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.

Reducing the considerable carbon emissions of the built environment industry is vital to addressing the current climate crisis. Though there have been substantial advancements in cutting emissions from building operations (operational carbon), addressing buildings’ embodied carbon is a newer focus, with ongoing research driving industry transformations. Embodied carbon refers to the greenhouse gas emissions arising from the manufacturing, transportation, installation, maintenance, and disposal of building materials. Since a large portion of embodied carbon emissions are upfront, rapid adoption of improved practices is critical. Thus, many architecture, engineering, and construction (AEC) firms around Seattle have initiated a focus on reducing the embodied carbon from their designs and practices. We have selected three Seattle-based AEC firms, and are analyzing each firm’s methods of improving emissions from their practices while considering the lessons and applications of these methods going forwards. The team is conducting in-person tours of firm offices and interviewing key sustainability-focused team members about their approach to improving their projects’ environmental impacts with particular focus on embodied carbon. Beyond broad qualitative data about firm practices, quantitative data on specific low-carbon buildings is collected. This is being reviewed, analyzed, and synthesized into deliverables summarizing common trends, best practices, and future research needs. Due to the relatively recent innovations in reducing embodied carbon, many practices are yet to be adopted. For this reason, we expect that many firms will be following longer-term plans as they gradually integrate sustainable practices into their workflows. Through an advanced understanding of the changing landscape around the impacts of embodied carbon, designers and researchers will be better prepared to eliminate carbon emissions from all sectors of the built environment.

The Khmer Empire was one of the largest preindustrial settlements in the world and is known for its vast temple complexes such as Angkor Wat. Although we have a functional understanding of the Angkor empire, the relationship between the Angkor state expansion as it relates to human-environment dynamics is unclear. Archaeological evidence from the site of Prasat Basaet, northeast of Cambodia demonstrates that there was human activity even before the Angkorian Period. Through our geological analysis of sediment samples excavated from ancient households at Prasat Basaet, we can get insights into the practices and activities of people and the surrounding environment. The preliminary results of our sediment particle analysis and organic matter measurements show change in human activity in Prasat Basaet as it relates to the expanding state. Using geoarchaeological methods such as; loss on ignition, magnetic susceptibility, and color, we have found an early, pre-Angkorian settlement at the site, followed by a long period of little to no activity, and then an increase in activity during the Angkorian period. We anticipate our research to be a pathway for understanding human-environment dynamics during expansion and contraction of the empire.

The Paleolithic era saw the beginning of the development of human language. Furthermore, the Paleolithic is characterized by the appearance of non-figurative, or geometric art. There are extensive examples of signs commonly seen in Paleolithic art, and while over 40 distinct signs have been recorded, we focus on the five most abundant signs in the SignBase dataset (cf. doi: 10.1038/s41597-020-00704-x): notch, line, obline, hatching, and dot. Here we investigate Bacon’s recent (2023, doi: 10.1017/S0959774322000415) hypothesis that geometric signs were a way of recording a phenological calendar. We found an abundance of representation of the notch symbol (254), followed by the line (174). To assess their proposed calendrical function, we report on the distribution of frequencies of the symbols to identify if seasonal patterns are represented. Our study sheds new light not only on the characteristics of geometric art, but also on the likelihood that they encode a phenological calendar as proposed by Bacon et al. (2023).

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.

The choice to terminate a pregnancy is rarely an easy one. It is critical that the experience of pregnancy termination is made as comfortable and convenient as possible as women grapple with the mental and physical challenges that arise from their choice to abort. The current standard protocol for at-home medical termination of pregnancy involves the patient-mediated oral misoprostol delivery 24-48 hours after mifepristone. This timed delivery ensures that misoprostol can trigger contractions after the cervix is dilated by mifepristone. This presents a challenge for patients, who have to manage the side effects of mifepristone while also timing the delayed dosage window for misoprostol. Combining these two medications into the single dose modality that instantly releases mifepristone and ensures the delayed release of misoprostol will improve the at-home medical abortion process for patients globally. The nanoparticle encapsulation of misoprostol for oral delivery offers the prospect of delayed release and release modulation through the alteration of variables such as molecular weight and co-polymer polymerization and other formulation parameters. Herein, we aim to use established protocols for nanoparticle encapsulation to fabricate and characterize misoprostol nanoparticles. By varying molecular weight and copolymerization parameters, we aim to tailor misoprostol release and evaluate the efficacy of different encapsulation approaches. We intend to investigate the particle size, entrapment efficiency, shelf-stability, and in vitro release of misoprostol nanoparticles in PBS and simulated gastrointestinal fluid. In doing so, we aim to provide a proof-of-concept of formulating misoprostol into nanoparticles to demonstrate encapsulation and sustained release. This understanding can contribute to the development of a single-dosage modality to meaningfully improve the comfort and ease of at-home medical abortions.

