The Effects of Cortisol on Blood Coagulation in Zebrafish

By Bailey Bashara

Faculty Mentor: Professor Dianne Baker

In humans, increased cortisol can lead to pathological increases in blood coagulation, in turn leading to thrombotic events such as deep vein thrombosis and pulmonary emboli. Cortisol appears to promote coagulation by upregulating the transcription of coagulation factors, thereby increasing the likelihood of the coagulation cascade. It is possible that cortisol has a similar effect on clotting in zebrafish, via effects on transcription of coagulation factors. If so, this would support the use of zebrafish as a model organism for testing anti-coagulant medications. I hypothesized that cortisol induces transcriptional upregulation of genes encoding coagulation factors in zebrafish, as in humans. Additionally, that transcript levels of the coagulation factor genes will be higher in stressed females than stressed males. Male and female zebrafish (n=34) were exposed to acute stressors for 7 days, resulting in an unpredictable chronic stress environment. Additional males and females (n=34) were not exposed to the stress environment and were used as control. Twenty-four hours after the seven-day treatment, the fish were euthanized, and their livers transferred to TriReagent. The RNA from the livers was isolated and cDNA synthesized for qPCR. qPCR measured transcript levels of clotting factors X and VII. Mean (SE) Factor VII transcript levels of the control group (-0.211) were not significantly different from stressed groups (-0.367), nor in Factor X control group (-0.100) and stressed groups (-0.576) (p>0.05). Difference in transcript levels between each sex were also determined, with no significant difference in Factor VII control females (0.063), stressed females (-0.583), control males (-0.063), or stressed males (-0.004). No significant difference was found between transcript levels of Factor X control females (0.051), stressed females (-1.361), control males (0.1) or stressed males (0.122) (p>0.05). There is no evidence that increased cortisol causes differences in the transcription of genes coding for coagulation Factors VII or X.

Quantifying Microplastics Found in Canada Geese from Various Fredericksburg Locations

By Zhanna Leavitt

Faculty Mentor: Professor Andrew Dolby

Plastic pollution has accumulated to a concerning degree in many of the world’s ecosystems. Microplastics are some of the smallest plastic debris and are generally defined as plastic fragments less than 5mm in diameter. Given their small size, microplastic fibers and fragments can easily enter aquatic ecosystems and the species that feed in or near the water, such as waterfowl. Studies have found microplastics present in the gastrointestinal tract and in fecal samples of several freshwater bird species. Since Canada Geese are commonly found in the Fredericksburg, VA area, I decided to quantify microplastics found in Canada Geese (Branta canadensis) fecal samples in several Fredericksburg locations. I wanted to test the hypothesis that microplastic concentration was correlated to sample location. I obtained fecal samples from Ficklen Island, Old Mill Park, and the parking lot of the Outback Steakhouse in Central Park. The samples underwent digestion in 20 mL of aqueous 0.05 M Fe(II) solution and 40 mL of H2O2 and then vacuum filtrated to isolate microplastics. I then quantified microplastic under a dissecting microscope. There was no correlation between microplastic concentration and sampling location however, the majority of samples contained microplastics. Blue fibers were the most common microplastic type. Microplastics were present in nearly all of the samples supporting the evidence that waterfowl are regularly ingesting microplastics from their environment.

