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1001. The role of McProf’s ESX-like TA cassette in M. chelonae antibiotic resistance.
Undergraduate Presentation. Author(s): Anna Schumann, Jaycee Cushman, Sarah McCallister, Keith Hutchison. Mentor(s): Sally Molloy
Abstract: Pathogenic mycobacteria are the leading cause of death worldwide. Mycobacterium abscessus is the most isolated pathogen from cystic fibrosis patients and often completely resistant to antibiotics. Increased research on antibiotic resistance mechanisms is important for developing new treatments. Prophage, viral genomes integrated into bacterial genomes, contribute to virulence in most pathogens. Their role in mycobacterial virulence is not known. We showed that the naturally occurring prophage, McProf, increases the antibiotic resistance and expression of whiB7, a transcriptional activator of antibiotic resistance genes, in pathogenic Mycobacteria when exposed to stress. It has not been reported how prophage change bacterial gene expression and antibiotic resistance. We hypothesize that prophage gene products alter bacterial gene expression. McProf expresses high levels of genes in a secreted polymorphic toxin system. Polymorphic toxins are known to play a role in the bacteria’s responses to stress and are known to trigger changes in gene regulation in other bacteria of the population to increase the overall population’s fitness. This project aims to determine if the McProf polymorphic toxin system is responsible for altered whiB7 expression and antibiotic resistance when the bacteria are exposed to stress. We have PCR amplified the genes of the polymorphic toxin system and cloned them into an integrative mycobacterial expression plasmid. If the polymorphic toxin system is responsible for changes in antibiotic resistance and whiB7 expression, mycobacteria carrying the plasmid-encoded polymorphic toxin genes will have whiB7 expression and drug resistance profiles similar to that of mycobacteria carrying the McProf prophage.

1002. Role of the Accessory Domain in CpsA Function and Capsule Production in GBS
Undergraduate Presentation.
Author(s): Gina DiFederico Mentor(s): Melody Neely
Abstract: Group B Streptococcus (GBS) can be found in the vaginal and genitourinary tract of females, as well as the genitourinary tract of males, where it behaves as a commensal organism. However, as an opportunistic pathogen, GBS has the capability to infect the immunocompromised, making it a major threat to neonates and fetuses. The pathogen can be passed from mother to baby either in utero or during birth. The capsule, which is a polysaccharide coating on the outside of the cell, is considered the most important virulence factor in GBS. Expression of capsule plays a role in evasion of the host immune response to GBS infection. The presence of capsule on GBS depends on the CpsA protein, which is involved in the attachment of capsule to the cell wall. CpsA is a multi-functional protein containing an intracellular domain and two extracellular domains including the accessory and the LytR domains. Previous data demonstrates a small region within the accessory domain of CpsA that, when expressed separately, can have a negative effect on the amount of capsule on the cell. In this study, the deletion of the extracellular accessory domain of CpsA will help to determine the role of the domain on CpsA function and capsule production. Cell morphology analysis, capsule assays, and virulence studies will all be used to analyze results of a deletion of the accessory domain.

1003. The COVID-19 public health response and alcohol abuse: A comprehensive analysis
Graduate Presentation.
Author(s): Anthony Posuniak Mentor(s): Deborah Saber
Abstract: The COVID-19 pandemic and alcohol abuse represent significant threats to public health in the United States. This capstone seeks to provide a comprehensive analysis of what is known about SARS-COV-2, examine prior pandemic strategies, define the current pandemic mitigation efforts and their scientific efficacy, consider potential societal impacts of the response, and determine the role of the nurse practitioner (NP) as a leader in public health. Process Much is known about SARS-COV-2 (COVID-19) and there are decades of research on coronaviruses like SARS. The COVID-19 response was a derivation from other pandemic responses by quarantining healthy individuals and locking down the economy. Modeling and mechanistic studies support the widespread use of masks, school closures, and lockdowns, albeit with varying degrees of contrary evidence and socio-economic concerns. Prior evidence on pandemic responses and current trends indicate that mental health problems, domestic violence, and suicide rates will likely increase due to the pandemic and its public health response. Alcohol abuse is also likely to increase as a means of coping with pandemic stress and depression. NPs are well-situated to lead future public health responses due to their leadership ability, safety, efficacy, and ability to build rapport with patients. Outcomes The public health response to COVID-19 is having a disparate impact on low-wage and socioeconomically disadvantaged Americans. The mental health, social, and economic impacts are likely to be vast. Accordingly, the current and future pandemic responses should be narrowly tailored, protect the most vulnerable, and allocate resources accordingly.

1004. Repurposed Therapeutics for Treatment of JC Polyomavirus Infection
Graduate Presentation.
Author(s): Avery Bond, Avery Bond, Mason Crocker, Michael Wilczek Mentor(s): Melissa Maginnis
Abstract: Progressive multifocal leukoencephalopathy (PML) is a fatal disease of the brain caused by JC polyomavirus (JCPyV), a virus that infects 50-80% of the human population. JCPyV persistently and asymptomatically infects the kidneys of healthy individuals. In severe cases of immunosuppression, JCPyV can spread from the kidneys to the brain, resulting in PML. Since there are currently no effective treatments for PML, many individuals will quickly succumb to this disease or become severely debilitated. Therefore, continued research surrounding JCPyV infection and PML is essential. Recently, the Maginnis laboratory performed a large-scale drug screen using the National Institutes of Health Clinical Collection (NIH-CC) and assessed viral infectivity by utilizing high-throughput In-Cell Western assays. Results from the drug screen revealed that multiple FDA-approved drugs, from several drug classes, are able to reduce JCPyV infection. Categories that contained the most drug “hits” include receptor agonists/antagonists, calcium signaling-related drugs, and enzyme inhibitors. Further investigation of calcium-signaling drugs and inhibitors supports a role for calcium signaling in JCPyV infection. Potential antivirals for JCPyV infection and PML will be identified through characterization of the mechanism of inhibition of drug hits during the viral infectious cycle. Importantly, drugs in the NIH-CC have previously been approved for other uses, and repurposing existing therapeutics is an efficient method to hasten discovery of antiviral treatments.

1005. AAV9-Ighmbp2 gene therapy significantly improves motor performance in severe SMARD1-like mouse model, nmdem3, and CMT2S mouse model,  nmdem5
Graduate Presentation.
Author(s): Sarah Holbrooks, Amy Hicks, Greg Cox Mentor(s): Dr. Greg Cox
Abstract: Autosomal recessive mutations in IGHMBP2, a ubiquitously expressed DNA/RNA helicase, have been linked to childhood neuromuscular degenerative diseases (NMDs). C57BL/6J-Ighmbp2em3Cx is a SMARD1-like strain, or Spinal Muscular Atrophy with Respiratory Distress, created via CRISPR-Cas9 targeting of the IGHMBP2 gene and hereafter referred to as em3. SMARD1 is characterized by muscle weakness starting in the distal extremities and diaphragmatic paralysis leading to respiratory failure. Most patients are diagnosed in early infancy and die in early childhood. The em3 mouse has more severe muscle atrophy than the historical SMARD1-like model (nmd2J) in the hind limb, diaphragm, and intercostal muscles. The em3 mouse model also has an average lifespan of ~3 weeks compared to the 2J’s ~3 month lifespan. C57BL/6J-Ighmbp2em5Cx is a Charcot-Marie-Tooth disease type 2S model that has not impact on lifespan but does impact motor and sensory function beginning around the 4 week timepoint.
Gene therapy has shown promise in another NMD, Spinal Muscular Atrophy (SMA). In collaboration with the Meyer lab at Nationwide Children’s Hospital in Columbus, OH, we are testing 2 different AAV9-Ighmbp2 vectors. Each has a different promoter with one having a Chicken β-Actin (CBA) Promoter [higher expression levels than endogenous levels] and the other having a truncated Methyl-CpG binding protein 2 (MECP2 aka P546) promoter [expression levels close to endogenous levels expressed by muscles and neurons]. We did p1 intracerebroventricular injections on em3 and em5 mutants and unaffected sibling pups to determine the efficacy of each treatment, respectively, and if there are toxic effects associated with overexpression of IGHMBP2 in wild type mice.

1006. Characterizing the role of prophages on whiB7 expression and antibiotic resistance in Mycobacterium chelonae
Graduate Presentation.
Author(s): Jaycee Cushman, Emma Freeman, Sarah McCallister, Anna Schumann, Dr. Keith Hutchison, Dr. Sally Molloy
Mentor(s): Sally Molloy
Abstract: Mycobacterium abscessus causes lung and disseminated infections that are most common among immunocompromised individuals. It is the most intrinsically antibiotic-resistant species known, making infections difficult to treat with a success rate of only 45%. While some extensively resistant isolates are caused by mutations in drug targets, others appear to be a result of increased intrinsic drug resistance. Common among these strains is presence of integrated viral genomes (prophage) that are known to contribute to fitness and antibiotic resistance in other pathogens but whose roles are largely unknown in mycobacteria. We have demonstrated that the presence of an M. abscessus cluster R prophage, McProf, in M. chelonae, increased resistance to antibiotics, such as amikacin, relative to strains lacking the prophage. The presence of McProf also enhances amikacin resistance in response to sub-lethal concentrations of antibiotics, or other cellular stresses such as infection by a second phage, BPs. Relative to strains carrying only one of the prophage or no prophage, the strain carrying two prophages, BPs and McProf, had the highest amikacin resistance. This strain also showed increased expression of the transcriptional activator, whiB7, which promotes expression of intrinsic antibiotic resistance genes. This work suggests that prophages play a role in increasing intrinsic antibiotic resistance and stress adaptation in pathogenic mycobacteria. Given that most pathogenic mycobacteria carry one or more prophages and encounter drug gradients in tissues during infection, characterizing how prophages regulate antibiotic resistance genes and adaptation to stresses will provide insight for developing more effective therapies for mycobacterial diseases.

1007.  Discovering Virulence Factors of Candida albicans that Affect Host Immune Responses
Undergraduate Presentation.
Author(s): Emma Bragdon, Bailey Blair, Nnamdi Baker Mentor(s): Robert Wheeler
Abstract: Candida albicans (C. albicans) is a commensal yet opportunistic pathogen. It is typically found in the oropharyngeal, gastrointestinal, and reproductive tract of humans. While healthy adults are not infected by the pathogen besides the common vaginal yeast infection, immune-compromised individuals such as those going through chemotherapy and bone marrow transplants are susceptible to life-threatening infection with mortality rates reaching up to 40%. Understanding the mechanisms as to how this pathogen causes disease may help us develop new strategies to prevent and treat these lethal infections. We sought to identify new virulence genes in C. albicans that regulate innate immune response. Since hyphal growth is tightly linked to virulence for C. albicans, we reasoned that novel immune evasion genes might be identified as mutations which affect hyphal growth or virulence, but not both. Previous work in the Wheeler Lab identified 133 mutants in the Noble mutant library collection which had previously been characterized to encode known secreted factors, or be required for hyphal growth or virulence, but not both. Using a zebrafish hindbrain infection model, where it is known that survival is closely linked to rapid immune responses, mutant C. albicans strains lacking single genes were injected into the hindbrain of the zebrafish, and infection was observed and analyzed to draw conclusions on virulence factors that are most important in C. albicans infection. This method identified two genes, CHT2 and ORF19.5547, that appear to be important in virulence. Further directions include determining the mechanisms by which these genes enhance virulence.

1008. Understanding the Role of Perivascular Adipose Tissue Differentiation in Cardiovascular Disease
Graduate Presentation.
Author(s): Caitlin Stieber Mentor(s): Dr. Lucy Liaw
Abstract: Obesity is a major risk factor for cardiovascular disease. Cellular interactions between adipose tissue and the vasculature may explain this link. Perivascular adipose tissue (PVAT) surrounds most vessels and exerts a paracrine effect on the underlying blood vessel. In metabolically healthy individuals, PVAT secretes factors that decrease constriction and expansion of smooth muscle in the vessel. However, in metabolic disease, PVAT-secreted factors increase constriction and expansion of the smooth muscle leading to restricted blood flow and disease. We study two human populations with different levels of cardiovascular disease to assess PVAT phenotype. Our interest is how the PVAT phenotype varies within populations and how this is regulated by the trafficking molecule RAB27A. We hypothesize that there will be differences in BMI, gene, and protein expression between the two populations. PVAT and subcutaneous adipose tissue (SubQ) were collected from donors undergoing coronary artery bypass graft (CABG) or those underdoing mitral valve repair (VR). We assessed demographic and clinical differences in the donor populations. Using single nuclei RNA sequencing and immunoblot we assessed the expression of RAB27A and adipose markers. Additionally, pre-adipocytes were isolated, grown in vitro, and assessed using immunoblot. The average BMI and diabetes prevalence was increased in the CABG population, indicating they are more likely to be metabolically unhealthy. RAB27a gene expression was increased in donors with a lower BMI, while protein expression was decreased. Moving forward, we will continue to examine molecular differences between donors, as well as strive to understand Rab27a’s mechanistic control of adipose differentiation.

1009. Understanding the Mechanism of Cancer Treatment Induced Ovarian Toxicity
Author(s): Monique Mills, Monique Mills, Chihiro Emori, Parveen Kumar, Zachary Boucher, Ewelina Bolcun-Filas Mentor(s): Dr. Ewelina Bolcun-Filas
Abstract: Genotoxic cancer treatments – such as radiation – induce DNA damage, preferentially killing cancerous cells but can also kill healthy cells. Primordial follicles (PFs), a finite population of ovarian follicles containing immature oocytes, are highly susceptible to DNA damage. Depletion of PFs through cancer treatment induced DNA damage can cause premature ovarian failure and infertility. Existing fertility preservation methods are invasive, delay cancer treatment, and are insufficient for prepubescent females. The goal of this project is to elucidate the DNA damage response in PFs to identify targets for a non-invasive pharmacological inhibition strategy to preserve ovarian function without delaying cancer treatment. Our lab previously showed that CHK2 kinase and its downstream target TAp63 are key mediators of the DNA damage response in oocytes. Inhibition of CHK2 or TAp63 activity improved PF survival after low-dose radiation. However, at high-dose radiation, TAp63-/- mice, but not Chk2-/-, were depleted of PFs indicating a TAp63-independent mechanism. Oocyte’s response to DNA damage is still poorly understood. To identify factors and processes involved in DNA damage induced oocyte elimination, we conducted single cell sequencing of irradiated and non-irradiated ovaries from wild type and Chk2-/- mice. We observed a strong CHK2-dependent response in oocytes and identified three novel factors that may be mediators or targets of this response. Defining the role of these proteins in oocytes, in response to radiation induced DNA damage, will allow us to design a pharmacological inhibition strategy to prevent premature ovarian failure and infertility in female cancer survivors.

1010. Characterization of Dual Specificity Phosphatase 1 (DUSP1) Activity in JCPyV Infection in Primary Astrocytes
Undergraduate Presentation.
Author(s): Aiden Pike, Michael Wilczek Mentor(s): Melissa Maginnis
Abstract: The human JC polyomavirus (JCPyV) causes a persistent, asymptomatic infection in the kidneys in up to 80% of the population. In immunocompromised individuals, JCPyV can spread to the brain and cause the fatal, demyelinating disease known as progressive multifocal leukoencephalopathy (PML). The method by which JCPyV causes PML remains poorly understood and there are no current treatments for the disease. Immortalized brain cells known as SVGAs have been utilized to define the interplay of cellular signaling pathways and viral infection; however, preliminary data suggests that JCPyV infection in normal human astrocytes (NHAs) can also lead to the activation of innate immune response components, not recognized during viral infection of SVGAs. Previous research has demonstrated that JCPyV infection in the kidneys leads to an interferon response in the host, resulting in an asymptomatic infection, yet these immune responses have not been well characterized in the brain. Dual specificity phosphatase 1 (DUSP1) is a well-described protein known to regulate a cellular pathway used by JCPyV, but is also involved in the immune response during infection by other viruses. DUSP1 has known interactions with genes that specifically activate downstream immune response pathways, which can play a key role in the outcome of viral infection. Utilizing bioinformatic tools such as EdgeR, STRING DB, and other statistical analyses we have further characterized the differential expression of genes involved in the immune response. This research will further our understanding of cellular factors that drive JCPyV infection, the immune response, and identify potential targets for antiviral treatments.