Seventy-five years after the forced removal of Palestinians from their homes and villages during the nakba, the permanent effects on the physical and geographical landscape of the region is not entirely understood. I investigate how much of the erasure of Palestinian villages has been made permanent through reforestation and repopulation. Prior work, specifically by Walid Khalidi in All That Remains: The Palestinian Villages Occupied and Depopulated by Israel in 1948, attempted to quantify the loss, but has not been made current. Using quantitative data from aerial photographs and previously collected records as well as qualitative data in the form of narratives from Palestinian refugees, I examine the permanent impact of the forced expulsion of Palestinians from their homes and villages and the destruction of those places. I focus on comparing geographical and population data from 1945 with recent data, and examine in particular the al-Ramla and Jerusalem subdistricts. These results are generalizable to other areas impacted by recent and ongoing wars, expulsions, and genocides and may add to the understanding of the ongoing impact of these events.

Archaeological sequences are the successive organization of artefact-bearing stratigraphical units spanning thousands of years. Due to vast periods of time represented by each unit, sometimes analysis of the sequence results in the appearance of little cultural change over time. This can make it hard to distinguish between random drift, directional selection, and stabilizing selection of ancient technologies. We demonstrate a new statistical method to test hypotheses about drift and selection using archaeological time series data. Through the analysis of Log-rate log-interval plots computed using R, we compare the median slope values of an archaeological sequence to experimentally determined time series values. We demonstrate this method using a case study from Gua Talimbue, a cave in Southeast Sulawesi, Indonesia. Using the artifact data spanning the late Pleistocene and Holocene, we analyze the rate of artifact change over three depositional phases to determine whether the cultural evolution of the artifacts is indicative of stabilizing, random, or directional selection. Previous work at this site hypothesized stabilizing selection, which is confirmed by the results of our new work. We formally evaluate this using a new statistical method borrowed from palaeontology, originally developed by Gingerich in his book "Rates of Evolution" (https://doi.org/10.1017/9781316711644). This research is vital in the building of a better understanding and deeper insight into rates of change of lithic technologies generally, and more specifically the skills employed by early people, specifically within pre-Neolithic Indonesia.

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.

Iron centers which feature metal ligand multiple bonds can be powerful group transfer agents, for example, terminal Fe-oxo intermediates in soluble methane monooxygenase can perform oxo-atom transfer for the selective oxidation of methane to methanol. Abiologically, ligand constructs which enforce desirable electronic and structural configurations have been shown to enhance group transfer to a range of organic substrates. We developed and studied an iron (Fe) molecular complex with two aminophosphine selenide ligands (Se=PPh₂NTol; Ph=Phenyl, Tol=4-Tolyl) that chelate the metal center via the selenium and nitrogen. The iron complex (FeL₂) was synthesized by a reaction between Fe(HMDS)₂ (HMDS = bis(trimethylsilyl)amide) and the aminophosphine selenide. Characterization shows a tetrahedral, high spin, symmetric compound. We hypothesized that FeL₂ can activate and transfer heteroatoms and explored the reactivity of FeL₂ with oxidants, oxo atom donors, and organic azides. Treatment with iodine (I₂) results in oxidation of the iron center (Fe(II) to Fe(III)) and coordination of the iodide counterion results in structural reorganization to a five-coordinate square pyramidal complex. Reactivity with oxo atom donors shows that either the ligand or Fe center are oxidized, and we identified a µ₂-oxo dimer, which is the first Fe-O-Fe dimer to have selenium in its first coordination sphere. We found that FeL₂ forms Fe-nitrenoid intermediates and can perform nitrene transfer to form diazos or do C-H amination, when treated with aromatic and aliphatic azides, respectively. The presented complexes are characterized by single crystal X-ray diffraction (XRD), Evan’s method, nuclear magnetic resonance (NMR), and Ultraviolet-Visible Spectroscopy (UV-Vis). This research builds upon the knowledge of transition metal complexes for heteroatom transformations.

