UT Southwestern Graduate School of Biomedical Sciences

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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.

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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.

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Now showing 1 - 20 of 1597

Carbon Starvation Metabolically Regulates Chromatin for Transcriptome Rewiring
(2022-05) Hsieh, Wen-Chuan; Conrad, Nicholas; Kraus, W. Lee; Orth, Kim; Tu, Benjamin
Cells robustly rewire their transcriptomes to survive under stress conditions. Yet, how does such reprogramming of gene expression occur? Under favorable nutrient conditions, acetyl-CoA normally promotes histone acetylation to activate genes required for cell growth. However, glucose starvation significantly reduces the availability of acetyl-CoA. And it is unclear how such a change impacts genome-wide histone acetylation and gene expression. In this study, I set up a robust glucose starvation model in budding yeast to discover a mechanism by which cells preserve acetyl-CoA, a key intermediate in energy metabolism, in order to sustain histone acetylation for gene activation even under stress conditions. I demonstrate a dramatic redistribution of histone acetylation upon glucose starvation. Mechanistically, I determined that a major histone deacetylase (HDAC) releases acetyl groups from histones at growth-promoting genes, which can subsequently be used to acetylate histones at a distinctive set of stress-responsive genes. Strikingly, bioinformatic analysis revealed these genes to be required for gluconeogenic and fat metabolism, which are metabolic pathways that generate acetyl-CoA for oxidation and ATP synthesis. Genetic deletion of histone modifiers mediating this reallocation, including the key HDAC or histone acetyltransferase (HAT), disrupts proper transcriptome rewiring for survival. Given the importance of acetate for recycling the acetyl- group, I next characterize acetyl-CoA synthetases (Acs), metabolic enzymes that convert acetate to acetyl-CoA. I demonstrate that Acs2 is required for maintaining global histone acetylation, yet its nuclear localization appears to be dispensable for such regulation. I observe that the catalytic activity of Acs2 governs the intracellular acetyl-CoA level and global histone acetylation amounts. Compromising its activity leads to up-regulation of ergosterol biosynthetic pathways in addition to gluconeogenic and fat metabolism genes upon glucose starvation. In summary, I reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting. I have elucidated how transcriptome rewiring is driven by reallocation of histone acetylation. My findings present a mechanism by which cells recycle acetyl groups to differentially acetylate histones for activation of key genes required for metabolism and survival.
Structural and Molecular Mechanisms of Centrosome Assembly and Strength
(2024-05) Rios, Manolo Uriel; Doubrovinski, Konstantin; Rosen, Michael K.; Woodruff, Jeffrey B.; Seemann, Joachim
The outermost layer of centrosomes, called pericentriolar material (PCM), organizes microtubules for mitotic spindle assembly. The molecular interactions that enable PCM to assemble and resist external forces are poorly understood. To answer such question, we use cross-linking mass spectrometry (XL-MS) to analyze PLK-1-potentiated multimerization of SPD-5, the main PCM scaffold protein in C. elegans. In the unassembled state, SPD-5 exhibits numerous intramolecular interactions that are eliminated after phosphorylation by PLK-1. Thus, phosphorylation induces a structural opening of SPD-5 that primes it for assembly. Multimerization of SPD-5 is driven by interactions between multiple dispersed coiled-coil domains. Structural analyses of a phosphorylated region (PReM) in SPD-5 revealed a helical hairpin that dimerizes to form a tetrameric coiled-coil. Mutations within this structure and other interacting regions cause PCM assembly defects that are partly rescued by eliminating microtubule-mediated forces, revealing that PCM assembly and strength are interdependent. We propose that PCM size and strength emerge from specific, multivalent coiled-coil interactions between SPD-5 proteins.
Factors That Influence Murine Norovirus Stability and Tropism
(2024-05) Budicini, Melissa Renee; Gammon, Don B.; Schoggins, John W.; Pfeiffer, Julie K.; Orchard, Robert C.
In this work I investigated factors that influence murine norovirus stability and tropism through two independent projects. Murine norovirus (MNV) is a model system used to study human noroviruses due to its robust replication in cell culture and mouse model. I first investigated the interactions between MNV and bacteria in vitro by determining what bacteria and bacterial components could impact viral thermostability. I found that that specific Gram-positive bacteria and conditioned medium from Gram-positive bacteria could stabilize MNV against heat inactivation. However, I found that Gram-negative bacteria and conditioned medium had no impact on viral stability. These stabilizing effects of bacteria may play a role in viral transmission due to the fact that the virus must remain stable in the environment to transmit to a new host. In my second project I used a forward genetic approach to select for MNV variants with increased host cell range. I found that by serially passaging murine norovirus in human HeLa cells I could select for mutant viruses that increased replication as compared with the parental strain in a non-natural host. The passaged viruses had many mutations spanning the viral genome, however I determined three specific mutations in the NS1/2 protein that allowed for the virus to grow better in human cells. I determined that the adapted viruses have increased replication because they overcame a post-entry replication block in HeLa cells, not because they have increased attachment. This was surprising given that HeLa cells do not have the MNV receptor. These studies show that MNV tropism is not only determined by receptor availability. Overall, these studies illuminate unique aspects of MNV biology that may be applicable to other viruses.
