Cancer is a leading cause of death worldwide and has propensity for rapid mutation,which constantly challenges our ability to treat aggressive forms. There is an unmet need for next generation cancer medicine capable of overcoming chemotherapeutic resistance in cancer. Ferroptosis is a form of regulated cell death that is caused by the iron dependent peroxidation of lipids1,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols3,4. Ferroptosis has been implicated in the cell death that underlies several degenerative conditions2, and induction of ferroptosis by inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death5. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines6, suggesting that additional factors govern resistance to ferroptosis. Here in this dissertation, we employ a synthetic lethal CRISPR/Cas9 screen, and we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q10 (CoQ), generating a lipophilic radical-trapping antioxidant that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumor xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a new ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutics. In addition, we screened more than 120,000 compounds using an in vitro FSP1 activity assay and identified 168 compounds that directly inhibit FSP1 oxidoreductase activity. We further characterized 19 FSP1 inhibitors, belonging to 7 structurally distinct groups, and find that these compounds exhibit synthetic lethality with loss of GPX4 in H460 lung cancer cells. The most potent of these FSP1 inhibitors, ferroptosis sensitizer 1 (FSEN1),

acts specifically through inhibition of FSP1 to sensitize cancer cells of different tissueorigins to ferroptosis induced by multiple compounds targeting the GSH-GPX4 pathway. Together, our results define several structurally unique FSP1 inhibitors that sensitize cancer cells to ferroptosis, providing tools for the study of FSP1 biology and enabling further exploration of the potential of FSP1 as a therapeutic target.

Orthodontic treatment impedes the oral hygiene of patients, leading to plaque accumulation and the development of white spot lesions (WSL), which are visible areas of demineralized enamel. Our study aims to investigate the efficacy of different commercially available fluoride varnishes to treat these white spot lesions after the completion of orthodontic treatment and evaluate their effect on the oral microbiome. The present pilot study used a split-mouth design, with each of the eight subjects receiving different treatment on the left and right sides of their mouth. Subjects were assigned to one of three groups using stratified permuted block randomization, with each group receiving two of the following three options: placebo varnish (petroleum jelly), 5% sodium fluoride varnish (Acclean, Henry Schein), and a resin-modified glass ionomer light-cured fluoride varnish (Vanish XT Extended Contact Varnish, 3M). There are four total visits for this study: T1 (baseline taken within one month of braces removal), T2 (day 30), T3 (day 90), and T4 (day 180). Several series of photographs were taken at each visit to assess their oral health and the appearance of the WSL. Stimulated saliva was collected, and the severity of the WSL was assessed using laser fluorescence (DIAGNOdent pen, Kavo), after which the varnishes were applied according to their group allocation.

Clinical photographs were assessed by a trained and blinded scorer using the Turesky-modified Quigley Hein Plaque Index (TQHPI) and the International Caries Detection and Assessment System (ICDAS). These index measurements, along with the DIAGNOdent scores, were used to assess the efficacy of the different interventions at remineralizing WSLs. Salivary loads of total bacteria, Candida albicans, and Streptococcus mutans were determined by plating saliva samples on agar plates containing Brain Heart Infusion (BHI), Sabouraud Dextrose (SAB), and Mitis Salivarius-Bacitracin (MSB) respectively.

Although the small sample size of this pilot study made it difficult to achieve statistical significance, several notable trends were identified. Average TQHPI levels stayed relatively stable, with modest decreases seen in the placebo and light-cured fluoride varnish (LCFV) groups. DIAGNOdent scores decreased in all groups, with the 5% varnish group showing a significant decrease from T1-T3 and the LCFV showing the greatest percent change from T1-T4. ICDAS scores increased in the 5% and placebo groups and decreased in the LCFV group. CFUs representing total bacterial load, S. mutans, and C. albicans all decreased by an average of 60.9 – 99.9 percent, showing decreased oral colonization in the 6 months after completion of orthodontic treatment. These preliminary trends need to be verified in future studies that include more subjects to reach statistical significance.

A central goal of genetics is to define the phenotypic consequences of genetic perturbations. Single-cell CRISPR screens such as Perturb-seq (pooled single-cell RNA-sequencing CRISPR screens) represent an emerging tool to systematically construct genotype-phenotype maps by pairing high-dimensional genetic perturbations with rich phenotypic readouts in single cells. However, to date, these screens have been deployed at a limited scale.

