 inhibitor and butyrolactone did replicate roscovitine’s effects. These observations suggest that cdks are involved in fibrotic protein upregulation and scar formation, and that both cdk 9 and one other cdk (1, 2, or 5) is required for matrix formation. Results support the conclusion that roscovitine reduces fibrotic protein levels through a cdk-dependent pathway involving inhibition of both cdk 9 and another cdk. Future directions include cdk kinase assays to quantify kinase activity. A limitation of the current study is that measurement of cdk activity was not able to be done. If these in vitro effects are confirmed in a preclinical in vivo model, then roscovitine may be a useful agent for treating fibrotic diseases.

Jillian Jaycox

Jillian Jaycox
Mentor: Subha Das

Hak-Jin Kim

Hak-Jin Kim
Mentor: Dannie Durand

Evolution of Sedoheptulose-1,7-bisphophatase (SHB17)

In eukaryotes the canonical mode of ribose synthesis is via the pentose phosphate pathway, but yeast is also capable of producing ribose via a newly discovered alternative, non-oxidative pathway driven by sedoheptulose-1,7-bisphosphatase (Shb17). It is unknown whether Shb17 exists in other genomes because Shb17 homologs are annotated as phosphoglycerate mutases or simply as members of a very large histidine phosphatase superfamily. I used PSI-BLAST and PSSM to construct a model that distinguishes Shb17 from its relatives and used significant matching sequences to build the maximum likelihood tree. Results showed Shb17 homologs are present only in various fungal species and a subset of non-monophyletic bacterial species. Phylogenetic analysis showed evidence of horizontal gene transfer from bacteria to fungi as the most likely origin of Shb17 in yeast. A history of symbiosis between plants, bacteria, and fungi suggests reasonable settings for the hypothesized inter-phylum gene transfer and is consistent with our phylogenetic results. Since ribose production via Shb17 requires no oxidative steps, we further investigate Shb17’s possible role in growth in fungal biofilm, where cells have limited access to oxygen. Biofilm growth assays of fungal pathogen Candida albicans Shb17-homolog deletion mutants showed reduced biofilm dry-weight, which may explain widespread presence of Shb17 in fungi. The origin of Shb17 through a horizontal transfer bypassing plants and animals suggests Sbh17’s possible role as a drug target against fungal plant and human pathogens.

Annette Ko

Annette Ko
Mentor: Veronica Hinman

Lazar LaLone

Lazar LaLone
Mentor: Veronica Hinman

Olumide Martins

Olumide Martins
Mentor: Jon Minden

Erica Moore

Erica Moore
Mentor: Jon Minden

Alanna Moss

Alanna Moss
Mentor: Maria Kurnikova

 Mark Nicholas

Mark Nicholas
Mentor: Sandy Kuhlman

Amritha Parthasarathy

Amritha Parthasarathy
Mentor: Jon Minden

Analyzing the Differences between APC2 mutants and Wildtype in Drosophila using 2D DIGE

Colorectal cancer is the second leading cause of death among cancers in the United States. In about 80% of colon cancers, there is a mutation in the genes encoding a particular set of proteins - Adenomatous polyposis coli (APC). APC is a tumor suppressor gene and also plays a vital role in how cells divide, cell-to-cell attachment and cell movement. APC2 functions as part of a destruction complex. Normally, the complex serves to destroy ß-catenin and prevent excess transcription in the cell. When the protein is mutated, the complex does not form leading to abnormal transcription and cancer. We performed proteomic screens of mutants null for APC2 to detect protein changes between mutant and wild-type embryos and to detect common and unique changes between APC2 mutants. 2-Dimensional Difference Gel Electrophoresis (2D DIGE) was used to detect the candidate proteins. APC2 mutant extracts and wild-type extracts were tagged with two different color fluorescent dyes and electrophoresed on the same 2D gel. The resulting 2D gels were fluorescently imaged with our Structured Illumination Gel Imager, which allows for the detection of extremely low abundance proteins. In order to control for genetic variations, we also used a full length rescue of APC2 to restore APC2 function to APC2 mutants. Cross-referencing the changes observed in the two gels, we found eleven distinct isoform changes that are caused by APC2 mutations. The proteins will be identified in the future through mass spectrometry.

