Schedule of Events
Science Showcase Poster Session
Friday, April 26, 2024
11:00 am - 12:30 pm
Soiland Recreation Center (Gym)
Students from the Division of Natural Sciences will present results from their research and class projects.
Student Abstracts
Characterizing the Role of the EmrAB-TolC Efflux Pump in E. coli using Transcriptomics and a Genetic Approach.
Multidrug efflux pumps found in Gram-negative bacteria contribute to resistance to several classes of molecules including antibiotics. In addition to their well characterized role in antimicrobial resistance, they have been demonstrated to be involved in regulation of metabolism in E. coli. Specifically, deletion of the main efflux pump in E. coli, AcrB, leads to global changes in metabolism as well as an increase in motility. We observed that deletion of another efflux pump, EmrB, has a similar increase in motility observed in the acrB-deleted strains. In this study, we aim to understand how the EmrB efflux pump controls motility and which metabolic pathways are altered when the pump is deleted. It has been demonstrated that the efflux pump regulator AcrR is the main mediator of increased motility when acrB is deleted. However, our results suggest that AcrR may not sense and respond to metabolic changes induced by deletion of emrB. To discover which metabolic pathways are altered by deletion of emrB and to understand the link between the EmrB pump and motility, we have prepared samples for global transcriptional analysis of the emrB mutant and parental strains to elucidate the role of this pump in regulating metabolism and motility. After the transcriptional samples were analyzed we selected six genes that were significantly upregulated in the emrB deleted mutant to begin preliminary analyses. The genes sdhA, sdhC, sdhD, fepD, metF, and entC, are currently involved in experiments examining the changes in Motility and Biofilm production.
Student(s):
Klay Adamson
Faculty Mentor:
Dr. Dana Harmon
Microfiber Content in Pacific Oysters (Crassostrea gigas) from Morro Bay, California
Plastics are a major source of marine pollution. One form of plastic pollution is microfibers, which are synthetic fibers five micrometers or smaller that are shed by artificial clothing. The size of microfibers enables them to easily be ingested by a number of marine organisms, including oysters. Oysters are filter feeders and a major aquaculture asset, which presents a concern for the effects of microfiber ingestion on human health. Very few studies have been conducted quantifying microfibers using Pacific oysters (Crassostrea gigas) sourced from California. This study quantifies microfiber content in the Pacific oyster farmed for human consumption in Morro Bay, California. Oyster samples were digested using 30% hydrogen peroxide, creating a heterogenous mixture from which microfibers were isolated by vacuum filtration. The microfibers were then manually quantified using a microscope. An average of 9.12 microfibers were recovered per oyster sample. Smaller oysters were found to contain more microfibers per sample than large oysters, however based on the quantity of MF/sample this difference was not significant. There also was no significant difference between the quantities of black and blue microfibers. However, there was a significant increase in quantities of black microfibers compared to green or red microfibers. The results of this study indicate that a large amount of microfibers are present in commercial oysters, but more research needs to be conducted to determine how this will impact human health.
Student(s):
Julia Bures
Faculty Mentor:
Dr. Andrea Huvard
The “Fattening” Phenomenon in Male Squirrel Monkeys and its Relationship to Male-Male Competition
Sexual dimorphism describes the difference in traits between males and females of the same species. Body size is a common sexually dimorphic trait in primates and can aid with male-male competition over females. Squirrel monkey males undergo a hormonally-induced “fattening process” in the mating season, which further increases sexual size dimorphism. The function of this trait or its relationship to male-male competition has not been investigated. Our field study examined whether more seasonally robust (“fatter”) males had higher mating success due to higher competitive abilities. In our six-week study (N=143 contact hours) in Eastern Amazonia, Brazil, we observed two social groups of wild squirrel monkeys (Saimiri collinsi); groups averaged 33 individuals, nine of which were adult males. Males were classified on a scale of grade 1 to grade 3 (1 = least fat, 3 = most fat). Focal animal samples were used to construct activity and nearest neighbor budgets for different males. We recorded the initiator, recipient and winner of male-male agonistic interactions, as well as copulation occurrences. We observed 11 copulations, nine of which involved grade 3 males. Males spent less than 20% of their time near males of the same fatness grade (N=104 observations). During male-male aggression occurrences, females were present 73% of the time (N=63 observations). Grade 3 males won 84% of competitive interactions, while grade 1 males won none. Our data suggest that the degree of fattening of male S. collinsi affects their competitive ability. Data from additional mating seasons are needed to confirm these results.
