Digital Commons @ Winthrop University

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  Diet Analysis of Gastrotricha

  Mikhail Anfinson

  The phylum Gastrotricha is comprised of small aquatic organisms that live in between sand grains. It has been shown that meiofauna communities can play valuable roles in ecosystems and serve as a food source for animals higher up the trophic levels. However, little is known about their own feeding behavior which this study is concerned about. Over the summer developments were made in the methodical sections for diet diagnostic PCR for marine bacterial strains, unicellular marine eukaryotes, and investigating different types of immunofluorescence for marine bacteria. We also plan at looking at the number of sarcomeres within the pharynx of these animals to see if there is a correlation between the number of sarcomeres and their feeding habits.

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  Bootstrap Percolation in the Random Geometric Graph

  MeiRose Barnette and John T. Herndon

  Bootstrap percolation on a graph is a process which models the spread of an "infection" given an initially infected set of vertices of the graph. To state the problem more precisely, suppose G is a graph, k is a natural number, and I_0 is a set of initially infected vertices. Then for any discrete time t, we define I_t to be I_{t-1} along with any vertex outside of I_{t-1} which has at least k edges to vertices of I_{t-1}. This type of process may be used to model the spread of a disease by taking people as vertices, interactions between people as edges, and assuming a rate at which the infection spreads. That is, if the rate of spread is 10%, we would expect that if a person is in contact with 10 infected people, then the person will become infected. Other applications of this type of model involve the spread of rumors and the fault tolerance for distributed computing.

  The random geometric graph is formed by fixing an r value and choosing n points from the unit square uniformly at random. We then join a pair of these points by an edge if their distance is less than r. This kind of random graph seems particularly relevant in the current socially distant world in which people attempt to only interact with others when necessary (i.e. distance-based edges roughly model this kind of relative isolation). Random geometric graphs have been well-studied. Bootstrap percolation on random geometric graphs has been examined although prior results in this direction cover limited regimes of the parameters. In our project, we extend previous work to study other ranges of values of the parameters. Along the way we use similar ideas to identify the threshold for connectivity in the random geometric graph which is a problem of independent interest.

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  Photoredox Mediated Alkylation of Imines with Potassium Organotrifluoroborates in the Presence of an Organic Photocatalyst

  Jackson W. Barrett, Evan H. Thibodeaux, Molly A. Quetel, and Eric J. Walters

  Our group is investigating the application of an organic photocatalyst, 9-mesityl-10-methyl acridinium tetrafluoroborate (Mes-Acr-Me), to the alkylation of aryl imines with potassium organotrifluoroborates. In the past, our group has employed an potassium isopropyltrifluoroborate for model reactions, but we desired to expand our BF₃K scope to see the effects of variation in the alkyl group of the organotetrafluoroborate. In addition, we also wanted to study the substituent electronic effects in the imine, which was done using a series of competition experiments.

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  Modelling the Effects of Complacency and Educational Countermeasures on the Spread of HIV

  Brooklyn M. Clive and Ryan Lumbert

  In this project, we consider a system of five ordinary differential equations which describe the population dynamics of HIV/AIDS when individuals are tested for the virus and then moved onto antiretroviral therapy. We include a Holling Type-II response to model the complacency of the population in response to the number of AIDS cases. We prove global stability of the disease-free equilibrium when the basic reproductive ratio is less than one. We then derive an optimal control problem and solve it theoretically using Pontryagin's Maximum Principle and numerically using the Forward-Backward Sweep Method. We conclude with a discussion on the impact of optimal educational strategies to combat complacency regarding the AIDS/HIV epidemic.

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  Exploring the Mechanisms Regulating Toroid Formation by Adipose-Derived Stem Cells Cultured on 3D Collagen Hydrogels

