Conjugative plasmids are extrachromosomal genetic elements commonly found in bacteria and are capable of being shuttled between different bacterial cells via a process called conjugation. The Luria-Delbruck Method (LDM) is a stochastic based modeling framework used to estimate the conjugation rate of a particular plasmid between bacterial strains. In my experiment, I am probing the theoretical experimental assumptions made by the LDM; in particular, I am testing that when there is variation in the precision of the selection assay, the application of a theoretical correction can result in accurate quantification of the conjugation rate. In the early phases of the experiment, I performed experimental assays in liquid medium to determine antibiotic concentrations in which donors (strains hosting a conjugative plasmid) and recipients (strains able to receive plasmids from donors via conjugation) die and transconjugants (recipient cells that have received plasmid from donor) grow. To test the theoretical correction, I chose two antibiotic concentrations that differ in the amounts of extinction occurring in the transconjugant population. I executed the LDM conjugation assay with these two conditions which produced equivalent conjugation rate estimates, as would be predicted if the correction factor is effective at mitigating the bias produced from variable amounts of transconjugant extinction in the selective conditions. My experiments demonstrate that the LDM continues to be robust in the face of violations to experimental assumptions which affirms the viability of applying the method to a wider range of bacterial populations with variable selective conditions and thereby broadens our ability to understand the dynamic movement of conjugative plasmids.

Describing genetic variation in deep-sea organisms is key to understanding ecological and evolutionary processes shaping biological diversity in these fragile ecosystems that are vulnerable to anthropogenic activity. Among deep-sea species, tubeworms (Class Polychaeta) often dominate faunal biomass in hydrothermal vents where they tend to grow in towering colonies. As foundation species, tubeworms create structural habitats that support assemblages of diverse biological communities across the seafloor. However, heterogeneous environmental conditions throughout their range, such as pH and temperature, and geographic isolation, may cause population subdivision by restricting connectivity between sites. This study aims to further our understanding of tubeworm population structure by investigating genetic relationships between Ridgeia piscesae subpopulations within a deep-sea hydrothermal vent system. At the Axial Seamount, a regularly surveyed area in the study of hydrothermal vent processes on the Juan de Fuca Ridge, our knowledge of the genetic connectivity of R. piscesae subpopulations remains limited. Here, we examined genetic variation in R. piscesae individuals collected from structurally-different sites within the Axial Seamount. Specifically, we genotyped mitochondrial genes and constructed SNP-based phylogenetic trees to elucidate evolutionary relationships between worms inhabiting a chimney and diffuse-flow locations. Genetic differentiation is expected to be low when gene flow is high between populations, but these outcomes may also be influenced by other aspects, such as environmental conditions. These results allow us to evaluate the degree of genetic diversity in tubeworms within this important area and provide insight into the potential impact of human activities, such as seabed mining, on deep-sea vent dynamics.

The population structure of species occupying deep-sea hydrothermal vents is strongly influenced by geological processes. The formation and maintenance of vents can create a patchwork of habitats across the sea floor, which are subject to catastrophic events that can extirpate whole communities. Therefore, the population structure of vent species is not only influenced by adaptation, life history, and dispersal but also by geological processes and oceanographic processes. Characterizing the genetic diversity and connectivity of species between vents can provide information about these processes. The Axial Seamount, located on the Juan De Fuca Ridge located off the Pacific Northwest coast of the USA, is a well-studied hydrothermal vent system. Here, sulfide worms (Paralvinella sulfincola) can be found on and around chimney vents, building tubes that contribute to chimney formation and habitats for other organisms. We predict low gene flow and low connectivity between different vent populations of this species because their reproductive life history strategies depend on pheromone signaling. Such signaling likely results in individuals mating more frequently with their closest conspecifics. However, little is known about the larval dispersion dynamics of this species. Sulfide worms were collected to investigate the connectivity and genetic diversity of their populations. Mitochondrial and nuclear genes were amplified from DNA extracted from worms that were collected from two locations within the Axial Seamount (Inferno and El Guapo chimneys, located within the ASHES and International District vent fields, respectively), to determine genetic diversity at each site and to test whether these sites constitute one or two separate populations. This research will ¬¬¬¬assist in understanding the connectivity and genetic diversity of sulfide worms within the Axial Seamount system across multiple vent fields and contribute to a broader understanding of how populations are established and maintained in this unique and dynamic ecosystem.

