Recorded five-minute presentations for the Undergraduate Scholarly Showcase in Category D: New Frontiers, Projects D-01 through D-35.
D-01: 1H NMR Investigation of Concentration and pH of Amino-Acid Derived Surfactant System Behavior
Claudia Macri, Chemistry
Project Advisor: Dr. Gerald Kasting
Amino-acid derived surfactants are of increasing interest in the personal care industries for their environmentally friendly properties and as an alternative to "sulfates", as the market deems them too harsh. The goal of this project was to provide supporting insight in the investigation of thickening mechanisms of amino-acid derived surfactant compositions. Our approach was to use proton NMR to identify interactions between sodium laurel sarcosinate (SLSar), an amino-acid derived surfactant, and cocoamidopropyl hydroxysultaine (CAHS), a zwitterionic surfactant, as they interact in mixed micelle systems. Amphoteric surfactants like CAHS and the closely related betaines are often used in anionic-based formulations to mitigate harshness and also to contribute to thickening of the formulation.
Aqueous solutions of SLSar and CAHS at their natural pH (7-8) were prepared in deuterated water at concentrations of 0.1, 1, 10, and 100 times the critical micelle concentration (CMC) of each surfactant and analyzed at room temperature by 1H-NMR. One SLSar solution was titrated to a pH range of 4.8-5.0 with citric acid and reanalyzed, and a 1:1 molar mixture of SLSar and CAHS at pH 4.8-5.0 was also studied. At the time of this writing we are analyzing and interpreting the 1H-NMR chemical shifts due to (1) protonation of the sarcosinate at low pH and (2) interaction of the sultaine with the sarcosinate at low pH. We hope to have more information by the time the project wraps up.
D-02: A Mutant Fibrinogen that is Unable to Form Fibrin can Improve Renal Phenotype in Mice with Sickle Cell Anemia
Blair Hoeting, Biological
Project Advisor: Dr. Md Nasimuzzaman
Sickle cell anemia (SCA) causes nephropathy, including tubular pathology, manifests as urine concentrating defect and glomerulopathy, manifests as albuminuria that progresses to kidney failure. Besides clot formation, fibrin(ogen) is responsible for inflammation, tissue injury, and wound healing. However, the precise roles of soluble fibrinogen vs insoluble fibrin polymers in the disease processes were impossible to investigate until the generation of FibAEK mouse that bears a germ-line mutation in the fibrinogen Aα chain at the thrombin cleavage site. Thrombin cannot cleave and release fibrinopeptide-A from this mutant fibrinogen that fails to form fibrin polymer. However, the role of fibrinogen vs fibrin in kidney damage in SCA is not investigated. To determine if soluble fibrinogen (FibAEK) can prevent kidney damage in mice with SCA, we performed bone marrow transplantation (BMT) of Berkeley sickle mice into irradiated wild-type fibrinogen (FibWT) and FibAEK mice, and analyzed kidney functions and pathology. We found reduced albuminuria in SS FibAEK mice compared with SS FibWT mice at 12 months post-BMT. Kidney sections of SS FibAEK mice had reduced kidney fibrosis, ischemic lesions, and increased survival of podocytes in the glomeruli compared with SS FibWT mice. However, urine concentrating ability was not improved in SS FibAEK mice compared to SS FibWT mice. Therefore, FibAEK mutation offers protection from the development of glomerulopathology resulting in reduced albuminuria in mice with SCA but does not affect urine concentrating ability. FibAEK mutation may represent a novel therapeutic target to protect against kidney damage.
D-03: Inhibition of Solar Ultraviolet Radiation-Induced Oxidative Stress in Human Melanocytes by MC1R Agonists
Madeleine Peck, Biological Sciences
Project Advisor: Dr. Zalfa Abdel-Malek
Melanocytes are skin cells found in the epidermal basal layer, the border of the upper skin layer and the lower dermis. Melanocytes produce melanin, the skin pigment that aids in protecting skin cells from harmful ultraviolet radiation (UV) from the sun. Exposure of cells to UV increases the production of reactive oxygen species, which results in oxidative stress that can damage cellular lipids, proteins, and DNA, causing mutations that lead to melanoma, the deadliest form of skin cancer, or to vitiligo, the hypopigmentary disorder, due to death of melanocytes. The Melanocortin 1 receptor (MC1R) is the main regulator of melanocyte function, survival, DNA repair, and antioxidant capacity and is activated by alpha-melanocyte stimulating hormone (alpha-MSH). Alpha-MSH reduces DNA damage of melanocytes by activating the transcription factor nuclear factor erythroid 2 (Nrf-2), the master regulator of antioxidant genes, and p53, a regulator of DNA repair pathways. Heme oxygenase-1 (HO-1) and peroxiredoxin 1 (PRDX-1) are proteins that aid in reducing oxidative stress and are Nrf-2 targets. The Abdel-Malek laboratory has developed short peptide analogs of alpha-MSH, and demonstrated their efficiency in activating MC1R, stimulating melanin synthesis, and repairing UV-induced DNA damage in human melanocytes. These peptides are now being tested for their ability to reduce oxidative damage caused by solar UV and their role in antioxidant genes expression using Western blot and flow cytometry analysis. The ultimate goal is to develop these peptides for topical application to mitigate the harmful effects of solar UV and prevent melanoma and vitiligo.
