UT Southwestern Graduate School of Biomedical Sciences

Permanent URI for this collectionhttps://hdl.handle.net/2152.5/203

Welcome to the UT Southwestern Graduate School of Biomedical Sciences’ electronic theses and dissertations (ETD) collection.

Most UT Southwestern ETDs are subject to a default embargo period of two (2) years from the date of degree conferral. These embargoed ETDs are unavailable until the embargo expires.

It might be possible to contact the author for more information before the end of the embargo. For assistance or more information, please contact the UT Southwestern Graduate School of Biomedical Sciences by email.

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Print theses and dissertations from 1943 to 2004 are located in the Library's Special Collections and Archives (Room E3.314) and are available by appointment. (Note: Former students may request a digitized copy of their work by email, but other users may submit an Interlibrary Loan request.) For more information, contact archives@utsouthwestern.edu.


Recent Submissions

Now showing 1 - 20 of 1571
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    On Sulfur Sensing in Saccharomyces cerevisiae
    (December 2021) Johnson, Zane Miller; Nijhawan, Deepak; De Martino, George; Yu, Hongtao; Tu, Benjamin
    The unique chemistry available to sulfur compared to oxygen, such as the ability to exist in numerous oxidation states and greater nucleophilicity, makes many of the biochemical reactions requisite for cellular life possible. As a result of this critical importance, organisms have developed several mechanisms for sensing and maintaining levels of sulfur-containing metabolites. In the yeast Saccharomyces cerevisiae, regulation of sulfur metabolism can be distilled down to the actions of two proteins; the F-box protein Met30, and the transcriptional coactivator Met4. Met30 belongs to the family of SCF (Skp1-Cul1-F-box protein) E3 ubiquitin ligases, and negatively regulates the transcriptional activity of the master transcriptional activator of sulfur metabolism genes, Met4, via oligo-ubiquitination when sulfur metabolite levels are high. When yeast are starved of sulfur, Met30 ceases to ubiquitinate Met4, releasing it to be deubiquitinated and transcriptionally active to boost levels of a network of sulfur metabolic genes known as the MET regulon to restore sulfur metabolite levels. While the molecular activities of both Met30 and Met4 have been extensively studied over the last two decades, the biochemical basis for sulfur-sensing by the Met30 E3 ligase has remained unknown. Herein, I reveal the biochemical details by which Met30, the master regulator of sulfur metabolism, senses the availability of sulfur metabolites to modulate its E3 ligase activity to regulate sulfur metabolism in yeast. Utilizing a combination of yeast genetics and biochemical assays, I show that Met30 uses redox-active cysteine residues in its C-terminal WD-40 repeat region to modulate binding between itself and its substrate Met4 in accordance with the availability of sulfur metabolites. These insights represent significant advances in the understanding of sulfur metabolic regulation in yeast.
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    Vanishing Act: Lymphatic Vessels and Disappearing Bones
    (December 2021) Monroy, Marco Antonio; Jewell, Jenna L.; Dellinger, Michael T.; Cleaver, Ondine; Brekken, Rolf A.
    Generalized lymphatic anomaly (GLA) and Gorham-Stout disease (GSD) are related diseases of the lymphatic system. Patients with GLA or GSD develop ectopic lymphatic vessels in bone and gradually lose bone. Despite growing interest in the development of tissue-specific lymphatics, the cellular origin of bone lymphatic endothelial cells (bLECs) is not known, and the development of bone lymphatics has not been fully characterized. In this dissertation, I review the latest advances in research on the development of the lymphatic system and human lymphatic diseases. I also present my work on the development of bone lymphatics in mouse models of GLA and GSD. I show by lineage-tracing that bLECs arise from pre-existing Prox1-positive LECs. I demonstrate that bone lymphatics develop in a stepwise manner, where regional lymphatics grow, breach the periosteum, and then invade bone. I also show that osteoclasts are closely associated with invading lymphatics and that lymphatic invasion of bone is impaired in mice that lack osteoclasts. Additionally, I demonstrate that rapamycin suppresses the formation of bone lymphatics in mouse models of GLA and GSD. These findings shed light on the development of bone lymphatics, a process that has puzzled investigators since Gorham and Stout published their landmark paper over 60 years ago. They also show that an emerging treatment for GLA and GSD patients can inhibit lymphatic invasion of bone.
