Poster Session B

Neuroscience/Physiology/Immunology/Developmental and Cellular Biology Alphabetical by last name Referred hip joint and lower extremity pain from a dermoid cyst of the ovary Ruslan Abdukalikov, OMS4 1, David Shbeeb OMS2 1, Terence K. Gray, DO 1,2 1University of New England College of Osteopathic Medicine, Biddeford, Maine, USA 2 Mercy Pain Center, Mercy Hospital, Portland, Maine, USA Referred pain patterns, which are associated with embryological development, can complicate treatment and management of pain, especially in the presence of visceral pathological processes. A dermoid cyst of the ovary can refer pain to the hip joint and lower extremity mimicking pain patterns associated with hip joint disease. It can also present with pain that persists after corticosteroid and local anesthetic joint injections. In such cases of pain refractory to interventional pain procedures, referred pain patterns secondary to undiagnosed pathology should be explored as a potential diagnosis. Elucidating the mechanisms governing neuromuscular phenotypic variation in a zebrafish muscular dystrophy model Bailey, E., Goody, M., Astumian, M., Drinkert, D., Henry, C. 1Graduate School of Biomedical Science and Engineering, University of Maine, Orono, ME, 2School of Biology and Ecology, University of Maine, Orono, ME, 3Orono High School, Orono, ME erin.bailey1@maine.edu Dystroglycanopathies are a subset of incurable muscular dystrophies that are challenging to study because they present with a broad range of symptoms and severities. Variability observed in these conditions does not correlate with specific genetic mutations, suggesting that environmental factors may play a role. In order to characterize neuromuscular phenotypic variation in these conditions, we engineered a novel zebrafish gmppb mutant dystroglycanopathy line and studied larval zebrafish development for five days post-fertilization. Gmppb homozygous mutant larvae consistently present with aberrant MTJ development; however, larval zebrafish harboring homozygous and heterozygous GMPPB mutations possess a range of increased susceptibilities to fiber damage and compromised locomotion. Subjecting gmppb mutants to oxidative stress exacerbates the severity of MTJ failure and fiber damage. These results demonstrate that genotype does not necessarily influence phenotype in gmppb mutants and that stress may influence disease severity, suggesting that variation observed this model reflects that observed in human dystroglycanopathies. CRISPR enabled screen of Candida albicans factors influencing host immune response Blair, B.1,2& Wheeler, R.1,2

Poster Session B

1Graduate School of Biomedical Science and Engineering, University of Maine, Orono, Maine, 2Department of Molecular and Biomedical Sciences, University of Maine, Orono, Maine. bailey.blair@maine.edu CRISPR has shown to be a powerful gene editing technology that has many advantages over previous methods in zebrafish. We are using CRISPR to generate fish with deficient phagocyte NADPH oxidase. Previous work in our lab has shown that NADPH oxidase promotes phagocyte recruitment in the larval zebrafish to a Candida albicans infection in the hindbrain. Surprisingly, NADPH oxidase was not required for phagocyte recruitment to a yeast locked mutant. This suggests that changes C. albicans makes during the yeast to hyphal transition help limit phagocyte recruitment. Because this transition is accompanied by many changes it is difficult to determine whether this effect is hyphae specific or due to other virulence factors that are co-regulated with this process. Therefore, we can use the NADPH oxidase deficient zebrafish as an in vivo model to screen mutant Candida strains for phagocyte recruitment and determine whether the affect is hyphae or virulence specific. Targeting DNA methylation to enhance memory function Boitnott, A.1, Zengeler, K.1, Gettens, C.2, Zhang, X.1, Smith, H.1, Malachowsky, B.1,2, Kennedy, A.1,2 1Neuroscience Program, Bates College, Lewiston, ME 2Department of Chemistry and Biochemistry, Bates College, Lewiston, ME aboitnot@bates.edu DNA methylation in the adult central nervous system is necessary for cognitive processes, such as learning and memory. In this study, we tested the idea that increasing the longevity of DNA methyl marks encoded onto the genome after experiential learning will increase the strength and fidelity of that memory. We found that cytosine hypermethylation improves and rescues aspects of hippocampal-dependent spatial learning and memory in mice with an ultra-rare autism spectrum-like disorder, known as Pitt-Hopkins Syndrome (PTHS). This suggests that DNA methylation may be a useful therapeutic target for PTHS and other monogenetic intellectual disabilities. Methicillin-resistant Staphylococcus aureus (MRSA) evades destruction by the macrophage autophagy system Bond, A., Harris, K., Burkholder, K. Department of Biology, University of New England, Biddeford, ME abond1@une.edu MRSA is a human pathogen that infects and persists in human macrophages, although precise mechanisms of intramacrophage survival remain unclear. We investigated the interaction of MRSA with the macrophage autophagy system, an innate host defense used to destroy some intracellular pathogens. Using confocal microscopy, we observed that during macrophage infection the majority of intracellular MRSA associated with the

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autophagosome membrane protein LC3. However, MRSA infection did not lead to autophagic degradation of p62, a protein that is normally destroyed when autophagy is active and functional. Moreover, inhibition of macrophage autophagy significantly decreased MRSA survival inside macrophages (p < 0.05). Collectively, findings suggest that MRSA activates, but perturbs, the normal degradative process of the macrophage autophagy system. Future work will further characterize the maturation of MRSA-containing autophagosomes in human macrophages and will focus on the role of specific Staphylococcal virulence factors in autophagy activation and bacterial survival within autophagosomes. FOXD1 Promotes Stromal Investment in Clear Cell Renal Cell Carcinoma Bond, K.1, Fetting, J.2, Gupta, A.2, Duarte,C.2, Karolak, M.2, Congdon, C.B.3, Emery, I.2, O hAinmhire, E.4, Humphreys, B.D.4, Oxburgh L.2 1University of Maine, Orono, ME; 2Maine Medical Center Research Institute, Scarborough, ME; 3Bowdoin College, Brunswick, ME; 4Washington University School of Medicine, St. Louis, MO kyle.bond@maine.edu We investigated the transcription factor FOXD in clear cell renal cell carcinoma (ccRCC). Using ccRCC tumor microarrays (TMAs), FOXD1 was found to be positive in 65% of tumors. A direct correlation was found between FOXD1 and PDGFRb, suggesting fibroblast recruitment by FOXD1+ cells. RNA-seq analysis showed increased expression of FOXD1 correlated with increased stage and reduced survival. Potential FOXD1 target binding sites were determined using the TRANSFAC FOXD1 binding site matrix, uncovering SLIT2 as a target. SLIT2 was found to be downregulated in response to FOXD1 overexpression in renal epithelial cells. Scratch assays were performed and showed that SLIT2 reduced PDGFBB induced fibroblast cell migration. To model stromal invasion in vitro, we devised a novel 3D invasion assay using silk scaffolds. In conclusion, we show that FOXD1 expression has prognostic relevance to patient survival in ccRCC, and that this may be due to modulation of SLIT2 expression. Development of a High-throughput Screen for Analysis of JC Polyomavirus Infection Crocker, Mason1, DuShane, Jeanne1, and Maginnis, Melissa S.2 1Department of Molecular and Biomedical Sciences, The University of Maine, Orono, ME 2Graduate School in Biomedical Sciences and Engineering, The University of Maine, Orono, ME JC polyomavirus (JCPyV) infects 50-80% of the population, causing a persistent infection of the kidneys in healthy hosts. In severely immunocompromised individuals the virus can migrate to the central nervous system (CNS) leading to a fatal, demyelinating disease, progressive multifocal leukoencephalopathy (PML). We have adapted a new methodology, the In-cell western (ICW) analysis, utilizes immunodetection of target proteins with antibodies tagged with a near infrared fluorescent dye and analyzed using a LI-COR Odyssey CLx Infrared Imaging System.

