Synthesis of Sulfonyl Fluorides and the Total Synthesis of the Rhodocorane Natural Products

The selective reactivity of different functional groups is at the forefront of modern synthetic chemistry. As predictable and controllable reactivity are the key to the synthesis of many bioactive and structurally challenging molecules, synthetic methodologies to install and understand these reactive moieties is always of interest to organic chemists. However, there is a fine line between too reactive and not reactive enough. Our studies on the development of new catalytic methods to synthesize sulfonyl fluorides via two separate photocatalytic methods and the synthesis of the rhodocorane family of bioactive natural products are described. In the first chapter, an overview of the development and the understanding of the reactivity of sulfonyl fluorides as a second generation "click" reaction is discussed. Various studies from the literature show that these moieties can undergo a selective activation and become reactive in both chemical and biological instances. The potential applications of these interesting functionalities are limitless, with the only hindrance being the lack of easily accessible syntheses. The understanding of that activation is discussed along with a variety of ways to synthesize both aliphatic and aryl sulfonyl fluorides. The second chapter describes our studies in the development of a photocatalytic method to convert anilines into sulfonyl fluorides through an activated and isolated substrate class of diazonium salts. Our approach allows for a mild and selective method to synthesize aryl sulfonyl fluorides from a widely available functional group while being tolerant of a range of other functionalities. We also present various experimental and theoretical studies to analyze the mechanistic pathways involved in the methodology. In the third chapter, we describe our effort to develop a separate organophotocatalytic methodology to synthesize aliphatic sulfonyl fluorides from potassium trifluoroborate salts. Our synthetic strategy leverages the activated boronate species to generate aliphatic radicals with the highly oxidizing organophotocatalyst, which undergoes a three-component coupling with a sulfur dioxide source and a fluorine source. The challenges associated with this methodology along with photophysical measurements and evidence for the mechanism are discussed. In the fourth chapter, we discuss our approach to the asymmetric synthesis of the rhodocorane family of bioactive natural products. These natural products possess a range of spirocyclic, fused rings, and highly oxygenated systems that allow for structurally diverse and complex molecules. We propose a common synthetic intermediate that can access a majority of this family through the addition of other commercially available molecules. The challenges and optimization of this synthesis and our approaches to solve these problems are discussed

Systemic Impact of Insulin-Sensitized Adipocytes and Their Precursors

Maintenance of healthy adipose, especially when challenged with a prolonged unhealthy diet, is crucial for the prevention of diabetes and metabolic disease. Previous studies have shown that healthy expansion of adipose tissue occurs via hyperplasia, rather than hypertrophy. Therefore, a better understanding of the adipocyte precursor can potentially lead to interventions that favor new adipocyte formation over unhealthy, hypoxic adipocyte enlargement. Insulin signaling is central to the development of type 2 diabetes mellitus; resistance within this pathway leads to global metabolic dysfunction and cardiometabolic disease. Downregulation of adipocyte PTEN, a downstream inhibitor of the insulin signaling pathway, can lead to insulin sensitization of the adipose tissue and, as a result, in whole body insulin sensitivity. Previous work from our lab has shown that insulin sensitization of mature adipocytes, or of only thermogenic adipocytes, is sufficient to improve global metabolic health. We now report that by sensitizing murine adipocyte precursor cells to insulin action, we can induce durable improvements in glucose tolerance, lower circulating insulin levels, and eliminate steatohepatitis, without a change in overall body weight, even in high fat diet-fed mice. Remarkably, transplantation of small amounts of murine gonadal adipose tissue, containing insulin-sensitized precursors, is sufficient to induce improvements of global glucose handling and insulin signaling. The transplantation of such an insulin-sensitized fat pad can induce a durable reduction in circulating insulin even in high fat diet-fed mice. These studies demonstrate that targeting the relatively small cell population of adipocyte precursors is sufficient to induce widespread improvements in metabolic health, and that these cells may hold the key to future therapies for diabetes and metabolic disease

Investigation of Divergent Metabolic Programs in the Tumor-Immune Microenvironment