The first chapter of this thesis addresses a major technological hinderance to the scalable application of Perturb-seq: reliance on indirect indexing of single-guide RNAs (sgRNAs). I present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. I then demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, I also show that targeting individual genes with multiple sgRNAs per cell improves the efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for large-scale single-cell screens. Last, I show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.

The second chapter builds on the first chapter by applying direct capture Perturb-seq and multiplexed CRISPR interference (CRISPRi) to perform the first genome-scale Perturb-seq screens. From these data, I yield a blueprint for the construction and analysis of rich genotype-phenotype maps. I show that genes can be clustered by transcriptional phenotypes across many essential cellular processes and reveal new roles for poorly characterized genes in ribosome biogenesis, transcription, and respiration. Beyond clustering genes, these data enable in-depth dissection of the functional consequences of genetic perturbations on a remarkable array of complex, composite phenotypes—including RNA processing, differentiation, and chromosomal instability. Leveraging this ability, I comprehensively identify genetic drivers and consequences of aneuploidy, and I uncover unanticipated perturbation-specific regulation of the mitochondrial genome. This thesis establishes Perturb-seq as a scalable tool for the principled exploration of multidimensional cellular behaviors, gene function, and regulatory networks.

As connected devices play an ever-growing role in our society, there is a subsequent need for advances in multi-user communication systems. In a network, senders and receivers are connected via a series of intermediate users who share information represented as sequences of bits or elements of some other finite alphabet. By allowing users to transmit functions of their inputs, as opposed to simply relaying received data, the information throughput of a network can be increased. Network codes in which these functions are linear are sub-optimal in general but are of practical interest due to their mathematical tractability and low implementation complexity. The study of linear network coding has primarily been limited to finite field alphabets. In this work, we consider linear network codes over more general algebraically-structured alphabets, namely finite rings. We contrast linear network codes over finite fields, commutative rings, and non-commutative rings, and we discuss cases where non-linear codes attain higher information rates than even very general linear codes. Our results show that finite fields are, in some sense, the best ring alphabets for linear network coding, but in certain instances, it may be advantageous to use linear coding over some other ring alphabet of the same size. Specifically, we prove results related to:

(i) network solvability: whether or not a network’s receivers can obtain their desired information using codes over a given alphabet. We characterize the commutative rings for which there exists a network that is linearly solvable over the ring but not over any other commutative ring of the same size. We show that these rings are, in some sense, the best commutative rings of a given size for linear network coding. We then present an infinite class of networks that are linearly solvable over certain non-commutative rings but not over any commutative rings. We also prove that vector linear codes over finite fields minimize the alphabet size needed for linear solvability, which is desirable from an implementation complexity standpoint.

(ii) network capacity: how much information per channel use can be sent to the network’s receivers in the limit of large block sizes for transmission. We show that the linear coding capacity of a given network cannot be increased by looking beyond finite fields to more general rings.

This dissertation is a historical investigation of the urban politics and mythic spaces produced in southeastern Nigeria, particularly in the port city of Old Calabar. As a city with a distinctive, and what some call “decentralized” and fragmented urban geography, this project draws connections between contemporary zoning strategies and these historical socio-spatial constructs, or “protozones”. These protozones, which were loosely tied together by secretive juridical systems and cultural codes, can be seen as a precursors and potential anticipatory diagrams for the splintering forms of urbanism emerging globally today. Borrowing the concept of “paradigmatic spaces”, this dissertation is organized around a series of spatial designations, or diagrams of spatial relationships that were exemplary during a given time period in Old Calabar’s urban history. This dissertation employs four spaces as representative of particular socio-historical relationships in Old Calabar—the compound, the masquerade, the offshore, and the zone. The historical imbrication of these four models combined with a flexible application of their spatio-temporal boundaries, provide a useful matrix for understanding the architectural and urban history of Old Calabar and challenging the novelty attributed to contemporary neoliberal free trade zones. The dissertation provides a productive case study for theorizing architecture and urbanism from the historicized periphery.

