Long-Term Single-Cell Imaging of Live Microbes by Correlative Fluorescence and Raman Microscopy & Time-Resolved Stark Effect Spectroscopy of Protein Crystals

Understanding the behavior of a microbe requires not only an understanding of molecular mechanisms, e.g. DNA replication, transcription, and translation, but also knowledge of the cell’s chemical composition over time. There are powerful tools for probing the mechanisms of core cell-biological processes. However, the existing tools to measure cell composition have significant limitations. Information-rich methods, such as mass spectrometry, are lethal, while fluorescence methods provide good time resolution and work on live cells, but are limited in what they can measure. Prior work has demonstrated that spontaneous Raman spectroscopy can be a powerful tool to measure cellular composition. However, low throughput has limited its potential for discovery. Here, I describe my contributions to correlated epifluorescence and laser scanning Raman microscopy to enable the collection of single-cell spectra from many single cells in long-term time-lapse experiments, with imaging every 15 minutes. I then establish that this single-cell Raman imaging (scRaman) system can be used without substantial phototoxic effects to image the yeast Saccharomyces cerevisiae. To benchmark the ability to track biologically important changes in cellular composition, I study S. cerevisiae under conditions of nitrogen starvation and repletion. I demonstrate the ability of this system to resolve the dynamics of compositional changes at a single-cell level, mapping the observed dynamics to known biological mechanisms. I describe the analysis pipeline required for this work and the advances in combined Raman and microscope control software to achieve these experiments. Finally, I describe a separate project in which I developed an analytical framework for interpreting time-resolved Stark effect spectroscopy data obtained from single protein crystals.Engineering and Applied Sciences - Applied Physic

Zealots of Justice: Coercive Officials in Late Medieval Italy

This dissertation considers how, between about 1200 and 1350, cities in the upper half of the Italian peninsula experimented with a new kind of official whose primary task was coercion. There is a rich scholarship on institutions of public justice in the self-governing “communes” of thirteenth- and fourteenth-century Italy, but little of it has engaged with the coercive apparatus that served the courts and their users. It was easily the most sophisticated in Europe since the fall of the western Roman Empire. Italy’s coercive officials originated as messengers—comparable to process servers in the modern American legal system—in the years around 1200. But as communal courts sponsored new procedures that enabled creditors to swiftly recover debts, court messengers were tasked with coercive services for which creditors could pay—namely, the seizure of debtors’ property or, if necessary, the arrest and incarceration of debtors themselves. The earliest coercive officials, although known by different terms from city to city, closely resembled one another by the end of the thirteenth century. I call this prototypical coercive official a “sergeant.” Sergeants had their analogues elsewhere in the medieval Mediterranean, even in northern Europe, but the surpassingly rich archives of a few Italian cities make it possible to study sergeants at a near-ethnographic level of detail. I use one such city in western Tuscany, Lucca, as my central case study. Drawing upon the tools of institutional history, prosopography, anthropology, and social network analysis, I seek to explain why coercive officialdom took an especially brutal turn in Lucca, and possibly other cities like it, in the decades after 1300. Chapter 1 examines the legislative compendia, or “statutes,” that defined coercive officialdom in sixty-five Italian cities between about 1175 and 1360. These reveal how the office became increasingly brutal as it responded to the demands of creditors and communal administrations. Chapter 2 turns to the case of Lucca, concentrating on the 1330s and early 1340s. Here, we can see how the dictates of the office could be tempered by the social environment in which sergeants—called “heralds” (nuntii) in Lucca, as in other Tuscan cities—were embedded. However, the Lucchese evidence also reveals how the technology of coercive officialdom could directly serve the state. In Lucca, the exaction of taxes, fines, and other debts owed to the commune itself was not left to sergeants, but to officials called “retainers” (familiares). I use digital techniques to study the social networks that formed among sergeants, retainers, and the sureties they presented for office, showing that these two communities of coercive officialdom experienced the city around them in fundamentally divergent ways. Retainers, unlike sergeants, were largely excluded from Lucchese society and free to be shaped into “zealots of justice,” to use the memorable expression that one retainer applied to himself and his colleagues. In practice, “zeal” often meant brutality. Chapter 3 delves into the world of retainers and their culture of brutality, which I argue was fostered by the institutions they served. Indeed, even as the Lucchese commune made a show of condemning the worst of this brutality, a regime of impunity prevailed. Chapter 4 shifts perspective and considers the resistance that brutality engendered, especially in Lucca’s countryside. Examining a decade of resistance, from 1334 to 1344, I show that the Lucchese commune authorized techniques of coercion that evoked the predatory tactics of marauding armies. Brutality of this kind often met with collective, organized resistance. While this dissertation charts the intensification of state-sponsored violence in the cities of late medieval Italy, it also shows that the trend toward brutality was far from inevitable or sustainable.Histor