The current immune metabolism field has been using the term lactate and lactic acid interchangeably, with the notion that lactate and lactic acid are waste products of highly glycolytic cancer cells that negatively affects TIL function and viability to promote tumorigenesis. However, it is yet unclear whether lactate or its protonated counterpart lactic acid is responsible for the dampening of immune cell functions within the TIME. The in vitro cell culture studies aimed to delineate the impact of lactate and lactic acid in the context of cancer growth. Data showed that lactate alone significantly impairs cancer cell metabolism independent of the acidic pH environment. Additionally, therapeutic lactate treatment significantly increased the glycolytic activity and effector functions of CD8+ T-cells, contrary to the current understanding that lactate dampens immune cell functions. The comparison study between lactate and lactic acid further demonstrated that CD8+ T-cells undergo cell death that is dependent on pH levels, with approximately 50% of T-cell death occurring at pH level below 6.0, whereas cancer cells were able to metabolically adapt to the acidic environment and sustain growth. These findings provide a new perspective that challenges the current notion that lactate is considered a "pro-tumor" metabolite. In vivo metabolomics study of tumor cells and tumor-infiltrating CD8+ T-cells revealed that lactate initiates a divergent metabolic reprogramming in the two cell types. Systemic lactate treatment suppressed tumor glycolytic activities while boosting CD8+ T-cell glycolytic rate and effector function. Such divergent changes in two cell types provide a basis for developing potent antitumor responses in the complex heterogeneous tumor microenvironment. The impact of lactate on glycolytic flux modulation on both tumor cells and CD8+ T-cells, therefore, exposes a previously undefined difference in metabolic programs between the two cell types that can be exploited in the immune-suppressive tumor microenvironment

Intracellular Lipid Surveillance Through Interplay Between a Nuclear Receptor and Rab GTPases

The general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.Cells continuously experience fluctuations in resource availability and therefore require adaptive mechanisms to recognize these changes and respond accordingly to ensure metabolic health. Signaling mechanisms like the AMPK and mTOR pathways enable cells to monitor energy and amino acid availability, respectively, and cholesterol sensing by SREBP provides a means for cells to maintain homeostatic lipid saturation. Lipids comprise a vast class of energy-rich molecules that, altogether, are essential for most every cellular process. Yet independent of sterol sensing, it remains unclear how cells sense and respond to global lipid depletion. While starving cells initiate fatty acid β-oxidation via mammalian nuclear receptors, PPAR and HNF4, it is unknown how cells couple this catabolic process with mechanisms to increase nutrient intake and prevent further starvation. Comprised predominantly of lipid droplets, the C. elegans intestine is an excellent model for elucidating the relationship between lipid depletion and nutrient absorption in the context of lipid sensing. The work presented herein defines a novel signaling mechanism through which the transcriptional regulator of β-oxidation in C. elegans, Nuclear Hormone Receptor 49 (NHR-49), senses intracellular lipid availability and restores lipid homeostasis following starvation-induced lipid depletion by monitoring abundance of a single de novo-synthesized lipid, geranylgeranyl pyrophosphate. In this pathway, lipid availability inactivates NHR-49 through its sequestration to cytosolic transport vesicles by the geranylgeranyl-conjugated RAB-11.1 GTPase. Lipid depletion prevents NHR-49 vesicular association, thereby promoting its nuclear translocation and activation of genes involved in nutrient absorption and catabolism. Importantly, activation of endocytic recycling regulator, rab-11.2, enhances nutrient transporter residency on the cell surface. Upon investigation of how this intracellular lipid surveillance pathway relates to other homeostatic mechanisms, the findings described in the succeeding chapter establish a cooperative relationship between NHR-49 and the regulator of cytosolic protein folding, Heat Shock Factor (HSF-1), in mediating metabolic health and age progression. Through stabilizing the intestinal actin network, HSF-1 ensures proper vesicle trafficking and acts as an upstream regulator of NHR-49 in promoting lipid homeostasis. Overall, this work expands our understanding of lipid sensing and details a novel mechanism by which cells increase nutrient intake in times of metabolic demand

Please Die in the Sun

The author submitted this entry in the Open Verse Poetry category (Amateur division) for the 2025 On My Own Time™ (OMOT) Art Show.A dying patient staring longingly out of the window

