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Friday, April 24th

Adipose Derived Stem Cell Morphology and Gene Expression in Two-Dimensional versus Three-Dimensional Environments


Streaming video available

Caroline G. Hammond, Winthrop University

Adipose-derived stem cells (ADSCs) are a population of mesenchymal stem cells with multipotent differentiation ability. Three-dimensional cell culture environments are particularly valuable for observing stem cell behavior as they represent a more accurate model of in vivo conditions than simple two-dimensional culture. The purpose of this research was to explore the potential to use several different three-dimensional culture systems to better understand how ADSCs cultured in three-dimensions behave relative to ADSCs cultured using traditional two-dimensional cell culture. The present study hypothesized that both ADSCs both on top of and inside of collagen hydrogels and as spheroids could be cultured to better understand how the cells behave in those environments. It was also hypothesized that cell sorting could be used to obtain a rare subpopulation of ADSCs known as Muse cells. Muse cells have greater developmental potency and the ability to differentiate at a faster rate than non-Muse mesenchymal stem cells. After both two-dimensional and three-dimensional cell culture, real-time RT-PCR was used to analyze gene expression of the housekeeping gene Mrp19 and genes of interest Cxcl12 and Cxcr4. It was observed that ADSCs are amenable to spheroid culture and self-organize into toroids when cultured on top of collagen hydrogels. Several differences were also observed in Cxcl12 and Cxcr4 gene expression between two-dimensional and three-dimensional cultures and between different three-dimensional culture platforms. In addition, the present study was able to identify Muse cells within the ADSC population, which will allow comparison between the behavior of Muse and non-Muse ADSCs in three-dimensional culture in future experiments.

Developing a Multicomponent, Three-Dimensional Culture Model of Esophageal Cancer

Connor B. Hogan, Winthrop University

Faculty Mentor: Matthew Stern, Ph.D.

Esophageal cancer is an uncommon form of cancer, making up just 1% of cancer diagnoses in the United States. While rare, a diagnosis of esophageal cancer carries a poor prognosis, with only 45% of patients surviving five years. One of the ways to improve cancer treatment is to improve the experimental models used to study cancer and to test different treatment strategies. Despite a recent trend toward the use of three-dimensional culture models in cancer research, few such options exist for esophageal cancer. We hypothesized that a three-dimensional, multi-component tissue model could be created utilizing commercially available advanced cell culture platforms. We initially attempted to use cell sheet technology to assemble a three-dimensional model; however, the human esophageal epithelial cells we intend to employ could not be effectively collected and transferred as cell sheets. We moved on to exploring the use of the RAFT system by Lonza, which concentrates cell-seeded collagen hydrogels in a way that facilitates constructing composite tissue models. We have succeeded in culturing multiple cell types within the RAFT system. One of the key features of our theorized model is the fluorescent labeling of each individual cell type in order to monitor the fate of different cell types within the model and facilitate the use of fluorescence-activated cell sorting to isolate and study each cell population separately. We are currently using a combination of selective culture and cell sorting to generate esophageal cancer cell lines that express a fluorescent marker protein for use in our novel model.

Effects of Brain Training on Brain Function


Jeanae Williams

Faculty Mentor: Aaron Aslakson, M.A.

Over one million Americans are diagnosed with brain diseases or disorders each year. This means brain disease is very common, and because most brain diseases are incurable, it is vital that more information on prevention is provided. Brain training is a program of regular activities that claims to maintain or improve one’s cognitive abilities. Cognitive abilities are brain-based skills required to perform various mental tasks, from the simplest to the most complex. This presentation will discuss the effects that brain training activities have on cognition and brain function.

Exploring the Link Between Iron Homeostasis and PhpP Activity

David Brandyburg

Faculty Mentor: Nicholas Grossoehme, Ph.D.

RitR (repressor of iron transport) is an orphan two-component signal transduction response regulator in Streptococcus pneumoniae that is central to iron homeostasis. RitR, however, lacks the amino acid (aspartic acid) that serves as the phosphate acceptor in traditional response regulators – consequently, it does not function in the same way that other response regulators do. Since its discovery, it has been shown that this protein is indeed the target of a Ser-Thr kinase-phosphatase pair, StkP and PhpP. STK has been shown to phosphorylate RitR in response to extracellular iron. This phosphorylation impacts the DNA binding ability of RitR, leading to expression of iron uptake genes. PhpP is an intercellular phosphatase that hydrolyzes the phosphate and effectively reverses the signal. This project aimed to determine whether PhpP could be metal sensitive and, if so, to test the sensitivity. This was accomplished using a standard pNPP (para-nitrophenylphosphate) assay. By utilizing this assay, we were able to determine that PhpP is indeed capable of dephosphorylating a chemical substrate and does so in an iron-dependent manner. Steps were taken to develop an assay to monitor the hydrolysis of phospho-RitR; however, optimization is still necessary.

Global Dynamics of the HIV Latent Reservoir with Latency Reversing Agents and Immune Response

Claire Berchtold, Winthrop University
Hannah Mitchum, Winthrop University

Faculty Mentor: Kristen Abernathy, Ph.D., and Zachary Abernathy, Ph.D.