Role of the Pvr signaling Pathway in a Fly Model for Myotonic Dystrophy Type 1

By Maya Takashima, Huda Al-Bana, Grace Holcomb

Faculty Mentor: Professor Ginny Morriss

Myotonic Dystrophy Type 1 (DM1) is a multi-systemic genetic disorder that causes severe muscle weakening and wasting. The phenotype is caused by a CTG repeat expansion in the 3’ untranslated region in the DMPK gene. Studies have shown several different signaling pathways implicated in the DM1 muscle phenotype; however, one pathway that has not been implicated in DM1 is the platelet-derived growth factor receptor beta (PDGFRβ) signaling pathway. PDGFRβ is involved in cell growth, survival, and skeletal muscle hypertrophy. With this involvement, the pathways deregulation could contribute to the muscle wasting seen in DM1. This project uses a fly model to understand the role of the pvr signaling pathway (PDGFRβ fly equivalent) in muscle wasting due to DM1. Through this fly model, it will be determined which downstream pathway is primarily affected during deregulation. The two downstream pathways targeted are PI3K/Akt and Ras/Mek/Erk. Flybase was utilized to determine the downstream gene targets and fly lines needed to start the fly model. After selecting the gene targets, fly lines were picked that were either overexpression of the gene (UAS – upstream activating sequence) or a knockout. Gal4 promoter lines and CTG repeat lines are also needed. Gal4 promoters drive the expression of the desired gene, and the CTG repeat represents the CTG repeat of DM1. With the fly lines chosen, mating schemes were planned to determine what crosses needed to occur to get the desired progeny. The goal was to end up with a progeny with the Gal4 promoter, CTG repeat, and stock line all in the same fly. In the future, using these progeny, different tests will be completed to look at how the overexpression or knockout affects the muscle wasting phenotype.

Effects of starvation and time of day on crayfish foraging behaviors

By Kylie Sunamoto

Faculty Mentor: Professor Abbie Tomba

Crayfish foraging behaviors can alter aquatic ecosystems. Starvation and time of day are two potential factors that can influence those foraging behaviors, but the interaction between the two variables has not been studied. In this study, we observed the movement of fed and starved crayfish during the day and at night in the presence of both water and food odor. We calculated both total movement and change in movement and predicted more activity when starved and at night. Crayfish did not show a preference for day or night, nor did they display significantly more movement when fed or starved. These results do not match previous literature, meaning that further research on these factors is necessary, especially in Cambarus acuminatus.

Differential Myokine Expression in a Model for Myotonic Dystrophy Type I

By Huda Al-Bana, Maya Takashima, Grace Holcomb

Faculty Mentor: Professor Ginny Morriss

Myotonic dystrophy type 1 (DM1) is a multi-systemic disease resulting in severe muscle weakening and wasting. Skeletal muscle wasting is the predominant cause of morbidity and mortality and is responsible for 60% of DM1-associated deaths. DM1 is caused by CTG repeat expansion in the 3’ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Myokines are proteins that are produced and released by muscle cells in response to muscular contractions. Previous studies indicated that myokine signaling is deregulated in a mouse model expressing expanded CUG repeat (CUGexp) RNA. The goal of this work was to assess muscle-specific differential expression of myokines in mice expressing CUGexp RNA, relative to non-CUGexp RNA expressing control mice. Previously published RNA-seq datasets were compared to determine which putative myokines, identified in skeletal muscle samples of healthy subjects, were differentially expressed in CUGexp expressing mice. Myokines that display significant differential expression at 6, 12, and 20 weeks of repeat expression include the Cx3cl1, Cxcl10, and Gdf5. Primers were designed customary for these genes and specified for regions with constitutive exons to allow consideration of overall expression levels. Primers were then optimized using RT-PCR. The developed primers will be used to validate differential expression levels of these myokines using quantitative RT-PCR. Validated myokines will be assessed in unaffected and DM1 human myoblast cell lines to determine the contribution of these myokines to the skeletal muscle phenotype in DM1.

