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ABSTRACT OF THE DISSERTATIONRespiratory Virus Diagnostics with DNAzyme-Based Detection byDaniel Nay SchuderDoctor of Philosophy in Biological SciencesUniversity of California, Irvine, 2024Professor John Charles Chaput, ChairThe coronavirus disease 2019 (COVID-19) pandemic has caused over 7 million reported deaths to date, according to the WHO. This estimate is considered to be lower than the reality, as many cases were left unreported either due to a lack of diagnosis or attributed to other factors. In the wake of this devastating virus, the field of respiratory viral diagnostics grew at an unprecedented rate. Of a primary concern was the lack of available resources in order to keep up with testing demand, which led to a push for alternative testing methods that are more available in resource limited conditions, whether this is through use of alternative reagents, higher throughput, or increased usability at the point of care. Here we will describe the expansion of our lab’s COVID-19 diagnostic assay, known as REVEALR, to both a genotyping assay for COVID-19 variants of concern, as well as a respiratory virus diagnostic panel that detects not only COVID-19, but influenza and RSV as well. Chapter 1 reviews the current diagnostic options for respiratory viruses, with a focus on those used historically and those that recently gained emergency use authorization from the FDA for use during pandemic conditions. Immunoassays will be described alongside the current gold standard, qPCR. These are followed by Isothermal amplification strategies, focused on RPA, TMA, and LAMP, which are preferentially used for EUA approved techniques. We will then describe assays that build on these techniques with sequence specific detection, both CRISPR-based and REVEALR-based methods.In Chapter 2, we design REVEALR into a novel genotyping assay that detects single-base mismatches corresponding to each of the major SARS-CoV-2 strains found in the United States. Of 34 sequence-verified patient samples collected in early, mid, and late 2021 at the UCI Medical Center in Orange, California, REVEALR identified the correct variant [Wuhan-Hu-1, alpha (B.1.1.7), gamma (P.1), epsilon (B.1.427/9), delta (B.1.617.2), and omicron (B.1.1.529)] with 100% accuracy. The assay, which is programmable and amenable to multiplexing, offers an important new approach to personalized diagnostics.In Chapter 3, we demonstrate the expansion of our REVEALR diagnostic assay to a respiratory virus panel that detects influenza A (IAV) and B (IBV), SARS-CoV-2 (CoV2), and respiratory syncytial virus (RSV). The assay has a limit of detection of ≤10 aM IVT, and was tested against 39 clinical samples, showing perfect detection of all available samples including IAV, CoV, and RSV, with 0 false positives. This assay is amenable to multiplexing, as well as a lateral flow readout, which provides a promising and adaptable form of viral diagnostic to supplement diagnostic efforts in resource limited conditions.

Traditional gene regulation models focus on protein-coding genes within the paradigm of DNA → mRNA → protein. However, recent insights have shifted this perspective, highlighting the crucial regulatory roles of noncoding regions of the genome. Protein-coding regions constitute only about 1.5% of the genome, while many noncoding RNAs (ncRNAs) are transcribed from other regulatory elements and play significant roles in regulating complex biological processes such as development, differentiation, and metabolism. This emerging understanding underscores ncRNAs as vital molecular players, necessitating advanced tools to study their dynamic expression and activity.The first three chapters of this thesis present recent advancements in metabolic labeling techniques to profile RNA expression dynamics to help elucidate novel RNA activity and dynamics. Metabolic labeling involves incorporating chemically modified nucleosides into nascent RNA, enabling detailed tracking of RNA synthesis, decay, and transient expression through pulse-chase experiments. This addresses the limitations of bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) methods, which are limited in their abilities to accurately capture RNA dynamics and cell-type-specific gene expression. The approaches outlined in this thesis provide cell-specificity to traditional metabolic labeling approaches to capture RNA activity and dynamics for a cell type of interest within complex cellular contexts. Chapter 4 explored the non-canonical regulatory roles of ncRNAs, focusing on the development of synthetic riboswitches to control mRNA translation. Riboswitches regulate gene expression through ligand-binding aptamer domains. The thesis details the creation of photoriboswitches using in vitro SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to evolve RNA aptamers that bind to photo-labile ligands. Since typical SELEX methodologies cannot efficiently ensure the selection of gene-regulating riboswitches, we sought to generate a more efficient regulatory riboswitch selection platform. Therefore, we took this evolved RNA pool after in vitro SELEX for a modified ribosome display pathway, called Capture the FLAG, to select functional mRNA translational regulators from our evolved RNA pool. Overall, this thesis advances our understanding of ncRNA functions and introduces novel methodologies for profiling and manipulating RNA dynamics. The integration of metabolic labeling and synthetic riboswitches provides powerful tools for studying RNA biology, enhancing our ability to investigate cellular processes and develop therapeutic strategies.

Alzheimer's disease (AD) is a multifactorial neurodegenerative condition characterized by heterogeneous molecular alterations across various brain cell types, posing significant challenges for the development of effective treatments. To address this complexity, we undertook two complementary projects aimed at advancing precision medicine approaches for AD, each building on the other to create a robust foundation for targeted therapies.The first project serves as a proof of concept for an innovative drug discovery strategy, utilizing a network correction approach grounded in direct human evidence and real-world data. By integrating diverse datasets, including single-cell human transcriptomics, drug perturbations, and electronic clinical records, we identified the combination of letrozole and irinotecan as potential therapeutics designed to correct gene expression alterations across multiple cell types implicated in AD. Rigorous validation in AD mouse models demonstrated that this combination therapy, targeting both neurons and glial cells, significantly ameliorated memory deficits and other AD-related pathologies, outperforming the single-drug treatments targeting either neurons or glial cells alone. The success of this project underscores the potential of cell-type-directed network-correcting therapy, demonstrating that targeting the transcriptomic landscape at a cell-type-specific level may offer a more efficacious approach to treating AD. Building on this foundation, the second project was designed to expand the knowledge foundation for precision medicine by comprehensively characterizing the molecular influences of major AD risk factors, such as age, apolipoprotein E4 (APOE4), and sex using AD mouse models. By analyzing single-nucleus RNA-sequencing (snRNA-seq) data from the hippocampus of human APOE4 and APOE3 knock-in (KI) female and male mice across different ages, we identified significant variations in cell type abundance and gene expression patterns, particularly driven by sex differences and the interplay between age and APOE genotype. This detailed molecular profiling not only enriches our understanding of the disease but also provides a valuable dataset for future applications of the network correction method. Specifically, it enables the mapping of distinct risk profiles and the identification of tailored therapeutic interventions for individuals based on their unique transcriptomic signatures.Together, these two projects are highly complementary: the first project validates the efficacy of network correction therapy, while the second project supplies the essential molecular data needed to understand disease heterogeneity, enabling the application of this therapeutic approach in a precision medicine framework. By integrating these insights, we can more accurately tailor treatments to individuals with distinct risk profiles, thereby advancing the development of personalized therapies for AD.