Experience-Dependent and Input-Specific Regulation of Neocortical Circuit Development by Genes Linked to Neurodevelopmental Disorders
(2022-05) Zhang, Zhe; Volk, Lenora J.; Huber, Kimberly M.; Gibson, Jay R.; Roberts, Todd; Chahrour, Maria
Abnormal structural and functional brain connectivity has been widely observed in human neuropsychiatric diseases. Specifically, patients with neurodevelopmental disorders like autism often show an imbalance in the local versus long-range connectivity for cerebral cortex. Whether and how genes implicated in neurodevelopmental disorders regulate development of cortical synaptic connectivity in a pathway-specific manner remain largely unknown. Furthermore, environmental sensory experience can determine or significantly remodel the postnatal development of synaptic connections and neural circuits in sensory cortices. Knowledge on what intracellular proteins or mechanisms can mediate experience-dependent development of specific cortical synaptic connections is also lacking. In this work, I studied the roles of two neurodevelopment disease implicated genes, namely, fragile X mental retardation 1 (Fmr1) and myocyte enhancer factor 2c (Mef2c) in the postnatal experience-dependent development of input-specific synaptic connections. I report that postnatal, cell-autonomous deletion of Fmr1 in postsynaptic L2/3 or L5 neurons results in a selective weakening of AMPA receptor-, but not NMDA receptor-, mediated callosal synaptic function, indicative of immature synapses. Sensory deprivation by contralateral whisker trimming normalizes callosal input strength, suggesting that experience-driven activity of postsynaptic Fmr1 KO L2/3 neurons weakens callosal synapses. Unlike callosal inputs, synapses originating from local L4 and L2/3 circuits are normal with postsynaptic Fmr1 deletion, revealing an input-specific role for postsynaptic Fmr1 in regulation of synaptic connectivity within local and callosal neocortical circuits. Opposite to Fmr1 KO, postnatal deletion of Mef2c in L2/3 neurons leads to a cell autonomous and selective weakening of excitatory synapses from L4, whereas ipsilateral or contralateral long-range excitatory synaptic inputs are unaffected. Postsynaptic Mef2c only promotes the development but not the maintenance of L4-to-L2/3 excitatory synaptic connections and Fmr1 is not required for this process, in contrast to predictions from work in CA1 hippocampal neurons. Weakening of L4-L2/3 synaptic strength by sensory deprivation can be rescued by postnatal postsynaptic expression of a transcriptionally active form of MEF2C (MEF2-VP16), suggesting that MEF2C transcriptional activation drives experience-dependent development of L4-L2/3 synapses. Together, my findings on Fmr1 and Mef2c demonstrate an interaction of experience and gene functions in regulation of specific synaptic connections with important implications for neurodevelopmental disorders.
The Crosstalk Between DNA Mismatch Repair and Replication
(2022-05) Zhang, Junqiu; Davis, Anthony John; Castrillon, Diego H.; Erzberger, Jan; Li, Guo-Min
DNA replication fidelity relies on DNA mismatch repair (MMR) and the proofreading nuclease activity of DNA polymerases. Normally, biosynthetic errors can be removed by the polymerase's proofreading nuclease activity upon their incorporation, and those errors that have escaped the proofreading nuclease are corrected by MMR. However, this model is challenged by the fact that cells expressing a proofreading-deficient P286R polymerase ɛ (Polɛ-P286R) display a hypermutable phenotype usually seen in MMR-deficient cells, implying the blockage of MMR function by Polɛ-P286R. We show here that consistent with frequent misincorporation by Polɛ-P286R, elevated levels of MMR proteins were found in replicating DNA/chromatin in Polɛ-P286R cells, but this does not result in a reduced mutation frequency, suggesting that cluster binding of MMR proteins at the replication fork inhibits MMR. Instead, the high-level binding of MMR proteins blocks the recruitment of fork protection factors FANCD2 and BRCA1 to replication forks, and promotes MRE11-catalyzed nascent strand degradation. This MMR-dependent degradation causes DNA breaks and chromosome abnormalities, thereby promoting an ultramutator phenotype. Therefore, our findings identify a novel MMR function in triggering replication stress response to promote genome instability when replication forks are filled with biosynthetic errors. The importance of MMR in maintaining genome stability prompts us to further study the mechanism of MMR in vitro, particularly how the MMR initiation complex is formed in response to misincorporation. Using purified recombinant proteins, we assembled MMR initiation complex in vitro and visualized protein-protein and protein-DNA interactions under transmission electron microscopy. These analyses allowed us to gain molecular insights into the mechanism of MMR initiation.
