2009 Summer Undergraduate Research Program

Paul Bailey

I worked with Greg Robbins, a 5^th year graduate student in Dr. Katherine Knight’s lab. We studied an early developmental check point during B cell development where the newly recombined immunoglobulin heavy chain (IgH) must pair with a light chain-like molecule called surrogate light chain ( SLC ).  Successful formation and surface localization of the preBCR is required for B cell survival and expansion.  This checkpoint ensures that the IgH chain is functional and will likely pair with conventional light chain later in development. My research focused on an IgH chain, VH81X, previously shown not to pair with SLC .  We used site directed mutagenesis to substitute evolutionarily conserved residues into VH81X and determine if SLC pairing was restored.  Thus far, our results indicate that multiple residues will be required to restore pairing between VH81X-utilizing heavy chains and SLC . 

Sarah Cook

In eukaryotes, acetylation is known to modify proteins and affect transcription; however, the effect of acetylation on transcription in bacteria has not been studied.  The Wolfe lab determined that excess carbon can increase the transcription of certain genes by increasing the concentration of acetyl-coenzyme A.  One of these excess carbon-responsive genes is rprA, which encodes a small RNA.  My project studies whether an acetyltransferase is required for transcription of rprA in the excess-carbon response.  From a screen of 23 known and predicted E. coli acetyltransferases, we expect to identify a list of candidate acetyltransferases required for excess carbon-induced transcription.

Elyssa Katz

This summer I worked in Dr. Gallagher’s lab studying the family of tripartite motif (TRIM) proteins under the tutelage of MD/PhD student Taylor Heald-Sargent. There are ~70 different TRIMs, and while each family member may have specific functions, one view is that the TRIMs generally provide immunity to infections.  Indeed, TRIM5a restricts HIV by targeting the capsid and blocking virus uncoating.  To find out whether the various TRIM proteins might have additional antiviral effects on a range of viruses, I expressed plasmid DNAs encoding different TRIMs and then challenged with coronaviruses, which are enveloped RNA viruses causing respiratory and gastrointestinal diseases.  Thus far, I have screened multiple TRIM proteins for inhibition of mouse coronavirus entry and gene expression using a virus that produces luciferase during infection.  Amazingly, some of the TRIM proteins potently suppressed luciferase expression.  I hypothesize that the TRIMs might be decreasing levels of the mouse coronavirus receptor, CEACAM, on the surface of the cells.  TRIMs are known to be ubiquitin ligases, and they could be targeting CEACAM for lysosomal degradation.  This would be a novel finding indicating that TRIM family members operate in disparate ways to effect antiviral responses.  I have really enjoyed being part of this new study.

Andrew Kilianski

This summer I worked with Ana Shulla, a graduate student in Dr. Gallagher’s lab, to elucidate the mechanism by which a plant lectin called griffithsin blocks virus infections.  Griffithsin is known to be a potent inhibitor of human immunodeficiency virus (HIV), blocking HIV entry by binding to sugars appended to viral surface glycoproteins and thus inhibiting HIV interaction with host cells.  As such, griffithsin may substantially increase the efficacy of topical virucides.  Griffithsin was recently shown to inhibit viruses other than HIV, notably coronaviruses, but it is not clear whether griffithsin works similarly to block coronavirus interaction with host cells.  My project was to find out whether griffithsin inhibits coronaviruses at the level of virus binding to host cells and if so, to find out whether griffithsin binds to coronavirus particles at the regions where the virus meets the cell receptors.

Paulina Kozupa

This summer, I was privileged to work with Dr. Basile Siewe in the Knight lab. The Knight lab studies B-cell development in rabbits. Dr. Siewe studies different factors that regulate B cell lymphopoiesis.  In rabbits, B lymphopoiesis is arrested in animals older than 8 weeks.  My project was to examine whether the extracellular matrix protein periostin plays a role in the arrest of B lymphopoiesis. Previous studies showed that the gene OSF-2, which encodes for periostin, is dramatically reduced in adult rabbits. To study B cell development in vitro, we make use of the OP9 co-culture system. OP9 is a stromal mouse cell line, that express the OSF-2 gene and promotes B cell differentiation and proliferation.  When OSF-2 is knocked down in OP9 cells using siRNA, there is a dramatic decrease in B cell differentiation and proliferation compared to the wild-type OP9 cells. The goal of my research project was to determine if the observed decrease in B cell proliferation and differentiation was due to the reduction in periostin expression. This was to be tested by reintroducing periostin in periostin knock down OP9 cells, and then investigating whether this reinstates their potential to support B cell differentiation and proliferation. I have learned a vast amount of knowledge in the past 10 weeks and am truly grateful for this wonderful experience.

Nicole Lehpamer

Under the direction of Dr. Dennis Lanning, I studied the expression of the chemokine CXCL12 and its receptor, CXCR4, in rabbit appendix follicles. I synthesized a riboprobe for CXCR4 and used it in in situ hybridization in order to study its expression and localization in rabbit appendix. I also made a CXCL12-Ig fusion protein to examine CXCR4 protein expression by immunhistochemistry and FACS analysis. My results suggest that the CXCL12-CXCR4 interaction is stimulated by select intestinal bacteria and is critical for appendix follicle development. 

