Senior Project Abstracts 2015

Investigation of Keap1 and Nrf2 Expression in Canine Osteosarcoma Cell Lines
Thomas S. Albanesi, 2015 (Advisor: Dr. Ann Kleinschmidt)

Osteosarcoma, a primary bone cancer, is a devastating disease that has a poor long-term prognosis and affects adolescent humans as well as Canis lupus familiaris (the domestic dog).  The profile of the disease is similar between both species, and thus, studying canine osteosarcoma may lead to a better prognosis for dogs as well as humans.  The Keap1/Nrf2 pathway regulates oxidative stress in cells, and through the protection Nrf2 provides, it may act as both a tumor suppressor gene and an oncogene.  Thus, dysregulation of Keap1 and Nrf2 may contribute to oncogenesis.  A form of gene expression regulation, DNA methylation, is a possible causation of such dysregulation.  Thus, in this project, the methylation status of the Keap1 promoter and its effects on Keap1 gene expression were analyzed in osteosarcoma cell lines.  Quantitative PCR did not show significant differences in the Keap1 mRNA levels between the cell lines, but there was a significant difference in the Nrf2 mRNA levels.  Minimizing the variability between the trials, the ratio of Nrf2/Keap1 mRNA levels showed significant differences between some cell lines.  Keap1 promoter methylation was detected in three of the four osteosarcoma cell lines, but the methylation status of the control cell line was inconclusive.  The data supports a possible role of Nrf2 and Keap1 in osteosarcomagenesis.  However, the results of the DNA methylation analysis were inconclusive.

An Investigation of the Thermodynamics of Cytosine Bulges at the Splice Site of Influenza A in the Double Stranded RNA Region of Hairpin Loops
Bethany M. Crile, 2015 (Advisor: Dr. Marty Serra)

Biofilm characterization of Haemophilus ducreyi’s sexually transmitted versus chronic limb ulcer strains
Andrew J. Crofford, 2015 (Advisor: Dr. Tricia Humphreys)

Haemophilus ducreyi, the causative agent of the sexually transmitted infection chancroid, has two sexually transmitted ulcer strains: class I (35000HP), class II (HMC112), and a newly discovered chronic limb ulcer (CLU) strain (NZS3). CLU are transmitted by non-genital interaction such as casual contact with a limb abrasion. It is unknown if these strains form biofilms, but it is hypothesized the CLU strain has greater biofilm components present because of its seeming ease of transmission. Therefore, the three strains were compared for biofilm growth using real time RT-PCR and fluorescent and confocal microscopy. Three open reading frames within a polycistronic operon that codes for fimbria-like proteins (flp) and tight-adhesion proteins (tad) were compared in biofilm and planktonic cells. Microscopy images identified extracellular DNA and carbohydrates (α-mannopyranosyl and α-glucopyranosyl), common biofilm formation indicators, which form a matrix around the cells. HMC112 typically had more proficient growth than 35000HP, which had better growth than NZS3. There was no statistical significance between biofilm and planktonic mRNA levels for flp3, orfB, or tadG between classes (P=0.49, 0.88, and 0.27 respectively). However, HMC112 formed the most prolific biofilm, but it had the least amount of mRNA from the flp/tad operon. NZS3 did not form as three-dimensional biofilms as the other two strains, but it had the greatest mRNA levels. This study supports the idea that H. ducreyi is a biofilm-forming bacteria when observed in a pure liquid culture.

Investigation of the Modification of Cysteine Residues in Brassica rapa APX1 by CysNO
Patrick P. Ottman, 2015 (Advisor: Dr. Ann Kleinschmidt)

There is evidence that modification of cysteines by oxidation and nitrosylation affect protein structure and function. While S-nitrosylation of cysteines has been researched, no studies have ever investigated how multiple cysteines in a single protein are differentially nitrosylated and only predictive models exist. It has been shown that nitrosylation at cysteines of the cytosolic ascorbate peroxidase of Arabidopsis thaliana (AtAPX1) affects its activity. To investigate the modification of the cysteines on Brassica rapa APX1, a protocol including reduction of the protein with TCEP, treatment with different concentrations of CysNO, followed by labeling unmodified cysteines with DyLight 550 were developed. Five transitions of fluorescence were observed between concentration ratios (CysNO/BrAPX1) 0.08/1, 0.11/1, 0.13/1, 0.22/1, 0.34/1, and 3.36/1. The activity of the corresponding nitrosylation events were also investigated and results showed that as more S-nitrosylation events occurred, the activity of the protein varied. From this research it is theorized that different cysteines are modified at different concentrations of CysNO and thus all the cysteine residues do not have the same redox potential because of differing microenvironments. This could mean that each cysteine modification has its own separate effect on the proteins activity and thus the protein’s activity could be redox controlled.