1011. Identification of Potential Antiviral Drug Targets in JC Polyomavirus Infection
Author(s): Nicholas Leclerc, Avery Bond, Mason Crocker, Melissa Maginnis Mentor(s): Dr. Melissa Maginnis
Abstract: JC Polyomavirus (JCPyV) infects up to 80% of the global population and causes a persistent, asymptomatic infection of the kidneys. This progresses to a lytic infection of astrocytes and oligodendrocytes in immunocompromised individuals, resulting in development of the fatal neurodegenerative disease known as progressive multifocal leukoencephalopathy (PML). Currently, there is no available standard therapy for the treatment of PML in clinical settings. Recent development of a high-throughput In-Cell Western (ICW) drug screen allows for the analysis of a substantial number of pharmaceuticals and their effect on viral infection. This method utilizes a high-throughput infrared scanner to quantify the percentage of virus-infected cells in culture after administration of over 700 drugs from the NIH Clinical Collection and subsequent exposure to JCPyV. Through normalizing these ratios and determining z-scores, the Maginnis lab has identified drugs that significantly reduce viral infection and prioritized drugs for further characterization based on their mechanisms of action. Identification of the characteristics of these drugs and analysis of preliminary data will allow for their prioritization in terms of their relevance to JCPyV infection. Through cell-based assays, the effects of these drugs on JCPyV infection will be more accurately elucidated, and the step in the JCPyV infectious cycle that is specifically affected by the treatment will be determined. Overall, this research could serve as the platform for identification of novel antiviral treatments to treat or prevent PML.

1012. Temporal Dynamics of Viral Load and False Negative Rate Influence the Levels of Testing Necessary to Combat COVID-19 Spread
Graduate Presentation.
Author(s): Katherine Jarvis Mentor(s): Joshua B. Kelley
Abstract: The world wide outbreak of COVID-19 in March 2020 forced college campuses to close and resort to online learning for the remainder of the Spring 2020 semester. But for the Fall 2020 semester, colleges and universities looked toward computational predictive models to assess the amount of testing and preventative measures in order to open college campuses without causing an outbreak. Some predictive models do not take into account the infectivity and false negative rate of the test changing over time based on the day of symptom onset. In order to prove dynamics of viral spread are crucial in simulating an epidemiology model, we developed a stochastic agent based model of COVID-19 spread in a university sized population and determined the amount of testing required to not cause an outbreak. The amount of testing required to prevent an outbreak with uniform infectivity and false negative rate of testing distributions was significantly less than the model accounting for the dynamics of infectivity and false negative rate. From this comparison, it shows the viral dynamics is an important factor when modeling viral spread.

1013. Defining the Role of GRK2 in JC Polyomavirus Entry
Undergraduate Presentation.
Author(s): Paige Giffault, Colleen Mayberry, Kashif Mehmood Mentor(s): Melissa Maginnis
Abstract: Defining the Role of G-Protein Receptor Kinase 2 in JC Polyomavirus Entry Giffault, PK1,2, Mayberry, CL1, Mehmood, K1, and Maginnis, MS1,3 1Department of Molecular and Biomedical Sciences, 2The Honors College, 3The Graduate School of Biomedical Science and Engineering, The University of Maine, Orono, ME. JC polyomavirus (JCPyV) is a human pathogen that targets kidney epithelial cells, resulting in an asymptomatic infection of the kidneys in healthy individuals in over half of the world’s population. However, in immunocompromised individuals, the initial infection in the kidneys can progress to a lytic infection in the brain through the dissemination of JCPyV into the central nervous system. Within the central nervous system, JCPyV infects and destroys human glial cells, astrocytes and oligodendrocytes, leading to the development of progressive multifocal leukoencephalopathy (PML), a fatal disease for which there is no current treatment. There is a limited understanding of the mechanisms that JCPyV uses to enter these target glial cells within the central nervous system. Previous research by Mayberry et al. has shown that serotonin 5-hydroxytryptamine subfamily 2 receptors (5-HT2Rs), G-protein coupled receptors (GPCRs), mediate JCPyV entry into target cells by clathrin-mediated endocytosis. JCPyV internalization and infection also requires the scaffolding protein β-arrestin, which has been shown to bind to a conserved amino acid motif, Ala-Ser-Lys (ASK), within the intracellular loops of the 5-HT2Rs. G protein receptor kinases (GRKs) are known to phosphorylate serine and threonine residues on G-protein coupled receptors, leading to the recruitment of scaffolding proteins such as β-arrestin. The aim of the research is to explore the role of GRK2 in the initiation of JCPyV entry by mediating the interactions between the ASK motif within the 5-HT2Rs and the β-arrestin scaffolding protein through quantitative biochemical and confocal microscopy-based analyses. The results of this research will provide a deeper understanding of the complex mechanisms involved in JCPyV entry into target cells and could highlight potential targets for therapeutic approaches in the treatment or prevention of PML.

1014. Multiscale Image Colocalization
Undergraduate Presentation.
Author(s): Aidan Greenlee, Jeremy Juybari Mentor(s): Dr. Andre Kahlil
Abstract: The increase in quality and quantity of biomedical images led to frequent use of Pearson’s Correlation Coefficient (PCC) for image colocalization, a common metric for quantifying overlap between a pair of images. In practice, PCC is often used with two-photon excitation microscopy data. Our previous work explored some fallbacks of PCC such as misleading PCC values when there is no overlap between images. By investigating Gaussian blurring at multiple size scales in conjunction with PCC we added a whole new analytical dimension by developing a multiscale version of the PCC. Our proposed approach mitigates limitations of the traditional PCC approach by providing a series of statistics rather than a single point from PCC. The multiscale PCC approach is a flexible tool, developed with the open-source mindset in FIJI and Python. Specifically, we found that when blurred, fiber width and approximate separation distance could be determined using the minimum measured PCC value across blurring scales.

1015. Spatial Frequency Domain Imaging Applications in Polyneuropathy
Graduate Presentation. Author(s): Christian Crane Mentor(s): Karissa Tilbury
Abstract: Our research is focused on yielding better results for patients in the form of early detection and/or screening for polyneuropathy. We will be collecting imaging data of study participants’ feet using Spatial Frequency Domain Imaging (SFDI) to analyze tissue perfusion. SFDI projects distinct spatial patterns of visible and near infrared light onto tissue and images of these patterns and the tissue are captured using a camera. These images contain information about tissue scattering and absorption properties which can be separated. The absorbed light is then used to calculate the amount of oxy/deoxyhemoglobin in the tissue as a measure of tissue perfusion. Altered tissue perfusion is a key factor in the development of diabetic neuropathy. In this study, we build upon study design protocols that measured pressure and temperature differences in normal and diabetic patients during prolonged periods of standing. Here we are building upon previous studies in which tissue metrics were measured during prolonged periods of standing Furthermore, other literature has found that diabetic patients on average take fewer steps during a single day. In this preliminary study, we seek to observe and quantify tissue perfusion differences between healthy and diabetic study participants during a series of defined tasks including standing, walking, sitting, and reclining to determine if there are transitory differences that may be used to detect early alterations in tissue perfusion which may provide opportunities for development of new therapeutics.

1016.  Effects of oral-care antimicrobial cetylpyridinium chloride on tyrosine phosphorylation: a potential mechanism for mast cell inhibition assessed via In-Cell Western
Undergraduate Presentation. 
Author(s): Jessica Bruno, Bailey West Mentor(s): Julie Gosse
Abstract: Effects of oral-care antimicrobial cetylpyridinium chloride on tyrosine phosphorylation: a potential mechanism for mast cell inhibition assessed via In-Cell Western Bailey West, Jessica Bruno, Michael Wilczek Mentor: Julie Gosse Cetylpyridinium chloride (CPC) is a quaternary ammonium salt antimicrobial found in many oral-care products such as Colgate Total mouthwash and toothpaste. There are no published studies on CPC effects on the immune system; however, the Gosse laboratory recently found that this antibacterial agent, ironically, interferes with the functioning of the mammalian immune cell type mast cells. Mast cells are dispersed throughout human tissues and are involved in many diseases and physiological processes. Mast cell degranulation occurs following a cascade of tyrosine phosphorylation events of various enzymes which contain charged protein motifs such as Src homology 2 (SH2) domains. We hypothesize that the positively charged CPC may interfere electrostatically with these enzymes. To analyze CPC effects on the tyrosine phosphorylation cascade, we will utilize an In-Cell Western (ICW) assay which is a high-throughput alternative to Western blotting. We are developing an In-Cell Western assay for use in rat basophilic leukemia (RBL-2H3) mast cells using an anti-phosphotyrosine primary antibody. Optimization of these experimental details will ultimately allow us to determine whether CPC interferes with early tyrosine phosphorylation events in the mast cell degranulation pathway, shedding light on the mechanism by which this popular antimicrobial inhibits mast cell function.

1017. Genetic Sequencing of CYP2C19 and CFTR Mutations and Their Application to Personalized Medicine
Undergraduate Presentation.
Author(s): Rebecca Collins, Aiden Pike, Claire Bourett, Claire Nowak, Michaela Gervais, Paige Giffault Mentor(s): Benjamin King
Abstract: Whole genome sequencing allows for the development of individual drug therapies for those resistant to standard treatment, in a practice known as personalized medicine. Personalized medicine based on an individual’s DNA can be particularly useful for those with genetic diseases like cystic fibrosis (CF). The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein responsible for transporting chloride ions across epithelial cell membranes. Mutations in the CFTR gene can lead to symptoms associated with CF. CYP2C19 is a cytochrome P-450 enzyme responsible for metabolizing at least 10% of pharmaceuticals. Mutations in this gene can alter drug metabolism capabilities, including those used to treat CF. This research analyzed patient genomic DNA for mutations in the CYP2C19 enzyme, and the dF508 and M470V mutations in the CFTR protein. DNA was extracted from the sputum of a diverse group of 16 people at Dartmouth; the samples underwent PCR, a restriction enzyme digest, and Sanger sequencing. Data analysis showed no indication of the dF508 mutation in patient sampling; however, the M470V mutation appeared in at least one allele for each patient. For CYP2C19, results were more varied, including mutated and unmutated genes. These results suggest that genetic mutations, even within a small sample size, are common. On this basis, it is advisable that whole genome sequencing and personalized medicine become more widely available and affordable. Future research should be done into other mutations that may cause drug resistance and an experiment with a larger sample size to assess the greater need for personalized medicine.

1018. Evaluation of the Changes of Nerve Function Following a Sciatic Nerve Crush in CD137 Ligand Knockout Mice Using Electromyography Measures
Graduate Presentation.
Author(s): Rebecca Peters Mentor(s): Dr. Ling Cao
Abstract: The sciatic nerve crush (SNC) model is a widely used rodent model for neuropathic pain and studying nerve regeneration following peripheral nerve injury. Our lab is currently investigating the role of CD137 ligand (CD137L) in the peripheral nerve injury-induced neuropathic pain-like behaviors using SNC. The long term goal is to identify drug targets for treating peripheral neuropathies. Behaviorally, CD137L knockout (KO) mice have shown reproducible reduction of mechanical hypersensitivity and faster sensory and motor functional recovery following SNC compared to wild type (WT) C57BL/6 mice. To obtain an objective, quantitative assessment of SNC-induced neuropathy that will be used to further delineate the role of CD137Lin this process, we have begun testing with an electromyography measure along the sciatic nerve. Using the UltraPro S100 from Natus Neurology we have established a working protocol and will start to conduct preliminary measurement with CDL137L KO mice following both SNC and sham surgery. Both CD137L KO naive mice and CD137L positive mice will be included as controls. Nerve conduction velocity will be determined by stimulating at both proximal and distal to the crush site. Both response latency and maximal combined action potential will be recorded. Measurements will be taken before surgery, and at days 1, 3, 7, 10, 14, 17, 21, 28, 35, 42, 49, 56, 63, 70 and 77, which are the same sampling times as our previous behavioral tests. We expect to observe a reversal or reduction of SNC-induced decrease in nerve function in CD137L KO mice compared to CD137 positive mice.

1019. Macrophage Response to Bone Marrow Adipocyte Expansion in C57BL/6J Mice
Graduate Presentation.
Author(s): Samantha Costa, Gisela Pachon-Pena Mentor(s): Dr. Clifford Rosen
Abstract: Adipose expansion is a characteristic feature of aging as well as obesity, but little is known how adipocyte expansion effects immune cell function within the bone marrow (BM). BM adipocytes secrete adipokines, such as adiponectin, leptin, resistin, and IL-6 that regulate immune response and inflammation. Within the BM, macrophages play a critical role in the resolution of inflammation through phagocytosis and immunomodulation. In this study, 8-week-old male C57BL/6J mice were fed a high fat diet (HFD, 60 kcal% fat) or chow diet (control) for 12 weeks. PIXImus body composition scans revealed HFD males had significantly more fat mass and percent fat than the controls (p<0.0001 and p<0.0001, respectively). Glucose tolerance testing showed HFD caused increased fasting blood glucose levels and a significant decrease in the overall glucose handling (p<0.05). Femoral microCT analysis showed no significant changes in trabecular and cortical bone parameters in the HFD males. Gene expression of BM adipocytes revealed significant increases in TNF-alpha and IL-6. Macrophage specific genes, SEMA3E/PLEXID1 and F4/80 were also significantly increased which indicated macrophage migration and infiltration to the BM (p<0.05 and p<0.001, respectively). Interestingly, we observed a significant decrease in resisitn gene expression from the BM adipocytes (p<0.01). This may be due to resistin activating the transcription of pro-inflammatory cytokine genes, resulting in the observed increase in TNF-alpha. Our data suggests BM adipose expansion increases macrophage infiltration in this specific niche. Further analysis is needed to understand how adipocyte expansion affects macrophage function and if macrophage pliability may provoke BM tissue inflammation.

1020. Antioxidant Effects of Winterberry Leaf Extract
Undergraduate Presentation.
Author(s): Cara McKinnon, Brendan Moline, Weaam Al Hallaf, Dr. Brian Perkins, Dr. Sam W. Caito Mentor(s): Jennifer Newell-Caito
Abstract: The holly plant genus Ilex contains 15 species native to North America. Native American tribes in the Northeast have historically used Ilex verticillata or winterberry leaves as a medicinal tea. For that reason, it was hypothesized that these winterberry leaves may have antioxidant properties. Polyphenols are secondary metabolites in plants which have been recently explored as antioxidants. Epidemiological studies have shown that long-term consumption of diets rich in plant polyphenols offer protection against development of cancers, cardiovascular diseases, diabetes, osteoporosis, and neurodegenerative disease. A polyphenolic extract was created by drying and distilling the winterberry leaves and the total phenolic content (TPC) was determined to be 524.45 ± 1.82 μg gallic/mL of extract. Using C. elegans as an in vivo model organism, a survival curve using the extract yielded an LD50 of 5.87%. Using this data, worms were pre- treated with 1/10%, 1%, and 5% extract and then the known oxidants manganese(II) chloride (MnCl2) and jugulone. The antioxidant effect of the extract was examined by measuring total reactive oxygen species (ROS). In the presence of the extract, there was a two-fold dose- dependent decrease in the total ROS generated with MnCl2, and a significant reduction in total ROS generated in the jugulone treatments. These are the first results suggest that the polyphenolic compounds in winterberry leaves act as antioxidants in vivo.

1021. Role of Non-Coding RNAs in Adaptation to Environmental Stressors Through the Study of Natural Populations
Graduate Presentation.
Author(s): Kayla Barton, Andrienne Kovach, Brian Olsen Mentor(s): Dr. Benjamin King 
Abstract: The long-term goal of our research is to understand the genomic architecture of adaptation to environmental stressors. The ability to maintain cellular homeostasis under a spectrum of stressors is critical to survival. Tidal marsh sparrow species have adapted to live in a harsh environment relative to inland species. We are studying three tidal marsh and three inland sparrow species to determine which genes and pathways are under selection in tidal marsh populations using comparative genomics, population genomics, and transcriptomics. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression of target genes. The set of miRNAs in animals is proportional to organismal complexity. As such, they can be used as characters to study evolution. Prior studies have examined the differences in miRNAs between broad ranges of taxa. We aim to determine whether the sets of miRNAs differ among a group of closely related species, allowing us to better understand the contemporary evolution of these species. In addition we will study sequence variation among individuals in two populations of introgressed Saltmarsh and Nelson’s sparrows to look for evidence of selection in miRNA loci. This will be done by opportunistically sampling sparrow chicks, extracting RNA, sequencing small RNA, annotating miRNAs, analyzing sequence variation, and comparing miRNA between species. To our knowledge, this would be the first study to examine the evolution of miRNAs among these species.

