2015 Presenters

Presenter Topic
Adam Korte Ric-8a Regulation of Intercellular Interactions in the Developing Brain
Interneurons are important cellular components in the brain and they are mainly responsible for inhibitory actions in the central nervous system. Investigation of how interneurons develop and function is necessary to understand neuronal circuitry and certain diseases which could be caused by excitatory/inhibitory imbalance. Our laboratory utilizes mouse genetics to study interneuron development by removing the ric8a gene from interneurons during embryogenesis. After joining the lab, I made progress through my preliminary data showing developmental defects in interneuron migration in ric8a mutation as seen in graphs 1 and 2, although morphologically there is no obvious difference between the mutant and wild type interneuron as demonstrated in figure 1. My further research will investigate the interneuronal developmental effects of ric8a excision using the 1 year old mouse mammalian model.
Kayla McKaveney A study of current gel electrophoresis techniques in pharmaceutical quality assessment
Pharmaceutical compounds are highly regulated throughout and following the manufacturing process. A variety of gel electrophoresis techniques can be used to evaluate the purity, potency, and stability of biopharmaceuticals in conjunction with other analytical techniques. This study explores the effectiveness and quantization limits of these highly prevalent methods.
Pamela Xiyao Song Zinc Deficiency Increases Intragenic Transcriptional Initiation In Yeast Cells
We are studying the transcriptional response to zinc deficiency. Regulated gene expression is important for normal cell function and response to stress such as nutrient deficiency. Disruption of this control will disrupt cell physiology. To study this, we mapped transcription start sites in zinc limited and zinc-replete cells using RLM-RACE combined with Illumina sequencing. I found that while many genes were unaffected, transcription within open reading frames increased for about 40 genes. Results show that zinc deficiency disrupts gene expression, and this could be the reason that zinc limited cells grow slowly. We also hypothesize the mechanism for the increase in transcription starts sites is due to the loss of histone deacetylase (HDAC) activity.
Katherine Kokkinias In vitro Enrichment and Analysis of Cellulose-Degrading Bacterial Communities from Various Natural Inocula
The degradation of cellulose is vital for the production of biofuels. Most bacteria can’t break down cellulose, but certain bacterial communities found in nature have evolved this skill. We hypothesize that environmental inoculum from cellulose-enriched environments such as decomposed plant material and ruminant feces would have bacterial communities that can degrade cellulose with a high efficiency. To test this, we selected 10 different types of inocula such as decomposing leaves, horse feces, and moss and enriched the material in replicate on cellulosic filter paper for 7 transfers. We measured how quickly the communities broke apart the cellulosic filter paper and quantified the amount of degradation at the initial and final transfer. One major trend we observed was that bacterial communities from degraded plant material, moss, and algae inocula degraded cellulose at higher percentages than ruminant fecal bacterial communities. For example, 4 of 20 microbial communities from decomposing leaf samples were found to degrade more than 80% of the cellulose while the microbes in the horse feces enrichments degraded at most 71% cellulose. In spite of this, the mean degradation from decomposing leaves (59.4%, SE 1.5%) was only slightly greater than the mean degradation from horse feces (54.8%, SE 0.9%) suggesting a wider variation in the ability of leaf replicates to degrade cellulose. From these data, we can determine which bacterial communities break down cellulose most efficiently. Future work will identify the bacteria and cellulase enzymes present in samples of interest. By studying the most highly cellulolytic systems, we can reduce the cost of biofuel production by increasing its efficiency.
Jackson Moran MGAT2 Deficiency Alters Plasma Bile Acid Concentrations in Mice (Mus musculus)
Acyl CoA:monoacylglycerol acyltransferase 2 (MGAT2) is an enzyme mainly expressed in the small intestine of both mice (Mus musculus) and humans. Deletion of Mogat2, the gene coding for MGAT2, is known to increase energy expenditure and decrease fat deposition in mice. The purpose of this study was to determine whether bile acid (BA) metabolism in the small intestine is also affected by MGAT2 deficiency. To determine the effects of MGAT2 deletion on BA metabolism, BA concentrations were measured via colorimetric analysis on samples of liver tissue, plasma, and tissue from the ileum of several mice both with (Wild Type) and without the Mogat2 gene (Mogat2-/-). Further investigation was conducted to determine whether long-term diet interacts with MGAT2 deficiency by measuring BA concentrations for mice on long term Low Fat or High Fat Diet. There was no significant difference in BA concentration in the liver or distal small intestine (ileum) due to diet or genotype, but a significant difference in the mean plasma BA concentration was detected in Mogat2-/- mice when compared to wild type mice. This suggests that BA is either reabsorbed to a greater extent, or degraded to a lesser extent in Knockout mice, which may lead to increased BA signaling. Increased BA signaling has the potential to produce body wide changes in energy expenditure, bile synthesis, and fat distribution. Our results spark further investigation into the possible role of BA signaling in the whole-body effects of Mogat2 deletion in mice.
Nicholas Pflum JAK1 Activity in Activated B-cell-like Diffuse Large B-cell Lymphoma
Lymphoma is the most prevalent blood cancer in the United States, and the most common form, diffuse large B-cell lymphoma (DLBCL), represents 30 to 40% of newly diagnosed lymphomas. The most lethal DLBCL subtype is activated B-cell-like (ABC). An important feature of ABC DLBCL is autocrine cytokine signaling, which activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT). The lone signaling effects of JAK have long been attributed to its activation of transcription factor STATs. However, recent studies have demonstrated a new function for JAK2 that can phosphorylate the histone protein H3, resulting in the displacement of an inhibitory heterochromatin protein and regulating gene transcription. In preliminary results, the activated kinase downstream of the cytokine signaling in ABC DLBCL is JAK1, which can phosphorylate both STAT3 and H3. JAK1 induces expression of around 2,500 genes through the latter epigenetic mechanism. My research focuses on investigating the function of these genes in ABC DLBCL. Specifically, the AP-1 genes JUN and JUND are of interest because their role in lymphomagenesis has not been studied. The goal of the research is to define whether JUN and JUND could potentially serve as biomarkers for the disease or as molecular targets for therapeutic treatment.