Structural Biology in Cellular Environments Using Sensitivity Enhanced NMR

This dissertation describes the general applicability of DNP solid state NMR to mammalian cells. DNP NMR can detect proteins at their physiological concentrations within cellular environment in tractable times. However, these experiments were limited to cellular lysates, which did not recapitulate the intracellular environment completely. Studying proteins within their native cellular environment required in-cell NMR. This work focuses on developing methodology for in-cell DNP NMR and applying the technique to investigate the conformational ensemble of Tau. Chapter 1 provides an overview of in-cell NMR in biological systems and intrinsically disordered proteins. A brief primer of NMR, DNP and EPR is also provided in this chapter. Chapter 2 describes the development of methodology to maintain biological integrity of mammalian cells during DNP NMR. Description of a novel method to insert samples into a spectrometer is added as a supplementary chapter 2. Chapter 3 is based on a comparative study between different cryoprotectants to optimize biological integrity for in-cell DNP NMR. Chapter 4 focusses on the characterization of the reduction process of a polarizing agent, AMUPol inside intact and lysed HEK293 cells using electron paramagnetic resonance (EPR) spectroscopy. In the supplementary chapter 4, a novel DNP radical AsymPol-POK and its reduction kinetics within intact HEK293 cells is discussed. Chapter 5 deals with the application of in-cell DNP NMR to study the conformational ensemble of tau protein within cellular environment. The in-cell DNP NMR methodology developed in this work can be potentially applied to various biological systems. Additionally, the strategies and experiments described here to investigate the conformational ensemble of an intrinsically disordered protein, tau, within cellular environment, can be tailored to any other IDP or protein of interest within normal or pathological cellular milieu. Also, the EPR experiments described here will be a useful guide to test reduction kinetics of novel polarizing agents for in-cell DNP NMR. Overall, this dissertation should serve as a useful literature for technical advancement and biological application of in-cell DNP NMR

Kinetic and Thermodynamic Allostery in the Ras Protein Family

The general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.An understanding of allosteric communication is necessary for interpreting protein function and regulation. For proteins involved in disease, such knowledge can lead to discovery of new sites for therapeutic targeting. However allosteric mechanisms have proven to be diverse, and allosteric communication in many proteins cannot currently be explained by their structure or dynamics. Progress has been made in elucidating the ensemble nature of allosteric communication, especially using MD simulations to provide structural specificity of previously averaged conformations. Here I show how kinetic (i.e. temporal) correlations encode information that is orthogonal to existing work on thermodynamic correlations. I performed atomistic simulations on H, K, and NRas isoforms in various states in the Ras cycle, for a total of 0.5 milliseconds. I show that Ras' most important structural motifs, switch I and switch II, are the primary members of my calculated thermodynamic and kinetic allosteric networks, consistent with the known roles of these two motifs in Ras function. I also reveal how these communication networks are altered by the presence of the -phosphate, as well as binding of the downstream effector Raf kinase. Strikingly, these communication networks correspond to structural motifs that are functionally engaged in the Ras cycle step being simulated. I find that kinetics-based allosteric communication is not restricted to the boundaries of secondary structure elements and can occur across long distances. I show that known features of Ras regulation, such as activation of allostery upon Raf binding, necessitate kinetic allostery. These data explain experimentally observed allosteric relationships by revealing the kinetic and thermodynamic communication pathways, and show how both modes of communication are needed to relay information within proteins. This work suggests that kinetics-based communication is a key mechanism of protein function and regulation

Development of the Multicultural Curriculum Appraisal (MCCA): A Process to Promote Multicultural Infusion into APA Accredited Doctoral Program Curriculum