The Role of TNRC6 in RNA Interference
(2022-05) Johnson, Samantha Tori; Liu, Yi; Conrad, Nicholas; Kraus, W. Lee; Corey, David R.
Small RNAs can influence translation, splicing, transcriptional activation, and transcriptional repression, through the RNA interference (RNAi) pathway. TNRC6, also known as GW182, and Argonaute (AGO) are the core proteins of RNAi. TNRC6 is a scaffolding protein that associates with AGO and bridges its interactions with other proteins to control gene expression in many different processes. There are three paralogs in mammalian cells, TNRC6A, TNRC6B, and TNRC6C. These paralogs share approximately 40% amino acid sequence identity. Whether the paralogs have unique or redundant functions is unclear. Much is known about the mechanisms of cytoplasmic RNAi but the world of nuclear RNAi remains murky. We understand that RNAi factors are present and active in the nucleus, but endogenous nuclear RNAi functions are unknown. I have used a suite of gene knockout cell lines for the TNRC6 paralogs to learn more about TNRC6 paralogs and their roles in RNAi. I examined if TNRC6 paralogs were required in several functions of small duplex RNA-mediated control of gene expression, including translational silencing by miRNAs, translational silencing by siRNAs, and transcriptional activation. On a global scale, I used high-throughput RNA sequencing, mass spectrometry, and enhanced crosslinking immunoprecipitation (eCLIP) to gather definitive answers about the TNRC6 paralogs, their roles in the cell, and their relation to AGO knockout cell lines. I found that that despite less than 40% sequence identity, the TNRC6 paralogs are functionally redundant and can replace one another for core RNAi functions. Each subsequent TNRC6 paralog knockout caused more gene changes and few genes overlapped between the single TNRC6 paralog knockouts. Changes in levels of gene expression in TNRC6 knockout cell lines are well-correlated with those observed in AGO knockout cell lines, emphasizing the important regulatory function of the partnership between AGO and TNRC6 in endogenous RNAi. Further, I found TNRC6 plays a role in splicing changes initiated by small RNA binding to intronic regions. Taken together these data further define the roles of the TNRC6 paralogs as part of the RNA interference machinery. TNRC6 is a close protein partner of AGO and serves as a cooperativity coordinator for RNAi.
Elucidating the Secreted Bacterial Kinome
(2022-05) Lopez, Victor Antonio; Alto, Neal; Cobb, Melanie H.; Tagliabracci, Vincent S.; Henne, W. Mike
In the post-genomic era, millions of amino acid sequences have been acquired and revealed a remarkable diversity among protein families. This is especially apparent in pathogenic species where host and pathogen participate in an intense evolutionary arms race, and horizontal gene transfer is common. We have taken a bioinformatic approach to identify and characterize distant members of the protein kinase superfamily that share little sequence similarity, yet retain a kinase fold, and possibly catalytic activity. Using this approach, we identified the uncharacterized HopBF1and MavQ families of bacterial type III and type IV secretion system effectors, respectively, as remote members of the eukaryotic protein kinase superfamily. We demonstrate that the HopBF1 kinases are eukaryotic-specific HSP90 kinases. HopBF1 phosphorylates HSP90 on a strictly conserved serine that potently inactivates its ATPase activity, and inactivation by phosphorylation prevents the maturation or folding of various HSP90 clients, including immune receptors necessary for the activation of the hypersensitive response in plants. Consequently, HopBF1 kinase activity is sufficient to induce severe disease symptoms in plants infected with the bacterial plant pathogen, Pseudomonas syringae. Moreover, we show that MavQ is a phosphatidylinositol (PI) 3-kinase required for full Legionella virulence in a eukaryotic host. MavQ and the Legionella PI 3-phosphatase SidP drive rapid PI 3-phosphate turnover on the ER and spontaneously form traveling waves that spread along ER subdomains and induce vesicle/tubule budding. Our results reveal a novel mechanism by which self-organizing bacterial effectors remodel host cellular membranes for survival and uncover a family of bacterial effector kinases which act to compromise the host immune response through a "betrayal-like" mechanism.
β-Globin Enhancers Regulate Bistable Gene Expression States
(2022-05) Herbert, Jeremiah David; Collins, James J.; Hon, Gary C.; Buszczak, Michael; O'Donnell, Kathryn A.