Nathan Carpenter A targeted RNAi-CRISPR screen identifies novel regulators of synapse formation and function
An intricate knowledge of neuronal synapse formation is crucial to understanding how neurons develop, interact, and grow. In an attempt to gain a deeper comprehension of these synapses, we performed a three-part screen using gal-4 driven RNAi drosophila melanogaster lines and identified three novel genes, which might play crucial roles in remodeling of the synapse. The three novel genes displayed one or more of the following: abnormal bouton count at neuromuscular junction four, abnormal electroencephalogram in the retina, or abnormal locomotion behavior. The genes CG14010, CG31708, and CG8909 were identified and then deleted individually using CRISPR/Cas9 genome editing and their RNAi phenotypes were confirmed.
Chris Miller Liquid Micro-Jet Production
High vacuum pressure beam scattering experiments provide a useful way to probe the dynamics of the interaction between gases and liquids at the liquid gas interface. High vapor pressure liquids, which have historically proved to be particularly difficult to study, can be investigated by introducing the liquid into the vacuum chamber via a liquid micro jet. Several methods for the fabrication of these liquid micro jets have been employed, but involve significant disadvantages that limits the practicality associated with each individual method. In this project several new methods of borosilicate liquid micro jet production are proposed and analyzed after successive trials in various beam scattering experiments.
Daniel Vogt Assessing the impact of the route of infection on the immunosuppressive phenotype of Xenorhabdus nematophila
The gram-negative soil bacterium Xenorhabdus nematophila (Xn) engages in a mutualistic association with Steinernema carpocapsae (Sc) nematodes. After the nematode-bacterium complex is formed, the nematodes infect lepidopteran insects by entering through the alimentary tract. Once inside the insect, Sc nematodes disrupt gut tissue to gain access to the insect hemolymph. Upon reaching the hemolymph, Sc defecate Xn into the insect body cavity. Furthermore, when gut tissue is disrupted, members of the insect’s gut microbiota are released into the insect body cavity. Tissue damage and the presence of invading microorganisms trigger a variety of immune responses aimed at warding off potential pathogens. Therefore, to overcome the insect immune responses, Xn secretes secondary metabolites that both suppress the host immune system and kill the insect. This immunosuppression and insect-killing is necessary for nematode reproduction and the completion of its lifecycle. However, our knowledge that Xn is able to suppress insect immunity is based on laboratory experiments where Xn has been directly injected in the insect body cavity. Accordingly, the goal of this project was to determine if the route of infection has an impact on the immunosuppressive capabilities of Xn against the humoral immune responses of the insect, Manduca sexta. Insects injected with colonized nematodes died at a faster rate than insects naturally infected with colonized nematodes. In addition, nonpathogenic E. coli incubated in hemolymph collected from insects that were injected with colonized nematodes had a higher rate of growth than E. coli that were incubated in hemolymph collected from insects naturally infected with colonized nematodes. These results indicate that the route of infection may impact insect virulence and immunosuppression and that direct injection of colonized nematodes enhances insect virulence and immunosuppression when compared to the natural route.
Christopher Stancic Using a High-Throughput NMR Ligand Affinity Screen to Help Characterize Proteins
An issue in proteomics that needs to be addressed is the current inability to reliably and rapidly characterize proteins with unknown functions. The National Magnetic Resonance Facility at Madison (NMRFAM) has been developing a metabolite-based, high-throughput NMR ligand affinity screening platform to expedite the protein characterization process. 1H NMR spectroscopy will be used to examine the binding of metabolites to a selected protein in order to construct a ligand binding profile. Protein functions are defined by their interactions and by constructing a ligand binding profile, potential functions of a protein could be inferred. This study will seek to evaluate this methodology’s ability to characterize the unknown function of a selected protein. Expected results will show the ligand binding profile of the selected protein as well as any issues that were encountered in the methodology. Conclusions drawn from the results will analyze the benefits and challenges of using this methodology and will possibly indicate the selected protein’s function, providing direction for future studies.
Julie Fischer Quantifying intestinal leakage in Drosophila melanogaster following traumatic brain injury
Traumatic brain injury is a major cause of death in the USA. Consequences of TBI are due to primary mechanical injuries to the brain and subsequent secondary injuries that can occur elsewhere in the body. One of the known secondary injuries from TBI is intestinal permeability, which allows unnatural molecular flow of glucose and bacteria to occur between the intestines and the circulatory fluid (blood in humans, hemolymph in Drosophila melanogaster). Excess glucose in the blood can potentially damage blood vessels that supply the vital organs, resulting in organ failure and nerve damage. It is known that both humans and rodent models display intestinal dysfunction due to TBI, followed by elevated glucose levels in the blood beyond the initial point of injury. Our lab hopes to develop a sensitive and quantitative method for measuring the leakage of molecules from the intestines following TBI. This will allow us to determine when leakage begins relative to a primary injury and also to calculate the rate of leakage at times after TBI.
Ryan Prestil Visualizing epigenetic & morphological change via custom multi-transgenic human pluripotent stem cells
Pluripotent stem cells possess the unique ability to differentiate into any somatic cell type, and several methods now exist to reprogram adult cells to a state of pluripotency. During these cellular transition processes, dramatic changes occur to the shape, size, and organization of both the nucleus and the cell body. Using RNA-guided Cas9 and lentiviral transgene integration techniques in human pluripotent stem cell models, we have created cell lines with simultaneous mCherry-tagged histone H2B, GFP-labeled actin filaments, and doxycycline-induced reprogramming. These cells allow real-time observation of genetic material repackaging and cytoskeletal remodeling during cellular transitions and permit high-throughput screening of substrates and soluble factors to optimize these processes. Further studies will utilize these cells to better elucidate the timeline and subcellular mechanics underpinning differentiation and reprogramming.
Tianxiao Han Challenges of International Science: An Interdisciplinary Analysis of Stem Cell Research
Stem cell therapies promise to advance human health. However, translating the science to clinics is challenging due to policy and ethical issues that differ across locales. Governments struggle to negotiate local needs and priorities while competing in the global stem cell economy, and must determine their regulatory environments accordingly. Compared to Asia, North American regulations are seen by researchers as cautious and cumbersome, while the environments in China, South Korea and Singapore are often viewed as lenient. This attracts researchers to pursue trials there, yet there is concern about increased risk to patients and the continued lack of interoperability across regulatory regimes. I analyze the ethical, social, political and scientific issues that arise in regulatory environments in the US and Asia and propose policy solutions.