1022. Investigation of the Extracellular Matrix in DMD Zebrafish Under Inactive and Endurance Exercise Treatments
Graduate Presentation.
Author(s): Kodey Silknitter, Kodey Silknitter, Elisabeth Kilroy, Benjamin King, Clarissa Henry Mentor(s): Dr. Clarissa Henry
Abstract: Duchenne Muscular Dystrophy (DMD) is a debilitating neuromuscular disease that affects roughly 250,000 people globally. This disease arises from mutations in the dystrophin gene, which produces a protein responsible for linking actin filaments to the extracellular matrix (ECM). Individuals diagnosed with DMD present muscle wasting and face few treatment options to combat the disease, being advised to not exercise and stay inactive. While it is known that, in healthy individuals, inactivity is damaging to muscle structure and health, less is known for patients with DMD. We investigated the role of inactivity and exercise (eNMES) in a zebrafish model that presents the DMD phenotype. This study specifically looks at the impacts inactivity and eNMES have on the ECM. We hypothesize that zebrafish that undergo inactivity treatment will show poorer ECM structure and homeostasis than their eNMES counterparts. To investigate this, we used a combination of bioinformatic and molecular techniques. Using RNA-sequencing of both wild-type and DMD mutant zebrafish under both inactive and eNMES conditions, we noticed differential regulation in several genes associated with the ECM. Concurrently, we are also using immunohistochemical staining and high-resolution, confocal imaging to look at muscle and ECM structure of both wild-type and DMD mutant zebrafish under either inactive or eNMES conditions. We expect to find that wildtype and DMD zebrafish that have undergone inactivity treatment will have poorer muscle and ECM structure than their eNMES counterparts. These findings will broaden the field of knowledge of DMD and could potentially lead to future therapies.

1023. Investigating the Presence of Putative RNA Thermometer-like Structures in the Zebrafish (Danio rerio) Mitochondrial Genome.
Undergraduate Presentation.
Author(s): Abigail Muscat Mentor(s): Dr. Nishad Jayasundara
Abstract: Bacterial RNA thermometers (RNATs) help to regulate the creation of heat-shock protein (Hsps), cold-shock proteins, and virulence factors in bacteria. These RNATs are typically found upstream, in the 5’-UTR (5’-untranslated region) of heat shock genes in mRNAs. Two common groups of bacterial RNATs are ROSE (repressor of heat‐shock gene expression) and fourU elements. Described as hairpin structures, with both zipper and switch-like compositions, these RNATs are vital in combating cellular damage caused by drastic temperature changes and in maintaining cellular homeostasis. In eukaryotes, small heat shock proteins have been suggested to aid the mitochondria by providing cardio-protection and protection against cellular injury, oxidative stress, and pathogens. However, these proteins are not in the mitochondria and are instead transported from the nucleus. Since evolutionarily mitochondria originated in bacteria it is possible that thermometer-like structures were retained in the mitochondrial genomes. This project aims to investigate whether thermometer-like structures can be found in the D. rerio mitochondrial genome. NCBI database searches and BLAST analyses were used for characterizing sequence similarity of existing bacterial RNATs and for comparing structures to the D. rerio mitochondrial genome to identify sequence homology. If structures are found in the mitochondria, the next step will be to investigate downstream mitochondrial genes and look for any indication of functional significance.

1024.  Description of A Novel CRISPR Vertebrate Model of Dolichyl-Phosphate Mannosyltransferase Subunit 3-Associated Secondary Dystroglycanopathies
Undergraduate Presentation.
Author(s): Claire Schaffer Mentor(s): Clarissa Henry
Abstract: Muscular dystrophies are a devastating group of genetic diseases that cause muscle wasting and weakness in patients. A subset of these, secondary dystroglycanopathies, occur when there are defects in the glycosylation, or sugar-residue modification, of α-dystroglycan (αDG). Α-DG is a transmembrane protein essential in anchoring muscle tissue. Glycoside chains extending off the sides of αDG allow it to interact and bind with laminin in the extracellular matrix. This association is integral in enabling the muscle to generate force without tearing. Dolichyl-phosphate mannosyltransferase 3 (DPM3) is a protein within the glycosylation pathway. When DPM3 is mutated, it cannot perform its job properly and carriers of these mutations can develop muscular dystrophy. DPM3 mutations, and secondary dystroglycanopathies in general, are an understudied and mysterious group of diseases. They vary in expression and severity, but there is no cure. The Henry lab has developed a line of CRISPR-Cas9 modified zebrafish to model DPM3-related secondary dystroglycanopathies. We were able to uncover structural and functional changed between the wild type and mutant fish through several metrics. Birefringence microscopy let us follow disease progression over time, phalloidin imaging allowed us to look at muscle fibers, and movement tracking allowed us to quantify and compare average muscle usage between the groups. Combined with sequencing data, we were able to develop a clear picture of our DPM3 mutant zebrafish phenotype. Going forward, we will use this model to pursue future studies to test therapies for DPM3-related secondary dystroglycanopathies that will support the damaged tissue at the molecular level.

1025. Investigating the Cell-Type Dependent Host Signaling Mechanisms of JC Polyomavirus
Undergraduate Presentation.
Author(s): Remi Geohegan, Michael Wilczek Mentor(s): Melissa Maginnis
Abstract: Characterizing the host genes needed for viral replication is essential to the study of infection and disease progression. JC polyomavirus (JCPyV) causes a persistent, asymptomatic infection of the kidneys in up to 80% of the population. In immunocompromised individuals the virus can reactivate, traffic to the brain, and cause progressive multifocal leukoencephalopathy (PML), a disease that destroys glial cells and is fatal. JCPyV only infects human cells, limiting research of PML to cell culture glial cell models. My project investigates cell signaling mechanisms of JCPyV in primary normal human astrocytes (NHAs) compared to immortalized cells. JCPyV activates extracellular regulated kinases (ERKs) within the mitogen activated protein kinase (MAPK) pathway, a cell signaling pathway exploited by viruses for its role in cellular differentiation and proliferation. Research from the Maginnis laboratory demonstrated that during JCPyV infection, ERK was activated early in immortalized cells, however preliminary evidence suggests that ERK activation was delayed in NHAs. The goal of this project was to further validate these findings, quantifying ERK protein expression with increasing JCPyV infection and through infectivity assays, following the treatment of chemical activators to ERK. Preliminary data suggests that increasing JCPyV infection may not enhance ERK activation in NHAs. Additionally, upregulating ERK using chemical activators may influence JCPyV infection in NHAs compared to infection in immortalized cells. This data corroborates initial findings; during JCPyV infection of NHAs the MAPK-ERK pathway is regulated and exploited differently in NHAs providing a deeper understanding of how JCPyV reprograms these cells and results in the fatal disease.

1026. Surface Acoustic Waves for Manipulation of Fluid and Biomolecules
Graduate Presentation.
Author(s): Joel Tewksbury, Caitlin Howell, Mauricio Pereira da Cunha Mentor(s): Dr. Caitlin Howell
Abstract: Small biomolecules can be challenging and expensive to isolate and manipulate, but they may be the key for developing more efficient diagnostic tools for pancreatic cancer. Pancreatic cancer has a survival rate of less than 5% five years after diagnosis. For this reason, the research and establishment of cheap, effective screening tools are highly desired. One promising method of screening is separating and identifying biomarkers in a patient’s blood that are indicative of pancreatic cancer. The purpose of this project is to develop a system using surface acoustic waves (SAWs), in conjugation with microfluidics and surface functionalization, to separate and manipulate small biomolecules in blood. The separation and manipulation are achieved by passing the targeted fluidic material, in this case blood, through a microfluidic channel on top of a SAW device. The SAWs are generated by using an array of interdigital transducers (IDTs) photolithographically printed on a piezoelectric crystal to convert the electrical signal into acoustic waves. The SAW propagates, guided at the top surface of the material, and interacts with the fluid, exerting a force on the fluid and on particles in solution. The possibility of using open microfluidics to manipulated fluid droplets as an alternative to a continuous flow stream in a microfluidic channel was also explored. The surface was functionalized using a fluorinated silane and coated with a fluorinated oil to create a SLIP surface for easy droplet manipulation. The results of implementing this procedure showed significant dewetting of the oil layer, but despite this when properly coated did provide a surface hydrophobic enough to allow for easy droplet sliding, and thus easy manipulation of droplets on the surface. This procedure combined with a heat source allows for concentration of particles at specific locations for easier particle detection. The development of the device discussed is expected to yield a new and consistent approach to pancreatic cancer screening methods that is both compact and extremely sensitive to facilitate the detection of pancreatic cancer long before symptoms appear.

1027. Characterizing the role of IRS1/2 in osteocyte differentiation and function in bone development
Graduate Presentation.
Author(s): Vivin Karthik, Victoria Van Berlo, Li Tian, Jennifer Daruszka, Victoria DeMambro Mentor(s): Anyonya Gunur
Abstract: Osteocytes are the most abundant cell type in skeletal bone and are present in the bone matrix. They form a network for sensing mechanical cues coming from the bone remodeling units which involve both the osteoblasts and osteoclasts. Osteocytes have been studied in the context of mechano-transduction biology and signaling, there are very few studies elucidating the role of osteocytes in metabolic homeostasis and bioenergetics. Insulin receptor substrate proteins 1 and 2 (IRS1/2), are scaffolding proteins involved in insulin and insulin growth factor 1 (IGF1) signaling necessary for the anabolic regulation of bone. In this study, we aim to delineate the role of IRS1/2 in osteocyte metabolism and bioenergetics by knocking out IRS1/2 in osteocytes in mice using osteocyte-specific Dentin Matrix Protein (DMP1) Cre. Preliminary results through dual X-ray absorptiometry to measure bone mineral content in 9-week-old mice show that Cre-specific males and females have reduced areal and femoral bone mineral density (BMD) compared to wildtype (wt) littermates. However, there were no changes in the fat mass and lean mass parameters. Micro CT analysis of aged mice that measure trabecular bone, cortical bone, and mechanical strength parameters show that Cre-specific males and females have reduced BMD, trabecular thickness, cortical area, cortical thickness, and polar moment of inertia (pMOI) compared to wildtype (wt) littermates. Future studies would evaluate the bioenergetic profile of the IRS1/2 knockout osteocytes in vitro by analyzing the metabolic flux of glycolysis and oxidative phosphorylation, in conjunction with identifying the genes involved in metabolism in the insulin signaling pathway of osteocytes.

1028. Design of Theoretical Modeling to Support a Regenerative Radiation Shield for Deep-Space Exploration
Undergraduate Presentation.
Author(s): Benjamin Chasse, Ainslie Allen, Damon Williams, Riley Drummond Mentor(s): Caitlin Howell
Abstract: Radiation shielding is an essential consideration for human deep-space exploration, as prolonged exposure serious threats to critical human-physiological processes. Current solutions include the use of materials like polyethylene to scatter cosmic radiation; however, these materials have a restricted lifetime as an effective radiation shield. To address this issue, research shows that the fungus Cladosporium sphaerospermum can act as a living and regenerative protective layer, capable of absorbing harmful radiation in a deep-space environment. Yet to create a continuous growth system, C. sphaerospermum would need a continuous supply of nutrients. In this project, we theoretically optimize such a system, building on our previously-developed vascularized networks. Optimization of the vascular system was performed in Solidworks and COMSOL to generate predictive models for rapid experimentation. The minimum nutrient delivery rate for sustaining C. sphaerospermum growth was determined to be 1.11*10-9 mol*mm-2*s-1 and the minimum surface-level concentration was found to be 1.1*10-4 mol/m3. To meet these needs and minimize wasteful diffusion of nutrients, the geometric configurations of the vascular channels and the concentration of the nutrient diffused from the channels were controlled. By adjusting these two variables, many theoretical models were generated and compared, and the best performing model was selected on its ability to sustain only the minimum livable conditions for C. sphaerospermum. Thus, with an optimized model of nutrient delivery, this work has set the foundation for a living and regenerative radiation shield to protect astronauts in deep-space exploration.

1029. Caloric Restriction and Fasting Diets have Negligible, or Potentially Damaging, Effects on Cognition in Diversity Outbred Mice
Graduate Presentation.
Author(s): Andrew Ouellette, Niran Hadad, Andrew Deighan, Kristen O’Connell, Adam Freund, Gary Churchill Mentor(s): Catherine Kaczorowski
Abstract: Aging remains the greatest risk factor for developing Alzheimer’s disease (AD). Given the projected increase in lifespan across the globe, the risk of developing AD is expected to increase, along with significant economic and social burden. As such, the demand for interventions that increase cognitive longevity in parallel with lifespan is high. Several studies have reported that caloric-restriction (CR) or fasting increase lifespan and improve cognition in humans and models of aging, but others have reported no beneficial effect of CR on cognition. These discrepancies may be explained by uncontrolled environmental factors inherent in large human studies, and the lack of genetic diversity in previous animal studies where effects of CR or fasting on cognitive longevity studies in one inbred strain of mice may not generalize to other mouse strains, let alone to humans. In this study, we compare the effects of Ad Lib, 1 day fast, 2 day fast, 20% CR and 40% CR on Y-maze working memory and contextual fear memory (CFM) in a genetically diverse population of mice – the Diversity Outbred (DO). We observed no benefit of CR or fasting on working memory or CFM at 24 months, despite expected increase in %live mice at 24 months. Actually, the 40% CR group exhibited impaired long-term CFM compared to groups fasted for 1 and 2 days. These results emphasize the importance of identifying therapeutics to enhance cognitive longevity, as CR enhancement of lifespan may not generalize to cognitive abilities.

1030. Rapid Local Anesthetic Warmer
Undergraduate Presentation.
Author(s): Benjamin Chasse, Joshua Hamilton, Nathan St. Jean, Omar Alsamam Mentor(s): Dr. Michael Mason
Abstract: The quality of patient care is obstructed by the pain post-injection of local anesthetics. Healthcare providers have attempted to resolve this issue by mixing the anesthetics with sodium bicarbonate, but it has been shown through clinical research that injection discomfort can be further reduced by warming the liquid to approximately 103 °F. The purpose of this work is to address the need for a rapid local anesthetic warmer so that healthcare providers may further decrease patient discomfort. To do this, we have developed plans for the construction of a rapid local anesthetic warmer, and plan to validate its function with three primary experiments. The first experiment’s purpose is to demonstrate the precision and accuracy of temperature control within the anesthetic vial to reach 103 °F, which will be done by generating a calibration curve and comparing the internal and external temperatures of the vial during heating and cooling. The second experiment will assess the container’s thermal stability by studying the deformation of the 3D printed filament. The final experiment will demonstrate the reliability of the system controlled by pre-programmed user inputs. Preset heating cycles will be programmed into the microcontroller of the system, and their accuracy in reaching the desired temperature of 103 °F will be assessed. The results of these tests will provide evidence of its efficacy as a rapid local anesthetic warmer for applications in a healthcare setting to reduce discomfort in patients and improve quality of care.

1031. The Crichton Mouse: A New Model for ADHD Derived From ENU-Mutagenesis Screen
Graduate Presentation.
Author(s): Michayla Moore, Yehya Barakat, Michayla Moore, Vivek Kumar Mentor(s): Vivek Kumar
Abstract: Animal models studied within the field of neuropsychiatric research have led to significant advancements in understanding the pathological changes driving many behavioral disorders and has been crucial in the development of new treatments. Despite the high heritability and growing prevalence of Attention-Deficit/Hyperactivity disorder (ADHD), gaps remain in the biological pathways underlying ADHD and the current availability of relevant pre-clinical models are lacking [1, 2]. A polygenic mouse model was developed in our lab through a N -ethyl- N -nitrosourea (ENU) mutagenesis screen selected for hyperactivity that develops in an age-dependent manner and is a mirrored clinical phenotype seen in patients diagnosed with ADHD [3, 4]. This mouse model, newly identified as the Crichton model, develops increased hyperactivity in the Open Field assay measured by total distance traveled and peaks at ~28 weeks of age. Key features identified in clinical studies of ADHD include dysregulation of specific brain regions such as the pre-frontal cortex, cerebellum, and striatum [5]. With this, we investigated neuronal changes within these three regions of Crichton brains compared to wild-type using immunohistochemistry as well as performed RNA-seq to assess transcriptional differences. Together, we found significant neuronal loss in the pre-frontal cortex of the Crichton mice compared to wild-type confirmed by anti-NeuN staining and significant transcriptional differences within these regions identified by RNA-seq. We did not see significant differences in either assay within the cerebellum or striatal regions. These findings provide additional evidence for pathological changes leading to hyperactivity phenotypes as well as introduces the Crichton mouse line as a new model to study the distinct molecular mechanisms involved in ADHD.