Multiple enhancers can coordinately regulate a target gene to ensure robust expression. Under the failure rate model, each redundant enhancer contributes a probability of target gene expression in the cell, and genetic deletion will cause single-cell expression phenotypes. However, this model has been challenging to test in human cells. To examine the role of the human β-globin enhancers in controlling single-cell expression phenotypes, we engineered a haploid locus in K562 cells that fluorescently reports the expression of HBG1. We isolated combinatorial enhancer deletion clones having single-cell expression phenotypes. Genetic and epigenetic analysis shows that deletion of HS3 or HS4 yields clonal cells having bimodal expression of HBG1. Time course analysis indicates that clonal cells can transition between bimodal expression states, and that GFP+ cells have more open chromatin at the HBG1 promoter than GFP- cells. Finally, we derive failure rate contributions for several enhancers. Thus, β-globin enhancers regulate bistable gene expression states, supporting the failure rate model of enhancer redundancy in human cells.
Hippocampal Output to the Septum Regulates Locomotion and Exploration
(2022-05) Chen, Yuh-Tarng; Pfeiffer, Brad E.; Xu, Wei; Meeks, Julian P.; Tamminga, Carol
The cognitive maps are established in the dorsal hippocampus (DH) when the animal explores the environment. During exploration, the DH combined extrinsic inputs from multiple sensory and motor-related structures to generate locomotion-dependent activity; however, the hippocampal mechanisms of locomotion and exploratory behavior remain controversial and unclear. By using optogenetics and chemogenetics, we selectively manipulated the DH pan-inhibitory interneurons (INs) and revealed that disrupting the DH inhibitory circuit is sufficient to acutely induce hyperlocomotion. Furthermore, we found that the DH-septum inhibitory pathway is critical for regulation of locomotion and exploratory behavior. Activation of the DH inhibitory output to the medial septum complex (MSc) decreased animals' distance traveled and increased immobility in the open field. Alternatively, inhibition of the DH inhibitory output to the MSc increased maximum speed and exploratory behavior. Moreover, activation of the septum-projecting DH INs is sufficient to decrease locomotion and exploratory behavior. On the other hand, activation of a subset of retrosplenial cortex-specific projecting DH INs or the entorhinal cortex-projecting DH neurons did not change animal locomotion. For the clinical application, targeting the DH-MSc inhibitory pathway can help us to determine the neuronal mechanisms of locomotion in animal models. Therefore, we may be able to ameliorate spatial processing deficits and motor symptoms in patients with memory impairment or psychosis and find therapeutic treatments for them.
UBE2J1 Is the E2 Ubiquitin Conjugating Enzyme Regulating AR Degradation and Resistance to Antiandrogen
(2024-05) Rodriguez Tirado, Carla Sofia; O'Donnell, Kathryn A.; Pan, Duojia; Tagliabracci, Vincent S.; Mu, Ping
Prostate cancer (PCa) is primarily driven by aberrant Androgen Receptor (AR) signaling. Although there has been substantial advancement in antiandrogen therapies, resistance to these treatments remains a significant obstacle, often marked by continuous or enhanced AR signaling in resistant tumors. While the dysregulation of the ubiquitination-based protein degradation process is instrumental in the accumulation of oncogenic proteins, including AR, the molecular mechanism of ubiquitination-driven AR degradation remains largely undefined. We identified UBE2J1 as the critical E2 ubiquitin conjugating enzyme responsible for guiding AR ubiquitination and eventual degradation. The absence of UBE2J1, found in 5-15% of PCa patients, results in disrupted AR ubiquitination and degradation. This disruption leads to an accumulation of AR proteins, promoting resistance to antiandrogen treatments. By employing a ubiquitination-centric AR degrader to adeptly restore AR ubiquitination, we reestablished AR degradation and curtailed the proliferation of antiandrogen-resistant PCa tumors. These findings underscore the fundamental role of UBE2J1 in AR degradation and illuminate an uncharted mechanism through which PCa maintains heightened AR protein levels, fostering resistance to antiandrogen therapies.
Novel Functions of the Transcription Factor Aryl Hydrocarbon Receptor (AHR) and Its Tryptophan-Derived Ligands in Cancer Cells
(2024-05) Perez Castro, Lizbeth; Green, Carla B.; Shay, Jerry W.; Conacci-Sorrell, Maralice; Friedman, Jonathan R.