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    Molecular Dissection of Bsc2: A Novel Negative Regulator of Triglyceride Lipolysis for a Lipid Droplet Subpopulation
    (December 2023) Speer, Natalie Ortiz; Goodman, Joel M.; Henne, W. Mike; Friedman, Jonathan R.; Nicastro, Daniela
    Eukaryotic cells store lipids in the form of triglyceride (TG) and sterol-ester (SE) in cytoplasmic organelles called lipid droplets (LDs). Distinct pools of LDs with unique surface proteomes exist in cells, but a pervasive question is how proteins localize to and convey functions to specific LD subsets. Here, we show the yeast protein Bsc2 localizes to a specific subset of TG-containing LDs, and reveal it negatively regulates TG lipolysis. Mechanistically, Bsc2 LD targeting requires TG, and LD targeting is mediated by specific N-terminal hydrophobic regions (HRs) sufficient for Bsc2 function. Molecular dynamics simulations reveal these Bsc2 HRs interact extensively with TG on modeled LDs, and adopt a specific conformation on TG-rich LDs versus SE-rich LDs or a modeled ER bilayer. Bsc2-deficient yeast display no defect in LD biogenesis, but exhibit enhanced TG lipolysis dependent on the major TG lipase Tgl3. Remarkably, over-expression of Bsc2, but not LD protein Pln1, causes TG accumulation without altering SE levels. Finally, we find that Bsc2-deficient cells display altered LD accumulation during stationary phase growth. We propose that Bsc2 is a novel regulator of TG lipolysis that localizes to a subset of TG-enriched LDs and locally regulates TG lipolysis.
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    Deciphering AXL-Driven Molecular Mechanisms of EMT
    (December 2021) Arner, Emily Nicole; Raj, Ganesh V.; Brekken, Rolf A.; Cobb, Melanie H.; Kim, James
    Cellular plasticity, a feature associated with epithelial-to-mesenchymal transition (EMT), contributes to tumor cell survival, migration, invasion, and therapy resistance. Across human cancer, tumors that are high grade, poorly differentiated, and have undergone EMT carry a worse prognosis with a high likelihood of metastasis and poor outcome. AXL, a receptor tyrosine kinase (RTK), drives EMT and is implicated in tumor progression, metastasis, and therapy resistance in multiple cancer types including pancreatic cancer (PDA) and breast cancer. We investigated the contribution of TANK-binding kinase 1 (TBK1) to PDA progression and report that TBK1 supports the growth and metastasis of KRAS-mutant PDA by driving an epithelial plasticity program in tumor cells that enhances invasive and metastatic capacity. We identified that the receptor tyrosine kinase AXL induces TBK1 activity in a Ras-RalB-dependent manner. Furthermore, we report that AXL activation stimulates TBK1 binding and phosphorylation of the specific AKT isoform, AKT3 at S472. Activation of AKT3 drives the binding of AKT3 to slug/snail, where the complex is translocated into the nucleus. The binding of AKT3 to slug/snail protects the EMT-TFs from proteasomal degradation thus leading to an increase in EMT. These data suggest that the translocation of AKT3 to the nucleus is required for AXL-driven EMT and metastasis. Congruently, nuclear AKT3 expression correlates with worse outcome in aggressive breast. These results suggest that selective AKT3 targeting represents a novel therapeutic avenue for treating aggressive cancer that may avoid toxicity associated with pan-AKT inhibition. Additionally, our findings suggest that interruption of the AXL-TBK1-AKT3 cascade, has potential therapeutic efficacy in AXL positive metastatic cancer.
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    Investigating the Mechanism and Mode of Action of Golgi Toxins
    (December 2023) Cervantes, Christopher Luis; Liszczak, Glen; Posner, Bruce A.; Wang, Fei; Nijhawan, Deepak
    Auxin-inducible forward genetics uncovered point mutations within Golgi Brefeldin A Resistant Guanine Nucleotide Exchange Factor 1 or GBF1 following lethal dose selection with a synthetic disubstituted pyrimidine toxin called Golgitox (GTOX). Resistant clones were also cross-resistant to the fungal toxin, Brefeldin A (BFA), and synthetic GBF1 inhibitor, Golgicide A (GCA). Like BFA and GCA, GTOX triggered Golgi disassembly via GBF1. Given that BFA is a reported molecular glue, we profiled Gbf1-Arf interactions in 293T cell lysates pre-treated with Golgi toxin. Both GTOX and GCA promoted GBF1-dependent interactions with Arfs 4 and 5, whereas BFA also interacted with Arf1. GBF1 domain mapping revealed that the HUS-SEC7-HDS1 domains were sufficient for promoting GTOX-dependent engagement with Arfs 4 and 5. Meanwhile, structural activity relationship studies showed that modifying the methyl group on the benzimidazole ring preserved GTOX activity and interactions between Gbf1 and Arfs 4 and 5. To assess which Arfs regulate BFA and GTOX cytotoxicities, genome-wide CRISPR/Cas9 compound enrichment screens were carried out, which identified ARF4 as being the most enriched hit. Next, we validated that ARF4 loss-of-function partially confers resistance to BFA and GTOX. Next, we asked whether GTOX preferentially interacts with ARF4-GDP versus ARF4-GTP. We found that exogenous ARF4 T31N (GDP-locked mutant) sensitized HCT116 scramble control cells 4-fold to GTOX, interacted with Gbf1 just as well as WT Arf4, but failed to rescue Arf4-mediated BiP retrieval. Collectively, these results suggest that the Gbf1-GTOX-Arf4-GDP complex is functionally inactive but deleterious to cell viability. Taken together, GTOX may act as a molecular glue to suppress GBF1 functions through downstream effector substrates like Arfs 4 and 5.
