Biological Sciences Honors Theses

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    Isolation and Purification of ClearColi Outer Membrane Vesicles with mutant Transferrin-Binding Protein B
    Richards, 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.
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    Expressions of students’ disagreement in life sciences teambased online discussions
    Menon, 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.
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    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.
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    The role of araucan in nymphalid wing color pattern formation
    Siegel, 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.
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    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.
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    Blue vs. Yellow Color Preference of Hyles Hawkmoths in Foraging Visitations ofVarious Real and Artificial Flowers
    Palmadessa, 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.
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    Investigation of potassium tetraborate resistance in Dickeya spp.
    Lou, Alice (2022-05)
    Dickeya spp. are common plant pathogens associated with bacterial soft rot, potato blackleg, and slow wilt, which are plant diseases that account for major losses in the agricultural industry. The diseases caused by these bacterial species are not yet fully managed with existing techniques, and new approaches need to be considered to minimize future crop loss. Previous research has shown that the inorganic salt potassium tetraborate tetrahydrate (PTB) can inhibit the growth of Dickeya species; however, disk diffusion assays result in a unique phenotype with two zones of inhibition. In this study, I investigated the effects of PTB on the growth of four Dickeya spp. I hypothesized that the production of phage is responsible for the two zones of inhibition. I used disk diffusion assays and growth curves to confirm the impact of PTB on Dickeya and attempted to directly isolate phage from the strains. To determine the mechanism of action of PTB, I used Tn-Seq libraries to determine which genes are required for growth in the presence of PTB. Tn-Seq libraries showed that different Dickeya strains shared seven overlapping genes including stress-related genes that increase bacterial resistance to PTB. I used gene expression studies to determine the changes in gene expression that result from PTB exposure. Preliminary results showed that exposure to PTB induces the expression of stress-related genes in Dickeya to increase survival in the presence of the compound. Further research is needed to better understand the implications of observed changes in bacterial gene expression.
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    Assessing Differences in Desiccation Tolerance in Two Species of Hawaiian Swordtail Cricket
    Lam, Vincent (2022-05)
    Two species of Hawaiian swordtail cricket, L. orientalis and L. makaio, inhabit different locations on the island of Maui in the Hawaiian archipelago, generally with L. orientalis residing in a region of higher, and L. makaio inhabiting a region of lower, rainfall. This difference in habitation exposes the two species to different levels of humidity, with L. orientalis being subjected to greater levels of moisture than L. makaio, when controlling for elevation. It may be possible that these species’ distinct habitats are a result of different humidity preferences. These distributions in turn may be influenced by the different compositions of cuticular hydrocarbons (CHCs) that coat the surface of their bodies and prevent desiccation. To test whether such differences in desiccation resistance may exist, individuals from each species were subjected to a series of desiccation tolerance experiments, which measured the length of time in which each species could survive arid conditions. Additionally, the CHC profiles of each species were analyzed by extracting the CHCs from individuals of each species and running them through gas chromatography. The results of these experiments do not reveal a statistically significant difference in the survivability of L. orientalis and L. makaio in arid environments, though a difference in CHC profiles has been observed.
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    A Review of Serotonin’s Role in Depression and Modern Perspectives
    McLaughlin, Liam (2022-05)
    Major depressive disorder (MDD) is a highly prevalent and debilitating illness in the modern world. In the 1960s, the theory that low serotonin (5-HT) was a primary cause of MDD emerged due to the efficacy of 5-HT restoring drugs in treating depression. The 5-HT deficiency hypothesis of depression has since been criticized through studies not being able to directly tie low serotonin to MDD. The discovery of the antidepressant efficacy of the glutamatergic stimulant ketamine led to a reevaluation of depression pathophysiology. Modern perspectives view depression as an issue of disrupted neurocircuitry resulting from stress induced atrophy of certain limbic and cortical brain regions, such as the hippocampus and PFC, and hypertrophy in the fear evaluating amygdala, the reward evaluating nucleus accumbens, and the orbitofrontal cortex. Depression may be treated by potentiating neuroplasticity, that when combined with psychotherapy, helps individuals relearn negative emotional associations and restores dysfunctional neurocircuitry. The functioning of the serotonergic system may be viewed as a vulnerability factor in developing depression due to its involvement in stress, as well as a treatment target which indirectly primes neuroplasticity. Other neurotransmitter systems similarly represent depressive risk factors and antidepressant targets, namely the noradrenergic and dopaminergic systems. Serotonergic antidepressants such as SSRIs see high rates of prescription due to their minimal side effects. They demonstrate slower efficacy than ketamine, whose dissociative side effects and potential for abuse are unideal, demanding further research of its mechanism to find safer and more effective antidepressant targets.
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    Studying Arbuscular Mycorrhizal Fungi, Their Endosymbiotic Bacteria, and Their Spores: Challenges and Solutions
    Tzng, Eileen (2022-05)
    Arbuscular mycorrhizal fungi (AMF) have broad applications in agriculture and land restoration due to their significance in forming symbiotic relationships with most terrestrial plants. However, the field of AMF is understudied, often extending results from a handful of AMF species to all AMF. Spores are a highly significant but often overlooked aspect of the AMF life cycle. In this honors thesis, I summarize the current knowledge surrounding the significance of spores in the AMF life cycle. Great strides have been made in understanding AMF spore germination, dormancy, and life strategies, but there are also gaps in the knowledge that, if filled, would further advance the various applications and studies of AMF. In particular, past research done on AMF must be revisited in order to: (1) redefine terms and standardize experiments; (2) reinforce genetic work in AMF; and (3) use knowledge from plant and seed experiments to better inform AMF experiments. I then empirically demonstrate that the endobacterium ‘Candidatus Moeniiplasma glomeromycotorum’ can be lost or gained during serial culturing practices, although the mechanisms by which endobacteria are lost or gained. I also show that routine spore processing methods, such as sonication and chemical washes during surface decontamination, can be detrimental to spore survival in some species, and therefore the interpretation of spore viability studies should be carefully considered.