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This technique removes operator bias and significantly reduces analysis time, enhancing productivity. Our goal is to adapt this method to quantify JCPyV infectivity and determine the correlation between the ICW and manual immunofluorescence analysis to illustrate the effectiveness of the ICW assay and improve productivity. This assay can also be used to identify viral and host protein expression patterns during infection and in response to chemical inhibitor, siRNA or potential therapeutics treatments. Isolation and Host Characterization of Mycobacteriophage and Nontuberculous Mycobacteria from the St. John River Daniels, K.1, Hodsdon, B.1, and Borges, K.1 1Biology Program, University of Maine at Fort Kent, 23 University Drive, Fort Kent, ME 04743 kborges@maine.edu Non-tuberculous mycobacteria (NTM) are natural inhabitants of waterways and soil that can colonize municipal drinking water and plumbing systems, some species being opportunistic human pathogens. Mycobacteriophages are viruses that infect and kill mycobacteria. Out of thousands identified few mycobacteriophages have been isolated from aquatic systems. The purpose of this study was to cultivate and characterize mycobacteriophages of water and sediment from the St. John River in Aroostook County. Twelve mycobacteriophages were isolated from river sediment, and two NTM strains were isolated from river water. Based upon partial 16s rRNA sequencing, one NTM is a close match to Mycobacterium asiaticum,and the other is closely related to M. montefiorense, both known to be opportunistic pathogens. Preliminary studies of the phages indicate a moderate host range, being able to infect three other species in addition to the isolation host M. smegmatis. Experiments to determine whether the St. John River phages can infect the river NTM are in progress. Detection of human pathogens on sugar kelp using microbiological and molecular methods Demers, M.1*, Barberi, O.2*, St. Gelais, A.2, Byron, C.2, Burkholder, K.1 1Department of Biology, 2 Department of Marine Sciences, University of New England, Biddeford, ME mdemers3@une.edu *Authors contributed equally to the work Sugar kelp (Saccharina latissima) is growing in popularity as a food product, and its production is a burgeoning Maine industry. However, there are no methodological guidelines for detecting human pathogens in kelp harvested for food. We compared microbiological and molecular methods, and the necessity of microbiological enrichment medium, for detecting pathogens on kelp inoculated with varying doses (0, 102, 104 or 106 cfu/ml) of Vibrio parahaemolyticus or E. coli O157:H7. Pathogens were detected by plating on selective media and by single-plex qPCR. Plating methods only consistently detected V. parahaemolyticus and E. coli following enrichment (102-106 cfu/ml), while qPCR detected both pathogens in unenriched (106 cfu/ml) and enriched samples (102-

Poster Session B

106 cfu/ml). Findings indicate that while plating and qPCR can detect kelp-associated pathogens, qPCR provides increased sensitivity and may eliminate the need for an enrichment step. Future work will develop a multi-plex qPCR method for rapid, simultaneous detection of pathogens in kelp. The imaging of RNA localization in E. coli and B. burgdorferi cells Disler, E. Bates College In this study, three sRNAs were used to examine the localization within E. coli cells so that we enhance our understanding of RNA synthesis, function, and degradation within the cell. Two silencing sRNAs, OxyS and RyhB, and one activating sRNA, GlmZ, were used and placed on plasmids in E. coli. Localization of the sRNAs was measured by RNA Fluorescent in-situ Hybridization (FISH). After we confirmed that we can visualize our fluorescent probes within the E. coli using the confocal microscope, we used the STED (stimulated emission-depleted) microscope to observe a more defined and resolute image of the RNA localization patterns. With this foundation, we have gone forward with applying this method with Borrelia burgdorferi cells, the bacteria that causes Lyme disease, to provide insight into the mechanisms underlying Lyme disease. Impact of anticonvulsant Ethosuximide on Huntington’s Disease modeled in Caenorhabditis elegans Duquette, H1 and Howard, A1 1University of Maine at Augusta, Augusta, ME heather.l.duquette@maine.edu Huntington’s disease is an autosomal dominant genetic disorder resulting in polyglutamine expansions on the huntingtin protein leading to misfolding and aggregation within neurons. Huntington’s disease typically presents between 30 and 40 years of age with individuals experiencing decreased movement, uncontrolled movements, and cognitive impairment. Disease progression ultimately leads to death. Ethosuximide (EthS) is a cognitive drug currently being used for epilepsy and acts as an anticonvulsant. EthS works on the electrical impulses in the brain by reducing abnormal firings which arise during petit mal seizures. EthS is known to extend lifespan in C. elegans but has not previously been used in the treatment of Huntington’s disease. We hypothesized that a C. elegans strain modeling Huntington’s disease would show improved health and delayed disease progression when treated with EthS. We determined that polyglutamine aggregate number decreased, aggregate size increased, and the health of diseased individuals were altered after EthS treatment. Characterization of MAPK-induced transcription factor activation during JCPyV infection