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    Molecular Underpinnings of Human Brain Evolution and Cognition at Cellular Resolution
    (December 2023) Caglayan, Emre; Chahrour, Maria; Hon, Gary C.; Madabhushi, Ram; Sun, Lu O.; Konopka, Genevieve
    Molecular and functional characterization of the human brain is challenging due to its experimental inaccessibility. Most of our understanding about human brain function relies on the assumption that biological processes uncovered in model organisms are conserved in humans. Comparisons of the humanii brain with non-human primate brains offer to both uncover the novelties in human brain evolution and better evaluate the insights obtained from model organisms about human brain function. To achieve this, highthroughput sequencing methods on post-mortem brain tissues provide a rewarding readout to understand human brain evolution at the molecular level. In addition to their use in comparative studies, these technologies were also utilized with a hope to understand molecular underpinnings of measurable human brain activity metrics. During my dissertation, I read relevant literature extensively (Chapter 1) and sought to understand human-specific epigenomic and transcriptomic changes at cellular resolution in the cortical brain (Chapter 2). Additionally, after in-depth analysis of many human brain single-nuclei RNA-seq datasets, I found a pervasive ambient RNA contamination problem, and devised in silico solutions to tackle this problem. My efforts improved the analytical approach in the field as well as in my research (Chapter 3). I have also been involved in efforts to identify transcriptomic correlates of brain activity in human subjects (Chapters 4-5). After detailing these efforts, I discuss the implications of these findings, weigh their impact on our understanding of human brain function and offer ideas for further research (Chapter 6).
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    An Optical Flow Based Methodology for Visualizing Dynamic Sucellular [sic] Organiztion [sic] Demonstrated Through Profilin and Rho GTPase Microdomains
    (December 2021) Jiang, Xuexia; Doubrovinski, Konstantin; Jaqaman, Khuloud; Danuser, Gaudenz; Rajaram, Satwik
    Live cell imaging has enabled the collection of movies of subcellular protein dynamics at a submicron resolution. Statistical time series analysis can greatly expand our understanding of subcellular interactions in minimally perturbed systems. This was previously achieved for the leading edge of migrating cells in select cases. Importantly no strategy existed to simultaneously analyze every subcellular location. Building on existing optical flow based non-linear image registration we developed an approach to remap a migrating cell to a common cell footprint while preserving the characteristics of our signal of interest at a spatial granularity necessary for understanding micron scale biological interactions. This tool enabled us to discover that Profilin fluctuations are organized in living cells. This organization was found to be dependent on cell polarization and actin binding capability. Expanding on this ability to query all subcellular locations, we developed a feature set and feature projection strategy to map molecular biosensor movies of Rho GTPase signaling into micron scale regions of internally consistent signaling dynamics or "microdomains". Microdomains of GTPases match literature descriptions of signaling organization and in an optogenetic study were found to almost precisely match the perturbation footprint.
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    Technical Development of Hyperpolarized [1-13C]Pyruvate Imaging for Clinical Translation
    (December 2021) Ma, Junjie; Madhuranthakam, Ananth; Park, Jae Mo; Malloy, Craig R.; Pinho, Marco Da Cunha; Chopra, Rajiv
    Magnetic resonance imaging with hyperpolarized [1-13C]pyruvate is an emerging tool for assessing in vivo metabolism noninvasively. Over the past few years since the first clinical study with prostate cancer patients, translational studies using this imaging technique have been focused on demonstrating the feasibility of applying to healthy subjects and patients under several pathophysiological conditions. However, there are multiple remaining technical limitations to overcome for translating the technique to clinics. In this study, I made three technical advances in imaging hyperpolarized [1-13C]pyruvate and products in humans. First, I measured in vivo T2* of hyperpolarized signals, one of the key parameters that directly affect 13C acquisition methods and image quality. Measuring the in vivo T2*'s can be useful for optimizing the acquisition parameters and improving the signal-to-noise ratio. I proposed a dynamic 13C metabolite-selective multi-echo spiral imaging sequence for the T2* measurement of hyperpolarized 13C-labeled metabolites. The feasibility and reproducibility of the method were confirmed by phantom and rat studies. Moreover, from healthy volunteers, in vivo T2*s of hyperpolarized [1-13C]pyruvate, [1-13C]lactate and [13C]bicarbonate were measured from cardiac tissue compartments using the sequence. Second, I developed a cardiac-gated multi-phase 13C imaging sequence for the human heart. To demonstrate cyclic changes in cardiac metabolic profiles seen by hyperpolarized signals, dual-phase cardiac imaging of hyperpolarized [1-13C]pyruvate, [1-13C]lactate, [1-13C]alanine and [13C]bicarbonate was conducted at end-systole (ES) and end-diastole (ED) on the short-axis and vertical long-axis planes. Significantly smaller myocardial signal of [13C]bicarbonate relative to [1-13C]lactate was observed at ED compared to that at ES (p < 0.05). Third, a patch-based algorithm (PA) was developed to improve spatial resolution of hyperpolarized 13C images by exploiting the co-registered high-resolution 1H images. The spatial resolution of hyperpolarized 13C imaging is genuinely poor, compromising the overall image conspicuity and limiting accurate assessment of the hyperpolarized 13C metabolites. The PA was validated in simulation and phantom studies, and was further applied to low-resolution human brain metabolite maps of hyperpolarized [1-13C]pyruvate and [1-13C]lactate with three compartment segmentation (grey matter, white matter and cerebrospinal fluid). The results demonstrated that the PA can enhance low-resolution hyperpolarized 13C images in terms of spatial resolution and contrast while preserving quantification accuracy and intra-compartment signal inhomogeneity.