The drug rapamycin can increase lifespan in a variety of model organisms by repressing the activity of the mTOR complex, a cellular component required for growth and development. Other than one small study that looked at genetic variation in the effect of rapamycin on lifespan in fruit flies (Rohde et al., 2021), little is known about how natural genetic variation affects the response to rapamycin. Previous work by the Promislow lab, utilizing developmental time to indicate rapamycin’s affect, has shown that some strains of fruit flies are completely resistant to rapamycin while others are sensitive. These genotypic differences are also reflected in the metabolome, the complete set of small molecules and metabolites present within cells. Metabolome analysis of these strains revealed significant differences in metabolite concentrations between resistant and sensitive lines. Interestingly, when we treated sensitive strains with rapamycin, their metabolome profiles were like those of starved larvae. I hypothesize that rapamycin is affecting larval ability to take up nutrients and that the starved metabolome is a result of actual starvation. To test my hypothesis, I am designing a starvation assay to compare the death rate of sensitive and resistant larvae. Measurement of resistance to starvation is taken two days after rapamycin treatment by transferring larvae to nutrient deficient food. The duration of time for individual larvae to die is recorded, and the death rates between the two populations are compared. If my hypothesis is true, the sensitive larvae will have a higher death rate than the resistant larva. If death rates are similar however, that could mean that rapamycin does not cause a nutrient deficit and there is another explanation for its effects on the metabolome. This study will provide insights to the underlying mechanisms of sensitivity to rapamycin, and why it might differ between individuals.

It is well-established that mutations have impacts on an organism’s fitness; however, the fitness effects of mutations are not static, and can vary depending on environmental contexts, such as the species in which a mutation is found. Evolution of the same gene in different species could thus lead to the evolution of different phenotypes, as different species would favour different sets of mutations. If that gene could be exchanged between species, it could lead to increased evolutionary possibilities, as high-fitness genotypes that require a prerequisite deleterious mutation in one species could become accessible if the mutation is not deleterious in another. Our research examines how the presence of two bacterial hosts, Escherichia coli and Klebsiella pneumoniae, could affect the evolution of an antibiotic resistance-conferring TEM-1 β-lactamase gene located on a conjugative plasmid. If different hosts confer different mutational effects to TEM-1, the process of horizontal gene transfer (HGT) that allows mutations to be shared between species could open up more mutational possibilities than those accessible in either single-species population alone. We tested this hypothesis through three rounds of experimental evolution in the presence of the antibiotic cefotaxime, where we evolved two single-species E. coli and K. pneumoniae populations and one multi-species population where HGT was simulated with a shared plasmid pool. We are now reconstructing the genotypes found in all three populations after each round to assess how much antibiotic resistance they confer in both species, and hope to see if the genotypes acquired under HGT treatment provide higher resistance compared to the single-species populations. Our results have practical implications for the predictability and nature of antibiotic resistance development in the real world, a current global health crisis, and potentially motivate further study in predicting resistance emergence in clinically encountered multi-species populations.

In the era of the internet, numerous individuals participate in fan communities on online social platforms to pursue their interests. Despite all members sharing genuine passions, these communities exhibit varying levels and types of engagement, with some attracting millions if not billions of active users daily, while others show little to no activity. Additionally, users tend to engage in discussions on certain topics more frequently in specific communities. This research aims to explore the reasons behind this difference by investigating the interaction between community size and openness. While previous works focus on the impact of either size or openness, this study develops hypotheses on how their interaction affects member engagement. A survey study is outlined to test these hypotheses, examining members' sense of belonging, level of hostility, and variation of opinions in four distinct communities representing different interactions between community size and openness: large and open, large and closed, small and open, and small and closed. The survey will be distributed among TFBoys fans on Sina Weibo in the Spring of 2023. The expected results will provide insights into how community size and openness interact to influence member engagement in online fan communities, offering recommendations for online social platforms to design features that promote member engagement and contribute to scholarly knowledge in Computer-Mediated Communication and Social Media Studies.

CRISPR Activation (CRISPRa) is a powerful discovery-based tool to evaluate gain-of-function en masse. Applied successfully to emerging cell therapies, this technology offers tremendous promise to inform next-generation therapy design. Despite this potential, translation of CRISPRa to primary cells, including T lymphocytes, has been impeded by poor transgene expression. Based on prior reports of dCas9 genotoxicity, we suspected that CRISPRa could be exerting a toxigenic effect on CAR T cells, and thereby selecting against clones with high expression. To test this hypothesis, three separate constructs were designed with inhibited transcription and/or translation of the CRISPRa transgene. Following delivery of the constructs to donor T cells, analysis by flow cytometry revealed similar levels of cell yields and no net increase in dCas9 marker positivity across all CAR T cell subsets. Further epigenetic experiments and drug studies with anti-silencing compounds revealed that transcription of the CRISPRa transgene was severely inhibited. Collectively, these findings suggest that the CRISPRa transgene does not exert a toxigenic effect on CAR T cells; rather, low CRISPRa expression is caused by transgene silencing. Targeted efforts to mitigate silencing of the CRISPRa transgene are thus warranted to achieve adequate implementation to therapeutic cell subsets.