Aging is an important problem in biomedical research. Given the increased risk of death with age, techniques to delay aging hold substantial promise for human well-being. The premise of my research is that rapamycin, a drug commonly used in transplant patients which is hypothesized to have development-slowing effects, can slow development of fruit flies. One finding from a previous study, however, is that there is enormous genetic variation within the Drosophila population (akin to the variation between dog breeds) which results in varying sensitivity to rapamycin. Earlier measurements indicated that strains of fruit flies that were sensitive to rapamycin had higher levels of histamine, a molecule that participates in metabolism, when on the drug. We set out to determine whether histamine could effectively extend sensitivity to the drug to a wider range of genotypes within Drosophila. Thus, I hypothesize that introducing histamine to the food that fruit fly strains that are genetically resistant to rapamycin are consuming will increase sensitivity. To test this hypothesis, I added solutions with different concentrations of histamine to food with or without rapamycin. I then placed eggs from a resistant strain onto the food to observe the time it takes from egg to pupa. Because I hypothesize that increasing histamine levels will make a resistant strain of Drosophila sensitive to rapamycin, the strain should become sensitive and consequently show an increase in development time compared to the conditions without histamine. Each test condition of a histamine solution and rapamycin has a corresponding control with no rapamycin. Hence, if the histamine-treated conditions show slower pupation times than the histamine-free controls, the hypothesis is validated. If validated, this work could help researchers understand ways to provide the benefits of rapamycin to individuals who might otherwise be genetically resistant to its impact in both aging and medical contexts.

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.

Trauma is the 6th leading cause of mortality among Americans aging 65-79 years and outcomes after traumatic injury are worse among older patients. However, identifying which older adults will have worse outcomes after trauma is difficult. Frailty, an aging-related syndrome of physiological decline, has been associated with worse outcomes post traumatic injury. Most tools used to identify frailty rely on subjective data, which is often unattainable if patients are unconscious, delirious, or suffering from mental health illnesses. The aim of this study is to determine the utility of ultrasound and CT measurements of the masseter muscle (jaw muscle) in quantifying frailty by diagnosing sarcopenia, analyzing its correlation with clinical measures of frailty, and its association with geriatric patient outcomes after traumatic injury. Following IRB approval, I screen trauma patients for eligibility. Patients must be adults, have had a head CT taken in the last 24 hours, have no injury to the masseter muscle, and are able to answer questions about daily life activities. After obtaining consent, I interview to assess nutritional status and overall health using frailty questionnaires. Then, I perform a bedside ultrasonographic exam of the patients’ masseter muscle on both left and right sides. Lastly, I analyze the masseter muscle on the head CT scan. I measure the masseter’s width, depth, and cross-sectional area (CSA) using the Centricity Software. Our preliminary data shows that changes in masseter muscle size are detectible in radiological studies. I found that a representative patient’s masseter muscle width on ultrasound decreased by 29% eighteen days post trauma. Additionally, I collected four CT measurements on the same patient during their hospital stay: masseter muscle CSA decreased by 28% over 12 days and by 41% over 29 days. Results from this study regarding the association of masseter muscle size and frailty are still being analyzed. If significant, these measurements will allow for quick detection of frailty, allow physician to take appropriate treatment decisions, and improve patient outcomes.

We are doing research that supplements a larger archaeological study on the impacts of contact with 19th-century Russian colonists on the Indigenous Sugpiaq community of the Kodiak Archipelago, Alaska. We are experimenting with dendrochronological methods to determine the most appropriate strategies for collecting data from wood samples, and whether the results will improve chronological resolution at the Ingyuq archaeological site that straddles Russian conquest of the region. Dendrochronology is a dating method that compares patterns in tree ring growth between living wood samples and wood found in the archaeological record. In the lab, we first measured the tree ring growth patterns on dwarf willow, birch and alder samples collected from living trees in the region around the site. Patterns were estimated using a microscope, magnifying glass, and photographs to count annual rings and measure their relative thickness. We then compared ring thickness across samples to determine if there is a shared growth history among trees around this site. This project forms the first and foundational piece in a longer project that will overlap growth patterns in living wood samples to those found in the archaeological record at this site. At the end of the project, we hope to find patterns across charcoal samples excavated from this site that allow us to develop a firmer understanding of the sequence of and temporal relationships between individual stratigraphic layers to better focus research on broader questions about the impact of Russian colonization on the Sugpiaq people.