The Peso Perspective: Understanding Risk and Return in Global Currency Markets

The drivers of currency excess returns remain poorly understood despite the foreign exchange market’s size and liquidity. Using five measures of risk — novel text-based measures (from newspaper articles and firm earnings calls) alongside traditional risk indices that capture a country’s economic, financial, and political risk — I show that financial risk is the dominant predictor of volatility in emerging market currency returns, while geopolitical risk is positively associated with excess returns, supporting a risk premium explanation for the profitability of currency trades. Firm-level risk perceptions, especially from foreign firms, outperform political/economic risk in forecasting returns. However, country-specific risks explain only a fraction of exchange rate movements, revealing fundamental limits to forecasting exchange rate movements. The results highlight financial stability as a stronger determinant of risk premia than political uncertainty, with implications for currency speculators and policy-making in emerging economies.Applied Mathematic

Evaluating Migration of Human Monocytes in an Inflammatory Environment Mimicking Burn-Induced Inflammation in Blood Donor Samples

In the U.S., more than half a million people are hospitalized for burn wounds every year. These wounds can progress into serious and even fatal injuries due to the immune system’s defense against harmful pathogens with access to bodily tissues through the burn injury. This invasion into bodily tissue can lead to systemic inflammation. One of the by-products of this inflammation is the activation and migration of white blood cells, specifically monocytes, to the site of injury and/or infection to eliminate the microbes and protect the surrounding tissues. While relatively small, inactive monocytes can drastically increase their size in the case of activation via pathogenic infection. Although the general mechanisms are understood, there are significant components of monocyte activity that have not been clearly elucidated. However, it has been shown that these monocytes boast significant prognostic capabilities. The severity and potential outcome of patient injuries can be predicted with considerable accuracy using Monocyte Distribution Width (MDW), or monocyte anisocytosis, which is the difference in monocyte sizes within an individual. In this research, we investigate the activity and size distribution of monocytes under various conditions. It was found that, as expected, the trigger protein (SDF-1α) induced monocyte migration dependent on the concentration used. However, the inhibitor (AMD-3100) markedly reduced migration when used in conjunction with SDF-1α. Also, the activators lipopolysaccharide (LPS) and Pam3CysSerLys4 (PAM) were shown to increase MDW.Extension Studie

Frame to Foreground: Translating Documentary Landscapes in Braddock, PA

The project posits that critical photography should be adopted as a design methodology – starting from the representation of a diagnostic relationship between a subject and their setting to grasp at the ineffable affect that artists capture beautifully, and often revealing social issues, left in tension within the frame through photographic techniques. This thesis project argues the translation of critical photography into spatial elements is crucial for our understanding of place. Furthermore, this new methodology reveals the concept of defining landscape as a set of relationships between living beings. The project is about preserving what every local in Braddock, Pennsylvania holds—a strong relationship to place. Using Frazier’s framing techniques, this thesis injects photography directly into the method of design. The project imagines a system of public landscapes that are centered in belonging, gathering, and reclaiming space. A riverfront park, a gathering pavilion, a public plaza, and a garden work as one to knit together a post-industrial steel mill town that has been disinvested for forty years. This thesis is advised by Sara Zewde.Department of Landscape Architectur

Logistics in the Line of Fire: A Stochastic Programming Model for Contested Environments

As military doctrine evolves to address the challenges of large-scale combat operations (LSCO), logistics planning must adapt to ensure the continuous sustainment of dispersed and contested forces. This thesis presents a two-stage stochastic mixed-integer programming (MIP) model designed to optimize military resupply under uncertainty. The formulation incorporates key doctrinal priorities, such as predictive logistics, prepositioned supply, and distribution network resilience, by explicitly modeling disruptions to supply routes and storage nodes. Using scenario data created from the Russo-Ukrainian War, the model evaluates the impact of adversarial attacks, storage costs, and vehicle availability on overall logistical performance. The results provide quantitative insight into the tradeoffs between transportation and storage-based supply strategies, revealing nonlinear thresholds that influence the model's behavior. The proposed framework offers a rigorous, extensible tool for analyzing logistics strategies in adversarial environments and contributes to the broader effort to formalize military logistics planning using mathematical modeling and data-driven decision making.Applied Mathematic