Poster Session B

DuShane, Jeanne1, Mehmood, Kashif1,2, Crocker, Mason1 and Maginnis, Melissa S.1,3 1Department of Molecular and Biomedical Sciences, 2Department of Physics & Astronomy, 3Graduate School in Biomedical Sciences and Engineering, The University of Maine, Orono, ME 04469 JC polyomavirus (JCPyV) infects 50-80% of the human population, presenting as an asymptomatic persistent infection in the kidney. In infected individuals who are immunocompromised, JCPyV can reactivate, spread to the central nervous system and cause a lytic infection resulting in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). Viral infection and pathogenesis is predicated on host-cell invasion and reprogramming of normal cellular functions in order to prioritize viral replication. Our lab has demonstrated that JCPyV infection activates the mitogen-activated protein kinase (MAPK) signaling pathway leading to phosphorylation of extracellular signal-regulated kinase (ERK) early in the infectious lifecycle. ERK activation is required for JCPyV infection and specifically promotes viral transcription. Using a MAPK-specific signaling array, we have identified MAPK-regulated transcription factor targets activated in response to JCPyV infection. These findings demonstrate how JCPyV activation of the ERK signaling cascade drives the infectious cycle and viral pathogenesis. Primary cilia of cardiac neural crest cells orchestrate multiple aspects of cardiovascular development Lindsey Avery Fitzsimons1,2, Adrian M Moran3, Kerry L Tucker1,2 1Graduate School of Biomedical Science & Engineering, University of Maine, Orono, ME; 2College of Osteopathic Medicine, Dept. of Biomedical Sciences, University of New England, Biddeford, ME; 3Department of Pediatrics; MMC Division of Pediatric Cardiology, Maine Medical Center, Portland, ME Previous work in our laboratory has shown that primary cilia of cardiac progenitor populations are critical to the septation and alignment of the developing outflow tract (OFT) and interventricular septum (IVS). Our current in vivo models have refined the elimination of primary cilia to cardiac neural crest cells (CNCC) of the embryonic mouse heart using multiple neural crest-specific Cre drivers (Wnt1:Cre; Wnt1:Cre2) crossed with the Ift88flox/flox mouse. The resultant mutant phenotypes include OFT defects, impaired septation of the membranous IVS, non-compaction of the developing ventricular myocardium, and perinatal lethality. Use of a Tdtomato reporter and various immunohistochemical approaches revealed the presence of transient CNCC contributions to the ventricular portion of the developing myocardium, outside of the OFT and endocardial cushion regions where CNCC are normally found to migrate. These data suggest a potential role for primary cilia of CNCC in the development and maturation of the ventricular myocardium. Stress-induced developmental programming of the immune system Ian Gans1,2, Elli Hartig1, Shusen Zhu1, James Coffman1,2 1MDI Biological Laboratory, Davis Center for Regenerative Biology and Medicine, Salsbury Cove, ME 04672

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2Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME 04469 igans@mdibl.org Chronic stress during development is associated with an increased susceptibility to disease throughout the lifetime, but the nature of this elevated risk is not fully understood. The stress response in humans is mediated primarily by the hormone cortisol, a steroid hormone that binds to the glucocorticoid receptor (GR), a transcription factor found in cells throughout the body. Once bound by cortisol, the GR regulates transcription of a complex network of stress-responsive genes and the resulting physiological functions, including immunity and inflammation. This mechanism is conserved in the Zebrafish, which has emerged as a useful model organism for the study of immune system development. Our lab has shown that zebrafish embryos directly exposed to elevated cortisol during early development mature into adult fish with immune system dysregulation, and our research is now focused on determining the specific cellular, molecular, and genetic mechanisms of this altered immune function. Distinguishing the developmental pathways of three teeth in the zebrafish pharynx. Gordon, C. M.1, Jackman, W.1 1Bowdoin College, Department of Biology, Brunswick, ME cgordon@bowdoin.edu In the zebrafish pharynx, the first three teeth to form, 3V1, 4V1, and 5V1, have distinct adult and embryonic morphometries. Previous studies of gene expression profiles and mutant phenotypes in 3V1, 4V1, and 5V1 have identified four genes that might be involved in dissociating these tooth modules: pitx2b, eve1, pbx1a, and pbx1b. To test how each of these genes might contribute to the observed divergence in tooth morphology between the three teeth, and obtain a better understanding of how these three teeth develop, I have performed CRISPR/Cas9–mediated knockouts in each of these genes, and observed the resulting tooth germs and mineralized tooth structures in WT and mutant embryos via fluorescence microscopy. Work thus far suggests a complementary role for pbx1a and pbx1b in distinguishing the developmental pathways of the three teeth, and a previously undescribed role for pitx2 in tooth mineralization. Injury-induced electrophysiological changes in the nociceptor Hale, C. 1, Brann, C. 2, Ganter, G. 2 1 University of Maine, Graduate School of Biomedical Science and Engineering, Orono, ME, 2 University of New England, Department of Biology and Center for Excellence in the Neurosciences, Biddeford, ME christine.hale@maine.edu Nociceptive sensitization underlies and perpetuates chronic pain, a condition that affects hundreds of millions of people worldwide. Recently, an injury-induced nociceptive sensitization model has been developed using Drosophila melanogaster,

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and novel pathways that produce sensitization have been revealed. In the present study, we aim to build upon the knowledge of this sensitization pathway, and to further explore unknown sensitization mechanisms, by investigating the electrophysiological consequences of ultraviolet light and thermal injury on fruit fly nociceptors. To carry out this aim, we use a genetically encoded calcium indicator that is targeted to the nociceptors of Drosophila larvae. Using confocal microscopy, we are examining the signaling properties of the nociceptors following injury. By investigating injury-induced electrophysiological changes in the nociceptor, novel mechanisms of sensitization could be revealed, providing valuable information that may lead to improved treatment options for chronic pain. Affective behavior in withdrawal seizure prone and withdrawal seizure resistant mice during protracted abstinence Hartmann, M.C.1,2, Holbrook, S.E.1,3, Crabbe, J.C.4,5,6, Rosenwasser, A.M.1,2,3 1Graduate School of Biomedical Science and Engineering, 2Department of Psychology, and 3School of Biology & Ecology, University of Maine, Orono, ME, USA; 4Department of Behavioral Neuroscience and 5Portland Alcohol Research Center, Oregon Health & Science University, and 6VAMC, Portland, OR, USA matthew.hartmann@maine.edu While the acute alcohol withdrawal syndrome has been well-characterized in both human clinical studies and in experimental animals, much less is known regarding the long-term affective disturbances that persist in some individuals over the course of protracted abstinence. In the present study, male and female Withdrawal Seizure Prone (WSP-2) and Withdrawal Seizure Resistant (WSR-2) mice were exposed to a chronic-intermittent ethanol vapor protocol (CIE) or plain air, and administered weekly behavioral assays for affective behavior. Across dependent-measures, ANOVA revealed complex effects of CIE, line, sex, and test week, as well as numerous significant interactions among these factors. Overall, CIE produced time-dependent effects on the SPT and LDT, while WSPs displayed more anxiety-like behavior in the LDT and appeared more sensitive to CIE (relative to WSRs). Together, these results suggest that mice selected for sensitivity to acute ethanol withdrawal may also differ in affective behavior during protracted abstinence. Investigating distinct sensory neuron populations that mediate cancer-induced ongoing and breakthrough pain Havelin, J.1,3,.1Lambrecht, M1, King, T. 1,2,3 1Center for Excellence in the Neurosciences, University of New England, Biddeford, ME, 2College of Osteopathic Medicine, University of New England, Biddeford, ME, 3Graduate School of Biomedical Sciences and Engineering, University of Maine Orono, Orono, ME Joshua.havelin@maine.edu Cancer-induced bone pain is characterized by two discernable phenomena; constant ongoing pain that increases in severity over time and breakthrough pain that is reported as severe pain that occurs in the setting of medication controlling ongoing pain.