Brian Maunze

I am an undergraduate student from Zimbabwe currently studying at Beloit College. I am going into my senior year as of fall 2009, and I am majoring in biochemistry and minoring in Computational Visualization and Modeling. For the summer of 2009, I am working in Dr. Visick’s lab performing research on the symbiotic relationship between the Hawaiian squid and the bacteria Vibrio fischeri. Under the mentorship of Satoshi Shibata, I am biochemically characterizing the components of biofilm, which may be central to the symbiotic relationship between the Hawaiian squid and bacteria Vibrio fischeri. Using preliminary electron microscopy, we now know that outer membrane vesicles (OMVs) are a component of biofilms, however, we are yet to determine what molecules are associated with these OMVs. The purpose of my research project thus is to characterize the molecules that are associated with OMVs found within the biofilm.

Josh Muniz

This summer, I had the privilege of working in the lab directed by Dr. Qiao. Under the tutelage of a grad student named Zhenyu Zhong, I undertook the project of determining if and how BPV VLPs and HPV-16 VLPs activate the inflammasome in THP-1 cells. We hypothesized that both of these VLPs activate the inflammasome and that they require endocytosis, cathepsin-B, and Caspase-1 to be properly activated. To test this hypothesis, we needed to see if these VLPs triggered the secretion of IL-1beta from the THP-1 cells. To determine if the cells secreted IL-1beta, we ran several ELISA in which we measured the amount of IL-1beta present in the supernatant of stimulated THP-1 cells. We saw from several ELISA results that HPV-16 VLPs and BPV VLPs did indeed trigger significant IL-1beta secretion compared to the control. After knowing this, we ran several ELISA in which the THP-1 cells were given different drugs to inhibit certain things, such as endocytosis or caspase-1. From these ELISAs, we were able to conclude that both HPV-16 VLPs and BPV VLPs needed endocytosis, caspase-1, ROS, and cathepsin-B to properly activate the inflammasome in THP-1 cells, by showing that these inhibitors reduced IL-1beta production. To verify if caspase-1 was indeed activated, we ran a western blot to show that it was. In further studies, the lab plans to delineate a specific pathway of activation for both VLPs, and also see how the nalp3 protein is involved in the whole activation progress, as it has been shown using other stimulants to be crucial to inflammasome activation.

Ryan Murphy

I worked with Dr. Dennis Lanning this summer, studying the role of chemokines in diversification of the primary antibody repertoire in rabbit gut-associated lymphoid tissues. I made a riboprobe for the CXCR4 gene, a chemokine receptor that mediates cellular homing to the chemokine CXCL12, and used it for in situ hybridization on rabbit appendix samples. I found that CXCR4 mRNA is upregulated in B cells that have been activated by signals from intestinal commensals in rabbit appendix. These activated B cells then home to the basolateral CXCL12 ⁺ region of the B cell follicle, where they diversify their VDJ genes and proliferate. I also cloned the rabbit CXCL12 coding sequence into an Ig fusion vector and made a CXCL12-Ig fusion protein to detect CXCR4 protein expression on B cells by immunohistochemistry and flow cytometry. 

Shannon Newman

I worked in Dr. Karen Visick’s lab this summer studying the role of the regulatory protein SypG in transcription of the syp gene locus in the bacterium Vibrio fischeri.  The syp genes are particularly important for symbiotic colonization by the bacteria, due to their ability to promote biofilm formation. Four operons within the syp locus are regulated by SypG. Upstream of sypA, the first gene in the locus, there is a conserved 22bp sequence hypothesized to be the SypG binding site.  My experiments this summer worked to further elucidate SypG’s role in activating sypA transcription. I asked whether or not SypG is directly binding to this enhancer sequence, and which base pairs of that sequence are necessary for SypG-mediated transcription of sypA. So far, I have determined that the first four bases of the sequence do not appear to be necessary for transcription of sypA.

Carol Rowley

This summer I have had the opportunity to work in Dr. Baker's lab studying the PLP2 protease of Human Coronavirus NL63. We are looking for areas of the PLP2 viral protein that are involved in interferon antagonism. I used alanine scanning mutagenesis to introduce changes in surface residues of PLP2. I then transfected the plasmid DNA encoding the wild type or mutant PLP2 into 293T cells, and determined if the mutant had the same level or a reduced level of interferon antagonism. Knowing where PLP2 disrupts the IFN response can lead to antiviral coronavirus therapy.

David Studer

During the summer of 2009, I was fortunate to be a member of the Baker Lab and study Coronaviruses (CoVs).  Throughout the summer, I studied the Murine Hepatitis Virus (MHV) and an antiviral compound, 0346S, which successfully blocks MHV replication by inhibiting the 3CLprotease which is necessary for viral replication.  For my project, I wanted to determine if I could generate 0346S resistant MHV, and identify possible mutations that confer drug resistance.  We hypothesized that because the antiviral drug (0346S) specifically targets the 3CLprotease of MHV, we expect to see mutations in this region of 0346S resistant viruses.  Over the course of the summer, we were able to successfully generate drug resistant viruses (DRVs) by passaging MHV infected cells in the presence of the 3CLpro inhibitor 0346S.  We then plaque purified our DRVs, PCR amplified the 3CLpro region, and performed sequence analysis.  Our data revealed that of ten isolates analyzed, a mutation at position 10285 with in the 3CLprotease region was consistently mutated resulting in an amino acid change from threonine to isoleucine.  Our future goals are to move this mutation into the MHV reverse genetics system so that we can study the effects of this mutation on virus replication.

Osama Zayyad

Currently, the mechanism of nonenveloped virus penetration of host cell membranes is poorly defined. During my summer experience in the Wiethoff lab, I examined the ability of various mutant forms of the membrane lytic protein of adenovirus to bind to and disrupt membranes in vitro. These results were correlated with the ability of adenoviruses containing these mutant proteins to penetrate endosomal membranes to infect cells.