1032. Understanding the role of prophage encoded polymorphic toxins in mycobacterial superinfection immunity and drug resistance
Undergraduate Presentation.
Author(s): Dakota Archambault, Jaycee Cushman, Maddie Kimble, Sarah MacCallister, JC Ross, Colin Welch Mentor(s): Dr. Sally Molloy
Abstract: Mycobacterium abscessus is the leading cause of lung infection in cystic fibrosis patients and is often resistant to all antibiotics. There is an urgent need for alternative therapies such as lytic bacteriophage (viruses that infect bacteria). Phage therapy has been successful in the treatment of a drug-resistant M. abscessus infection in a teenaged cystic fibrosis patient but there are challenges to broad use as most M. abscessus isolates are highly resistant to lytic bacteriophage infection. Prophage, or integrated bacteriophage genomes within the M. abscessus genome, likely defend against phage superinfection via prophage-encoded viral defense systems. The Molloy lab has shown that the Mab cluster R prophage McProf increases bacterial resistance to both bacteriophage superinfection and antibiotics. To better understand the role of cluster R prophage in antibiotic resistance and superinfection immunity, we identified and characterized 25 novel cluster R genomes in sequenced clinical M. abscessus isolates. These strains encod a type VII secreted polymorphic toxin (PT) system that we hypothesize play a role in drug resistance and superinfection immunity. The PT cassettes include a small ESXA-like protein, a large PT with a WXG100 motif and a cognate immunity protein. There are two types of PT systems represented in the cluster R genomes: a Tde1-like DNAse and a second PT with no recognizable toxin motif. To determine the role of PT in superinfection immunity and drug resistance, I will construct an expression plasmid that encodes the second PT and test its effect on superinfection immunity and drug resistance in pathogenic mycobacteria.

1033. Progression of Cardiomyogenesis from Embryonic Stem Cells in a Three-Dimensional gel Matrix
Graduate Presentation.
Author(s): Jonathan Bomar Mentor(s): Rosemary Smith
Abstract: Cardiovascular disease (CVD) accounts for about one in three deaths in the United States. Traditional therapies aim to minimize damage caused by CVD, but do little to address the loss of healthy heart tissue following acute injury. Engineered cardiac tissue has emerged as a promising therapy for cardiac tissue repair. In this project, HM1 mouse embryonic stem cells suspended in a three-dimensional gel matrix are differentiated in vitro into functional cardiac tissue with a highly reproducible, complex spatial cooperation of multiple cardiac cell types. A field of proliferative fibroblasts grows out from the cell-seeded matrix onto a glass substrate at approximately day 3 of differentiation. A spontaneously contracting network of myosin heavy chain (MHC)-positive cardiomyocytes self-organizes on top of the fibroblasts directly outside the original matrix at approximately day 10.5. This self-organization holds the key for the mechanism behind in vitro cardiomyogenesis. We hypothesize that boundary cues and cell-cell interactions signal the organization and outgrowth of fibroblasts from the matrix onto the glass substrate, followed by cardiomyocyte precursors which require the fibroblast outgrowth in order to further differentiate. Short term specific aims are to map the spatiotemporal development of cell types in the tissue, identifying self-organization induced by boundary conditions. Long term goals are to use these mechanisms of in vitro cardiomyogenesis to engineer cardiac tissue for in vitro drug testing and in vivo cardiac tissue grafts.

1034. Analyzing and Characterizing the Strictly Lytic Infection of BPsΔ33 in Mycobacterium chelonae
Undergraduate Presentation.
Author(s): Matthew Cox, Jaycee Cushman, Sarah McCallister, Emma Freeman, Maddie Kimble, Colin Welch Mentor(s): Sally Molloy, Benjamin King
Abstract: Mycobacterium abscessus is a major causative agent of soft tissue and lung infection, especially in cystic fibrosis patients, and are oftentimes multi-drug resistant. Prophage, bacteriophage integrated into the host genome, play a role in antibiotic resistance in many pathogens, but the mechanisms by which this occurs are still unknown. Understanding the mechanisms of multidrug resistance will aid in the development of new treatments, such as improved drug regimens and/or phage therapy. The Molloy Lab recently demonstrated that two co-habitating prophage, McProf and BPs, increase antibiotic resistance and expression of antibiotic resistance genes relative to strains that carry only one or no prophage. We hypothesize that induction of the BPs prophage and lytic gene expression activates genes in the second prophage that leads to changes in downstream gene expression and antibiotic resistance. To better understand how BPs lytic gene expression interacts with the second prophage, we aim to determine the temporal lytic gene expression profile of a strictly lytic mutant of BPS (BPsΔ33) during early, intermediate, and late phases of lytic infection of M. chelonae.

1036. Development of Image Collection Apparatus for Color Band Analysis
Undergraduate Presentation.
Author(s): Theo Erikson, Alia Parsons, Oisin Biswas, Raymond Perry, Caitlin Howell Mentor(s): Caitlin Howell
Abstract: With the spread of the SARS-CoV-2 virus, attention to the rapid assessment of surface cleanliness has intensified. Current monitoring methods of surfaces are both lengthy and expensive; however, nanostructured materials have recently garnered interest in quickly assessing the state of a surface. Structural color, or the bands of color that appear as different wavelengths of light reflected off a textured surface varies as a surface becomes contaminated with dust, dirt, or oil. To quantitatively analyze this phenomenon, a tri-axially rotating apparatus was constructed to dynamically measure the change in the structural color of samples that were either clean or contaminated. This apparatus, also known as TARDIS (Tri-axial Apparatus for the Rotation of Discrete and Independent Samples) is made of mostly 3D printed parts. It uses a phone camera to capture images and an Arduino to run three stepper motors to manipulate each of the three axes. The nanostructured material sample rotates on a platform exactly at the height of each arm’s center of rotation. Both the camera and LED are centered about the sample and rotate with a set radius around said sample. This permits the generation of the hundreds of thousands of images at different angles required to analyze the structural color reflected off nanostructured material in an effort to detect the degree of contamination. The use of nanostructured surfaces and light to detect contamination has the potential to help slow the spread of the SARS-CoV-2 virus and other infectious materials via commonly used surfaces by providing a low-cost, timely detection method of surface contamination.

1037. Developing New Skills and Approaches to Authentic Research Experiences During COVID-19
Undergraduate Presentation
Author(s): Jacob Cote Mentor(s): Sally Molloy
Abstract: Providing authentic student research experiences during COVID-19 presents a challenge for mentors and their learners in both the classroom and the research lab. The year-long UMaine SEA-PHAGES course typically supports 60 students in the isolation and characterization of novel bacteriophages, viruses that infect bacteria, using wet bench and bioinformatic approaches. The effects of COVID-19 on student safety inspired a novel research component for the 2020/2021 UMaine SEA-PHAGES cohort that could be conducted safely in the remote classroom. This year, students identified and characterized prophages, integrated viral genomes, in clinical strains of the drug-resistant pathogen Mycobacterium abscessus. For my CUGR summer research project in the Molloy lab, we collaborated with the Hatfull laboratory to develop new methods of prophage genome extraction for the new SEA-PHAGES research component. The outcomes of this research were the successful adaptation of the research component to the UMaine SEA PHAGES class and the development of my independent research project, characterizing a novel group of M. abscessus prophages that belong to the Mab cluster R. The Molloy lab has demonstrated that the presence of prophages, or bacteriophage genomes integrated into bacterial genomes, increases antibiotic resistance in pathogenic mycobacteria. Recent studies in our lab have shown that prophage-prophage interactions may be a novel mechanism of antibiotic resistance. Understanding the role prophages play in this resistance hinges upon deepening our understanding of the prophage communities in clinical bacterial isolates.

1038. An allelic series of spontaneous mutations in Rorb causes a gait phenotype, retinal abnormality, and gene expression changes relevant to epilepsy, bipolar and autism spectrum disorders. 
Graduate Presentation.
Author(s): George Murray, Robert W. Burgess, Abigail Tadenev Mentor(s): Abigail Tadenev
Abstract: Mutations in RAR-related orphan receptor b (Rorb) cause a characteristic “high-stepper” phenotype in mice arising from dysfunction of interneurons in laminae III, V, and VI of the dorsal horn of the spinal cord. Loss of rods and changes in cone photoreceptor abundance in the retina have also been reported in mice carrying Rorb null alleles. More recently, a critical role for Rorb in the formation of cortical barrels of the somatosensory cortex and thalamocortical afferent (TCA) projections was described. There are several clinical reports of RORB variants segregating with epilepsy, often with comorbid conditions such as intellectual disability and affective disorders. Here we describe five spontaneous mutations in Rorb identified at The Jackson Laboratory due to the overt gait phenotype that they cause in mice. These mutations affect different domains and splice variants of Rorb, which may explain the spectrum of anatomical and physiological abnormalities in these mice. Gene expression analysis in the most severely affected mutants implicates pathways associated with development and nervous system function and indicates decreased expression of genes involved in molecular circuits whose components are strongly associated with epilepsy, bipolar and autism spectrum disorders (ASD). These findings support the role of Rorb in nervous system development, provide further evidence for an association between RORB and epilepsy, and describe a group of Rorb mutant mice that might be useful to researchers interested in cortical development and animal models of transcriptomic shifts related to those occurring in neurological conditions such as epilepsy, bipolar disorder, and ASD.

1039. Red raspberry consumption counteracts pathological conversion of perivascular adipose tissue in rat model of metabolic syndrome.
Graduate Presentation
Author(s): Marissa McGilvrey, Natalie VandenAkker, Kimberly Malka, Penny Clum, Lucy Liaw, Dorothy Klimis-Zacas Mentor(s): Dorothy Klimis-Zacas
Abstract: Metabolic syndrome (MetS) is a collection of physiological and metabolic abnormalities that increase risk of obesity, type-2 diabetes and cardiovascular disease (CVD). In these pathologies, systemic inflammation initiates a signaling cascade between the vasculature, adipose tissue, liver, and other organs. The local vascular environment is heavily influenced by perivascular adipose tissue (PVAT) by secreting metabolically active molecules to either support vasorelaxation or promote pathological pro-contractile vascular activity, which plays a critical role in progression of CVD. As an alternative to traditional pharmacotherapy strategies to reduce inflammation, diets enriched in bioactive-rich plant foods, like raspberries and blueberries, are increasingly being used to prevent or reverse development of MetS and related pathologies. To study red raspberry (RR) enriched diets in MetS, male obese Zucker rats (OZR) containing homozygous leptin receptor mutation, and their lean littermates (LZR) were placed on a control (C) or 8% w/w RR-enriched diet for 8 weeks. Results published by VandenAkker et al documented that RR-enriched diet restores impaired vascular tone of OZR by enhancing vasoconstriction and attenuating vasorelaxation through eNOS and COX-2 pathways; and dramatic improvement of hepatic lipid deposition in OZR on RR diet. This study builds on these previous findings by evaluating the associated thoracic PVAT of these animals. Molecular evaluation of PVAT shows obesity induced increases in eNOS, as well as markers of signaling, inflammation, and adipocyte proliferation, are attenuated by RR-enriched diet. These results indicate that RR-enriched diets may play an important role in modifying PVAT to influence progression of CVD in metabolic syndrome.

1040. Characterizing neuropathy phenotypes and gene expression changes caused by peripheral myelin protein 22 overexpression in mouse models of Charcot-Marie-Tooth Disease 1A
Graduate Presentation
Author(s): Audrie Langlais, George C. Murray, Timothy Hines, Abby Tadenev, Kathy Miers, Courtney Hatton Mentor(s): Robert Burgess
Abstract: Charcot-Marie-Tooth Disease (CMT) is a heterogeneous group of inherited peripheral neuropathies affecting approximately 1 in 2,500 individuals. CMT1A, which comprises 30% of all CMT cases, is caused by a duplication in human chromosome 17 containing peripheral myelin protein 22 (PMP22). Patients with CMT1A experience a demyelinating neuropathy characterized by distal muscle weakness and reduced fine motor coordination and proprioception. PMP22 is expressed by myelinating Schwann cells, where its overexpression impairs axon myelination and causes regulatory changes in the EGR2/SOX10 coexpression gene network. Our lab is investigating points of therapeutic intervention within this network that may modify neuropathy using validated transgenic mouse models of CMT1A including C22-PMP22 mice, with high expression of PMP2, and C3-PMP22 mice with lower PMP22 expression, albeit higher compared to wild-type. In this study, we investigated pathological phenotypes and transcriptomic changes associated with myelin and the integrated stress response using electrophysiology, histology and a published RNA-seq data set (Zhao et al, 2018). We observe visible myelin thinning and abnormal nerve conduction in C3-PMP22 mice. Differential expression analysis in C22-PMP22 spinal cord indicates time-dependent differential expression of SOX10/EGR2 targets and other myelination related genes as well as gene sets associated with cellular stress. These findings provide a basis to understand the molecular changes underpinning PMP22 neuropathy, identify potential points of therapeutic intervention, and will enable us to interrogate future RNA sequencing data from C3-PMP22 mice and human GWAS data in a hypothesis driven manner.

1041. Impact of Exercise on Dystrophic Skeletal Muscle
Graduate Presentation
Author(s): Amanda Ignacz, Elisabeth Kilroy, Ahmed Almaghasilah, Kodey Silknitter Mentor(s): Clarissa Henry
Abstract: Duchenne muscular dystrophy (DMD) is a fatal, progressive muscle-wasting disease that affects 1 in 5000 live male births. DMD is caused by mutations, usually deletions, in the gene coding for dystrophin production. Dystrophin acts as a link between the actin cytoskeleton and the extracellular matrix in myocytes. With a lack of functional dystrophin, as observed in DMD patients, muscle becomes more susceptible to damage as the cells contract and relax resulting in muscle degeneration and weakness. In a zebrafish model for this disease (sapje) it has been shown that the dystrophic phenotype can be ameliorated through endurance neuromuscular electrical stimulation (eNMES), providing insight on the effect of exercise on muscle structure and function. In DMD, slow-twitch muscle fibers tend to be more refractory to damage than fast-twitch muscle fibers. The first step in this analysis was assessing if there was a correlation between fast-twitch and slow-twitch muscle fibers per myotome. From this any difference in fiber correlation between eNMES-treated and untreated fish could be determined. This will allow us to deduce whether eNMES plays a role in muscle fiber improvement or degeneration, and if the effect is seen in fast fibers, slow fibers, or both. Through quantitative scoring of myotomes, it was determined that eNMES-treated fast and slow muscle improvement/degeneration correlated less than that in the untreated muscles. This implies that eNMES impacts fast and slow fibers differently, and whether this is due to improvement or degeneration in one fiber type will be assessed in future analysis.

1042. Interferon Response Genes Expressed During the Innate Immune Response to Influenza A Virus
Undergraduate Presentation
Author(s): Haley Foreman, Julianna Grampone, Riley Grindle, Sarah Foust Mentor(s): Benjamin King
Abstract: The Influenza A virus (IAV) is one of four types of Influenza viruses and is the predominant cause of flu pandemics. Changes in the viral genome through antigenic drift and shift continue to pose a significant threat to global health. The objective of our study is to understand innate immune response to IAV by comparing differentially expressed interferon response genes using a larval zebrafish model of IAV infection. Zebrafish larvae are an excellent model for the innate immune system as it is functional by 24-48 hours post fertilization and the adaptive immune system does not develop until 2-4 weeks later. The innate immune response to virus infection includes the interferon pathway. Interferons are proteins secreted by an infected cell that signal to nearby cells to resist further infection. Interferons can induce the expression of interferon response genes (IRGs). The corresponding protein products of these IRGs are responsible for mediating the antiviral response. We analyzed the set of 1,096 genes differentially expressed at 6 hours post IAV infection that were characterized using RNA sequencing. We compared these genes to homologous human interferon responses genes expressed from the Interferome database (http://www.interferome.org) to determine candidate IRGs. We used homology mappings between zebrafish and human genes provided by Ensembl (https://www.ensembl.org). 74 candidate IRGs were found to be up-regulated, and 97 IRGs were found to be down-regulated. Future directions of our research includes validating the expression of the candidate IRGs by quantitative PCR. These studies will expand our knowledge of interferon signaling following IAV infection.