Student(s):
Jacob Carrell, Jillyn Kinsley
Faculty Mentor:
Dr. Anita Stone
Markov Chains and the Game of Life
The Game of Life was the US's first popular parlor game. The game simulates a person's travels through their life, from early adulthood to retirement, with college if necessary, jobs, marriage, and possible children along the way. This study models the Game of Life using markov chains to see on average how much a player earns depending on which decision they make. It analyzes if going to college or going straight into a career is the optimal choice to make the most money. The results show that the expected value for money earned when going to college is significantly higher in comparison to going straight to career.
Student(s):
Charles Nevynne De Leon
Faculty Mentor:
Dr. Christopher Brown
Shoulder Strength and CKCUEST Performance In Division III Collegiate Softball Player.
Shoulder strength measured with a handheld dynamometer (HHD) and the Closed Kinetic Chain Upper Extremity Stability Test (CKCUEST) are clinical tools that have been used to measure athlete’s performance and track their progress. The specific aim of this study were to describe softball (SB) infield and outfield players isometric strength measures and their performance on the CKCUEST; examine the relationships between strength and the CKCUEST; compare isometric strength measures of the throwing and on throwing arms; compare the strength and the CKCUEST measures between infield and outfield SB players. This experiment was conducted using an observational cohort study. Participants included 15 DIII SB players. Shoulder strength for the internal and external rotators were measured using HHD and the CKCUEST was performed. The CKCUEST score and power were calculated. Descriptive statistics and paired t tests were used to compare throwing and on throwing shoulder strength. Independent t-test was used to compare infield and outfield players shoulder strength and the CKCUEST measures. (RESULTS SECTION)(CONCLUSION SECTION)
Student(s):
Jacqueline Diaz, Dr. Louise Kelly
Faculty Mentor:
Dr. Louise Kelly
Diversity of Arctic Ostracods
The goal of this research is to test if ostracod distribution and diversity are different in two relatively close but physically different areas of the Chukchi Sea, Arctic Ocean. The U.S. Geological Survey collected 14 surface samples (~2cm) from the continental shelf (38 -110 meter water depth) from the Alaskan Chukchi Sea, 7 from the northern near-shore region off-Barrow, and 7 from the northwest offshore region, Hanna Shoal. The samples were processed using traditional micropaleontological methods. Ostracods were found in 5 of 7 samples from the Barrow area, whereas all samples from the Hanna-Shoal area contained ostracods. At the Barrow sites, the number of different taxa per sample ranged from 1 to 7 (average = 4.25). While at Hanna Shoal, the number of different taxa per sample ranged from 1 to 4 (average = 2). The difference in dominant species and diversity of these two areas shows that the bottomwater environmental conditions were different between the two sites. Waters in this area also exemplify lower salinity and higher organic matter than offshore areas. Therefore, each area offers a specialized niche for bottom-dwelling organisms with specific ecological preferences. The Hanna-Shoal region has a higher abundance (number) of ostracod specimens in the samples, which may be indicative of a quieter offshore ostracod habitat. This work is a continuation of previous ostracod studies and allows scientists to better understand past environments to then provide insights into future environments.
Student(s):
Cristiann Dipietrantonio, Dr. Laura Gemery
Faculty Mentor:
Dr. Megan Fung
Binding Thermodynamics in Desferrioxamine D: Cooperative Sites without Cooperativity
Siderophores, critical iron-acquisition molecules for microbes, are synthesized via several pathways, notably the relatively understudied NIS pathways. Critical to the NIS pathways are NIS Synthetase enzymes, including DesD from Streptomyces coelicolor, which are increasingly linked to bacterial virulence, making them appealing drug targets. While structural insights into DesD-cofactor interactions have been established, understanding the binding thermodynamics remains crucial for inhibitor design.
In this research, we explored the binding behavior of DesD with ADP and AMP using Isothermal Titration Calorimetry (ITC). Surprisingly, a two-site binding model, with one site exhibiting significantly higher affinity than the other, was observed. Despite DesD being a homodimer, this unexpected binding asymmetry between the two sites challenges the previously established absence of cooperativity in catalytic turnover.
Initial assays have established binding conditions, paving the way for future studies exploring substrate recognition by DesD. Structural insights have revealed ATP in both sites but limited substrate presence, underscoring the complexity of DesD's substrate binding. Our current hypothesis proposes a strategic advantage for catalytic efficiency, ensuring an available site for subsequent catalytic events while releasing products. Establishing the structural distinction will be pivotal in directing future studies for effective substrate or inhibitor dosing.