D-04: Adeno Associated Virus Transforming Gene Therapy
Grace Fowler, Biological Sciences
Project Advisor: Dr. Daniel Buchholz
I did research at Nationwide Children's Hospital on the Flow Cytometry floor, specifically for gene therapy. Gene therapy introduces new genetic material into cells to replace nonfunctioning genes to make a specific protein. The purpose of my research was to study how Adeno Associated Virus (AAV), specifically AAV9 has impacted gene therapy. AAV9 is a particular virus that does not cause disease and is used to deliver normal copies of genes to the tissues and organs that are not functioning properly. In the Flow Lab, we performed ELISA assays to validate the presence of AAV9 antibodies in patients. ELISA assays have been validated to be both safe and effective tests. These assays were important because if the patient had the AAV9 antibodies, they did not qualify for the gene therapy. Using Adeno Associated Virus, we take out the viral components at the ITR sites and replace the DNA to target specific genes. The ELISA assays were also important to perform on patients undergoing the gene therapy because it allowed us to monitor their immune response. During my research, I was trained to perform the assays and found my patient to test positive for having AAV9 antibodies. Adeno Associated Virus has become the leading platform for gene therapy.
D-05: Annotation of Microbial Genome with Potential Extracellular eEectron Transport Capabilities
Waleed Amir, Biological Sciences And Neuroscience
Project Advisor: Dr. Annette Rowe
The diversity of microbial metabolisms is constantly being expanded and characterized. This includes understanding of how microbes use different inorganic materials under different environmental conditions. To better understand the metabolic capabilities of Idiomarina loihiensis str. FeNA, isolated from marine sediment cathodes, we sequenced and annotated its genome. The genome was annotated and assessed using various bioinformatic resources. I. loihiensis has 2.9 MB genome that encodes approximately 2,786 genes. Comparing these genes against the Kyoto Encyclopedia of Genes and Genomes (KEGG), we see that this organism has a wide range of carbon utilizing pathways, consistent with eating organic carbon, but lacks a complete respiratory pathway for anything but oxygen (e.g., nitrogen or sulfur). In this work we aimed to investigate the microbes ability to oxidize metals through the investigation of novel (uncharacterized) cytochrome containing genes since these are not well characterized. We so far have identified 8 cytochrome containing genes, 2 of which are localized on the membrane or extracellularly which supports a potential role in extracellular electron transfer. Further investigation of these genes could provide insight into extracellular electron transfer which is important because it plays a key role in bioremediation and microbe-electrode technologies.
D-06: Assessing Sex Differences in Threat Responding to Posttraumatic Stress Disorder (PTSD)-- Relevant Challenges in Mice
Allison Wilson, Biological Sciences
Project Advisor: Dr. Renu Sah
Post-traumatic stress disorder (PTSD) is a fear-associated disorder that afflicts approximately 6 million individuals annually with higher prevalence in war veterans and females. PTSD is characterized by maladaptive threat responding and persisting trauma-associated fear memories. There is considerable interest in identifying pre-trauma vulnerability factors that may promote PTSD. Pre-deployment studies in veterans showed a role of sensitivity to CO2 inhalation, a homeostatic stressor, in PTSD risk. We developed a mouse model of CO2 associated fear dysregulation where only CO2-sensitive mice develop enhanced fear phenotypes. Our previous study was limited to male animals and studies are currently lacking in females. Here we assessed sex differences in threat responding to CO2 and footshock fear conditioning, a PTSD relevant paradigm. Passive (freezing) and active (rearing) were measured for coping behaviors and neuronal activation (ΔFosB immunostaining) in fear-regulatory amygdala nuclei was quantified. Our data revealed heterogenous freezing and rearing behaviors to CO2 in male and female mice. Females demonstrate significantly higher rearing in both CO2 and fear conditioning phases indicative of active coping behaviors. This was accompanied by alterations in amygdala FosB counts, an effect not observed in male mice. Our data indicate that both male and female mice elicit differential CO2-sensitive distribution similar to humans but significant differences in defensive coping, with females showing active threat responding. Differences in fear circuit recruitment might contribute to these effects. In conclusion, our CO2-fear conditioning paradigm provides a useful model to investigate mechanisms promoting PTSD risk in a sex-dependent manner. Funding Support: VA Merit award (RS)
D-07: Understanding the Biology of the Freshwater Brain-eating Amoeba
Aqsa Raja, Biological Sciences
Project Advisor: Dr. Yoshi Odaka
Naegleria fowleri is a pathogenic unicellular protist of free-living ameba commonly found in warm freshwater and undergoes three stages of the life cycle, amebic trophozoite, flagellate, and metabolically dormant cyst, depending on the environmental conditions. However, the trophozoite is the only form that invades the central nervous tissues through the nasal cavity and causes highly fatal primary amebic meningoencephalitis (PAM). In this study, the non-pathogenic species, Naegleria gruberi (NEG-M strain, ATCC 30224), was assessed its sensitivities to Streptomyces-derived macrolides rapamycin and FK506, and the ATP-competitive TOR (target of rapamycin) inhibitor, Torin-1. Although expressing the common macrolide receptor, FKBP12, N. gruberi was resistant to the allosteric inhibitors for TOR kinase (rapamycin) and calcineurin phosphatase (FK506) in terms of its viability and locomotion, while showing the vulnerability to Torin-1 in a dose-dependent manner (IC50 = 7.79 nM). Moreover, Torin-1 treatment induced the formation of cyst-like structure and greatly affected ameba's locomotion, suggesting TOR's potential regulatory role in the morphological differentiation and actin filament polymerization in the Naegleria genus.
