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    Helly-Type Theorems on Support Lines for Families of Congruent Disks in the Plane
    (2023-12) Russ, Tyler R.; Soltan, Valeriu
    In this dissertation, we consider the problem to determine Helly-type numbers for support lines of nonoverlapping families of congruent disks in the plane. This problem, originally posed by R. Dawson for the case of disjoint families of convex bodies and by V. Soltan for the case of disjoint families of unit disks, has been recently solved. This research generalizes to the case of non-overlapping families of congruent disks. An essential part of the argument is based on the study of "critical" families of congruent disks.
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    Studying School Gardens as Habitat for Urban Butterflies and Outcomes of Involving Elementary School Students in Data Collection during a DC Summer Program
    Pontarelli, Katherine; Smith, Cynthia
    Schoolyard gardens are increasing in cities to simultaneously provide students with experiential learning opportunities and local communities with increased food security. These gardens may also provide urban habitats for pollinators and opportunities for students to interact with urban wildlife. Here we assess how schoolyard gardens may provide habitat for large-bodied butterflies and discuss how they may be designed to support more butterfly diversity. Due to their charismatic nature and presence in urban spaces, butterflies can be a flagship species to reconnect urban residents with the natural environment. Therefore, we designed the project to be student-led and assessed students' participation in the data collection process. Three elementary schools with rising first and third-grade students observe and capture large-bodied butterflies in their gardens during the summer of 2022. The species richness and abundance at school gardens were compared to butterflies caught by researchers in a corresponding natural area near each school. An N-mixture model was used to estimate the correlation between tree canopy, site area, and impervious surface to eastern tiger swallowtail (Papilio glaucus) abundance. Results showed that swallowtail abundance was negatively related to the percent of impervious surface at a site regardless of the site's area and proportion of tree cover. Our results indicate that urban schools with limited green space can increase butterfly abundance by planting more vegetation around the garden and decreasing impervious cover. Student discussions provided program feedback and increased interest in butterfly ecology within urban environments. These results indicate that involving K- 12 students in urban ecological research within their school grounds may increase their awareness of interactions with nature.
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    Assessing Thermal Imagery Integration into Object Detection Methods on Air-Based Collection Platforms
    Gallagher, James; Oughton, Edward
    Object detection models commonly focus on utilizing the visible spectrum via Red-Green-Blue (RGB) imagery. Due to various limitations with this approach in low visibility settings, there is growing interest in fusing RGB with thermal long wave infrared (LWIR) (7.5 - 13.5 μm) images to increase object detection performance. However, we still lack baseline performance metrics evaluating RGB, LWIR and RGB-LWIR fused object detection machine learning models, especially from air-based platforms. This study undertakes such an evaluation finding that a blended RGB-LWIR model generally exhibits superior performance compared to traditional RGB or LWIR approaches. For example, an RGB-LWIR blend only performed 1-5% behind the RGB approach in predictive power across various altitudes and periods of clear visibility. Yet, RGB fusion with a thermal signature overlayed provides edge redundancy and edge emphasis, both which are vital in supporting edge detection machine learning algorithms.
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    Urban Stream Restorations Increase Floodplain Soil Carbon and Nutrient Retention along a Chronosequence
    Napora, Katrina; Ahn, Changwoo
    Stream restoration is a common management practice to meet regulatory or voluntary efforts to improve water quality via carbon and nutrient retention, including in the Chesapeake Bay watershed. However, restoration projects have few quantifiable measures of project success, no standard metrics, and rarely collect pre-restoration data. Storage of nutrients, such as phosphorus (P), and carbon (C), in floodplain soils of restored streams can act as an easily quantifiable indicator of restoration success, particularly when the project goals include improved water quality. To determine how floodplains of restored streams change in their phosphorus and carbon storage as time since restoration increases, floodplain surficial soil samples (10 cm depth) were collected from 18 streams in the urbanized Piedmont region of northern Virginia, representing a chronosequence of time (1-10+ yrs.) since restoration as well as unrestored and reference streams. The samples were analyzed for total carbon (TC), total nitrogen (TN) and total phosphorus (TP) storage, whereas CO2 mineralization potential and equilibrium phosphorus concentration (EPC0) were measured as metrics of nutrient and carbon loss. These metrics were compared to time since restoration and potential environmental drivers, including soil moisture, pH, particle size, organic matter content, and degree of phosphorus saturation. These stream restorations demonstrated increasing nutrient storage for TC, TN, and TP along the chronosequence to values greater than both unrestored or reference streams, as well as decreasing C mineralization turnover and no significant changes in EPC0. Soil wetness and organic matter, key drivers in nutrient retention, also increased as restoration projects aged increasing nutrient and C storage. Overall, stream restoration did improve carbon and nutrient retention in floodplains as compared to unrestored sites and exceeded those of low urbanization 'reference' sites.