Poster Session B

Previous work performed by our group demonstrated IB4-binding fibers play a critical role in mediating breakthrough pain in a rat model of cancer-induced bone pain. We are using pharmacological and optogenetic techniques targeting MrgD+ nociceptive fibers that have a high degree of overlap with IB4-binding fibers, to further delineate role in these pain phenomena and potential pharmacological targets. Mu and delta opioid receptor agonists are being used to evaluate affective components of pain relief and movement evoked pain. Experiments using virally mediated ArchT in MrgD-cre-ER2 mice will examine the hypothesis that these fibers selectively mediate breakthrough but not ongoing pain in our mouse model of cancer-induced bone pain. This research was supported by a COBRE award (P20GM103643). The correlation between psychosocial factors and pain sensitivity in healthy and chronic pain patients Heinrich, R, OMS II1, Shbeeb, D, OMS II1, Hull, S, M.D.2, Cao, L, Ph.D, M.D.1 1University of New England College of Osteopathic Medicine, Biddeford, Maine 2Mercy Hospital, Pain Center, Portland, Maine Introduction: Our preliminary research has shown a correlation between pain threshold and psychosocial screening tests, which include testing for fatigue, depression, anxiety and a pain catastrophizing scale, in chronic pain patients but not in healthy controls. Our current research analyzed the relationships between individual psychosocial factors that contribute to pain and pain sensitivities tested via various methods in both control and pain patients. Our hypothesis was that chronic pain patients and normal healthy controls display differential relationships between specific pain sensitivities and individual pain related psychological conditions. Further, opioid-induced hyperalgesia (OIH) is a paradoxical phenomenon that can sometimes result from long-term opioid therapy. While opioid therapy aims to increase a patient’s pain threshold, OIH results in a decreased pain threshold. The few validated methods of testing for OIH can be difficult for physicians in a primary care setting due to time constraints and lack of special equipment. The sphygmomanometer test is being investigated as a mode of testing for muscle hyperalgesia in osteoarthritic patients. Our research compares the sphygmomanometer test to previously validated OIH testing methods that test sensation to cold, heat and pressure to see if it can effectively differentiate between a healthy control group and chronic pain patients on opioid therapy (experimental group) with a long-term goal of investigating the use of the sphygmomanometer test for identifying OIH. Methods: Upon enrollment, subjects were assigned to one of two groups. The control group consisted of healthy subjects without pain-related therapy or history of persistent pain in the past six months, and the experimental group consisted of chronic pain patients who were currently on regimented therapy. The participants underwent a series of sensory tests to determine their pain threshold including a pressure algometer, cold pressor, heat thermode, and sphygmomanometer test. After the sensory tests, information known to be associated with chronic pain was collected from the subjects, and the subjects’ psychosocial screening test scores were also combined to create a summative pain score. Sensory test results were analyzed and compared between the

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control and the experimental group, and each psychosocial test was analyzed in their relation to each sensory test. Results: All psychosocial tests individually and combined were significantly higher for the chronic pain patients than for the control group, but no significant differences were detected with sensitivity testing. Our data also revealed a positive correlation between psychological test scores and pain sensitivities across all sensitivity modalities in the chronic pain patients. However, the control group did not show consistent positive correlation. Conclusion: Our research provides evidence showing a differential relationship between pain related psychosocial factors and specific pain sensitivity when comparing normal healthy controls and chronic pain patients. It also supports further investigation into the validation of the sphygmomanometer as a sensory test for OIH. This information again demonstrates the importance of managing patients’ psychological conditions while managing both healthy and chronic pain patients. Neurogenic muscular degeneration in SMARD1-like mouse model em3 Holbrook, S.1, Martin, P.1, Cox, G.2 1University of Maine, Orono, ME 2The Jackson Laboratory, Bar Harbor, ME sarah.holbrook@jax.org Neuromuscular degenerative (NMD) diseases are devastating conditions that can lead to pre-mature fatality or life-long suffering. Autosomal recessive mutations in IGHMBP2, a ubiquitously expressed DNA/RNA helicase, have been linked to childhood NMDs. em3 is a SMARD1-like strain, or Spinal Muscular Atrophy with Respiratory Distress, created via CRISPR-Cas9 targeting of the IGHMBP2 gene. SMARD1 is characterized by muscle weakness and respiratory failure in early infancy usually leading to early fatality. We have preliminary muscle fiber area and neuromuscular junction innervation data comparing wild type, em3, and a historical SMARD1-like model (nmd2J) in the hindlimb and intercostal muscles. These preliminary results suggest that em3 mice suffer from more muscle atrophy and denervation than both wild type and nmd2J mice especially in the intercostal muscles. Characterizing the degree and range of neurogenic muscular atrophy in the em3 model will allow us to identify if this is a clinically relevant model of SMARD1. Identification of TNFAIP8L1 binding partners through co-immunoprecipitation and mass spectrometry Hoyle, A1. Mayhue, S1. Sullivan, C1,2. 1Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME, 2 Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME audrey.hoyle@maine.edu Expanding our understanding of the gene families and mechanisms governing tumorigenesis pathways has potential to improve cancer therapies and patient prognoses. Members of the tumor necrosis factor alpha- induced protein 8 (TNFAIP8)

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gene family, which is comprised of TNFAIP8, TNFAIP8L1, TNFAIP8L2, and TNFAIP8L3, have been shown to participate in the regulation of tumorigenesis. The TNFAIP8L1 gene is understood to function as a tumor suppressor, but the mechanisms have yet to be explained. We hypothesize that the TNFAIP8L1 protein achieves its tumor suppressor role through protein-protein interactions that regulate tumorigenesis. The H1299 non-small cell lung cancer cell line was engineered to overexpress TNFAIP8L1 protein to identify protein binding partners through co-immunoprecipitation and mass spectrometry assays. We are developing mammalian two-hybrid assays to further characterize and validate putative interactions identified by co-ip/MS. We anticipate that TNFAIP8L1 binds proteins involved in alternative tumorigenesis pathways that may hold clinical relevance in future cancer treatments. Research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423. Additionally, this project was financially sponsored by the INBRE- Honors College Comparative Functional Genomics Thesis Fellowship. A functional analysis of PAZ 5 A protein Acyltransferase Jones, A. Gundersen, R. Department of Molecular and Biomedical Science, College of Natural Sciences, Forestry and Agriculture, University of Maine alyssa.j.jones@maine.edu Cell signaling proteins are regulated by numerous modifications, one of which is palmitoylation. Palmitoylation is performed by enzymes known as protein acyltransferases, which add a palmitic acid onto proteins post-translationally. This enzyme contains a conserved DHHC amino acid sequence in a cysteine rich domain. Analysis of residues located within the cysteine rich domain will help determine activity of the enzyme at specific locations. The model organism D. discoideum contains 14 known protein acyltransferase genes, labeled Paz 1-14. Previous work has shown that when Paz 5 is “knocked out”, there is a discernible change in phenotype from the wild type, which is restored upon reinsertion of the gene. This phenotypic change of the Paz5 knock-out will be used to assess the role of highly conserved amino acids in the DHHC domain. Site-directed mutagenesis was used to change specific amino acids, which will be re-inserted into the Paz 5 “knockout” model. After insertion, the D. discoideum will be observed for a rescue in phenotype or no change, depending on the role of the mutated site on Paz5 function. Determining the role of SalY of Streptococcus pyogenes in immune evasion using fluorescence microscopy Kiidli, T.1,2, Neely, M.N.1,3 1Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME, 2Honors College, University of Maine, Orono, ME, 3Graduate School of Biomedical Science and Engineering, University of Maine, Orono, ME taaniel.kiidli@maine.edu