Kaitlin Weisshappel Identifying Genes Involved in Dopaminergic Neuron Loss in Drosophila
Parkinson’s disease is a neurodegenerative disease characterized by motor deficits due to a loss of dopaminergic (DA) neurons. However, much is still unknown about the causality of the disease. We conducted a screen in drosophila to identify mutants that lose DA neurons. We isolated drosophila strain HR-7 and mapped the mutation to chromosome 3 using dominant markers. Then, through recombination mapping, the mutation was isolated on the right arm of the chromosome. Deficiency mapping was used to narrow the region further, allowing us to generate a candidate list of genes. We can then sequence probable genes for the mutation and test any existing alleles. After the mutation is isolated, the role in which this mutation contributes to the loss of DA neurons can be determined.
Samuel Schmitz The Effects of Alum on Bacterial Communities
In an attempt to control excess phosphorus which results in algal blooms, the City of Madison added alum into a storm water pond in the UW Arboretum. To better understand how the addition of alum affects the bacterial populations of the ecosystem, we conducted our own study alongside the City of Madison. The results received from the study are unable to determine any correlation between changes in bacterial biodiversity and the addition of alum. Variability and evenness of bacteria in the lake system also appear unaffected by the alum in the ecosystem. However, it would be ideal to have a full year’s worth of data before concluding whether the alum has a significant impact on a lake’s bacterial communities.
Danielle Day Numerical Understanding in Mind, Brain and Education Relations (N.U.M.B.E.R.)
Past studies have demonstrated that the human brain can process numerical information with increased accuracy and ease when presented in multiple senses, but the neural mechanisms that support this ability are unknown. We will investigate how the parieto-frontal network processes numbers when presented in auditory and visual modalities. In two behavioral tasks, participants will 1) estimate the number of auditory or visual stimuli presented and 2) discriminate which of two intervals contained the larger number of items. The third experiment will use fMRI to test whether the parieto-frontal network contains populations of neurons tuned to specific quantities, independent of sensory modality. We will repeatedly present a specific number (e.g., 7 or 9) to adapt neurons that are sensitive to that number. We will then present deviant numerosities (e.g., 6, 8, and 10) in either the same or different modality. We hypothesize that there will be larger responses to 6 and 10 rather than 7 (the habituating numerosity), regardless of modality. This research could provide insight into the brain’s ability to integrate information about number from multiple senses, and, in turn, lead to improvements in the educational system and enhance teaching techniques.
Methyl-CpG binding domain protein 1 (MBD1) is known to be a reader of DNA methylation. By binding to methylated DNA, MBD1 mediates gene repression, and has been shown to play a role in adult neurogenesis, the formation of new neurons. In previous studies, our lab published that MBD1 deficiency leads to reduced differentiation of adult neural stem cells and impaired neuronal dendritic development. However the role of MBD1 in regulating neuronal maturation is unclear. The goal of my project is to determine whether MBD1 regulates maturation through γ-protocadherins (γ-PCDH), a transmembrane protein previously shown to influence neuronal maturation. Through the use of ChIP and RNA sequencing we have identified gamma-PCDH (10 and 12) as potential targets of MBD1, and using qPCR confirmed it is upregulated in MBD1-KO tissue. To determine if the MBD1 regulates neuronal maturation through γ-PCDH, the intracellular domain (ICD) of PCDH will be overexpressed in cultured neurons as they mature. Morphological differences between these in vitro neurons and their respective wild-type neurons will be assessed using neuron tracing and Scholl analysis.
Andrew Steinberger Determination of Ethanol Generation by 19 Cellulomonas Species
Bacteria in the genus Cellulomonas are Gram-positive, facultative anaerobes characterized by their ability to hydrolyze cellulose. Importantly, this genus contains the only bacterial species reported to be capable of degrading and using cellulose aerobically and anaerobically. They are known to degrade cellulose at rates comparable to other cellulolytic bacteria currently used in ethanol generation like Clostridium thermocellum, while using fewer cellulases. Due to the increasing attractiveness of cellulose as a potential source of biofuels, these unique characteristics may present a novel method for increasing ethanol production from cellulosic sources. To better understand the prevalence and usefulness of this ability in the genus, we characterized the ethanol production potential of 19 Cellulomonas species and mapped these properties to their phylogenetic tree to determine if any evolutionary patterns for this ability exist.
We investigated the ethanol generation abilities of the isolates by analyzing the amount of ethanol present in spent media taken from each isolate after being grown on cellulose in either aerobic or anaerobic conditions. Ethanol amounts were compared to media controls to determine the amount of ethanol generated by each isolate. We then compared ethanol generation levels to a multi-locus Bayesian phylogenetic tree of the tested isolates to examine conservation patterns for ethanol generation. We confirmed that the majority of the Cellulomonas isolates were capable of degrading cellulose aerobically and anaerobically and that most were able to generate ethanol from cellulose in both growing conditions. We also discovered that there was large
variation in the amounts of ethanol generated by isolates, both within and between oxidative growth conditions. This variation did not map back to the Cellulomonas phylogeny, suggesting that the amount of ethanol production is not driven by evolutionary constraints. Given our finding that the majority of these species are capable of generating ethanol from cellulose under both aerobic and anaerobic conditions, we propose that this unique ability is highly conserved in this genus.
Meghan Turner Mechanism of the Antimicrobial Peptide Indolicidin on the Cytoplasmic Membrane of Escherichia coli
The current antibiotic-resistance epidemic has led to increased focus on antimicrobial peptides (AMPs), short, host-defense peptides multi-cellular organisms, as potential prototypes for the design of novel antibacterial drugs. Bulk biochemical assays have helped elucidate possible mechanisms by which these AMPs attack, but make it difficult to separate the sequence of events due to limited spatial and temporal resolution. Single cell, time-lapse fluorescence microscopy was used to determine the importance of mechanosensitive channels on membrane permeabilization, cell growth, and cell survival from the attack of the AMP indolicidin on Escherichia coli. We hope to provide a detailed mechanistic explanation of indolicidin’s attack on the membrane system of E. coli to further improve designs of synthetic AMPs for use as alternatives to antibiotics.
