• Establishing a GFP Marker in Zebrafish to Study the Localization of Tinagl1

      Blackburn, Helena; Department of Biological Sciences (Augusta University, 2020-12)
      Tinagl1 is a secreted protein found in the basement membrane under epithelial cells. The LeMosy lab previously showed that tinagl1 knockdowns resulted in abnormal spinal development and heart orientation during zebrafish development. These data, together with changes in length of motile cilia, suggested that tinagl1 is involved in cilia function during development. The mechanism of this interaction is unknown, and it is unclear whether tinagl1 is only in basement membranes at the basal side of cells, or if it also localizes to the apical side of cells where most cilia project. A deeper understanding of the localization of tinagl1 during development is a logical next step in understanding how this protein functions. Zebrafish provide an excellent model for studying this localization as they display strong phenotypic effects that can be easily imaged. The localization of tinagl1 will be tracked using a tinagl1-GFP fusion construct developed through PCR and insertion into a Tol2 transposon vector. This construct will be injected into early embryos together with transposase mRNA to create mosaic fish showing tinagl1-GFP in selected tissues. Successful germline integration of the tinagl1-GFP DNA will lead to the development of a transgenic line of zebrafish allowing imaging of tinagl1 localization during development.
    • Does a Negative Emotional State Decrease Plasticity Related Gene Expression in the Hippocampus?

      Bonilla, Joselyn Amadiz; Department of Biological Sciences (Augusta University, 2020-12)
    • The Application of Low-Cost, Close-Range Photogrammetry in Dentistry

      Patel, Mohit; Mettenburg, D.; Biological Sciences, Restorative Sciences (Augusta University Libraries, 2020-05-05)
      This item presents the abstract for a poster presentation at the 21st Annual Phi Kappa Phi Student Research and Fine Arts Conference.
    • Atypical Magnesium Requirements in a Phyllite Population of Rare Plant Species, Pediomelum Piedmontatum

      Zimmerman, Matthew; Biological Sciences (Augusta University Libraries, 2020-05-04)
      This item presents the abstract for an oral presentation at the 21st Annual Phi Kappa Phi Student Research and Fine Arts Conference.
    • Development of Defined Culture Conditions for Human Wharton’s Jelly Stem Cells

      Shaikh, Arika; Biological Sciences (Augusta University Libraries, 2020-05-04)
      This item presents the abstract for a poster presentation at the 21st Annual Phi Kappa Phi Student Research and Fine Arts Conference.
    • Salty or Slightly Salty: Is Fish Species Richness Affected by an Obsolete Navigational Cut?

      Patterson, Rebecca; Mathews, Loren; Saul, Bruce; Biological Sciences, Georgia Southern University (Biology) (Augusta University Libraries, 2020-05-04)
      This item presents the abstract for a poster presentation at the 21st Annual Phi Kappa Phi Student Research and Fine Arts Conference.
    • Fast- Track Approach Following Heart Surgery in Infancy and Early Childhood: Outcome Analysis and Predictors of Failure

      Esquivel, Raquel; Department of Biological Sciences (Augusta University, 2020-05)
      Early extubation (EE) has become a critical determinant in perioperative management following congenital heart surgery (CHS) during early childhood. Fast track (FT) strategies and EE, when feasible, can have beneficial effects on clinical outcomes. The authors sought to determine the impact of EE on clinical outcomes, total hospital costs, identify predictors of failure and suggested criteria for new patients. A retrospective chart review of children ≤6 years old (n=64) who underwent CHS between January-December 2017 was performed. EE was defined as successful removal of the endotracheal tube in the operating room or upon arrival in intensive care unit (ICU). Groups were identified as (A):EE/Fast track and (B):no EE. Determinants for EE failure were assessed, and cost analysis pursued. The authors found 39 patients with EE compared to 25 that were not. Children who were EE (mean=6.795 days, sd= 4.250) spend significantly less (p < 0.0001) overall time in the ICU compared to non-EE patients (mean=19.960 days, sd=13.081). The authors also found that the total hospital stay for patients who were EE (mean=6.976 days, sd=4.090) was significantly reduced compared to those who were not (mean=21.783 days, sd=13.450) (p <0.0001). Furthermore, the authors found that children who were EE had a significant reduction (p <0.0001, sd= 23,196.203) in total hospital cost than patients who were not EE. Based on our analysis, we concluded that EE is feasible following CHS during early childhood but requires team approach and thoughtful use of FT protocols.
    • Fast-Track Approach Following Heart Surgery in Infancy and Early Childhood: Outcome Analysis and Predictors of Failure

