Summer 2009 Research Projects

During the summer of 2009, Dimensions supported twelve faculty/student research projects.

Abby Schultz '11
Bailee Beauman '11

Supervisor: Dr. Jeff Cardon S

Identification of Swarming Genes of Psuedomonas aeruginosa

Swarming is a complex motility mechanism, utilizing a large amount of cooperating genes (Overhage, 2006). According to Overhage et. al., over 400 genes are responsible for swarming, these genes are either up-regulated or down-regulated during swarming.  In  our research, we are attempting to further identify the genes responsible for swarming. More specifically we are randomly inserting Tn5 transposon into the genome of non-swarming mutant P. aeruginosa in order to reinstate the swarming phenotype in hopes to identify the genes involved in swarming repression.  By analyzing the genes involved in the repression of swarming, advances can be made in inhibiting the virulent phenotypes in P. aeruginosa, such as biofilm formation, swarming and antibiotic resistance. Clinical applications of this research could allow for treatments for people afflicted with infections caused by P. aeruginosa, including prolonging the lives of cystic fibrosis patients.

Alisa Vickrey '11
Michelle Ellison '10

Supervisor: Dr. Brian Nowak-Thompson

The Analysis of Compounds Produced by the Pseudomonas fluorescens Strain Pf-5 Compounds In Vitro

The strain of Pseudomonas fluorescens, Pf-5, is known to produce metabolites that inhibit rhizosphere plant pathogens, therefore acting as a natural biocontrol agent and increasing plant survival (Raaijmakers et al., 2002). The P. fluorescens Pf-5 strain produces 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin (PLT), and pyrrolnitrin (Duffy & Defago, 1999; Bender, et al, 1999; Sarniguet, et al., 1995). These act as natural fungicidal agents toward certain fungal pathogens common to the rhizosphere of commercial crops (Raaijmakers et al., 2002; Duffy & Defago, 1999). To further understand the biosynthesis and regulation of these antibiotics, analytical methods, such as High Performance Liquid Chromatography (HPLC), column (flash) chromatography and NMR can be used. In this experiment a screening process was constructed using HPLC for analysis of the compounds produced by Pf-5 grown in liquid culture. Column chromatography and NMR were utilized to try and isolate compounds produced by the organism. Once an HPLC screening process was established of Pf-5 liquid culture extracts, isolated fungal extracts were added to Pf-5 liquid cultures to analyze difference in compound production within the strain. A difference in production of specific Pf-5 compounds was positively identified in one of the Pf-5 liquid cultures exposed to a fungal extract. This in vitro approach contributes to the evidence that fungal exudates may stimulate the production of Pf-5 antibiotic compounds (Duffy & Defago, 1999).

Chuck Hay '10
Marvin Leopold '10
Aye Mon '11

Supervisor: Dr. Craig Tepper

Continuing the research on Millepora alcicornis and Millepora complanta, our lab started a new project to determine if there is host-specificity for Sybiodinium within coral. 

C.J. Ronhovde '11
Ellie Hutchison '11
Yongik Lee '11

Supervisor: Dr. Charley Liberko

Synthesizing Organic Dyes for Solar Panels

Ronhovde: I worked on synthesizing a push-pull poly-ene organic dye for the solar cells made by the Physics summer researchers. The dye was reported in a research article to have an efficiency of approximately 5% and could be used to compare solar cells made at Cornell to solar cells used by other research groups. Hutchison: Organic dye –sensitized solar cells have emerged as an inexpensive and efficient alternative to inorganic and organo-metallic solar cells. Azo dyes with both an amine electron donating group and a carboxyl electron withdrawing group were synthesized for use in these solar cells. P-aminobenzoic acid was diazotized in tetrafluoroboric acid to produce a diazonium-fluoroborate intermediate, which was coupled to triphenylamine to produce the reddish brown 4-[2-(diphenylamino)phenylazo] benzoic acid. A similar procedure produced the reddish brown 3-[4-(dimethylamino)phenylazo] naphthalic anhydride from 3-amino-1,8-naphthalic anhydride and N,N-dimethylaniline.

Lee: Traditional solar cells have been too expensive, as organic dyes used to cover them did not allow preventing the loss of the energy and sunlight on its way to solar cells. iPr-PMI is just one out of many potential solutions that could substantially increase the power conversion efficiency of solar cells. Synthesizing iPr-PMI for energy purposes could produce positive effects on the quality and amount of solar energy generated by solar cells, but without a detailed research and extensive investment, solar energy may forever remain an unachievable dream.

Kirsten Gierach '10

Control Mechanisms of ELF4 Downregulation in CD8+ T-cells

Research on CD8+ T-cells in Dr. Lacorazza’s lab has focused on understanding the signaling pathway that regulates proliferation and homing of naïve T-cells. During the immune response, CD8+ T-cells are activated by an antigen presenting cell (APC) and undergo a rapid expansion producing cytotoxic T-cells as well as memory cells. Previously, Dr. Lacorazza’s lab published findings that ELF4 negatively regulates quiescence in naïve CD8+ T-cells by activating Klf4, a tumor suppressor, inducing cell cycle arrest.