This comprehensive study delves into the metabolic reprogramming of cancer cells, focusing on the proto-oncogene MYC and the aryl hydrocarbon receptor (AHR). The role of MYC in regulating tryptophan (Trp) metabolism was investigated in colon and liver cancer cells using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our findings reveal that MYC enhances the intracellular levels of Trp and its metabolites in the kynurenine pathway, specifically increasing the expression of Trp transporters SLC7A5, SLC1A5, and the enzyme AFMID. Elevated levels of these components were observed in immortalized colon cancer cells lines and patient tissues, with a significant increase in kynurenine. Blocking enzymes in this pathway led to the preferential death of cancer cells, suggesting a potential therapeutic strategy. Furthermore, our research highlights the role of AHR in cellular detoxification and proliferation, particularly in MYC-overexpressing cells. We found that AHR knockdown reduced the expression of genes crucial for metabolic pathways necessary for cell proliferation, such as LDHA, DHODH, and UMPS. Additionally, we identified SCIN, an actin-severing protein, as a key target of AHR in colon cancer cells, necessary for cell proliferation and activation of the WNT pathway through β-catenin. In liver cancer, we discovered that MYC-driven tumors have a critical dependence on Trp, with a diminished utilization of the Trp via the Kyn pathway. Depriving these tumors of Trp inhibits their growth, while supplementation with the Trp metabolite I3P restores growth, presenting a novel therapeutic target. Overall, our findings underscore the importance of MYC and AHR in regulating amino acid metabolism in cancer and open new avenues for targeted cancer therapies.
Identification and Characterization of a Nuclear Export Pathway for GC-Rich RNAs in Mammalian Cells
(2022-05) Thomas, Anu; Chen, Zhijian J.; Conrad, Nicholas; Yu, Hongtao; Mendell, Joshua T.
Splicing promotes nuclear export of RNAs by recruiting export factors to nascent transcripts. Nevertheless, many intronless RNAs are efficiently exported to the cytoplasm through mechanisms that remain poorly characterized. To identify new RNA export factors, we performed a genome-wide CRISPR screen to investigate the mechanism of export of NORAD, an intronless cytoplasmic long noncoding RNA. This screen revealed an RNA binding protein, RBM33, that directs the nuclear export of NORAD and numerous other transcripts. RBM33 directly binds substrate transcripts and recruits components of the NXF1-dependent RNA export pathway. Interestingly, high GC-content, not intron number or splicing efficiency, emerged as the feature that specifies RBM33-dependent nuclear export. Accordingly, RBM33 directly binds GC-rich elements in target transcripts. Importantly, these targets are nuclear enriched in RBM33 deficient cells, thereby establishing the role of RBM33 as a hitherto unknown nuclear export factor. These results provide a broadly applicable approach for the genetic dissection of nuclear export mechanisms and reveal the existence of an alternative RNA export route for GC-rich transcripts, providing a mechanistic basis for the long-standing observation that GC-rich sequence composition boosts gene expression and cytoplasmic localization.
Glutamine Antagonism and Its Utlity [sic] as a Therapeutic Modality in Cancer
(2022-05) Rosales, Tracy Ibarra; Kim, James; DeBerardinis, Ralph J.; Whitehurst, Angelique Wright; Conacci-Sorrell, Maralice
Glutamine metabolism is important in cancer as it fuels the TCA cycle, plays a role in redox homeostasis, and contributes to the production of nucleotides, amino acids, and lipids for survival, making glutamine metabolism a promising target in cancer therapy. The work outlined in this dissertation focuses on understanding the mechanism of the broad glutamine metabolism inhibitor, 6-diazo-5-oxo-L-norleucine (DON) and its prodrug, JHU-083, while comparing them to the effects of CB-839, a specific glutaminase inhibitor. DON is one of the oldest and well-known glutamine antagonists and can effectively limit tumor growth in a preclinical setting. Unfortunately, DON was removed from early phase clinical trials due to unacceptable toxicity in the gastrointestinal tract (GI). Thus, DON prodrugs were recently developed to be inactive until cleaved by cathepsins enriched in the tumors or by plasma esterases, bypassing toxicity in the GI tract. Using isotope tracer studies in cancer cells and mouse xenograft models, I found that DON and JHU-083 mainly inhibit glutamine-derived nitrogen labeling in purines but unexpectedly does not limit the contribution of glutamine-derived carbon labeling of tricarboxylic acid (TCA) cycle metabolites. Additionally, I found that DON and JHU-083 can limit the levels of purines but not the levels of most TCA cycle metabolites. These findings suggest that these drugs are poor inhibitors of glutaminase in the cancer cell lines tested and that DON and JHU-083 mainly target purine metabolism. Recognizing DON and JHU-083 as effective purine metabolism inhibitors can offer insight into which cancer patients could benefit from these drugs. Relapsed small-cell lung cancer (SCLC) is characterized by an upregulation of de novo purine biosynthesis and have few durable therapies. Using metabolic tracing and untargeted metabolomics, I found that DON can inhibit purine metabolism in treatment-naïve and chemoresistant pairs of SCLC. In a mouse xenograft model of relapsed SCLC, JHU-083 induces a delay in tumor growth without overt side effects. My work provides an opportunity to explore JHU-083 as an anti-cancer therapy for diseases that depend on purine biosynthesis.
A Molecular Basis for Macrophage Control of Enteric Nervous System Function
(2022-05) Pendse, Mihir Vidyadhar; Reese, Tiffany A.; Hooper, Lora V.; Kliewer, Steven A.; Chen, Zhijian J.