The retinal pigment epithelium (RPE) is the primary site of injury in age-related macular degeneration (AMD), the leading cause of permanent central vision loss in the elderly. Our work seeks to understand the mechanisms driving RPE atrophy to identify novel therapeutic targets for AMD. Using super-resolution and high-speed live imaging, along with a mouse model of disease and AMD human donor tissue, this thesis examines how mitochondrial integrity regulates cellular pathways involved in macular degeneration.Cholesterol regulates essential cellular processes, but its dysregulation in the RPE can lead to disease. Our lab has demonstrated that complement and cholesterol pathways, which are major genetic and biological risk pathways for AMD, collaborate to damage RPE mitochondria. We demonstrated how allelic variants of apolipoprotein E, a cholesterol transporter implicated in AMD, regulate cholesterol homeostasis in the RPE. The AMD-risk ApoE2 accumulates cholesterol, leading to trafficking defects and mitochondrial injury, whereas the AMD-protective ApoE4 efficiently transports cholesterol and maintains mitochondrial health. ApoE and cholesterol are primary components of drusen, a hallmark of AMD. We identified a novel mechanism linking RPE mitochondrial injury with drusen biogenesis: fragmented mitochondria drive redox state-related liquid-liquid phase separation (LLPS) of ApoE2, the AMD risk isoform.This thesis also explores how mitochondrial integrity is regulated and how these processes are disrupted in AMD. We report excess oxidative stress and fragmented mitochondria in models of macular degeneration, suggesting a loss of mitochondrial quality control (MQC). Mitochondria undergo repeated cycles of fusion and fission, as well as degradation through mitophagy, to maintain their integrity. However, how the MQC machinery activates to induce mitochondrial fragmentation in diseased RPE remains unclear. Here, we identified three potential mechanisms driving mitochondrial fragmentation in diseased RPE: 1) defects in mitochondrial fusion proteins: mitofusin 2 (MFN2) and optic atrophy 1 (OPA1); 2) increased expression of a mitochondrial fission regulator, mitochondrial fission process 1 (MTFP1); 3) phase separation of a mitochondrial dynamics regulator, mitochondrial rho GTPase 1 (Miro1). This thesis further investigates mechanisms regulating drusen deposition in the RPE. Ceramide accumulation impairs intracellular trafficking, leading to endosomal abnormalities that activate exocytosis machinery. We observed an upregulation of proteins involved in different aspects of exocytosis, including Rab11, Rab27a, and the endosomal sorting protein HRS.Overall, these studies highlight the crucial role of mitochondrial health in RPE function and retinal health. Our research reveals that targeting upstream pathologies like excess cholesterol and ceramide to protect RPE mitochondria can prevent drusen formation and RPE atrophy in models of macular degeneration.

Cell-based assays are crucial for identifying lead molecules suitable for clinical advancement by providing a more physiologically relevant model. However, the field faces significant challenges due to the lack of robust screening technologies and efficient compound library generation, resulting in high attrition rates. In this dissertation, I discuss the development of two novel cell-based platforms aimed at enhancing the effectiveness of early-phase drug screening and accelerating the delivery of promising small molecules.Chapter 2 focuses on the development and application of a targeted protein degradation system using an engineered variant of the DNA-repair protein O6-alkylguanine-DNA-alkyltransferase (hAGT) and novel benzylguanine (BG) substrates, demonstrating versatility across proteins in different cellular localizations. Chapter 3 refines this system, enhancing degradation efficiency and exploring practical chemistry without requiring specialized equipment. Chapter 4 introduces a 3D cellular screening platform that integrates DNA-encoded libraries (DELs) with intracellular assays, showcasing its potential to transform early drug discovery through efficient, scalable interrogation of complex intracellular pathways with novel chemical matter.All together, this dissertation explores advancements in cell-based assays through targeted protein degradation and DNA-encoded library technologies, improving our ability to manipulate and understand cellular functions at a molecular level, thus enhancing the scope of targets and tractability of small molecules identified in early drug discovery efforts.

Atherosclerotic Cardiovascular Disease (ASCVD) remains the leading cause of death in diabetes mellitus (DM) adult patients. I investigated the current status of ASCVD and DM risk factor control for DM adult patients in the UCI health system, stratified by sex, race/ethnicity, and ASCVD status, A total of 34,207 DM adult patients were identified in the UCI electronic health records (EHR) through ICD-10 code during the timeframe of January 1st, 2022 – June 30th, 2023. Of these patients, within a year after their last DM diagnosis, while 91.3% received a blood pressure (BP) measurement, only 55.3% received a hemoglobin A1c (HbA1c) measurement and less than 50% received any kind of lipid profile measurement (total cholesterol, HDL-C, LDL-C, and triglycerides). Only 56.5% of patients were on any kind of DM drugs, with just 34.1% on metformin, even lower (29.6%) for patients with ASCVD. Only 15.6 % and 10.7% were on newer DM therapies of SGLT2i and GLP1-RA drugs, respectively. Only 46.6% were on statins, and non-statin use was infrequent (

Sickle cell disease (SCD) is the most common inherited disease, characterized by severe organ damage, often life-long pain, and a shortened lifespan. A hallmark of SCD is vaso-occlussive crisis (VOC), the blockage of small blood vessels by rigid and sticky sickled red blood cells which results in intense and unpredictable episodes of acute pain requiring hospitalization, reduced survival, and treatment with high-dose opioids. Cannabidiol (CBD) is one of the major non psychotropic constituents of cannabis and is generally considered safe and non-toxic. CBD has recently been approved by the Food and Drug Administration (FDA) for the treatment of certain types of epilepsy. Preclinical studies have shown CBD may have many benefits including for pain, inflammation, neuroinflammation, oxidative stress, ischemia/reperfusion injury, and asthma. All of these conditions occur in SCD, and so we considered using CBD for the treatment of SCD to support FDA-led translational studies for clinical translation.In this dissertation, I show the effects of CBD treatment in a humanized HbSS-BERK mouse model of SCD, which recapitulates the pathobiology and characteristic features of pain observed in persons with SCD. We found that treating both male and female sickle mice with CBD led to significant reduction in chronic pain-like behaviors, oxidative stress, and inflammation, which was sustained for more than a week after discontinuation of CBD suggesting a disease-modifying effect in the treatment of SCD. Pre-treatment with CBD for two-weeks also prevented acute hyperalgesia from occurring in sickle mice following the incitement of hypoxia/reoxygenation to simulate VOCs. CBD decreased IL-5 in both spinal cords and skin releasate, suggesting that CBD’s anti-hyperalgesic action in SCD acute pain may be due to attenuation of pro-nociceptive and/or anti-inflammatory activity in the periphery and the central nervous system (CNS). Furthermore, long-term CBD use did not result in impairment of motor coordination or negatively affect memory in sickle or non-sickle control mice, providing further evidence that long term CBD use may be without major side effects. While more investigation is required, our data provide support for clinical trials to evaluate the effect of CBD for the treatment of pain in SCD and other painful conditions such as cancer.

The Human Genome Project ultimately aimed to translate DNA sequence into drugs. With the draft in hand, the Molecular Libraries Program set out to prosecute all genome-encoded proteins for drug discovery with automated high-throughput screening (HTS). This ambitious vision remains unfulfilled, even while innovations in sequencing technology have fully democratized access to genome-scale sequencing. Why? While the central dogma of biology allows us to chart the entirety of cellular metabolism through sequencing, there is no direct coding for chemistry. The rules of base pairing that relate DNA gene to RNA transcript and amino acid sequence do not exist for relating small molecule structure with macromolecular binding partner and subsequently cellular function. Obtaining such relationships genome-wide is unapproachable via state-of-the-art HTS, akin to attempting genome-wide association studies using turn-of-the-millennium Sanger DNA sequencing. Our laboratory has been engaged in a multi-pronged technology development campaign to revolutionize molecular screening through miniaturization in pursuit of genome-scale drug discovery capabilities. We employed DNA-encoded library (DEL) synthesis principles in the development of solid-phase DELs prepared on microscopic beads, each harboring 100 fmol of a single library member and a DNA tag whose sequence describes the structure of the library member. Loading these DEL beads into 100-pL microfluidic droplets followed by on-line photocleavage, incubation, fluorescence-activated droplet sorting, and DNA sequencing of the sorted DEL beads reveals the chemical structures of bioactive compounds. This scalable library synthesis and screening platform has proven useful in several proof-of-concept projects involving current clinical targets. Moving forward, we face the problem of druggability and proteome-scale assay development. Developing biochemical or cellular assays for all genome-encoded targets is not scalable and likely impossible as most proteins have ill-defined or unknown activity and may not function outside of their native context.In this dissertation I describe the utility of selective translation modulation as a modality that circumvents canonical druggability constraints while also raising the prospect of in vitro transcription translation (IVTT) as a universal biochemical assay for screening. In Chapter 2, I present the development of an IVTT activity assay by fusing a GFP reporter to various gene sequences that are known to be selectively inhibited by compound PF846. Wheat germ IVTT was miniaturized to microfluidic droplets and demonstrated to exhibit excellent assay quality both in response to positive control PF846 and beads displaying a photocleavable broad-spectrum translation inhibitor, hygromycin B. Chapter 3 describes the screening of a 5,348-member DEL of custom translation inhibitor analogs. Screening a proof-of-concept PCSK9-GFP reporter yielded several hits that selectively inhibited PCSK9-GFP IVTT. Chapter 4 details the screening of an unbiased 55,296-member DEL to discover novel translation inhibitor chemotypes. These studies set the stage for scaling microfluidic IVTT DEL screening to the human proteome, irrespective of mature protein function, to fulfill the Genome Project's vision of proteome-wide control over cellular pharmacology.

