ItemA Cytokine Receptor Masked IL-2 Prodrug Selectively Activates Tumor-Infiltrating Lymphocytes for Potent Antitumor Therapy(August 2021) Hsu, Eric Jonathan; Zhang, Chengcheng "Alec"; Farrar, J. David; Malladi, Srinivas; Yan, Nan; Fu, Yang-XinCancers are very difficult to treat, and many cancer patients fail to respond to numerous standard of care therapies. Many of these tumors have been observed to lack functional CD8 T cells, which have been observed to be correlated with improved patient prognosis. One of the main strategies to combat the lack of functional tumor infiltrating immune cells is to treat patients with immune stimulating cytokines such as interleukin-2 (IL-2). As a potent lymphocyte activator, IL-2 is an FDA approved treatment for multiple metastatic cancers. However, its clinical use is limited by short half-life, low potency, and severe in vivo toxicity. Current IL-2 engineering strategies exhibit evidence of peripheral cytotoxicity. Here, limitations of both recombinant IL-2 and these next generation IL-2 variants are addressed through the engineering of a novel IL-2 prodrug (ProIL2). Numerous designs of ProIL2 were designed, engineered, and tested until a final optimal construct was synthesized. The activity of a CD8 T cell-preferential IL-2 mutein/Fc fusion protein is masked with IL2 receptor beta linked to a tumor-associated protease substrate. ProIL2 restores activity after cleavage by tumor-associated enzymes, and preferentially activates inside tumors, where it expands antigen-specific CD8 T cells. This significantly reduces IL-2 toxicity and mortality without compromising antitumor efficacy. ProIL2 also overcomes resistance of cancers to immune checkpoint blockade. Furthermore, neoadjuvant ProIL2 treatment can eliminate metastatic cancer through an abscopal effect. Lastly, ProIL2 can also synergize with radiation therapy to more effectively control both primary and metastatic cancer. Further protein engineering strategies are being implemented to overcome potential limitations of ProIL2. Taken together, this approach presents an effective tumor targeting therapy with reduced toxicity. ItemCompensation Between Foxp Transcription Factors Maintains Proper Striatal Function(August 2023) Ahmed, Newaz Ibrahim; Tsai, Peter; Chahrour, Maria; Roberts, Todd; Konopka, GenevieveSpiny projection neurons (SPNs) of the striatum are critical in integrating neurochemical information to coordinate motor and reward-based behavior. Mutations in the regulatory transcription factors expressed in SPNs can result in neurodevelopmental disorders (NDDs). Paralogous transcription factors Foxp1 and Foxp2, which are both expressed in the dopamine receptor 1 (D1) expressing SPNs, are known to have variants implicated in NDDs. Paralogous transcription factors are thought to have the ability to compensate for each other and previous work published by the lab supports the hypothesis that Foxp1 and Foxp2 have compensatory roles in D1 SPNs as well. For my dissertation work, I utilized mice with a D1-SPN specific loss of Foxp1, Foxp2, or both and a combination of behavior, electrophysiology, and cell type specific genomic analysis to address if there was compensation occurring. It is only upon the loss of both genes that motor behavior was impaired whereas Foxp1 mediated social behavior impairments were exacerbated upon the further loss of Foxp2 (Chapter Two). I also found that while loss of Foxp1 resulted in KLeak mediated hyperexcitability of D1-SPNs, this too was further impaired with the additional loss of Foxp2 (Chapter Three). Viral mediated re-expression of Foxp1 in the double knockouts was sufficient to restore both behavioral and electrophysiological impairments to baseline. I further studied the contribution of Foxp1 and Foxp2 to regulation of downstream targets genes using single-nuclei RNA-seq and found that in both juvenile and adult D1-SPNs, loss of both transcription factors resulted in differential expression of hundreds of genes (Chapter Four). I was able to use these experiments to also investigate how loss of these transcription factors from the D1-SPNs impacted gene expression in other cell-types (Chapter Five). I also utilized single-nuclei ATAC-Seq and again found that loss of both genes resulted in large scale dysregulation of chromatin state not seen in the single knockouts, including in regions enriched for Fox motifs (Chapter Six). I also began to address the open question of what the direct binding targets of Foxp1 and Foxp2 are using the newly developed CUT&RUN technique (Chapter Seven). The findings from my experiments point towards a form of compensation between Foxp1 and Foxp2 where one transcription factor maintains striatal function upon the loss of the other, which I discuss more in depth (Chapter Eight). I also discuss my involvement in a project where we further study the role of Foxp1 in D1- and D2-SPNs, which I am working on in collaboration with Dr. Nitin Khandelwal (Chapter Nine). I conclude by discussing the implications of my findings and suggest recommendations for further study (Chapter Ten). ItemAspartate Is Limiting for Hematopoietic Stem Cell Function in vivo(August 2021) Qi, Le; Xu, Jian; Morrison, Sean J.; Hill, Joseph A.; Thomas, Philip J.A key function of the electron transport chain is to promote aspartate synthesis, which is required for cancer cell proliferation. However, it is unclear whether aspartate is also limiting in normal stem cells, which divide intermittently. We found that