Alex Dwyer Docetaxel and PSA-TRICOM in Castrate-Resistant Metastatic Prostate Cancer
Background: Anti-tumor vaccines have demonstrated clinical efficacy in patients with castration-resistant metastatic prostate cancer. One such vaccine, Prostvac-VF®, which uses a heterologous prime-boost strategy with vaccinia and fowlpox viral vectors encoding PSA, is currently being evaluated in a registration phase III multinational clinical trial. The current trial was a randomized phase II trial designed to specifically assess the clinical efficacy of this vaccine in patients with castration-resistant metastatic prostate cancer receiving subsequent docetaxel chemotherapy.

Methods: 144 patients with castration-resistant, metastatic prostate cancer, with a predicted survival of at least 18 months, were the planned subject population. Patients were randomized to receive docetaxel chemotherapy alone, versus treatment with Prostvac-VF (days 1, 15, 29, 43, 57) followed by docetaxel chemotherapy beginning at month 3. The primary endpoint of the trial was overall survival, and secondary endpoints included time to radiographic progression and immunological response.

Study conduct: The trial was opened within the Eastern Cooperative Oncology Group, but due to slow accrual was closed by CTEP after only 10 patients were enrolled within 13 months.

Results: Presented here are the safety, clinical, and immunological results from 8 eligible patients who underwent treatment. While no definitive conclusions can be reached from this underpowered study, other trials remain ongoing to evaluate the role of anti-tumor vaccination in sequence with other traditional anti-tumor therapies.

Conclusions: Primary endpoint of overall survival not assessed due to early closure of trial. Secondary endpoints of time to radiographic progression and immunological responses assessed for 8 patients with no significant deviations in PSA levels observed in patients from either treatment group

Trial Registration: NCT01145508, Eastern Cooperative Group Protocol 1809

Nicole Rademacher Absolute Quantitation of Sirtuin-3 in Metabolically Active Tissues: An application of Mass Spectrometry
Sirtuin-3 (SIRT3) is a mitochondrial deacetylase with a critical role in metabolism. Using isotopic labeling, we obtained absolute quantitation of SIRT3 in metabolically active mammalian tissue such as the heart, lung, liver, kidney, and cancerous breast tissue. To calculate absolute quantitation, a known amount of an isotopically labeled protein can be compared to an unknown amount of an unlabeled version of that protein. We grew recombinant E. coli in 15N enriched minimal media to express isotopically labeled (heavy) SIRT3. Endogenous (light) SIRT3 was isolated from mammalian tissue cultures. Both heavy and light SIRT3 were combined to directly compare ion intensities given by mass spectrometry. Absolute quantitation of SIRT3 levels in metabolically active tissues will have implications for prevalent health topics including type II diabetes, tumor growth, and aging.
Michael Diny Investigating the Role of Kdm3b, a Histone Demethylase, in the Acquisition of Pluripotency
Induced Pluripotent Stem Cells (iPSCs) are somatic cells that have been reprogrammed to acquire the properties of embryonic stem cells by the overexpression of a small set of proteins. iPSCs have the unique ability to self-renew and maintain pluripotency, allowing them to differentiate into any of the three primary germ layers. This makes them suitable tools for regenerative therapy and disease modeling. Since somatic cells and their corresponding iPSCs share the same genome, iPSCs have acquired pluripotency by modifying their epigenome, the chemical marks on their DNA and histone tails. These epigenetic modifications are performed by various epigenetic enzymes. These enzymes have shown to be highly specific in their activity, but the mechanism by which they operate is not well understood. In order for safe, controllable and efficient iPSC therapy to be achieved, the molecular mechanism underlying the transition to the pluripotent state must be better understood. The goal of this proposal is to help delineate the molecular mechanism of an important epigenetic enzyme, Kdm3b, during the reprogramming process.
Zoe Morgan Establishing basal somatic mutation rates of phosphatidylinositol N-acetylglucosaminyltransferase subunit A in peripheral blood mononuclear cells
Recent research has suggested that targeted immune therapies, such as Ipilimumab, may hold promise for treating certain metastatic melanoma patients. However, the majority of patients do not show a durable response, and side effects can be severe. It is therefore necessary to develop a probe to identify which patients have the highest chances of responding positively to this treatment. The phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PIG-A) gene encodes a protein involved in the proper folding of Glycosylphosphatidylinisotol (GPI) linkages, and is X-linked. Mutation of this gene was used as a surrogate marker for activated anti-melanoma t-lymphocytes. Normal donor basal rates of this somatic mutation in PIG-A were necessary to obtain as controls. Normal donor peripheral blood mononuclear cells were analyzed using rare-population negative-selection flow cytometry analysis with key markers against GPI-anchors. Overall average frequency was 7 putative mutants per million T-cells, with a standard deviation of 3.8 for normal donors.
Kayla McKaveney Activity of yeast chaperones Ssb and Zuo1 regulates viability and population growth
Molecular chaperones are ubiquitous among prokaryotes and eukaryotes and play a central role in protein homeostasis. Recent evidence demonstrates that the function of yeast chaperone protein partners Zuo1 and Ssb extends to growth regulation. Deletion strains continue growing to high cell densities in low nutrient environments and have longer chronological life spans than wild-type yeast. However, it remains unclear whether these phenotypes are a result of Zuo1 and Ssb-mediated growth arrest or from programmed cell death. I will investigate which of these cellular processes are influenced by Zuo1 and Ssb using Zuo1 mutant H128R, which exhibits less severe growth phenotypes than deletion strains. This project investigates whether partial phenotypes are a result of intermediate enzymatic activity, which may lend insight into growth arrest and programmed cell death. These findings in the Zuo1 and Ssb yeast chaperone system may give insight into growth regulation by orthologues in higher eukaryotes.