Over the past five decades, there has been a significant increase in the discovery and applications of nucleic acids. Much has expanded since RNA was first postulated to act as an intermediary between DNA and proteins. The rapid discovery of RNAs many roles and functions has allowed researchers to interrogate and uncover new technologies and approaches to not only create therapeutic intervention for multiple disease prognoses, but also expand the repertoire for synthetic and chemical biology pursuits. The inherent complexity of nucleic acids is evolving as more information is uncovered, which in turn allows for the creation of novel approaches to answer fundamental questions. The work presented in this thesis focuses on the discovery, evolution, and development of nucleic acids for multiple applications. First, I discuss the in vitro selection and evolution of synthetic photoriboswitches that bind to a photoactive molecular switch (e.g., amino trans methoxyazobenzene) to act as an optogenetic tool for regulating mRNA translation. Second, I present the development of a novel high-throughput platform that elucidates and demarcates the functional RNA domains from heterogonous in vitro selected populations of aptamers and riboswitches. Third, I discuss the in vitro selection and discovery of human cerebral spinal fluid (CSF) aptamers with the future goal of developing an aptamer-based, point of care detection device. Lastly, I present the development of a ribosome display platform, coined capture the flag, to serve as a translation assay to select for the discovery of translational riboswitches and photoriboswitches.

DEL technology has gained popularity as an adjunct to high-throughput screening techniques, offering expanded capabilities for investigating novel chemical entities. Typically, DELs comprise mixtures of DNA-small molecule conjugates, in which the DNA tag reflects the synthesis history of the combinatorial library member. These DELs, termed "on-DNA" DELs, undergo screening using affinity selection, and the DNA is sequenced to decode compounds which bind to specific protein targets. With modern DNA sequencing technologies, DEL can explore unique binding interactions in a single experiment, which has led to the discovery of ligands for various targets and the identification of numerous clinical candidates. More recently, the use of DELs in drug discovery has also spurred computational intervention, as the inherent properties of combinatorial chemistry yield highly structured and modular datasets ripe for informatics and machine learning-based analyses.We have previously pioneered solid-phase one-bead-one-compound (OBOC) DEL technology, introducing an innovative approach to DEL synthesis that unlocks new avenues for screening. In this method, a DEL library member is polyvalently coupled to a bead via a photolabile bond, alongside covalently bound DNA-encoding tags. Upon UV-exposure, the library member is released into the surrounding solution where it may engage with and modulate the activity of a target of interest. We have previously demonstrated the efficacy of activity-based OBOC-DEL "off-DNA" screening for novel enzyme inhibitors, in competition-based fluorescence polarization screening, and for the prediction of compound potency.In this dissertation, I build on this prior work and present advances in DEL library design and synthesis principles alongside the development of assays that enable the use of DEL in in vitro systems. Chapter 1 contains a detailed protocol summarizing improved synthesis and quality control procedures for OBOC-DELs, accessible by both academic and industry groups. Chapter 2 presents design considerations for a diversity-oriented DEL, proposing principles that increase the compatibility of DEL members for lead optimization processes. Chapter 3 details the development of a miniaturized in vitro transcription and translation assay and the design of a bespoke scaffold-oriented DEL, culminating in the discovery of novel translation inhibitors. Lastly, Chapter 4 discloses the development of a cell-based phenotypic screening platform for OBOC-DELs, and reports preliminary screening results from a library inspired by agonists of the immunomodulatory cGAS-STING pathway.

Nature challenges healthy mammals with constant risk of infection by a wide variety of pathogens and with degradation of healthy tissue and control systems. I have been interested in drug delivery and improved delivery of therapeutics primarily to attack pathogens but with incidental value in assessing vital functions of a healthy mammal. My recent work uses protein design to approach such problems. Delivering biomacromolecules remains important both for therapeutics and in discerning and shaping functions of cells.My first thesis project focused on designing a better glomerular filtration rate (GFR) marker to facilitate assessment of renal function and seeking a marker reflecting water distribution in the body, which is relevant to distribution of highly soluble drugs [1]. Tritiated polyethylene glycol (PEG) was known to clear the kidneys effectively and correlated well with a GFR standard assay. A better radioactive label for PEG would allow for easy detection, including imaging. At the time of the project, only one biological conjugation with PEG was reported.Attaching an iodinatable moiety to polyethylene glycol (PEG) polymers of different sizes enabled tracking the compound using radioactive iodine. I made a series of related compounds and studied the pharmacokinetics (PK) and pharmacodynamics (PD) of these in rodents and ultimately in a dog. Using relatively long PEG polymers of molecular weight (MW) 5,000 to 6,000 daltons, the PEG dominated the behavior of the compounds, clearing rapidly through the kidneys. With shorter PEG polymers, the chemistry of the iodinatable group was more significant and the compounds were more likely to clear through the bile, to a degree making them unsuitable for a GFR marker but possibly useful to study liver function.Chapter 1, the published manuscript from my first thesis project, is cited in 20 scientific publications and 51 issued US patents. Variations on the design principles of my project have been used widely in the pharmaceutical industry.During a break in my PhD studies, I improved the formulation of a human Phase-2-ready antifungal drug and designed and organized extensive testing in mice and dogs to show that a sustained-release formulation would overcome PK limitations and made the drug much more potent. This work is discussed briefly in Chapter 2.My second thesis project studied brilacidin activity against 40 fungal isolates from 20 different species, showing useful activity against several important human pathogens [2]. The human and many other innate immune systems includes a variety of peptides known as defensins that weaken or kill a variety of pathogens, including bacteria, fungi, and viruses. Brilacidin is a synthetic defensin-mimic, designed to exhibit the physicochemical properties of defensins as a class. Brilacidin is in human Phase 2 trials. Despite its potential, Brilacidin's efficacy against fungi had not been comprehensively explored until my studies, which showcased its viability as a therapeutic agent against challenging-to-treat fungal infections, thereby offering a beacon of hope for future clinical interventions.Chapter 3, the published manuscript based on this second thesis project, has recently been submitted for review, available online in Preprints.