Peristaltic movement of the intestine propels food down the length of the gastrointestinal tract to promote nutrient absorption. Interactions between intestinal macrophages and the enteric nervous system regulate gastrointestinal motility, yet we have an incomplete understanding of the molecular mediators of this crosstalk. Here we identify complement component 1q (C1q) as a macrophage product that regulates gut motility. Macrophages were the predominant source of C1q in the mouse intestine and most extraintestinal tissues. Although C1q mediates complement-mediated killing of bacteria in the bloodstream, we found that C1q was not essential for immune defense of the intestine. Instead, C1q-expressing macrophages were localized to the intestinal submucosal plexus where they closely associated with enteric neurons and expressed surface markers characteristic of nerve-adjacent macrophages in other tissues. Mice with a macrophage-specific deletion of C1qa showed changes in enteric neuronal gene expression, increased peristaltic activity, and accelerated intestinal transit. Our findings identify C1q as a key regulator of gastrointestinal motility and provide enhanced insight into the crosstalk between macrophages and the enteric nervous system.
Intestinal Epithelial Cell Intrinsic IL-1R Signaling in Host Defense and Inflammation
(2022-05) Overcast, Garrett Ray; Satterthwaite, Anne B.; Pasare, Chandrashekhar; Koh, Andrew Y.; Zaki, Hasan
Intestinal epithelial cells (IECs) constitute a critical first line of defense against microbes by providing a physical barrier and producing antimicrobial peptides (AMPs) and cytokines. While IECs are known to respond to various microbial signals, the precise upstream cues regulating diverse IEC responses are not clear. Here we discover a dual role for IEC intrinsic interleukin-1 receptor (IL-1R) signaling in regulating both intestinal homeostasis and inflammation. Specifically, absence of IL-1R in epithelial cells abrogates a homeostatic antimicrobial program including production of AMPs. Mice deficient for IEC intrinsic IL-1R are unable to clear Citrobacter rodentium and have impaired AMP gene expression during infection. Mechanistically, IL-1R signaling enhances IL-22R induced STAT3 phosphorylation in IECs leading to elevated production of AMPs. A similar synergy between IL-1β and IL-22 was found in human IECs. We find that IL-1R signaling on IECs directly induces expression of chemokines as well as genes involved in the production of reactive oxygen species in IECs. Absence of IL-1R in IECs protected mice from chemically induced colitis, suggesting that IEC intrinsic IL-1R signaling contributes to worsened inflammation and pathology during acute inflammation. Our findings establish a protective role for IEC intrinsic IL-1R signaling in combating infections, and a detrimental role of IEC intrinsic IL-1R signaling during colitis induced by epithelial damage.
Re-Designing Cancer Immunotherapy to Enhance Anti-Tumor Immunity
(2022-05) Moore, Casey Elizabeth; Yan, Nan; Fu, Yang-Xin; Qiao, Jian; Hannan, Raquibul; Chen, Zhijian J.
Immunotherapy has transformed the field of oncology. However, clinical challenges of toxicity and low response rate offer opportunities to develop novel, rational strategies to integrate into current clinical oncology treatment. To address these challenges, we designed a novel local mesenchymal stromal cell (MSC) based immunotherapy to study the immune tumor microenvironment (TME), and optimized radiotherapy delivery with cGAMP and systemic α-PD-L1 immunotherapies. Using MSC, we delivered various forms of anti-CD3 and/or co-signals to explore their contribution to re-activate T cells inside the TME. Combined α-CD3 and CD40L rather than CD80 leads to superior anti-tumor efficacy compared to either alone. Mechanistically, TCR activation of pre-existing CD8+ T cells synergizes with CD40L activation of DCs inside the TME to control tumor growth. We found exogenous TCR signals can better re-activate TIL, but distal tumor control required T cells from the draining lymph node. This study supplies further evidence that TCR signaling for T cell re-activation in the TME is defective but can be rescued by proper exogenous signals, indicating the importance of the local tumor microenvironment on anti-cancer immunity. To approach the problem from another angle, we chose to optimize delivery of radiotherapy for enhanced immune stimulation. Treating radiotherapy like a vaccination in situ, we tested the hypothesis that spacing radiation doses by 10 days would allow for enhanced anti-tumor immunity. This new ablative radiation dosing scheme "personalized ultra-fractionated stereotactic adaptive radiotherapy" (PULSAR) was tested in combination with α-PD-L1 therapy and cGAMP treatment in immune activated and resistant syngeneic immunocompetent mouse models of cancer. We report that both radiation and immunotherapy sequencing as well as radiotherapy fraction spacing affects the combination treatment response. Better tumor control was achieved by giving α-PD-L1 therapy during or after radiation. Spacing fractions 10 days apart (PULSAR) achieved better tumor control when combined with α-PD-L1 and cGAMP immunotherapies than traditional single day spacing. These results illustrate that radiotherapy dosing and scheduling impacts tumor control in combination with two different immunotherapies. These pre-clinical investigations demonstrate that modifying the tumor microenvironment toward immune stimulation by local delivery of exogenous TCR signaling and radiation can lead to novel effective immunotherapy.