Diana Lewis Effects of Withaferin A on Mouse Models of Alexander Disease
Alexander disease is a rare neurodegenerative disorder which is caused by the accumulation of glial fibrillary acidic protein (GFAP) a main cytoskeletal protein in astrocytes, resulting in aggregates termed Rosenthal Fibers (RFs) in the central nervous system. This creates disruptions in normal intermediate filament development, and astroglial cell function. A therapeutic approach is to reduce the amount of GFAP in the astrocyte. TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA binding protein that is involved in the pathology of disorders such as amyotrophic lateral sclerosis (ALS) as well as Alexander disease. Withaferin A (WA) is a plant derivative and known inhibitor of NF-ĸB involved in which is a protein, p65, that interacts with TDP-43. It is hypothesized that with deregulated TDP-43, binding to p65 is increased and co-activates NF-ĸB. Activated NF-ĸB targets the GFAP promoter, therefore treatment of the mice with WA we propose should down-regulate the GFAP promoter. To test this, we treated 1 and 6 week old Alexander disease mutation GFAP knock-in mice with Withaferin at varying time intervals and analyzed the amount of mRNA and protein concentrations present.
Ryan Rebernick PAD4 Does Not Affect the Transcriptional Regulation of Inflammatory Cytokines in Murine Neutrophils
Rheumatoid Arthritis is an autoimmune diseased characterized by chronic inflammation of the small joints. Peptidal Arginine Deiminase-IV (PAD4) is a citrullinating enzyme with implications in the pathogenesis and exacerbation of rheumatoid arthritis, but the mechanism by which it acts is unclear. PAD4 has been shown to act as a transcriptional regulator in some cell types through histone citrullination and arginine methylation but its role as transcriptional regulator in neutrophils is unknown. We hypothesized that PAD4 was altering the transcription of several inflammatory cytokines including TNFα, IL-1β, CCL3, and BAFF in stimulated neutrophils. To test this hypothesis, PAD4+/+ and PAD4-/- TNFα-overexpressing mice were injected with thiogycolate intraperitoneally, a treatment which recruits and modestly stimulates neutrophils. The gene expression was then analyzed using RT-qPCR. No significant difference in gene expression between genotypes was found for TNFα, IL-1β, CCL3, or BAFF. These findings indicate PAD4 does not play a significant role in the expression of TNFα, IL-1β, CCL3, or BAFF in neutrophils, however it may function as a transcriptional regulator elsewhere in the immune system.
Ryan Johnson Bcl-2 Expression in the Endothelium: Impact on Vascular Development and Neovascularization
Bcl-2 is the founding member of a family of proteins that influences apoptosis, or programmed cell death. It is an anti-apoptotic protein that plays an important role in vascular angiogenesis and homeostasis. Previously, we have shown that mice globally lacking Bcl-2 (Bcl-2 -/-) displayed reduced retinal vascular development and neovascularization. In vitro studies have shown that Bcl-2 expression also affects cell adhesion, migration and extracellular matrix production in addition to modulating apoptosis. However, it is not clear the role Bcl-2 expression plays in the endothelium during vascular development, remodeling, and neovascularization. Mice were bred to carry a conditional Bcl-2 allele (Bcl-2Flox/Flox) and VE-cadherin-cre (Bcl-2EC mice). Analysis of the retinas indicated that these mice displayed some of the retinal vascular defects previously observed in Bcl-2 -/- mice, including decreased numbers of endothelial cells and retinal arteries and premature primary branching of the retinal vasculature. However, spreading of the superficial vascular layer proceeded normally unlike in the global Bcl-2 knockout mice. In addition, choroidal neovascularization was attenuated in Bcl-2EC mice, although retinal neovascularization accompanying oxygen-induced ischemic retinopathy was not. Thus, we concluded Bcl-2 expression in the endothelium impacts artery numbers and pathological neovascularization.
Sin Chan Expression and Spectroscopic Analysis of Cobalt-Substituted DGCR8 RNA-Heme Binding Domain
Analysis of DiGeorge Critical Region 8 (DGCR8) protein bound to a cobalt protoporphyrin IX (CoPPIX) may provide information about how the native iron protoporphyrin IX (heme) functions in the DGCR8 protein. DGCR8 plays an important role in microRNA (miRNA) processing; miRNAs are single-stranded RNA that regulate gene expression by interacting with messenger RNA (mRNAs). Improperly processed miRNAs may result in various human disorders. DGCR8 contains heme but its role in protein function and regulation is still unclear. Determining how DGCR8 utilizes its heme may provide information on this protein’s role in miRNA processing. My project seeks to develop a stable E. coli cell line that uptakes cobalt and inserts it into PPIX in iron-poor, cobalt-rich minimal media. These cells can then be transformed with an expression construct for DGCR8 RNA-heme binding domain (Rhed) to express DGCR8 Rhed with CoPPIX bound. The objective of this project is to understand and analyze the effect of replacing the iron with cobalt in the heme, on both protein function and native heme ligation.
Cody Q Lane Social structure and temperature influence the daily foraging patterns of wintering birds
During the winter months, Black-capped Chickadees (Poecile atricapillus) form flocks structured by dominance hierarchies with dominant and subordinate individuals experiencing different levels of risk for predation and starvation. These two risk factors represent a major behavioral trade-off potentially influencing individual variation in foraging patterns and resource acquisition. As winter is a time of shifting environmental pressure, these trade-offs could also be mediated by fluctuations in daily temperature. I hypothesize that foraging strategies by winter birds is driven by a combination of social status and dynamic winter temperatures. To test this hypothesis, I recorded pairwise interactions at a fixed feeding station to establish dominance classes and used radio-frequency identification (RFID) to record the feeding activity of individual birds.