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    Creation of a Novel Fusion Protein WGA-eNpHR3.0 for Circuit Dissection in The Brain
    Kinsey, Lucas; Dumas, Theodore
    Over the last decade, optogenetic manipulation of neuronal activity has redefined functional circuit analysis in the central nervous system. Combined with promoters for specific neuron types, optogenetic actuators permit activation or inhibition of select neurons within intact circuits. A major advance in this field would be to enable movement across synapses to direct the optogenetic actuator to specific afferents of the target neuronal population. Fusion constructs of wheat germ agglutinin (WGA) have been shown to move retrogradely across synapses. In this thesis project, a transsynaptic optogenetic-construct was created by fusing WGA to the N-terminus of halorhodopsin (eNpHR3.0). This was done so by first amplifying the WGA sequence using mutagenic primers in a polymerase chain reaction (PCR) to flank the WGA fragment with restriction sites that match the insert location in eNpHR3.0. The PCR product was then ligated to the N-terminus of eNpHR3.0 and samples were transformed into E coli. Positive clones were verified to include the WGA sequence via restriction digest and electrophoresis. Positive plasmid samples were commercially sequenced in order to verify the WGA orientation. This novel WGA-eNpHR3.0 plasmid serves as a powerful tool to traffic eNpHR3.0 retrogradely both in vivo and in vitro.
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    Extracellular Vesicle Subpopulations From HTLV-1 Infected Cells Induce Differential Effects in Recipient Cells
    Cuba, Zachary; Kashanchi, Fatah
    Human T-cell Leukemia Virus Type 1 (HTLV-1) is the causative agent of Adult T-Cell Leukemia/Lymphoma (ATLL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Extracellular vesicles (EVs)—membrane-bound vesicles excreted by cells into the microenvironment which play an extensive role in cell-to-cell communication—have been implicated in contributing to these two conditions by carrying cellular materials from donor cells to recipient cells. Our lab has previously shown that EVs from ATLL cells contain viral protein and mRNA and promote cell-to-cell contact when placed on uninfected recipient CD4+ T-cells, enhancing HTLV-1 infectivity. Another recent paper involving ATLL EVs resulted in mesenchymal stem cell proliferation and was attributed to helping aid in leukemic progression. Because ATLL is a leukocytic cancer, ATLL EVs have the potential to interact with any cells that blood or biofluids interact with. However, a gap in knowledge exists relating to how ATLL EVs interact with differentiated non-leukocytes. In addition, many of the conclusions made about the effects of cancer “extracellular vesicles” look at a small subpopulation within the total EV population, and have not been generalized. Therefore, in this study, we sought to better understand the different impacts of individual ATLL EV subpopulations upon non-leukocytes. We found that differential ultracentrifugation subpopulations (2K, 10K, 100K, 167K (4hr) and 167K (16hr)) of HuT102 EVs contained different distributions of viral proteins, viral mRNA, and vesicle-associated proteins. We saw that HeLa cells treated with EV subpopulations had no change in cell viability but had preservation of normal cell morphology post-confluency and had different patterns of anchorage-dependent proliferation. Furthermore, we found that less-dense EV subpopulations 100K and 167K (16hr) helped promote anchorage-independent proliferation. Collectively, our data suggests that there may be a correlation between subpopulations with high amounts of vesicle markers and anchorage-dependent proliferation, while subpopulations with low amounts of viral markers may promote anchorage-independent proliferation. These findings prompt further research into how the contents of each ATLL subpopulation affect recipient cell proliferation and whether these recipient cells are pushed towards cancer development.