This dissertation presents a multifaceted study exploring critical aspects of drug development, focusing on the transporters on Blood-Brain Barrier (BBB) and a novel approach for Non-Alcoholic Fatty Liver Disease/Steatohepatitis (NAFLD/NASH).The first segment of this research offers an in-depth analysis of the BBB transporters, focusing on age-related changes in protein expression and the functional impact of polymorphisms. The Blood-Brain Barrier (BBB) serves as a selective barrier for a variety of small molecules, including chemical carcinogens, environmental toxins, and therapeutic drugs. This barrier is constructed from brain capillary endothelial cells, pericytes, and astrocytic end-feet, working in unison to protect neurons and maintain brain homeostasis throughout life. The selective nature of the BBB is attributed to the tight junctions within the capillaries, which prevent the free passage of small molecules, and also to an array of transporters that regulate the influx of essential nutrients and the exclusion of many xenobiotics. The goal of this part of the dissertation is to elucidate the effect of aging from neonates to elderly on the human BBB, with an emphasis on transporters. A secondary goal is to examine in detail the determinants of function of a key transporter in the human BBB, ATP-binding cassette transporter, ABCG2 (BCRP). This part of the dissertation begins with an overview of the current understanding of elements that influence the operation of the BBB, particularly transporters. It starts with a review of the BBB's structural components and their joint function in sustaining barrier integrity. It then summarizes how the BBB controls the movement of nutrients and medicines into the brain through various transport methods. The discussion includes the development of the BBB, how it changes as we age, and how diseases may affect it, emphasizing the dynamics of BBB and the challenges it creates for creating brain-targeted drugs. The chapter then shifts to how genetic variants in transporters influence BBB function and drug disposition, focusing particularly on the prominently expressed ATP-binding cassette transporters ABCB1 and ABCG2. After the overview, this dissertation provides a rich set of comprehensive analyses how age and genetic variations in transporters impact the functionality of BBB to advance our understanding of the multifaceted regulatory framework that controls BBB in physiological and pathological contexts. Major gaps in our understanding of the BBB, which are addressed by this dissertation research, are highlighted.Chapter 2 examines changes in the human BBB proteome throughout a human lifespan, noting the significant shifts in protein expression that influence barrier permeability and the transport of nutrients and drugs. It is acknowledged that the BBB matures after birth, adjusting its transport mechanisms to align with each stage of development, and later alters due to aging and neurodegenerative disorders. Yet, fully grasping these modifications in the human BBB is an ongoing challenge. This chapter introduces a comprehensive proteomic analysis of the evolution and senescence of proteins in brain microvessels (BMVs). Samples from healthy individuals across a wide age spectrum and Alzheimer’s disease patients were analyzed using LC-MS/MS. A plethora of proteins, including numerous SLC and ABC transporters, were identified. Network analysis of the BMV proteome suggested potential alterations in BBB permeability over time and pinpointed transporters crucial for nutrient supply and drug penetration that exhibit age-dependent expression patterns. This investigation sheds light on the dynamic regulation of BBB proteins, emphasizing how transporter variations with age can affect drug permeability. These findings are crucial for refining pharmacokinetic modeling and therapeutic approaches across different stages of life.The dissertation (Part A) then pivots to explore how genetic factors may alter the functionality of transporters, potentially causing variances in drug distribution within the brain. It focuses particularly on ABCG2, a transporter highly expressed at the BBB, noting that genetic variations leading to functional changes can result in differing drug responses. Utilizing deep mutational scanning (DMS), an innovative technique that combines next-generation sequencing (NGS) with functional outcomes of numerous variants, this study evaluated 12,724 variants of the ABCG2 gene. Our experimental setup was crafted to assess over ten thousand of missense, synonymous, and deletion variants of ABCG2 in a high-throughput manner. The abundance of ABCG2 was quantified, its surface expression was measured, and the functional effects of each variant were examined using the anti-cancer drug, mitoxantrone. The resulting detailed functional map, visualized through heatmaps and integrated with the structural data of ABCG2, helped identify crucial residues essential for ABCG2's function and poly-specificity. This study enhances our understanding of ABCG2 and lays the groundwork for future investigations into other ABC transporters. It underscores the value of DMS in dissecting the intricacies of pharmacogenetics and the mechanisms underlying drug resistance.In summary, this part of the dissertation presents a comprehensive examination of the factors important for the functionality of BBB, shedding new light on transporters. Importantly, we unveil the BBB's dynamic protein regulation across the human lifespan, demonstrating how age-related changes affect drug permeability through a detailed proteomic analysis. Furthermore, the dissertation explores how mutations influence transporter functionality, ABCG2 as an example for developing the platform. The innovative use of Deep Mutational Scanning (DMS) to assess thousands of ABCG2 variants provides a rich functional map, revealing key insights into the transporter's operation and offering a valuable resource for future pharmacogenetic and drug resistance research. Overall, these studies highlight the necessity of understanding the BBB's complex mechanisms to enhance drug delivery strategies and overcome barriers in treating neurological disorders including neurodegenerative diseases.The second part of the dissertation (Part B) shifts focus to the global health issue of NAFLD and its more severe form, NASH. It investigates Cis-Regulation Therapy (CRT) as a novel treatment approach, utilizing nuclease-deficient gene-editing technologies to modify gene regulatory elements for therapeutic ends. Approximately 30% of people worldwide are affected by NAFLD, and about 25% of these cases may advance to NASH. NASH represents a more serious stage of NAFLD, marked by liver inflammation and damage due to fat accumulation in the liver. About 25% of those with NAFLD progress to NASH, characterized by significant liver inflammation and damage due to fat accumulation. Currently, the pharmacological treatment options for NAFLD/NASH are severely limited. Our study investigates the potential of CRT as an innovative treatment strategy. In our research, we explored the effectiveness of CRT as a promising new treatment strategy. CRT employs nuclease-deficient gene-editing technologies, such as dead Cas9 (dCas9) combined with transcriptional modulators, to alter the activity of gene regulatory elements for therapeutic purposes. The goal of this part of the dissertation research specifically focuses on the nuclear receptor-like protein 1 (NURR1, NR4A2), a transcription factor critical in regulating inflammation which is a hallmark of NASH.This part of the dissertation initiates with an overview of existing treatment options for NAFLD/NASH, pinpointing their limitations and the urgent need for more effective interventions. It further explores contemporary strategies in drug and therapeutic development targeting NAFLD/NASH, with a particular emphasis on animal models. After the overview, we present our findings that activating Nurr1 through CRISPR activation (CRISPRa) offers a promising therapeutic strategy for NAFLD/NASH within FATZO mouse models. This technique has shown efficacy in improving glucose metabolism abnormalities and reducing the CCL2-CCR2 axis, a critical inflammatory pathway, both before and after the onset of the disease. Our findings introduce a promising new therapeutic avenue for NAFLD/NASH, highlighting the capability of Nurr1 activation to control and possibly reverse the disease's progression.In summary, this dissertation delivers a comprehensive analysis of the variables impacting the functionality of BBB transporters and presents a promising therapeutic approach for NAFLD/NASH. Through this research, we aim to pave new pathways for the advancement of treatments for neurological and hepatic disorders.

Addiction, including alcohol use disorder, is a maladaptive form of learning and memory. Initially, the effects of an addictive substance are rewarding, reinforcing the drug-seeking behavior. Over time, repeated exposure leads to changes in neuroanatomy, altering the brain’s circuitry and resulting in cycles of drug binge, withdrawal, and craving, undermining the individual’s health and functioning. This dissertation aims to examine the role of Rac1 in maladaptive learning and Prosapip1 in normal learning and memory. The first chapter examines a protein that has not previously been studied in mammals in the context of AUD, Rac1. In this chapter, we present evidence that Rac1 is activated in the DMS in response to repeated cycles of alcohol binge and withdrawal. This subregion-specific activation leads to phosphorylation of downstream proteins and promotes F-actin assembly, which then causes increased dendritic arborization and dendritic spine maturation. We also show that Rac1 in the DMS is involved in alcohol-associated goal-directed behavior, and therefore likely contributing to the progression of AUD. The second chapter investigates the physiological role of a protein previously associated with AUD, Prosapip1. We developed a Prosapip1 neuronal knockout mouse line to examine its mechanism of action in vivo. We present data to suggest that Prosapip1 is vital in regulating the PSD scaffold. Disruption of the PSD leads to a loss of LTP. Finally, these biomolecular changes result in a spatial learning and memory deficit, which is localized to the dorsal hippocampus. In summary, this dissertation contributes to the knowledge of molecular mechanisms controlling normal and maladaptive learning, and identifies potential targets for the treatment of AUD.

