2013 HHMI Summer Scholar Participants
Sarah Horner, Carnegie Mellon University
Mentor: Aaron Mitchell
The Role of ZFU2 in Biofilm Formation in Candida albicans
Candida albicans (C. albicans) is the predominant form of fungal infection in the United States, causing upwards of $2.6 billion in treatment costs each year. Infection is linked to the formation of biofilms on medically implanted devices, such as artificial joints and catheters. Furthermore, the increased resistance of C. albicans to current antifungal drugs complicates treatment. Therefore, we are looking to disrupt the first step in biofilm formation, which is the adherence of yeast form cells to a substrate. Prior to this work, Professor Jonathan Finkel identified thirty transcription factors essential for adherence. We assayed a knockout strain of each of these transcription factors in spider, YPD, and RPMI media. This showed zfu2Δ/Δ as defective in biofilm formation in YPD medium. Next, the zfu2Δ/Δ biofilm was compared to a wildtype biofilm using confocal microscopy. This showed zfu2Δ/Δ was significantly inhibited in producing a biofilm. A zfu2Δ/Δ complement will also be imaged using the confocal to determine if biofilm formation is adequately restored. Then zfu2Δ/Δ was assayed in vivo where it was also biofilm defective but restored by the zfu2Δ/Δ complement. Now we are determining the role of ZFU2 in biofilm formation. We know that in the absence of ZFU2, YWP1 is overexpressed, and YWP1 has been shown to inhibit adherence. Therefore, our hypothesis is that zfu2Δ/Δ is adherence defective because it overexpresses YWP1. We are creating a knockout of ZFU2 and YWP1, to determine if adherence and therefore biofilm formation is restored in order to test this hypothesis.
Rachel Sewell, Carnegie Mellon University
Mentor: Eric Ahrens
19F MRI Assessment of Systemic Inflammation Following TBI and Combined TBI and Hemorrhagic Shock in Mice
Traumatic brain injury (TBI) is one of the leading causes of mortality and morbidity in the United States, and has gained attention due to the conflicts in Iraq and Afghanistan. It is now understood that mortality is effectively doubled following a secondary insult, such as hypotension and hypoxia. This research proposal takes advantage of a perfluorocarbon emulsion MRI visible contrast agent (VS580H) to quantify systemic inflammation following experimental TBI and hemorrhagic shock (HS) in a mouse model. With the contrast agent, it is possible to label macrophages in situ and then noninvasively detect the trafficking and accumulation of these cells in vivo with MRI. This is due to the fluorine providing a unique signal for tracking these cells. Mice were divided into five groups: sham, controlled cortical impact (CCI with a velocity of 5m/sec at a depth of 1mm), CCI + HS, Severe CCI (velocity of 6m/sec at a depth of 1.4mm), and Severe CCI+HS. Mice undergoing HS sustained a shock period of 35 min with a mean arterial blood pressure of 25-27 mmHg, followed by resuscitation. The mice were injected with VS580H 48 hours post injury. Organs (brain, heart, liver, lungs, etc.) were excised and subjected to 19F NMR spectroscopy using a 10% TFA (trifluoroacetic acid) standard for quantification. There was a significant increase in macrophage accumulation in the brain for all of the conditions involving trauma when compared to shams (p<0.05). Mice with severe CCI were found to have higher total fluorine signal levels when compared to moderate CCI animals regardless of whether HS was imposed. A better understanding of the systemic response to these injuries will hopefully improve the efficacy of first responders.
Isaac Shamie, Carnegie Mellon University
Mentor: Manoj Puthenveedu
Characterizing the Trafficking and Signaling of a Clinically Relevant Allele of the mu-Opioid Receptor
Opioid analgesics, such as morphine or codeine, are the most powerful and widely used drugs to manage pain. However, there is tremendous variability among patients in terms of pain relief, side effects, and tolerance. Several allelic variants have been described in the mu-opioid receptor (MOR), the main target of opioid drugs in the brain. To understand whether and how these variants change the opioid response, I focused on the A118G, the most prevalent allele. Clinically, A118G correlates with lower opioid efficacy and higher tendency for addiction. The goal of this study was to test whether this allele changed the function and regulation of opioid responses at a molecular level. Specifically, I focused on the agonist-induced internalization of the receptor, which has been established to be important in controlling both short-term and long-term effects of opioid signaling. Internalization rates were initially analyzed in live cells using wild type and A112G (the mouse variant corresponding to A118G) receptors tagged with a pH-sensitive GFP. Because its fluorescence is quenched in acidic environments, this provides a novel biosensor to measure receptor movement from the plasma membrane to acidic endosomes with high time resolution. Agonist-induced internalization of A112G was significantly slower than the wild type, suggesting less desensitization of the A112G variant upon activation. To understand the functional significance of this delay, I will use TIRF microscopy to analyze individual steps in MOR endocytosis, and measure differences in Extracellular signal-Regulated Kinase, a downstream effector of MOR activation. Understanding these differences between the wild type and mutated receptor could facilitate the development of novel and personalized treatments for the management of pain and addiction.
Catherine Stephenson, Carnegie Mellon University
Mentor: Alison Barth
Characterization of TrpM8 Axons in the Mouse Spinal Cord
Cold sensation in the range 5-25 degrees Celsius relies on the TrpM8 receptor, which is expressed in a specific set of neurons in the peripheral nervous system. However, TrpM8 neurons are not uniformly distrubuted across the body. In thermal sensation experiments being performed in the Barth lab, the mouse hindpaw is stimulated with menthol, the chemical ligand for the TrpM8 receptor. We wanted to determine the best area to maximize the number of TrpM8 fibers stimulated. If the cervical spinal cord has more TrpM8 afferents, it might be best to stimulate the forepaw rather than the hindpaw in thermal sensation experiments. We were also interested in the laminar and medial/lateral distribution of these axons. Therefore, my project was to image, characterize, and quantify expression of the TrpM8 receptor across different locations in the spinal cord.
To characterize the distrubution of TrpM8 axons in the mouse spinal cord, I used TrpM8GFP transgenic mice. These mice carry a transgene encoding the green florescent protein (GFP) in the region under the control of the TrpM8 promoter. The neurons, including the axons, emit green florescence under light with a wavelength of 488nm.
I found that there are more neurons expressing the TrpM8 receptor terminating in the cervical rather than in the lumbar spinal cord. This suggests that the mouse forepaw can sense cold more robustly than the hindpaw; however, it is unclear why this might be the case. In addition, I discovered deeper TrpM8 fibers than had been previously described. Therefore, my experiments uncovered interesting features of TrpM8 axons that are important for continuing to study thermal sensation.