Apoptosis in Crithidia fasciculata

By Kaelynn Parker and Abigail Delapenha

Faculty mentor: Professor Swati Agrawal

Crithidia fasciculata belongs to a group of parasites called kinetoplastids that comprise many important human pathogens. Evidence of apoptosis has been found in these parasites with pathways that appear to be different than in mammalian cells. Therefore, careful characterization of these pathways can provide ways to manipulate parasite infection which could be used to create better treatments for these diseases. In this study, potential apoptosis genes conserved across all kinetoplastid parasites were identified using gene prediction programs in Tri-TrypDB and BLAST searches. Homologous genes were identified in C. fasciculata and a comprehensive q-PCR analysis showed differential upregulation upon induction of apoptosis. One of the genes significantly changed was Bax1 inhibitory gene (Bax1i), an inhibitor of the putative apoptosis promoting Bax1. In order to characterize this gene further we made gene modification constructs for tagging and gene deletion using the CRISPR-Cas-9 system. A homologous repair template was created for Bax1i using 500 bp homology arms and a drug resistance gene using a fusion PCR protocol. Constructs were made using both Puromycin and Blasticidin resistance genes. We have successfully created and optimized the fusion PCR protocol for generation of 3.5Kb drug repair cassettes. The same process was repeated for Phosphoglycerate mutase family member 5 (PGAM5). Drug selection trials using Puromycin found the optimum concentration of drug is 50 µg/mL. Blasticidin trials are still being performed. The optimization of the fusion PCR protocol and drug selection procedure, along with the identification of genes done in this project will be important for continuing work.


Influence of Social Status on Behavior and Stress in Mice

by Samantha Everett

Faculty mentor: Professor Parrish Waters

Mice are socially aggressive animals and tend to interact in ways that are representative of a social hierarchy. Their interactions and behaviors determine their position in the social hierarchy, i.e., dominant, subordinate, or somewhere in-between. The present study examined the effect of social rank on behavior and stress/anxiety. Mice were given access to a running wheel, an important resource because it provides the mice with stress relieving exercise, in both their home cage and their accessory cage. The mice’s daily activities, along with specific tests, were recorded to measure each mouse’s anxiety and identify them as dominant or subordinate. While the social rank of the mice was determined, none of the physiological or behavioral tests performed provided conclusive results demonstrating significant differences in anxiety or response to stress. This could be attributed to several factors, such as the spacious home cages, the accessibility of the isolated accessory cages, and the availability of two running wheels. Said factors possibly created a less stressful environment for the mice. The lack of significant results could also point to the behaviors and tests observed were not appropriate for detecting the effects of social stress in mice.

Toxoplasma gondii SUB4 Gene Deletion Mutants and Their Viability in Host Cell Invasion

by John Asmus

Faculty mentor: Professor Swati Agrawal

Toxoplasma gondii is a microscopic parasitic protist. It is responsible for the disease toxoplasmosis which can cause severe health problems in immunocompromised individuals and babies whose mothers become infected while pregnant. T. gondii is an obligate intracellular parasite that can infect almost any warm-blooded mammal. Because of this, host cell invasion is a key function that T. gondii must perform. Vital to this process is a group of secretory organelles including rhoptries, micronemes, and dense granules. Research has shown that subtilisin-like proteases act in processing and cleaving certain sites on proteins associated with these secretory organelles. T. gondii has a set of 12 genes that code for these proteases. While much research has been done on SUB1 and SUB2, little to none has been done on other genes such as SUB4. To determine the vitality of SUB4 in T. gondii host-cell invasion, I am creating gene knockout mutants using CRISPR-cas9 and will observe their ability to invade mammalian fibroblast cells. I created a drug repair cassette by adding SUB4 homology arms to a drug marker amplified from pJET plasmid. I designed and am currently in the process of amplifying two sgRNAs that flank the gene on both 5′ and 3′ ends. Once these are constructed, I will transfect T. gondii ΔKU80 cells, which cannot perform nonhomologous end-joining repairs, with these vectors to knockout the SUB4 gene. Following drug selection and screening to determine if the parasites successfully recombined the SUB4 locus, I will characterize mutant parasites using microscopy and biochemical techniques. Finally, I will conduct invasion and egress assays to determine the ability of Toxoplasma gondii to invade host cells without expression of the SUB4 gene. I expect that they will invade the fibroblast cells but at a slower rate compared to the wild-type parasites, elucidating the role of SUB4.

Turtle Population Sex Ratios at Urban vs. Rural Locations

by Abigail Conklin

Faculty mentor: Professor Bradley Lamphere

In many turtle species, the sex of an individual is strongly influenced by the environmental temperatures it experiences prior to hatching. Climate change and urbanization may raise the temperature of nesting habitat enough to strongly skew sex ratios in freshwater turtles, but data on that question are lacking. I sampled multiple urban and rural sites to examine the effect of urbanization on the sex ratio of turtle populations. Three urban sites and three rural sites were sampled.