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Streptococcus pyogenes, the etiologic agent for several life-threatening invasive diseases, utilizes numerous mechanisms to evade the host immune response and establish a successful infection. The bacterium causes necrotizing fasciitis in both humans and zebrafish, with extensive necrotic damage but surprisingly lacking in inflammation, suggesting that recruitment of inflammatory cells is inhibited. To observe this in real-time, we used the zebrafish model of Streptococcal pathogenesis to analyze macrophage recruitment in the attenuated putative ABC transporter permease mutant, SalY, to explain the difference in virulence as compared to wild-type. Fluorescence microscopy using transgenic zebrafish with fluorescent immune cells will be used to determine the interactions between the host and pathogen to determine the role of SalY in evasion of the immune response. Further characterization of SalY and other genes of the Salivaricin locus in relation to their function in immunity will potentially lead to the development of novel antimicrobials. Acknowledgements: This project was financially sponsored by Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institute of Health. Purification and characterization of the Calpain 5 Catalytic Core. Landry, A., Karunasiri, C., Croall, D.E. Department of Molecular and Biomedical Sciences, University of Maine, Orono ME anna.landry@maine.edu Calpain-5 (CAPN5) is a calcium activated protease that is highly expressed in the central nervous system and retinal tissues of humans. An ortholog of CAPN5 participates in pathways of normal signaling events, necrosis, and apoptosis. Mutations within CAPN5 cause retinal pathologies such as blindness and retinal detachment. Our overall goal is to determine possible substrates of CAPN5 to better understand its biological functions. The specific aims of this project were to isolate and purify the catalytic core of CAPN5 (C5-CC) and to try to demonstrate its proteolytic activity. Subcloning cDNA encoding C5-CC into the pMAL-C5X vector greatly improved expression of soluble protein. Adding a his-6 affinity tag allowed for purification of C5-CC. Assays show Anxa1 can be cleaved by C5-CC suggesting C5-CC is folded properly. Completion of these aims was important because it allows us to proceed with affinity capture of biological substrates using catalytically incompetent C5-CC. Bone morphogenetic proteins and tanycyte function in the hypothalamus Leon-Palmer, N.1, Fudge, C.1, Greco, C.2, Jensen, G. 2, Townsend, K.1,2 1 School of Biology and Ecology, 2 Graduate School of Biomedical Science and Engineering; University of Maine noelle.leon@maine.edu Energy balance is maintained through the actions of the hypothalamus, or the central coordinator of appetite and energy expenditure in the brain. Bone morphogenetic proteins (BMPs) are a family of multi-functional growth factors, which we have shown to be important for various aspects of energy balance regulation, including controlling

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appetite and energy expenditure in the hypothalamus. For example, central BMP7 is able to reduce appetite and increase sympathetic drive to adipose tissues, thus increasing thermogenesis and lipolysis. The type 1 BMP receptor BMPR1A is tightly co-localized with a specialized cell type called tanycytes, which line the hypothalamic third ventricle. These cells are known to be involved in glucose sensing and transport, and given their proximity to circulating cerebrospinal fluid (CSF) and their long extensions reaching in to the hypothalamic parenchyma, these cells also likely communicate between CSF and hypothalamic nuclei. In addition, tanycytes are putative adult neural stem cells. Given the ability of BMPs to also influence neural plasticity and neurogenesis as well as hypothalamic energy balance, the fact that BMP7 circulates in the CSF, and the understanding that the adult hypothalamus is able to undergo nutrient-related changes in neural plasticity, we hypothesized that tanycyte BMPR1A may be playing a role in these processes. To investigate this, we have utilized Cre-Lox technology to ablate BMPR1A in either alpha-tanycytes (along the dorsal portion of the third ventricle, using GLAST-Cre) or beta-tanycytes (along the ventral portion of the third ventricle, using RAX-Cre). Since both populations of tanycytes express a novel adult neural stem cell marker that our lab has identified, as well as expressing BMPR1A, we sought to determine which genetic deletion affects hypothalamic plasticity and energy balance regulation more strongly. Investigating consequences of sarcomere length to heart rate and force produced by cardiac muscles of the American lobster (Homarus americanus) Maguire, M.1, Cho, S.1, Ellers, O.1, Johnson, A.1 1Bowdoin College, Brunswick, ME Crustaceans have a wide variety of sarcomere lengths in different structures with different functions requiring high speed or high force. Longer sarcomeres produce larger forces whereas shorter sarcomeres produce higher strain rates. For example, in the lobster, Homarus americanus, sarcomere lengths are longer in strong crusher claws (8 micrometers) compared to fast cutter claws (3 micrometers) (Jahromi and Atwood, 1971), and sarcomere lengths are even shorter (2 micrometers) in the high frequency flagellum abductor muscle on a crab maxilliped (Stokes & Josephson, 1994). The lobster heart is mechanically anisotropic, where contraction force is greater along the transverse than the longitudinal axis of the heart (Dickinson, Johnson et al., 2016). We measured sarcomere lengths of variously oriented muscles in the lobster heart under a variety of degrees of contraction and found that heart sarcomere lengths varied from 2-3 micrometers, a length consistent with the observed 1 Hz heartbeat. b-arrestin turns the key, opening the door to JC polyomavirus infection Mayberry, CL.1, Soucy, AN.1, Lajoie, CR.1, DuShane, JK.1 , and Maginnis, MS.1, 2. 1 Department of Molecular and Biomedical Sciences, The University of Maine, Orono, ME, 2 The Graduate School in Biomedical Sciences and Engineering, The University of Maine, Orono, ME colleen.mayberry@maine.edu