Cancer is a complex disease with high incidence and mortality rates. Conventional therapies, such as radiation or chemotherapy, often come with harmful side effects and have limited effectiveness against aggressive forms of cancer. Newer approaches, including immunotherapy and targeted therapy, have shown promise in improving therapeutic outcomes and sparing normal healthy cells from damage. While advanced sequencing technologies and research have uncovered cancer-driving molecular targets, specifically modulating those targets have remained challenging. Many proteins lack robust biochemical characterization or obvious binding pockets that traditional drugs target, rendering them “undruggable”. Chemoproteomics and fragment-based covalent ligand libraries have emerged as innovative tools for addressing these challenges, offering a more efficient and rational path to drug discovery. Furthermore, covalent drugs provide pharmacological advantages such as enhanced potency, selectivity, and prolonged duration of action.Dysregulated oxidative stress plays a major role in cancer pathogenesis, offering a unique vulnerability for intervention. Targeting glutamate-cysteine ligase (GCL) to inhibit the synthesis of the major antioxidant glutathione (GSH) may be a promising therapeutic strategy for certain cancer types that are particularly sensitive to oxidative stress. In this dissertation, I use fragment library screening and chemoproteomic approaches for identifying new covalent inhibitors against GCL. We have discovered an acrylamide-containing cysteine-reactive ligand, EN25, that covalently targets cysteine C114 on GCLM, the modifier subunit of GCL. We show that EN25 allosterically reduces GCL activity, lowers cellular GSH levels, and impairs cell viability in ARID1A-deficient cancer cells that are particularly sensitive to glutathione depletion. We also performed a small-scale SAR study and identified additional covalent ligands with moderately enhanced binding affinity in vitro. Our studies reveal a new ligandable site within GCLM that can be targeted to inhibit the GSH synthesis in vulnerable cancer cell types.

Rational drug design is a multimodal optimization problem where most of the control nodes are at least partially obscured. Fundamentally, rational drug design is predicated on a deep understanding of biological molecular recognition. Arguably, biological molecular recognition is an amalgamation of two principles. Chiefly, biological molecular recognition relies on a so-called metabolic code of logic. This code defines a logic for all the chemical moieties that biological systems use to achieve molecular recognition. At some level, biological interactions are iterations and combinations of only a small set of chemical interactions between a very exclusive set of chemical functional groups. Harnessing this underlying logic to rationally design drugs is the primary investigation of this thesis. This inquiry is supported by another old and well developed theory of biological interactions, that of pre-organization. Pre-organization supposes that biological systems that have evolved specific molecular recognition have done so by paying an energetic cost during folding to pre-organize an energetically unfavorable binding site that relaxes dramatically upon the desired complexation event. One promising approach to tackling this immense problem is by employing computational models of ligand-protein interactions using coarse physical models of the complexation event.The focus of this dissertation is to understand molecular recognition for a clinically relevant and widely generalizable model system, the membrane embedded mu opioid receptor, a 7-pass transmembrane protein receptor that plays a fundamental role in pain perception and analgesia. In this thesis, I employ molecular docking, a computational approach that assesses physical complementarity between a ligand and a protein target to screen humongous chemical space. I then experimentally characterize a number of new to the planet opioid small molecules, and even solve a structure of the most potent compound in complex with the human opiate receptor. In so doing, I not only uncover new to the planet opioid therapeutics, but also learn some fundamental new qualities regarding molecular recognition at the human mu opioid receptor.Also included in this dissertation is a commentary on the state of education in the biomedical enterprise. This PhD experience was unique because it happened amidst a racialized global pandemic and a period of extended social uncertainty and unrest. During this time, student leaders capitalized on the heightened societal focus on social justice. In the final chapter of this dissertation, I share my experiences in this domain and offer some of my personal insights for addressing historical injustices in the biomedical enterprise and making science more representative of our population and more efficacious in solving difficult problems in the domain of human health and wellness.

A table-top microdevice was used to produce flavopiridol-loaded ultrasmall particles for pulmonary delivery to reduce pulmonary fibrosis and inflammation. Microparticles were image with the help of scanning electron microscopy to visualize their structure. The Loading capacity and encapsulation efficiency of flavopiridol in the microparticles were determine using a UV-Visible spectrophotometer. Commercial microfluidic piezo devices with different orifice diameter were optimized to get the preferred inhalable particle morphology. Optimized microparticles were subject to aerodynamic performance testing and moisture content analysis. The stability of microparticles were perform for up to 24 weeks. A high-speed camera with a long working distance lens was mounted to visualize the droplets size formed by a microfluidic piezo device. Synthesis parameters were optimized to produce microparticles, whose morphology, size, physio-chemical properties, aerodynamic performance, moisture content and release profiles met the criteria for inhalation.

Solution state NMR was used to study two DNA-binding proteins. Chapter one covers the different NMR techniques used, including hydrogen exchange, relaxation, chemical shift analysis, and temperature coefficients. In addition, I also implement circular dichroism and protein sequence analysis to supplement the NMR studies. Chapter two covers the p53 project, in which we compare protein dynamics between WT and rescue mutants to understand the mechanism of stabilization. We measured and compared dynamics from NMR hydrogen exchange rates of backbone amides and find that both rescue mutants impose a global stabilizing effect that dampens their motions in the beta-sandwich compared to WT DBD. We also compared NMR relaxation results to obtain flexibility information in the ps to ns timescale regime. Chapter three covers the dynamics of the DinB homolog polymerase (Dbh). This polymerase belongs to the Y-family of translesion DNA polymerases that can synthesize using a damaged DNA template. Since Dbh comes from the thermophilic archaeon Sulfolobus acidocaldarius, it is capable of functioning in a wide range of temperatures. Here we use NMR and circular dichroism to understand how the structure and dynamics of Dbh are affected by temperature (2-65°C) and metal binding in solution. We measured hydrogen exchange protection factors, temperature coefficients, and chemical shift perturbation analysis with and without magnesium and manganese. HX reveals that both the thumb and finger domains are very dynamic relative to the palm and LF domains. These trends remain true at high temperatures with dynamics increasing as temperatures increase from 35 °C to 50 °C. Notably, NMR and CD spectra show that Dbh tertiary structure cold denatures beginning at 25 °C and increasing in denaturation as the temperature is lowered to 5 °C with little change observed by CD. Chemical shift perturbation analysis in the presence and absence of magnesium and manganese reveals three ion binding sites, even in the absence of DNA.

Pharmacokinetic models, i.e., the models describing distribution and elimination kinetics of drugs within the body, have become an integral part of any drug discovery and development process. Unfortunately, the difficulty of measuring drug concentration levels in-vivo has restricted the pharmacokinetic modeling field to working with sparse datasets. The recent development of continuous molecular monitoring platforms such as electrochemical aptamer-based (EAB) sensors presents an unprecedented opportunity to overcome this obstacle by providing fast in-vivo drug concentration measurements.In this work, I present our work on how such high-temporal resolution datasets can help us specify individualized pharmacokinetic models and the implications of having such high-precision models. We first developed a system identification-based approach to test physiological assumptions about drug transport kinetics that are almost universally accepted but rarely tested. Because the precision of the identification process is an important factor in testing these assumptions, we further developed a way to design experiments to maximize the information content of the measured data to improve the precision of model identification.The availability of individualized compartmental pharmacokinetic models also offers a chance to improve clinical practices. In particular, we first discuss how such models can improve the sample collection process by developing a way to estimate plasma concentration levels based on easier-to-measure subcutaneous interstitial fluid (ISF) measurements. These individualized models can also help in the treatment of a patient by enabling feedback-controlled drug delivery. Particularly, we design an adaptive feedback control mechanism to adjust the drug intake rate to keep the measured drug concentration levels at a targeted value to improve the therapeutic impact while avoiding the toxicity of the drug.