D-08: Characterizing Blast Waveforms for Traumatic Brain Injury
Mei-Ling Liber, Biological Sciences
Evan Reeder, Pharmaceutical Science
Project Advisor: Dr. Matthew Robson
The increased prevalence of improvised explosive device (IED) encounters in Iraq and Afghanistan has greatly increased the number of blast-induced traumatic brain injuries (bTBI) among military personnel. Reliable models of TBI are critical to understanding the underlying molecular mechanisms of acute and long-term comorbidities associated with bTBI. Compressed gas-driven shock tubes have been used by laboratories as a common approach to replicate explosive blast waves to model bTBI in animal subjects. Blast waves scaled for animals should expose subjects to the highest possible peak pressure while minimizing the blast duration. The effects of driver gas selection on the resulting blast-waves have not been well characterized, although previous research has shown that the driver gas has strong influence on the blast wave parameters: magnitude (peak overpressure), duration (positive phase duration), and shape. To demonstrate this, we utilized a murine model of bTBI in conjunction with a variety of driver gases: helium, ambient air, carbon dioxide, argon, and nitrogen. We hypothesized that helium, due its low molecular weight, would produce the closest pressure time profile to the desired blast wave shape. Driver gas molecular weight was a strong predictor of mean peak overpressure (R2 = 0.98) and mean positive phase duration (R2 = 0.91), where helium had the highest mean peak overpressure (1162.16 kPa) and the shortest mean positive phase duration (1.03 ms) compared to other gases. In conclusion, we recommend using helium to produce scaled blast waves that can be used as reliable models for blast-induced TBI in military-affiliated populations.
D-09: Contact Model for Mobile Robot Swarming
Benjamin Russ, Mechanical Engineering
Project Advisor: Dr. Tamara Lorenz
Mobile robots have entered society at a crucial moment as powerful and flexible robotic tools have become a necessity for many complex problems; dynamic spaces being one of the most difficult. Current methods for dynamic spaces range from single robot mapping and navigation to multi-robot collaborative systems. This research hopes to expand on multi-robot collaborative systems through self-organizing behavior which utilizes contact information created between connected robot agents. The connected system will then navigate as a connected body to an object which they will attempt to push to a target position. The coupling of self-organization and moving as a connected body will create a swarming system capable of reconfiguring the connected body based on the sensed contact information. As a result, this swarming system will be able to push the object which would be individually impossible. How would this work when the system initializes and there is no contact between the robots? Using ultrasonic sensing they will randomly move around until they are able to successfully detect another agent and drive closer to them. This will repeat until most or all have assembled into one larger body and will drive towards the target maintaining contact and collectively moving together. This behavior will all be completed without any global information and actions will be decided purely on local sensing information. The benefit of this research has many layers and hopes to encourage the field towards contact behavior development.
D-10: Creating a Pipeline for Recurrent Respiratory Papillomatosis
Adrean Carlile, Biological Sciences
Project Advisor: Dr. Mary Bedard
The beginning purpose of this project was for me to become familiar with simple lab techniques, so I can confidently execute needed experiments. Once confident in my techniques I moved on to working on a project dealing with Recurrent Respiratory Papillomatosis (RRP). This disease has recurrent tumors on the air way that is driven by human papilloma virus (HPV) that has no curative options. This is a very understudied disease so we established a cell population that mimicked patient biopsies. For this project I ran Quantitative Polymerase Chain Reaction to test the ability of the cell cultures to retain HPV. I also ran Immunofluorescence (IF) on 3D rafts for characteristics similar to RRP diseased cells. We found that our 2D cells did retain the HPV gene expression that was consistent across patients. We also found that our 3D rafts had the same organization of the tissue of origin. These finding will develop a pipeline for further understand the disease.
D-11: Creation of a Null ADAM17 Cell Line to Probe for Details in Molecular Function
Nicholas Shaw, Medical
Project Advisor: Dr. Tom Seegar
Ectodomain shedding is an essential biological process in human development that results in a non-reversible release of membrane-tethered substrates, by a protease, into the luminal or extracellular space. A disintegrin and metalloproteinase 17 (ADAM17) is one of 21 human ADAM family proteins that function in ectodomain shedding in cell adhesion. Specifically, ADAM17 is responsible for the shedding of tumor necrosis factor α (TNFα), a potent cytokine, and precursory epidermal growth factor molecules. Accordingly, dysfunctional ADAM17 activity is implicated in chronic inflammatory disease and cancer progression. While ADAM17 has essential roles in human development and disease pathogenesis, a clear molecular understanding of its regulation and substrate selectivity remains unknown. To help us study ADAM17 regulation, we will make an ADAM17 null cell line using CRISPR/Cas9 genome-editing technology and validate the loss of the ADAM17 gene and protein. With the ADAM17 null cell line, we will use structure-guided mutations to study the importance of specific regions in ADAM17 on the processing TNFα. This study will identify regions of ADAM17 that are required for biological activity.
D-12: Developing Nerve Guidance Conduits for Peripheral Nerve Injury Treatment using Piezoelectric Scaffolds
Corinne Smith, Chemical Engineering
Project Advisor: Dr. Greg Harris
More than 20 million people in the United States suffer from peripheral nervous system (PNS) injuries, often due to car accidents or crushing injuries after falls. PNS injuries possess a limited amount of regenerative potential, mainly due to the presence of Schwann cells within the PNS, which can transdifferentiate into a regenerative cell type to guide axons across nerve lesions and support nerve recovery. However, despite this innate regenerative capacity of the PNS, there are limited clinical solutions for traumatic PNS damage over approximately 2 cm in size. In recent years development of nerve guidance conduits has been explored as an alternative clinical solution to eliminate nerve size mismatch, multiple surgeries, and comorbidity associated with autografts. To address this need, we have developed and analyzed the effectiveness of a piezoelectric electrospun scaffold as a potential building block for nerve guidance conduits, as well as investigated the response of Schwann cells to the scaffolds as an avenue for promoting nerve regeneration. Our results demonstrated that the scaffolds exhibited appropriate mechanical and piezoelectric characteristics to produce a microenvironment suitable for regeneration. Additionally, scaffolds supported cell adhesion and alignment on scaffolding, illustrating biocompatibility. This ongoing work will ultimately contribute to an increased understanding of Schwann cell response to different scaffold properties, which will provide novel insight to the development of biomaterials for nerve repair. The development of conduits using piezoelectric materials can ultimately lead to the production of safer and more effective peripheral nerve injury treatments.