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Progressive multifocal leukoencephalopathy (PML) is a fatal, demyelinating disease caused by JC polyomavirus (JCPyV), a common human pathogen. In healthy individuals JCPyV establishes a life-long asymptomatic infection within the kidney. However, during immunosuppression, JCPyV can reactivate within the central nervous system (CNS) leading to PML. JCPyV internalization is facilitated by the serotonin family of receptors (5-HT2Rs). Entry of JCPyV is thought to occur by clathrin-mediated endocytosis (CME) but this process is poorly understood. We have determined that JCPyV enters by CME and that clathrin scaffolding proteins b-arrestin, dynamin, and AP2 are required for JCPyV entry. Furthermore, we have characterized a b-arrestin-binding domain within the receptor that is essential for JCPyV infection, likely acting to scaffold b-arrestin and CME proteins to facilitate viral entry. Furthermore, G-protein receptor kinases (GRKs), responsible for stabilizing 5-HT2R-b-arrestin interactions, are important for infection. Collectively, these data further define JCPyV internalization, increasing our understanding of JCPyV pathogenesis. Prolonged endoplasmic reticulum stress causes downregulation of TNFAIP8L1 expression in cancer cells Mayhue, S.1 Mildrum, S.1 Sullivan, C.1,2 1Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME, 2Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, ME sari.mayhue@maine.edu Cancer cells must adapt to cellular stresses to survive and proliferate. Protein folding and cellular homeostasis are essential functions of the endoplasmic reticulum (ER). Excessive protein accumulation within the ER jeopardizes cellular homeostasis causing ER stress, which consequently activates the unfolded protein response (UPR). The UPR allows the cell the capacity to overcome cellular instability through sensor-driven pathways. The tumor necrosis factor alpha induced protein-8 type 1 (TNFAIP8L1) gene plays a role in tumor proliferation, although its participation as a mediator of ER stress has not been established. We hypothesize TNFAIP8L1 is regulated through IRE1𝛼 in an ER sensor-driven pathway. Our data reveal that prolonged induction of ER stress elicits downregulation of TNFAIP8L1 transcription in cancer cells. Additionally, pharmacological inhibition of IRE1𝛼 limits the negative regulation of TNFAIP8L1 upon ER stress, indicating cancer cells downregulate TNFAIP8L1 expression through IRE1𝛼 in order to respond to ER stress and promote cell survival. Research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423. Characterization of 5-HT2 receptor subtypes in JCPyV infection using super-resolution localization microscopy

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Mehmood, K.1, DuShane, J.1, Parent, M.2, Hess, S.2 and Maginnis, M.1 1Department of Molecular and Biomedical Sciences, 2Department of Physics & Astronomy, The University of Maine, Orono, ME 04469 Kashif.mehmood@maine.edu JC polyomavirus (JCPyV) asymptomatically infects most of the human population with lifelong persistence in the kidney. In immunosuppressed individuals, it causes a fatal demyelinating disease called progressive multifocal leukoencephalopathy (PML). Previous studies demonstrated that JCPyV requires α2,6-linked sialic acid for attachment and the 5-hydroxytryptamine 2 (5-HT2) receptor subtypes for entry into host cells. These interactions are poorly understood largely due to resolution limitations in conventional microscopy techniques. This project aims to define JCPyV interactions with 5-HT2 receptors utilizing super-resolution fluorescence photoactivatable localization microscopy (FPALM), delivering resolution beyond the diffraction limit of conventional microscopy techniques. Our preliminary results suggest all three 5-HT2R-Dendra2 photoactivatable constructs show cluster distribution with sizes from approximately 0.1 to many microns. Utilizing FPALM would help visualize the interactions between JCPyV virus and receptors. These studies will improve our understanding of JCPyV attachment and invasion of host cells and help discover new targets for potential antiviral therapies. Differential gene expression during sex change in the black sea bass (Centropristis striata) Greenlaw, K.1, Montgomery, J.1, Kenter, L.2, Berlinsky, D.2, Breton, T1. 1Division of Natural Sciences, 173 High Street, University of Maine at Farmington, Farmington, ME 04938 2Department of Biological Sciences, University of New Hampshire, 38 College Road, Durham, NH 03824 Jacob.Montgomery@Maine.edu, Katherine.Greenlaw@Maine.edu Black sea bass (Centropristis striata) is a commercially valued teleost fish that has been well-studied in aquaculture. Little is known about how these fish change sex from female to male, which is an occurrence often accelerated in captive individuals. In this study, juvenile brain and gonadal transcriptome assemblies and RNA-seq were analyzed, and putative genes were identified that may be involved in sex change. Partial gene sequences were verified, and expression was assessed using quantitative PCR. Four gene assays were conducted with previously synthesized brain and gonadal cDNAs from fish induced to change sex with the aromatase inhibitor exemestane. Gonadal sequences for estrogen receptor beta and RIF1 exhibited higher male expression, while brain expression of synGAP was stable. Microtubule-associated protein 6 was upregulated 2-fold in the brain between female and sex change. These preliminary results may be used in further studies to characterize sex change processes in black sea bass. Investigating the antiviral effects of Hemocyanin derived from Homo americanus Nichols, S.1, DuShane, J.1, Bayer, R.2, and Maginnis, M.3

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1Department of Molecular and Biomedical Sciences, The University of Maine, Orono, ME, 2Lobster Institute, The University of Maine, Orono, ME, 3The Graduate School of Biomedical Sciences and Engineering (GSBSE), The University of Maine, Orono sarah.l.nichols@maine.edu JC polyomavirus (JCPyV) establishes a persistent infection in the kidneys in 50-80% of the population. In immunocompromised individuals, JCPyV can spread to the central nervous system (CNS), and cause progressive multifocal leukoencephalopathy (PML). PML is a fatal neurological disease for which there are no effective treatments. Hemocyanins are large biomolecules acting as oxygen-transporting proteins in arthropods and mollusks, that promote host defenses against bacterial, fungal, and viral infections. Hemocyanin derived from Homo americanus, or the American lobster, has been demonstrated to exhibit antiviral properties against a number of viruses including herpes simplex virus. The goal of this project is to determine whether hemocyanin can inhibit JCPyV infection. Through preliminary studies we have defined working concentrations of hemocyanin that are non-toxic to cells and have an inhibitory effect on JCPyV infeciton. Future work will determine whether hemocyanin derived from lobster has potential antiviral effects on JCPyV infection. Studying the effects of injecting Streptococcus pyogenes into the Duct of Curvier in young zebrafish Patenaude, K. The University of Maine Streptococcus pyogenes (Group A streptococcus or GAS) is a bacterium that can lead to infections such as necrotizing fasciitis, pharyngitis, myositis, and post-streptococcal glomerulonephritis. Infections from GAS will likely occur in the respiratory tract, bloodstream, or as skin infections. My project focuses on injecting different concentrations of GAS mixed with cell-tracker Red CMTPX through the Duct of Cuvier in two-day old zebrafish to observe different stages of a systemic infection at different time points. The Duct of Cuvier is a vein on the side of the yolk sac leading in the heart. I use a zebrafish strain called “Caspers”, that lack pigmentation. At two days old, the Duct of Cuvier is visible in the zebrafish and the embryos have fully functional neutrophils whose responses to the infection can be studied. Having a better understanding of how the immune system fights these bacteria could help in developing more effective anti-microbials. The effect of embryonic arsenic exposure on the sensorimotor behavior of zebrafish (Danio rerio) Paye, L., Bowser, T., Van Beneden, R. J. University of Maine, Orono, ME laura.paye@maine.edu The goal of this study is to determine the effect of arsenic on vision in the zebrafish (Danio rerio). Zebrafish are small, readily available and their eye morphology is very similar to humans, making them ideal for toxicological studies. Previously, our lab has

