Tag: Natural/Physical Science

Systemic Inflammation Increases Expression of Pro-Inflammatory Interleukin-1 Receptors On Neurons And Astrocytes, but Not Microglia, in the Cervical Spinal Cord

Presenter(s): Kelly Royster − Human Physiology

Faculty Mentor(s): Adrianne Huxtable, Austin Hocker

Poster 64

Research Area: Natural/Physical Science

Funding: Adrianne Huxtable’s Parker B. Francis Fellowship, University of Oregon

Inflammation is a component of all diseases, whereby key pro-inflammatory signaling molecules and receptors increase systemically and in the central nervous system (CNS). Systemic inflammation activates astrocytes and microglial cells in many regions of the CNS, which alter neuronal function and lead to behavioral changes. Our work has shown systemic inflammation impairs respiratory function through activation of the pro-inflammatory interleukin-1 receptors (IL-1RI) in the cervical spinal cord, but the roles of different cell types are unknown. To better understand how activation of IL-1RIs undermines breathing, we first need to determine what CNS cell types express IL-1RIs and how systemic inflammation changes IL-1RI expression. Based on the expression of IL-1RIs in other CNS regions, we hypothesized systemic inflammation would increase IL-1RI expression on identified neurons, astrocytes and microglia in the cervical spinal cord. Using immunohistochemistry to fluorescently label cell types and IL-1RIs, we first determined the optimal concentration of the IL-1RI antibody (1:250) by
a dilution series (n=5) in the hippocampus, where IL-1RI was known to be expressed after systemic inflammation. Further, preliminary data (n=2) suggest systemic inflammation increases IL-1RI expression on neurons (labeled by NeuN, 1:500) and astrocytes (labeled by GFAP, 1:500), but not microglia (labeled by Iba1, 1:1000). These results suggest neurons and astrocytes are likely the key cells undermining respiratory function after systemic inflammation. Understanding the mechanisms by which systemic inflammation undermines respiratory function may lead to targeted therapeutic interventions to promote breathing.

Utilizing a Fusion Protein for Sequence Specific Nucleosome Shifting in Chromatin

Presenter(s): William Reed-dustin − Biology, Human Physiology

Faculty Mentor(s): Jeffrey McKnight

Poster 81

Research Area: Natural/Physical Science (Molecular Biology)

Chromatin refers to the organization of DNA in eukaryotic organisms. Chromatin is organized such that DNA wraps around protein groups called histones. The units of histones wrapped in DNA are called nucleosomes, nucleosomes are connected by short stretches of linker DNA. DNA in nucleosomes is relatively inaccessible to RNA polymerase and transcription factors and thus, is effectively turned off. The goal of this research was to move nucleosomes onto specific DNA sequences by producing a fusion protein that would combine the binding domain from a specific transcription factor, XBP1, and the active domain from a known chromatin remodeler protein, CHD1. A procedure originally developed by Dr. Jeffrey McKnight was used to produce a plasmid that coded for a protein with the binding domain of XBP1 and the active domain of CHD1. This plasmid was then transformed into yeast. The cells’ DNA was then digested into mono-nucleosomes which were sequenced and compared to yeast without the plasmid inserted. This was done to see if the fusion protein had altered the nucleosomes’ locations.
The goal of this research is to show that the strategy for fusion protein production can be applied to diverse transcription factors across the yeast genome. Ultimately, this strategy could be useful in cancer treatment, silencing oncogenes by moving nucleosomes onto their binding sites.

Strain-Dependent Differences in Mouse Norovirus Capsid Determine Cell Death and Inflammation