Land Transitions to Regenerative Vegetable Farming: Assessing Opportunities for Established Traditional Farms and Small Vegetable Growers

Economic challenges facing farmers in the United States are well-documented. Among these are systematic overproduction and low crop prices, competition on a global scale, high costs related to industrialized farming inputs and fluctuating weather (Baines, 2017). The average age of all U.S. farmers in 2022 was 58.1, up 0.6 years from 2017, highlighting a trend towards aging of farmers (USDA, 2022). Despite financial challenges of farming, there are people interested in getting into the business of farming, both in the United States and abroad (de Wit et. al., 2019). While this set of new farmers may seem a ready-made solution to the question of who will take over farms of the future, there exists a multifaceted transition gap, characterized by barriers that prevent aging farmers from exiting farming, and barriers to new farmers that would enter the profession. The most severe barrier is lack of access to land (Shute, 2011). My research examined the mismatch between land held by traditional farms and land needed by new farmers, the economics of small-scale direct market vegetable farming, and proposed and analyzed a business model based on the success of farm incubators to address this mismatch while meeting economic needs of farmers. While a national issue, I focused on New England, where traditional farms are often on the scale of 50-100 acres, growing commodities such as hay. New direct market vegetable growers will typically only need a small plot of land on the order of one or a few acres. This project considered a hypothetical farm that shares features of an incubator farm as an organizational model and reviewed existing benchmark studies to understand the economics of small-scale direct market vegetable farming. The central research questions were: Is it economically feasible for a traditional land-owning farm producing a commodity such as hay, to repurpose a portion of land in production and lease it to individual vegetable farmers? Could this proposed model be economically viable for both parties, and could this help reduce the barriers to entry and increase longer term viability for new farmers? This project used scenario analysis to forecast financial feasibility of a model where an existing traditional farm transitions a portion of their land to individual growers, leasing the land and a set of farm related services. I generated a set of outcomes both for the participating individual growers and for the traditional farm that would participate in this multi-farm project. I assessed scenarios for the individual growers referencing published research data on costs and benefits of selling via different market channels. Scenarios for the traditional farm varied in the numbers of acres transitioned, as well as the number of individual vegetable growers to whom they would be leasing. Results showed that the proposed model is economically viable for individual growers when selling via direct market channels but would not be viable for vegetable growers selling via a wholesale market. Results also indicate that the model is marginally financially viable for the traditional landowning farm. This research may be of interest to traditional land-holding farms, and small growers that are considering alternative approaches to land use and to policy makers. The CBA from this project may also have value as a forecasting tool to new or existing incubators looking to plan financial roadmaps for their own organizations, or for a collective group of individual farmers looking to participate in a joint venture.Extension Studie

Mutable Ghosts: A Collection of Short Stories

Mutable Ghosts is a collection of three short stories that feature unreliable narrators in the first person. These works explore the fickle nature of the self and its ability to create its own reality. They are in some way, a study of solipsism. Each narrator resides intensely in his or her own reality. These stories investigate the conditions needed to either expand or contract a person's understanding of themselves and likewise a person's understanding of their intrinsic responsibility to those around them. These stories explore a host of conditions that affect these insular worlds. Among them include personal hauntings, that is the people and experiences that bedevil us, as well as place and environment. These stories offer a tour of the Eastern Seaboard from Massachusetts to West Virginia to Florida. I am inspired by the cast of unreliable narrators that have defined my experience of fiction; from the Underground Man to Humbert Humbert and Nick Carraway, from Holden Clawfield and Richard Papin to Ava Bigtree and Amy Elliot Dunne. I am consistently moved by fiction that finds the universal truth in the utterly untrue; that which holds authentic in even the most skewed and insular of human spirits. I have always taken solace in fiction that acknowledges the subjectivity of every human being and so too, the remarkable sameness of the human experience. I hope these works are, in some small way, an addition to that project.Extension Studie

Mechanistic Characterization of Acyltransferases that Modify the Bacterial Cell Envelope