This research lays the groundwork for understanding DesD binding thermodynamics, providing insights into substrate recognition. Future endeavors will focus on refining experimental conditions for cleaner data and exploring substrate binding dynamics, essential for effective drug design and catalytic efficiency studies.
In this research, we explored the binding behavior of DesD with ADP and AMP using Isothermal Titration Calorimetry (ITC). Surprisingly, a two-site binding model, with one site exhibiting significantly higher affinity than the other, was observed. Despite DesD being a homodimer, this unexpected binding asymmetry between the two sites challenges the previously established absence of cooperativity in catalytic turnover.
Initial assays have established binding conditions, paving the way for future studies exploring substrate recognition by DesD. Structural insights have revealed ATP in both sites but limited substrate presence, underscoring the complexity of DesD's substrate binding. Our current hypothesis proposes a strategic advantage for catalytic efficiency, ensuring an available site for subsequent catalytic events while releasing products. Establishing the structural distinction will be pivotal in directing future studies for effective substrate or inhibitor dosing.
This research lays the groundwork for understanding DesD binding thermodynamics, providing insights into substrate recognition. Future endeavors will focus on refining experimental conditions for cleaner data and exploring substrate binding dynamics, essential for effective drug design and catalytic efficiency studies.
Student(s):
Ashley Dumas
Faculty Mentor:
Dr. Katherine Hoffmann
Potential of a Novel pH-Switchable Antibody that Targets MUC16 in Patients with Ovarian Cancer
Ovarian cancer is one of the most fatal gynecological malignancies, with over 70% of cases left undiagnosed until late-stage development. In the late-stage development of ovarian cancer, the abnormal cells metastasize, or spread, to other parts of the body such as the liver, stomach, and lungs. The five-year survival rate for metastatic ovarian cancer of all types is approximately 30.8%. Biomarkers, biological molecules that can be observed through a patient’s fluids, can indicate these abnormal processes in the body. The membrane-bound protein, CA125/MUC16, is a common biomarker for detecting ovarian cancer and it is overexpressed in patients with metastasized ovarian epithelial cancer. Ovarian cancer tumors create an acidic environment for the cancer cells, at a pH around 6.7-7.1, in comparison to the normal tissue pH of 7.4. A novel study revealed the binding of the immunoglobulin-binding protein M, derived from the bacterium Mycoplasma genitalium, to various human antibodies in a pH-dependent manner. Due to the aggressive nature and late diagnosis of ovarian cancer, novel therapeutics that target important biomarkers is needed; therefore, this novel pH-switchable antibody could be delivered with high specificity to MUC16 and be an effective treatment for ovarian cancer.
Student(s):
Jessica Farfan
Faculty Mentor:
Dr. Chad Barber
Structural Depiction of RNA Thermometers via Selective 2′ Hydroxyl Acylation and Primer Extension
ATP-binding cassette (ABC) transporters are membrane proteins that translocate substrates across cell membranes, playing key roles in cellular processes such as the virulence of pathogenic bacteria. In response to thermal stress, bacteria may upregulate ABC transporters, and RNA thermometers (RNATs) are thought to mediate this process. RNATs are noncoding RNA elements within the 5' untranslated regions of genes that modulate gene expression by altering their secondary structure in a temperature-dependent manner, exposing the Shine-Dalgarno (SD) sequence at higher temperatures and enhancing translation initiation. ROSE-like RNATs are distinguished by a unique motif where nucleotides U(U/C)GCU are anticipated to form an imperfect base pairing with the SD sequence, resulting in a stem-loop structure with a protruding G nucleotide. This project aimed to elucidate the structural dynamics of ROSE-like RNAT sequences using the Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) technique to determine their viability as thermosensors. This methodology involves the selective modification of the RNA molecule by employing an electrophilic chemical reagent that reacts with the nucleophilic 2′-hydroxyl group (2′-OH) of the ribose sugar in the RNA backbone, resulting in an acylation reaction that maps the reactivity and flexibility of each nucleotide. Following treatment with an electrophilic SHAPE reagent and mapping nucleotide modifications through primer extension, secondary structures of the sequences were compared at two temperatures to detect heat-induced conformational changes. This study reveals a temperature-mediated structural shift of these RNATs, suggesting an adoption of conformations at elevated temperatures that facilitate gene expression.