Michael Zaiken Impact of flanking residue modifications on binding specificity of H3K9 methylation readers; Dppa3 and HP1 family members
Somatic cells can be converted to an embryonic stem cell (ESC)-like state by transcription factor-mediated reprogramming. These induced pluripotent stem cells (iPSCs), like ESCs, have the ability to divide indefinitely and differentiate into any tissue under the correct stimuli. This property makes them valuable for regenerative therapy. Chromatin modification is known to play a role in maintaining the plasticity of pluripotency. Histone modifications are recognized by specific “reader” proteins for further downstream function. The recognition and binding by the reader protein to a specific site can be influenced by modifications on flanking amino acid residues. High levels of Histone H3 lysine 9 methylation (H3K9me) is a barrier to the late stage of reprogramming. We are therefore interested in assessing the impact of flanking residues on the binding of Dppa3 and members of the HP1 family, both of which have been implicated as important for reprogramming, to the H3K9 methylation site. To this end we have purified recombinant versions of these proteins and determined the impact of flanking residue modifications to their binding specificity using an in vitro histone peptide array.
Joseph Connolly, Alexandra Mechler- Hickson, and Nick Mathers Exploring Environmentally Relevant Gene Families in the Genome of a Common Copepod
The transition from marine to freshwater environments represents one of the most challenging evolutionary transitions in the history of life. Eurytemora affinis, a calanoid copepod, is unique in its ability to frequently invade across this barrier: it has done so at least eight times across three different continents independently in the last century. Crossing this biogeographic boundary places particular stress on the copepod in many ways, but particularly regarding temperature shifts, changes in the osmotic composition of the environment and the presence or absence of environmental toxins. The recent availability of the comprehensive genomic sequence of Eurytemora affinis allowed for a study of gene families relating to these challenges: heat shock proteins (HSPs, temperature change resistance), ion transport enzymes (respond to shifts in osmotic composition), and Cytochrome p450 enzymes (CYPs, key components of detoxification systems). Gene candidates in E. affinis were identified and annotated via Blast algorithms using multiple previously identified orthologs as the query. This was done in the genome browser Web Apollo, an online genome annotation interface maintained by the i5k Genome Sequencing Initiative for Insects and other Arthropods (National Agricultural Library, USDA). 38 Heat shock genes were identified in the genome, including members from families 10, 27, 40, 60, and 90.Thirteen V-type H+ ATPase (VHA) subunit genes, seven novel sodium (or potassium)/hydrogen antiporter (NHA) genes, and ten sodium/hydrogen exchanger (NHE) genes have been preliminarily annotated in E. affinis. 36 CYP genes were discovered during annotation, belonging to four clans: CYP2, 3, 4 and mitochondrial. Their distribution represented a proportional decrease in the CYP4 clan and a proportional increase in the CYP2 clan compared to similar taxa (e.g. Drosophila melanogaster, Daphnia pulex). This study will provide significant insight into the mechanism by which E. affinis executes and survives its freshwater invasions.
Claire Armstrong Development of TOXGFP Assay to Measure Transmembrane Helix Interactions
Association of transmembrane α-helices is a fundamental part of membrane protein structure. The development of an assay (TOXGFP) allows for the efficient study of transmembrane helix oligomerization in live cells. The N-terminal DNA binding domain ToxR is a dimerization-dependent transcriptional activator, which is fused to the transmembrane domain of interest. Association of the transmembrane domains leads to activation of the reporter gene encoding green fluorescent protein (GFP). The level of fluorescence indicates the amount of GFP expressed, which correlates with the strength of the transmembrane association. Using a known dimerizing transmembrane domain glycophorin A as a model, as well a mutant variant known for low dimerization, the sensitivity of the TOXGFP system is shown. The TOXGFP assay allows for the high throughput analysis of a large range of transmembrane domain oligomerization motifs.
Tim Krueger JQ1-mediatied inhibition of the Bromodomain and extraterminal (BET) family decreases expression of immune genes in prostate cancer
With 233,000 new cases in 2014, prostate cancer is the most common cancer in men in the United States. Novel approaches to prostate cancer treatment harness the immune system to target and destroy tumor cells. A large body of evidence has shown that the epigenome, the collection of chemical modifications such as methyl or acetyl groups on the DNA or histone core proteins, plays a large role in the aberrant regulation of gene expression in prostate cancer. This includes genes involved in immune recognition such as antigens as well as antigen processing and presenting machinery (APM). Proteins that add or remove epigenetic marks, termed “writers” and “erasers”, have been targeted by drugs, often resulting in reversal of the aberrant repression of such genes in preclinical studies. Similarly, the molecular “readers” that link DNA modifications to the proteins that carry out the appropriate regulatory function have recently been targeted. JQ1 is a small molecule inhibitor of the bromodomain and extraterminal (BET) family of proteins, which contain bromodomains that allow them to bind to acetylated lysine residues of histone N-terminal tails. Through quantitative PCR, we here provide the first characterization of the effect of JQ1-mediated BET inhibition on expression of APM as well as antigens relevant to prostate cancer in prostate cancer cell lines and patient biopsy tissue. We found that the majority of genes tested had significantly decreased expression at most JQ1 concentrations in most cell lines and patient tissue following JQ1 treatment. Although JQ1 treatment would therefore likely decrease immune targeting of prostate cancer cells, this study shows that inhibition of bromodomain proteins can have a strong effect on immune gene cellular expression in prostate cancer cells. Inhibitors of other BET family members or other epigenetic readers may increase rather than decrease expression of these genes and result in a more robust immune response.
Maliya Lor Characterization of microbes capable of growing on different plant materials
Microbes have evolved in nature alone and in communities to break down plants. The worldwide pressure in fuel production to meet the current and future energy needs opens research to finding ways to use these evolutionarily microbe-plant relationships for biofuel. One goal of this study was to identify the microbes or microbial communities that can degrade different plant biomasses relevant to biofuel. To study this, three Streptomyces strains and two microbial communities were inoculated on different biomasses. First, most of the Streptomyces strains showed consistently high growth on corn stover compared to poplar and cactus. Although growth was qualitatively characterized qualitatively, these overall initial findings will encourage more exploration to identify the specific enzymes these microbes use to efficiently degrade tough plant cell wall components. Second, our communities from the soil and leaf-cutter ant refuse dump grew best on cactus and corn stover compared to poplar. These results from the communities may suggest that cactus and corn stover are less resistant to degradation than poplar. Alternatively, these results could also describe that our soil and dump microbes are better at degrading cactus and corn stover than poplar. Another more quantitative comparison used optical density (OD) readings to detect the growth and biomass degradation of our communities on corn stover. Our initial results showed that our soil and dump cultures increased in OD after two weeks. These data may suggest that there are microbes in these environments that may be efficient at breaking down corn stover. In the future, our results will help to 1) understand the role of microbes in plant degrading processes and 2) identify more efficient biofuel production pathways.