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    Modeling Tumor Neoantigens for Predicting Patients' Clinical Outcomes
    (December 2021) Lu, Tianshi; Hoshida, Yujin; Wang, Tao; Xiao, Guanghua; Ahn, Chul; Aguilera, Todd A.
    Tumor neoantigens are critical targets of the host antitumor immune response and their presence play an important role in affecting tumor progressions and immunotherapy treatment response. Neoantigens showed a lot of potential of being applied to clinical treatment. However, systematic study of neoantigens' impact on tumors and patients is still challenging due to the huge diversity of neoantigens, heterogeneity within tumors, and the model to study the pairing between neoantigen-MHC and T cells to identify the neoantigens that truly elicit T cell response. To study the impact of neoantigen-T cell interaction on tumorigenesis, I developed a Bayesian hierarchical model to infer the history of neoantigen-cytotoxic T cell interactions in tumors.
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    The Molecular Basis of Tissue Elasticity and Force Balance During Drosophila Gastrulation
    (December 2021) Goldner, Amanda Nicole; Collins, James J.; Doubrovinski, Konstantin; Chen, Elizabeth; Douglas, Peter
    The mechanics of folding any material rely on two things: the physical forces forming the fold and the material properties of the substance being folded. When working in biological tissue such as an early Drosophila embryo, there is no existing way to directly measure morphogenetic forces, and the relative contributions of forces in various cellular domains remain unknown. To begin, I studied gastrulation in a genetic background where basal membranes never form and cells remain open to the yolk sack throughout the course of VF formation. Strikingly, the VF is still capable of forming in this background. I extensively characterize this phenotype by a combination of electron microscopy and immunofluorescence. My observations rule out a class of popular models of VF formation that would generically predict no folding in the absence of basal membranes. To address this discrepancy, we propose that viscous shear forces play a major role in allowing the furrow to form. We have developed a new computational model that takes cytoplasmic viscous shear into account. In accordance with our observations, our model predicts that basal membranes are dispensable for VF formation. Tissue material properties such as elasticity are also key to fold shape. In vivo tissue deformation experiments show that embryonic tissue is elastic in the stages leading up to gastrulation. Inhibiting F-actin polymerization severely decreases elasticity. I propose that different characteristics of F-actin networks - e.g. branching, remodeling, and crosslinking - are variably responsible for conferring elasticity. It is unclear whether the presence of active forces along actin filaments contributes to tissue elasticity. To this end, I engineered the auxin-inducible degron system to degrade the foremost source of active forces in F-actin networks: myosin II. My design allows us to specifically degrade Drosophila myosin II in under 1hr in vivo. This will allow us to precisely quantify the contribution of myosin II to not only tissue elasticity, but any other feature or developmental process of interest.