      Geister, Emma; Department of Biological Sciences (Augusta University, 2020-05)
      Early extubation (EE) has become a critical determinant in perioperative management following congenital heart surgery (CHS) during early childhood. Fast track (FT) strategies and EE, when feasible, can have beneficial effects on clinical outcomes. The authors sought to determine the impact of EE on clinical outcomes, total hospital costs, identify predictors of failure and suggested criteria for new patients. A retrospective chart review of children ≤6 years old (n=64) who underwent CHS between January-December 2017 was performed. EE was defined as successful removal of the endotracheal tube in the operating room or upon arrival in intensive care unit (ICU). Groups were identified as (A):EE/Fast track and (B):no EE. Determinants for EE failure were assessed, and cost analysis pursued. The authors found 39 patients with EE compared to 25 that were not. Children who were EE (mean=6.795 days, sd= 4.250) spend significantly less (p < 0.0001) overall time in the ICU compared to non-EE patients (mean=19.960 days, sd=13.081). The authors also found that the total hospital stay for patients who were EE (mean=6.976 days, sd=4.090) was significantly reduced compared to those who were not (mean=21.783 days, sd=13.450) (p <0.0001). Furthermore, the authors found that children who were EE had a significant reduction (p <0.0001, sd= 23,196.203) in total hospital cost than patients who were not EE. Based on our analysis, we concluded that EE is feasible following CHS during early childhood but requires team approach and thoughtful use of FT protocols.
    • Identification of the CAP1-Binding Domain of Adenylyl Cyclase 3 in Humans

      Gunby, Kimberly; Department of Biological Sciences (Augusta University, 2020-05)
      My research is aimed at finding the cyclase-associated protein 1 (CAP1) binding domain on human adenylyl cyclase 3 (AC3). Previous studies in our lab show that the interaction between CAP1 and AC3 inhibits migration and invasion of pancreatic cancer cells. The inhibitory mechanism is thought to involve the binding of AC3 and CAP1, causing the inhibition of globular-actin polymerization needed for filopodia formation and cell motility. A better understanding of this interaction will help facilitate the discovery for drugs that inhibit the migration and invasion of pancreatic cancer cells. To locate the binding region, we constructed mutants of WT AC3 plasmid using a Site-Directed Mutagenesis kit. We substituted a highly conserved proline residue at position 307 for an arginine residue (P307R) and a glutamate residue at position 308 for an alanine residue (E308A). The mutations were confirmed by sequencing. We then transfected pancreatic cancer cell line PANC-1 with WT and mutant AC3 plasmids and confirmed the expression using Western-blotting. To test whether the mutated AC3 could still interact with CAP1, we performed co-immunoprecipitation. We found that the residues proline and arginine in AC3 are not required for the interaction with CAP1. Further substitutions of other conserved residues are underway.
    • Profiling the HCA Receptor Family through BRET Analysis of GPCR-G-Protein and GPCR-Arrestin Interactions