1043. Integrating Molecular Profiles with Clinical Outcomes in Cancer Patients from Rural Maine
Graduate Presentation.
Author(s): Michael J. Babcock, Marek Skacel, Benjamin King Mentor(s): Benjamin King
Abstract: Michael J. Babcock1,2, Marek Skacel2, Benjamin King1 1. Graduate School for Biomedical Sciences and Engineering, University of Maine , Orono, Maine. 2. Dahl-Chase Pathology Associates, Bangor, Maine. Background: Current clinical pathology information systems are not well-structured to incorporate high-throughput genomic data. In addition, patient electronic medical records (EMRs) are ill-equipped for the integration of molecular profiling data forcing clinicians to rely on “paper documents” to support patient management. Furthermore, low resource, rural health care facilities do not have the budget for expensive storage systems, third party software, and/or specialized expertise. However, pathology laboratory information management systems (LIMS) can be queried and results collated with molecular profiles obtained from cancer patient tumors integrated into a separate relational database to support clinical data management. Methodology This pilot study demonstrates how genomic data can be integrated with discrete synoptic pathology reports from a pathology LIMS and EMRs to read, extract, and analyze data. Using the RODBC package, an R script was developed to extract discrete pathology results from an anatomic pathology database (Sunquest PowerPath®). Data was then collated with patient genomic data obtained from a separate novel relational molecular pathology database using SQL. Data analytics can be performed for compliance with the College of American Pathologists (CAP) requirements. Outcomes Results of clinical and overall molecular characteristics of cancer patients from rural Maine can be compared to those reported in the literature. In the future we would like to incorporate genomic data with clinical findings obtained from both the patient EMR and pathology LIMS to support clinical decision making.

1044. The effects of BTS induced inactivity on a zebrafish model of Duchenne Muscular Dystrophy
Graduate Presentation
Author(s): Sean Driscoll Mentor(s): Clarissa Henry
Abstract: Duchenne’s Muscular dystrophy (DMD) is a congenital disease of the muscle characterized by muscle atrophy, weakness, and a lower quality of life. Often diagnosed in children, it affects about 1 in every 5,500-7,700 males. A patient diagnosed with DMD is often told to avoid physical activities outside the required amount needed to go on with their day in order to preserve the muscle fibers and integrity. Inactivity in a healthy person leads to decreased muscle mass and increased weakening of the muscle, so the effects may be exacerbated in a person diagnosed with DMD having already weakened muscle. In this project, we explored the effects of inactivity on a zebrafish model of DMD. We used N-Benzyl-p-toluenesulfonamide (BTS), a myosin heavy chain inhibitor, to induce total inactivity for 72 hours consecutively at disease onset (2 days post fertilization), followed by a 72 hour recovery period out of BTS where normal activity is resumed. We then analyzed the effects on muscle structure (via birefringence and immunohistochemistry staining) and function (via swimming distance and velocity). We found that while muscle structure and function is improved immediately following the period of inactivity, upon resumption of normal activity, muscle structure and function was drastically reduced. This data can be used to help inform patients diagnosed with DMD, and potentially other forms of muscle disease, about the potential hazards of inactivity on the muscle structure and function.

1045. Nociceptive Sensitization in Larval and Adult Drosophila
Graduate Presentation.
Author(s): Christine Hale, Samia Pratt, Julie Moulton, Geoffrey Ganter Mentor(s): Geoffrey Ganter
Abstract: Nociceptive sensitization underlies and perpetuates chronic pain, a condition that affects ~50 million adults nationwide. With many treatment options for chronic pain, such as opioid analgesics, carrying numerous deleterious side effects, research into safer and more effective treatment options is crucial. Recently, a nociceptive sensitization model was developed using Drosophila melanogaster larvae, in which UV-injured or genetically modified animals become hyper or hypo-sensitive. Using this model, the necessity and sufficiency of a novel pathway, the Bone Morphogenetic Protein (BMP) pathway, functioning in the nociceptors, was revealed. We have continued to build upon this knowledge to reveal a more complete mechanism for how nociceptive sensitization occurs by investigating into the potential roles of other novel genes/signaling pathways including, arm, a component within the Wnt/Wg signaling pathway. At present, our findings indicate Arm to be a facilitator in controlling nociceptor sensitivity. In addition to our focus on mechanisms that promote hypersensitivity, we also propose to expand our efforts by bringing new focus to more chronic nociceptor sensitization conditions. To facilitate these efforts, because the larval stages of fruit fly development are relatively brief, we are also developing a methodology that allows longer term experimentation of nociceptive sensitization after injury in adult fruit flies. Recent efforts toward this goal include mapping the adult fruit fly nociceptor distribution by confocal microscopy and developing thermonociception methods for use with adult flies. Our ultimate aim is to better understand the mechanisms of nociceptive sensitization so that better treatment options for chronic pain may be revealed.

1046. The role of cholinergic interneurons in impaired cognitive flexibility in chronic pain
Graduate Presentation.
Author(s): Megan Tomasch, Makaela Rice Mentor(s): Christoph Straub
Abstract: Pain is a multidimensional experience with sensory-descriptive, motivational-affective, and evaluative-cognitive components. The impairments in cognitive functions associated with chronic pain contribute to the diseases disabling impact, but, despite their clinical importance, the mechanisms underlying these non-sensory aspects remain largely unexplored. One major cognitive impairment associated with chronic pain is decreased cognitive flexibility, i.e., an impaired ability to update the decision-making process in response to changes in the environment. Chronic pain is marked by structural and functional changes in prefrontal cortex (PFC) and nucleus accumbens (NAc), brain areas that are also implicated in cognitive flexibility. Within striatal tissue (including NAc), levels of the neuromodulator acetylcholine (ACh) are inversely correlated with cognitive flexibility both in humans and animal models. I hypothesize that chronic pain alters PFC-NAc signaling to increase ACh levels in the NAc, thereby decreasing cognitive flexibility and contributing to impaired cognitive function. To test this hypothesis, we use an established mouse model of chronic, the spared nerve injury (SNI). In agreement with previously published work, we find that SNI mice display reduced cognitive flexibility when assessed in a T-maze assay, confirming the suitability of this model. Electrophysiological assessment of cholinergic interneurons (CINs, the sole source of ACh in NAc) revealed increased spontaneous activity levels, in agreement with my hypothesis. Ongoing work aims to mechanistically characterize the pain-induced CIN hyperactivity and assess NAc ACh levels in vivo. Future work intends to test the casual role of CINs through in vivo modification of CIN activity during the cognitive flexibility assessment.

1049. Characterizing The Diversity of Cluster R Prophage in Mycobacterium abscessus
Undergraduate Presentation
Author(s): Colin Welch, Maddie Kimble, Dakota Archambault, Claire Bourett, Hector Orellana, Andre Daigle Mentor(s): Sally Molloy
Abstract: The opportunistic pathogen Mycobacterium abscessus is one of the most resistant bacterium to antibiotics and maintains a low cure rate of 25%–58%. Investigating the mechanisms of antibiotic resistance in M. abscessus may lead to more effective treatments. Prophage, viral genomes that are integrated into bacterial genomes, are known to enhance the virulence of many bacterial pathogens. Yet, their role in pathogenic mycobacteria virulence is not yet known. The Molloy lab determined that the prophage McProf increases resistance in pathogenic mycobacteria through the upregulation of the antibiotic resistance gene WhiB7. This upregulation was enhanced whenever there was a cohabiting prophage with McProf. To understand how prophage influence drug resistance in M. abscessus, we have identified and extracted eight novel McProf-like that form a novel Mab cluster R. We also identified six unique cohabiting prophage. We identified sixteen duplicates of the eight MabR prophage, each containing duplicates of at least one of the six cohabiting prophage. To understand the relationships between the MabR prophage and the cohabiting prophage, we analyzed the genome content of the prophage and the frequency and distribution of the MabR and cohabiting prophage within the M. abscessus isolates.

1050. Collagen Microscopy Image Analysis Using Second Harmonic Generation (SHG).
Undergraduate Presentation
Author(s): Christopher Roberts, Betelhem S. Abay Mentor(s): Karissa Tilbury
Abstract: The tumor microenvironment is a complex hub of cellular and non-cellular signaling cascades that often are protumorigenic. Fibrillar collagen is the most abundant protein in the tumor microenvironment. Numerous studies have demonstrated the importance of collagen remodeling and have linked unique morphological signatures of collagen fibers to patient survival using a collagen-specific, label-free imaging technique called Second Harmonic Generation (SHG) microscopy. SHG is a non-linear process in which 2-photons are scattered from a non-centrosymmetric structure creating a single photon with half the wavelength and twice the frequency to conserve energy. The direction of SHG scattering, Forward or Backward, is dependent on phase matching. Here we employ both Forward and Backward SHG detection to probe collagen fiber remodeling in a 3D tumor microenvironment. Low density collagen gels (2 mg/mL) were seeded with both breast cancer (MCF7A and MDA-MB-231) and fibroblast cells (cAMP activated +/-) to recapitulate the interaction of breast cancer cells and neighboring normal fibroblasts. The 3D tumor models were fixed with PFA after 72 hours for both Forward and Backward SHG, and 2p fluorescent cell-specific imaging studies. A custom image analysis algorithm is currently under development using Ilastik to segment cells to use for a localized assessment of collagen remodeling around the cells. Analysis is still underway; however preliminary results are supportive of the hypothesis that activated fibroblasts are more impactful in remodeling the tumor microenvironment and may be more influential in the protumorigenic tumor microenvironment than the aggressiveness of the tumor cells themselves. Further development and refinement of this model and analysis pipeline is important in understanding the biophysical interactions of collagen remodeling in tumor microenvironments.

1051. Characteristics of Neural Electrical Activity Recorded Beneath the Skin in Diabetic Mice
Graduate Presentation.
Author(s): Joshua Passarelli, Lydia Caron, Sarrah Marcotte, Leonard Kass Mentor(s): Dr. Kristy Townsend
Abstract: Peripheral neuropathy (PN) affects over 20 million Americans, with diabetic peripheral neuropathy (DPN) being the largest subcategory. PN is characterized by the progressive loss of innervation from distal to proximal tissues of the peripheral nervous system. Although there is no cure for PN, early detection may allow treatments to help slow disease progression, especially in DPN where glucose regulation may be important to mitigate the neuropathy once detected. Unfortunately, current tools used for a functional diagnosis of PN tend to detect denervation of larger nerves, which occurs late in disease progression. There is not currently a medical device for the diagnosis of small fiber neuropathy using nerve function. Here, we have developed a device to measure the electrical activity of nerves beneath the skin, which are the first to be affected by DPN. We hypothesize that early detection of DPN can be achieved by identifying a loss or change in the activity of these more distal small fiber nerves. Pre-clinical proof-of-concept studies in mouse models of diet-induced diabetic neuropathy revealed multiple characteristics of neural activity that may be a signature of DPN onset. Neural activity recorded from animal models using our methods were categorized into 3 types: (1) “spike trains”, or evenly-spaced sequences of spikes (which are putative action potentials, or APs); (2) “spike bursts,” or APs occurring periodically; and (3) “individual spikes,” which occur in a singular fashion. Electrophysiological results obtained in murine models with this device favor the potential utility of this product for the early detection of DPN in humans. Such a product could provide early diagnosis that may help prevent the progression of PN for millions of affected individuals and improve prognosis.
Acknowledgement: We thank Julia Towne for her help in constructing the microneedle array. NOTE: data herein are covered by a provision patent and are private and confidential.

1052. Infection and Diet-induced Gut Dysbiosis: Impact on Sleep Fragmentation in Danio Rerio
Undergraduate Presentation
Author(s): Ben Williams Mentor(s): Robert Wheeler
Abstract: A known bidirectional relationship between intestinal microflora and the central nervous system, coined the gut-brain-axis, has provoked researchers to examine the correlation between gut dysbiosis, inflammation, and sleep quality. Previous studies within the Hayes Lab have reported that a high fat (HF) diet yielded an association with the mean number of immobile phases and sleep duration. These findings are concordant with previous studies which report that Western Diet is associated with activation of a gut dysbiosis-induced acute phase response (Zinöcker and Lindseth, 2018), which is known to alter clock gene transcription and upregulate NREM sleep (Cavadini et al, 2007). Within this Honors Thesis, an existing protocol for Danio rerio sleep analysis (Sorribes et al., 2013) was modified in order to, for the first time, assess the correlation between inflammatory pathways, induced via direct infection by the human fungal pathogen Candida or via a HF diet-induced gut dysbiosis mechanism, and sleep quality. Preliminary results indicate that there is high variability in zebrafish sleep analysis but that 3/4 of infected zebrafish larvae (n = 42) cohorts yielded a greater total sleep duration than the respective control groups (n = 42), as well as a greater mean sleep bout length, and a smaller fragmentation index. Despite these findings, preliminary qPCR analyses do not support a correlation between inflammatory gene expression (e.g. TNF-ɑ, IL-1β, or IL-6) and differential sleep quality. Further research will include repetition of the infected cohort analysis as well as identical experimental methods administered to the HF diet cohort.

1053. 3D Tracking of Muscle Precursor Cell Movements in Zebrafish
Undergraduate Presentation.
Author(s): Sabrina Varga, Jared Talbot Mentor(s): Jared Talbot
Abstract: Cell migrations are vital to normal embryonic development, but when they go awry it can lead to disease. The cues that control cell migrations are not understood fully and can vary between different biological contexts. The Talbot lab conducts research on how muscle precursor cells migrate in zebrafish embryos. First, a timelapse is taken using a confocal microscope on the developing zebrafish. These images are collected in 3D by imaging a stack of XY slices, and new image stacks are collected every two to three minutes. The data is then put into an artificial intelligence driven program, Aivia, for 3D cell tracking. Traditional cell tracking programs, without artificial intelligence, use nuclear labels to track the cells and require extensive manual input; however, we lack nuclear labels for the cells in question. Instead, I “teach” the program how to identify cell centers by painting the appropriate areas of a subset of images and applying machine learning algorithms to find the rest. Once taught, the software can identify the center of cells based on their membrane labels and uses a custom-optimized algorithm to track those identified cell-centers like they were nuclei. Using this program, we will test how FDA-approved drugs influence cell movements in whole living organisms, thereby revealing precisely how these drugs influence cell behaviors.

1054. Epigenetic Effects of Dio3 Knock-Out Paternal Lineage on Fertility in Female Mice
Graduate Presentation.
Author(s): Logan Douglas Mentor(s): Dr. Arturo Hernandez
Abstract: This study concerns the transgenerational epigenetic effects of thyroid hormone. Its objective was originally to investigate the phenotype of CD-1 mice born from mothers heterozygous for an inactivating mutation of Dio3 gene, which codes for a protein that catalyzes the breakdown of thyroid hormones. These mothers were born from either phenotypically normal heterozygous fathers or from Dio3KO fathers, which were overexposed to thyroid hormone during development. These two groups of females were crossed with wild type males and we aimed to analyze the offspring for growth and neuroendocrine molecular markers related to adipose tissue function and the leptin-hypothalamic system. Most mothers from the control lineage got promptly pregnant and a complete set of serum and RNA samples from hypothalamus and adipose tissues were isolated from their 2-weel old offspring. However, the KO paternal lineage mothers did not give birth to any pups after 3 months with the male. The project pivoted to investigate the fertility impairment of these females. Previous lab data has shown that these females exhibit increased adiposity, reduced bone mass and altered hypothalamic gene expression compared to control females. The phenotype difference despite their identical genotype and mode of inheritance suggests an epigenetic cause. Data from daily vaginal smears on available females over 16 days suggested a deficient estrous cycle, but this was not statistically significant. For future analyses of gonadal axis function, we used some females to collect serum and ovaries, which appeared cystic and swollen especially in those most obese.