The current American Psychological Association's standard for Diversity Education and Training in doctoral-level accredited Health Service Psychology programs is sparce and lacks a measurable expectation of programs. There is no published research on broad application or outcomes of this standard across Health Service Psychology curricula. To address any variation in the interpretation of the diversity education and training standard, we created an audit tool, the Multicultural Curriculum Appraisal (MCCA) Tool, to capture the amount of multicultural education and training present in today's psychology programs. One important step is to understand the how much multicultural content psychology instructors are incorporating into their course syllabi. Another essential step is to identify the areas, within a syllabus, that contain multicultural content. Lastly, it is critical to understand any potential barriers to multicultural infusion identified by psychology instructors. Our study surveyed doctoral level instructors (n=55) and audited their course syllabi (n=92). The primary aim was to explore the influence of instructor identity on the amount of multicultural content in a course. We expected instructors who identified with historically marginalized groups to have more multicultural infusion into their courses. This studies secondary aim examined barriers to multicultural infusion using three types of barriers: institutional, instructor and student. We anticipated participants who identified instructor barriers to have lower scores than participants who selected other barrier types. Contrary to expectations, participants with marginalized identities and those who selected instructor barriers did not have significantly different scores than their peers. Participants who were trained as counseling psychologists, taught at counseling psychology programs, were younger, ranked themselves lower on a national subjective socioeconomic scale, and in the early career stage demonstrated higher scores on the Multicultural Curriculum Appraisal. Additionally, participants who identified institutional barriers also demonstrated higher scores on the Multicultural Curriculum Appraisal. This study lays the groundwork for future research designed to better understand and correct for the variation between instructors, academic programs, and psychology disciplines and understand the potential influences of instructor identity and barriers in multicultural education and training in psychology curricula

RNA Exonuclease Xrn1 Regulates TORC1 and Autophagy in Response to SAM Availability

During methionine deprivation, yeast cells experience loss of S-adenosyl methionine (SAM), resulting in globally reduced histone methylation levels. Investigations into the location of the enduring histone methylation initiated our studies into Y' elements, subtelomeric noncoding RNAs that we have found are highly expressed under this condition. Their accumulation following loss of methionine is caused by reduced degradation by the conserved 5'-3' exonuclease Xrn1, rather than increased transcription during this condition. The finding that Xrn1 has altered activity under methionine deprivation led to investigate how Xrn1 may sense the change in nutrient availability. Autophagy is a conserved process of cellular self-digestion that promotes survival during nutrient stress. In yeast, methionine starvation is sufficient to induce autophagy. One pathway of autophagy induction is governed by the SEACIT complex, which regulates TORC1 activity in response to amino acids through the Rag GTPases Gtr1 and Gtr2. However, the precise mechanism by which SEACIT senses amino acids and regulates TORC1 signaling remains incompletely understood. We identified Xrn1 as a surprising and novel regulator of TORC1 activity in response to methionine starvation. This role of Xrn1 is dependent on its catalytic activity, but not on degradation of any specific class of mRNAs. Instead, Xrn1 modulates the nucleotide-binding state of the Gtr1/2 complex, which is key for its interaction with and activation of TORC1. This work identifies a critical role for Xrn1 in nutrient sensing and growth control that extends beyond its canonical housekeeping function in RNA degradation and indicates an avenue for RNA metabolism to function in amino acid signaling into TORC1

Increased Risk of Cutaneous Immune-Related Adverse Events in Patients Treated with Talimogene Laherparepvec and Immune Checkpoint Inhibitors: A Multi-Hospital Cohort Study

The general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.BACKGROUND: Previous studies have shown that combining immune checkpoint inhibitors (ICIs) with Talimogene Laherparepvec (TVEC) may improve antitumor responses. However, the risk of developing cutaneous immune-related adverse events (cirAEs) in patients treated with ICI and TVEC has not been studied. OBJECTIVE: To evaluate the differences in cirAE development between patients treated with ICI alone and both ICI and TVEC (ICI+TVEC). METHODS: Patients with cutaneous malignancy receiving ICI with or without TVEC therapy at the Massachusetts General Brigham healthcare system were included. CirAE development, time from ICI initiation to cirAE, cirAE grade, cirAE morphology, and survival were analyzed. Pearson's chi-squared test or Fisher's exact test for categorical variables and t-test or Kruskal-Wallis test for continuous variables were used. To account for immortal time bias, we performed adjusted time-varying Cox proportional hazards modeling. RESULTS: The rate of cirAE development was 32.3% and 38.7% for ICI only and ICI+TVEC, respectively. After adjusting for covariates, ICI+TVEC was associated with a 2-fold increased risk of cirAE development (HR: 2.03, p=0.006) compared to patients receiving ICI therapy alone. LIMITATIONS: The retrospective nature and limited sample size from a tertiary-level academic center. CONCLUSION: These findings underscore potential opportunities for dermatologists and oncologists in counseling and monitoring patients