Student(s):
Michael Hannani, Elisha Tong, Alina Tong
Faculty Mentor:
Dr. Michael Abdelsayed
A Methodology for Efficient and Accurate Recovery of Desferrioxamines from Assay Conditions
The NIS synthetase family of enzymes is a potential drug target for virulent bacteria. Some limitations to biochemically and structurally characterizing NIS synthetase DesD include the use of large amounts of substrates that are either unavailable commercially, or very expensive. A ready supply of substrates or ability to recover substrates would be valuable for repeated experimental use or to explore a mechanistic hypothesis. The focus of this current study is to present the established chromatography-based protocol for separation and identification of Desferrioxamine B from cell contaminates including (a) strong cation exchange chromatography, (b) UV/Vis spectra and absorption, and (c) reverse phase chromatography. This protocol circumvents an interesting phase separation problem and explores different ways to detect desferrioxamines. Future work includes scaling up to a cation exchange column in a on a high-performance liquid chromatography instrument and using liquid chromatography-mass spectrometry (post-separation) to successfully identify intermediates in the mechanism.
Student(s):
Jocelin Hernandez
Faculty Mentor:
Dr. Katherine Hoffmann
Determination of H+ Dependence of NIS Synthetase Mechanism via pH Kinetics
Our bodies contain metal chelators which absorb iron and process it, the same goes for bacteria within our body. Iron is crucial for aerobic organisms, and pathogenic bacteria secrete siderophores to compete for host metals. Primarily these siderophores are synthesized via two pathways: one much more well-known nonribosomal peptide synthetase (NPRS)-dependent pathway, and NPRS-independent siderophore (NIS) pathway, the less studied pathway. In spite of the two pathways having the similarity of synthesizing siderophores, the host is able to intercept NRPS siderophores and keep them from retrieving iron. NIS siderophores evade the human immune system and therefore are virulence factors for pathogenic bacteria.
Streptomyces coelicolor, a bacterium, can synthesize two NIS siderophores, Desferrioxamine B and Desferrioxamine E (dfob and dfoE), which are both produced in their final steps by the enzyme Desferroxiamine D (DesD). DesD, is a model enzyme for the NIS synthetases and their pathways.
The Hoffmann Lab is pursuing structure based drug design targeting these understudied NIS synthetases, including establishing the basic biochemical mechanism and function. The focus of my current research is to finish a kinetic profile at different pHs to explore the proton-dependence of DesD and its substrate Desferroxiamine G (dfoG). Utilizing a single injection kinetics assay created previously in lab, I will use an Isothermal Titration Calorimeter (ITC) and GraphPad Prism Software, to analyze the changes in kinetics at systematically varied pH.The kinetic curves and constants discovered will be used to hypothesize the mechanism of catalysis, and in the future, to design catalytic inhibitors.
Streptomyces coelicolor, a bacterium, can synthesize two NIS siderophores, Desferrioxamine B and Desferrioxamine E (dfob and dfoE), which are both produced in their final steps by the enzyme Desferroxiamine D (DesD). DesD, is a model enzyme for the NIS synthetases and their pathways.
The Hoffmann Lab is pursuing structure based drug design targeting these understudied NIS synthetases, including establishing the basic biochemical mechanism and function. The focus of my current research is to finish a kinetic profile at different pHs to explore the proton-dependence of DesD and its substrate Desferroxiamine G (dfoG). Utilizing a single injection kinetics assay created previously in lab, I will use an Isothermal Titration Calorimeter (ITC) and GraphPad Prism Software, to analyze the changes in kinetics at systematically varied pH.The kinetic curves and constants discovered will be used to hypothesize the mechanism of catalysis, and in the future, to design catalytic inhibitors.
Student(s):
Alec Lopez
Faculty Mentor:
Dr. Katherine M. Hoffmann
Effect of Ball Drag Characteristics on Drag Flick Shot Velocity
The drag flick is a dynamic and skillful technique used in field hockey, primarily used during penalty situations. This intricate maneuver involves a combination of exceptional coordination, strength and timing as players drag the ball along the ground and flick it towards the goal in one swift motion. The drag flick is one of the most important offensive techniques, accounting for between 30 - 50% of total goals. Research on the drag flick shot strategy is notably limited, emphasizing the need to conduct further research on this topic. The purpose of this study was to determine the effects of ball drag characteristics on shot velocity. Five players from the United States Men’s National Field Hockey Team were recruited for this study. Participants were asked to perform drag flick shots under three conditions: aiming towards their preferred corner of the net (P), opposite (non-preferred or NP) corner, and shots focused solely on speed (S). The ball was covered with reflective tape and tracked with a seven camera Vicon motion capture system (120 Hz). Ball path length (overall, anterior, and mediolateral) was computed from ball pick up until release for multiple trials for each condition (P, NP, S). Ball path characteristic differences, as well as the relationship between these characteristics and release velocity were determined with t-tests and Pearson correlation coefficients, respectively (p < 0.05).