Trypanosoma brucei has been responsible for quite a few epidemics. It affects both humans and animals, and it is fatal is left untreated. Though drugs have been developed to treat T. brucei, the growing rate of resistances to these drugs sparks the need for further research into new compounds. To assist the drug discovery process, several analogs of carmaphycin B, a marine cyanobacterium extract, were screened against T. brucei after seeing positive results against Plasmodium falciparum, the parasite that causes with malaria. A structure-activity relationship was developed with the purpose of being able to optimize the production of drugs that can target the parasite’s proteasome (Tb20S) with minimal cytotoxicity.

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common known cause for Parkinson’s disease (PD) and are associated with several other diseases, including cancer and autoimmune disease. Excitingly, several strategies to modulate LRRK2 activity in the brain are currently being tested in clinical trials for PD. Yet, there remain many unanswered questions as to how LRRK2 mutations affect cellular homeostasis and ultimately cause disease. Increasing our understanding of the cellular biology surrounding LRRK2 function is certain to aid in the acceleration of drug discovery and development in PD. In Chapter 1, I broadly summarize the role of LRRK2 in PD and reported LRRK2 molecular and cellular functions. In particular, I describe the unanswered question of how PD-causing mutations in the kinase and GTPase domains of LRRK2 differentially affect observed LRRK2 kinase activity in cells. In Chapter 2, I present my published work detailing distinct cellular effects of PD-causing mutations in either the GTPase or kinase domain of LRRK2. Specifically, I found that GTPase-inactivating mutations strongly increase LRRK2 localization to endosomes upon inhibition of endosomal maturation. Under the same conditions, kinase-activating mutations only modestly affect LRRK2 localization and wild-type LRRK2 localization is minimally affected. I further demonstrate that the extent of LRRK2 endosomal localization is directly related to observed levels of LRRK2 substrate phosphorylation. Through this work, I provide a rationale for why PD-causing mutations across LRRK2 domains lead to differing levels of substrate phosphorylation in cells. Overall, my work highlights the importance of LRRK2’s GTPase domain for the protein’s cellular localization and activity and suggests that therapeutic strategies targeting LRRK2 GTPase function or localization may be beneficial for PD.

Xeno-nucleic acids (XNA) are artificial genetic polymers that have potential impact in synthetic biology, biotechnology, and molecular diagnostics or therapeutics. These polymers structurally mimic deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) composed of a base, sugar and acid moieties. XNAs, specifically vary in the sugar moiety, while retaining naturally occurring bases and acid moieties. Similar to DNA and RNA, specific XNAs (threose, hexitol, arabino, 2′-fluoro-arabino) are able to hybridize via a Watson-Crick base pairing and form heteroduplexes with DNA. Natural polymerases have demonstrated inert ability to recognize and incorporate XNAs across DNA template by primer extension. However, the rate of synthesis is inefficient and unable to generate oligonucleotide lengths intended for the discovery of aptamers. Thus, engineering natural polymerases is required to enhance activity and function. Laboratory-evolved polymerase have demonstrated the ability to synthesize with intended XNA, however, evaluations were not performed for substrate specificity and thermal stability. Upon evaluation, laboratory-evolved polymerases demonstrate an unbiased recognition for XNAs and reduction in thermal stability with an increased mutational load. Characterization of established evolved polymerases empower insight to specified regions to be further modified for enhancement. Development of methodology is required to further enhance evolved polymerases for increased function or to discover new functions of interest. A robust methodology for evolution requires molecular biology techniques to mutate the gene of interest and a screening platform to isolate variants of interest. With intensions to further engineer a threose nucleic acid (TNA) polymerase, a library was developed by combining homologous recombination and error-prone PCR. Upon library assembly and verifications, variants are expressed in E. coli and screen through droplet-based optical polymerase sorting by a fluorescent readout to separate active and inactive variants. After 10 rounds of directed evolution, an enhanced TNA polymerase 10-92 was discovered to be ~25 times more active than Kod-RSGA, along with enhanced ability to incorporate based modified tUTPs. To utilize an enhanced TNA polymerase for the discovery of TNA aptamers, a platform is required to increase the screening capabilities and reduce the time to discover functional aptamers. Hydrogel particles were developed as a solid support system to synthesize, isolate, and characterize TNA aptamers in a screening process that can be performed in 96 well format in a matter of days compared to months. By developing a general strategy to perform directed evolution on polymerases and a platform for screening TNA aptamers, these strategies are tools to empower the discovery of TNA aptamers for diagnostic and therapeutic applications.

To understand and treat Alzheimer’s disease (AD), we need to understand the structures of the Aβ peptide aggregates that cause neurodegeneration. In this dissertation, I address this need by applying triangular trimers derived from Aβ to create antibodies for immunohistochemical studies and as vaccines against AD. Chapter 1 outlines the exponential growth of people who will live with AD and current therapies to alleviate symptoms or slow down the progression of AD. Here, Chapter 2 demonstrates the application of a conformationally defined Aβ-derived peptide (the 4AT-L trimer) to better understand the composition of Aβ pathology in people who lived with Late-Onset Alzheimer’s disease (LOAD). Chapter 3 expands on the subject of current peptide vaccines and immunotherapies against AD by discussing the composition of each therapy, the current state of clinical trials, and the possible future of these therapies. Chapter 4 demonstrates how a structurally defined Aβ antigen may be useful in treating AD-like symptoms in mice. Chapter 5 shows preliminary work demonstrating the recognition of pathology in people who lived with Down Syndrome and Alzheimer’s disease (DSAD). Lastly, Chapter 6 discusses the conclusions collected from the previous chapters and lists future directions that can be pursued by junior members of the Nowick Group.This body of work is one of the earliest collections of studies in the Nowick Group which provides a biological and therapeutic application to structurally defined triangular trimers derived from Aβ. Additional studies exploring the biological and therapeutic application of trimers of Aβ and associated antibodies will continue to address the need to understand the structures of the Aβ peptide aggregates that cause neurodegeneration. These efforts will contribute to a better understanding of neurodegeneration and develop therapies to slow down the progression of AD.