The goal of my research was to determine what pathways were involved in downregulation of ELF4 during TCR (Tell Cell Receptor) activation. The methods I used were to isolate murine CD8+ T-cells from spleens using negative selection, in vitro stimulation with and without inhibitors of specific pathways, RNA isolation, reverse transcription, and quantification of ELF4 expression using real time PCR. I looked at three different pathways: MAPK, PI3K, and mTOR. If these pathways are involved in suppression of ELF4, selectively inhibiting them should reverse downregulation of ELF4 during activation. To activate the CD8+ T-cells, I used two techniques: TCR cross-linking and antigen stimulation using ovalbumin peptide and OT-1 transgenic mice. Preliminary results suggest that the mTOR pathway, downstream of PI3K and MAPK, is involved in ELF4 downregulation during TCR activation. These findings could be important in improving immunological memory during vaccinations by targeting the activity of ELF4. Future directives will focus on understanding how mTOR regulates ELF4 expression.

Jennifer Bays '10

Gene Expression in Corneal Epithelium 24 Hours Post-Injury

Dr. C Wayne Smith’s laboratory focuses on inflammation and wound healing with an objective of determining the mechanism for leukocyte accumulation at sites of inflammation and tissue injury. One of the studies of his lab, and the study that I worked with, uses a mouse cornea model with an induced inflammatory response to an injury on the ocular surface. The inflammation develops as a cascade of sequentially dependent cellular emigrations into the corneal epithelium with precise temporal and molecular relationships. There are unique features in the cornea not predictable from studies of other vascular beds, and defects in this cascade delays corneal wound healing.

Past works have shown γ/δ t cells and the ICAM-1/ LFA interaction critical to leukocyte emigration. Use of knockout mice for ICAM-1 and δ-TCR evaluated these genes’ specific molecular contributions to wound healing. Temporal studies attest that γ/δ t cells peak at 24 hours after injury thus this time period was investigated. A PCR Super Array was ran using isolated RNA pooled from six CD57BL/6 mice corneas. This array permits the simultaneous analysis of multiple genes related to the inflammatory response. The technique was chosen to show a range of cytokines and growth factors present at 24 hours. Unfortunately, for unknown reasons the data from the arrays indicated contamination or denaturing within our samples or probes and thus data collected was unreliable. Using prior research, specific chemokines and growth factors were chosen for their roles in the attracting leukocytes to the injury site. The gene expression of each was evaluated by amplifying the sequence of interest with quantitative PCR. Preliminary data shows that an increased expression of the CCl4 and Nerve Growth Factor beta (NGF-b) at 24 hours. There is a marked decrease in CCl4 in the δ-TCR-/- mice compared with the WT, suggesting that γ/δ t cells release this chemokine. Further experiments using immuno-fluorescence will better explain this observation.

Mikelle Wortman '10

Underprivileged Patients Treated for Traumatic Brain Injury (TBI) at Iowa Methodist Medical Center

The purpose of this study is to identify outcomes of underprivileged patients treated for Traumatic Brain Injury (TBI) at Iowa Methodist Medical Center.  While most research on TBI is done in larger, more diverse cities nationwide, there are minimal studies done on patients of ethnicity and lower socioeconomic status who experience TBI in small, rural, historically less diverse cities such as Des Moines.  This study identifies and compares treatments and outcome discrepancies of patients among differing ethnicity and varying income levels.  The anticipated outcome of this study can lead to the development of new measures to ensure equal access to treatment and help provide the best TBI outcomes for all patients specifically in central Iowa. 

Sean Lehman '10

Supervisor: Dr. Cindy Strong

In Vitro Analyses of Human Copper-Zinc Superoxide Dismutase: Protein Chemistry Studies of Metal-Binding Properties

Amyotrophic lateral sclerosis (ALS), or more commonly known as Lou Gehrig’s disease, is a neurodegenerative disease that usually causes eventual respiratory failure due to motor neuron death. Approximately 90% of all ALS cases are classified as sporadic ALS, meaning that there is no known cause. The remaining 10% of cases are classified as familial ALS, and of these, 2% have been linked to the gene for SOD1. This gene encodes the copper-zinc superoxide dismutase protein, an antioxidant enzyme which catalyzes the reduction of the superoxide radical (O2-) to hydrogen peroxide, which can then be further broken down into water and diatomic oxygen via hydrogen peroxidases. In vivo, metals bound to the wild-type protein are the catalyst for the reduction of superoxide, but certain mutants exhibit different metal-binding properties. In our experiments, we explored these properties through a variety of applications. I produced the S134N mutant in E. coli expression bacteria, in addition to our previously generated variants, and then purified the proteins using hydrophobic interaction and ion exchange chromatography. Metals were removed from SOD1 via the standard EDTA dialysis method. Metal content was assayed using an inductively coupled plasma-optical emission spectrometer (ICP-OES). Apoproteins were selectively titrated with metals to form the Cu2Co2SOD derivatives, with metal-binding monitored by UV-Visible spectrophotometry. Results from these experiments have furthered our body of evidence suggesting that proper metallation is possible under the correct experimental conditions. Furthermore, these studies contradict previously obtained results about metal-binding, possibly suggesting an issue of a thermodynamic versus kinetic product. Future experiments will seek to elucidate this, and provide answers to these questions.