Student(s):
Kylie McMasters, Ethan Smith
Faculty Mentor:
Dr. Michele LeBlanc
Spectropolarimetric Analysis of Two Emission Line B-Type Stars
Both classical Be stars and rapidly-rotating, magnetic Bp stars show emission in hydrogen lines throughout their visible and infrared (IR) spectra due to the presence of circumstellar material. While the physical process that places the material around the star, creating the emission, are different, it can be nearly impossible to distinguish between an edge-on Be star disk and magnetically trapped, centrifugally supported plasma. A study by Chojnowksi et al. (2015) identified several stars with high (>500 km/s) velocity IR emission. Optical spectroscopy of these stars was obtained to understand the relationship between emission location and stellar rotation. The data led to two promising magnetic candidates, HD 51477 (B3) and HD 167401 (B4), that could have centrifugally-supported magnetospheres. To investigate, both stars were observed with CFHT/ESPaDOnS to obtain spectroscopic and polarimetric data to search for the presence of a magnetic field. The spectra of both stars were normalized (continuum set to 1) and processed through a series of programs, SpecPolFlow. SpecPolFlow is a pythonic workflow that takes normalized spectra and a numerical technique called Least-Squares Deconvolution (LSD) to produce line profiles that can be used to calculate the line of site of the magnetic field or Bz of the star. If the longitudinal magnetic field is statistically significant (>3x the standard deviation) then that would indicate the presence of a magnetic field. Because both stars had Bz values that were statistically equal to zero, and also showed emission in Fe lines, we can conclude that they are Be stars.
Student(s):
David Meleney
Faculty Mentor:
Dr. Mary Oksala
Metabolic Elucidation and Modeling of Agrochemicals Towards Time- and Dose-Dependent Biomarker Screening
This study focused on exploring untested links between agrochemical structure,metabolic breakdown via cytochrome P450, and hazard characterization using toxicityrating [lethal dose 50 (LD 50 )] concepts. Formation and separation of agrochemicalstructures (organochlorine and organophosphorus pesticides) were performed throughbiomarker screening instrument gas chromatography-mass spectroscopy (GC-MS).Abundance and mass-to-charge determinations were used to confirm their presence instandards of organic solvents (i.e. hexanes: toluene, methanol, and acetone) and toconstruct relevant calibration graphs on the GC-MS. These results, along with themechanistic approach, allowed us to better elucidate the breakdown of these compounds and how they may be presented/detected in human urine and blood samples for future biomarker research. (Quantitative) structure–activity relationships [(Q)SAR] modeling proved to be a valid alternative to in vivo assays for assessing acute toxicological hazard.
Student(s):
Chelsea Munoz, Dr. Grady Hanrahan
Faculty Mentor:
Dr. Grady Hanrahan
Crystallization of the Glu467Ala Residue and Its Co-Complex in Desferrioxiamine D
Iron chelation has been an issue with pathogenic bacteria and new classes of antibiotics have not been discovered in recent years. While humans use iron for transporting oxygen through the bloodstream, bacteria repurpose it for performing metabolic processes to further infect its host. Antibiotics need to be developed to prevent these virulence factors from taking place and the first step towards this path is uncovering the structure of the NIS synthetases, a pathway used to catalyze the production of the siderophore Desferrioxamine E. To determine how one of the catalytic residues (Glu467Ala) in a precursor (Desferrioxamine D) interacts with the substrate, crystallography screenings were performed to test a wide variety of conditions for complexing substrate and ATP into their respective binding sites. Previous binding studies with E445A complexing two ATP to DesD implied there may be two conformations for hydrolyzing ATP, however, were unable to complex with HSC. One hypothesis implies that complexing both ATPs may reduce the enzyme’s affinity for HSC, preventing the binding from taking place. To investigate this, each of the crystals were soaked in high concentrations of HSC and lower concentrations of ATP. However, in addition to the HSC substrate loop being in a closed conformation, the active site for ATP was also empty with an R value of 0.19 and an R free value of 0.24. Future directions for exploring the conformation changes may require the crystals to be grown in higher temperatures to improve the chances of interaction.
