Biological Sciences Honors Theses
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Item Investigating Feedback Loop Between ROS, Microtubules, and Mechanical Stress in Robust Organ DevelopmentShapland, Emily (2024-02-26)Arabidopsis thaliana sepals grow to the same shape and size consistently, allowing them to function properly to protect the immature flower. Cell growth rate and direction is heterogeneous during sepal development, but heterogeneity accumulates over time to be more uniform across the tissue. The ftsh4-5 mutant has a loss of robust development of sepal size and shape which is caused by decreased heterogeneity in cell growth rate and direction, leading to uneven accumulation of growth. Elevated levels of reactive oxygen species (ROS) cause this phenotype, but the mechanism by which ROS affects growth heterogeneity is unclear. It is possible that a feedback loop is involved in growth heterogeneity, connecting ROS, microtubules, and cell growth direction, and mechanical stress. I find that the microtubule arrangement is disrupted in ftsh4-5, with an increase in areas of dense microtubule signal, areas of intense microtubule crossover, and overall signal intensity as compared to wild type. These results suggest that the microtubules of ftsh4-5 are more highly bundled than wild type and are likely less able to reorient during development, which would affect averaging of growth direction. I also find that ftsh4-5 produces increased ROS after mechanical stress, to a greater extent than wild type. This result suggests that the elevated ROS levels in ftsh4 may be partially due to an over-active response to mechanical stress. These results are consistent with my hypothesis that a feedback loop controls robust development and suggest that the overactivity of this feedback loop may disrupt development, resulting in the ftsh4-5 phenotype.Item Characterizing the Unique Reliance on Translesion Synthesis in Embryonic Stem Cells in Response to ssDNA AccumulationWang, Jerry Kaizhong (2025-01-21)All replicating cells face an innate amount of replication stress. Stem cells, due to their role as progenitors, must also utilize efficient and high-fidelity pathways to minimize mutagenesis or delays in replication. Embryonic stem cells (ESCs) are rapidly proliferating stem cells isolated from an early embryo and have previously been shown to lack cell cycle checkpoints as well as accumulate much higher amounts of single-stranded (ssDNA) gaps. These gaps are thought to arise from PrimPol restarting stalled forks by repriming downstream from sites of damage, leaving ssDNA that must be filled in post-replicatively by translesion synthesis polymerases Polζ and Rev1. In this study, we utilized a flow cytometry assay that quantifies ssDNA accumulation by BrdU incorporation. Using this assay on cells treated with translesion synthesis (TLS) inhibitor JH-RE-06, we demonstrate the unique reliance of ESCs on TLS polymerase Polζ for ssDNA gap filling in G1 and S phases. Applying this assay revealed differential utilization of TLS in other cell types. Using immortalized mouse embryonic fibroblasts (MEFs), we demonstrate differentiation causes less reliance on TLS, implying reduced ssDNA stress and utilization of other pathways for repair. Through these experiments, we reveal another direction to understand DNA damage response pathways in vivo. We corroborate evidence that reliance on TLS is vital for ESCs. TLS is historically regarded as a low-fidelity pathway, but because ESCs have a much lower mutation rate, we propose that reliance on TLS, specifically Polζ-dependent repair, is in fact a high-fidelity response pathway that is essential for resolving ssDNA gaps.Item Identification and Characterization of Rad54 HypomorphsSridalla, Krishay (2025-01-21)The maintenance of genomic stability is critical for the proper functioning of all organisms. Various DNA repair processes are involved in safeguarding the integrity of the genome, including homologous recombination (HR), a process that repairs double-stranded breaks in the DNA. The DNA motor protein Rad54 is a highly conserved DNA translocase that is crucial for HR function. In the human context, mutations in its homolog, Rad54L, are known to be associated with many cancer types. This study utilized the model system of Saccharomyces cerevisiae to examine the effects of mutations in Rad54 on important cellular phenotypes. rad54R272Q and rad54R272A demonstrated significant growth defects in response to DNA damage, exhibiting a gradient of activity when compared to the WT. Intriguingly, diploid rad54R272A/rad54R272A displayed a growth defect even under normal conditions. Additionally, the study demonstrated no significant difference in template switching frequencies between the WT and mutants. Together, these results provide greater insights into Rad54’s function in HR and illuminate how mutations within this protein can lead to genome instability, which can contribute to cancer development in humans.Item Role of an RNA granule in the jus-dependent mechanism of epileptogenesis in DrosophilaSharma, Anirudh (2024-05-24)This work aims to identify the role of an RNA granule in the development of epilepsy in Drosophila melanogaster. Prior studies have shown varying degrees of evidence of granule-associated macromolecules co-localizing with