1055. Loss of LOTR-1 Affects Germ Granule Movement in Germ Cells
Graduate Presentation
Author(s): Madeleine Nowak Mentor(s): Dustin Updike
Abstract: Germ granules are ribonuclear proteins that localize to the cytoplasmic surface of germ cell nuclei. One constitutive component of germ granules, from nematodes to humans, is a protein that contains both LOTUS and Tudor domains. In mammals, these proteins (TDRD5 and TDRD7) are critical for spermatogenesis and are also associated with familial glaucoma and cataracts, yet their molecular functions are difficult to attain. The homolog in C. elegans, LOTR-1, can be visualized and manipulated in this genetic model to understand its role further. Immunoprecipitation of LOTR-1 followed by quantitative mass spectrometry revealed the potential for the LOTUS domains of LOTR-1 to interact with the cytoskeleton. A strain where germ granules are labeled with a red fluorescent protein (RFP) makes it easy to visualize germ granule dynamics. We sought to test whether cytoskeletal interactions through the LOTUS domain account for these dynamic germ granule movements. Using a spinning disc confocal, I imaged germ granules dynamics over time in four-cell embryos. Images were taken every 500 ms for two minutes. After creating videos, germ-granule movement was tracked using Imaris image analysis. The number of granules per embryo, mean granule track length, and mean granule speed was collected and analyzed using t-tests in Excel. While the wild-type and LOTR-1 deletion strains did not differ in the average amount of germ granules per embryo, the mean track length for germ granules was shorter in mutants by a slightly significant margin. Surprisingly, the mean track speed of germ granules in the LOTR-1 mutant strain was also slightly faster. Overall, these results seem to indicate that germ granules in LOTR-1 mutant embryos interact more loosely with the cytoskeleton, which could cause both these shorter path lengths and higher mean track speeds.

1056. Study on the synergistic activity of Fluconazole and Cyclosporine A on Candida albicans
Undergraduate Presentation
Author(s): Maria Vina Lopez Mentor(s): Robert Wheeler
Abstract: Candida albicans is an opportunistic pathogen that can cause mild yeast infections but is also able to get into the bloodstream and cause severe illness. It is life-threatening in immunocompromised individuals and we aim to improve the therapeutic options. Fluconazole is an antifungal azole drug that causes membrane stress and is commonly used to treat candidiasis. However, it is fungistatic–it inhibits the growth of Candida but does not kill it–and this encourages emerging drug resistance in C. albicans. We have observed a fungicidal synergy–killing of Candida–between fluconazole and cyclosporine A (CSA). CSA is an immunosuppressive drug that also exhibits antifungal activity by inhibiting calcineurin, a phosphatase, that contributes to fungal stress resistance. In this project, I will test how CSA works so we can understand how the two drugs synergize, with the ultimate goal of discovery of better treatments for Candida infections.

1057. Investigating the Interaction of Streptococcus agalactiae and Candida albicans in vitro and in vivo
Graduate Presentation
Author(s): Kathryn Patenaude, Siham Hattab, Robert T. Wheeler, Melody N. Neely Mentor(s): Melody N. Neely
Abstract: Treatment of opportunistic infections can be problematic because we do not have a complete understanding of how factors or other organisms found in the localized environment play a role in the progression of infection. One factor that has been poorly studied is how bacteria and fungi interact together in the host, and how that may be influencing the progression or virulence of pathogens in an infection or even in causing co-infections. Both the fungus Candida albicans (Ca) and the bacteria Streptococcus agalactiae (Group B Strep or GBS) are commensals that reside in the vaginal tract. While usually harmless in the human host, both of these organisms can cause opportunistic infections in immunocompromised patients, which can also progress to co-infections. What we don’t understand is how interactions between these organisms can affect the progression of infections caused by each pathogen or how they affect treatment outcomes. Previous in vitro work has sought to analyze synergistic and antagonistic interactions between other commensal bacteria and Ca, but there is not much known about how GBS and Ca interact in co-infections and what influence that interaction may have on the effectiveness of current treatments for each of the individual pathogens. Through both in vitro and in vivo experimental methods, we will investigate in depth the influence each have on each other in their individual growth, their virulence, and their resistance to treatment.

1058. Contribution of Sox11 in Nav1.8-positive neurons to nerve regeneration after acute and chronic corneal injury.
Graduate Presentation.
Author(s): Cara Sullivan, Jun Lee Mentor(s): Ian Meng
Abstract: Corneal nerve injuries are a common source of ocular pain and discomfort that can result from disease, injury and ocular surgeries. Delayed healing of the corneal epithelium and nerves can lead to extended pain and discomfort. This study investigated the role of the nerve regeneration-associated gene Sox11 in corneal wound healing and reinnervation following acute and chronic injury. Two injury models were used to create corneal nerve injury, double lacrimal gland excision (LGE) which induces chronic dry eye by eliminating the aqueous component of tears and corneal abrasion (CA) which mechanically removes the corneal epithelium and axon terminals. LGE results in persistent corneal epithelial cell damage and retraction of corneal afferent nerve terminals, while the abraded axon terminals in CA are allowed to recover. Corneal fluorescein was used to examine the severity of epithelial damage, mechanical sensitivity was evaluated using a corneal aesthesiometer and nerve terminal density was imaged and Sholl analysis was performed. This study found that Sox11 knockout animals exhibited delayed epithelial healing following both acute and chronic injury. Post-injury mechanical hyposensitivity was observed across phenotypes and nerve regeneration was significantly reduced in Sox11 knockout animals. Expression of key regeneration factors, while normally shown to increase following corneal injury, was diminished following injury where Sox11 expression was knocked down. In summary, our results provide evidence of Sox11’s intrinsic role in nerve development and the impact of its absence on nerve regeneration and epithelial healing.

1059. Uncovering Candida albicans Factors that Modulate the Host Phagocyte Response
Graduate Presentation
Author(s): Bailey Blair, Emma Bragdon Mentor(s): Robert Wheeler
Abstract: Opportunistic fungal infections caused by Candida and Aspergillus primarily affect immunocompromised patients, but they can be quite deadly in the hospitalized patient population. The ability of Candida albicans to evade the host immune system helps it to be a successful pathogen, but little is known about how C. albicans achieves this. Previous work in our lab suggests that the ability of C. albicans to switch to the hyphal growth form may help to limit the recruitment of phagocytes to the infection site, but the mechanism responsible for this is not yet understood. In order to further understand this, we are using the larval zebrafish hindbrain infection model to screen a large number C. albicans mutants for their virulence. This is the first screen of this scale to look at the virulence of individual C. albicans mutants in a live host. This identified a number of mutants that show a virulence defect, including many that have not previously been characterized for their virulence. Transgenic zebrafish with fluorescent immune cells are now being used to investigate the phagocyte response to hypovirulent mutants. This will allow us to identify important genes and pathways for limiting the host phagocyte response, including immune cell recruitment and pathogen engulfment. Preliminary data with one hypovirulent mutant suggests that deletion of a single fungal cell wall protein enhances uptake by the host. This work will allow us to gain valuable insight into C. albicans factors that allow it to evade immune attack.

1060. Antimicrobial agent Cetylpyridinium Chloride interferes with Phosphatidylinositol 4,5-bisphosphate-protein interactions in influenza infection fibroblast model and in mast cells
Graduate Presentation.
Author(s): Prakash Raut, Sasha Weller, Bright Obeng, Bailey West, Christian Potts, Julie Gosse Mentor(s): Samuel T. Hess
Abstract: As we endure through this pandemic, vaccines are very important to curb the pandemic and save millions of lives. In addition to the vaccines, other anti-viral therapeutics or drugs that can help to save lives become crucial moving forward. In order to find alternative such anti-viral therapeutics, it is crucial to understand the viral lifecycle and the interactions between viral proteins and the host cell. Influenza A virus (IAV) Hemagglutinin (HA) is a viral glycoprotein responsible for viral binding and entry. HA clusters at the plasma membrane of the host cell, and these clusters need to be high in density in order to catalyze membrane fusion for viral entry. However, the mechanism by which HA forms clusters remains unknown. We recently showed clustering of HA is modulated by phosphatidylinositol (4,5) bisphosphate (PIP2) [N.M. Curthoys et al. Biophysical Journal, 2019]. Targeting this interaction could lead to possible alternative anti-viral therapeutics. CPC (cetylpyridinium chloride) is a positively charged quaternary ammonium compound used in mouthwashes and personal care items. CPC has been previously shown to have antibacterial and antiviral properties. While both the antibacterial and antiviral properties are well understood at high concentrations (millimolar), the effect of CPC on cell function at relatively low (micromolar) concentrations is not well understood. In this study, we use the super resolution microscopy technique FPALM [S.T. Hess et al., Biophysical Journal, 2006] to study the effect of CPC on phosphatidylinositol (4,5) bisphosphate (PIP2) binding proteins, and to illuminate the mechanism of the antiviral properties of CPC at the these much lower, non-cytotoxic micromolar concentrations in the cell model. Results show that CPC at these concentrations significantly modulates PIP2 clustering and HA clustering more importantly reducing the HA density and also significantly reduces their co-clustering. These results are important because dense HA clusters correlate with the efficient viral entry and infectivity and modulating PIP2 clusters reduces HA clustering. In addition, we also show CPC at mircomolar concentration can improve survival of zebrafish infected with IAV.

1061. Defective CXCR4 Signaling Decreases Survival in a Zebrafish Model of Influenza A Virus Infection
Graduate Presentation
Author(s): Brandy Soos, Con Sullivan, Paul A. Millard, Carol H. Kim, Benjamin L. King Mentor(s): Benjamin L. King
Abstract: The World Health Organization has estimated that up to 650,000 deaths occur per year from respiratory diseases associated with seasonal influenza infections. Influenza A virus (IAV) causes severe disease in older adults and individuals with chronic health conditions. The long-term goal of our studies is to understand the molecular mechanisms of the innate immune response to IAV infection and find new antiviral therapeutic targets. Neutrophils have essential roles in innate immunity to bacterial and fungal infections, but their roles in antiviral responses are understudied. Recently, it was demonstrated that IAV infection can be established in zebrafish (Danio rerio) larvae. The zebrafish are powerful vertebrate models that have been used to study infection and innate immunity. We are using this model to study the roles of neutrophils in controlling IAV infection and how over-activation during IAV infection triggers a damaging hyperinflammatory response. First, we are examining pathways which regulate respiratory burst function and how reactive oxidative species control neutrophil function during IAV infection. 12 members of the CXCR4 signaling pathway were differentially expressed at 6 hours post-infection. Survival studies of IAV-infected WHIM (Tg1(-8mpx:cxcr4b-EGFP)) mutants that overexpressed a truncated cxcr4b transgene had decreased survival compared to sibling controls that underscore the roles of neutrophils. Second, we are investigating how hyperinflammation occurs during IAV infection so therapeutic measures that preserve the antiviral response, yet contain the associated inflammation, can be developed. This work is supported by the National Institute of Allergy and Infectious Disease of the National Institutes of Health (R15AI131202).

1062. Optimizing RT-LAMP for use in COVID Wastewater Testing
Undergraduate Presentation.
Author(s): Audrie French, Kettie Rose Cormier, Harrison Cyr Mentor(s): Robert Wheeler
Abstract: RT-LAMP (reverse transcriptase loop-mediated isothermal amplification) is a simple, rapid detection method for the SARS-CoV-2 virus. Currently, most tests for the virus use RT-qPCR (reverse transcriptase quantitative PCR) – this includes both swab samples and wastewater RNA extracts. Both RT-qPCR and RT-LAMP amplify a target nucleic acid sequence for specific detection. RT-LAMP is far more adaptable to use on site at wastewater treatment plants or other non-lab testing facilities as it is done at a single temperature and it can be scored positive by eye based on a color change. However, using a thermocycler or a plate reader in conjunction with a fluorescent nucleic acid dye allows for more quantitative analysis of the sample. Using the qPCR machine yields real-time quantitative data on the samples, and the fluorescent dye may allow detection of positive results not detected by a visual color change, while still allowing the colorimetric results to be visually analyzed. Using a fluorescent dye post-protocol and viewing with a plate reader or using the dye with a qPCR machine may yield more sensitive results than the colorimetric LAMP itself.

1063. Neurological and Muscle Fiber Health in Zebrafish Dystrophy and Dystroglycanopathy Models
Graduate Presentation.
Author(s): Mary Astumian, Prakash Raut, Sam Hess, Clarissa Henry, Erin C. Bailey Mentor(s): Clarissa Henry
Abstract: Muscular dystrophies and dystroglycanopathies are progressive diseases, with varying severity, affecting neurological and muscle health. In functional muscle fibers, alpha-dystroglycan and integrin-alpha7 proteins attach the actin cytoskeleton to the extracellular matrix (ECM). To function, integrins must cluster in the muscle membrane and alpha-dystroglycan must be glycosylated. GMPPB and DPM3 genes are essential for glycosylation. GMPPB helps glycan production while DPM3 (Dolichyl Phosphate Mannosyltransferase Subunit3) stabilizes glycosylation. Both are mutated in human dystroglycanopathy subsets. Dystroglycan and integrin-alpha7 are receptors for the extracellular matrix laminin. Previous work revealed oxidized nicotinamide adenine dinucleotide (NAD+) treatment improved laminin deposition and muscle health in dystroglycan and integrin-alpha7 zebrafish mutants. NAD+ remains to be tested in DPM3 mutants but did not improve muscle health in GMPPB mutants. One hypothesis explaining these contrasting results is hypoglycosylated dystroglycan lacks sugar sidechains so cannot cluster, interfering with integrin-alpha7 clustering. The localization of dystroglycan and integrin-alpha7 proteins relative to each other at the muscle cell membrane is hypothesized to be important. Using superresolution microscopy, images of Dystroglycan, GMPPB, and DPM3 mutants treated and untreated with NAD+ will be analyzed to assess the average distance between the molecules of dystroglycan and integrin-alpha7 at the myofiber membranes. Neurological and muscle health will be measured with synapse staining, birefringence, swimming behavior, and cell adhesion.

1065. Yeast Actin Cytoskeleton Regulation by RGS
Undergraduate Presentation
Author(s): Lucas Craig, Cory Johnson, Joshua Kelley Mentor(s): Joshua Kelley
Abstract: Yeast release pheromone into their environment to signal to nearby yeast that they can mate. The receptors for this pheromone are G-protein coupled receptors (GPCRs). Once these GPCRs detect this pheromone yeast cells grow towards the pheromone gradient to contact the possible mate and begin the mating process. GPCRs are found throughout the human body and are common targets for many drugs, so understanding how they are regulated is essential, particularly with the Regulator of G-protein Signaling (RGS) Sst2. This RGS has previously been shown to also interact with actin polymerizing proteins (formins) leading us to ask if Sst2 mutants affect polymerization of actin and how the regulation of these formins by Sst2 alters mating efficiency. Using actin staining with phalloidin and confocal microscopy we investigated the localization of actin when Sst2 has been mutated at its activating phosphorylation site. Using mating assay techniques, we also investigated the impact on yeast mating. We find that actin, which is usually localized to the site of growth in yeast responding to pheromone, is poorly organized in yeast with mutated Sst2. We also find that yeast mating efficiency is decreased with mutated proteins. This indicates that Sst2 is important in regulating the localization of actin which is a key component in the cell cytoskeleton and is important in allowing the cell to continue to grow, therefore, decreasing mating efficiency. This allows us to better understand how GPCRs are regulated and how these regulators affect cell health.

1066. Regulation of septins through receptor-epsin interactions
Undergraduate Presentation
Author(s): Sarah Latario, Cory Johnson Mentor(s): Joshua Kelley
Abstract: Yeast utilize a G-protein coupled receptor (GPCR) signaling pathway for mating, that recognizes mating factor and leads to cell polarization and elongation. For directed polarization to occur, endocytosis of the mating receptor is needed to internalize regions of the cell’s membrane where mating factor has bound. Epsins are proteins that support membrane curvature and assist in selective endocytosis. They also contain a domain that is known to interact with septin regulators. Therefore, we suspect that epsins are involved in coupling endocytosis to septin deposition. Septins are cytoskeletal components that provide membrane stability, restrict transmembrane protein diffusion, and require precise regulation for proper cell polarization. To test the role of epsins in septin distribution, we engineered yeast deletion strains that lacked epsins (ENT1 and ENT2), exposed them to pheromone using microfluidics devices and monitored them over 12 hours to track polarization and elongation. Additionally, we deleted ENT1 in a hyperactive Gα strain that manifests improper septin deposition and an inability to track a gradient, to further investigate our hypothesis. Our data suggests that Ent1 is partially involved in coupling endocytosis to septin deposition, that epsins may be involved in proper septin distribution, and that yeast lacking epsins may have an altered mating response.

