2008 Summer Undergraduate Research Program

Jill Beamon

This summer I worked in Dr. Alan Wolfe’s Lab studying iron uptake in E. coli. Iron is important because it is essential for viability and it plays a role in bacterial virulence. Vertebrates have learned to withhold iron, which limits the capacity of pathogenic bacteria to multiply. To combat this iron deficiency, bacteria produce siderophores. A siderophore is an iron chelator that removes iron from the host sources by its superior binding constant. One example is enterochelin, which is part of the enterochelin iron uptake system.

When grown in iron-rich media, ompR mutants form an orange pellet. Since the response regulator OmpR is known to repress the transcription of the ent genes that are responsible for enterochelin production, I hypothesized that the formation of orange pellets is due to derepression of the ent genes in ompR mutants. To test this hypothesis, I made ompR ent double mutants and observed the pellet color. I also investigated the impact of the central metabolite acetyl phosphate on this orange phenotype by making mutations that alter the levels of acetyl phosphate in the cells. My experiments showed that the formation of an orange pellet in ompR mutants did not depend on acetyl phosphate or the enterochelin system. Another explanation for the orange phenotype could be that the iron might simply leak into the cells because ompR mutants are known to have impaired outer membranes. Being able to work on this project provided me with a great opportunity to get hands on experience in a research lab as well as it allowed me to develop my critical thinking skills.

Lisa Choi

This summer, I was privileged to work with Dr. Britte Beaudette-Zlatanova in the Knight lab, which studies B-cell development. Dr. Beaudette-Zlatanova focuses on generating precursor B cells from umbilical cord blood in vitro. Cord blood contains hematopoietic stem cells (HSC), which differentiate into lymphocytes or myeloid cells. Her research centers on pushing these HSCs to common lymphoid progenitors to supplement cord blood HSC transplants with human precursor B cells. This would lead to a faster development of normal diversified B cell repertoires in cord blood transplant patients. This summer, my project was to examine whether the cytokines stromal cell-derived factor (SDF), insulin-like growth factor (IGF), and leukemia-inhibitory factor (Lif) enhance B-cell development from cord blood HSCs. In addition, previous studies showed that the combination of thrombopoietin, stem cell factor, and FLT3 ligand (TSF) expanded HSCs three to four-fold. I tested to see if this expansion could be augmented with the addition of a feeder layer. In order to test these scenarios, I sorted CD34 lineage positive cells from umbilical cord blood and set up in vitro cultures of 293T cells to act as feeder layers. I then plated the cells with the prospective cytokines. My preliminary data revealed that SDF and IGF with a cocktail of known proliferative factors did not further enhance B cell expansion compared with other known growth factors. However, SDF in combination with the cytokines IL-3, SCF, and HGF improved B-cell development. TSF in the presence of a feeder layer did not yield varying results from cultures without stroma. My preliminary data also showed that Lif alone boosted B-cell development. This suggests that Lif acts with factors specific to 293T cells that enhance B-cell development. I learned more in these past ten weeks than in any other laboratory I have worked in and am so thankful for having been a part of this special opportunity.

Nada Daher

My summer work was carried out in Dr. Iwashima's laboratory and focused on the mechanism of T lymphocyte trafficking. Previous work of the lab showed that a surface antigen called CD28 plays a critical role in tissue invasion by aggressive self-reactive T cells and contribute to lethal disease progression in a form of autoimmune disease. We studied whether CD28 is sufficient to make T cells invasive. THe data showed that CD28 can enhance proliferation of invading T cells but is not essential for invasion.

Dana Dieringer

This summer I worked on a research project with Kari Severson, a graduate student in Dr. Katherine Knight’s lab. We observed that the antibody IgM binds to Bacillus anthracis spores. We saw that IgM from three different species, rabbit, mouse, and human binds to spores. Because of this observation, we believe IgM binds to spores in an antigen non-specific manner and that the binding could serve an innate immunological purpose. 

We hypothesized that spores coated with IgM would be more readily phagocytosed by macrophages than spores not coated with IgM. To test this hypothesis, we purified, fluorescently labeled, and coated spores with IgM. We then incubated spores with macrophages and measured the resulting fluorescence of the macrophages using flow cytometry. As compared to macrophages incubated with non-IgM coated spores, we saw a decrease in the fluorescence when macrophages were incubated with IgM-coated spores, suggesting that IgM inhibits the uptake of spores. Based on these results, we predict that upon binding to spores, IgM may block a molecule on the spores that is necessary for the recognition and subsequent uptake by macrophages. Experiments are underway to test this prediction.

Jennifer Jensen

I had the amazing opportunity to work in Dr. Susan Baker's lab this summer, aiding her student Mark Clementz in his research on a mutant form of MHV (Mouse Hepatitis Virus), a coronavirus. The aim of the Baker lab is to study coronaviruses: a family of RNA viruses known for their unique crown of glycoprotein spikes covering the virion, or virus particle.

As I studied my mutant strain of MHV, I learned that it was temperature sensitive, meaning replication of the virus was inhibited at higher temperatures. Mark had previously shown through electron microscopy photographs that after two hours exposure to a non-permissive higher temperature, our virus tsAlb6 would lose most of its double membrane vesicles (sites of RNA replication) and mitochondria would expand and bleb. I performed time course experiments to determine if two hours in the non-permissive temperature was truly needed for this effect, using electron microscopy to photograph cells.

I also investigated whether our mutant virus could be supplied with a protein (specifically non-structural protein 4) to rid of the temperature-sensitive phenotype. We supplied wild type nsp4 to determine if it would facilitate DMV assembly when tsAlb6-infected cells were incubated at the non-permissive temperature.

Because it took a full two hours in the non-permissive temperature for DMVs to decrease and mitochondria to bleb, and because wt nsp4 did not complement tsAlb6 and eradicate the temperature sensitive phenotype, we believe that the p150 precursor to nsp4 is required for complementation. Mark will continue investigating tsAlb6, making an effort to determine the localization of a mutant form of p150, considering whether it induces mitochondrial blebbing, and deciding if its expression will complement tsAlb6.

Jessie Panks

This summer I worked in the Wiethoff laboratory examining the inflammatory response to adenovirus. My work involved understanding the mechanisms involved in adenovirus activation of the inflammasome. The inflammasome is a multiprotein complex which mediates the release of interleukin-1beta from cells in response to infection by various pathogens or other danger signals. Interleukin-1beta is a potent proinflammatory cytokine released from cells upon infection which triggers as well as directs the innate and adaptive immune response. My work determined that reactive oxygen species were generated in macrophages infected with adenovirus and that these species were required for adenovirus activation of the inflammasome. 

Mona Patel

I am very fortunate to have been chosen to work with Dr. Dennis Lanning for the past ten weeks. I worked on a smaller project branching from the larger idea that Dr. Knight's Lab focuses on. My project involved the analysis of CD5 expression. CD5 is expressed in all adult rabbit B cells, unlike in humans and mice. This is a unique characteristic of the rabbit and hence the reason I choose to study CD5 expression in the rabbit. It is hypothesized that CD5 is a scavenger protein that has a putative role as a co-receptor with BCR. The B cell population expresses an initial antibody repertoire of limited diversity via VD(J) gene arrangements. After birth, the B cells migrate to the GALT, of which the appendix is a major site, where select intestinal commensals stimulate somatic diversification of Ig genes. My methodology consisted of three main steps leading to my ultimate analysis. During my research internship, I was able to successfully produce DNA templates for riboprobe synthesis as an initial step for in situ hybridization experimentation. I have really enjoyed my experience and grown into a more scientifically knowledgeable and confident individual.