      Saj, Dalia; Department of Biological Sciences; Department of Pharmacology & Toxicology (Augusta University, 2020-05)
      Increasing obesity rates have put the American population at higher risk for developing obesity-related medical conditions such as hypertension, heart disease, and diabetes. The hydroxycarboxylic acid (HCA) receptor family is a family of G protein-coupled receptors (GPCRs) that are expressed in adipose tissue and function as metabolic sensors, making them potential pharmaceutical targets in the treatment of obesity and other metabolic disorders. The HCA receptor family consists of the HCA1, HCA2, and HCA3 receptors, which are activated by hydroxycarboxylic acids such as lactate and 3-hydroxybutyric acid. We utilized bioluminescence resonance energy transfer (BRET) to study agonist-induced coupling of luciferase-tagged HCA receptors to Venus fluorescent protein-tagged G protein heterotrimers or arrestins. Our results indicate that the three HCA receptors couple to the Gαi/o subfamily of G proteins. The data additionally confirms a lack of coupling to the other G protein subfamilies (Gαs, Gαq, and Gα12/13), and lacks evidence of arrestin recruitment to HCA receptors. Overall, our study highlights the use of BRET as a powerful tool for analysis of GPCR signaling and demonstrates its possible use for future studies to determine the potency of potential drugs targeting HCA receptors as a therapy for health-related problems such as obesity.
    • Development of Chemically Defined Culture Conditions for in vitro Expansion of Human Wharton’s Jelly Stem Cells

      Shaikh, Arika; Department of Biological Sciences (Augusta University, 2020-05)
      Mesenchymal stem cells (MSCs) are multi-potent and capable of differentiating into a variety of cell lineages. While MSCs have commonly been isolated from bone marrow for treatment of various diseases, Wharton’s Jelly (WJ), an extra-embryonic umbilical cord tissue rich from hyaluronic acid (HA), represents an alternative source for a safer and less invasive isolation of MSCs. Typically, WJ-MSCs are isolated and cultured in undefined media containing fetal bovine serum (FBS), of which use has been associated with different complications, including reproducibility of studies, transmission of infectious agents, and induction of immunologic reactions. To overcome these complications, and thus to facilitate clinical applications of WJ-MSCs, this project aimed to develop chemically defined and safe culture conditions for human WJ-MSCs. We hypothesize that undifferentiated growth of WJ-MSCs will be supported by an HA-based extracellular matrix and fortified DMEM/F12 supplemented with defined macromolecules, antioxidants, lipids and growth factors found in platelet lysate. This hypothesis was tested by comparing the growth kinetics and morphology of WJ-MSCs cultured in defined and undefined media. WJ-MSCs were isolated via enzymatic digestion from discarded human umbilical cords. Following phenotyping and sorting by evaluating expression of relevant markers (i.e., CD105, CD73, and CD90) using flow cytometer, WJ-MSCs were randomly distributed and cultured in five different defined media plus an undefined control medium. The best alternative in terms of cell morphology and proliferation was the medium 3 consisting of DMEM/F12 supplemented with glutamine, ITS (define), antioxidant mixture, lipid mixture, and growth factor mixture. Medium 3 was further improved by adding increasing concentrations of ethanolamine. These results are of significance for therapeutic applications of MSCs. Further research is needed to optimize compositions of extracellular matrix and growth factors while examining the plasticity of MSCs.
    • Characterization of a Cyclic Peptide (ADO5) as a Novel Inhibitor of the Hsp90 Chaperoning Machine

      Fang, Wayne; Department of Biological Sciences (Augusta University, 2020-05)
      Protection of oncogenic proteins is the foundation of many hallmarks of cancer. Based on this, hsp90 inhibitors have emerged as a potentially potent strategy for cancer treatment. The clinical efficacy of the earlier Hsp90 inhibitors remains unsatisfactory, in part due to their induction of heat shock response and anti-apoptotic mechanisms in cancer cells. To identify alternative therapeutic agents without these effects, we have developed a cell-free high-throughput screen (HTS) platform based on the folding of progesterone receptor (PR) by the core components of the Hsp90 chaperoning machine. During our initial screening of 175 natural products from North African medicinal plants, we discovered the cyclic peptide AD05 as a novel Hsp90 inhibitor. AD05 has shown a powerful antitumor activity against various cancer cell lines including HeLa, Hs578T, MDA-MB231, MDA-MB453, E0771, THP1, and U937. Western blot analysis revealed that AD05 destabilizes Hsp90 client proteins without inducing heat shock response as indicated by lack of upregulation of Hsp70, Hsp40 and Hsp27. Remarkably, AD05 does not induce apoptosis but rather triggers autophagy in various cell lines.
    • The Effect of PFOA on ERα+ and ERα- Human Breast Cancer Cell Lines