1067. Analysis of Cluster MabD Prophage
Undergraduate Presentation
Author(s): Andre Daigle, Colin Welch Mentor(s): Sally Molloy
Abstract: Bacteriophages are viruses that infect bacteria and comprise an estimated 1031 particles in the biosphere. Prophage, viral genomes integrated into the bacterial genome, encode genes that often increase the fitness of the bacterial cell, including viral defense systems and toxin/antitoxin systems. Mycobacterium abscessus causes lung infections in patients with lung diseases such as cystic fibrosis and is often extensively drug-resistant. The relationship between the drug resistance of M. abcsessus and prophages is understudied. The Molloy lab established that cohabitating prophage alter drug resistance in pathogenic mycobacteria. The aim of this project is to characterize M. abscessus prophages that co-habitate with a second type of prophage ( Mab Cluster R prophage) that is known to alter drug resistance in mycobacteria. Two Mab cluster D prophages were identified and extracted from clinical isolates of M. abscessus that carry cluster R prophage genomes. The prophiFLQ01-2 and prophiFSQJ01-3 genomes are 503226 and 50715 base pairs in length and encode for 79 and 85 genes respectively. MabD cluster genomes are highly conserved through the structural genes in the right arm. At the left attachment site, both prophages encode a tyrosine integrase gene. Near the integrase are a divergently transcribed immunity repressor and likely Cro gene, although the protein Phams differ between the two genomes. At the right attachment site, both prophages carry Type VII-secreted polymorphic toxin systems that include an ESAT-6 protein, a polymorphic toxin, and an immunity protein. This cassette is being studied further as genes that potentially increase the fitness of M. abcessus.

1068. Notch signaling regulates perivascular adipose tissue (PVAT) function during diet-induced obesity
Graduate Presentation.
Author(s): Chenhao Yang, Anne E. Harrington, Larisa M. Ryzhova, Lucy Liaw Mentor(s): Lucy Liaw
Abstract: Obesity is an established risk factor for cardiovascular diseases (CVD) and possibly shares molecular and cellular disease mechanisms as CVD. As a component of the vasculature, perivascular adipose tissue (PVAT) is as a critical regulator of vascular function due to its anatomical proximity to the vascular wall. In addition to a broad role in embryonic development, Notch/Rbp-jk signaling plays a crucial role in regulating metabolic homeostasis. Suppression of Notch signaling components was reported to induce beige phenotypes within white adipose tissue. We studied how Notch/Rbp-jk signaling and potential downstream pathways regulate PVAT phenotype. We generated mouse models with adipose tissue specific knockout of Rbpj genes or overexpression of Notch intracellular domain using Adipoq-Cre driver and examined their physiology, histology, and expression of metabolic and vascular relaxation pathway components as compared to control non-Cre mice. Our data showed that Notch signaling was activated in PVAT during high fat diet (HFD) treatment. Expression of Notch signaling component including RBPJ-k was increased during differentiation of PVAT. Moreover, PVAT from adipocyte-specific RBPJ-k conditional null mice showed alterations in the vasorelaxation pathway including eNOS, with a potential link through PI3/AKT signaling. We are conducting vessel wire myography experiment to further assess the influence of Notch signaling in PVAT on vascular reactivity. Our studies show that Notch signaling regulates metabolic homeostasis of adipose tissue including PVAT. In addition, Notch signaling potentially regulates PVAT-mediated vasorelaxation through eNOS pathways.

1069. Anti-fungal Treatment Efficacy during Polymicrobial Infection
Graduate Presentation
Author(s): Siham Hattab, Jennifer Quezada-Loja, Anna Maria Dagher, Avery Bond Mentor(s): Robert Wheeler
Abstract: Polymicrobial infections are challenging to treat because of lack of understanding of how pathogens interact during infection and how these interactions affect drug efficacy. Candida albicans is an opportunistic fungus that can cause invasive candidiasis with 40 % mortality in hospitals. Pseudomonas aeruginosa is the most important pathogen responsible for respiratory tract infections in cystic fibrosis (CF) patients. Both pathogens can be found in the lungs of CF and mechanically ventilated patients. In our research project, we investigated the efficacy of anti-fungal treatment during C. albicans-P. aeruginosa co-infection. We have performed in vitro and in vivo experiments using the zebrafish swimbladder infection model. This infection model is a powerful tool that provides a simple environment that mimics mucosal infection in human lungs. Because of the zebrafish’s transparency, this allows us to monitor complex interactions between host and pathogens. Our results suggest that treatment with fluconazole (Flc), an antifungal drug that stops the growth of C. albicans but doesn’t kill the fungus, is effective at killing C. albicans during co-infection. This potentiation of antifungal efficacy during co-infection suggests a synergy between Flc treatment and the bacterium. Our current goal is to understand how P. aeruginosa causes this synergy with the anti-fungal drug. Our preliminary data suggest that iron chelation, stripping iron from the environment, might be one of the mechanisms involved in this synergy. This work will help us identify future therapeutic targets to treat C. albicans infection and gain new insights into how the presence or lack of iron affect response to antifungal treatment

1070. Development of a Novel RAB27a Knockout Strain to Study Adipose-Vascular Signaling in Vivo
Author(s): Ashley Soucy, Larisa Ryzhova, Anne Harrington, Benjamin Tero, Bethany Fortier, Lucy Liaw Mentor(s): Dr. Lucy Liaw
Abstract: Perivascular adipose tissue (PVAT) is a specialized adipose depot that surrounds the vasculature. During obesity, PVAT exhibits a lipid storage phenotype and secrets inflammatory factors which can be detrimental to vascular function. When comparing endogenous protein expression between healthy and obese mice, we found a dramatic increase of RAB27a protein levels in PVAT. A member of the Ras-related protein family, RAB27a is a trafficking protein that regulates the formation and secretion of exosomes. Dysregulation of exosome secretion has been linked to various diseases including type II diabetes and increased heart muscle deterioration. However, the role of RAB27a has yet to be defined in PVAT regarding exosome secretion and cardiovascular disease. We hypothesize that increased RAB27a expression in PVAT during obesity alters PVAT-derived exosome signaling.
To test our hypothesis, we developed a novel Rab27a global null mouse, characterized by an ashen coat color that mimics type II Griscelli’s syndrome in humans. These mice are a good model for studying how obesity alters PVAT-vascular communication without RAB27a, as the strain remains susceptible to diet-induced weight gain. Additionally, isolated aortas with intact PVAT from Rab27a global null mice exhibit a reduced contractile response when treated with phenylephrine compared to wild type mice – suggesting that loss of RAB27a may regulate vascular contractility. With these mice, we now have the tools necessary to investigate how obesity utilizes RAB27a to regulate adipose-vascular communication via exosome-mediated signaling.

1071. Mycobacterium abscessus Cluster R Prophage Genomic Analysis
Undergraduate Presentation
Author(s): Madeline Kimble, Colin Welch, Dakota Archambault, John Ross, Jacob Cote Mentor(s): Sally Molloy
Abstract: Mycobacterium abscessus (Mab) is an emerging lung pathogen of cystic fibrosis patients. It is one of the most drug-resistant pathogens and infections typically result in high morbidity and mortality. Understanding mechanisms of antibiotic resistance is critical for developing more effective treatments. Prophage, integrated viral genomes, are known to contribute to bacterial virulence and antibiotic resistance, yet Mab prophages remain largely uncharacterized. My research aims to characterize the diversity of novel cluster Mab R prophage genomes. The Molloy lab has demonstrated that the prophage McProf increases mycobacterial resistance to antibiotics. Using the McProf prophage genome, we probed the PATRIC M. abscessus database to identify bacterial strains that carry prophage genomes related to McProf. We identified 25 related genomes, 8 of which were unique. This group of prophages are genetically distinct from prophages already described and we assigned them to a new Mab cluster, R. Prophage genome ends were defined, and prophage sequences were extracted from bacterial genomes. MabR genomes are highly conserved, particularly across the structural genes in the right arm and the immunity cassette in the left arm. All nine genomes share a tyrosine-integrase and nearly identical attachment sites. Interestingly, the immunity repressor is unique to known Mab prophages but is found in tthe M. tuberculosis phage, DS6A. In future research we will investigate the role of novel genes within cluster MabR prophages. All nine members share a secreted polymorphic toxin system adjacent to the right attachment site.

1072. Characterization and Comparative Analysis of Prophage in Antibiotic Resistant Mycobacterial Genomes
Undergraduate Presentation
Author(s): John Ross, Madeline Kimble, Colin Welch, Anna Schumann, Dakota Archambault Mentor(s): Sally Molloy
Abstract: Mycobacterium abscessus is a highly resistant nontuberculous opportunistic pathogen, that causes pulmonary and disseminated infections in individuals with underlying diseases, such as cystic fibrosis. Infections are incredibly difficult to treat due to extensive drug resistance. Understanding intrinsic and acquired resistance mechanisms is important for developing effective treatments. Prophage, integrated viral genomes, are known to alter drug resistance in Escherichia coli, but their role in mycobacterial drug resistance is not yet known. Oftentimes prophage carry genes which increase the overall fitness of their bacterial hosts. In M. abscessus however, the prophages remain poorly characterized. Characterization of these prophage and the genes they encode may provide more understanding of how prophage could contribute to virulence and drug resistance. The Molloy Lab recently demonstrated that the mycobacterial prophage, McProf, alters expression of intrinsic resistance genes in M. chelonae. McProf is included in the cluster MabR, a novel cluster of M. abscessus bacteriophage, which have not yet been characterized. In this project, we compare the genomes of two novel MabR phages, prophiFSQJ01-1 and prophiFSIL01-1 to McProf. These genomes are 76,665 and 66,497 base pairs long and encode 119 and 97 genes, respectively. Like McProf, these novel prophage encode Esx secreted polymorphic toxin (PT) systems however the C-terminal polymorphic toxin motif of prophiFSQJ01-1 differs from the Tde-like DNAse motif found in the McProf and prophiFSIL01-1 genomes. We hypothesize that these PT systems may allow the bacterium to respond to stress, such as antibiotics, in the environment.

1073. Antimicrobial Cetylpyridinium Chloride Inhibits Immune Mast Cell Function 
Author(s): John Burnell, Bright Obeng, Christian M. Potts, Bailey E. West, Julie A. Gosse Mentor(s): Dr. Julie A. Gosse
Abstract: Cetylpyridinium chloride (CPC) is a positively charged antimicrobial in consumer products such as mouthwashes at concentrations up to 3 millimolar, thus exposing humans to high concentrations. Minimal information on eukaryotic toxicology of CPC exists; hence, there is need for information since humans and wildlife are exposed. Mast cells, ubiquitous throughout the body, are central in numerous physiological processes and diseases. We have demonstrated that CPC potently inhibits antigen-stimulated RBL-2H3 mast cell functioning, including their ability to degranulate, which is the release of bioactive substances including histamine, from intracellular granules. Degranulation inhibition occurs at non-cytotoxic CPC doses as low as 1 micromolar, ~1000-fold lower than the concentrations of consumer products. We have investigated the molecular mechanisms underlying the inhibition of mast cell degranulation by CPC. We have shown that CPC inhibits the antigen-stimulated influx of Ca2+, a core mediator of the degranulation pathway, via plasma membrane channels, into the cytosol. Elevated Ca2+ concentration in the cytosol is required to activate various Ca2+-dependent enzymes and processes, including microtubule polymerization. Microtubules serve as “railroad tracks” to transport the granules to the plasma membrane for fusion and degranulation. Here, we show that 5 or 10 micromolar CPC shuts down microtubule polymerization within 60 minutes. This research provides biochemical mechanisms underlying the effects of CPC on immune signaling and allows prediction of effects on cell disparate cell types that share similar signaling and cytoskeletal elements.

1074. Distance Vitals
Undergraduate Presentation
Author(s): Kiana Goodwin, Callie Witt, Allie Nutting, Ethan Saville Mentor(s): Michael Mason, Robert Bowie
Abstract: In recent years, popularity has grown for search and rescue teams to utilize drones to detect and locate a missing person(s). Without physically coming into contact with the individual, the necessary technology and ability are lacking to precisely measure an individual’s vital signs (i.e., breathing rate, pulse rate, and temperature). To assist in rescue efforts, a device has been designed to analyze videos and photographs that determine the vital signs of incapacitated individuals. A program was constructed to utilize the Eulerian video processing technique and a Raspberry Pi, allowing for rescue scene information to be extracted and analyzed from a secure digital (SD) memory card. Five critical aspects of the device were tested to ensure quality and functionality and are as follows: (1) testing of the primary programs written to split a video into frames for analysis, (2) testing the pulse rate program, (3) testing the body temperature detection program, (4) determining the functionality of the video stabilization program, and (5) testing the reliability of the breathing rate program all against a control where the vitals are found using equipment known to produce reproducible values. To tie the device together, all programs are compiled and tested for functionality. The results demonstrate that patient vital analysis can be performed via remote access in search and rescue environments.

1075. Low Dose Arsenic Exposure Alters the Expression of MicroRNAs in the Innate Immune Response to Pseudomonas aeruginosa Infection.
Graduate Presentation
Author(s): Liz Saavedra Perez, Brandy L. Soos, Carol H. Kim Mentor(s): Benjamin King
Abstract: The innate immune system is essential for responding to different types of infections, injuries and that response can be altered by environmental toxicants. Low-dose arsenic exposure can perturb and dysregulate the innate immune response. In Maine, it is estimated that 10% of private wells are contaminated with arsenic. Exposure to arsenic can lead to decreased overall innate immune function and decreased host’s ability to fight infections by opportunistic pathogens, like Pseudomonas aeruginosa, but the mechanisms are not well understood. We utilize the zebrafish (Danio rerio) as a model system to study how arsenic alters the innate immune response to infection. To further understand these mechanisms, our lab focuses on the highly conserved non-coding RNAs, microRNAs, responsible for regulating the protein-coding genes behind these processes. The expression of microRNAs was characterized in 48 hours post-fertilization (hpf) embryos infected with Pseudomonas aeruginosa at 6 and 18 hours post-infection (hpi) following exposure to 0, 2, and 10 ppb arsenic. The highest number of differentially expressed microRNAs in response to infection at 6 and 18 hpi were in embryos exposed to 2 ppb arsenic with 81 and 87 microRNAs, respectively. Dre-Mir-722-P1_3p, previously described to reduce neutrophil chemotaxis to sites of injury or infection, is normally upregulated in response to infection. At 6 and 18 hpi, it was downregulated with increasing concentrations of arsenic. These studies, combined with survival and bacterial burden studies, will provide new insight into the mechanisms of innate immune responses and inform on the dysregulating effect of arsenic.

1076. Computational Analysis and Classification of SHG Images of Cancerous Pancreatic Tissue Based on Collagen Fiber Alignment
Undergraduate Presentation
Author(s): Gerren Welch Mentor(s): Karissa Tilbury
Abstract: Pancreatic cancer is a deadly disease, with a low five-year survival rate partly due to the difficulty in diagnosing the cancer early in its development, as it shares symptoms with more common and less lethal conditions. Using Second Harmonic Generation (SHG) microscopy and computer analysis, our knowledge of the biophysics of the pancreatic tumor microenvironment increases which may lead to the development of more effective therapies. In collaboration with Maine Medical Center Research Institute (MMCRI), we have identified 20 pancreatic cancer patients. In these 20 pancreatic cancer patients, Dr. Jones, a pathologist at MMCRI has identified normal adjacent pancreas, fibrotic pancreas tissue, and tumorous pancreas tissue. Using SHG imaging microscopy with an 890 nm excitation laser and collection via a 445/20 bandpass filter in the forward direction, we imaged the collagen primarily around pancreatic ductal structures, which studies have shown to be common tumor origin sites1. OrientationJ, a Java plugin in ImageJ, uses a structure tensor to quantify morphological changes in the collagen fibers within the extracellular matrix. In this analysis, the quantitative orientation measurement function built into OrientationJ provides orientation and coherency values for regions of interest selected by the user. Collagen within the ECM generally has a random, basketweave pattern, but in other cancerous tissues there are studies which show a correlation between progression of cancer and the increased aligning of the collagen fibers2,3. We anticipate finding quantitative biophysical alterations that are distinct between cancerous, fibrotic and normal adjacent collagen organizations.