The melanin-concentrating hormone (MCH) system, composed of the hypothalamic neuropeptide MCH and its receptor MCHR1, is a critical regulator of several functions, including energy homeostasis, food intake, sleep, stress, mood, aggression, reward, and cognition. The MCH system is expressed primarily in the lateral hypothalamus and zona incerta and projects throughout the central nervous system. MCHR1 is widely distributed in several brain regions, particularly in the frontal cortex, amygdala, nucleus accumbens, and hippocampus, suggesting that MCH may modulate social, emotional, and cognitive functions. MCHR1 is a G protein-coupled receptor that is located in the primary neuronal cilia.Primary cilia are small microtubule backbones, hair-like structures that protrude from the plasma membrane of almost every cell, including neurons. They serve as sensory organelles that detect and transduce extracellular signals, such as mechanical and chemical stimuli, into intracellular signals that regulate cell signaling pathways and gene expression. Ciliopathies are a group of inherited disorders caused by defects in cilia structure or function, and many of the genes implicated in these disorders have been linked to neurological deficits, including cognitive impairments. Despite the evidence suggesting that cilia dysfunction may play a role in psychiatric disorders, such as schizophrenia, autism spectrum disorder, bipolar disorder, and major depressive disorder, the specific mechanisms underlying this association remain poorly understood. Alongside, almost all brain cells have cilia that are made of microtubules that play critical roles in brain functions. They are essential for brain formation and maturation during neurodevelopmental stages, and in the adult brain, they act as signaling hubs that receive and transduce various signals, regulating cell-to-cell communications. Cilia are intricate and adaptable sub-cellular systems that work in a coordinated way to perform their structural and functional roles. These roles involve sensing environmental stimuli that follow circadian rhythms, which suggests that genes that encode the components of cilia might also have circadian patterns of expression.G-protein-coupled receptors (GPCRs) are crucial to the neurobiology of psychiatric disorders, as they mediate the effects of most neurotransmitters implicated in these disorders and are the primary targets of psychotropic drugs. However, their precise role in the development and progression of psychiatric disorders remains poorly understood. The MCH system is a critical player in several physiological and behavioral functions, and the MCHR1 receptor's distribution in neuronal primary cilia suggests that MCH may regulate these functions by modulating cellular signaling pathways. Further research is necessary to understand the exact mechanisms by which MCH exerts its action and how modulation of the MCH system could be utilized for therapeutic purposes. This thesis investigates the dysregulation of cilia genes in psychiatric disorders, with a focus on circadian patterns, age-related changes, and region-dependent functions. Additionally, the thesis examines the involvement of brain primary cilia in MCH signaling and its role in the manifestation of behavioral deficits related to social and cognitive impairments in animal models with time-dependent ciliary MCHR1 deletion. Finally, we begin to explore the potential of MCH fragment analogues as treatments for ciliopathies or psychiatric disorders. To begin we identified patterns of cilia gene dysregulation in psychiatric disorders by analyzing differentially expressed genes from publicly available databases. We found that a significant portion of brain-expressed cilia genes were differentially expressed in these disorders, indicating that cilia signaling pathways may be involved in their pathophysiology. Additionally, we revealed that genes encoding proteins of almost all sub-cilia structural and functional compartments were dysregulated in these disorders, suggesting that cilia dysfunctions may be involved in various aspects of disease pathology. We also found that genes encoding for certain cilia proteins were differentially expressed across multiple psychiatric disorders, indicating that cilia signaling may be a common pathway in their pathophysiology. Overall, this study represents the first step towards understanding the role that cilia components play in the pathophysiological processes of major psychiatric disorders. By shedding light on the role of cilia signaling in these disorders, this study may lead to the development of novel therapeutic targets for these disorders. Disruptions to the cilia-circadian rhythm connection have been linked to various diseases and disorders, such as obesity, diabetes, and sleep disorders, highlighting the crucial role of cilia in maintaining proper circadian rhythm and overall physiological function. By analyzing the gene expression atlas of primates using computational techniques, we found that approximately 73% of cilia transcripts showed circadian rhythmicity in at least one of the 22 brain regions studied. Furthermore, cilia transcriptomes in 12 brain regions were enriched with circadian oscillating transcripts compared to the rest of the transcriptome. Notably, cilia circadian transcripts shared between the basal ganglia nuclei and prefrontal cortex peaked in a sequential pattern similar to the order of activation of the basal ganglia-cortical circuitry, which is essential for movement coordination. These findings suggest that the spatiotemporal orchestration of cilia genes expression might play a critical role in the normal physiology of the basal ganglia-cortical circuit and motor control. It is unknown if MCH system activation in vivo causally regulates cilia length, which is highly dynamic in morphology and length. To investigate this, we used different experimental models and methodologies, including organotypic brain slice cultures from rat prefrontal cortex (PFC) and caudate-putamen (CPu), in vivo pharmacological approaches, germline and conditional genetic deletion of MCHR1 and MCH, optogenetic, and chemogenetic methods. Our results revealed that activation of the MCH system through MCHR1 agonism or optogenetic and chemogenetic excitation of MCH-neurons caused cilia shortening, while MCH signaling inactivation via MCHR1 antagonism or genetic manipulation resulted in cilia lengthening. Our findings indicate that the MCH system plays a significant role in ciliary signaling and highlight MCHR1 located at primary cilia as a potential therapeutic target for pathological conditions associated with abnormal primary cilia function and modification of its length. Next, we wanted to understand cilia’s role in higher-order brain functions as it remains largely unknown. Acting as a hub that senses and transduces environmental stimuli to generate appropriate cellular responses, cilia-rich brain structures, such as the striatum, receive and integrate various types of information to drive appropriate motor responses. In this study, we employed loxP/Cre technology to remove cilia from the dorsal striatum of male mice and observed the behavioral outcomes. Our results suggest a critical role for striatal cilia in the acquisition and brief storage of information, specifically in learning new motor skills, but not in the consolidation of long-term information or the maintenance of learned motor skills. Moreover, the deficits observed in the behavior of mice without striatal cilia were clustered around the clinical manifestations of neuro-psychiatric disorders that involve striatal functions and timing perception. Therefore, striatal cilia may act as regulators of the timing functions of the basal ganglia-cortical circuit by maintaining accurate timing perception. MCHR1's role in primary cilia is not yet fully understood, but has been implicated in regulating a range of physiological processes, such as appetite and energy balance, as well as behaviors related to reward, motivation, and mood. To better understand the role of ciliary MCHR1 in social and cognitive deficits, we utilized an inducible knockout model. Our results revealed that late deletion of ciliary MCHR1 does not significantly affect sociability but leads to an increase in hyperactivity and deficits in cognition and sensorimotor gating. On the other hand, early deletion of ciliary MCHR1 leads to deficits in both social and cognitive function, as well as sensorimotor gating deficits. Additionally, we quantified the amount of ciliary and non-ciliary MCHR1 that localizes to primary cilia to better understand the role they play in these deficits. Our findings suggest that the MCH system's disruption interferes with neurodevelopmental processes, which could contribute to the pathogenesis of schizophrenia. Lastly, we began to design MCH analogues with improved binding affinity for MCH receptors to potentially develop new therapies for these conditions. We used various in vitro binding techniques to analyze the affinity of the MCH analogues for MCH receptors. In the in vivo experiments, we injected MCH and MCH analogues intracerebroventricularly in mice to study their effects on feeding behavior, energy homeostasis, and cilia length. We discovered an MCH fragment analogue with a reduced number of amino acids and molecular weight that showed potential to bind in vivo. This MCH fragment analogue had a potency comparable to the full MCH peptide and caused cilia shortening in the adult mouse brain and was reversed when administered with an MCHR1 antagonist. We also found that when administered i.c.v similarly to MCH, the mice gained weight. When given simultaneously with an antagonist, it resulted in weight loss. This suggests that MCH fragment analogues could potentially be used as potential treatments for conditions associated with abnormal MCH signaling, such as ciliopathies or psychiatric disorders. In conclusion, our study provides new insights into the design of MCH analogues with improved binding affinity for MCH receptors. We believe that these findings will contribute to the development of new therapeutic approaches for conditions associated with MCH signaling abnormalities. By providing new insights into the underlying mechanisms of schizophrenia and other neurological disorders, the studies in this thesis may pave the way for the development of novel therapeutic targets for the treatment of these conditions.

Drug transporters are membrane proteins known for their role in handling pharmaceutical products. However, recent work suggests they contribute to physiology by transporting endogenous metabolites. This understudied function may explain some aspects of organ crosstalk, inter-organismal communication, and adverse drug side effects. This dissertation focuses on determining the endogenous compounds that renal organic anion transporter 1 (OAT1/SLC22A6) interacts with using in vivo, in vitro, and in silico methodologies. Chapter 1 presents the current state of drug transporter research. Chapter 2 of this dissertation is a reprint of published work that describes the role OAT1 plays in mediating the relationship between the host and the gut microbiome via regulation of circulating small molecule metabolites in a pre-clinical model. These in vivo results were supported with in vitro binding and transport assays. Chapter 3 of this dissertation is a reprint of published work that explores the role that a common OAT1-inhibiting drug, probenecid, has on the plasma and urine metabolomes of healthy humans. This revealed dozens of plasma and urine drug-metabolite interactions caused by short-term exposure, including multiple likely occurring at OAT1. Chapter 4 of this dissertation is a reprint of published work that combines genomic and metabolomic data to determine associations between single nucleotide polymorphisms and circulating small, polar, bioactive molecules. This work reveals that numerous drug transporter and drug metabolizing enzyme genes play important individual and combined roles in physiology. Chapter 5 describes in silico models used to better characterize the nature of protein-ligand interactions involving OAT1. By analyzing molecular descriptors of compounds known to interact with OAT1, we generated predictive ligand-based models. We then explored potential binding mechanisms for different classes of compounds using a predicted OAT1 structure. Chapter 6 is a summary of the contributions to the field and future directions in drug transporter research.