  Mary-Catherine Cobb

  Mesenchymal stem cells (MSCs) are a huge topic of interest to many researchers due to their therapeutic potential. MSCs can be harvested from various body tissues, including bone marrow, blood, and adipose tissue. Adipose-derived mesenchymal stem cells (ADSCs) are of particular therapeutic interest due to their great abundance and ease of accessibility relative to other types of MSCs. We have observed that ADSCs will migrate and self-organize into a toroid when applied to the top of a 3D type I collagen hydrogel. ADSCs fail to form toroids when embedded within collagen hydrogels, which raises the question of what specific environmental factors and cellular mechanisms regulate toroid formation? The binding of the ligand CXCL12 to the chemokine receptor CXCR4 initiates chemotactic signals in ADSCs. The goal of this study is to determine if the CXCL12:CXCR4 signaling axis is essential to the migration and self-organization required for ADSCs to achieve toroid formation. We hypothesized that the binding of the ligand CXCL12 to the chemokine receptor CXCR4 is essential for toroid formation by the telomerase immortalized human ADSCs used in our culture model. The selective inhibitor AMD3100 can be used to block the interaction between CXCR4 and CXCL12. To test our hypothesis, we compared toroid formation by ADSCs cultured in two concentrations of AMD3100 to control conditions. We used phase-contrast microscopy to qualitatively monitor toroid formation and integrated software to measure the geometry of any structures formed 24 hours after plating. We found that there was no observable effect caused by the inhibitor. These results suggest that the CXCL12:CXCR4 signaling axis is not essential for toroid formation under the conditions tested. Future directions include testing different numbers and sources of ADSCs in our system and using selective inhibitors of downstream pathways known to be important for cellular migration and self-organization.

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  Recellularization of Porcine Internal Thoracic Arteries as a Tissue Engineered Small-Diameter Vascular Graft Alternative

  Carlos E. Escoto-Diaz, Jesse B. Kooistra, Nicholle E. Lewis, and Holdyn C. Ferguson

  Small-diameter vascular grafts are the leading treatment for myocardial infarctions resulting from atherosclerosed coronary vasculature. A potential alternative to using patient-derived grafts is the use of tissue engineered vessels. The removal of native cells from xenogenic vessels allows for recellularization with human cells types; however, the decellularization process depends on the use of cytotoxic reagents that must be removed prior to recellularization. The recellularization process must produce a functional graft that contains the multiple cell types found within arteries. We hypothesized that porcine internal thoracic arteries decellularized using a combination of detergents could be processed to support human endothelial and smooth muscle cell growth. We also hypothesized that in order to optimize in vitro co-culture conditions during recellularization, a mix of media types would be necessary to simultaneously support endothelial and smooth muscle cell growth. A cytotoxicity assay was performed to assess the effects of residual detergents on endothelial cells seeded onto the scaffolds. A relationship between the degree of detergent rinsing and cellular viability was identified via a resazurin reduction assay with more extensive rinsing significantly enhancing cell viability. This same cell viability assay was used to identify media combinations that supported growth of both endothelial and smooth muscle cells. Both cell types were able to grow in a 50:50 mixture of their media types without any loss of viability or effect on morphology. Furthermore, endothelial cells grown in the mixed medium maintained their characteristic CD31 expression. Taken together, these results show that human cells native to arteries can remain viable within the extracellular matrix of porcine internal thoracic artery scaffolds after thorough scaffold detoxification. Additionally, the co-culture conditions established can support the growth of both endothelial and smooth muscle cell types found within arteries. Future work will focus on simultaneously culturing the cells within scaffolds to build toward the goal of tissue engineering small-diameter vascular graft alternatives.

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  Mutating Lpar4 in the Visual System using CRISPR

  Thomas A. Gonzalez

  The visual system is developed throughout the embryonic stage and is dictated by axon guidance molecules that tell the growing fibers of nerve cells, axons, where to grow. The ends of axons, called growth cones, continuously sprout out or collapse based on the axon guidance molecules. Research has shown that lysophosphatidic acid (LPA) causes growth cone collapse, so it is hypothesized that it is an axon guidance molecule. LPA binds to specific receptors, known as Lpars of which there are at least five known. Previous research indicates that Lpar 1,2, and 3 are not required in mice for growth cone responses to LPA. Furthermore, Lpar5 is not expressed in retinal tissue, so our research has focused on Lpar4. Our research is trying to determine how axon guidance molecules help shape the visual system that we have today so we start at the embryonic stage where it begins. By working with an embryo that is somewhat similar to ours, we can further figure out how the system works and how our visual system builds itself. We are working to mutate the receptor that we believe to do a substantial amount of axon guidance by using CRISPR as a tool and then see how growth cones respond to LPA without Lpar4. Chicken eggs are used since we easily could take them out at embryonic ages in an artificial culture system in a cup. We have validated guide RNAs that can mutate chicken Lpar4 by using CRISPR. We will inject plasmids expressing these guide RNAs as well as Cas9 along with a tdTomato fluorescent tag into the developing embryonic chicken eye. After developing further, we dissect the chicken retina to see if retinal cells still respond to LPA with growth cone collapse in the absence of Lpar4. We have been optimizing injection and electroporation of plasmid DNA into early embryonic chicken retina as well as dissecting and removing embryonic retina tissue to determine if our sample has transfected properly. We have also been obtaining baseline data on growth cone collapse by different concentrations of LPA.