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    The Structural Distribution of Epistasis in a Pair of Essential Metabolic Enzymes
    (December 2021) Nguyen, Thuy Ngoc-Thi; Hibbs, Ryan E.; Reynolds, Kimberly A.; Rice, Luke M.; Yu, Hongtao
    Interactions between proteins provide the basis for cells to perform metabolism, grow, divide, move, and appropriately respond to external stimuli. Because proteins do not act as independent entities, the genetic background influences the effect of a mutation in unexpected ways. This context-dependence of mutational effects is epistasis. Extensive progress has been made in our ability to identify epistasis between proteins. However, how the epistasis between a pair of proteins is distributed across the amino acid sequence is less clear. Previous work characterized this sequence-level epistasis between proteins that bind to form a physical complex. Until now, the structural pattern and magnitude of epistasis between pairs of mutations spanning interacting metabolic enzymes remained uncharacterized. In my dissertation work, I deeply examined the context dependence of mutations for two essential enzymes in the bacterial folate metabolic pathway, Dihydrofolate Reductase (DHFR) and Thymidylate Synthase (TYMS). To achieve this goal, I used deep mutational scanning assays on DHFR in the context of varying activities of TYMS. The result is a rigorous dataset with epistasis measurements over the entire amino acid sequence of DHFR. I found that the positions with the greatest magnitude of epistasis within the structure of DHFR lied at the active site. However, the sign of epistasis at the DHFR active site was dependent on whether TYMS was active. Beyond the active site, the distribution of positive epistasis among the positions of DHFR was also context- dependent on the state of TYMS. Therefore, we can think of the active site as a non-physical "interface" between protein pairs that do not form a physical complex but share an intermediate. The potential consequences of this dataset on the epistasis between DHFR and TYMS are profound. This dataset is fundamental towards our understanding of how epistasis mechanistically emerges in nonlinearities between catalytic activity in enzymes, protein abundance, and cellular growth rate. This experimental dataset is also necessary to credibly validate predictions of epistasis from models of statistical co-evolution.
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    The Immunosuppressive Function of VEGF Signaling in the Tumor Microenvironment
    (December 2021) Zhang, Yuqing; Aguilera, Todd A.; Brekken, Rolf A.; Castrillon, Diego H.; Dellinger, Michael T.
    Angiogenesis, a hallmark of cancer, is induced by vascular endothelial growth factor-A (VEGF). As a result, anti-VEGF therapy is commonly employed for cancer treatment. However, anti-VEGF therapy generally provides modest efficacy in cancer patients and therapy-induced hypoxia results in a less differentiated mesenchymal-like tumor cell phenotype, which reinforces the need for effective companion therapies. Cyclooxygenase-2 (COX-2) inhibition has been shown to promote tumor cell differentiation and improve standard therapy response in pancreatic cancer. Here, I evaluate the efficacy of COX-2 inhibition and VEGF blockade in preclinical models of pancreatic cancer and identity it as a strategy to overcome therapy-induced resistance in pancreatic cancer. Combination therapy reverses anti-VEGF-induced epithelial-mesenchymal transition, collagen deposition and promotes an immune stimulatory microenvironment. Recent studies have also found that VEGF expression is also associated with immune suppression in cancer patients. This connection has been investigated in preclinical and clinical studies by evaluating the therapeutic effect of combining anti-angiogenic reagents with immune therapy. However, the mechanisms of how anti-VEGF strategies enhance immune therapy are not fully understood. We and others have shown selective elevation of VEGFR2 expression on tumor-associated myeloid cells in tumor-bearing animals. I further investigate the function of VEGFR2+ myeloid cells in regulating tumor immunity and find VEGF induces an immunosuppressive phenotype in VEGFR2+ myeloid cells including directly upregulating the expression of programmed cell death 1-ligand 1 (PD-L1). Moreover, I demonstrate that VEGF blockade inhibits the immunosuppressive phenotype of VEGFR2+ myeloid cells, increases T cell activation and enhances the efficacy of immune checkpoint blockade. These studies highlight the function of VEGFR2 on myeloid cells and provide mechanistic insight on how VEGF inhibition potentiates immune checkpoint blockade.
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    ADAP1 Promotes Latent HIV-1 Reactivation by Tuning the KRAS-ERK-AP-1 Signaling-Transcriptional Axis
    (December 2021) Ramirez, Nora-Guadalupe Piña; Schoggins, John W.; D'Orso, Iván; Pfeiffer, Julie K.; Alto, Neal
    Immune stimulation fuels cell signaling-transcriptional programs that induce biological responses to eliminate virus-infected cells. Yet, retroviruses that integrate into host cell chromatin, such as HIV-1, co-opt these programs to switch between latent and reactivated states. However, many regulatory mechanisms are still unfolding. As such, here I take advantage of the unique intrinsic reliance HIV-1 has on host cell signaling-transcriptional programs to discover undescribed cell signaling regulators. Specifically, I implemented a functional screening platform, given HIV-1 gene expression relies on CD4+ T cell activation state, to identify host factors modulating CD4+ T cell signaling-transcriptional axes and consequently HIV-1 fate. Among the hits, I focus on ADAP1 (ArfGAP with Dual PH Domains 1), a previously thought neuro-restricted factor, and discover it is an amplifier of select human CD4+ T cell signaling programs. Using physiological models, I characterize ADAP1 expression is low in naïve and memory CD4+ T cells, but largely induced upon immune stimulation where it interacts with the immune signalosome. Using complementary biochemical and cellular assays, I demonstrate ADAP1 directly stimulates the GTPase activity of KRAS to amplify CD4+ T cell signaling through targeted activation of ERK-AP-1 axis. In primary CD4+ T cells which I have genetically ablated ADAP1, I show loss of ADAP1 function blunts gene expression programs in response to stimulation thereby reducing CD4+ T cell expansion and dampening latent HIV-1 reactivation. Supporting the impact of these findings, I propose the reduced CD4+ T cell programs and proliferation upon ADAP1 loss validates Genome-wide Association Studies linking ADAP1 single nucleotide polymorphisms in non-coding enhancers to an altered T lymphocyte count trait, potentially attributed to ADAP1 haploinsufficiency. Through these combined experimental approaches, I was able to define ADAP1 as an unexpected tuner of CD4+ T cell activation programs and co-opted by HIV-1 to escape latency.