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    Sentinel-1 Synthetic Aperture Radar Burned Area Detection Using Expectation Maximization in a Multiscale Approach
    Heying, Louis D; Yang, Ruixin
    This research explores wildfire burn mapping using Sentinel-1 Synthetic Aperture Radar (SAR) imagery for the 2021 Woods Creek Fire in the Helena-Lewis and Clark National Forest in Montana and the 2021 French Fire near Lake Isabella in Kern County California. Sentinel-1 SAR imagery is used since it can be collected during most weather conditions as well as in heavy smoke and is useful in the upper latitudes where wildfires often occur. Both the ascending and descending orbits as well as the co-polarity (VV) and cross-polarity (VH) are evaluated. The increase of wildfire occurrence is the result of lower precipitation and fuel moisture content as a result of climate change. The methodology by which SAR imagery detects wildfire burns is adapted to use SAR imagery from Google Earth Engine (GEE) and a method provided by the Alaska Satellite Facility (ASF). This method utilizes a stationary wavelet transform and math morphology to process imagery at various scales and expectation maximization in order to generate change classes. The resulting burn area is compared to Sentinel-2 differenced Normalized Burn Area (dNBR) and MODIS Burned Area. The ascending orbits of Sentinel-1 burned areas provided the best results compared to those of the descending orbits likely due to the limited ability of GEE to radiometrically terrain correct SAR imagery.
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    Neural Effects of Estrogen on Mitochondrial Lipids and Cardiolipin Levels and Distribution
    Lebert, Nadege; Kabbani, Nadine
    A variety of lipid classes play important roles in neuronal structure and function. One of these lipids is cardiolipin, a phospholipid exclusive to the mitochondria and important to processes such as mitophagy and apoptosis through its differential oxidation. Estradiol (E2), a steroid sex hormone, is known to exert antioxidant effects in various cell types. I will establish a baseline lipidome in a line of neural cells and use E2-treatment conditions to determine if estrogen antioxidant effects are mediated through lipids, with a particular focus on cardiolipin. The results could contribute to understanding how hormonal changes can impact neural cell health and neurodegeneration.
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    Mammal Reactions to Novel Objects in Their Environment
    Wood, Hannah; Luther, David
    The way in which mammals respond to novel objects can inform experts and managers about how urbanization is impacting species. The behaviors of three common North American mammal species with great behavioral adaptability, raccoons (Procyon lotor); red foxes (Vulpes vulpes); and white-tailed deer (Odocoileus virginianus), were observed using remotely triggered wildlife cameras and novel objects. The amount of time an individual spent within view of the camera, the number of behaviors exhibited, and the time it took an individual to approach novel objects and control sites were analyzed as a function of canopy cover, impervious cover, and human population density. Correlations between canopy cover, impervious cover, and population density and response variables, and the direction of effects differed between species, but trends emerged among similar taxa (carnivores). This study can inform plans and protocols to mitigate human-wildlife conflicts while considering how different species respond to urbanization.
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    The Geographic Roots of Cultural and Liturgical Differences Amongst American Catholics
    Conti, Joel; Burtch, Nathan
    This paper addresses the geographic components of American Catholicism from 1972-2020. The study found significant, previously undiscussed geographic variations in the character and expression of the Catholic faith within the United States. This project fills a geography-sized gap in existing literature, which hitherto has only discussed regional differences at the broadest geographic scale. This paper found significant spatial variations amongst Catholic settlement and growth patterns, regional ancestral composition, age, rural/urban divisions, as well as inter-regional migration. Relationships and intersections between the variables were also examined, as well as the temporal evolution of these spatially distributed attributes. The study found that the modern Catholic Church in America was far from spatially uniform, varying across regions in a myriad of ways. Additionally, an examination on sociopolitical attitudinal differences between Catholics of different regions towards controversies within the Church is conducted using statistics from studies undertaken throughout the late 20th and 21st centuries. Opensource data on the locations of parishes categorized by liturgical styles were examined to discern spatial differences in traditionalist and modernist approaches to the Mass, as well as the distribution of parishes offering the Traditional Latin Mass. Data from the General Social Survey, Pew Research Center, county-level religious censuses of the United States, as well as a variety of survey data on Catholics from the previous thirty years were examined at the smallest geographic scale possible. The results of county, state, diocese, and census-division level analysis found that previous assessments of the geographic trends and characteristics of Catholics were missing key nuances which were only discernable by examining the data at these scales.