Presenter(s): Ruth Vanelle Nouboussi − Human Physiology

Faculty Mentor(s): Sierra Dawson

Poster 39

Research Area: Natural/Physical Science

The Murine Norovirus is a common virus used in mice experiments in many research facilities. The virus alters the cellular morphology in hematopoietic cells and it is transmitted through fecal or oral routes. This study focuses on CW3 & CR6 strains of the mouse norovirus. The purpose of this experiment was to determine if the strains that use the CW3 capsid produces more IL-1α (cytokine released as a measure of the degree of inflammation) and cell death compared to the CR6 capsid. We performed a Mouse IL-1α Elisa on virally infected BMDCs (Bone Marrow-derived Dendritic Cells), a virus-mediated cytotoxicity assay performed on BV2 cells (mice microglia cell line) using CW3, CR6, CW3-VP1^CR6 (CW3 virus strain with the CR6 capsid; VP! shows that there was a capsid swap) and CR6-VP1^CW3 chimeric viruses to determine the necessity for VP1^CW3 in IL-1α secretion and cytotoxicity. Using two-way ANOVA testing, we found that there was more LDH (Lactate DeHydrogenase) released in the supernatant of cells infected with strains that use the CW3 capsid compared to CR6 capsid at MOI (Multiplicity Of Infections) 10, 1 and 0.1, and there was less LDH present in the supernatant of cells infected with strains that use the CR6 capsid compared to the CW3 capsid. We also found significantly more IL-1α in the supernatant from cells infected with strains that use CW3 compared to wild type and CR6 (P < 0.05). These findings demonstrate that the CW3 capsid produced more IL-1α and also caused more cell death compared to CR6.

Synthesis of 7,14-Diarylfluoreno[3,2-b]fluorenes

Presenter(s): Tristan Mistkawi − Biochemistry

Faculty Mentor(s): Josh Barker, Michael Haley

Poster 74

Research Area: Organic Chemistry (Natural/Physical Science)

Funding: UROP Mini-grant

The Haley group is interested in a class of organic molecules, known as the indenofluorene (IF) scaffold, for potential use as organic semiconductors (OSCs) in electronic devices. IFs show great promise as OSCs because of their ability to easily and reversibly accept electrons. Similarly to the well-known class of acene OSCs, we are interested in studying the effect of extending IF scaffold -conjugation to discover trends in electronic properties. While other researchers in the literature have studied compounds with similar properties, no one has performed a rational, systematic study. This work requires exploring the synthesis of several 7,14-diarylfluoreno[3,2-b]fluorenes (FFs) to compare to structurally related molecules in the IF scaffold. Along with affecting the optoelectronic properties of FFs, substituting different aryl groups at specific positions on the molecule is important for crystal engineering, which will help improve our understanding of this novel scaffold. Many derivatives have not been explored yet, and studying solid-state packing interactions may improve device performance and influence our ability to implement these compounds as organic semiconductors.

Assay of Insulin-Stimulated Signaling by Flow Cytometry: Key Points of Regulation

Presenter(s): Shawn Melendy − Biochemistry

Faculty Mentor(s): Carrie McCurdy, Byron Hetrick

Poster 75

Research Area: Natural/Physical Science

Funding: UROP mini-grant, American Physiological Society Undergraduate Summer Research Fellowship Program

Type 2 Diabetes, an increasingly prevalent disease worldwide, is partially caused by a progressive loss of insulin response in adipose tissue and skeletal muscle. Multicolor flow cytometry is a powerful tool that can be used to measure multiple signaling events simultaneously in specific cell types within mixed populations. The objective of this study is to design a sensitive and high-throughput assay to measure key points of regulation in the insulin signaling pathway for myocytes using flow cytometry. We have developed a multicolor flow cytometry panel to measure the insulin stimulated phosphorylation of Akt(S473) and the transport of the insulin responsive glucose transporter, GLUT4, to the plasma membrane. C2C12 myoblasts were stained with primary conjugated antibodies for pAkt(S473) and an extracellular region of GLUT4, indicative of translocated GLUT4 present
in the plasma membrane. Both C2C12 myoblasts, an immortalized cell line, and primary myoblasts, isolated from non-human primate muscle, responded to insulin with increased pAkt(S473) and plasma membrane GLUT4 with an EC50 of <10nM, similar to physiological response. Future work will expand the panel to measure phosphorylation of insulin receptor substrate and phosphoinosital 3-kinase (PI3K) activity by quantitating phosphoinosital (3,4,5) phosphate (PIP3) production. The sensitivity of the assay will be demonstrated by inhibiting key insulin-activated kinases including PI3K by Wortmannin, and Akt activation by MK- 2206 and measuring insulin signaling at points up and downstream of inhibition. We anticipate that this will provide a powerful method to rapidly dissect the insulin signaling cascade for a specific cell type within mixed populations of cells.