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    Identification of EWS-FLI1 Regions Necessary for Ewing Sarcoma Proliferation
    (December 2021) Bremauntz Enriquez, Alberto; Amatruda, James F.; McFadden, David G.; McKnight, Steven L.; Yu, Hongtao
    Ewing sarcoma is pediatric bone malignancy defined by a translocation between EWS and ETS family transcription factor. EWS-FLI1 (EF) is the most common translocation and codes for a novel transcription factor that combines the N-terminus of EWS, which contains LC domain comprised of tyrosine rich peptide repeats, and C-terminal portion of FLI1, which contains the ETS DNA binding domain. EF is a key transcriptional regulator known to both activate and repress genes. While understanding of the molecular mechanism by which EF controls transcription have become clearer, targeted therapeutic interventions against EF or its transcriptional program have yet to make clinical impact. This is due in part to a poor understanding of how the N-terminus of EWS is contributing to the oncogenic program and a disparate range of reported effects after EF depletion on Ewing sarcoma cells. This report shows the adaptation of two inducible degron systems, Small Molecule Assisted Shut-Off (SMASh) and Auxin-inducible degron (AID), into the endogenous locus of EF in a series of Ewing sarcoma cell lines to define the phenotype of EF depletion. Across multiple cell line and degradation mechanisms, EF depletion in Ewing sarcoma cells leads to decreased cell proliferation through G1/S arrest that can be rescued through re-expression of EF. Having established a baseline for EF depleted cells, I developed a proliferation-based assay to test the functionality of mutant EF constructs on their ability to drive proliferation in the setting of endogenous EF depletion. I tested a series of EF truncations, with single or multiple exon deletions in the EWS portion of the translocation. Expression of EF constructs with loss of any single exon was tolerated and allowed for continued proliferation, but loss of at least exons 1-4 on the N-terminus and loss of exons 5-7 from the C-terminus resulted in non-functional EF constructs. Given that there appear to be redundant elements within the N-terminus of EF, I tested truncated and mutant version of a minimal rescue construct containing only exon 1-5 of the N-terminus of EF. Mutation of only 3 tyrosines to serine within the minimal construct was sufficient to prevent proliferation in Ewing cell lines.
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    Factors Governing Gastrointestinal Colonization of Candida albicans
    (December 2021) Mishra, Animesh Anand; Hendrixson, David R.; Hooper, Lora V.; Winter, Sebastian E.; Koh, Andrew Y.
    Candida albicans can colonize the human gastrointestinal tract (GI) and cause disseminated infections in immunocompromised hosts. Depletion of specific gut commensal microbiota is associated with or results in increased C. albicans burden in the gut and increased likelihood of dissemination in human patients and mice, respectively. The exact mechanisms by which gut microbiota mediate C. albicans colonization resistance in the gut, however, are unknown. Here, we show that gut microbiota-derived short chain fatty acids (SCFA) directly inhibit C. albicans growth in vitro. SCFA inhibit C. albicans hexose uptake and induce intracellular acidification. In contrast, SCFA promote C. albicans GI colonization resistance in vivo but only when an intact gut microbiome is present. SCFA induce gut microbiota composition changes that promote C. albicans colonization resistance. Commensal gut microbiota unable to produce SCFA have a diminished capacity to reduce C. albicans GI colonization. Prebiotic therapy results in increased GI SCFA levels which enhance C. albicans GI clearance. This work also describes two C. albicans isolates 529L and CHN1 that can stably colonize the murine GI tract without the use of antibiotics. These clinical isolates have a higher resistance to antimicrobial peptide CRAMP compared to the most commonly studied C. albicans laboratory strain SC5314. Thus, the work sheds light on mechanisms that might be critical in governing C. albicans gastrointestinal colonization levels. It provides mechanistic insights into the importance of gut microbiota-derived metabolites in maintaining C. albicans colonization resistance and may have therapeutic implications for modulating C. albicans gastrointestinal colonization levels in order to prevent invasive candidiasis in immunocompromised patients. Further, C. albicans strain-specific difference in colonization ability appears to depend on the sensitivity to these host immune effectors. The described isolates can further serve as valuable tools to probe the mechanisms of C. albicans gastrointestinal colonization without the intervention of any antibiotics.