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  Optimal control of an HIV model with gene therapy and latency reversing agents

  Andrew B. Grant and Paul Hazelton

  In this project, we study the dynamics of HIV under gene therapy and latency reversing agents. For constant treatment controls, we establish global stability of the disease-free equilibrium and endemic equilibrium based on the value of the basic reproductive ratio. We then consider time dependent controls and formulate an associated optimal control problem that emphasizes reduction of the latent reservoir. Characterizations for the optimal control profiles are found using Pontryagin's Maximum Principle. We perform numerical simulations of the optimal control model using the fourth-order Runge-Kutta forward-backward sweep method. We conclude with findings that suggest a combination treatment of gene therapy with latency reversing agents provides better remission times than gene therapy alone.

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  Discovery Research for New Optical Materials

  Kameron Johnson

  This project utilizes the MGI approach in order to discover new compounds at an accelerated rate. Novel materials for optical applications, such as luminescent scintillators, are desired for improvement of properties. Using density functional theory (DFT) calculations, new single-crystal scintillating materials can be optimized leading to more efficient scintillation. (5) Two existing compounds in the quaternary system Na-O-Si-Y were chosen and further modified using substitutions into proposed structures that can be investigated. The two original compounds, NaO4SiY and NaO26Si6Y9, were chosen and modified because of their already high density by substituting yttrium for lanthanum, sodium for potassium and yttrium for lanthanum, and lastly yttrium for zirconium and sodium for potassium. The potassium substitution allows the structure to slightly expand in order to yield more realistic bond distances. This yields a total of 8 structures, with 6 being proposed structures for study. Figure 1 shows the band gap for the NaO4SiY.

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  Generation of Fluorescent Esophageal Adenocarcinoma Cells for Lineage Tracing Within Composite 3D Culture Models

  Madeline G. Linker

  While relatively rare, the aggressive nature and poor prognosis associated with esophageal carcinoma make it a particularly dangerous form of cancer. One approach to discovering esophageal cancer treatment options includes the development of composite 3D culture models. This method of research allows cellular interactions and responses toward interventions to be evaluated within a more complex system than 2D cultures. Additionally, a composite 3D model provides a higher throughput and is more cost-effective than in vivo animal studies. To analyze the interactions between the model’s cell types, fluorescent labeling allows each cell type to be distinguished from the others, facilitating independent study via lineage tracing. Our goal was to generate fluorescent esophageal cancer cells. Of primary interest was the Homo sapiens esophageal adenocarcinoma cell line OE19 that was transfected with a plasmid carrying the mCherry (red) fluorescence marker and neomycin resistance gene. We hypothesized that if geneticin antibiotic (G418) was introduced to transfected OE19 cultures, the percentage of mCherry expressing cells would be enhanced due to G418 selection against cells lacking the plasmid. To test our hypothesis, 400 mg/mL of G418 was used for multiple passages during routine cell maintenance. Fluorescence-activated cell sorting (FACS) was then used to sort each cell based on mCherry expression. In addition, OE19 cultures under G418 selection were stained with Hoechst dye (cyan), to visualize nuclei as reference for mCherry appearance during analysis via fluorescent microscopy. The data obtained from FACS analysis indicated that 5-7% of OE19 sample cultures expressed the mCherry label. This was supported by fluorescent microscopy. Low cell viability following cell sorting did not allow for continued culture of sorted cells. Future directions include increasing the selection pressure on OE19 cells by raising the concentration of G418 and using modified FACS procedures to improve post-sorting viability.

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  Using nucleic acid-gold nanoparticle conjugate in the fight against bacteria that are resistant to tetracycline antibiotics

  Allen T. Livingston

  Antibiotic resistance is major problem in modern medicine. Seventy percent of bacterial strains are resistant to at least one antibiotic, making treatment of bacterial infections ever more expensive and difficult. Currently, we are investigating the therapeutic potency of nucleic acid-gold nanoparticle conjugates as treatments against bacteria that are resistant to the antibiotic tetracycline. We hypothesize that by attaching RNA that binds to tetracycline to silver or gold nanoparticles the resulting conjugates will work as a “Trojan-horse” tetracycline-delivery vehicle that smuggles the antibiotic into the cell without being detected by cellular defense systems. Moreover, we reason that silver or gold ions released by the nanoparticles add to the antimicrobial effects of tetracycline.