Legacy binaries need to continue functioning even when no source code has been preserved, to support the workflows of government and industry. The binaries often lack recent improvements in compiler design and software engineering practices, causing them to be slower and less secure than modern binaries. Binary rewriting seeks to patch, optimize, instrument, or harden binaries to bridge this gap, but existing practice is limited by the underlying static analysis. We created a framework, BinRec, to use dynamic analysis to lift binaries to LLVM IR then recompile them, which overcomes the limitations of static analysis.The protection of software against memory corruption exploits has a rich history, which this thesis both systematizes and extends. We present a study of the performance, precision, and security of control-flow integrity (CFI). Data-only attacks can bypass CFI, and so we present a defense against these attacks. The application of these hardening techniques to binaries deserves further study, and we discuss the extent to which security hardening can be applied to recompiled binaries.This dissertation presents building blocks for the securing of legacy binaries using dynamic analysis, which we hope will become a dominant paradigm in the secure software ecosystem of tomorrow.

Dye-sensitized solar cells (DSSCs) are a potentially low-cost alternative to silicon solar cells, however DSSCs have not been largely commercialized due to their poor solar to electric energy conversion efficiency, ~14%, compared to silicon, ~25%. One reason for their poor efficiency is that current record holding DSSCs employ dyes that absorb a less-than-ideal portion of the solar spectrum as dictated by the Shockley-Queisser Limit. To remedy this, infrared-absorbing osmium polypyridyl dyes were synthesized and their basic parameters characterized by ultraviolet-visible absorption spectroscopy, stepwise potential step spectroelectrochemistry, and cyclic voltammetry. The dyes appeared to have close-to-ideal optical pseudo-bandgaps and excited state energy levels, however, their performance in DSSCs employing the commonly used iodide-triodide redox shuttle was very poor. To better understand the behavior of these dyes in a working environment apparent diffusion coefficients of later self-exchange electron transfer between oxidized and reduced dyes bound to mesoporous titanium dioxide films was studied using several different techniques which produced different results. The different techniques were able to be harmonized by correct use of mathematical assumptions and measurement of non-ideal Nernstian behavior of bound dyes. Foot-of-the-wave analysis was modified for use on dyes bound to mesoporous films with substrate in solution. This revealed that several infrared absorbing osmium polypyridyl dyes performed very slow electron transfer from iodide with maximum second order electron transfer rate constants of 78 M-1 s-1, which could explain their poor performance in DSSCs using the iodide triiodide redox shuttle. Rates with 1,1’-dimethyl ferrocene were much faster, with rate constants as high as 74,000 M-1s-1. Nanosecond transient absorption spectroscopy and nanosecond transient microwave conductivity measurements were performed on DSSCs containing working benchmark dye N3 and infrared-absorbing osmium polypyridyl dyes. Osmium polypyidyl dyes were observed to successfully inject electrons into the TiO2 film with electron in TiO2 to oxidized dye recombination rates comparable to N3. Oxidized N3 rapidly regenerated in the presence of iodide, resulting in charge separated states that lasted for almost a second, but the infrared-absorbing osmium dyed did not. Several catalysts were fabricated to speed up iodide oxidation, but none worked in a solar cell.

Techniques to analyze and sort single cells based on complex phenotypes such as secreted products have the potential to transform our understanding of cellular biology as well as accelerate the development of next generation cell and antibody therapies. Microfluidic techniques have emerged over the last two decades that enable such assays by creating small compartments that can isolate individual cells. Despite these technical advances, adoption of this technology has been slow due to the complexity of the approaches and the requirement of specialty instruments. To address this issue, we developed a microparticle based approach to create uniform sub-nanoliter water in oil compartments using only standard lab equipment. We refer to these droplets formed with microparticles, dropicles.

In this dissertation I will provide an overview of various strategies used to create microscale compartments, specifically to perform single-cell secretion assays. I will then discuss in detail an approach to fabricate and use 3D structured microparticles to perform single-cell secretion assays massively in parallel with standard lab equipment. By making this microparticle approach compatible with existing high-throughput flow cytometers we are able to analyze and sort of over 100,000 single cells based on their secreted products. Lastly to create a truly democratized platform there is a need to be able to manufacture these particles in a scalable manner. In these last chapters I will discuss approaches to fabricate microparticles with different morphologies in high-throughput.