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    #COVID-19 Searching for a Relationship between Twitter Sentiment and Infectious Disease
    Stassinos, James Odysseus Van Der Loo; Anderson, Taylor
    Digital health data such as social media data has shown potential for identifying outbreaks faster than official records of disease incidence. The objective of this thesis 1 is to examine the relationship between COVID-19-related Tweet sentiment and COVID-19 cases over space and time and assess the extent to which Twitter-derived sentiment can be used for local COVID-19 surveillance in the United States. To our knowledge, there is no existing study that examines the relationship between Tweet sentiment and infectious disease cases at a spatially local level. The sentiment is computed using 56,755,894 Tweets from the TBCOV dataset for US counties over time. Tweet sentiment is examined with COVID-19 cases for each county globally, over time, and space using Pearson's R correlation. A negative association was observed between COVID-19 cases and the sentiment polarity of COVID- 19 tweets, but only in some regions of the US and only for some duration of the period of study. Further research is needed to understand the cause of the spatial and temporal non-stationary correlations between Twitter sentiment and COVID-19 cases. This would allow for the identification of when and where Twitter sentiment could be used as a signal for early disease outbreak warning.
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    Advancements in Computational Digital Pathology for EGFR and pd-l1
    Hoo-Fatt, Nicholas; Ullah, Aman
    Annually Inova Health Care System treats more than two million individuals through their integrated network. With the increasing need for fast and accurate results for its cancer patients, the Inova Laboratories Healthcare System’s reference laboratory, has acquired a Rapid Molecular Testing System, called BioCartis Idyllatm which is a real-time PCR bases molecular testing system. This system is user friendly and will help improve the turnaround time for diagnosis and treatment of cancer patients. Most of the existing molecular testing for Next-Generation Sequencing (NGS) platforms such as whole genome sequencing, Exon sequencing and Disease diagnosis panel takes approximately fourteen days or longer to obtain results. This is due to the need for high volume batched samples, specimen procurement, transportation and complex interpretation. While there have been great expansion of knowledge on molecular changes occurring in the cancer development, clinical utility of molecular tests for the diagnosis and especially for the treatment of cancers have been limited. The FDA has approved some treatments targeted for specific molecular changes such as EGFR mutation and KRAS mutation in lung cancers and BRAF mutation in melanomas. Molecular testing aids the pathologist to check for certain changes in a gene or chromosome that may cause or affect the chance of developing a specific disease or disorder, in this case lung cancer. Another area of rapid progress is the advancement of immune targeted therapy aimed at PD-L1. Initially applied to lung cancers and some GI cancers, and has been approved by FDA- for multiple cancer types recently. To be able to apply these advanced therapeutics there is a growing need for more efficient targeted biomarker testing, including fast TAT (turnaround time), user friendliness, remarkable accuracy, specificity and sensitivity which are equivalent to the NGS test results The objective of this project is to determine the use of rapid molecular tests for tumor mutations and the application of AI-based digital scoring methods of IHC (immunohistochemistry) test for PD-L1 for Keytruda® (Pembrolizumab).
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    Color and Temperature Are Not Related in the Crepuscular Reptile, Eublepharis macularius
    Hastings, Brandon; Chiari, Ylenia
    Ectothermic organisms depend on external sources for heating and cooling to regulate their internal body temperature. Some ectotherms rely on physiological color change by adjusting melanistic body coloration to increase or decrease heat absorption. Furthermore, ectotherms with a greater proportion of stable melanistic body coloration seem to have better thermoregulatory performance in cold climates due to better absorption of solar energy to buffer body temperature in relation to environmental temperatures. Together with its influence on thermoregulation, body coloration is also used in ectotherms for other ecological functions, from escaping predators to communicating with conspecific individuals. As fluctuations in extreme temperatures due to global warming are becoming more frequent, understanding the relationship between coloration and suboptimal temperature is essential to predict the influence that this may have on other functions. In this study, we tested if the exposure to suboptimal low temperatures produces a physiological color in the crepuscular reptile Eublepharis macularius. Furthermore, we tested if the proportion of melanism on the body of these animals is related to differences in heating and cooling rates of the internal body temperature during suboptimal temperature exposure. Temperature measurements were taken using infrared photography and temperature loggers, and coloration was obtained using objective photography and analyzed using a newly developed custom software package. As the dorsal part of the body is the one mostly involved in physiological color changes in ectotherms, we tested if dorsal coloration would change as a response to lowering environmental temperatures, analyzing melanistic and non-melanistic areas of the body separately for twelve geckos. We expected a higher degree of color change in non-melanistic versus melanistic coloration and a relative higher average body temperature in geckos with a higher proportion of melanistic coloration. We found that body temperature reflects substrate temperatures, but that the proportion of melanistic coloration has a moderate negative correlation with cooling rates of body temperature. We also did not observe a relationship between body temperature and physiological color change for melanistic and non-melanistic color. These findings suggest that in E. macularius melanistic coloration may be used for other purposes such as predator avoidance and that physiological color change may not be used for thermoregulation to increase heat absorption, but may occur for background matching, similarly to what described for other nocturnal/crepuscular reptiles. Future research should further test these hypotheses to elucidate the function of melanistic coloration in crepuscular and nocturnal geckos and to understand the evolution of pattern development in organisms active in low light conditions.