  To demonstrate the viability of this idea, we set out to generate a nuclease resistant variant of the tetracycline binding RNA ykkCD and attach it to gold nanoparticles. During the summer we furthered the progress of this research in there major ways. First, we optimized conditions used for the polymerase chain reaction that generates the DNA template for RNA synthesis. Second, we found the most efficient way to synthesize RNA containing modified nucleotides. Usage of modified nucleotides is necessary to prevent degradation of the tetracycline-binding RNA by cellular nucleases. Third, we optimized a stability assay to verify that the modified RNAs are stable in cellular environments. Fourth, we attached the tetracycline binding RNA to gold nanoparticles.

  The next steps in the process would be to test the potency of this modified RNA-gold nanoparticle conjugate against a tetracycline-resistant strain of the bacteria E. Coli.

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  Testing the Allelic Strength of Drosophila melanogaster mus109 Alleles

  Erica V. Nestore

  Drosophila melanogaster, commonly referred to as fruit flies, possess a group of genes that when mutated can cause sensitivity to DNA damaging agents. These mutagen sensitive (mus) genes are likely involved in DNA repair, and one of these genes, mus109, was the focus of this study. To perform the experiment, complementation crosses were set up between the three mus109 alleles: mus109^lS, mus109^D1 and mus109^D2. The wild-type DGRP-59 was used as the control. For each cross, twenty vials were scored. Brood one contained ten vials that were mock treated with distilled water, and brood two contained ten vials that were treated with the alkylating agent methyl methanesulfonate (MMS). The relative survival was calculated as the ratio of mutants and non-mutants in brood 2, normalized to brood 1. An ANOVA analysis indicated that there was a significant difference in survival rate between the various allelic combinations. Pairwise analysis revealed that the mus109^D1allele demonstrated greater allelic strength compared to mus109^D2. Crosses with the hypomorphic mus109^D2 allele yielded a higher relative survival rate as compared to mus109^D1 and this value was not statistically different from DGRP-59.

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  Investigating Cytotoxicity and Defense functions of bacteriophage Larva genes in host Mycobacterium smegmatis

  Dallas K. Nivens and Laela A. Walker

  It is estimated that approximately 75% of gene functions within any given phage cluster remain unknown. Two phage traits that can be measured phenotypically were investigated: cytotoxicity, which causes host cell lysis, and superimmunity, which results in protection of the host from infection by similar phages. Five genes (35, 42, 46, 49, and 59), from a subcluster K5 bacteriophage named Larva, were amplified by the polymerase chain reaction (PCR) and assembled into a cloning plasmid (pExTra) using isothermal assembly. Initially, chemical transformation of Escherichia coli with the pExTra + gene insert, enabled amplification of the plasmids. Clonal PCR verified the presence of the inserts that were selected for on Kanamycin plates. Host bacteria M. smegmatis cells were electroporated with pExTra + insert and the inducer molecule anhydro-tetracycline (aTc) was used to induce expression of the relevant genes. The expression of Larva genes 35, 42, 46, and 59 were non-toxic to the host. However, Larva gene 49 was toxic, causing the bacteria to lyse. Evidence of this was further revealed in the defense assay where expression of gene 49 caused an absence of bacterial lawn growth. The expression of Larva genes 42 and 59 highlight a defense mechanism associated with these gene products that protects the host from attack by other phage in the same cluster. Further work to elucidate which host-parasite proteins are in contact to cause the phenotypic changes will be investigated using protein-protein interaction (PPIs) assays and hopefully reveal important clues towards understanding phage gene function.