Development of an IL12 Prodrug to Treat Solid Tumors with Minimal Toxicity
(2022-05) Moon, Benjamin Ik; Gao, Jinming; Hammers, Hans; Li, Bo; Fu, Yang-Xin; Qiao, Jian
Cytokines are secreted molecules that guide the immune system to respond correctly to various challenges. Among all cytokines, IL12 is perhaps the most powerful at polarizing the immune response into a Type 1, cell-mediated phenotype. Cell-mediated immunity plays a critical role in cancer immunoediting, allowing CTLs to recognize and kill aberrant cells. Because of this, IL12 has been tested in many different preclinical and clinical studies for its potential use as an anti-tumor therapeutic agent. However, IL12 also causes severe, dose-limiting systemic toxicity due to on-target, off-tumor activation of peripheral immune cells. Newer attempts at IL12-mediated delivery focus on restricting IL12 activity to within the tumor as much as possible, but they each have their own limitations. To address these problems, we developed a novel IL12 prodrug, pro-IL12, that is actively blocked until it is preferentially activated within the TME. We achieved this by using portions of the IL12 receptor attached with a flexible linker to sterically block the active site of IL12. The linker contains a substrate site that can be cleaved by tumor-specific proteinases, thereby releasing the blocker and activating the prodrug. Pro-IL12 successfully maintained anti-tumor efficacy with reduced toxicity compared to its non-prodrug counterpart. We determined that the mechanism of anti-tumor immunity was predominantly through pre-existing, intratumor CD8+ T cells that produce IFNγ after direct binding of the prodrug to cell surface IL12 receptor complexes. Pro-IL12 also worked in combination with TKI and ICB to achieve even more potent tumor control. In a follow up study, I propose that a higher dose of pro-IL12 might use distinct cellular and molecular mechanisms. Indeed, high dose pro-IL12 more effectively controls large tumors at the cost of reintroducing systemic toxicity. Mechanistically, this dose used a broader, T cell-dependent mechanism that was independent of IFNγ. Further analysis determined that IFNγ was responsible for all manifestations of toxicity and that IFNγ blockade given concurrently with pro-IL12 could limit toxicity with no effect on efficacy. Additionally, the absence of IFNγ signaling on T cells had no effect on their phenotype or ability to control the tumor. As a whole, these studies document the development of a next generation, IL12 immunotherapy for the treatment of solid tumors with an emphasis on its mechanisms of tumor control that are distinct from toxicity.
Efficient and Intelligent Radiotherapy Planning and Adaptation
(2022-05) Ma, Lin; Gu, Xuejun; Lu, Weiguo; Jiang, Steve B.; Jia, Xun; Wang, Jing
Treatment planning--inverse planning on volumetric tomography images--is the foundation of modern radiotherapy. Contouring planning structures and optimizing plan dose distributions are the two most important components of treatment planning. Treatment planning happens in two stages. The initial treatment plan is first designed before the commencement of treatment course. With the on-line images acquired before each fraction of treatment, treatment plan may be adapted off-line/on-line accounting for inter/intra-fractional changes. This dissertation presents innovative research to improve the efficiency and intelligence of treatment planning, with a special focus on its application in on-line adaptive planning. Four standalone, but related, techniques are developed in this dissertation. * Registration-guided on-line image segmentation. This technique targets the segmentation of on-line images. Based on the previous methods for on-line image contouring such as registration-based contour adaptation and deep learning-based image segmentation, we proposed a technique that can combine the merits of individual method. * Volumetric dose extension and isodose tuning. Interactive plan dose tuning is essential to on-line plan adaptation. We developed a dose painting algorithm that can output volumetric dose distribution from two isodose surfaces in real time. Then we applied the algorithm to interactive plan dose tuning, which allows for tuning volumetric dose by dragging isodose lines. * Fluence map prediction. Plan optimization is the central part of inverse planning. We proposed a deep learning-based fluence map prediction method, to achieve inverse planning without optimization. Compared to fluence map optimization, fluence map prediction is as accurate and much faster because of its nature as a direct inference calculation. * On-line proton range verification. Proton therapy is sensitive to motion and anatomy variation. For the previously proposed range-guided proton therapy strategy, we conducted an end-to-end Monte-Carlo simulation of the on-line proton measurement process. The key parameters of range measurement, such as the mapping from measurement result to range and measurement uncertainty, are obtained by simulation. Clinical data has been used to train and evaluate each technique. The results show the feasibility, generalizability, as well as limitations of the developed techniques. Future directions for on-line planning were discussed.