      Gaw, Victoria; Department of Biological Sciences (Augusta University, 2020-05)
      Perfluorooctanoic acid (PFOA) is a man-made chemical that belongs to a large group of fluorotelomers. PFOA is used to manufacture both industrial and consumer products and individuals can be exposed to PFOA through ingesting PFOA-contaminated water or food. While the long-term effects of perfluorooctanoic acid are largely unknown, there is increased evidence suggesting it to be an endocrine disruptor. Studies have shown that PFOA binds to and activates the peroxisomeproliferator-activated receptor α (PPARα), which can regulate the expression of other genes and receptors such as the other PPAR isoforms as well as estrogen receptor α (ERα). Previous experiments in our lab demonstrated that PFOA treatment of MCF-7 breast cancer cells (an ERα positive cell line) decreased ERα mRNA and protein levels, and decreased cell viability by ~20% within 48h of treatment. However, these cells were treated in the absence of fetal bovine serum (FBS), a cell culture additive that contains important growth factors. When we repeated these experiments without serum withdrawal, we initially noted a tendency towards increased proliferation in MCF-7 cells treated with 50µM and 100µM PFOA at both 24h and 48h compared to control. To further examine the role of ERα in PFOA-induced proliferation, we carried out additional experiments in another ERα positive cell line, T47-D, as well as an ERα negative cell line, MDA-MB-231. All three cell lines showed a tendency for increased viability. These data suggest that the PFOA-induced increase in cell viability in these cell lines is not dependent on ERα expression. In addition, the opposing effects of PFOA on proliferation in MCF-7 cells in the presence and absence of FBS demonstrates the importance of accurately and completely reporting cell culture and treatment conditions.
    • Diabetes Attenuation of the Estrogen-Mediated Increase in Endothelial Function is Associated with Circulating SIRT1

      O’Bryant, Sinéad; Department of Biological Sciences (Augusta University, 2020-05)
      Cardiovascular disease (CVD) is the most prominent killer within Type 1 Diabetes (T1D) with endothelial dysfunction as a major player in the development of CVD. Women with T1D experience an accelerated CVD risk despite the apparent sex-specific cardioprotection from circulating endogenous estrogen experienced by heathy pre-menopausal women. Animal models have shown the modulation of SIRT1, a NAD+ histone deacetylase, by estrogen as a CVD protector. This study sought to test the hypothesis that lower circulating SIRT1 is associated with reduced endothelial function in T1D women. Change in flow mediated dilation (FMD), a clinical measure of endothelial function, and SIRT1 over the menstrual cycle exhibited contrasting trends between T1D women and healthy women: increases of FMD and SIRT1 as estrogen increases in healthy women and decreases of FMD and SIRT1 as estrogen increases in T1D women. This provides evidence that signaling roles by circulating estrogen may be attenuated in T1D and that the effects of decreased SIRT1 contributes to endothelial dysfunction, resulting in determinant effects on vascular health in T1D women.
    • Characterization of Potential Proton Sensitive G Protein-Coupled Receptors