Shayna Welch Effect of Sub-Acute Ruminal Acidosis on Methane Emission Rates in Lactating Cows
Sub-Acute Ruminal Acidosis (SARA) is a condition that occurs when ruminal pH drops below 5.6 for a substantial duration, leading to lower milk production efficiency and feed conversion efficiency in dairy cattle. This study determined the impact of a SARA challenge on methane emission rates in lactating dairy cows fed two dietary forage levels. Eleven rumen cannulated lactating cows (mean ±SD 711±59 kg BW; 103±59 DIM) were subjected to two dietary treatments of high (HF) or low forage (LF), with a 67:33 and 45:55 forage to concentrate ratio respectively. Cows were adapted to either diet for 14 days in the pre-trial and assessed for their acceptance to consume feed from the Greenfeed methane measurement unit’s gas intake samplers. Four cows on each diet treatment were selected to undergo the SARA induction protocol, which consisted of four periods: the baseline (d 1 to 4), feed restriction (50% Avg daily DMI; d 5), challenge (baseline TMR plus wheat-barley pellet equal to 20% baseline DMI; d 6 and 7), and recovery (baseline TMR; d 8 and 9). Rumen pH was recorded in six cows (three per diet) every five minutes for seven days starting on day three of baseline treatment with Kahne Ltd wireless rumen sensors. Methane emission rates (g/h) were collected four times daily during the trial (12 PM, 5 PM, 9 PM, 6 PM). Results show that methane emission rates were lower (P<0.05) during the challenge (11.7 g/h) and recovery (10.1 g/h) periods compared to baseline (13.6 g/h) emission rates. Mean ruminal pH was not affected by diet during baseline (6.00), challenge (5.67), and recovery (6.09); but was lower for LF compared to HF diet during feed restriction (6.39 vs 6.55, respectively). Duration of ruminal pH below 5.6 increased from 1.5 to 6.4 hours/day between baseline and challenge periods. In this study, the SARA protocol reduced methane emission rates by 20% in LF and 38% in HF, but there was a complex relationship between ruminal pH and methane emission rates based on diet type during and after SARA induction.
Kayla Sippl Evaluation of Entomopathogenic Bacteria as a Novel Source of Mosquito Repellent
Aedes aegypti mosquitoes are known transmitters of diseases such as Yellow fever, Dengue fever, and Chikungunya. The prevalence and severity of these diseases has increased the use of repellents to reduce disease transmission. Currently, market repellents include synthetic chemicals and natural products, however as mosquitoes increase their geographic range, the need for cheap and effective repellents becomes critical. This study was designed to evaluate and identify the potential natural mosquito repellent compound(s) from microorganisms. Different bioassay methods were evaluated in order to precisely screen for the deterrent activity. The preliminary data acquired, along with research methods developed, proves that entomopathogenic bacteria can produce functional repellent compounds and will play an important role in advancing the study in the future.
Patricia Paskov Women’s Empowerment in Agriculture and Food Security
Across the globe, gender equalities persist in the agricultural realm, resulting in less food being grown, lower incomes, higher poverty, and higher levels of food insecurity (World Bank 2012). In this study, I investigate the relationship between women’s empowerment in agriculture and household food security in the central valleys of Oaxaca, a maize-producing region where one of every two households report having difficult accessing food. I construct an empowerment index (WEAI) using original data on central valley women’s capacities in production, income, resources, and leadership. I then use regression analysis to estimate the WEAI’s relationship with food security (as indicated by an index adapted from USAID’s Household Food Insecurity Access Scale), controlling for poverty, education, household size, and household headship. I present two key findings. First, food insecurity is significantly higher within female-headed households. Secondly, female-headed households are those in which the WEAI is the most strongly associated with the reduction of food insecurity. This study indicates that closing the gender gap in agriculture may offer one possible solution to hunger eradication among female-headed households in the central valleys of Oaxaca.
Sora Ji Elucidating the Molecular Mechanism of Dorsomorphin Action on SOD1 Mutant Caenorhabditis elegans, a Model of Amyotrophic Lateral Sclerosis
Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, causes progressive degeneration of the motor neurons in the brain and spinal cord. This gradually impairs mobility in patients and completely paralyzes patients in its later stages. In 2010, Dr. Atwood’s laboratory published a novel hypothesis on ALS progression that implicates increased accumulation of lactate at the neuromuscular junction being the major molecular event that triggers a vicious cycle of molecular events resulting in the exponential progression of ALS leading to paralysis. Recently, we tested that hypothesis in SOD1 mutant C. elegans worms (G85R; an ALS worm model) using dorsomorphin, an AMP-activated protein kinase (AMPK) inhibitor that could reduce glycolysis and thereby the conversion of pyruvate to lactate. Dorsomorphin treatment (≥10µM) for 3 days significantly increased the movement (measured as thrashes/minute) of G85R worms. As the animals aged, the beneficial effect was gradually lost, consistent with the dysregulation of the reproductive hormone axis that was proposed as an etiological component of ALS in the hypothesis. Thus, we will hypothesize the molecular mechanism behind the dorsomorphin effect on movement of the G85R worms and test whether higher doses of dorsomorphin increase worm movement after 3 days. The results will hopefully help us reveal whether lactate production is decreased with AMPK inhibitor and whether higher doses of the AMPK inhibitor is required with the dysregulation of the reproductive axis with aging.