Analysis of Interrelationships Among NAD+, PARP1, ADP-Ribosylation, and Splicing in Murine Embryonic Stem Cells
(2022-05) Jones, Aarin; Banaszynski, Laura; DeBerardinis, Ralph J.; Wang, Yingfei; Kraus, W. Lee
The differentiation of embryonic stem cells (ESC) into a lineage-committed state is a dynamic process involving changes in epigenetic modifications, gene expression, RNA processing, and cellular metabolism. Previous studies have implicated poly(ADP-ribose) polymerase 1 (PARP1), an abundant nuclear enzyme that plays key roles in a variety of nuclear processes, in ESC self-renewal and lineage commitment. Given the diverse molecular functions of PARP1, I sought to determine the potential regulatory role of PARP1 in determining ESC state. PARP1 functions both as an enzyme, through its NAD+-dependent ADP-ribosyltransferase catalytic activity, and as a structural protein, through its NAD+-independent nucleic acid binding activity. I observed a dramatic induction of PARP1 catalytic activity during the early stages of mESC differentiation (e.g., within 12 hours of LIF removal) leading me to query the regulation and outcome of PARP1-mediated ADP-ribosylation in mESCs. NAD+ is synthesized through three main pathways - De novo, Salvage, and Preiss-Handler - and is constrained within cellular compartments. I found that both pathway usage and subcellular localization were dynamic during differentiation in a PARP1-dependent manner, with transition from De novo to Salvage pathway usage and increases in nuclear NAD+ levels upon differentiation feeding PARP1 catalytic activity. Using an NAD+ analog-sensitive PARP (asPARP) chemical biology approach, I characterized the PARP1-mediated ADP-ribosylated proteome during mESC differentiation. PARP1-modified proteins in mESCs are enriched for biological processes related to stem cell maintenance, transcriptional regulation, and RNA processing. The PARP1 substrates include core spliceosome components, such as U2AF35 and U2AF65, whose splicing functions are modulated by PARP1-mediated site-specific ADP-ribosylation. In addition, I observed a genome-wide dysregulation of splicing events upon loss of PARP1 in transcriptomic analysis. These results demonstrate a role for the NAD+-PARP1 axis in the maintenance of mESC cell state, specifically in the splicing program during differentiation.
Enabling Structural Studies of the Yeast Prion Protein Within a Cellular Environment
(2022-05) Costello, Whitney Nicole; Lin, Milo; Rizo-Rey, José; Diamond, Marc; Frederick, Kendra
My motivation for the work in this thesis was to take steps towards bridging the gap in structural information between atomic models of metastable proteins in isolated and cellular environments. Most biophysical techniques are generally limited by either sample composition or resolution. One technique, Nuclear Magnetic Resonance (NMR) is not limited by sample composition and can provide atomic-level resolution. However, NMR is limited by sensitivity. Recent advancements in the field produced a sensitivity-enhanced solid-state NMR technique, namely Dynamic Nuclear Polarization (DNP) NMR. Using DNP NMR, I observed sensitivity enhancements of up to 90-fold increase in sensitivity, eliminating this barrier. Recent work on a metastable protein, Sup35, assembled in cellular lysates using DNP NMR demonstrates that the biological environment has a dramatic effect on the Sup35 protein structure. In this thesis, I sought to harness sensitivity gains from DNP NMR to identify strategies for the specific detection of isotopically labeled proteins at within a cellular lysate for structural analysis. First I present theoretical calculations, validated by experimental results, for the expected signal of detection ratio of an isotopically labeled protein within a cellular lysate. These results concluded that DNP NMR can specifically detect a 30 kDa, uniformly isotopically labeled protein at low micromolar concentrations. However, sensitivity is still a barrier to specifically detect proteins with lower molecular weights or non-uniform isotopic labeling. Therefore, I optimized sample preparation for maximum sensitivity for DNP NMR on cellular lysates. DNP sensitivity enhancement depends on sample composition. DNP NMR is performed at 100 K, and requires sample glassing, polarizing agent, and protonation for optimal DNP enhancement. Some of the first DNP NMR experiments on purified protein samples were optimized for a matrix of 60:30:10, d8-glycerol:D2O:H2O with 10 mM biradical. These matrix conditions became standard in the field, known as "DNP Juice". However, I found that these matrix conditions are not optimal for DNP NMR cellular lysate samples. In the presence of cellular lysate, sensitivity is improved by addition of lower cryoprotectant (15%) and biradical concentrations (5 mM). I also found that deuteration was unnecessary. Finally, I investigated methods to simplify DNP NMR spectra through segmental labeling of proteins. The strategies in this thesis benefit future research of structural studies on environmentally sensitive proteins, such as alpha-synuclein or tau, within their native environment at physiological concentrations.

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