      Nam, Alisha J.; Department of Biological Sciences (Augusta University, 2020-05)
      G protein-coupled receptors (GPCRs) are membrane-bound receptors that can stimulate an intracellular signaling pathway following activation by a ligand. According to the International Union of Basic and Clinical Pharmacology (IUPHAR) database, GPR4, GPR65, and GPR132 are Class A orphan GPCRs with protons reported as their putative endogenous ligand. Because these receptors are currently understudied, the purpose of our study was to investigate the interactions between GPR4, GPR65 and GPR132 and G protein subtypes (Gαs, Gαi, Gαq, and Gα12) as a function of pH. Using bioluminescence resonance energy transfer (BRET), we studied the coupling between luciferase-tagged GPR receptors and fluorescent protein (Venus)-tagged heterotrimeric G proteins in response to changes in proton concentration. We found that all three receptors responded to pH changes. Upon extracellular response to pH changes, the receptors activate different G protein subtypes and thus, different signaling pathways: GPR4 activates all four G protein subtypes but has the strongest activation with Gαs; GPR65 activates all four subtypes; and GPR132 activates Gαi and weakly activates Gαq and Gα12. Identifying these receptors as true proton sensors leads the way in understanding the role they play in maintaining acid-base homeostasis and will be critical for the development of novel drugs combatting acidbase related disorders, such as ulcers and reflux esophagitis.
    • Ecotoxicology of Yellow-Bellied Sliders (Trachemys scripta scripta) in Natural Wetlands

      Hammesfahr, Rachel; Department of Biological Sciences (Augusta University, 2019-05)
      Glyphosate is one of the active ingredients in many different herbicidal products such as Roundup. Preliminary research has suggested that glyphosate is a possible endocrine disruptor, can cause developmental defects, and is a potential carcinogen (Dallegrave et al., 2007; Daruich et al., 2001). Due to its potential harmful effects on different organisms, the researchers sought to monitor the levels of glyphosate in wetland areas. This was done by analyzing samples from a common indicator species, the yellow-bellied slider turtle, Trachemys scripta scripta. Research was done on turtles caught at Reed Creek Nature Center and Brick Pond Park. Physical measurements were taken, and blood was drawn from each turtle. Analysis of the glyphosate levels in the blood samples was completed using a glyphosate specific ELISA kit. Analysis from the kit showed detectable glyphosate levels in 34 out of 42 turtles tested, with concentrations ranging from 0.00 ppb to 0.59 ppb. No correlations could be found between glyphosate concentrations and the sex, mass, or age of the turtle, or the month the sample was collected, but glyphosate was found to be ubiquitous in turtles from both sampling areas. Further research could focus on different tissues such as fat or the scutes to see if glyphosate is stored in higher concentrations there than in the plasma. Other species, such as amphibians, living in the same areas could also be studied to determine if certain species are more susceptible to storing glyphosate than others.
    • Effect of night shift work on sleep memory

      Ravula, Ordain; Department of Biological Sciences (Augusta University, 2019-05)
      Sleep deprivation is a very common phenomenon world-wide and it has been known to affect motor and cognitive functions. In this multi-faceted study, we sought to assess the effect of sleep /deprivation on mood, cognitive performance, and memory, as well as any potential. Our sample was drawn from anesthesia residents at the Augusta University Medical Center. Our first focus was to determine the effects of sleep deprivation on mood, cognitive performance, and memory. Tasks were split amongst members of the research team, and I was charged with assessing memory retention and working memory via the Ospan test, a computerized test involving letter sequence memorization and mathematical problem solving. I administered the Ospan to residents at a baseline or control reading, then after their calls. If the residents had a night float (a stretch of calls over 5 days), then I would assess them after the first day of the float, right after the float, and 24 hours after the float. The higher the score on the Ospan, the better working memory and memory retention abilities were in that resident. Data collection began in April 2018 and continued into February 2019. For our secondary evaluation, we assessed how sleep deprivation affected gene expression, by looking at a set of genes known to be associated with sleep deprivation. These genes were: PER1, PER2, and ARNTL or BMAL. (Gene details explained in body paragraphs). Following similar protocol to the first study, expression was assessed before and after night calls and night floats. The testing involved sampling saliva from the residents who participated in the cognitive assessments in Part 1, isolating RNA from the samples, creating cDNA from the RNA, and then performing microarray analysis on the samples. To produce the cDNA samples from the RNA samples, reverse transcriptase PCR was used to create cDNA and amplify the number of copies of genes we had to work with. Microarray analysis was performed to measure the actual gene expressions of our genes of interest. These were certain genes which were previously found in another study by multiple researchers at the University of Surrey, in the UK (article title given below later) to affect sleep deprivation. These genes were then examined to determine if those specific genes increased, decreased, or maintained their expression, post-sleep deprivation.
    • Surveying Mosses for Fungicidal Activity