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    FishATLAS: Assessment of Organotropism Determination Through Imaging Informatics of Xenografted Zebrafish
    (December 2021) Saucier, David Hamilton; Fiolka, Reto; Danuser, Gaudenz; Amatruda, James F.; Whitehurst, Angelique Wright; Brekken, Rolf A.; Mason, Ralph P.
    Ewing sarcoma patients with metastatic disease have a 5-year survival rate of approximately 28%. The hallmark of this disease is an aberrant transcription factor made by a fusion of Chromosomes 11 and 22 called EWSFLI1. EWSFLI1 expression levels have been correlated with differing responses in cell metastatic propensity, but much remains to be elucidated. Indeed, many current models fail to meet the statistical rigor that is needed for exceedingly spontaneous, rare events like metastasis. To address this need, FishATLAS utilizes zebrafish human cancer cell xenograft images after high fidelity registration using a novel diffeomorphic transformation to display metastatic hot spots of different cancer cell conditions to begin to grasp the deeper underpinnings of organotropism in vivo with individual cancers. Utilizing a suite of statistical tests, FishATLAS determines at a global whole-fish scale and the local microenvironment, if there are statistically different cell hotspots when comparing two or more different conditions. As it stands, data for EWSFLI1, its target SOX6, a non-transformed cell line NIH3T3, TC32 subclones, and melanoma cell lines have all shown unique distributions of metastatic hot spots. These findings serve as a tool for drug discovery and later environmental re-mapping in FishATLAS by allowing transgenic fish images (such as vasculature and lymphatics) to be overlayed onto any historical data set. These can then be used to determine a given microenvironment's contributions to secondary sites of metastasis. In the case of EWSFLI1 and its target SOX6, there was a marked difference upon shRNA-mediated knockdown that removed a population of hotspots in the upper somitic veins while some were persistent post genetic perturbation. SOX6 shRNA KD data indicates that the somite and inter-somitic arteries are more sensitive for metastatic colonization. Previous studies and our current accumulator data suggest these to be regions with higher oxidative stress, guiding insights for oxidative-mechanistic therapy. These and other conditional accumulations of sparse metastatic hotspots demonstrate the power of FishATLAS as a longitudinal assay of cellular and genetic conditions across all cancers.
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    Exploiting Multi-Cell Type Cultures to Elucidate Tumor Cell Features That Impact Macrophage Phenotype
    (December 2021) Voth Park, Josiah Malachi; Kim, James; Minna, John D.; Brekken, Rolf A.; Akbay, Esra A.; Malter, James; Huang, Lily
    Lung cancer is expected to kill ~150,000 people this year, encompassing 25% of all cancer related deaths making lung cancer the leading cause of cancer-related mortality in men and women. Lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) which represent 80-85% and 15-20% of cases, respectively. My dissertation project focused on understanding how to model the interactions between lung cancer cells, fibroblasts and immune cells. Immune cells are critical components of the tumor microenvironment (TME) that contribute to tumorigenesis, angiogenesis and metastasis. Macrophages are key regulators of the immune landscape within the TME. The plasticity of macrophage phenotypes in the TME correlates with prognosis of NSCLC. Depending on their phenotype, macrophages in the TME can secrete pro-tumor cytokines and chemokines, ultimately suppressing the function of anti-tumor immune cells in the TME. The purpose of my project was to investigate if and how NSCLC cells alter macrophage phenotype in multi-cellular co-cultures and to relate effects on macrophages to the molecular characteristics of different NSCLCs. The central hypothesis of the project is, tumor cell characteristics drive macrophage polarization in the TME, and this can be captured using a multicellular co-culture model. Given the central importance of macrophages to the TME and the immune landscape of NSCLC, an understanding of the tumor cell characteristics associated with immune suppressive or immune stimulatory macrophage phenotype could be exploited from a therapy perspective in the future. To address this hypothesis, an in vitro co-culture system (NSCLC tumor cells, human cancer associated fibroblasts (CAFs), and mouse macrophages) was developed to interrogate cancer cell features driving heterogeneity of macrophage phenotypes across a panel of NSCLCs. We measured: mRNA expression in mouse macrophages with a panel of qPCR probes for genes associated with distinct macrophage phenotypes (Arg1, iNOS, Il-1β, Il-6, Ym-1, Socs3). This system was validated by comparison of macrophage phenotypes represented in the TME of lung cancer xenografts grown in athymic nude mice. Using our platform, we evaluated ~80 NSCLC patient derived lines for their effect on mouse macrophage phenotype. We identified three main macrophage phenotypes across this panel of NSCLCs. To identify cancer cell biomarkers for macrophage polarization, we interrogated molecular characteristics of the cancer lines. Additionally, we expanded the functionality of the platform to assess the effects of pharmacologic agents on macrophage phenotype. As a proof of principle, a small panel of known immune stimulating compounds was tested in the in vitro co-culture platform and validated in human tumor xenografts. Finally, we identified a few novel compounds that show selective cancer cell toxicity and reprogram macrophage phenotype. In conclusion, we built a reproducible in vitro platform to interrogate macrophage polarization in the TME. We leveraged this platform to identify three dominant macrophage phenotypes induced by NSCLC cells and CAFs. We found that no cancer cell molecular characteristic alone drives macrophage polarization. Finally, we illustrate the significance of this platform for immune stimulating drug identification; we identified two novel chemicals that repolarize macrophages and kill cancer cells simultaneously.