Building Zebrafish Gut Bacterial Communities From the Bottom Up

Presenter(s): Dylan Martins − Biology

Faculty Mentor(s): Raghuveer Parthasarathy

Poster 44

Research Area: Natural/Physical Science

The intestines of humans and other animals are home to tens of trillions of microbes. These microbial communities play important roles in health and disease, and are composed of dozens to hundreds of interacting species. While the factors that determine a particular species’ presence in the gut are largely unknown, both physical and biochemical interactions between species are likely important. Learning about these factors poses challenges due to the difficulty of performing controlled experiments with existing tools.

This project addresses these challenges by constructing five-species microbial communities in zebrafish, a model vertebrate animal to determine whether these model groupings are stable, and what inter-species interactions are evident. We use zebrafish as a model organism because they can live in a bacterially controlled environment and because their larval transparency allows for live microscopy. Experimentally, we introduce commensal intestinal microbes to larval zebrafish, initially raised germ-free to allow introduction of controlled combinations of bacterial species. Using a combination of conventional dissection and plating assays and three-dimensional live imaging, we have been able to demonstrate the existence of stable multi-species communities, and we can test whether outcomes from two-species competitions contain enough information to allow prediction of multi-species abundances and interactions, of key importance to creating predictive models of the human gut. Further, we find that individual species are differentially sensitive to the presence of other species, and that the community stability is sensitive to the presence of certain species. Correlations can also be identified between species and their spatial structure within the fish gut.

The microbiome is important to health and disease, but it is a complex system which is difficult to understand. By constructing a model system in a vertebrate gut that has an interesting and tractable number of species, we gain insights and reveal principles that might apply to the human microbiome.

Properties and Synthesis of Three Component Heterostructure: (BiSe)1+δ(Bi2Se3)1+ δ (BiSe)1+ δ (TiSe2)

Presenter(s): Alexander Lygo − Physics, Chemistry

Faculty Mentor(s): David Johnson,

Poster 22

Research Area: Natural/Physical Science

Funding: Vice President for Research and Innovation (VPRI) Undergraduate Fellowship, Presidential Undergraduate Research Scholars program

As potentially applicable in high-performance electronics and quantum computers, topological insulators and heterostructures containing them have recently garnered significant interest by materials scientists. Despite their imagined utility, these compounds have proven difficult to synthesize. In a recent study of a series of compounds, [BiSe1+δ]m[TiSe2] m with m = 1, 2, 3, it was observed that, for the m = 3 compound, the topological insulator Bi2Se3 formed upon deposition and was present at all annealing temperatures. To test if Bi2Se3 could be incorporated into a heterostructure, a series of (Bi- Se)3-TiSe2 precursors with varying Bi-Se ratios and layer thicknesses were prepared and annealed at various temperatures for 30 minutes. A combination of specular and in-plane diffraction indicated that select precursors formed a highly crystalline and crystallographically aligned compound containing BiSe, Bi2Se3, and TiSe2 and high-resolution electron microscopy revealed the stacking sequence of the constituents. X-ray fluorescence measurements reveal that the compound formed readily over a range of Bi-Se ratios. Electron transport measurements revealed metallic behavior and surprisingly high carrier mobility, compared to BiSe1+δ TiSe2. These results provide a synthetic route for preparing a high quality Bi2Se3 containing heterostructure with unexpected properties and with further research, a material with properties applicable to electronics or quantum computers may be discovered.

Tectonic Tremor And Seismic-Wave Attenuation in Cascadia

Presenter(s): Geena Littel − Geophysics

Faculty Mentor(s): Amanda Thomas

Poster 1

Research Area: Natural/Physical Science

Funding: UO Department of Earth Sciences: Walter Youngquist Fellowship, James C. Stovall Fellowship, UROP VPRI Fellowship, UROP Mini Grant