D-13: Engineering Flourescent Fusion Protein with Novel Electron Uptake Gene in Shewanella oneidensis
Anna Cunnane, Biological Sciences
Project Advisor: Dr. Annette Rowe
Mineral reduction via extracellular electron deposition onto solid phase minerals and/or electrodes has been well characterized in several organisms, but the reverse of this process has yet to be characterized. For example, the MTR respiratory pathway in the Shewanella oneidensis strain MR-1 is known to be involved in electron deposition, but only accounts for some of the electron uptake under aerobic cathodic conditions. Using S. oneidensis as a model system, our work recently identified five genes that are uniquely involved in electron uptake and not electron deposition, however the functional role of these proteins is unclear. One of these genes, SO3662, is thought to produce a ferredoxin (Fe-S protein) involved in extracellular electron uptake. We predict SO3662 associates at the cytoplasmic membrane and helps transfer electrons to the terminal cytochrome oxidase to allow for energy conservation. This research aims to characterize the localization of this protein by generating gene fusion with the fluorescent marker cyan fluorescent protein (CFP). SO3662 was PCR amplified and inserted into a custom E. coli plasmid that contained CFP. The plasmid was cut on the N-terminal end of the CFP and the gene was inserted in-frame at restriction enzyme sites. The plasmid was transferred from E. coli into S. oneidensis via conjugation. SO3662 will be localized in the cell using live fluorescent microscopy techniques, and possibly TEM. Results of this work will help inform the role SO3662 plays in electron uptake and could help better engineer or optimize this system for microbe-electrode technologies such as electrosynthesis.
D-14: Exploring the Biological Sulfur-Oxidation Cycle Using Genetic Engineering
Vageesha Herath, Biochemistry
Project Advisor: Dr. Annetta Rowe
Sulfur is an abundant non-metal found in the universe and plays an important role as a structural element, carbon carrier, and redox center. Microbial activity has a major impact on sulfur cycling globally, yet the biochemistry of many solid phase sulfur minerals remain somewhat elusive. The marine bacterium Thioclava electrotropha ElOx9 is known for its sulfur oxidation ability, via the Sox pathway The Sox pathway, is found in a variety of microbes and encodes for proteins involved in thiosulfate oxidation. These proteins are largely periplasmic and how electrons from extracellular and/or mineral sulfur species remains uncharacterized. The project examines the role of genes Sox C and Sox D within the Sox pathway and its potential role in extracellular sulfur oxidation. Sox(CD) is a periplasmic multiprotein enzyme complex comprised of two molybdoproteins (SoxC) and two diheme c-type cytochromes (SoxD). To understand the functions of these genes, a mutant strain of Thioclava was created where the Sox C and Sox D genes were deleted. The SoxCD knockout strain will be evaluated for the ability to oxidize inorganic sulfur compounds by plating mutant strains on agar plates containing thiosulphate or extracellular solid phase sulfur. The above methodology will provide insights into the function of SoxCD will regard to thiosulfate oxidation (diffusible to the periplasm) and extracellular sulfur. Continued experimentation will focus on the potential role of SoxCD in extracellular electron transfer with an electrode. Understanding the mechanism behind inorganic sulfur oxidation in Thioclava will help researchers to understand the extracellular electron transfer mechanisms.
D-15: Progress Towards Developing Gene Therapy to Treat Amyotrophic Lateral Sclerosis (ALS) Disease
Jessica Do, Biological Sciences
Project Advisor: Dr. John MacLennan
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that affects motor neurons (MNs) and impairs voluntary muscle movements, which ultimately leads to symptoms of paralysis and death. There is currently no treatment or cure, and the etiology of this sporadic disease is unknown. Evidence suggests that ciliary neurotrophic factor (CNTF) receptor signaling can protect MNs, promote their natural regeneration, and slow progression of neurodegenerative diseases involving MNs, including ALS. To investigate the functions of CNTF receptors in ALS, previous work in our lab disrupted the essential ligand-binding sub-unit of the receptor, CNTF receptor alpha (CNTFRα) in muscle. This muscle specific CNTFRα knockdown increased disease progression in superoxide dismutase 1 (SOD1G93A) ALS model mice indicating a role for muscle CNTFR in protecting MNs. Here we show that increasing muscle CNTFR with AAV gene therapy slowed disease progression without side effects in the SOD1G93A mice. When using the same approach and gene- manipulation technique on the CNTF receptor ligand, cardiotrophin-like cytokine (CLC), there were similar outcomes: CLC increased with AAV gene therapy it inhibited disease in the ALS mice. We conclude the increase of CNTFRα and CLC have positive clinical impacts on reducing ALS symptoms and regenerating MNs.
D-16: Genome Sequence of Idiomarina loihiensis, a Gram-Negative Novel Proteobacteria Capable of Mineral/Electrode Oxidation
Casey Norman, Biological Sciences
Project Advisor: Dr. Annette Rowe
At present little is known about the genetic mechanisms of mineral/electrode oxidation, in part due to the lack of microbes in culture that have this metabolism. Here we are diving into the genome sequence of Idiomarina loihiensis isolated from Catalina sediments, named for a species that was isolated from iron mates on the Loihi submarine volcano in Hawaii. This bacterium is capable of aerobic respiration on an oxidative anode, however, the genes that allow this process to occur remain unknown. I. loihiensis has a 2.8 megabase genome that contains 2,644 protein-coding genes. The genome has a GC percent of 47.02 % and an average nucleotide of 95 compared to the type strain 97.34. Our genomic investigations demonstrate this organism is metabolically flexible, containing many carbon utilizing pathways. However, no genes for Nitrogen and Sulfur metabolic pathways were observed. Investigating genes that contain heme motifs, we identified 9 genes found throughout the organisms genome that are potentially involved in extracellular electron transfer. Future work will test the activity of these genes in electrode-oxidation.