Nick Campbell '11
Phil Gallagher '11
Amanda Gilbert '10
Brock Nelson '11

Supervisors: Dr. Bob Black and Dr. Andy McCollum

Reproductive Biology of Ornate Box Turtles

During June and July of 2009, I joined the research group co-directed by Bob Black and Andy McCollum on the reproductive biology of ornate box turtles in eastern Iowa. We used a variety of techniques to study to reproduction of these turtles including the monitoring of the activities of female ornate box turtles using radio telemetry, daily searches of our study site for evidence of nesting and nest depredation, and experimentation with artificial nests that examined the cues that predators use to locate turtle nests.

See-yin So '10

Using fluorescent resonant energy transfer to measure membrane voltage of cells

This past summer I had the opportunity to spend eight weeks working at the University of Baltimore Biotechnology Institute (UMBI) in Baltimore, Maryland, as the Kao Fellow in Medical Biotechnology.  My project focused on testing and validating a new superior fluorescence method for measuring membrane voltage of cells using fluorescent resonant energy transfer (FRET) and fluorescent microscopy.

Vicki Levasseur '11

Supervisor: Dr. Richard Kraig, University of Chicago

Microglia and TNF-a Modulate Chronic Migraine

This summer at the University of Chicago I studied a neurological process called Spreading Depression (SD) and how it can lead to an increase in pain sensitivity associated with migraine headache; SD is believed to be the neurophysiologic basis of the sensory symptoms experienced during migraine aura. The three main characteristics of SD include a period of electrical silence and a Direct Current (DC) change, which propagates at 3 mm/min. Using electrophysiological techniques I observed the effects of recurrent SD on rat hippocampal slice cultures. I postulated that the progression of episodic to chronic migraine is due to reversible changes in neuronal excitability as a result of recurrent SD. If recurrent SD leads to an increase in excitability then it could be considered an upstream cause of chronic migraine. Additionally, since Tumor Necrosis Factor-a (TNF-a), a cytokine involved in the regulation of immune cells and cell signaling, causes an upregulation of AMPARs and a downregulation of GABAA receptors (Malenka, et al., 2005), I proposed that this increase in excitability is dependent on TNF-a. Therefore, my goals were to determine whether or not it is “easier” to fire spreading depression in slices that had previously experienced SD and to decipher whether or not TNF-a is involved in lowering the current threshold to fire CSD. I concluded that SD makes brain more susceptible to SD and that this increase in susceptibility is dependent on TNF-a signaling. Thus, the transformation of episodic migraine to chronic migraine may stem from migraine-induced alterations in innate cytokine signaling within brain.

Fadzai Fungara '10

Supervisor: Dr. Derin Sherman

Medical Physics: Imaging electrical activity within the body

There are some medical conditions that are electrical in nature. For instance, an electrically erratic piece of heart tissue can trigger an episode of ventricular fibrillation, which can be fatal. Treating this condition involves localizing and identifying the part of the heart which behaves erratically. One way of doing this would be to place electrodes on the patient’s body and using the measured voltages to reconstruct the pattern of electrical activity within the heart.  Our project this summer was to simulate this situation in 2 dimensions.

Brian King '10

Supervisor: Dr. Jason Rhodes, The Children's Hospital , Aurora, Colorado

Apparent equinus in children with spastic cerebral palsy

I was an intern at The Children's Hospital in Aurora Colorado for 10 weeks. I performed clinical research in the Gait and Movement Analysis Laboratory at the hospi.tal, with children with neuromuscular disabilities. With an Orthopedic surgeon Jason Rhodes and through the Muscular Skeletal Research Institute, under a larger study involving knee extension limitations in terminal swing (a formality for institutional review board legality purposes) we performed a pilot study and a relatively unknown gait disorder called in children with spastic cerebral palsy called apparent equinus. I compiled and analyzed a database of over 80 apparent equinus patients that were seen in the gait lab at the hospital. Dr Rhodes and I hope to co-author a paper publishing our findings in the near future. In addition to my research I was able to shadow Dr Rhodes and other orthopedic surgeons multiple times a week in clinic and surgery as a part of my internship experience.