In addition to fast, seismic slip during an earthquake, many subduction zones also host slow, largely aseismic slip. These “slow earthquakes” occur on timescales of weeks to months and are often accompanied by a weak seismic signal known as “tectonic tremor,” or simply “tremor.” Tremor behaves differently than regular earthquakes in that it is comprised of many small earthquakes that radiate low-frequency seismic energy and originate at the plate interface downdip of where large earthquakes typically occur. Ground-motion prediction equations (GMPEs) quantify ground-motion during an earthquake, and employ estimates of seismic-wave attenuation, that is, the decrease in amplitude of seismic waves as a function of distance from the earthquake source. Because tremor occurs frequently when compared to regular earthquakes in Cascadia, it presents an opportunity to better refine attenuation parameters for use in GMPEs. Here we quantify seismic-wave attenuation by performing an inversion using tremor ground motion amplitudes from three tectonic tremor episodes to determine the extent of regional variations and frequency dependence of seismic-wave attenuation in Cascadia. Inversion refers to the process of using tremor ground motion amplitudes, and a mathematical formulation relating seismic-wave amplitude and other known parameters, to solve for the unknown parameter- in this case, attenuation. Due to the large amount of tremor data, we can resolve spatial variations in the attenuation parameter along strike in Cascadia. As well, tectonic tremor exhibits the frequency dependence expected for attenuation, as seen in GMPEs developed from moderate to large magnitude earthquakes. Hence, tectonic tremor can be used to provide insight into the geological and physical factors manifested in attenuation and refine estimates of attenuation for ground-motion prediction, thus having important implications for hazard assessment.

Experimental Evolution of a Bacterial Symbiont to its Host’s Environment

Presenter(s): Helena Klein − Biology

Faculty Mentor(s): Karen Guillemin, Cathy Robinson

Poster 85

Research Area: Natural/Physical Science

Funding: META Grant

The bacteria that live in our guts, and those of other vertebrates, affect our health in a myriad of ways, from aiding in digestion to training our immune system. However, how bacteria first colonize the gut is little-understood. In particular, environment seems to play an important role in host colonization, especially in aquatic organisms. I proposed investigating environmental adaptation to find novel mechanisms for host colonization. I hypothesized that adaptation of a bacterial symbiont to its host’s environment increases host colonization. I tested this hypothesis via experimental evolution by serially passaging a strain of Aeromonas veronii, a zebrafish gut isolate, in fish-conditioned water to quickly and non-specifically find new genes that could affect host colonization. Surprisingly, I found that while the evolved strains grew to higher population densities in the water than the ancestor, these strains had variable gut colonization fitness. In fact, one strain had significantly reduced gut colonization fitness. Genome sequencing revealed that this strain had mutations affecting motility and Type I secretion system membrane protein genes. I recreated the latter mutation in the wildtype bacterial strain and found that it increased Aeromonas fitness in fish water, however gut colonization was comparable to the wildtype. This suggests that other mutations in the evolved isolate, presumably those in the motility genes, are responsible for the reduced host colonization. Future work will further investigate motility mutations among others. This work contributes to our understanding of host colonization dynamics and can lead to the development of probiotics to improve human health.

Characterization of Lrig1 Positive Stem Cells During Colitis Recovery

Presenter(s): Nicholas Jahahn − General Science

Faculty Mentor(s): Annie Zemper

Oral Session 4S

Research Area: Natural/Physical Science

Funding: National Institute of Diabetes and Digestive and Kidney Diseases, OURS program at the university of Oregon through NIH award

The intestine is a highly regenerative organ in humans and mice. Within the epithelium, structures called crypts contain epithelial stem cells that repopulate the intestinal mucosa. Lrig1 is an ErbB negative regulator that marks a population of stem cells in the base of the intestinal crypts. To study intestinal epithelium in a disease state, Dextran sodium sulfate (DSS) was used to induce ulcerative colitis in mice, characterized by inflammation of the distal colon epithelia. Here we examined the role of Lrig1 positive stem cells in colitis recovery. To accomplish this, we utilized transgenic mice that expressed Cre recombinase protein from the Lrig1 promoter and expressed YFP protein from the ROSA locus. Lineage tracing was carried out to observe what stem cells give rise to when the mouse is treated with DSS. Immunofluorescent analysis was conducted to visualize the localization of Lrig1 positive stem cells and their progeny in the wound healing process for comparison between homeostasis and different durations of recovery. It was observed that at both 36 and 48 hours after a weeklong assault of DSS there was a greater percentage of lineage traced cells found higher in the crypt compared to homeostatic conditions. We further observed that proliferation of the lineage traced cells followed the same trend. These results indicate that Lrig1 stem cells do actively participate in the recovery process. Where exactly the Lrig1 stem cells that participate in recovery originate from will be addressed with further lineage tracing closer to the time of cessation of DSS.