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shown that embryonic arsenic exposure alters the expression of genes involve in zebrafish eye development, affecting eye morphology. We hypothesized that arsenic may also affect vision. Zebrafish embryos were exposed to 4 treatments of sodium arsenite, 0-72 hours post fertilization. We tested at 5 days post hatch, when the eye was fully developed and fish were visible on camera. In order to test visual acuity, we recorded fish in a small tank surrounded by a black and white striped cylinder rotating at 10 rpm. The optomotor response indicates that they should follow the striped pattern parallel to the arena, exposing any visual defects due to arsenic. CRISPR/Cas9 gene editing of nematode germ granule protein genes Sean Barbosa1, Sabrina Douglas1, Seth Hannigan2, Samantha Johnes2, Savannah Levesque2, Autumn London2, Hunter Milliard2, Romario Romain2, Michaela Schwartz1, Ryan Tebo1, Judith L. Roe1, Jacob Theriault2, Dustin Updike3 1University of Maine at Presque Isle, Presque Isle ME 04769, 2University of Maine at Ft. Kent, Ft. Kent, Maine 04743, 3MDI Biological Laboratory, Salisbury Cove, ME 04672 Germ granules are important for fertility during animal development to establish the germline. In Caenorhabditis elegans these germ granules localize to the nuclear envelope and the RNA helicase protein GLH-1 is important in their formation and maintenance. We created a deletion mutation in the glh-1::GFP gene using a co-CRISPR/Cas9 method with ‘targeting RNAs’ for both the glh-1 and the dpy-10 gene. Unlike the original glh-1::GFP worms, 90.4% of the dpy worms targeted for deletion showed a de-localized expression of GFP and thus a high level of co-CRISPR efficiency. The glh-1 mutation was later confirmed as a loss-of-function deletion mutation by sequencing and the dpy mutation was segregated away. The phenotype of the glh-1 deletion mutant was investigated by testing it’s fertility. Compared to wild-type, which had high levels of fertility at both 20 and 26℃, the deletion mutant had greatly decreased fertility when grown at 26℃, suggesting that GLH-1 is important in fertility at temperatures such as 26℃. Interestingly, a dominant-negative allele of glh-1 also showed reduced fertility at 26 and not 20℃. We also obtained a substitution mutation in the conserved C-terminal acidic domain of unknown function which retained a localized expression of GFP in the germ granules, suggesting that this domain is not responsible for granule localization. Examining molecular markers of fertility in the male germline of the soapberry bug, Jadera haematoloma Smith, D.M.1 and Angelini, D.R.2 1Colby College, Department of Chemistry, Waterville, ME, 2Colby College, Department of Biology, Waterville, ME dmsmith@colby.edu

Polyphenism is a phenomenon in which individuals within a species exhibit a few distinct phenotypes due to environmental influences. Manifestations can be sex-specific, in which certain traits and their accompanying tradeoffs affect one sex while the other sex has limited plasticity and variation in displaying those traits with environmental cues. Examples of sex-limited polyphenisms include the development of

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many male weapons used to compete for social dominance as well as many wing polyphenisms were females may develop long wings for flight or greater reproductive ability. In some species, polyphenism affects both sexes, but does not confer equal advantages, leading to sexual conflict. For example, a dispersal/fecundity polyphenism in the red-shouldered soapberry bug Jadera haematoloma (Hemiptera: Rhopalidae) is dependent on juvenile nutrition, resulting in two discrete wing morphs in adults. Recent evidence has identified sexual conflict in the wing polyphenism of J. haematoloma. The short-winged morph appears to be maladaptive in males: not only are they unable to fly, but short-winged males have lower fertility than long-winged males. To understand the basis of these fertility differences, we have investigated cell populations and genetic markers of the male germline. We use in situ hybridization, immunohistochemistry, and RNA interference to describe male germline maintenance and spermatogenesis in J. haematoloma to characterize fertility differences between male morphs. Role of Kelch proteins in gradient tracking Sojka, S1, Hart, A.1, Simke, W.1, Kelley, J. 1, 2 1 Molecular and Biomedical Sciences, University of Maine, 2 Graduate School of Biomedical Science and Engineering, University of Maine. savannah.sojka@maine.edu Cancer metastasis and pathogen recognition by immune cells are examples of gradient tracking in the biological world. Baker’s yeast utilize gradient tracking during mating, in which a yeast cell elongates in the direction of a potential mate by sensing the relative pheromone amounts in the environment. Once the cells physically meet, they undergo cell fusion. Kelch proteins are ubiquitous in nature and serve a variety of biological functions. In this case, there are kelch proteins known to regulate cytoskeletal actin growth and facilitate cell fusion. This research aims to investigate the role of kelch proteins in gradient tracking and their known interactions with regulatory proteins involved in the pheromone signalling pathway. This is accomplished through use of fluorescence microscopy, microfluidics experiments, and computational image analysis. This will improve our understanding of the diverse roles of kelch proteins in biology and contribute to the mechanistic understanding of gradient tracking. Electrifying neuroscience, an electronics-based approach to teaching introductory neuroscience principles to undergraduate students Sullivan, C.1,2 & Ganter, G.1,2,3 1University of Maine, Graduate School of Biomedical Sciences and Engineering, Orono, ME, 2University of New England, Center for Excellence in the Neurosciences, Biddeford, ME, 3University of New England, College of Arts and Sciences, Biology Department, Biddeford, ME cara.sullivan@maine.edu The desire to understand how biological processes influence consciousness and behavior has directed scientific inquiry since ancient times. In recent years, neuroscience topics have dominated pop-culture and social media. In turn, interest studying neuroscience in a formal academic setting has surged and undergraduate