Jus, a previously-studied protein whose dysfunction during development is associated with a bang-sensitive phenotype that models human epilepsy. By assessing the bang-sensitivities (via vortex testing) of flies with altered expression of RNA-granule markers (via transposon-insertional mutation or GAL4-UAS driven RNA interference and overexpression), it was found that altered function of RNA granules is associated with the jus-dependent mechanism of epileptogenesis. In particular, the primary finding was that overexpression of the gene FMR1 (whose protein product contains numerous RNA-binding motifs) in jus-expressing neurons is associated with enhanced bang sensitivity in both wild-type flies and jus-mutant heterozygotes. Additional genetic crosses were conducted to support the finding that this effect was specific to jus-expressing neurons. However, western blot analyses show that Jus levels were not consistently altered in flies where FMR1 was overexpressed or knocked down, indicating that the role of the RNA granule in epileptogenesis needs closer examination and that FMR1 does not directly regulate jus expression. Additionally, a mutation of the gene CG31249, a known member of the RNA granule, was associated with enhanced bang sensitivity in jus-mutant heterozygotes and increased mean recovery times (an indication of seizure intensity) in jus-mutant homozygotes. Overall, this work provides multiple lines of evidence for the role of an RNA granule in causing seizures in Drosophila.Item Isolation and Purification of ClearColi Outer Membrane Vesicles with mutant Transferrin-Binding Protein BRichards, Margot (2024-01)Escherichia coli outer membrane vesicles (eOMVs) have potential as vaccine delivery systems. A possible constraint is their lipopolysaccharide (LPS) content, sometimes causing toxic effects in vivo. Here, I explore the ability of eOMVs from ClearColi, a genetically engineered strain of E. coli with only the lipid IVa portion of lipopolysaccharide (LPS), to be used as a vaccine delivery system. I investigate whether these OMVs that do not stimulate TLR4 can be used to express a surface-exposed protein. Transferrin-binding protein B (TbpB), a surface-exposed protein, is a potential vaccine candidate for Glaesserella parasuis due to its conservation across multiple serotypes. A major drawback of current G. parasuis vaccines is their failure to generate a cross-protective immune response and prevent Glässer’s disease. In our laboratory, I have applied engineered ClearColi to express a mutant TbpB. This study found that ClearColi could express the surface exposed mutant TbpB in the eOMV. Additionally, various methods were used to characterize ClearColi expressed OMVs (eOMVs); these included Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis (WB), and transmission electron microscopy (TEM) imaging. The eOMVs reported here represent a potentially safer and better cross-protective vaccine platform against G. parasuis for the prevention of Glässer’s disease. The methods here also present additional knowledge to the development of safe and straightforward vaccine delivery platforms using ClearColi outer membrane vesicles.Item Expressions of students’ disagreement in life sciences teambased online discussionsMenon, Priyanka (2023-01-11)Studying disagreement in STEM classrooms is one way to better understand students’ sense of belonging in a learning environment by linking the frequency of disagreement with how comfortable they are contributing their true opinions. I determined whether students’ frequency of expression of disagreement in an online STEM course is affected by group composition (gender, size, topic). Disagreement is measured in this study directly by quantifying how many students replied to prompted discussion questions with disagreement statements or when students ask and answer questions to clarify the disagreement. I predicted that students would express more disagreement when they were male, were in groups with less than four people, and discussed topics involving evolutionary processes and phylogeny based on conclusions from previous literature. The results showed that there was not a difference in disagreement rate across gender, there was more disagreement for "Population Genetics" and "Other Evolutionary Processes" compared to "Phylogenetics" and "Biodiversity," and there tended to be more disagreement for teams of five compared to smaller sized groups. These findings can be applied to future biology education research to improve the ways group activities are structured to be more inclusive of students of all backgrounds and to maximize true expression of opinions.Item The Role of Trehalase in Phenotypic Plasticity of the Buckeye Butterfly (Junonia coenia)Pires, Samantha (2023-01-11)The buckeye butterfly, Junonia coenia, shows seasonal variation in wing coloration. The duplicate genes treh-1a and treh-1b encoding a soluble form of trehalase are implicated in this environmentally induced phenotypic variation. treh-1a has been shown to be involved in the mechanism of ommochrome pigmentation plasticity in J. coenia but the role of treh-1b is unknown. In this study, I first aimed to characterize the role of treh-1b, a presumptive metabolic pathway enzyme, in both ommochrome pigmentation, and, second, to assess the potential adaptive role of