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    Mechanisms of Innate Immune Response Activation in Human Monocytes by Small Extracellular Vesicles Released During Infection with Burkholderia thailandensis
    Matulis, Graham; Hakami, Ramin
    The function of small extracellular vesicles (sEVs) released from host cells during bacterial infections is still largely uncharacterized, despite EVs representing key components of cell-to-cell communication. In particular, there is still a strong gap in knowledge about sEV released from innate immune cells that are infected with high-consequence pathogens such Burkholderia pseudomallei (Bp), including the molecular mechanisms by which these host-derived vesicles alter innate immune cell function. The Hakami laboratory had previously demonstrated that sEV released from monocytes that are infected with the Bp model organism Burkholderia thailandensis (EXi-Bt) induce specific phenotypic changes within naïve recipient monocytes. Specifically, they cause differentiation of the monocytes to macrophage-like cells, increased production of pro-inflammatory cytokines, and reduced intracellular bacterial burden if the cells are infected post sEV treatment. In this work, we have characterized the signaling pathways and cellular processes involved in these observed EXi-Bt-associated phenotypes. We demonstrate that the p38 MAP kinase pathway is involved for several of the observed effects, with chemical inhibition of p38 activity resulting in phenotype loss within EXi-Bt-treated cells. We also show that while IL-6 production is important for the reduced bacterial burden phenotype, it does not impact the monocyte-to-macrophage differentiation effect. Importantly, we show that EXi-Bt induces autophagy within the macrophage-like cell population, and that inhibition of autophagy by siRNA knockdown of Atg5 leads to a reversal of the reduced bacterial burden phenotype within the EXi-Bt-treated cells. These findings suggest that sEV released from Bt-infected monocytic cells prime recipient cells for enhanced response to bacterial challenge via the p38 and IL-6 signaling pathways and through induction of autophagy. The mechanistic characterization of these EXi-Bt-induced phenotypes provides further insight into the innate immune response that occurs during Bp infection, which may help inform the development of novel preventative and therapeutic strategies.
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    Using GEDI Data to Evaluate the Impact of the Australian 2019-2020 Fire Season on the Structure and Biomass of Gondwana Rainforests
    Flynn, Colin G; Wessels, Conrad
    Wildfires are increasing globally in their intensity, frequency, and range. A unique yet globally significant example is the devastating eastern Australia 2019-2020 fire season, which resulted in an unprecedented burnt forest area with > 21% of Australia’s temperate forests affected, and, in an unprecedented event, over 50% of Gondwana Rainforests in Australia were impacted. Currently, optical satellite imagery is used to detect active fires and assess burnt area, as well as impact. Assessing the impacts of fires solely through these techniques lacks information on vegetation structural changes, especially subcanopy changes. Lidar (light detection and ranging) can be used to evaluate the impacts of fire on vegetation structure more comprehensively. This study uses full waveform lidar data from NASA’s Global Ecosystem Dynamics Investigation (GEDI) to study the Gondwana Rainforests of Willi Willi National Park and Werrikimbe National Park, in New South Wales, Australia. GEDI footprints were filtered to remove those incident upon steep slopes (>25°) and collected more than 9 months before and after the MODIS recorded burn date. GEDI footprint level canopy structure measurements (Level 2A and 2B data products) were aggregated into 5 km2 grid cells across the study sites and showed an average decrease of 19% in Plant Area Index, and a 15% decrease in Canopy Cover Fraction. Vertical profiles of Plant Area Volume Density (PAVD) grouped into 5 meter vertical profile height bins showed an average decrease of -15.6% (STD = 8.1) for bins from 0-5 meters to 35-40 meters, with the largest decrease of -27.63% occurring in the 10-15 meter vertical profile height bin. GEDI relative height (RH) metrics were lower post fire: RH50 decreased by average of 12.7%, RH75 by 5.1%, and RH90, RH95, RH98, and RH100 all by <1%. GEDI footprint level aboveground biomass density (Level 4A product) showed a -1.5% decrease, correlating closely with percent change in RH75 and the 20-25 meter PAVD vertical profile height bin. The PAVD metric change therefore captured the impact of the fire more effectively than RH metrics, which showed limited change. Consequently, the footprint-level GEDI biomass which is largely derived from RH metrics show minor changes after the fire. These results indicate that GEDI was uniquely positioned to directly quantify the effects of vegetation disturbance on vertical canopy structure. The structural damage resulting from these fires ranged from entire stand-replacing disturbance to primarily crown or understory fires. We discuss the potential implications of these findings to understand the impact of fire severity and post-fire recovery on carbon stocks at regional scales.
