This dissertation, by Thomas Barton Suter, discusses enhancer function driving cellular senescence, DNA damage repair, differentiation, and nuclear organization. Enhancers are a major regulatory feature of epigenomic regulation, and play a diverse array of functions. In the first chapter, I discuss the background of cellular senescence and the epigenomic modifications that are known to play a role in this process, including marks associated with enhancer function. In the second chapter, I provide primary evidence of the key role enhancers play in driving the senescence phenotype. In the third chapter, I discuss the role that DNA damage and repair plays in nuclear receptor activity at enhancers. In the fourth chapter, I discuss the mechanisms by which regions in embryonic stem cells can be marked for later activation in future cell fates. In the fifth chapter, I present a study showing the role enhancers can play in nucleating nuclear organization.

Controlled and selective activation of immune cells is critical for ridding the body of disease-causing threats while limiting damage to healthy cells and maintaining normal tissue function. Though immune signaling can be initiated and mediated by soluble factors, many key immune responses require direct contact with a target to both evaluate the threat that it poses and initiate attack, if necessary. Macrophage phagocytosis is one such example of a contact-dependent effector function, in which macrophage Fc receptors (FcRs) directly engage with antigen-bound antibodies on a target cell. This drives FcR phosphorylation of intracellular ITAM motifs and initiates downstream signaling, ultimately leading to actin polymerization, membrane remodeling, and target engulfment. However, simultaneous to engagement of FcRs, inhibitory receptors—such as SIRPα—can bind their ligand on a target, shutting down phagocytosis through their intracellular ITIM motifs, despite the presence of activating signaling. While important for preventing auto-immune responses, these inhibitory checkpoints also reduce efficacy of tumor-targeting antibodies and other immunotherapies.

In the first Chapter of this dissertation, I probe how macrophages integrate contradictory signals from activating FcR-antibody and inhibitory SIRPα-CD47 interactions. Using reconstituted tumor cell-like target particles, I conclude that macrophage phagocytosis decisions are dictated by the ratio of activating ligand to inhibitory ligand over a range of absolute molecular densities. Lowering the antibody:CD47 reduces FcR phosphorylation due to inhibitory phosphatases recruited to CD47-bound SIRPα, thus preventing downstream phagocytic signaling from proceeding. I demonstrate that this ratiometric signaling is important in tumor cell phagocytosis and that it can be manipulated by blocking SIRPα engagement, demonstrating that this decision-making paradigm may be important for controlling macrophage phagocytosis in cancer immunotherapy.

In the next two Chapters, I discuss two strategies for probing and manipulating signaling at the macrophage-target interface. First, I probe the importance of ITAM and ITIM multiplicity on phagocytic receptors. By creating panels of synthetic receptors containing 0-4 ITAMs each, I conclude that increasing signaling motifs beyond two ITAMS does little to enhance phagocytosis and that additional ITAMs in series do not decrease inhibitory signaling. Second, I propose a novel therapeutic strategy that leverages spatial reorganization of the phagocytic interface to overcome inhibitory signaling. Using synthetic receptors, I show that tethering a protein with a short extracellular domain to one with a large extracellular domain can lead to exclusion of the short protein from a cell interface due to size-dependent segregation of the tall protein. I then propose a system for linking short SIRPα to tall CD45 to drive exclusion of SIRPα from a macrophage-target interface and reduce inhibitory signaling.

Next, I zoom out from the single macrophage-target interface to look more wholistically at macrophage signal integration. Here, I ask whether engagement with many inhibitory targets can impact phagocytosis of activating targets. I conclude that high levels of SIRPα-CD47 engagement does not significantly impact phagocytosis of other targets, highlighting how macrophage decisions are made locally.

Lastly, in the final Chapter of this dissertation, I zoom into the phagocytic interface to examine phosphorylation of ITAM motifs at a single receptor level. ITAMs contain two tyrosines that become phosphorylated and subsequently engage with Syk kinase, and the phosphorylation state of the ITAM alters Syk binding kinetics. By using variation in these binding kinetics to readout ITAM state, I present a strategy for spatial mapping of ITAM phosphorylation. With ITAM phosphopeptides, I demonstrate the ability to kinetically distinguish between ITAM phosphorylation states. I then show how this kinetic variation can be implemented for mapping phosphorylation of endogenous FcRs in macrophages.

Stendhal's conception of le naturel has long been defined in opposition to the theater: for the narrators and the authentic heroes and heroines of his fiction, there is nothing so vile as the histrionic personality who does not live life so much as perform it. But while most critics have interpreted the theatricality of Stendhal's fictional characters and that of his own authorial performance as an accidental lapse into the sort of hypocrisy he spends his oeuvre simultaneously denouncing, this dissertation argues that his naturalism was not an inadvertent "comédie de sincérité," as Valéry suggested, but rather a carefully constructed "Theater of Authenticity." This theater comprises three performances of authenticity: the social, the private, and the written. To each performance corresponds one part of the Stendhalian self, which simultaneously inhabits three roles: actor, spectator, and narrator. Before these performances were staged in his novels and short stories, they were both rehearsed and intellectually scrutinized in Stendhal's para-fictional writings--his "nonfiction," autobiographies, and private journals. I contend that these genres should be read as an atelier du roman--the literary space in which Stendhal worked out the règles du Je of the authentic self, and how these rules are most authentically transformed into literature. Unearthing this process of intellectualization dispels many Shibboleths of Stendhal's naturalism as well as of the concept of authenticity itself--namely, that both are predicated on the suspension of rational, analytical faculties in order to liberate the raw passion required to fuel spontaneity and improvisation. While authenticity has been understood to be a problem of modern subjectivity, I argue that it is first and foremost a crisis of literacy: a problem of self-consciousness in the relationship between subject and language, which may be traced back to the invention of the phonetic alphabet in Ancient Greece. This self-consciousness is a narcissism of the word rather than of the image, and the source of Stendhal's anxiety of authenticity is his fear of indulging in what he famously called "cette effroyable quantité de Je et de Moi." As such, Stendhal's para-fiction should also be read as an atelier du romancier, for they provide a narrative to the author's overcoming of this anxiety: after decoding the rules of authenticity, Beyle himself had to perform them in order to become Stendhal, the novelist.

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