Student(s):
Jeric Nhou
Faculty Mentor:
Dr. Katherine Hoffmann
Molecularly Mapping the Genomic Insertion Site of a Transgene with a Novel Expression Pattern
Transgenes are invaluable tools for exploring genetic mechanisms. Our research focuses on a novel expression of a white+ (w+) transgene in Drosophila melanogaster, named Swoosh (SW). When a w+ construct is integrated into the genome, it may report the transcriptional activity of genes near the insertion site (an enhancer trap). The Swoosh expression pattern consists of a band of w+ expression along the equator of the eye, known to be a key signaling center during compound eye development. We are mapping the genomic coordinates of SW since the w+ reporter may identify a pattern of expression of a developmentally important gene near the site of insertion. In previous lab studies, it was verified that the w+ transgene is next to a gene called rosy(ry), however; looking back at studies from Hazelrig in the 80s, it is shown that this particular gene was used as a vector in conjunction with w+ to insert into the genome. Our current hypothesis is that the w+ transgene has rearranged itself and we are working with one of the original vectors. We are recovering genomic sequences flanking the SW transgene insertion via an Inverse PCR approach. After Inverse PCR, forward PCR will be used to amplify the sequence of interest using sites from where Age1 cut into ry. Bioinformatic analysis of the SW insertion region, followed by studies of candidate genes driving SW expression will be conducted.
Student(s):
Kari Orr, Anthony Garay
Faculty Mentor:
Dr. David Marcey
Stair Descent Transition Mechanics in Adults with Symptomatic and Asymptomatic Femoroacetabular Impingement
Femoroacetabular impingement (FAI) is a pathological condition defined by an irregular intersection between the acetabular rim of the pelvis and the femoral head, causing friction which can lead to cartilage damage. Symptoms are not always present for all individuals with FAI. Previous studies have compared symptomatic FAI (FAI-s) and asymptomatic (FAI-a) participants in stair ascent tasks (Hammond, et al., 2017). No previous studies have analyzed the kinematics or kinetics of stair descent transition for individuals with FAI. PURPOSE: To determine how symptom status may alter ground reaction forces and lower body joint angles during stair descent transition to the floor. METHODS: Participants were selected by an orthopedic surgeon who used radiographs to identify the condition and their symptom status indicated their subgroup (FAI-s n=10, FAI-a n=11). All individuals signed a consent form approved by the university IRB prior to participation. Each participant descended a 3-step staircase at a self-selected speed, multiple times for each lead leg. Three-dimensional ground reaction forces and reflective marker data on key body landmarks were collected via a Kistler force plate embedded in the laboratory floor (1200 Hz) and 6 Vicon motion capture cameras (120 Hz), respectively. Motus software was used to calculate lower body joint angle values during the stance phase between the stairs and floor. The average over three trials per lead leg was used for analysis. Comparisons were made between groups using independent t-tests (p<0.05).
Student(s):
Sadie Peete
Faculty Mentor:
Dr. Michele LeBlanc
EmrAB-TolC Efflux Pump Influence on the Expression of the emrRAB Promoter and Motility.
Efflux pumps are tripartite integral membrane proteins that are capable of transporting substrates inside the cytosol or periplasmic space of the bacteria to the outside of the bacterial cell. Efflux pumps have risen in prominence due to their role in antibiotic resistance. Current research indicates efflux pumps are involved in the regulation of metabolism. EmrAB-TolC is an that efflux pump of the major facilitator superfamily (MFS) found in the Gram-negative bacterium E. coli. Little is known about the role EmrAB-TolC plays in metabolism, as well as what signals regulate the expression and function of the pump. Studies have shown that the function of efflux pumps also controls motility in E. coli. Thus, we have characterized the environmental conditions that control motility in an emrB-dependent manner. We found that the emrB mutant was hypermotile at low and high temperatures, as well as in multiple nutrient conditions. We also sought to characterize the genetic controls of the emrRAB promoter (emrRABp) by evaluating different chromosomal knock out strains of genes known to regulate efflux pumps. The knock strains were then transformed with a reported plasmid containing the emrRABp controlling the expression of gfp. Deletion of components of the EmrAB-TolC pump, such as emrB and tolC, as well as regulators for other pumps, such as marR and marA, all increased expression of the emrRAB promoter. This suggests that EmrAB-TolC role goes beyond antibiotic resistance, potentially having a role in metabolism, and that regulation of different efflux pumps may be interconnected.