Nicotine, the main psychoactive constituent in tobacco, is highly addictive and poses a particular vulnerability to adolescents. Adolescence is a critical period of neurodevelopment when nicotine use is initiated. Nicotine is the exogenous ligand for nicotinic acetylcholine receptors (nAChRs). The alpha()6 nAChR subunit (encoded by the CHRNA6 gene) reaches peak expression in dopaminergic neurons of the midbrain during adolescence. A ‘C’ to ‘G’ single nucleotide polymorphism (SNP) in the 3’-untranslated region (UTR) of the CHRNA6 gene (rs2304297) has been associated with nicotine addiction in adolescent humans. Discerning the genetic and neurobiological mechanisms impacting adolescent nicotine seeking could help with improved prevention and intervention strategies in humans. My findings illustrate that nicotine-seeking behavior could be assessed in adolescent wild type male and female Sprague Dawley rats using a self-administration and reinstatement paradigm, a model of relapse behavior in humans. Further, I show that the CHRNA6 3’-UTR SNP elicits sex- and genotype-dependent nicotine seeking behavior, with 6GG male adolescents most vulnerable. Mechanistically, I illustrate that dopaminergic mechanisms underlie deficits in nicotine seeking behavior, Taken together, the results of the present study will add to a growing body of literature that the CHRNA6 3’-UTR SNP sex- and genotype-dependently contribute to adolescent nicotine addiction with dopaminergic circuits involved.

The United States is currently experiencing its worst drug crisis, which is largely driven by opioid addiction and primarily due to fentanyl. It is therefore necessary to investigate the mechanisms mediating fentanyl's rewarding and reinforcing properties to contribute to the development of successful treatment strategies. Gut bacteria communicate with the brain, and vice versa, via the gut-brain axis to regulate brain function, mood, and behavior. Addiction is a chronic brain disorder that alters circuitry involved in reward, stress, learning, and motivation, all of which have a bidirectional influence between their associated behaviors and gut bacteria. Given the associations between opioid use, gastrointestinal distress, and microbial dysbiosis in humans and rodents, I tested the hypothesis that interactions between gut bacteria and the brain mediate the reinforcing and motivational properties of fentanyl. In this dissertation, I present my work that supports a bidirectional relationship between gut bacteria and fentanyl intravenous self-administration (IVSA) in Sprague Dawley rats. In the following studies, I implanted rats with intravenous catheters in preparation for fentanyl IVSA on an escalating schedule of reinforcement and analyzed gut microbiota by sequencing bacterial DNA from rat fecal samples. I demonstrate that based on sex and fentanyl dose, the diversity of gut bacteria is either increased or decreased following fentanyl IVSA and predicts progressive ratio breakpoint, a measure of motivation. Further, I show that depletion of gut bacteria via prolonged oral antibiotic treatment enhances fentanyl IVSA, and restoration of microbial metabolites with short-chain fatty acid administration decreases fentanyl IVSA back to controls at higher fixed ratio schedules of reinforcement. My findings highlight an important relationship between the knockdown and rescue of gut bacterial metabolites and fentanyl self-administration in adult rats, which provides support for a relationship between gut microbiota and opioid use. Further work in this area may lead to effective, targeted treatment interventions in opioid-related disorders.

Parasitic nematodes are globally widespread in human, animals and plants, infecting one-quarter of the world’s population and creating an economic burden on agricultural industries. With the fear or reality associated with the emergence of drug resistance, new anthelmintic compounds are needed. Therefore, to facilitate the drug discovery process, an infrared-based motility assay utilizing the free-living nematode, Caenorhabditis elegans, as a model for parasitic nematodes, was developed and validated. The assay was put to work to screen 400 compounds from two open-source small-molecule libraries distributed by the Medicines for Malaria Venture, namely, the COVID Box and Global Health Priority Box. The screening assay identified four compounds with previously undescribed activities against nematodes, underlying its utility as a drug discovery tool.

In recent years, the complexity of chemotherapies and their interactions with the peripheral nervous system have come into focus but limitations in experimental models have remained a significant challenge in the field. As evidence, despite most chemotherapy drugs being around for decades, there are currently no therapies approved that target chemotherapy-peripheral nervous system interactions as an anti-neurotoxic agent. Human pluripotent stem cells offer an appealing model system that, unlike rodent models, are compatible with high throughput, high content applications; techniques that reflect modern drug discovery methodologies. Thus, utilizing the key advantages of stem cell-based models in tandem with the strengths of traditional animal models offers a complementary and interdisciplinary strategy to advance chemotherapy-peripheral nervous system research and drug discovery. This dissertation begins with an overview of the current status of chemotherapy-peripheral nervous system research, describing examples of taxane chemotherapy-induced damage to the sensory nerves and platin chemotherapy-induced damage to the enteric nerves. Avenues where stem cell-based models may further advance the field are also presented. Based on this foundation, I established a stem cell-based model of chemotherapy-induced enteric neuropathy, focusing my efforts on the platin chemotherapy drug class as they are heavily prescribed and highly neurotoxic to the enteric neurons, which innervates and controls the gastrointestinal tract. To uncover the mechanism of platin-induced gastrointestinal neurotoxicity, I leveraged my scalable stem cell-derived enteric neuron model, performing high throughput screens and transcriptomic analyses to reveal excitotoxicity through muscarinic cholinergic signaling as a key driver of platin-induced enteric neuropathy. Single nuclei transcriptomics identified inhibitory nitrergic neurons as selectively vulnerable to platins, which we validated through histological analysis of the enteric nervous system in platin chemotherapy-treated patients. Lastly, we found that dampening muscarinic signaling through either pharmacologic or genetic methods is sufficient to prevent platin-induced excitotoxicity in vitro and platin-induced constipation and degeneration of nitrergic neurons in mice. Altogether, this work succeeds in defining the mechanism underlying platin-induced gastrointestinal neurotoxicity. Furthermore, it serves as an example and framework for how stem cell-based models of the peripheral nervous system can be utilized to rapidly deconvolute mechanisms of chemotherapy-induced neurotoxicity and power drug discovery pipelines to tackle numerous peripheral neuropathies that have been intractable to date.

Organoid culturing is a three-dimensional tissue culturing method with significant promise for increasing our ability to model cancer subtypes in the laboratory. We modified tissue processing methods and used a natural hydrogel (GrowDex) as well as basement membrane extract (BME) to optimize conditions for growth and immunostaining of breast cancer organoid cultures. We focused on breast cancers that were Estrogen Receptor (ER) and/or Progesterone Receptor (PR) positive, generating cultures of residual disease after treatment with neoadjuvant endocrine therapy in the Investigation of Serial studies to predict your therapeutic response with Imaging and Molecular AnaLysis 2 (NCT01042379, I-SPY2) clinical trial. Patients on an Endocrine Optimization Protocol (EOP) in I-SPY2 were treated with amcenestrant alone or in combination with an aromatase inhibitor or a CDK4/6 inhibitor prior to specimen collection and culturing. The goal of these studies was to generate conditions for successful propagation of residual breast cancer resistant to primary treatment, including verification of expected protein expression patterns, for future experiments to test new therapies for ER+ breast cancer resistant to endocrine therapy.