hematopoietic stem cells (HSCs) do not take up exogenous aspartate. To test if aspartate limits HSC function, we over-expressed the glutamate/aspartate transporter, Glast, or deleted glutamic-oxaloacetic transaminase 1 (Got1). Each increased aspartate levels in hematopoietic stem/progenitor cells, increasing the function of HSCs but not colony-forming progenitors. Conversely, deletion of glutamic-oxaloacetic transaminase 2 (Got2) reduced aspartate levels and HSC function but not colony-forming progenitors. Isotope tracing showed aspartate was used to synthesize asparagine and purines. Both contributed to increased HSC function as deletion of asparagine synthetase (Asns) or treatment with 6-mercaptopurine attenuated the increased function of GLAST over-expressing HSCs. Stem cell function is thus limited by aspartate in some contexts. ItemThe Effects of Chromatin Remodeling and Pseudokinase Activity on Liver Pathophysiology(August 2021) Moore, Austin Bradley; Agathocleous, Michalis; Hobbs, Helen H.; Hoshida, Yujin; Zhu, HaoThe liver exhibits a remarkable capability to regenerate itself in the face of injury; however, in the setting of sustained damage, this capability can be overwhelmed and eventually lead to chronic liver diseases such as non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. Although these processes are complex and not completely understood, specific genetic and epigenetic factors that drive aspects of this pathophysiology can shed further light on both how these diseases develop and on how normal, healthy regeneration differs from liver disease. In this body of work, we show that loss of Arid1a, a DNA-binding component of the SWI/SNF chromatin remodeling complex, shifts hepatocyte metabolism to promote lipid accumulation in a manner similar to that seen in non-alcoholic fatty liver disease. We further explore the dynamics of the SWI/SNF complex by examining a mutually exclusive homolog of Arid1a, Arid1b, and provide evidence to suggest that its role is to stabilize the hepatocyte SWI/SNF complex in the absence of Arid1a seen in regeneration and hepatocellular carcinoma. Finally, we harness the power of in vivo CRISPR screening within the liver to identify the pseudokinase STK31 as a positive regulator of hepatocyte proliferation and liver oncogenesis. Our findings underscore the important role that chromatin remodeling has in enforcing hepatocyte identity and functionality as well as allowing for plasticity. Additionally, our work with STK31 highlights the power of in vivo screening within the liver to identify new potential therapeutic targets not only for hepatocellular carcinoma, but other tumor types as well. ItemCrosstalk Signaling Between cAMP and mTORC1(August 2021) Melick, Chase Hunter; DeBerardinis, Ralph J.; Cobb, Melanie H.; Pan, Duojia; Jewell, Jenna L.The mammalian target of rapamycin complex 1 (mTORC1) senses multiple stimuli to regulate anabolic and catabolic processes. G-protein-coupled receptors (GPCRs) paired to Gs proteins increase cyclic adenosine 3'5' monophosphate (cAMP) to activate protein kinase A (PKA), which phosphorylates Raptor at Ser 791 resulting in potent mTORC1 inhibition. We identified a novel mTORC1-interacting protein called A-kinase anchoring protein 8L (AKAP8L). Using biochemical assays, we found that the N-terminal region of AKAP8L binds to mTORC1 in the cytoplasm. Importantly, loss of AKAP8L decreased mTORC1-mediated processes such as translation, cell growth and cell proliferation. AKAPs anchor protein kinase A (PKA) through PKA regulatory subunits, and we show that AKAP8L can anchor PKA through regulatory subunit I (RI). Full-length AKAP8L restored mTORC1-regulated biology, whereas AKAP8L missing the N-terminal region that confers interaction with mTORC1 did not. Additionally, we have shown that H89 (N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide), a well-characterized ATP-mimetic kinase inhibitor, renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors across multiple cell lines. Moreover, H89 prevented the dephosphorylation of AKT and S6K1 under nutrient depleted conditions. PKA and other known H89-targeted kinases do not alter the phosphorylation status of S6K1 and AKT. Pharmacological inhibition of some phosphatases also enhanced S6K1 and AKT phosphorylation. These findings suggest a new unknown target for H89 by which it sustains the phosphorylation status of S6K1 and AKT, resulting in mTOR signaling. Lastly, we identified A-kinase anchoring protein 13 (AKAP13) as a crucial scaffold involved in GPCR-Gs signaling to mTORC1. AKAP13 potently enhances Raptor Ser 791 phosphorylation and inhibits mTORC1 activity. Consistently, in cells where Raptor Ser 791 is mutated to Ala, AKAP13 is unable to supress mTORC1 activity. AKAP13 mediates mTORC1-induced cell proliferation, cell size and colony formation. Interestingly, AKAP13 expression inversely correlates with mTORC1 activation and positively correlates with overall lung adenocarcinoma patient survival. Our results place the GPCR-Gas signaling pathway to mTORC1 as a potential target that may be beneficial for human diseases with hyperactivated mTORC1. ItemLiquid-Liquid Phase Separations in Innate Immune DNA Sensing and NF-κB Signaling Pathways(August 2021) Du, Mingjian; Liu, Yi; Chen, Zhijian J.; Beutler, Bruce; O'Donnell, Kathryn A.The binding of DNA to cyclic GMP-AMP synthase (cGAS) leads to the production of the secondary messenger cyclic GMP-AMP (cGAMP), which activates innate immune responses. We have shown