In this project, we model the dynamics of HIV-1 latently infected cells under the effects of latency reversing agents (LRAs) to promote a natural immune response. We establish the existence of immune-free and positive equilibria and then utilize Lyapunov functions to prove the global asymptotic stability of each. Numerical simulations are performed to support and illustrate these results. We conclude with a discussion on the model’s predicted threshold for LRA effectiveness to stimulate a natural immune response and decrease the size of the latent reservoir.

Modeling the Latent Reservoir in the Dynamics of HIV Infection with CTL Memory

Sarah Fleetwood
Josiah Bauer

Faculty Mentor: Zachary Abernathy, Ph.D., and Kristen Abernathy, Ph.D.

In this project, we model the dynamics of HIV-1 latently infected cells under the effects of a natural immune response. Our purpose in this model is to study the long-term effects of CTL memory on viral load. We establish the existence of equilibria and the global asymptotic stability of the disease-free equilibrium based on the rate that cells are latently versus actively infected. We then perform numerical simulations to illustrate the stability behavior of immune-free and internal equilibria. Furthermore, we demonstrate that anti-retroviral therapy can stimulate a memory response and reduce the viral load in the case when all equilibra exist.

On Prime Labelings of Berge Hypergraphs of Nonprime Graphs

Chris Chamberlin, Winthrop University

Graph labeling problems, such as the Four Color Conjecture, date back to the beginning of Graph Theory itself. Roughly forty years ago, the notion of a prime labeling of a graph was introduced: a graph on n vertices has a prime labeling if its vertices can be labeled by the numbers 1, 2, …, n, so that each edge spans a coprime pair (i.e. the labels on an edge have greatest common divisor one). A hypergraph consists of “edges” on a vertex set where the edges may contain any number of vertices. Since greatest common divisor can be generalized to more than two numbers, it is natural to consider prime labeling hypergraphs. As an entry point to this problem, we focus on a subclass of hypergraphs referred to as Berge hypergraphs. Given a graph G, the hypergraph is Berge-G if there is a matching between edges of G and edges of the hypergraph in which, in this matching, each edge is within the corresponding edge of the hypergraph. The paper gives a condition based on how “close” G is to being prime, which implies that any hypergraph which is Berge-G is prime, and finds that a handful of G for which any hypergraph which is Berge-G is prime.

Optimization of RNA Isolation Methodology for Gene Expression Analysis of Self-Organizing Three-Dimensional Tissue Structures

Chandler E. Burt, Winthrop University
Nathan Kidd, Winthrop University

Faculty Mentor: Matthew Stern, Ph.D.

Three-dimensional culture systems allow for more complex cellular interactions and organization than traditional two-dimensional culture. It has been observed that cells placed on top of collagen hydrogels organize into a donut-like shape called a toroid, while cells mixed into the hydrogels do not organize into toroids. The ultimate goal of this project is to evaluate the signal transduction pathways and cellular mechanisms that are involved in toroid formation. The specific goal of the work described here was to optimize RNA isolation from cells cultured on or in collagen hydrogels – a procedure that is known to be technically challenging – and to use real-time RT-PCR to compare the expression of select genes during toroid formation. We tested and compared several different RNA isolation protocols and found that a method based on the use of cetyltrimethylammonium bromide (CTAB) prior to alcohol precipitation, which is more typically used in isolation of RNA from plants, proved to be the most consistently effective in our hands. These results allowed us to move onto using real-time RT-PCR to compare the expression of two specific genes, Cxcr4 and Cxcl12 in mouse adipose-derived stem cells over a 12-hour timecourse of toroid formation. Our results revealed some differences in gene expression during toroid formation. More importantly, they demonstrate the feasibility of conducting similar and/or larger-scale experiments using methods like RNA-sequencing to monitor changes in gene expression during toroid formation and to compare gene expression between different collagen hydrogel-based culture platforms.

Optimization of Smooth Muscle Cell Culture for Blood Vessel Tissue Engineering

Nicholle E. Lewis, Winthrop University

Over 1.5 million heart attacks occur annually in the United States. Vascular bypass surgery is a viable treatment option; however, grafts used in small-diameter bypass surgeries suffer from limitations. To combat those limitations, additional optimization is needed. Potential solutions include the use of tissue engineered conduits constructed using patient-specific smooth muscle cells (SMCs) and endothelial cells (ECs). This project tested the effects of 1) uncoated versus collagen-coated culture dishes, 2) different SMC media formulations, and 3) different mixtures of both SMC and EC media to identify optimal culture conditions for human aortic smooth muscle cells prior to and during recellularization of vascular scaffolds. The present study hypothesized that 1) collagen would enhance growth based on its abundance in the ECM, 2) both SMC media formulations tested would increase SMC growth, and 3) mixtures of SMC and EC media with more SMC media would enhance SMC proliferation. A parallel project was conducted using ECs cultured in mixtures of SMC and EC media to determine optimal conditions for simultaneous seeding. Results show that 1) collagen was not a significant enhancer of growth, 2) both types of SMC media promoted SMC growth, and 3) mixtures varied in their effect on SMC proliferation, but a 50:50 mixture showed no negative effect on SMC proliferation after 72 hours. These results indicate that SMCs grow successfully in several conditions, thus providing more leeway for simultaneous seeding of SMCs and ECs. Understanding factors needed to culture SMCs in combination with ECs provides valuable information for vascular tissue engineering.