1077. Investigation of the Relationship between Black Soldier Fly Larvae Pathogen Suppression and Growth Substrate in Relation to Maine Agricultural Industries
Undergraduate Presentation
Author(s): Haley Morrill, Marissa Kinney, Emily McLaughlin, Matthew Moyet Mentor(s): Edward Bernard
Abstract: Insects are promoted as cost-effective and sustainable protein sources for animal feed. Their utilization may help to avoid a predicted global protein shortage. Black soldier fly (Hermetia illucens) larvae (BSFL) grow on organic wastes, converting these wastes into larval biomass which can fulfill this purpose. BSFL suppress the growth of some gram-positive and gram-negative human pathogens in these substrates; though suppression of Bacillus cereus, a spore-forming bacterium that causes food-poisoning, has not been documented. Potential benefits of using BSFL to remediate organic wastes include reduction of waste mass and bacterial load, along with the sale of larvae as a protein supplement. This project focused on Bacillus cereus suppression by BSFL on byproducts (used as growth substrates) of 2 Maine agricultural industries: potatoes and blueberries. Colony counts on B. cereus selective media were higher for larvae fed on potatoes spiked with pathogen than pathogen alone on potatoes after 2 days. After 4 days, an opposite effect was observed, with lower colony counts observed for larvae fed on potato with B. cereus than pathogen alone on potato. Blueberry substrate was not capable of supporting B. cereus as colony counts for all treatments were below the detection threshold after 2 days of larval feeding. While 100% viability was observed for BSFL reared on blueberries, larval weight decreased by an average of 82% with pathogen compared to a 32% decrease without pathogen. Additional PCR analysis of pathogen presence is in progress.

1078. Comparative Genomic Analysis of Prophages in Clinical Isolates of Streptococcus agalactiae
Graduate Presentation.
Author(s): Caitlin Wiafe-Kwakye, Andrew Fournier, Katie Southworth, Hannah Maurais, Brandon Rockwell, Caiden Fraser Mentor(s): Melody Neely
Abstract: Prophages, viral genomes integrated into bacterial genomes, are known to enhance bacterial colonization, adaptation, and ecological fitness providing a better chance for pathogenic bacteria to disseminate and cause infection. Streptococcus agalactiae typically carries one or more prophages, yet their role in pathogen fitness and virulence has not yet been described. Approximately 50% of babies born through vaginal delivery from women colonized with S. agalactiae develop life-threatening infections, such as sepsis and meningitis. However, antibiotic treatment of these infections has long-term negative effects on neonatal microbiota. Characterizing the role of prophages in disease pathogenesis of S. agalactiae could lead to development of alternative treatments. We performed comparative genomics, of prophages extracted from the genomes of 8 previously uncharacterized S. agalactiae vaginal isolates and prophages extracted from published sequences of 5 clinical isolates of S. agalactiae. Dotplot analyses, to explore genome synteny of the extracted prophage sequences, found that they can be grouped into seven distinct clusters. Comprehensive annotation revealed that all S. agalactiae prophages encode paratox, a protein that prevents the uptake of DNA in Streptococcus along with an associated toxin, hol-tox. Furthermore, each prophage genome has at least one toxin-antitoxin system. Investigation is currently underway into the genomes of over 40 previously uncharacterized S. agalactiae vaginal isolates to identify and extract prophage genomes. Additional analyses of the prophage genomes are being performed to identify genes that may be associated with S. agalactiae pathogenesis. Ultimately, these findings may lead to the identification of potential targets for alternative therapeutic approaches.

1079. Common Environmental Contaminants and their Effects on Zebrafish Mitochondrial Function and Behavior 
Undergraduate Presentation.
Author(s): Audrie French, Noah Burby, Grace LaFrance, Patrick Fleming, Marc Thibodeau Mentor(s): Remy Babich
Abstract: Many individuals, including children across the state of Maine rely primarily on well water, which can have contaminants that contribute to a number of health deficits. Making sure the water is free of contaminants is important for the health of everyone who uses it. . While the US EPA sets limits for toxicity of individual contaminants, data on interactive effects of metals mixtures are lacking. To test for physiological effects of exposure to drinking water samples, we conducted a series of mitochondrial and behavioral assays using zebrafish, Danio rerio. Well water samples from locations across Maine and New Hampshire were collected then analyzed for key metal contaminants. Zebrafish embryos were exposed to a 50% dilution of the water sample starting at 1 or 24 hours post fertilization (hpf) for mitochondrial function and behavioral assays respectively. At 25 hpf, mitochondrial function was determined by measuring oxygen consumption rate (OCR), additionally a subset of embryos exposed from 1 day post fertilization (dpf) to 5 dpf were used to measure exposure impact on behavior via a light dark test, in which total distance traveled over a 25 minute period was extrapolated. OCR and behavioral data were compared to the levels of contaminants in the corresponding well water samples. There were no statistically significant effects on zebrafish mitochondrial respiration or behavior in single metal analysis at levels below the EPA thresholds, though there was a significant difference between single and dual metal toxicity in the water samples with regard to its effect on mitochondrial function and behavior. Our research confirms the validity of the established metal toxicity thresholds set by the EPA, but suggests that metals could be acting in mixture to cause problems such as altered mitochondrial function and behavior in developing zebrafish – effects which should be further investigated as they may apply to humans. Further research is necessary to evaluate the differences between single metal and mixture toxicity and its effects on embryonic development.

1080. The Optimization of RT-LAMP Testing from Wastewater for SARS-Cov-2 Detection
Undergraduate Presentation
Author(s): Harrison Cyr, Kettie Cormier, Audrie French Mentor(s): Robert Wheeler
Abstract: Having tragically ended the lives of over 500,000 people in the USA alone and having impacted many more, the COVID-19 pandemic has undoubtedly been one of the most influential events in recent history. One of the most important aspects of controlling this pandemic is effective and large-scale testing, and my project is focused on helping to improve the University of Maine’s testing capabilities. In addition to the nasal and saliva testing that is widely used, wastewater testing is capable of detecting an increase in infection before infected individuals even develop symptoms. Along with two other undergraduate researchers, this project has been focused on optimizing wastewater and saliva testing using RT-LAMP, an alternative technology to qPCR that allows for the detection of the virus. The allure of RT-LAMP is in its speed, reduced cost, and ease of use as compared to typical qPCR. My place in the project has been working on several smaller goals to help the project as a whole. Some of these goals include: generating and quantifying positive control DNA to ensure our assays are working correctly, developing a method of testing for the more infectious B.1.1.7 variant in wastewater, determining if the extraction of viral RNA can be replaced with a simpler heat treatment method, and analyzing data generated since the fall to establish the precision and variability of our quantification methods.

1082. Pseudomonas aeruginosa Infection Interacts With NF-κB Activation Through Integrin Signaling
Undergraduate Presentation
Author(s): Daisy Drinkert, Jon Donnelly, David Flewelling, Victoria Mayers, Sabrina Varga Mentor(s): Benjamin King
Abstract: Pseudomonas aeruginosa, a gram-negative aerobic bacillus, is an opportunistic pathogen that exhibits antibiotic resistance and commonly infects those with underlying conditions such as Cystic Fibrosis. Low dose arsenic exposure and CFTR mutations, such as those responsible for Cystic Fibrosis, may also contribute to more severe symptom presentation in Pseudomonas infections. Binding of P. aeruginosa pili have been implicated in increasing the expression of NF-kB, a signaling pathway involved in the innate immune response. It has also been shown that P. aeruginosa interacts with beta-integrins and disrupts normal integrin function. We hypothesize that normal response to Pseudomonas infection runs through the integrin signaling pathway. To test this hypothesis we evaluated RNA sequencing data from developing zebrafish embryos in an experiment where CFTR or control morphants were exposed to 0, 2 or 10 ppb arsenic and infected with Pseudomonas or phosphate buffered saline at 48 hours post fertilization. We analyzed differential gene expression in NF-kB regulated genes and beta integrins at 6 hours post infection. We found that NF-kB related genes and beta integrins were upregulated with Pseudomonas infection, CFTR knockdown, and arsenic exposure. This supports our hypothesis that Pseudomonas infection activates the integrin signaling pathway. Disruption of this pathway could decrease the virulence of Pseudomonas aeruginosa. Beta integrin knockdown models paired with these treatments should be studied to further confirm this hypothesis.

1084. Role of cAMP-pathway Regulators on Acute to Chronic Pain Transition
Graduate Presentation
Author(s): Zaid Al-Abbasi, Ramaz Geguchadze, Katherine J. Motyl, Derek Molliver Mentor(s): Derek Molliver
Abstract: Chronic pain is challenging to treat and maintained by neuronal plasticity in the peripheral nervous system (PNS) and central nervous system (CNS). Furthermore, diagnoses and treatments for chronic pain are also associated with skeletal disorders. One of the most significant molecular changes in the PNS caused by chronic-pain is the cAMP-signaling upregulation. cAMP pathway activation in the CNS produces hyperalgesia, an increase in sensitivity to noxious thermal or mechanical stimuli, while cAMP inhibition reduces hyperalgesia in inflammatory and other pain models. Hyperalgesia plays a critical role in acute to chronic pain transition. Many studies propose the cAMP signaling pathway as a potential target for developing chronic pain therapeutics. Proteins mediating cAMP signaling are organized into complexes by scaffold proteins called A-kinase anchoring proteins AKAPS, such as (AKAP12). Analysis of data in Molliver lab indicated that AKAP12 might be an essential scaffold for sensory neurons. There are three isoforms of this protein that may form different complexes. Therefore, I generated inflamed mice by injecting them with carrageenan to determine how AKAP12 is regulated by inflammation in sensory neurons. Also, during my current rotation in the Motyl lab, I treated osteoclasts with the β-adrenergic-receptor agonist isoproterenol and examined its effect on the regulation of AKAP12. We identified the three isoforms in multiple mice tissues, with varied responses of the isoforms to the carrageenan-generated inflammation, and to isoproterenol in osteoclasts, indicating a different cellular expression-pattern of each isoform. Following, I will expand the investigation of AKAP12 isoforms’ roles in regulating cAMP-induced chronic pain.

1085. Characterizing the Role of Neuroblastoma Suppressor of Tumorigenicity 1 (NBL-1) in Progression of Kidney Disease in Mice
Graduate Presentation
Author(s): Teresa Easterbrooks, Yuka Takemon, Kristina O’Neil, Andrzej Krolewski Mentor(s): Ron Korstanje
Abstract: The Krolewski laboratory at the Joslin Diabetes Center has identified circulating plasma levels of the protein NBL-1 to be associated with progression of end stage kidney disease (ESKD) in diabetic patients. However, whether the increase of NBL-1 is a cause or consequence of kidney disease is not known. To examine the timing of kidney damage and increased NBL-1, we analyzed NBL-1 plasma levels and albumin-creatinine ratio (ACR), a kidney disease marker, in a cohort of genetically diverse mice with a mutation in the Col4a5 gene, which causes kidney disease. We found a significant correlation between ACR and NBL-1 levels, supporting NBL-1 as a kidney disease marker in mice. Increased ACR generally preceded the increase in NBL-1, suggesting that the increase in NBL-1 is not causal to kidney disease.
We generated a transgenic mouse model that overexpresses Nbl1. These mice show increased gene expression in the liver and kidney, but their circulating NBL-1 does not differ from non-transgenic littermates. In addition, we are currently generating an Nbl1-knockout mouse.
We will use these two new mouse models to further explore the role of NBL-1 in kidney disease. We will induce kidney disease in transgenic, knockout, and wild type mice and measure plasma NBL-1 levels, as well as markers of kidney disease including ACR, to establish the causal relationship between kidney disease and NBL-1.

1086. Investigating the Link Between Aging-Related Vascular Dysfunction and Peripheral Neuropathy through Pressure Myography
Undergraduate Presentation.
Author(s): Bailey Woodard, Jake Willows Mentor(s): Kristy Townsend
Abstract: Peripheral neuropathy is any condition that reduces the innervation of peripheral tissues. Pathologies such as diabetes and aging are catalysts for the development of peripheral neuropathy starting in the skin. We previously showed that neuropathy with aging and obesity can worsen over time and reduce adipose innervation as well. Losing proper nerve communication between brain and adipose is detrimental to metabolic health and can worsen obesity or diabetes. Since aging increases the risk for these pathologies, a better understanding of neuropathy with aging is necessary to mitigate them. We have observed denervation around vasculature in subcutaneous white adipose tissue (scWAT) with aging, which is thought to contribute to microcirculation dysfunction and reduced cardiometabolic health. To test the integrity of scWAT vasculature with aging, we are developing a pressure myography system to apply to adipose blood vessels – the first time this has been performed in these tissues. Myography measures myogenic tone and vascular resistance, which may be affected by aging or neuropathy. We will test functional responses and vascular reactivity of an isolated artery from mouse WAT to quantify vascular function. As part of this project, I have spent the past year building and testing a custom pressure myography system and optimizing the microdissection of small blood vessels for mounting. Now that the system is fully built and functional, assessments across aging will commence.

1087. Concentration-dependency of Suppression of B. Cereus and MRSA using Black Soldier Fly Larvae in Potato Waste
Undergraduate Presentation
Author(s): Daniella Leal Espinal Mentor(s): Edward Bernard
Abstract: Municipal solid waste (MSW), of which the US is the top global producer, harbors a great number of drug-resistant pathogenic bacteria, making it one of the most important environmental reservoirs of infectious disease. This issue particularly affects marginalized communities who are most often exposed to this biohazardous waste. Gram positive bacteria make up approximately 38% of pathogenic load in all municipal solid waste. The Black Soldier Fly (Hermetia illucens), is a species of fly whose larvae (BSFL) have been shown to suppress pathogenic bacteria. This study sought to better understand BSFL-induced suppression of gram-positive bacterial growth during potato waste remediation under differing larval densities. We investigated suppression of Bacillus cereus and Methicillin-resistant Staphylococcus aureus, which are two of the most commonly found pathogens in MSW. Varying larval numbers in each treatment were used to quantify the impact of larval density on pathogen suppression. Some of the methods utilized to assay this impact were suppression disk assays, total and selective bacterial plate counts, and molecular methods. In a preliminary trial made using blueberry waste, the diameter of the zone of inhibition in treatments using 100 larvae decreased by an average factor of 15% when compared to treatments with only 50 larvae. While experiments and data analysis are ongoing, these results may suggest a direct correlation between suppression and larval density. Further investigation will elucidate this relationship.

1088. Receptor Endocytosis Regulates Septin Distribution in Yeast
Graduate Presentation. 
Author(s): Cory Johnson, Sarah Latario Mentor(s): Joshua B. Kelley
Abstract: G-protein coupled receptors (GPCRs) are the largest, most diverse family of receptors in eukaryotes, and are required for gradient tracking in many eukaryotic cells including neurons. Yeast polarize and grow toward a mating partner through GPCR signaling and the master regulator of cell morphology Cdc42. During polarized growth, yeast deposit cytoskeletal scaffolding proteins called septins at the periphery of the mating projection. In mutant yeast with defective desensitization of GPCR signaling, septin structures are aberrant. We hypothesized that septin distribution is regulated at the level of the receptor. To test this, we used live cell imaging and computational image analysis to observe and quantify septin localization relative to sites of polarity. In our defective GPCR signaling mutant, we find that septin structure changes are dependent upon two negative regulators of Cdc42, the GAPs Rga2 and Bem3. These proteins are known interactors with endocytic proteins called epsins. We next examined epsin signaling in septin deposition and found that the Ent1 exacerbates abnormal septin structures in our mutant strain. Since epsins are involved in receptor endocytosis, we investigated septin distribution in yeast lacking the receptor cytoplasmic tail, which are unable to undergo epsin-mediated endocytosis. We found that receptor mutants do not appear to rescue the septin defects found in our GPCR signaling mutant. Instead, receptor mutants are unable to selectively deposit septins, leading to aberrant septin distribution. Our results suggest that selective receptor endocytosis is critical to proper septin distribution, which may provide insight for future research in neuron development and disease.