D-17: How Supplemental Oxygen Affects Traumatic Brain Injury
Jordyn Torrens, Cell Biological Sciences
Project Advisor: Dr. Shelby Hetzer
Traumatic Brain Injury (TBI) affects 2.8 million people each year, Hypoxemia (low concentration of oxygen) is found to cause lesion enhancement in the brain, and Hyperbaric Oxygen Therapy is proven to reduce the risk of death and improves the level of coma. These statements have helped to guide our research question, "How supplemental oxygen affects TBI's." Using a traumatic brain injury weight drop model, the affects of 100% oxygen on these TBI's are studied through neurodegeneration and astrocyte activity. Oxygen is found to increase survival, decrease seizures, stimulate the brain's immune response, and decreases inflammation after a Traumatic Brain Injury.
D-18: Identifying Target Sites for Placental Therapeutics through the Comparison of Normal Term Pregnancies and Intrauterine Growth Restricted Proteomes
Amanda Bowman, Medical Laboratory Science
Project Advisor: Dr. Helen Jones
A variety of pathologies, including intrauterine growth restriction (IUGR), have been linked to placental insufficiencies as important causal factors, however, little remains understood in the way of molecular development of said conditions. In order to recognize developmental dispositions that may characterize pathologies, we observed the proteome of the syncytiotrophoblast membranes from a normal term pregnancy, identifying several key genes, proteins, and pathways that could play an important role in conditions such as IUGR. Distinct expression of membrane proteins (CDC42, CD59, RAB5C, and STBP) show promise in understanding the role of cytoskeletal development, inflammatory regulation, and cellular signaling in both healthy and adverse pregnancy outcomes. Also discovered were receptor proteins such as CALR and EGFR whose mechanistic functions may be critical to avoiding placental insufficiencies. Joined with supporting pathway identifications, critical points of interest involving Rho GTPase signaling, interleukin signaling, and IGF signaling pathways are seen as promising development sites that may lead to a better understanding of IUGR and potential therapeutic intervention.
D-19: Investigating the Kinetics of Solute Binding to Keratin: Preliminary Data of Theophylline Desorption
Emmalee Bane, Chemistry
Project Advisor: Dr. Gerald Kasting
Dermal absorption is important to the pharmaceutical, cosmetic, and personal care industries for its application in the development of topical products and transdermal drug deliveries, as well as to government agencies for safety regulation of occupational and environmental exposure to chemicals. Keratin is the predominant protein in the stratum corneum layer of skin, thus its binding to solutes remains a significant contributing factor to predictive models for transient dermal absorption that have been previously and ongoingly studied. Theophylline is currently being studied as the solute of interest for keratin binding as it poses relevant use in the skin care community. The goal of this project was to assist in the investigation of experimental methods and measurements of keratin binding kinetics that would aim to complement and expand existing data. This portion of the research demonstrated keratin dispersion purified through dialysis membrane cells, equilibrium uptake of theophylline, and sequential desorption of theophylline bound to fresh sodium acetate buffer. UV/Vis spectrophotometry was utilized in the measurement of desorbed theophylline. The investigation will be continued with analysis of equilibrium binding data and kinetics and with studying nicotinamide, methyl nicotinate, benzophenone, and avobenzone as solutes.
D-20: Metal Nanoneedle-Based Ion Channel Probes for Single-Molecule Detection
Kelly Klein, Biological
Project Advisor: Dr. Ryan White
Ion channels play an important role in cell functioning and signaling. Recently, ion channels have been used as nanopore sensors for the detection of biomolecules such as ssDNA, miRNA, and peptides. Metal nanoneedle-ion channel probes have recently been developed to support lipid bilayer formation and ion channel recordings. Preliminary data suggests that although gold nanoneedles can be used for detection of B-cyclodextrin molecules, they are limited by the current decay resulting from the double layer charging at the electrode surface. Here, we used electrochemically etched silver nanoneedles which allow for the formation of an AgCl layer, producing a more stable current. Afterward, the surface of these probes was modified to support an aqueous layer around the probe tip which is essential for bilayer formation. The bilayer was formed by contacting two lipid monolayers between the tip of the nanoneedle and the aqueous layer. Once a stable bilayer is acquired, ion channel proteins can be reconstituted into this membrane causing free ion movement between two electrolyte sides. When a target molecule binds to a protein pore, it will physically block the protein channel and blockage currents can then be characterized. By optimizing nanoneedle material, more stable channel current recordings can be obtained. Further, these probes will allow for better control over protein insertion and deinsertion across an aqueous mixture. This project is important because it will lead to the development of ion channel probes that overcome lipid membrane instability and maintain control over bilayer formation, allowing for more stable measurements.
D-21: MicroRNA Mediated Modulation of Electrographic Activity in a Cntnap2 Mouse Model
Rishav Mukherjee, Medical Sciences
Project Advisor: Dr. Durgesh Tiwari
Rationale: Cntnap2 encodes contactin-associated protein-like 2 and plays a vital role in neuron-glia interaction and action potential propagation. Mutations in Cntnap2 are associated with epilepsy and autism. Cntnap2 knockout mice exhibit seizures and autism related behavioral deficits however the mechanism leading to the network excitability is unknown. MicroRNAs (miRNAs) regulate post-transcriptional expression of mRNA and can be used to regulate gene expression. MiR-324-5p and miR-218-5p are notable miRNAs which are involved regulating seizure susceptibility in mouse models of epilepsy. We used antagomirs (antisense oligonucleotides) to explore the neuroprotective potential of miR-324-5p and miR-218-5p in Cntnap2 a mouse model and analyzed the changes in electrographic activity.