Orthopedic Injuries in Athletes


Cameron Adams

Faculty Mentor: Aaron Aslakson, M.A.

When thinking of athletes and orthopedic injuries, we often associate these injuries with more physical sports such as football, basketball, or even soccer. What we don’t realize is that an injury of any degree can occur in any sport, regardless of the level of physicality. An orthopedic injury is any injury to the bones, muscles, or joints of the musculoskeletal system. These injuries include, but are not limited to, ACL tears, meniscus tears, broken bones, muscle tears, sprains, dislocations, or fractures, obtained during a sport. Most injuries of this nature are hard to ignore, but depending on the area and severity of the injury, some may go unnoticed or be mistaken for another issue. If not properly examined and treated, the injury could severely worsen. Orthopedic doctors and surgeons typically examine, treat, help to prevent, and provide rehabilitation services for patients with orthopedic injuries. Since their main priority is to treat and rehabilitate patients, all treatments are provided with the intent of the patient returning to normal activities of daily living or his or her respective sport profession. This presentation will discuss the specific types of orthopedic injuries that can occur, how they are obtained, what age/gender group is most likely to obtain an orthopedic injury, the sports we most see them within, and lastly how they are examined, treated, and rehabilitated.

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

Evan H. Thibodeaux, Winthrop University

Faculty Mentor: James M. Hanna Jr., Ph.D.

Recently, the use of visible light combined with a suitable photocatalyst to promote key bond-forming steps in organic synthesis has emerged as a viable strategy to achieve a number of important synthetic transformations. The photocatalyst involved is often a ruthenium or iridium polypyridyl complex, which absorbs light in the visible range to give a relatively long-lived excited state, which may engage organic substrates in a series of single-electron-transfer (SET) events. The organic radicals thus generated participate in downstream reactions leading to the final product(s). During previous research, this strategy for the alkylation of aldimines with potassium organotrifluoroborates using transition-metal photocatalysts was deployed. However, because of the much lower cost of organic photocatalysts (approximately $50/mmol for acridinium-based catalysts versus $1,000/mmol for Ir-based catalysts), it was desired to explore the use of organic photocatalysts in this transformation. Optimization studies using the reaction of potassium isopropyltrifluoroborate with benzalaniline revealed that the photocatalyst 9-mesityl-10-methylacridinium tetrafluoroborate (Mes-Acr-Me) in dichloromethane gave the best yields of alkylation product, N-(2-methyl-1-phenylpropyl)aniline. In this presentation, the results of current efforts to expand the scope of the protocol to other imines and potassium organotrifluoroborates will be described.

Ramsey and Star-Critical Ramsey Numbers involving Generalized Fans


Streaming video available

Paul Hazelton, Winthrop University
Suzanna Thompson, Winthrop University

Faculty Mentor: Arran Hamm, Ph.D.

Ramsey Theory, one of the most well-studied branches of Combinatorics, can be paraphrased as the pursuit of "order amongst chaos." The Fundamental Theorem of Ramsey Theory (for graphs) states that, for any two graphs G and H, any large enough red/blue edge-colored complete graph contains a red G or a blue H. The Ramsey number for G and H, then, is the smallest complete graph with this "unavoidability" property. Recently, the star-critical Ramsey number was introduced, which is a slightly sharper measure on the unavoidability property. Our work focused on the generalized fan, which is formed by taking disjoint copies of a fixed graph H and joining each to a vertex. Recently, researchers have investigated Ramsey and star-critical Ramsey numbers involving this kind of graph which motivated our work. We computed both parameters for a type of generalized fan versus a complete graph, a type of generalized fan versus disjoint triangles, and a type of generalized fan versus a complete graph on four vertices.

The Efficacy and Convenience of Different Means for Improving Blood Pressure


Zain Anderson

Faculty Mentor: David Schary, Ph.D.

As of 2016, hypertension affects 33.2% of adults over 20 years old. Many people know that lifestyle changes, such as increasing physical activity through exercise, can improve their blood pressure. Sadly, more than 80% of adults do not meet the recommended guidelines for weekly aerobic and resistance training activities. People who do not already exercise might struggle with adherence to new, regimented programs, or they might find gyms to be intimidating, unfamiliar places. There is a need to inform this population of how they can improve their blood pressure most effectively without compromising other aspects of their lives. There are many forms of physical activity that can meaningfully improve blood pressure, including the use of hand grippers, aquatic training, and other more traditional forms of aerobic and resistance exercise. There are also passive means of improving blood pressure that do not require the use of pharmaceutical intervention, which can negatively impact an individual’s quality of life. This paper evaluates the efficacy and convenience of different means for improving blood pressure for a general population that does not regularly exercise, with the goal of providing approachable recommendations to get more people physically active while combatting hypertension.