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  The Effect of Semaphorin 3A on Chick Embryo Retinal Growth Cones

  Allison T. Reed

  During embryonic development, axons grow from the retina of the eye to the tectum of the brain which allows for visual information transfer. Axons travel to the tectum via axon pathfinding, which is influenced by axon guidance cues. Axon guidance molecules interact with the growing tips of retinal ganglion cell (RGC) axon, which are motile structures known as growth cones. Some inhibitory axon guidance molecules are known to cause a growth cone to collapse, where they cease growth and then retract, or turn away. One such inhibitory axon guidance molecule is Semaphorin 3A (Sema 3A). While Sema 3A’s importance is known in the nervous system, a study conducted by Luo et al. (1993) found that Sema 3A causes growth cone collapse of chick embryo dorsal root ganglion cells (DRGs) but not growth cone collapse in chick retinal ganglion cells (RGCs). However, we have found significant evidence that Sema 3A does indeed cause growth cone collapse of embryonic chick retinal ganglion cells, which is inconsistent with the Luo et al. (1993) finding. After further investigation, we have found that RGC’s have the ability to regenerate from collapse after a 15-20 minute treatment with Sema 3A, thus giving the illusion to Luo et at. (1993) that Sema 3A does not affect RGC’s because of the 60-minute allotted time window they used. We are currently investigating the effect of Sema 3A on different embryonic ages (E5, E6, E7, E8). We have found preliminary data that suggests that Sema 3A will give the same effect on RGC’s no matter the age.

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  Nutrient limitation in freshwater streams in the South Carolina Piedmont

  Rachael Rowe

  Availability of nutrients, particularly nitrogen (N) and phosphorus (P), is a key driver of microbial growth and metabolism in freshwater ecosystems. Microbial biofilms composed of both autotrophs and heterotrophs play crucial roles in freshwater food webs. These stream communities may be limited by N, P, or both N and P. This project examined nutrient limitation of biofilm communities in two small, freshwater streams in the Catawba river basin within the Catawba Nation in South Carolina. Stream conditions were monitored and nutrient concentrations for ambient ammonium and phosphate were monitored. We quantified nutrient limitation, community respiration, gross primary production, and ash-free dry mass of biofilms on two substrate types, selecting for autotrophs or heterotrophs, using nutrient diffusing substrates (NDS). NDS allow for slow, constant nutrient amendments for autotrophic and heterotrophic communities without affecting the rest of the ecosystem. Ambient nutrient concentrations in both streams were relatively low. Nevertheless, for all parameters except community respiration in one stream, microbial biofilms did not appear to be nutrient limited. In one stream, community respiration on substrates selecting for heterotrophs suggested those communities may be nutrient limited. Understanding how nutrients impact microorganism growth and metabolism in these streams is important for managing these resources and managing anthropogenic inputs in the surrounding watershed.

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  Elucidating the effect of the antibiotic tetracycline on the regulatory function of the guanidine sensing riboswitch ykkCD

  Thomas k. Sullivan

  The primary objective of this research is to develop an assay – one more feasible for undergraduate institutions than currently available methods – that will enable evaluation of the concomitant binding of, guanidine and tetracycline to the ykkCD bacterial guanidine sensor riboswitch. This noncoding regulatory RNA, in addition to guanidine, also recognizes tetracycline, a translation-inhibiting antibiotic, via a high-affinity aptamer domain. The resulting conformational change by the riboswitch upregulates the expression of genes that are downstream of this regulator, most of which are membrane transporter that expel toxins from bacteria. This phenomenon exemplifies the increasing issue of bacterial antibiotic resistance, by raising the possibility that bacteria uses its existing detoxifying machinery to expel antibiotics thus rendering them ineffective therapeutics.

  A binding assay is planned to see if the two ligands, tetracycline and guanidine bind to the riboswitch at roughly the same region, and to investigate the thermodynamics and/or kinetics by which the ligands bind, and to therein see whether or not the binding of the tetracycline antibiotic makes guanidine bind to the aptamer more efficiently. The RNA utilized in this project is from the soil bacterium Bacillus subtilis. Binding assays currently proposed in the literature have called for more cumbersome methods, including the use of radioactively labeled RNAs and the tedious, time-consuming running of sequencing gels, but the goal here is to develop an assay that utilizes the more universally accessible method of isothermal titration calorimetry (ITC) and/or surface plasmon resonance (SPR) to measure the efficiency of guanidine binding to the ykkCD riboswitch while tetracycline is bound. Due to limitations on in-lab experimentation imposed over this summer of 2020 by the ongoing COVID-19 pandemic, a literature review alone is presented here which aids our overall understanding of the project’s current standing and that provides insight as to how we can benefit from applying both or either ITC and/or SPR to make the desired measurements.

  When safe and advisable to do so, we intend to physically collect and analyze assay data (going forward with both methods for now) to closely consider the role of tetracycline binding in the life of the regulator and its effect on guanidine binding.