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institutions have created neuroscience courses and degree programs to accommodate these interests. Neuroscience is an interdisciplinary subject that requires knowledge spanning basic biology and genetics to electronics and computer programming. Designing and implementing hands-on neuroscience laboratory activities is challenging in terms of equipment needed, costs of supplies and limited time available during laboratory class sessions. In the spring of 2018, the University of New England, created a new neuroscience laboratory course to accompany ‘Introduction to Neurobiology’. This course focuses on the electrophysiological properties of neurons using previously validated neuroscience laboratory activities and introduces a novel variation of the classic Otto Loewi experiment using zebrafish. The zebrafish model of the immune response to Pseudomonas aeruginosa infection in cystic fibrosis patients Anna Struba1, Ben Tero1, Caroline Spangenberg2, James Seuch1, Sydney Green2, Dr. Melody Neely 1 Molecular and Biomedical Sciences, 2 School of Biology and Ecology, University of Maine Due to weakened immune responses, Cystic Fibrosis (CF) patients experience high mortality rates caused by opportunistic pathogens, such as Pseudomonas aeruginosa. CF patients experience lung and intestinal problems due to mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes an epithelial chloride channel. A zebrafish model of CF was created by knocking down CFTR expression in order to explore innate immune response mechanisms. Confocal microscopy demonstrated migration of neutrophils to the site of P. aeruginosa infection. Fewer neutrophils were observed at the infection site in CF fish compared to negative control fish. Similarly, after 48 hours post infection, a higher concentration (CFU/mL) of P. aeruginosa was observed in CF fish compared to negative control fish. These results indicate that CF is responsible for compromising innate immune responses in zebrafish. Further work should be done to explore treatments for CF to increase the ability to resist opportunistic pathogens. Acknowledgements: The research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health. Study the role of TIPE1 protein-lipid interactions in tumor suppression White, B. The University of Maine The tumor necrosis factor (TNF)-alpha-induced protein 8 (TNFAIP8) family is a group of proteins shown to regulate tumorigenesis. Belonging to the family are proteins TNFAIP8 (TIPE), TNFAIP8-like 1 (TIPE1), TNFAIP8-like 2 (TIPE2), and TNFAIP8-like 3 (TIPE3). Collectively, the TIPE family has been shown to function in immune homeostasis, as well as in the regulation of apoptosis to effectively promote or suppress tumor growth. The TIPE3 protein achieves tumor promotion through the upregulation of phosphoinositide signaling. Similarly the TIPE1 protein, a tumor suppressor, has been

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found to bind to various second messengers of phosphoinositide signaling, though these protein-lipid interactions have not been closely studied. We aimed to study the interactions of the TIPE1 protein with the second messengers phosphatidic acid (PA) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) to examine the role of TIPE1 in phosphoinositide signaling. In addition, we hope to show functional conservation of these interactions with the zebrafish protein so we may use zebrafish as a model for future studies. Research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423. Human JC Polyomavirus infection of primary astrocytes: A model for a deadly disease Wilczek, M.P. and Maginnis, M.S. The University of Maine, the Department of Molecular and Biomedical Sciences, Orono, Maine michael.wilczek@maine.edu JC polyomavirus (JCPyV) infects most of the population causing a persistent infection in the kidneys. In immunocompromised individuals, the virus reactivates in the CNS causing the disease progressive multifocal leukoencephalopy (PML). There is no treatment for PML, which can be fatal within 1-2 years. Currently, our understanding of PML pathogenesis is limited due to a lack of an animal model and restricted growth of JCPyV in cell culture. Due to these challenges, the majority of JCPyV research has been conducted using SVG-A cells, a mixed glial cell line. However, SVG-A cells present several limitations for studies of JCPyV infection and PML pathogenesis due to their immortalized characteristics. Thus, we have established an innovative model of JCPyV infection using primary human astrocytes (NHAs). Our research demonstrates that JCPyV infection in NHAs differs from SVG-As, and these differences will serve as the basis for future studies to elucidate mechanisms of PML pathogenesis. Imaging and analysis of whole adipose depot neuronal innervation Willows, J., Blaszkiewicz, M. , Johnson, C. , Townsend, K. Townsend Laboratory, University of Maine, Orono ME jake.willows@maine.edu Both white (energy-storing) and brown (energy-burning) adipose tissues are important for energy balance regulation and must communicate with the brain via peripheral nerves in order to maintain metabolic health. Surgical and chemical denervation studies that result in loss of brain-adipose communication have demonstrated the clear importance of adipose tissue nerves for processes like lipolysis, thermogenesis, and ‘browning’ (or conversion of white to brown adipose). All of these calorie-burning processes are negatively impacted when a proper nerve supply in adipose tissue is lost. While most studies have worked to understand the upstream pathways in that brain that influence sympathetic outflow to adipose depots, very little work has been done to investigate regulation of peripheral nerves at the level of the adipose tissue itself. Our

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work has revealed that certain conditions lead to adipose neuropathy, or the death of the peripheral nerves, and that certain interventions can reverse this by promoting adipose nerve plasticity. Local release of neurotrophic factors by adipose-resident immune cells appears to play an important role. Using new techniques, we have optimized in order to image, quantify, and analyze adipose peripheral nerves, we are now able to answer questions about brain-adipose communication, including: Which nerve types are important for proper metabolic function in adipose tissues? How are adipose nerves regulated at the tissue level? Where do adipose peripheral nerves synapse in the tissue? & Do both sensory and sympathetic nerves play a role in adipose tissue health? Network dynamics of microRNA co-expression during innate immune system responses Yang, C., King, B. L., Sullivan, C., Kim, C. H. Department of Molecular and Biomedical Sciences, Graduate School of Biomedical Science and Engineering, University of Maine, Orono, ME chenhao.yang@maine.edu Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF patients are susceptible to Pseudomonas aeruginosa infections. Our previous studies have shown that zebrafish infected with Pseudomonas aeruginosa have diminished innate immune responses with Cftr knockdown and low dose arsenic exposure. Here, we studied the co-influence of Cftr knockdown, arsenic exposure, and Pseudomonas infection on innate immunity of zebrafish. Arsenic-treated embryos and Cftr morphants showed defective responses to infection including increased bacterial burden and reduced respiratory burst capacity. We characterized microRNA expression in response to infection under normal conditions, arsenic exposure and Cftr knockdown. Clustering analyses of the microRNA co-expression networks revealed that sets of microRNAs normally co-regulated during infection were no longer co-regulated in the defective responses caused by arsenic exposure and/or Cftr knockdown. Our ongoing study advances our knowledge of innate immune response to infection in CF patients. The roles of the epidermal growth factor receptor in response to Candida albicans infection Zwirner, C.P.1, Gratacap, R.L.2, Wheeler, R.T.1,2 1Department of Molecular and Cellular Biology, College of Natural Sciences and Forestry, 2Department of Molecular and Cellular Biology, Graduate School of Biomedical Science and Engineering, University of Maine Candida albicans is an opportunistic fungus that can cause life-threatening infections. Approximately 46,000 cases of candidiasis, Candida infection, occur per year. Understanding the pathophysiology of C.albicans is crucial for combating this infection. The epidermal growth factor receptor (EGFR) regulates immune responses to pathogens by recruiting neutrophils and reinforcing epithelial barriers. Many human disease treatments suppress EGFR signaling, so investigating EGFR's roles in

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immunity to Candida is also linked to uncovering potentially deleterious side-effects for these treatments. We induced mucosal candidiasis in the zebrafish swim bladder and chemically inhibited EGFR to study its roles in regulating C.albicans pathophysiology in vivo . Confocal microscopy, transgenic zebrafish, and fluorescent C.albicans were utilized for analyses. Blockade of EGFR increased mortality rates and resulted in fewer recruited neutrophils. No significant increase in pathogen-epithelial interactions or fungal burden was observed, suggesting that EGFR suppression may increase mortality due to toxic side effects rather than enhancing infection.