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    AXL Targeting Restores PD-1 Blockade Sensitivity of STK11/LKB1 Mutant NSCLC Through Expansion of TCF1+ CD8 T Cells
    (December 2021) Li, Huiyu; Akbay, Esra A.; DeBerardinis, Ralph J.; Fu, Yang-Xin; Brekken, Rolf A.; Minna, John D.; Aguilera, Todd A.
    Mutations in STK11/LKB1 in non-small cell lung cancer (NSCLC) are associated with poor patient responses to immune checkpoint blockade (ICB) for unknown reasons. We found that introduction of a Stk11/Lkb1 (L) mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 (KP) resulted in an ICB refractory syngeneic KPL tumor. Mechanistically, this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype that was recapitulated in human STK11/LKB1 mutant NSCLCs. We found that systemic inhibition of Axl results in increased type I interferon secretion from dendritic cells that expands tumor-associated TCF1+ PD-1+ CD8 T cells, restoring therapeutic response to PD-1 ICB for KPL tumors. This effect was observed in syngeneic immunocompetent mouse models and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts. Anecdotal NSCLC patients with STK11/LKB1 mutant tumors also demonstrated responses to the combination of AXL inhibitor bemcentinib and pembrolizumab. We conclude that AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC.
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    Myogenic Effectors and Disease
    (December 2021) Ramirez Martinez, Andres; Sadek, Hesham A.; Mendell, Joshua T.; De Martino, George; Olson, Eric N.
    Skeletal muscle is essential for life. Inside muscle fibers, filaments of actin and myosin slide on each other to generate the mechanical forces that drive muscle contraction, movement, and breathing. Mutations in muscle-related genes can cause severe diseases in humans. Here we characterize the role of three understudied muscle-specific genes and their potential contribution to human disease. We show that constitutive and juvenile loss of the nuclear envelope protein Net39 in mice recapitulates different manifestations of Emery-Dreifuss muscular dystrophy. Deletion of Net39 caused disruption of nuclear envelope integrity and associated genomic, transcriptional, and metabolic changes that compromised muscle function. Mechanistically, Net39 regulates nuclear organization by associating with LEM proteins, and gene expression by controlling the transcription factor Mef2c. In contrast, global deletion of the Kelch protein Klhl41 in mice causes severe nemaline myopathy, including neonatal lethality and aggregation of contractile proteins in muscle, particularly Nebulin. Molecularly, Klhl41 acts as a chaperone for Nebulin, and N-terminal poly-ubiquitination of Klhl41 acts as a signal to regulate its activity. Finally, we identify a novel pathogenic mutation in the cell fusogen Myomixer. We show that patients with Carey-Fineman-Ziter syndrome lose a region of Myomixer required to destabilize opposing cell membranes during myoblast fusion. Overall, our findings here highlight the contribution of understudied genes to muscle biology and the molecular etiology of muscle disorders.
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    Investigating Vascular Patterning and Regression in Kidney Development and Organoids
    (December 2021) Ryan, Anne Regina; Marciano, Denise; Munshi, Nikhil; Dellinger, Michael T.; Cleaver, Ondine
    Chronic kidney disease (CKD) and end stage renal disease (ESRD) are increasingly frequent and devastating conditions that have driven a surge in the need for kidney transplantation. A stark shortage of organs has fueled interest in generating viable replacement tissues ex vivo for transplantation. One promising approach has been self-organizing organoids, which mimic developmental processes and yield multicellular, organ-specific tissues. However, a recognized roadblock to this approach is that many organoid cell types fail to acquire full maturity and function. I comprehensively assessed the vasculature in two distinct kidney organoid models as well as in explanted embryonic kidneys. Using a variety of methods, my work shows that while organoids can develop a wide range of kidney cell types, as previously shown, endothelial cells (ECs) initially arise but then rapidly regress overtime in culture. Vasculature of cultured embryonic kidneys exhibit similar regression. By contrast, engraftment of kidney organoids under the kidney capsule results in the formation of a stable, perfused vasculature that integrates into the organoid. This work demonstrates that kidney organoids offer a promising model system to define the complexities of vascular-nephron interactions, but the establishment and maintenance of a vascular network present unique challenges when grown ex vivo. The future of the field necessitates the inclusion of flow and perhaps additional factors into in vitro culture methods. Future studies investigating endothelial heterogeneity in the developing kidney will aid in forwarding our mission of creating a functional organoid vasculature.