Disruption of Haemophilus ducreyi biofilms by probiotic lactobacilli
Jenna L. Sandala, 2015 (Advisor: Dr. Tricia Humphreys)

Haemophilus ducreyi, an obligate human pathogen, is the etiological agent of the sexually transmitted infection (STI) chancroid, which is marked by soft, erythematous papular lesions that appear on or around the genitals. Though wide-spectrum antibiotics are generally successful in clearing H. ducreyi infections, this conventional treatment can be hindered by a variety of factors, including the formation of biofilms, communities of bacterial cells that organize into a matrix of extracellular polymeric substances (EPS).  Recently, the introduction of probiotic lactobacilli to other urogenital infections has been shown to not only disrupt biofilm formation, but also clear the infection on the whole.  Therefore, the purpose of this study was to determine what effect, if any, the introduction of probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 had on H. ducreyi 35000HP biofilm structure. Additionally, the H. ducreyi transcriptional response to the presence of lactobacilli was quantified by performing real time RT-PCR of the CpxRA stress response gene products.  Analysis of biofilm structures was performed using both light and fluorescent microscopy, and revealed that H. ducreyi biofilms that had been challenged with lactobacilli were consistently less dense and exhibited scattered growth in comparison to unchallenged biofilms. Real time RT-PCR analysis of the cpxA and cpxR gene products showed a decrease in expression of both genes in cells within a biofilm compared to planktonic cells; similarly, cells challenged with lactobacilli exhibited a down-regulation of both genes in comparison to unchallenged cells.  Because the CpxRA regulon inhibits transcription of virulence factors under normal conditions, this means that both biofilm formation and challenge with lactobacilli are correlated with an increase in virulence factor expression, suggesting that these conditions are associated with the H. ducreyi stress response.  Findings from this study support the idea that probiotic bacteria play an integral role in maintaining urogenital health and show promise for the use of probiotic lactobacilli for treating chancroid infections in the future.

The Enzymatic Degradation of Diblock Copolymers by Burkholderia cepacia lipase
Mark P. Seraly, 2015 (Advisor: Dr. Ryan Van Horn)

Oxyfunctionalization via Flavin-Dependent Monooxygenases and their Biomimetic Analogues
Will D. Shields, 2015 (Advisor: Dr. Shaun Murphree)

Effect of malathion and resveratrol treatment of SH-SY5Y cells on levels of SOD1, SOD2 and CAT mRNA
Katherine M. Shumway, 2015 (Advisor: Dr. Ann Kleinschmidt)

The effects of malathion with and without resveratrol on mRNA levels of the antioxidant enzymes SOD1, SOD2, and CAT were observed in SH-SY5Y human neuroblastoma cells. Several studies have shown that malathion induces oxidative stress, so it was hypothesized that treatment with malathion would induce oxidative stress and cause increased mRNA levels of the enzymes, and treatment with resveratrol would increase mRNA levels of SOD2 and CAT while SOD1 mRNA would remain unchanged.  Treatments with no chemical (control), 100 µM malathion, and 100 µM malathion and 100 µM resveratrol, were performed for 16 hrs, 1 hr, 2 hrs, and 3 hrs.  Total RNA was isolated from the cells and analyzed for RNA quality.  The 16 hr treatments resulted in degraded RNA, only 1-3 hr treatments were used for reverse transcription.  Real-time PCR was performed on resulting cDNA to measure mRNA levels of SOD1, SOD2, and CAT in control cells, those treated with malathion, and those treated with malathion and resveratrol.  Results suggest that neither malathion treatment nor malathion and resveratrol treatment had an effect on the levels of mRNA for these antioxidant enzymes.  It is possible malathion and resveratrol are unable to regulate the oxidative stress response in SH-SY5Y cells.

Total Synthesis of a Potential Antifouling Furanosesquiterpene and Analogues
Chad N. Ungarean, 2015 (Advisor: Dr. Shaun Murphree)