Methods: Mice were genotyped and investigated at younger (4-6 months) and older (12-16 months) age points to determine age-specific seizure development. Seizures were confirmed using the tail-suspension assay and cortical EEG recordings. Mice were implanted with electrodes and injected with miR-324-5p, miR-218-5p, or scrambled antagomir at both age points, and EEG monitored for a week. Younger mice underwent a kainic acid seizure onset challenge.
Results: At younger and older age point miR-324-5p antagomirs delayed the seizure onset and severity and miR-218-5p antagomir-treatment increased seizures and seizure susceptibility compared to SCR control. EEG waveform and spike analysis showed no significant difference in EEG waveforms between antagomir treatment and SCR control (preliminary data).
Conclusion: This study demonstrated the potential for miR-mediated regulation of seizures in Cntnap2 KO mice. Ongoing studies will aim to explore the localization of the effects using immunohistochemistry and sleep-wake patterns in EEG activity.
D-22: Genetic Mutation in Drug Transporter explains Side Effects in Pediatric Inflammatory Bowel Disease
Rishi Mehta, Medical Sciences
Project Advisor: Dr. Laura Ramsey
Low-dose methotrexate (MTX) is an immunosuppressant used to treat Inflammatory Bowel Disease (IBD). SLCO1B1 genetic variation has been associated with delayed MTX clearance and increased toxicity. The purpose of this study was to evaluate the association between genetic variation in SLCO1B1 and MTX-induced nausea in children.
A retrospective chart analysis of 278 patients <19 years of age who were prescribed MTX for IBD at Cincinnati Children's Hospital was performed. 202 patients had banked DNA and were genotyped for 3 SLCO1B1 single nucleotide polymorphisms (rs4149056, rs2306283, and rs11045819). Diplotypes were determined by combining the SNPs into *1a, *1b, *4, *5, *14, and *15 alleles. Nausea was abstracted from clinician notes. Prescriptions and demographics were extracted from the medical record.
The cohort was 69.5% male and 89.2% white with a mean age of 15.4 (±3.9) years. MTX intolerance and MTX-induced nausea were noted in 51% and 34% of the cohort, respectively. MTX-induced nausea was significantly associated with SLCO1B1 diplotype (p=0.006) after accounting for MTX dose group and whether treatment was initiated with concomitant ondansetron. MTX-induced nausea was dependent upon the number of reduced function *15 alleles (p=0.0344) and occurred 2.26x more often in patients with at least one *15 allele who did not initiate MTX treatment with concomitant ondansetron (p=0.034).
Our data demonstrate that the SLCO1B1*15 allele is associated with MTX-induced nausea in pediatric patients with IBD. Additionally, patients with at least 1 *15 allele could benefit from a dose reduction of methotrexate to reduce exposure and concomitant ondansetron to reduce nausea.
D-23: Stress and the Expression of PACAP in Basolateral Amygdala of Male and Female Rats
Anna Enders, NeuroBiological Sciences
Project Advisor: Dr. Ilya Vilinsky
Abstract provided in completed project.
D-24: Progress Towards Understanding the Relationship Between How the Brain Removes Plaque-Forming Beta-Amyloid Proteins and Alzheimer’s Disease
Owen Traubert, NeuroBiological Sciences
Project Advisor: Dr. Shailendra Patel
Alzheimer’s Disease is the 6th leading cause of death in the United States and cannot be cured, prevented, or slowed. A major pathological underpinning of Alzheimer’s is buildup of beta-amyloid (Aβ) plaques. Mechanisms affecting Aβ buildup, including its excretion, are therefore an important line of Alzheimer’s research, especially in identifying possible targets for treatment.
ABCG4 is a transport protein that is present in brain tissue. It has been demonstrated in samples of the blood-brain barrier to effectively transport Aβ and is thought to export it from the brain into the bloodstream. We used a mouse model of Alzheimer’s, a mouse line (J9) modified to express the Indiana and Swedish mutations, which cause types of human hereditary Alzheimer’s and result in Alzheimer’s-like behavior in mice. Some of these mice had ABCG4 production disabled. We tested the hypothesis that loss of the ABCG4 transporter would exacerbate the Alzheimer’s-like behavior.
We analyzed the following genotypes: ABCG4+/+, J9+ (control), compared to ABCG4-/-, J9+; ABCG4+/-, J9+; and ABCG4-/-, J9- mice with both sexes as separate groups. Three behavioral tests were performed on these groups: open field test (measures anxiety), novel object recognition (measures conceptual memory), and novel object placement (measures spatial memory). We found no significant differences between groups. This indicates that ABCG4 does not play a central role in Alzheimer’s, and that if it does have a role, it is redundant. Either its absence can be compensated for by another Aβ-transporting protein, or Aβ transport across the blood-brain barrier is not relevant to Alzheimer’s.
D-25: The Study of Biomarkers in Neurodegenerative Diseases
Alexa Amato, Neuroscience
Project Advisor: Dr. Alberto Espay
The purpose of this study is to learn what causes types of neurodegenerative diseases and learn why the diseases are happening. The two diseases focused on in the study are Parkinson's disease(PD) and Alzheimer's disease(AD). The patient will be asked to give samples, complete questionnaires, and use devices during their yearly visits/clinical assessments. The concentration is to look at the environmental and genetic factors specifically using biomarkers. The study is ongoing and hopes to end before 2029. The goal is to slow or stop the progression of biological defined-types of neurodegenerative diseases.