Student(s):
Rafael Poveda
Faculty Mentor:
Dr. Dana Harmon
Biogeographic Distribution of Modern Benthic Ostracodes from the Chukchi Sea, Arctic Ocean
Seventeen surface sediment samples (0-2cm) collected by the USGS were analyzed to better understand the distribution of ostracode assemblages in the Chukchi Sea, specifically from the north off-Barrow (DBO5) and in the southern Chukchi Sea (DBO3) regions. We expect to find a high abundance of ostracodes throughout both regions and a higher abundance of ostracodes in waters closer to the shoreline. In the southern DBO3 region of the Chukchi Sea, 6 out of 13 samples had ostracodes. Normanicythere leioderma was the most abundant species, found in 5 samples, Elofsonella concinna was found in 3 samples, and Sarsicytheridea bradii was found in 3 samples. In the DBO5 region, 4 out of 4 samples contained ostracodes. E. concinna was found in 3 samples and Munseyella kiklukhensis, S. bradii, and Heterocyprideis fascis were found in 2 samples. The distribution of ostracode assemblages is dependent on factors such as water mass (salinity, temperature), pH of water, sediment substrate, and food availability. Many ostracode species have specific ecological requirements, such as narrow salinity or temperature tolerances, and these species can be used to infer what past ocean/climate conditions were like when those ostracodes were alive. For example, N. leioderma was most prevalent in the DBO3 region in widely varying temperatures (-2°C to 10°C bottom water temperatures) and nearer shore areas with higher productivity (carbon food sources that sink to feed benthic-dwelling organisms). Documenting shifts in ostracode faunal assemblages through time can provide us with water mass history and other important paleoenvironmental changes.
Student(s):
Cheyanne Rodriguez, Cristiann Dipietrantonio
Faculty Mentor:
Dr. Megan Fung
Effects of Drag Flick Shot Location on Ball Velocities and Ground Reaction Forces
Drag flicks are one of the most influential offensive techniques in field hockey. A drag flick occurs during a penalty shot where a player will scoop and flick the ball towards a particular direction of the goal. The drag flick is a crucial part of field hockey, this technique was responsible for one third of goals scored in the men’s and women’s 2016 Rio Olympic Games (Ng et al., 2018). The purpose of this study was to determine the effect that shot location had on lead leg ground reaction forces and ball velocities. Five members of the United States Men’s National Field Hockey Team were recruited for this study. Participants were asked to aim their shots in their preferred corner and opposite corner (non-preferred) of the net. A trial was successful when the participant landed on a force plate, hit the correct target, and had a high rating by the athlete. Three-dimensional ball coordinates and ground reaction forces were collected via seven camera Vicon motion capture and Kistler force plates respectively. The four best rated successful trials for each condition were used for analysis. Dependent t-tests were used to determine the effect of shot direction (p<0.05). There were significant differences in forward and resultant velocities were greater for the preferred versus the non-preferred directions (p=0.030 and p=0.011, respectively). There were no significant differences in vertical, posterior, or medio-lateral ground reaction force values based on shot direction.
Student(s):
Ethan Smith, Kylie McMasters
Faculty Mentor:
Dr. Michele LeBlanc
Discovery and Validation of ROSE-like RNA Thermometers in the 5′-UTRs of ABC Transporter Genes
ATP-binding cassette (ABC) transporters are a large family of cellular transporters found across prokaryotes and eukaryotes, which use the hydrolysis of ATP to transport a large variety of substrates across the cell membrane. In bacteria, ABC transporters can play an important role in virulence and pathogenicity. In this study, we aimed to elucidate the molecular mechanism for the upregulation of ABC transporters under heat stress. RNA thermometers are noncoding RNA structural elements in the 5′ untranslated region (UTR) of genes. In most cases, they result in the upregulation of downstream genes in response to changes in temperature within a physiological range. The ROSE-like class of RNA thermometers is characterized by a specific motif in which U(U/C)GCU nucleotides are predicted to imperfectly base pair with the Shine-Dalgarno (SD) in a stem with a bulged G nucleotide. We utilized a bioinformatic structural search to discover ROSE-like RNA thermometers based on their characteristic motif. Manual curation of the search results led to our discovery of a multitude of predicted ROSE-like RNA thermometers in the 5′-UTRs of diverse ABC transporter genes in different bacteria, suggesting a potential widespread means of temperature-dependent regulation of these genes. Using β-galactosidase assays, we validated five predicted ROSE-like RNA thermometers upstream of different ABC transporter genes. Additional characterization of mutants revealed the dependence of thermoregulatory activity on the ROSE-like motif. Our study suggests that ROSE-like RNA thermometers may play a critical role in the upregulation of ABC transporters under heat stress.