The Effectiveness of Uzarigenin as an Antiseptic against Staphylococcus aureus

by Gabrielle Moore

Faculty Mentor: Professor Lynn Lewis

To determine the effectiveness of uzarigenin on S. aureus, the agar disk diffusion method was first completed to study the cytotoxicity effect of uzarigenin directly on the bacteria. A cytotoxicity assay was then completed to test the cytotoxicity of the uzarigenin on the human epithelial fibroblasts themselves. After, a TCID50 assay was used to test for a preventative dose of uzarigenin against S. aureus to determine at what dilutions the uzarigenin can protect the cells from infection at least 50% of the time. It was found that there was an average 8.5 mm zone of inhibition compared to a standard 26.4 mm zone of inhibition by tetracycline. Since there may be different diffusion rates, S. aureus may be resistant to uzarigenin. It was also found that the uzarigenin caused cytopathic effects (CPE, changes in the host cell morphology due to infection) on the human epithelial fibroblasts up until a dilution of 10^-6. Additionally, the 10^-6 and 10^-9 dilutions of uzarigenin were effective in combatting an infection from S. aureus, leaving little to no traces of cytopathic effect. However, dilutions further than 10^-9 seemed to be too diluted to combat an infection, which resulted in CPE in all or most of the wells. Furthermore, the preventative dose for uzarigenin against S. aureus was found to be 10^11.2. In conclusion, uzarigenin may be able to be used as an antiseptic.

Phage Hunting at the University of Mary Washington: Genome Annotation of Hari and JackRabbit

By Hannah Lee, Sabine Krall, John Tan, Raesa Zia, Charlotte Russell, Jayme Boudreau, Hannah Stottlemyer, Andrew Cooper, Elizabeth Sullivan, Madison DeWitt, Mitchell Freitag, Jacob Cantor, Arianna Chase, Vallarie Burge

Faculty Mentor: Professor Swati Agrawal

Bacillus thuringiensis subspecies Kurstaki (BTK) is often used as a microbial insecticide for pest control and as a simulant for Bacillus anthracis in biowarfare and bioterrorism studies. Students in 2021 Phage Hunters class at University of Mary Washington isolated nine bacteriophages using the host Bacillus thuringiensis subspecies Kurstaki. Two phages, Hari and Jackrabbit, were sent to SEAPHAGES for sequencing are currently being annotated in the lab during the Spring semester. Hari was found in a soil sample obtained from King George, VA while JackRabbit was isolated from Linton, VA. Both samples were isolated from enriched cultures. Hari has a genome length of 161,978 bp, which auto-annotated with 286 features, and a direct terminal repeat of 2,633 bp. Hari is most similar to DIGNKC, SBP8a and PPIsBest by BLAST. JackRabbit has a genome length of 161,552 bp, which auto-annotated with 288 features, and a direct terminal repeat of 2,821 bp.