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    Automated Electrochemistry for Measuring the Temperature Dependence of Various Cell Parameters via A Custom Built Microcontroller-Based Potentiostat
    Canon, Benjamin; Hussam, Abdul
    A $70 microcontroller based potentiostat with a temperature probe was designed, fabricated, and tested. The potentiostat was programmed to automatically repeat chronoamperometric experiments on a solution of potassium ferricyanide in potassium chloride at different temperatures under both kinetic and diffusion controlled conditions. Under kinetic control, the system proved to be quasi-reversible. Cottrellian behavior was observed under diffusion control. Currents due to the formation of an electric double layer were found to follow a power law. The temperature dependence of the heterogeneous rate constant, diffusion coefficient, double layer capacitance, and surface excess were all measured. From this data, the ionic mobility, activation energy and frequency factor for diffusion and heterogeneous electron transfer were all measured.
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    Vibrio vulnificus Type 6 Secretion System Exhibits Influence in Marine Aggregate Colonization
    Amiri, Navolle; Froelich, Brett
    Oysters are among the most economically beneficial species with regards to aquaculture, the process of farming aquatic species. As such, in order to ensure that oyster farming remains profitable, it is necessary to maintain a product that is safe for consumption. As oysters are oftentimes consumed raw, they have been known to cause seafood-based disease, particularly from bacteria in the genus Vibrio. Vibrio infections can be costly to treat and depending on the severity of disease, can even be fatal. With sea water temperatures rising due to global climate change, infection rates have also been on the rise. There is therefore a need to rapidly develop therapies that will prevent a further number of clinical cases. Gaining a better understanding of Vibrio ecology would aid with doing so, however, data examining this is limited. Previous studies have examined higher oyster uptake levels when bacteria are integrated onto marine aggregates. This project aims to observe the relationship between three different strains of pathogenic Vibrio vulnificus species both on artificially generated marine aggregates and within oyster matrices. This study found that within both aggregate and oyster competitions the three Vibrio vulnificus strains initially displayed an “rock-paper-scissors" effect. In competition between two Environmental (E) genotype strains VV JY1305 pVSV102 (hereby dubbed VV5-102) and SREL 106 pVSV208 (VV8-208), VV5-102 was outcompeted. Strain VV5-102 was able to outcompete the Clinical (C) genotype strain VV C7184 pVSV208 (VV2-208). Unexpectedly, VV2-208 was found to be more abundant than strain SREL 106 pVSV102 (VV8-102). However, this was later understood to be the occurring due to the presence of the plasmid. There was a novel discovery made that established the type 6 secretion system plays a role in integration of V. vulnificus into natural marine aggregates. These results expand upon our understanding of Vibrio interactions as well as highlight the need for more uptake experimentation in order to create viable treatments.