Treatment of individuals with HIV using antiretroviral therapy (ART) is highly effective, but effective clinical management depends on maintaining therapeutic drug concentrations. Antiretroviral (ARV) drug concentrations in patients with HIV can vary due to differences in drug metabolism, medication adherence, or interactions between multiple drugs. These individuals may have subtherapeutic or supratherapeutic drug concentrations, putting them at risk of treatment failure, acquisition of drug resistance, and risk of hospitalization or death. Current measurement of ARV concentration is done through liquid chromatography tandem mass spectrometry, which requires expensive equipment and requires a labor-intensive protocol. This restricts accessibility to specialized laboratories, making it difficult for persons with HIV to have routine measurements of ARV drug concentrations. The goal of the project is to develop an assay that is simple to perform and uses standard equipment to increase access to routine clinic-based drug level monitoring to improve HIV care. We designed an assay using a 2-step process of DNA strand transfer and quantitative polymerase chain reaction (qPCR) to quantify integrase strand transfer inhibitors (INSTIs). We tested for dolutegravir (DTG) and cabotegravir (CAB) in both buffer and plasma -- the latter to simulate patient blood samples. We were able to demonstrate that the assay could quantify clinically relevant drug concentrations of DTG and CAB. By developing an assay that can be readily integrated into most clinical laboratories, we will contribute to increasing access to routine HIV drug level monitoring to improve clinical HIV care and maintaining viral suppression in persons with HIV.

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.

The drug rapamycin has been shown to extend lifespan in model organisms ranging from mice to fruit flies, but little is known about how genetic variation affects the response to rapamycin. It is possible that some individuals might not respond to the drug, or may even respond negatively. To study the impact of genetic variation on rapamycin sensitivity, the Promislow lab has been using the fruit fly, Drosophila melanogaster, to test the ability of rapamycin to slow early development in different strains. They found a range of responses, from strains that were completely resistant to those that were highly sensitive. To better understand why resistant and sensitive strains differ, the Promislow lab analyzed their metabolome profiles. The metabolome consists of diverse small molecules, metabolites, that are fundamental to sustaining life in cells, and the relative abundance of metabolites is referred to as a metabolome profile. By comparing metabolome profiles, the Promislow lab found that the metabolome of sensitive larvae treated with rapamycin was similar to those of larvae starved of nutrients. One possible explanation for this observation is that sensitive larvae on rapamycin-treated food eat less food than resistant larvae. To test my hypothesis, I am measuring how much food they ingest, using dyed food, and scoring how much dye each larva ingests over time. If sensitive larvae eat less rapamycin-treated food than resistant larvae, then we can investigate why rapamycin affects feeding. If no significant difference is found between scores for sensitive and resistant larvae, then we can investigate why rapamycin affects early development in sensitive strains when their food consumption is not limited. By determining if sensitivity to rapamycin is accompanied by differences in feeding, then we could potentially manipulate feeding to sensitize flies, and possibly humans, to this beneficial drug.

Particle-laden turbulent flows are important in both natural and industrial contexts. The particles in many of these processes, such as the formation of ice crystals in clouds or the paper-making process, are anisotropic, with directionally-dependent drag coefficients. Generally, anisotropic particles are free to rotate as they are advected by the carrier fluid. However, external forcing from gravitational and magnetic fields, the larger scale flow, and active behavior can restrict the particles’ orientation, fixing their anisotropic resistance with respect to the reference frame. The dynamics and statistics of symmetric particles in isotropic turbulence are well-studied, but the effect of anisotropic forcing on the transport and behavior of asymmetric particles is less well-understood. We studied these dynamics by conducting Lagrangian particle tracking in simulated isotropic turbulence from the Johns Hopkins Turbulence Database. Computer simulations of 54,000 randomly-placed particle tracers were run in Python, with anisotropy introduced by directly scaling the velocity of the tracer-particle at each simulation time step. We examine how increasing a particle’s resistance to motion in one direction in isotropic turbulence impacts the transport and dispersion statistics in all three directions. We find that increasing tracer anisotropy decreases diffusivity in the direction of velocity scaling as expected, but the diffusivity in the unscaled directions increases such that the total diffusivity remains roughly constant. Studying the dynamics of these simulated anisotropic particles in turbulence will provide a better understanding of how turbulence mixes both particles and the fluid itself, which can then be applied to particle-turbulence interactions in both environmental and industrial contexts.