Development and Application of Covalent-Labeling Strategies for the Large-Scale Thermodynamic Analysis of Protein Folding and Ligand Binding

Thermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.

Comparison of pattern detection methods in microarray time series of the segmentation clock.

While genome-wide gene expression data are generated at an increasing rate, the repertoire of approaches for pattern discovery in these data is still limited. Identifying subtle patterns of interest in large amounts of data (tens of thousands of profiles) associated with a certain level of noise remains a challenge. A microarray time series was recently generated to study the transcriptional program of the mouse segmentation clock, a biological oscillator associated with the periodic formation of the segments of the body axis. A method related to Fourier analysis, the Lomb-Scargle periodogram, was used to detect periodic profiles in the dataset, leading to the identification of a novel set of cyclic genes associated with the segmentation clock. Here, we applied to the same microarray time series dataset four distinct mathematical methods to identify significant patterns in gene expression profiles. These methods are called: Phase consistency, Address reduction, Cyclohedron test and Stable persistence, and are based on different conceptual frameworks that are either hypothesis- or data-driven. Some of the methods, unlike Fourier transforms, are not dependent on the assumption of periodicity of the pattern of interest. Remarkably, these methods identified blindly the expression profiles of known cyclic genes as the most significant patterns in the dataset. Many candidate genes predicted by more than one approach appeared to be true positive cyclic genes and will be of particular interest for future research. In addition, these methods predicted novel candidate cyclic genes that were consistent with previous biological knowledge and experimental validation in mouse embryos. Our results demonstrate the utility of these novel pattern detection strategies, notably for detection of periodic profiles, and suggest that combining several distinct mathematical approaches to analyze microarray datasets is a valuable strategy for identifying genes that exhibit novel, interesting transcriptional patterns.

Globalizing the Sculptural Landscapes of the Sarapis and Isis Cults in Hellenistic and Roman Greece

“Globalizing the Sculptural Landscape of Isis and Sarapis Cults in Roman Greece,” asks questions of cross-cultural exchange and viewership of sculptural assemblages set up in sanctuaries to the Egyptian gods. Focusing on cognitive dissonance, cultural imagining, and manipulations of time and space, I theorize ancient globalization as a set of loosely related processes that shifted a community's connections with place. My case studies range from the 3rd century BCE to the 2nd century CE, including sanctuaries at Rhodes, Thessaloniki, Dion, Marathon, Gortyna, and Delos. At these sites, devotees combined mainstream Greco-Roman sculptures, Egyptian imports, and locally produced imitations of Egyptian artifacts. In the last case, local sculptors represented Egyptian subjects with Greco-Roman naturalistic styles, creating an exoticized visual ideal that had both local and global resonance. My dissertation argues that the sculptural assemblages set up in Egyptian sanctuaries allowed each community to construct complex narratives about the nature of the Egyptian gods. Further, these images participated in a form of globalization that motivated local communities to adopt foreign gods and reinterpret them to suit local needs.

I begin my dissertation by examining how Isis and Sarapis were represented in Greece. My first chapter focuses on single statues of Egyptian gods, describing their iconographies and stylistic tendencies through examples from Corinth and Gortyna. By comparing Greek examples with images of Sarapis, Isis, and Harpokrates from around the Mediterranean, I demonstrate that Greek communities relied on globally available visual tropes rather than creating site or region-specific interpretations. In the next section, I examine what other sources viewers drew upon to inform their experiences of Egyptian sculpture. In Chapter 3, I survey the textual evidence for Isiac cult practice in Greece as a way to reconstruct devotees’ expectations of sculptures in sanctuary contexts. At the core of this analysis are Apuleius’ Metamorphoses and Plutarch’s De Iside et Osiride, which offer a Greek perspective on the cult’s theology. These literary works rely on a tradition of aretalogical inscriptions—long hymns produced from roughly the late 4th century B.C.E. into the 4th century C.E. that describe the expansive syncretistic powers of Isis, Sarapis, and Harpokrates. This chapter argues that the textual evidence suggests that devotees may have expected their images to be especially miraculous and likely to intervene on their behalf, particularly when involved in ritual activity inside the sanctuary.