Zachary Boor Detecting Genes Implicated in Abscission Processes in Cold-Hardy Grape DNA
The field of phylogeography can provide a description of the climatic and geographic mechanisms driving speciation. One rising topic in the field of phylogeography are the patterns of biodiversity observed in the mountainous regions of northwest North America. Several studies investigating the evolutionary history of Pacific Northwest taxa support the theory that historical populations were forced into climatic refuge during Pleistocene glacial cycle resulting in genetic differentiation. This study investigates whether Pleistocene glacial cycling influenced the genetic structure of a group of flightless ground beetles occupying alpine habitats in the Cascade Mountains and Trinity Alps. Implementing genetic sequence data, we compare the evolutionary history of these beetles with patterns of demographics, genetic relatedness, and speciation expected of taxa influenced by glacial cycling. A genetic isolation by distance analysis reveals genetic structure amongst between populations in a general north-south pattern. These results support an incremental dispersal of ancient beetle populations, possibly but not conclusively resulting from habit flux due to glacial cycling. Genetic isolation of carabid beetle populations on a north-south axis supports a larger trend of genetic isolation of alpine species populations in the Pacific Northwest.
Andrew Maule Detecting Genes Implicated in Abscission Processes in Cold-Hardy Grape DNA
Abscission is a natural process whereby plants control the detachment of organs such as petals, leaves, seeds, and fruit. Regulated by environmental and genetic cues, it allows for effective dispersal of seeds and fruits when they are fully developed. An early target of crop domestication by humans, controlling abscission allowed for more efficient harvest by precisely timing abscission to clear physiological markers. When abscission processes in crops are unpredictable or early, harvest losses can be costly. Early and unpredictable abscission, also known as shattering, is a problem plaguing newer cultivars of cold-hardy grapes that are the backbone of a burgeoning midwestern viticulture economy. These new grape cultivars are the early results of breeding efforts of a cold-sensitive Eurasian species with several cold-hardy North American species. Correlating physiological markers and genetics to abscission behavior in these grapes is critical to understanding this problem. Despite no data available in cold hardy grapes, foundational research in Arabidopsis thaliana has revealed a host of gene and gene pathways involved in floral organ abscission. Genes in Arabidopsis that correlate highly with abscission include ethylene receptors, ethylene transcription factors, jasmonic acid receptors, jasmonic acid response factors, auxin response factors, non-hormonal small peptides involved in abscission signaling, and receptor kinases. Using abscission-related genes documented in literature, this study sets up the preliminary work for doing future gene expression studies on aforementioned abscission-related genes in these cold-hardy grapes by probing the DNA of cold-hardy grape cultivars for these genes. Specifically, DNA was extracted from four different cold-hardy grape cultivars: Frontenac, La Crescent, Canadice, and Somerset, representing shatter-resistant wine-, shatter-prone wine-, shatter-resistant table-, and shatter-prone table-grape cultivars, respectively. Using studies implicating an ethylene response 1 (ETR1) protein receptor in initiation of the abscission process, several DNA primers were designed using conserved motifs of ETR1 derived from interspecies protein alignments. Running a polymerase chain reaction (PCR) with DNA from the four cultivars and the designed primers, etr1 amplicon products were sequenced using the Sanger method. Results indicated that the etr1 sequence of these cultivars maintains high similarity with the etr1 sequence from Vitis vinifera, the Eurasian species, paving the way for simple RNA primer design of etr1 mRNA for future gene expression studies.
Melanie Roemer Wisconsin Active Schools Descriptive Statistics of Fifth Graders
Background: In Wisconsin, both obesity and physical inactivity levels are increasing in children. Both of these are associated with adverse health effects. Transform Wisconsin implemented an intervention program in elementary schools across the state to reverse these secular trends by increasing physical activity in children and baseline data was collected. Aims: The aim of this analysis was to evaluate the baseline data and determine the physical activity levels of Wisconsin 5th grade children, what times of the day they are most and least active, how these correlate with obesity as measured by Body Mass Index (BMI), and how these correlate with their fitness as measured by the timed PACER shuttle run. Methods: The final study population consisted of 691 children in the 5th grade (Boys n=380, Girls n=311), ages 9-11 (age 9 n=1, age 10 n=514, age 11 n=176) from 11 counties and 19 schools. Results: On average, this population took 9,564 total steps per day. Of these, an average of 841 steps were aerobic. A total of 4861 steps were taken during the school day (8am-3pm), and 4703 were taken out-of-school. The average PACER score averaged 28.1 laps and the average BMI was 20.2 kg/m2. Interpretations: The average steps per day in this population fell below the recommended 12,000 steps per day. The average obesity of this population fell in the overweight category (19.5 – 22 kg/m2). Conclusions: Wisconsin schools and families should strive to increase physical activity levels in children in order to achieve an activity level of 12,000 steps per day.
Lauren Frances Reil Mesenchymal stem cell-educated macrophages role in GVHD and radiation injury protection
Total body irradiation is typically used to condition patients for allogeneic hematopoietic stem cell transplant, leading to release of inflammatory cytokines from damaged tissues. After transplant, many patients develop graft-versus-host disease (GVHD) resulting from donor T cells attacking host tissues leading to further inflammation. Mesenchymal stem cells (MSCs) have immunosuppressive and tissue repair properties, but clinical trials using MSCs to treat GVHD have shown mixed results. Macrophages (MQs) are important regulators of immunity and can promote tissue remodeling. We have previously shown that MSCs can educate MQs toward a unique anti-inflammatory phenotype, however the implications for in vivo models of inflammation have not been studied. Using flow cytometry, we found that in comparison to MQs, mesenchymal stem cell-educated macrophages (MEMs) have increased expression of the inhibitory molecules PD-L1 (p=0.039), PD-L2 (p=0.0002), and markers of alternatively activated macrophages: CD206 (p=0.04) and CD163 (p= 0.0004). MEMs also show increased expression of TGF-β (p<0.0001), Arginase-1 (p<0.0001) and IL-6 (p=0.0006), and decreased expression of IL-12 (p=0.004) and TNF-α (p<0.0001) by RT-PCR. In two xenogeneic mouse models, we show that MEMs significantly enhance survival from lethal GVHD (p=0.028 vs MSC) and from lethal radiation (p=0.0044 vs MSC). MEMs could be a novel cellular therapy for the management of GVHD and protection from radiation-induced injury.

Contributors: Lauren Reil, Myriam N. Bouchlaka, Mike Martinez, Peiman Hematti, Christian M. Capitini

Affiliations: Department of Pediatric Hematology & Oncology, Department of Medicine, Hematology and Oncology, School of Medicine and Public Health