trehalase upregulation in cold tolerance. CRISPR mutagenesis of the treh-1b promoter, as well as the coding region, revealed various developmental phenotypes, but had no obvious effect on ommochrome pigmentation. I next used chill coma recovery time bioassays to ask how treh-1a -associated phenotypes affect cold tolerance. My bioassays revealed that Red line butterflies (red phenotype treh-1a allele) recover from cold-shock faster than Nijh line butterflies (tan phenotype treh-1a allele) when both groups come from a warm environment, but not when both groups come from a dark, cooler environment. Interestingly, both Nijh and Red butterflies show a large reduction in recovery times when coming from a dark, cooler environment. This suggests that Red butterflies may have increased thermal retention compared to Nijh butterflies, thus allowing them to recover from cold-shock faster than Nijh butterflies. This also suggests acclimation to cooler, darker conditions impacts J. coenia recovery after cold-shock.Item The role of araucan in nymphalid wing color pattern formationSiegel, Kate (2022-11-17)Despite the great beauty and diversity of butterfly wing color patterns, there is still much that remains unknown about how these color patterns are determined. Certain master regulator genes are known that can determine the scale type or pigment across a variety of color pattern elements, and their expression is determined through the combined activity of upstream pre-patterning genes. For one such key gene, optix, most of its regulatory network is still unknown, though candidates have been proposed. This study aims to take a closer look at the functions of one candidate upstream gene, araucan, a proximally expressed transcription factor that plays a role in wing vein specification in Drosophila. Using CRISPR-Cas9 gene editing, mutations were induced in the araucan gene in two nymphalid butterfly lineages, Junonia coenia and two co-mimetic species of Heliconius. Examining wings for phenotypes revealed some optix-typical mutations such as bright blue structural iridescence across J. coenia wings, as well as vein reductions in H. erato that suggest the function of araucan may be partially conserved relative to Drosophila. An unexpected novel phenotype affecting the iridescence of the eyespot center, or focus, also appeared, introducing a potential role for araucan in eyespot color patterning.Item The Putrescine Puzzle: How do Manduca sexta hawkmoths respond to scented nectar in Datura wrightii flowers?Cheng, Sheri (2022-05)The unusual large amounts of a polyamine, putrescine, in the nectar of the Datura wrightii flower add complexity to the mutualistic interactions between the flower and its pollinator, Manduca sexta, acting as a potential modulator in the pollinator’s foraging behavior. However, whether this non-sugar metabolite acts as an attractant or a repellent in the floral nectar is not well understood. I performed an innate preference bioassay using the Datura nectar and a synthetic sugar solution to analyze the possible effects of putrescine on Manduca preference during foraging. Based on the assessment of the animal’s first choice, the frequency of visits per flower, and the final volume of nectar consumption measurements, this study failed to find any statistically significant difference in the moth’s preference between Datura nectar and the synthetic sugar solution. However, while the absence of behavioral changes may suggest the animal’s indifference towards the additional polyamines in the nectar, it remains possible that physiological or fecundity implications may arise from a long-term study.Item Blue vs. Yellow Color Preference of Hyles Hawkmoths in Foraging Visitations ofVarious Real and Artificial FlowersPalmadessa, Matthew (2022-05)As one of the most important pollinators across North America, there is much incentive to further understand the foraging habits of Hyles hawkmoths. Recent research on the subspecies Hyles lineata has identified a preference for both blue and yellow colored light for flower visitations, with the stronger bias between the two being for blue. However, the blue LED used in this earlier experiment was later discovered to be more intense than the yellow, casting doubt upon this finding. Here, the idea of the former study was revisited, and a binary choice assay was created with these same two blue and yellow LEDs. However, a plastic filter was positioned below the blue one, such that its intensity was decreased to match the yellow. H. lineata moths were reared from eggs, and adults were individually released into a darkened flight chamber where each light projected its color down onto either a white Oenothera flower or false paper flower. Their first visitation was recorded as their preference. Additional testing without the filter was also conducted, as well as paper flower trials with the addition of fragrant Bergamot oil. When light intensities were equalized, the moths actually preferred yellow flowers to blue ones. However, when the filter was removed, a blue preference only reappeared for the moths tested with real flowers. This persistent yellow bias was also maintained through testing with paper flowers and Bergamot oil. Further investigations may identify whether fragrance plays a notable role in the flower color bias of H. lineata.