In the final two chapters, I consider sculptural programs and ritual activity in concert with sanctuary architecture. My fourth chapter focuses on sanctuaries where large amounts of sculpture were found in underground water crypts: Thessaloniki and Rhodes. These groups of statues can be connected to a particular sanctuary space, but their precise display contexts are not known. By reading these images together, I argue that local communities used these globally available images to construct new interpretations of these gods, ones that explored the complex intersections of Egyptian, Greek, and Roman identities in a globalized Mediterranean. My final chapter explores the Egyptian sanctuary at Marathon, a site where exceptional preservation allows us to study how viewers would have experienced images in architectural space. Using the Isiac visuality established in Chapter 3, I reconstruct the viewer's experience, arguing that the patron, Herodes Atticus, intended his viewer to inform his experience with the complex theology of Middle Platonism and prevailing elite attitudes about Roman imperialism.

Throughout my dissertation, I diverge from traditional approaches to culture change that center on the concepts of Romanization and identity. In order to access local experiences of globalization, I examine viewership on a micro-scale. I argue that viewers brought their concerns about culture change into dialogue with elements of cult, social status, art, and text to create new interpretations of Roman sculpture sensitive to the challenges of a highly connected Mediterranean world. In turn, these transcultural perspectives motivated Isiac devotees to create assemblages that combined elements from multiple cultures. These expansive attitudes also inspired Isiac devotees to commission exoticized images that brought together disparate cultures and styles in an eclectic manner that mirrored the haphazard way that travel brought change to the Mediterranean world. My dissertation thus offers a more theoretically rigorous way of modeling culture change in antiquity that recognizes local communities’ agency in producing their cultural landscapes, reconciling some of the problems of scale that have plagued earlier approaches to provincial Roman art.

These case studies demonstrate that cultural anxieties played a key role in how viewers experienced artistic imagery in the Hellenistic and Roman Mediterranean. This dissertation thus offers a new component in our understanding of ancient visuality, and, in turn, a better way to analyze how local communities dealt with the rise of connectivity and globalization.

HPA Axis Activation by Ethanol Dependence in Adult and Adolescent Rats

Alcoholism is a disorder marked by cycles of heavy drinking and chronic relapse, and adolescents are an age cohort particularly susceptible to consuming large amounts of alcohol, placing them at high risk for developing an alcohol use disorder. Adolescent humans and rats voluntarily consume more alcohol than their adult counterparts, suggesting that younger consumers of alcohol may be less sensitive to its aversive effects, which are regulated by the function of the hypothalamic-pituitary-adrenal (HPA) stress axis. While HPA axis dysfunction resulting from ethanol exposure has been extensively studied in adult animals, what happens in the adolescent brain remains largely unclear. In this study, chronic injections of ethanol was used to model alcohol dependence in adult and adolescent rats, and post-withdrawal anxiety behaviors were measured using light-dark box testing. Furthermore, corticosterone (CORT) release during treatment and after withdrawal was measured by collecting fecal and plasma samples from adults and adolescents. It was found that adults, but not adolescents, exhibit significant anxiety-like behavior following chronic ethanol withdrawal. Additionally, while the process of chronic ethanol treatment elicits an increase in day-by-day CORT release in both adults and adolescents, significantly sustained levels of CORT were not observed during withdrawal for either age group. Moreover, it was found that adults experience a longer-lasting CORT increase during chronic treatment, suggesting a larger and more robust period of dysfunction in the HPA axis for older consumers of alcohol. These results highlight CORT and glucocorticoids in general as a potential therapeutic target for treatment for alcoholism, especially that which has an onset during adolescence.