Alexander Post

Alexander Post
Mentor: Sandy Kuhlman

Alex Rodriguez

Alex Rodriguez
Mentor: Gordon Rule


Nicole Sansone
Mentor: Newell Washburn

Danielle Schlesinger

Danielle Schlesinger
Mentor: Jon Minden

Analyzing the Differences between APC2 mutants and Wildtype in Drosophila using 2D DIGE

Colorectal cancer is the second leading cause of death among cancers in the United States. In about 80% of colon cancers, there is a mutation in the genes encoding a particular set of proteins - Adenomatous polyposis coli (APC). APC is a tumor suppressor gene and also plays a vital role in how cells divide, cell-to-cell attachment and cell movement. APC2 functions as part of a destruction complex. Normally, the complex serves to destroy ß-catenin and prevent excess transcription in the cell. When the protein is mutated, the complex does not form leading to abnormal transcription and cancer. We performed proteomic screens of mutants null for APC2 to detect protein changes between mutant and wild-type embryos and to detect common and unique changes between APC2 mutants. 2-Dimensional Difference Gel Electrophoresis (2D DIGE) was used to detect the candidate proteins. APC2 mutant extracts and wild-type extracts were tagged with two different color fluorescent dyes and electrophoresed on the same 2D gel. The resulting 2D gels were fluorescently imaged with our Structured Illumination Gel Imager, which allows for the detection of extremely low abundance proteins. In order to control for genetic variations, we also used a full length rescue of APC2 to restore APC2 function to APC2 mutants. Cross-referencing the changes observed in the two gels, we found eleven distinct isoform changes that are caused by APC2 mutations. The proteins will be identified in the future through mass spectrometry.

Irini Sotiri

Irini Sotiri
Mentor: Phil Campbell

Anthony Spadaro

Anthony Spadaro
Mentor: David Geller

ìX-linked Associated Factor 1 (XAF1) Plays a Critical Role in Regulating Apoptosis During Ischemia Reperfusion(I/R) Injury in Mouse Hepatocytesî

XAF1 is a downstream effector of the transcription factor Interferon Regulatory Factor-1 (IRF-1). IRF-1 plays a role in regulating apoptosis during I/R injury in liver tissue, a common complication that occurs during liver transplantation. It has previously been shown that down regulating IRF-1 protects hepatocytes from apoptosis, which leads to increased survival after liver transplantation. My hypothesis is that IRF-1 regulates apoptosis through XAF1, and that by decreasing XAF1 expression, mouse hepatocytes will be protected from apoptosis and I/R injury. The goals for the project are to show that XAF1 expression is correlated with apoptosis in vitro and show that levels of XAF1 expression are correlated with the degree of I/R injury after isograph liver transplantation in vivo in mice. Overexpression of XAF1 will be accomplished by adenovirus transfection and underexpression by injection with shXAF1. Protein levels will be measured by Western Blotting, RNA levels will be measured by PCR, and apoptosis will be measured by TUNEL assay. I expect to discover that apoptosis is regulated by XAF1 in hepatocytes and that down regulating XAF1 will have protective effect for hepatocytes during transplantation. This will add to the current knowledge of liver inflammatory pathways and present a possible therapeutic target for better outcomes for liver transplants.  

Preethy Sridharan

Preethy Sridharan
Mentor: Manoj Puthenveedu

Alissa Ting

Alissa Ting
Mentor: Alison Barth

Wesley Wang

Wesley Wang
Mentor: Alan Waggoner

Sophia Wu

Sophia Wu
Mentor: Newell Washburn

Sowmya Yennam

Sowmya Yennam
Mentor: Veronica Hinman

Tammy Ying

Tammy Ying
Mentor: Fred Lanni

Nicolas Zuniga-Penaranda

Nicolas Zuniga-Penaranda
Mentor: Aryn Gittis