Student(s):
Alina Tong, Elisha Tong, Michael Hannani, Danna Santiago, Dr. Luiz Passalacqua (NIH)
Faculty Mentor:
Dr. Michael Abdelsayed
Discovery of a Putative Modifier of a Novel Transgene Expression in Drosophila melanogaster
This study aims to explain the differences in Swoosh (SW) expression in Drosophila melanogaster by examining if identifiable modifiers of transgene expression exist on the third chromosome. The SW phenotype represents the expression of a white+ transgene in a thick band along the D-V midline of the compound eye. Forty-three isogenic lines bearing isogenic recombinant or nonrecombinant SW chromosomes were created, examined, and compared with respect to the degree of expression of the SW transgene. We have identified a putative modifier of SW expression that maps to a distal region of chromosome 3R. Chromosomes containing this modifier, present on the original SW chromosome, produce a significantly broader band of white+ expression than SW chromosomes lacking the modifier. We are currently testing the hypothesis that the modifier may act to suppress position effect variegation.
Student(s):
Linda Tran
Faculty Mentor:
Dr. David Marcey
Design and Development of Hydrogen Peroxide and Free Radical Scavenging Assays from Natural Medicine Components
Green tea is consumed by a wide range of people around the world as a natural medicine and is known for its health benefits such as cancer prevention and anti-inflammatory and antidiabetic properties. The purpose of this experiment was to explore the metabolic breakdown of green tea catechins and identify the development of hydrogen peroxide and free radicals formed from the interaction of green tea catechins with the cytochrome p450 liver enzymatic system. A mechanistic scheme was developed for four green tea catechins and their interaction with the cytochrome p450 iron (III) oxide complex. The mechanistic scheme was further developed for the production of hydrogen peroxide. An extraction of green tea tablets was performed which revealed the green tea tablets were soluble in a 50:50 hexane/toluene solvent. A derivatization method was developed with a 1:1 ratio of BSTFA + 1% TMCS and green tea catechin mix and analyzed on GC/MS. The instrument displayed silyl groups interacting with ring-like structures such as those of the green tea catechins. A colorimetric assay of hydrogen peroxide was run as a comparison to the hydrogen peroxide production of green tea catechins. Future experiments could include efforts in the cell culture analysis of green tea catechins and artificial neural network development to compare factors of green tea catechins and their efficiency in the production of hydrogen peroxide and free oxygen radicals.
Student(s):
Aaliyah Valladolid
Faculty Mentor:
Dr. Grady Hanrahan
Meaningful Decision in the Game of Snakes and Ladders
During this research project, we examined the popular board game Snakes and Ladders with two alterations to introduce meaningful choice for the player and to simplify the analysis. First, players only advance one or two spaces, depending on a coin flip. Second, each player is given a token, which they are able to spend on any space of their choice. Spending a token allows the player to choose to move forward one or two squares. Our goal was to analyze different strategies for spending the token to decrease the mean time to complete the game. We performed different analyses, including simulations in Python and Markov chain analysis. We found that in many board configurations, the game has a unique strategy minimizing the mean completion time.
Student(s):
Ashley Vaughn
Faculty Mentor:
Dr. Christopher Brown
Role of the EmrAB-TolC Efflux Pump in Regulation of Metabolism in E. coli
Understanding the role of efflux pumps has potential applications for biotechnological advances and a solution to antibiotic resistance. We have analyzed the contribution of the EmrAB-TolC efflux pump to metabolic flux in E. coli by comparing the total metabolite pools of an emrB mutant to that of the parental strain. To prepare samples for analysis at the UC Davis Metabolomics core facility for chromatography and mass spectrometry analysis to identify specific metabolites found in both strains, we conducted growth experiments in M9 minimal media with glucose and EZ rich media at 30˚C and 37˚C. We found no significant difference in growth between either strain in these media conditions. To further characterize our findings from the metabolomics study, we will conduct minimal inhibitory concentration (MIC) assays to screen for metabolite toxicity. This study, and others, have shown a link between efflux and motility. To that end, we have shown that the emrB-deleted mutant cells have increased motility over the parental strain. Thus, we will use motility tests to screen hits from our metabolomics study for their participation in efflux. Through this work, we hope to better understand the role that EmrAB-TolC plays in metabolism in bacteria.
Student(s):
Joseph Wiersma
Faculty Mentor:
Dr. Dana Harmon