D-26: The Synthesis and Use of Rotaxane Molecules as Transfer Agents for Ions
Alyssa Tobin, Chemistry
Project Advisor: Dr. David Smithrud
Being able to selectively bind and transport varieties of particles to specific locations is an extremely useful strategy in areas such as targeting specific cells, such as cancer cells, and developing MRI contrast agents. Rotaxane molecules can selectively bind ions and act as this type of transport agent. We synthesized rotaxane molecules, specifically rotaxane-18-crown-6, in the laboratory in order to test its ability to transfer a variety of particles- in this case, metal cations. Different metal cations (positive ions) are transferred by these rotaxanes at different concentrations. To detect and measure cation concentrations, we need to use a sensitive instrument that can detect trace levels of ions. Inductively coupled plasma mass spectroscopy (ICP-MS) provides the necessary sensitivity to measure very dilute concentrations of elements in solution. We utilized ICP-MS to measure the levels of cation transfer to the rotaxane molecules synthesized in the lab. After analyzing transfers for calcium, sodium, lithium, and potassium cations to the rotaxane, we found that the calcium ion is transferred at the highest concentration.
D-27: Treatments for Amyotrophic Lateral Sclerosis (ALS)
Caitlin Pastor, Biological Sciences
Project Advisor: Dr. John MacLennan
Our laboratory is working on developing novel treatments for amyotrophic lateral sclerosis (ALS), an invariably terminal motor neuron disease with not currently effective treatment. We use mice that have genetic mutations that cause ALS in humans and the mice. Before I joined the laboratory, they identified natural, endogenous, growth factor signaling processes that inhibit ALS in the mice. We are now using the mice to test whether we can further inhibit the ALS by boosting this natural protective system with targeted gene therapy. The hypothesis driving the research is simply that boosting these growth factor systems will inhibit the ALS. I will be performing behavioral tests on the treated and control mice to determine how the treatments affect the disease. Our hope is to identify one or more treatments that significantly inhibit the disease in mice carrying different ALS causing mutations. This work may then justify the much more expensive and lengthy human trials that will be needed before the treatments can be approved for human ALS.
D-28: Using Innovative Sequencing Technology to Describe Nurse Macrophages Supporting Red Blood Cell Production
Dayle de Graaf, Biological Sciences
Project Advisor: Dr. Theodosia Kalfa
Millions of patients worldwide with cancer, sepsis, or autoimmune diseases such as rheumatoid arthritis, become anemic because the bone marrow shifts from producing red blood cells to white blood cells in an effort to combat the disease. This decrease in red blood cell production, known as anemia of inflammation, compromises significant quality of life for patients. It has long been known that maturing red blood cells rely on a nurse macrophage cell to aid in development, provide iron, and digest extruded nuclei prior to the final step of red blood cell maturation. Our lab has published findings that red blood cell and white blood cell maturation may be closely intertwined by use of the same nurse macrophage cell. We hypothesize that this cell acts as a balance between red blood cell and white blood cell production. In order to target nurse macrophages to increase red blood cell production in anemia of inflammation, a deeper understanding of their proteins, or units that allow a cell to carry out its specific function, and their mRNA, or blueprints for the protein, is needed. To accomplish this, we have employed a cutting edge sequencing technique called CITE-sequencing, which allows for simultaneous evaluation of the mRNA and protein in nurse macrophages. CITE-sequencing reveals protein and mRNA heterogeneity within populations of nurse macrophages supporting red blood cell maturation. Future studies of this data set will be focused on validating these results through antibody staining and qPCR and identifying potential targetable pathways.
D-29: The Methods and Implications of Chinese and French Foreign Direct Investment in Africa
International Affairs and History
Project Advisor: Dr. Albert Klein
With the shift in the modern era from conventional warfare to more impersonal forms of conflict, it is important to maintain awareness of the more prevalent methods in which nations engage in hostile actions. This study seeks to examine one of these newer methods, economic warfare, in the context of the French and Chinese governments' methods of Foreign Direct Investment (FDI) into developing states on the African continent. This was done through an analysis of both investment data provided by the United Nations Conference on Trade and Development (UNCTAD) and analysis of research articles other scholars have done regarding France's Françafrique region and China's Belt and Road initiative. FDI presents a concept with a lot of nuance because it is not an explicitly violent action to invest in a given economy, but opens the door for quiet methods of exerting control over them. Other scholars argue that both the "East" and "West" invest in resource rich and corrupt institutions for their own benefit, but viewing any type of economic warfare through a Cold War focused framework does the analysis no academic justice. The results in this study suggest that the repercussions of these investments either facilitate corruption (in the Chinese case) or shape an oppressive economic structure (in the French case), making both sides of the geopolitical "spectrum" unsavory in one way or another.
D-30: Understanding How Different Types of Exchanges Using Blockchain Technology Affect Price Transparency and Diffusion in Comparison to Traditional Exchange Markets
Nicholas Zimmerman, Finance and Economics
Project Advisor: Dr. Michael Jones
The purpose of this paper was to explore how/if exchanges on blockchain technology can offer a more beneficial option to users in comparison to how traditional markets operate. My process has been diving into how traditional markets work, as well as centralized and decentralized exchange markets that exist on the blockchain. The appeal to multidisciplinary audiences is that blockchain technology has the potential to be used in many fields. The audience may take this information to be applied to their own discipline. In addition, people of all audiences tend to either operate in the markets or have someone who operates in the markets for them. For that reason as well, my research has relevance to most everyone.
D-31: Chemistry 2.0: Connecting Molecules Together Without Solvent
Hunter Starbuck, Chemistry
Project Advisor: Dr. James Mack
Most pharmaceuticals are not found in nature but must be made through organic synthesis. Traditional organic synthesis of pharmaceuticals is a wasteful process, with most of that waste being attributed to solvent. Nucleophilic aromatic substitution reactions (SNAr) are popular reactions in medicinal chemistry that apart from being wasteful, also rely on polar aprotic solvents which are harmful to people and the environment. By implementing a mechanochemical system, these reactions could be carried out without solvent, solving both forementioned problems. The mechanochemical system was able to achieve higher yields faster than traditional solvent systems.