Metabolism Regulates the Fate and Function of T Lymphocytes

Proper balancing of the activities of metabolic pathways to meet the challenge of providing necessary products for biosynthetic and energy demands of the cell is a key requirement for maintaining cell viability and allowing for cell proliferation. Cell metabolism has been found to play a crucial role in numerous cell settings, including in the cells of the immune system, where a successful immune response requires rapid proliferation and successful clearance of dangerous pathogens followed by resolution of the immune response. Additionally, it is now well known that cell metabolism is markedly altered from normal cells in the setting of cancer, where tumor cells rapidly and persistently proliferate. In both settings, alterations to the metabolic profile of the cells play important roles in promoting cell proliferation and survival.

It has long been known that many types of tumor cells and actively proliferating immune cells adopt a metabolic phenotype of aerobic glycolysis, whereby the cell, even under normoxic conditions, imports large amounts of glucose and fluxes it through the glycolytic pathway and produces lactate. However, the metabolic programs utilized by various immune cell subsets have only recently begun to be explored in detail, and the metabolic features and pathways influencing cell metabolism in tumor cells in vivo have not been studied in detail. The work presented here examines the role of metabolism in regulating the function of an important subset of the immune system, the regulatory T cell (Treg) and the role and regulation of metabolism in the context of malignant T cell acute lymphoblastic leukemia (T-ALL). We show that Treg cells, in order to properly function to suppress auto-inflammatory disease, adopt a metabolic program that is characterized by oxidative metabolism and active suppression of anabolic signaling and metabolic pathways. We found that the transcription factor FoxP3, which is highly expressed in Treg cells, drives this phenotype. Perturbing the metabolic phenotype of Treg cells by enforcing increased glycolysis or driving proliferation and anabolic signaling through inflammatory signaling pathways results in a reduction in suppressive function of Tregs.

In our studies focused on the metabolism of T-ALL, we observed that while T-ALL cells use and require aerobic glycolysis, the glycolytic metabolism of T-ALL is restrained compared to that of an antigen activated T cell. The metabolism of T-ALL is instead balanced, with mitochondrial metabolism also being increased. We observed that the pro-anabolic growth mTORC1 signaling pathway was limited in primary T-ALL cells as a result of AMPK pathway activity. AMPK pathway signaling was elevated as a result of oncogene induced metabolic stress. AMPK played a key role in the regulation of T-ALL cell metabolism, as genetic deletion of AMPK in an in vivo murine model of T-ALL resulted in increased glycolysis and anabolic metabolism, yet paradoxically increased cell death and increased mouse survival time. AMPK acts to promote mitochondrial oxidative metabolism in T-ALL through the regulation of Complex I activity, and loss of AMPK reduced mitochondrial oxidative metabolism and resulted in increased metabolic stress. Confirming a role for mitochondrial metabolism in T-ALL, we observed that the direct pharmacological inhibition of Complex I also resulted in a rapid loss of T-ALL cell viability in vitro and in vivo. Taken together, this work establishes an important role for AMPK to both balance the metabolic pathways utilized by T-ALL to allow for cell proliferation and to also promote tumor cell viability by controlling metabolic stress.

Overall, this work demonstrates the importance of the proper coupling of metabolic pathway activity with the function needs of particular types of immune cells. We show that Treg cells, which mainly act to keep immune responses well regulated, adopt a metabolic program where glycolytic metabolism is actively repressed, while oxidative metabolism is promoted. In the setting of malignant T-ALL cells, metabolic activity is surprisingly balanced, with both glycolysis and mitochondrial oxidative metabolism being utilized. In both cases, altering the metabolic balance towards glycolytic metabolism results in negative outcomes for the cell, with decreased Treg functionality and increased metabolic stress in T-ALL. In both cases, this work has generated a new understanding of how metabolism couples to immune cell function, and may allow for selective targeting of immune cell subsets by the specific targeting of metabolic pathways.