Using BLAST to Detect Horizontal Gene Transfer in Pathogenic Fungi

By Christopher Good

Faculty Mentor: Professor Theresa Grana

Similarly to antibiotic resistance, antifungal resistance is a growing challenge for clinicians. Mechanistically, one method of antibiotic resistance acquisition is through horizontal gene transfer (HGT). Although associated more with prokaryotes, past studies show limited evidence of HGT in Candida yeast, warranting additional comparative, genomic and proteomic research on the evolutionary forces behind fungal virulence. This honors capstone project used the NCBI’s Basic Local Alignment Search Tool (BLAST) to quickly and statistically compare existing biological sequence data in conjunction with EMBL Multiple Sequence Comparison by Log-Expectation (MUSCLE) alignment and Molecular Evolutionary Genetics Analysis (MEGA) to visualize likely evolutionary relationships. Thus, the objective of this project was to use bioinformatics tools to identify potential instances of Horizontal Gene Transfer (HGT) between pathogenic yeast and viruses, specifically HGT as an evolutionary mechanism for antifungal resistance gene (ARG) acquisition. Of the subset of ARGs searched, BLAST showed more support for ERG3 (C-5 sterol desaturase) HGT between edafosvirus and C. glabrata and between orpheovirus and C. albicans and C. californica, respectively. However, the ML phylogeny contradicts these BLAST results and shows more support for ERG3 HGT between both viruses and Hypopichia burtonii. Thus, while BLAST showed limited evidence for ERG3 HGT between three Candida species and two viruses, the ML phylogeny fails to support these evolutionary events. For the purposes of HGT, BLAST might be better suited to certain organisms (i.e., prokaryotes) and its use should be reinforced as a non-definitive predictor of such evolutionary events. Furthermore, increased understanding of the numerous uncharacterized Candida genes could reveal new evidence of fungal HGT and guide treatment options. Word Count: 261

Quantifying Hyperforin in St. Johns Wort

By Chase Forster

Faculty Mentor: Dr. Josephine Antwi

Hyperforin is a known compound naturally produced by St. Johns Wort plants and has been proven to help treat mild depression. The purpose of this project was to determine optimal growing conditions for this plant to maximize efficiency of hyperforin production.

The Influence of Habitat Disruption and Induced Overstimulation on Working and Spatial Memory and Hippocampal BDNF Levels in CD1 Mice

By Katie Warlick, Chloe Dishong, Jada Ramos, Olivia Asbell

Faculty Mentor: Professor Parrish Waters

Our experiment aims to explore the influence of habitat disruption and induced overstimulation on working and spatial memory in CD1 mice, and consequently, hippocampal Brain- Derived Neurotrophic Factor (BDNF) levels. We hypothesized that, because of the significance of the role of BDNF in the regulation of neuroplasticity, overstimulation onset by habitat disruption will result in diminished cognition, including working and spatial memory, and decreased intracellular output of hippocampal BDNF. Following a two-week acclimation period, the second of which we began habitat disruption, we conducted a behavioral testing paradigm on a group of ten CD1 mice (n=5) to assess working memory known as the Y-maze test. Contrary to our hypothesis, results showed that mice who were subjected to overstimulation induced by habitat disruption performed better in the Y-maze and displayed an enhanced capacity for working memory compared to the control mice, but the difference in BDNF concentration between groups was not statistically significant. We therefore retained the null hypothesis that overstimulation does not influence hippocampal BDNF levels in CD1 mice.

The Influence of Olfactory Stressors on Anxiety-like Behaviors (ALB) and Amygdalar Brain-Derived Neurotrophic Factor (BDNF) Levels in CD1 Mice

By Mary Zagrobelny, Bradley Torrington, Olayemi Fadahunsi, Laiba Murad

Faculty Mentor: Professor Parrish Waters

Cat odors serve as stressful stimuli for mice, leading to profound anxiety-like behaviors (ALB). These ALB are possibly the result of decreased brain-derived neurotrophic factor (BDNF) levels in the amygdala. Although the anxiogenic effects of cat odors have been established extensively, the relationship between long-term cat urine exposure and amygdalar BDNF levels has yet to be studied. To explore this relationship, we conducted a 21 day experimental study, in which mice were intermittently exposed to urine-soiled cat litter. Our study determined that long term intermittent cat urine exposure induced ALB in mice, which were negatively correlated with amygdalar BDNF levels. However, we observed no significant changes in the amygdalar BDNF levels in response to stress.

Lab Manual by a Lab Aide, for Lab Aides

By Theresa Vierow

Faculty Mentor: Professor Swati Agrawa

For my Capstone project, I created an online lab manual. My intention with this document is to provide guidance for future lab aides of BIOL 125 and BIOL 126 – the series of introductory honors biology classes referred to as Phage Hunters. This document is online for easy access when in the lab, and contains information on where to find needed materials for labs in the spring and fall, from wet labs to observational labs to bioinformatics. Also included in this lab manual is information I have picked up as a lab aide for three years that will not be immediately obvious to newcomers to the job.