D-32: Understanding the Substrate Binding Specificity of ribonuclease MC1 through in Silico Mutagenesis
Shun Satake, Biochemistry
Project Advisor: Dr. Balasubrahmanyam Addepalli
RNA is the intermediate between the genetic information in DNA and the work horses of cell, proteins. The building blocks (ribonucleotides) of RNA are chemically or enzymatically modified during their lifetime. While some of them are beneficial, others are harmful to human health. Locating the position of these modification in RNA is the first step in understanding the function and the impact on human health. For this, scientists employ biochemical tools such as Ribonuclease MC1. It cleaves RNA at the uridine building block. For its effective utility as biochemical scissors, thorough understanding of its cleavage specificity is essential. Towards this goal an in silico mutagenesis (substituting the native amino acids with a different one) was performed at two different positions. I hypothesized that π- π interaction between two ring structures of enzyme and substrate can improve the binding. I altered leucine at position 73 to a phenyl ring containing amino acid. Secondly, one of the three histidine residues in the binding site, His83 was also altered. Subsequently, I assayed the interactions between enzyme and substrate, a defined ligand, using bioinformatic tools, PyMOL and Hermes. I will discuss the observed effects on substrate binding and future studies in this presentation.
D-33: A novel prefrontal neurocircuit regulating fear-associated defensive behaviors relevant to PTSD
Sachi Shukla, Neuroscience
Project Advisor: Dr. Renu Sah
Post-traumatic stress disorder (PTSD) associates with dysregulated fear responding. Recent studies suggest PTSD patients show heightened reactivity to CO2 inhalation, a homeostatic stressor that triggers acidosis and evokes fear- and anxiety-associated responses. Mounting evidence suggests CO2 sensitivity exists prior to pathology and could predict vulnerability to develop PTSD following later trauma. To investigate this, we developed a mouse paradigm to examine the effect of CO2-inhalation on PTSD-relevant behaviors and found prior CO2 exposure compromised fear extinction in a contextual fear conditioning paradigm one week later. This associated with reduced neuronal activation within the infralimbic (IL) cortex, a region strongly associated with fear extinction learning. We previously reported the subfornical organ (SFO) as a key site regulating CO2-evoked fear and recently identified direct SFO projections to the IL cortex. This suggests SFO-IL projections could contribute to the lasting effects of CO2 inhalation. Here, we used an intersectional chemogenetic strategy to test the hypothesis that inhibiting SFO-IL projections during CO2-inhalation would attenuate CO2-evoked defensive behaviors and later extinction deficits. We examined active (rearing) and passive (freezing) coping responses to these threats. Consistent with our hypothesis, DREADD-Gi mediated inhibition of the SFO-IL circuit significantly reduced CO2-evoked defensive behaviors and prevented contextual fear extinction deficits. These data elucidate a novel SFO to IL projection that promotes convergence of homeostatic threat sensing and long-term modulation of fear responding. Our findings highlight the SFO as a novel hub through which interoceptive triggers can regulate vulnerability to external threats and trauma of relevance to PTSD.
D-34: The Understanding of Thermodynamics under Mechanochemical Conditions
Samantha Fry, Chemistry
Project Advisor: Dr. James Mack
The purpose of this research was to determine the thermodynamics of mechanochemical reactions, such as the ball milling reaction. More specifically how the entropy is effected during these reactions when the temperature is varied. In order to due this we used a heat associated mixer mill to run a reaction involving p-nitro, Sulfer, EtOCN, and morpholine at various temperatures (50- 120 degrees Fahrenheit) and time lengths (2- 8 hours). A solvent extraction was then done on the final product, and a column with 12g of silica was run to find the different components of the final product. Those different components found, along with a crude sample of the final product, were then analyzed using NMR tests. After a series of different reaction conditions were tested, there have been two main discoveries. The first being that reactions ran under conditions of 120* F produced too low of yields due to the high temperature because the base Morpholine evaporating before being able to react. The second being that the highest yields are discovered at the end of the 2 hour mark, and thereafter the yields appear to not change much. Other bases besides Morpholine, such as potassium and cesium carbonate, have also been tried using these same conditions but no reaction was found.
D-35: Experimental Drug Effective in Reducing Growth in Pediatric Brain Cancer Cells in Tissue Culture
Oluwadamilola "Dami" Omojola, Medical Sciences
Project Advisor: Dr. Biplab Dasgupta
Diffuse intrinsic pontine glioma (DIPG) is an aggressive form of pediatric brain cancer, currently with no effective treatment options due to its location in the hindbrain. Minimal understanding of the biological and biochemical nature of these tumors results in a poor prognosis with only 1% of patients surviving 5 years after diagnosis. This project aims to develop an effective treatment option for DIPG by investigating the efficacy of an experimental drug known as SHIN1. SHIN1 is a small molecule inhibitor that is known to inhibit glycine production in cells. Glycine plays a crucial role in the biosynthesis of the building blocks of DNA. It also functions in antioxidative capacities to detoxify the cell of the toxic by-products that result from a high metabolic activity which is characteristic of cancer cells. An increase in the production of glycine would support the rapid cell growth needed for tumor development. We investigate the inhibition of the cellular pathway of glycine production which is increased in these cancer cells. We tested this drug on brain cancer cells in Petri dishes, comparing them to normal brain cells to ensure that the drug would only target cancer cells. In glycine-free conditions, this drug showed a significant reduction in the growth of these cancerous cells.
D-36: Surface-enhanced Raman Scattering Sensors Design in Python
Project Advisor: Dr. Pietro Strobbia
No abstract was submitted