      Yan, Stephanie; Department of Biological Sciences (Augusta University, 2019-05)
      The emergence of resistance to current fungicides is of serious concern because of the widespread diseases caused by fungi. One way to combat this problem is to discover new compounds that have antifungal properties. Plants are extensively attacked by fungi and many have evolved defenses, including fungicides and other compounds, such as a waxy cuticle, that make attack difficult. The mosses (Bryophyta) lack a cuticle. This makes them a likely group to survey for fungicidal activity because they may have additional chemical defenses. In this study, we made aqueous and ethanolic extracts from crushed mosses and tested their effect on the growth of the yeast Saccharomyces cerevisiae. These mosses were collected across a broad geographical range (locally, from Arkansas, and from Alaska) to test the hypothesis that resistance to fungal attack may correlate with the general suitability for fungal growth of the environment. Results include the demonstration of fungicidal activity in some, but not most, of the mosses. There was no correlation with geographical origin. Both solvents seem able to extract compounds that will suppress yeast growth. In addition, re-tests of some samples suggest that fungicidal properties may be lost during drying. Several of the mosses show strong enough antifungal activity that further investigation seems warranted.
    • The Mechanism of Inverse Agonists Binding to G-Protein Coupled Receptors, Histamine Receptor H1 and Histamine Receptor H2

      Patel, Shrey; Department of Biological Sciences (Augusta University, 2019-05)
      The thesis discusses the mechanism of an inverse agonist binding to receptors and how it is different from an agonist binding to the same receptor. The specific receptors that were focused on were histamine receptor H1 (HRH1) and histamine receptor H2 (HRH2) which are types of G-protein coupled receptors (GPCR). It is understood how an agonist binds to a GPCR and activates a signaling pathway within the cell, and that an inverse agonist can bind to the same receptor but elicit an opposite response. The current idea behind the mechanism of an inverse agonist is that it binds to the receptor, and the G-protein is not being recruited to continue the signaling pathway within the cell. The hypothesis was that the G-protein is recruited when the inverse agonist binds to the GPCR, but the G-protein would be in its GDP state or its inactivated state. To test the hypothesis, a luciferase assay was done in different conditions where the bioluminescence absorbance was measured and recorded to see if there was protein-protein activity between the GPCR and the G-protein. From doing multiple trials, it is still believed that the G-protein is not being recruited to elicit the signaling pathway when an inverse agonist binds to the receptors.
    • Investigating the requirement of HOB1 on the sensitivity of Schizosaccharomyces pombe after exposure to various DNA damaging agents

      Qureshi, Arman; Department of Biological Sciences (Augusta University, 2019-05)
      DNA encodes the genetic information for the growth and development of living organisms. As such, it is inherently important that when damaged, the DNA is repaired efficiently and accurately. BIN1 encodes a protein that plays a role in genomic stability, specifically in cell cycle regulation, chromatin remodeling, and DNA repair. Previous research has shown that the protein Bin1 exhibits an inhibitory role in the double strand break repair pathway of non-homologous end joining (NHEJ). The homolog of BIN1, HOB1, is found in the fission yeast, Schizosaccharomyces pombe. To understand the role HOB1 has on yeast survival after damage, two strains of S. pombe, a wild type strain (WT) and a strain without HOB1 (hob1Δ), were exposed to various DNA damaging agents. Each treatment introduced a different type of DNA damage that would require different DNA repair pathways. The treatments included UV radiation, hydrogen peroxide, bleomycin, and cisplatin. After treatment with each respective agent, the % of surviving cells at multiple doses was analyzed and graphed logarithmically. The data collected supports the idea that the presence of HOB1 has a role on the survival of yeast after DNA damage. The WT strains survived better than the hob1Δ counterparts when exposed to hydrogen peroxide or bleomycin. When exposed to UV radiation or cisplatin damage, no significant difference is observed between the strains regarding survival.