Bacteria are surrounded by a complex macromolecular structure called the cell envelope, which is essential for survival. The cell envelope structure varies depending on the bacterial species; however, there are conserved features present in all cell envelopes, like peptidoglycan. Because of its conservation and essentiality, peptidoglycan has long been the target for antibiotics, such as beta-lactams. Unfortunately, bacteria have developed resistance to these antibiotics, and their broad-spectrum activity is considered detrimental to commensal bacteria. With this in mind, it is critical to explore new antibiotic targets within cell envelope biosynthesis. Furthermore, it would be beneficial for these new drugs to have more specific activity, which could be achieved by targeting features that are unique to specific bacterial clades. This work explores the potential of cell envelope-modifying acyltransferases as new targets for antibiotic development. We focus on understanding the basic mechanism of these acyltransferases to lay the groundwork for future drug development campaigns. In this thesis, we determined the mechanism for teichoic acid D-alanylation in Gram-positive bacteria. We showed that the small membrane protein DltX uses a conserved C-terminal hexapeptide motif to help shuttle D-alanine across the membrane. This occurs through a cascade of covalent intermediates that promote thermodynamically favorable transfer. Additionally, we found that cell envelope modifying pathways that lack a DltX homolog use a similar hexapeptide motif; however, it is fused directly to a polytopic membrane protein akin to DltB. We then used cryoEM to structurally characterize the DltXBCD complex to further probe the mechanism. We developed a robust pipeline to determine this structure and applied it to a disulfide mutant that traps DltX in a DltB-bound state. Finally, we turned our attention to an acetyltransferase, TmaT, important for mycomembrane biogenesis in Mycobacteriales. We found that this acetyltransferase interacts with an arabinosyltransferase, AftD, involved in arabinogalactan biosynthesis. We also discovered that the acetyltransferase and the arabinosyltransferase regulate each other’s activities. This work led to a model where the TmaT/AftD complex serves to coordinate cell envelope assembly in Mycobacteriales. Taken together, this work uncovered fundamental mechanistic information about a range of cell envelope-modifying acyltransferases that will serve as a basis for future antibiotic development efforts.Chemical Biolog

Exploring Variations and Decision-making Processes of the Utilization of Modern Contraceptive Methods During Protracted Violence and Political Instability in Rural Haiti

Background Haiti's protracted sociopolitical instabilities greatly impact healthcare delivery, leading to low family planning coverage of 35%. Short-term contraceptive methods are predominant, making frequent and expensive visits to healthcare facilities necessary. These instabilities also cause disruptions in healthcare services, potentially resulting in missed follow-ups and unintended pregnancies ultimately. Methodology We investigated family planning utilization trends and decision-making in four Haitian communes during prolonged sociopolitical instabilities through a convergent mixed-method study. Secondary quantitative data on family planning service usage from healthcare facilities were collected, alongside data on major sociopolitical events from online newspapers. Linear regressions were employed to analyze the impact of these events on family planning usage. Qualitative data were gathered from in-depth interviews and focus group discussions across the communes. Then inductive content analysis was performed using Dedoose. Results During the study period, 163,179 women aged 15 to 49 utilized the six most common contraceptive methods across four healthcare facilities. Simultaneously, 188 sociopolitical events were recorded, with 80% being national, 10% local, and 10% regional events. In bivariate analysis, we found a negative correlation between stockout of common contraceptives and family planning service utilization -0.64 (95%CI: -0.79, -0.49), p.001. Strikes reduced family planning utilization -0.63 (95%CI: -2.6, 1.35) but, their effect was not statistically significant, p=0.53. Having more than two trained providers was positively correlated with family planning utilization, 1.99 (95%CI:1.8, 2.0). Surprisingly we found an increase in family planning service utilization during gang clashes 1.94 (95%CI: -0.02, 3.9), police-gang clashes 1.8 (95%CI:0.64, 2.9), and gang violence 1.8 (95%CI:0.68, 2.9). After controlling for confounders, the relationship between health facilities characteristics and family planning service utilization remained statistically significant. Inductive qualitative analysis revealed six main themes representing three barriers: 1) accessing healthcare facilities, 2) family planning side effects, and 3) workforce challenges. Additionally, three facilitators for family planning utilization emerged: 4) integrated services, 5) interpersonal influences, and 6) socio-economic challenges. Conclusion Prolonged sociopolitical events impact family planning access, creating both barriers and opportunities. These events may hinder the movement of people and goods, prompting proactive strategies by healthcare professionals and family planning users. Our recommendations include expanding storage capacity, promoting long-acting contraceptives, and improving community-based distribution. The observed positive correlation between family planning service utilization and sociopolitical instabilities suggests increased demand during crises. This highlights the need for timely provisioning of FP supplies to empower communities to navigate ongoing crises effectively.Graduate Educatio