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    Determining the Safety of Antimicrobial Peptides, GATR3 and DRGN1, Against Host Cells and Red Blood Cells A
    Carpenter, Ashley; van Hoek, Monique
    Up to half of all combat injured patients suffer from infectious complications, predominately with biofilm-forming, multidrug-resistant (MDR) bacteria. Biofilm management of chronic wounds is an increasingly recognized factor in the healing of these injured patients. Coupled with the lack of new antibiotics and the emerging antibiotic resistance crisis there exists an urgent need for new therapeutics. Antimicrobial peptides (AMPs) have shown potential as antibacterial and antibiofilm; therefore, AMPs are an attractive candidate for clinical development. The synthetic peptides DRGN1 and GATR3 are being studied, and have been Both peptides have shown to have antimicrobial and/or anti-biofilm activity against medically relevant bacteria including Staphylococcus aureus and Pseudomonas aeruginosa. This study aims to test the safety of these lead peptides, DRGN1 and GATR3, and their wound healing properties against host cells. To evaluate the cytotoxicity of the lead peptides, cell cytotoxicity and hemolysis assays will be performed using human epidermal keratinocytes, human red blood cells and a human liver cancer cell line, HepG2 cells. To quantitate the wound healing of HEKa cells a scratch assay will be performed and measured. The cytotoxicity, hemolysis, and scratch assays will be compared with the control peptides, LL-37 and ID-1018. The therapeutic index is the ratio between the concentration at which a drug is toxic versus the concentration at which a drug is effective. While the exact therapeutic index can not yet be measured, some estimation of it will be made for the peptides. The proposed study will test the safety of these antimicrobial peptides and help identify whether DRGN1 and GATR3 advance through the pre-clinical in vivo studies.
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    Affinity Functionalized Strings for Blood Purification
    Cachaco, Silvia; Luchini, Alessandra
    Pathogen reduction is a proactive approach to maximize safety of blood transfusion. Methods of bacterial reduction are available for plasma and platelets but not for red blood cell concentrates and whole blood. We addressed this technology gap by using a novel approach consisting of molecular probes immobilized on nylon filaments (affinity networks) as a means to capture bacteria in suspension. We dyed nylon filaments with 24 dyes with different chemical properties, including acidic, basic, fast, metallic, hydrophobic, and uncharged/polar dyes. We established quality control measures of acceptable ranges of dye incorporation in the nylon network to ensure reproducibility. We performed incubations and microbiology cultures to assess the depletion capability of different affinity networks. Four affinity networks incorporating basic and hydrophobic dyes (pinacyanol chloride, reactive blue 21, sudan IV, alizarin cyanin) depleted more than 90% of E. coli suspended in phosphate buffer saline (PBS, 10 and 100 CFU/mL). Safranin O, methylene blue, alcian blue pyridine variant, and reactive blue 21 dyed nylon networks removed more than 90% of S. epidermidis from PBS (10 and 100 CFU/mL). In several instances, affinity networks removed all bacteria present in suspension. Affinity networks efficiently captured >90% S. epidermidis spiked in plasma at a 10 and 100 CFU/mL concentration, and the combination of sudan IV, alcian blue pyridine variant, and reactive blue 21 functionalized affinity networks had synergistic effects. Affinity network sequestration of lipoteichoic acid (LTA), a major component of gram-positive bacterium cell wall, spiked in model solutions, suggested that LTA is involved in the binding and capture of bacteria. In conclusion, affinity networks functionalized with synthetic dyes is a promising medium to capture bacteria from suspensions and from biological matrices for future application as a pathogen reduction technology. Future work will include a prototype production for a pathogen reduction device.
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    A Floristic Checklist of Shenandoah River State Park Warren Co., Virginia
    Sheik, Matthew; Weeks, Andrea
    A floristic checklist of vascular plants was conducted at Shenandoah River State Park (SHSP) in Bentonville, Virginia during the 2021 and 2022 growing seasons. SHSP encompasses 665 hectares (1619 acres) of the northern Shenandoah Valley between the Massanutten Mountains and Shenandoah National Park. The park lies in Warren County which has been botanically underexplored, consistently ranking last in plant collection metrics among the five northern Shenandoah Valley counties. SHSP contains 11 community types that span 165 – 296 masl from inland wetlands and river scour communities to upland forests, including endemic Central Appalachian Shale Barrens. A total of 797 collections yielded 536 unique taxa from 109 vascular plant families. These collections were secondarily extended with in-field pictures that were aggregated on an iNaturalist project page. Quick Response (QR) codes were used to link herbarium vouchers to their respective iNaturalist entry. Sixty-four county records of taxa not yet observed in Warren County and one state record, Equisetum laevigatum A. Braun were documented. As a first for SHSP, this floristic checklist provides baseline botanical knowledge of the park and expands the available digital data of vascular plants for Warren County by 20%. This augmented knowledge, in tandem with the accompanying iNaturalist project page, will not only be useful to park managers for future development and conservation plans but will also serve as a tool for community scientists to enhance their knowledge of the local flora.