that DNA binding to cGAS robustly induced the formation of liquidlike droplets in which cGAS was activated. The disordered and positively charged cGAS N terminus enhanced cGAS-DNA phase separation by increasing the valencies of DNA binding. Long DNA was more efficient in promoting cGAS liquid phase separation and cGAS enzyme activity than short DNA. Moreover, free zinc ions enhanced cGAS enzyme activity both in vitro and in cells by promoting cGAS-DNA phase separation. These results demonstrated that the DNA-induced phase transition of cGAS promotes cGAMP production and innate immune signaling. Beyond cGAS-DNA phase separation, we sought to determine whether protein liquid-liquid phase separation is a ubiquitous mechanism across immune signaling pathways. NF-kappa-B essential modulator (NEMO), also known as IKBKG, is essential for the activation of IκB kinase (IKK) complex in NF-κB signaling, including Interleukin-1 (IL-1β), Tumor Necrosis Factor (TNFα) and Toll-like receptors (TLR) pathways. NEMO activates IKK complex by binding to polyubiquitin chains. Here we show that Lys63(K63)-linked or linear(M1)-linked polyubiquitin chains binding to NEMO robustly induced the formation of liquidlike droplets in which IKK was activated both in vitro and in cells. Both NEMO ubiquitin binding (NUB) domain and zinc finger (ZF) domain of NEMO contributed the multivalencies for binding to polyubiquitin chains. Long polyubiquitin chains were more efficient in promoting NEMO phase separation than short polyubiquitin chains. These results demonstrated that polyubiquitin chains induced phase transition of NEMO to promote IKK complex activation and NF-κB signaling. ItemCongenital Heart Defect-Associated Enhancers Shape Human Cardiomyocyte Lineage Commitment(August 2023) Armendariz, Daniel Alejandro; Xu, Jian; Hon, Gary C.; Johnson, Jane E.; Munshi, Nikhil; Wu, JunAdvancements in whole genome sequencing have identified thousands of disease-associated variants which land within enhancer boundaries. As enhancers play critical roles in orchestrating gene networks throughout development, variants which disrupt enhancer function have been shown to contribute to developmental defects. However studying enhancer variants within a developmental context has been limited by a few key challenges. First, thousands of enhancer variants have been identified which could be causal for disease. Thus, a high-throughput approach is necessary to feasibly interrogate these elements. Second, enhancers function in a cell-type specific and spatiotemporal manner to regulate target gene expression. Perturbation of these enhancers thus requires an in vitro model that can phenocopy the lineage and context in which they are active. Addressing these points, I first identify 25 putative cardiac enhancers harboring variants identified in patients with congenital heart defects (CHD). Using a CRISPRi repression system, I perturb these putative enhancers in human embryonic stem cells (hESC) followed by differentiation towards cardiomyocytes (CM). This allows for the study of enhancer activity throughout the specification of the vital muscle cells of the cardiac system. I then perform single-cell RNA sequencing to identify diverse CM cell populations and assess the impact of enhancer perturbations on lineage specification. My analysis revealed 16 enhancers of known cardiac genes which, when perturbed, result in deficient CM differentiation. Genetic knockouts of two enhancers near TBX5 phenocopied the single-cell data and revealed enrichment of early CM populations resulting from depletion of later stages. My thesis provides a framework for single-cell enhancer screens within a developmental context and provides support for the biological relevance of the approach. I expect that the throughput of this methodology and the ease at which it can be adapted towards diverse developmental systems will provide an invaluable tool for future studies. ItemDevelopment and Implementation of a Brief Video-Based Mindful Movement Intervention Prior to Pelvic Exams to Reduce Pain Intensity and State Anxiety(August 2021) Collins, Marielle Heather; Greer, Tracy L.; Evans, Harry M.; Kho, Kimberly A.; Trombello, Joseph M.; Carmody, ThomasMindfulness interventions have demonstrated efficacy for numerous health related outcomes, and been implemented in medical settings to reduce pain and anxiety prior to exams, surgeries, or procedures. This broad investigation begins with a theoretical review exploring a proposed mechanism of mindfulness, improved attention. It presents a theory of mindful movement as a type of mindfulness potentially more efficacious for individuals who struggle with mind-wandering, such as those with depression and anxiety. Mind-wandering and rumination are barriers to mindfulness, and movement has been shown to engage finite executive resources also utilized for mind-wandering. Mindfulness interventions with movement, may promote greater engagement for individuals with attentional challenges, compared to focused attention meditation. Mindful movement may provide a more accessible form of mindfulness for brief interventions, implemented in settings with a wide range of participants. The current study tests the efficacy of a 5-minute mindful movement video prior to pelvic examination in a sample of 99 women at an outpatient gynecology clinic, with the aim of reducing anxiety and pain. Participants were randomized into Treatment (5-minute mindful movement video; n=49) and Control (educative materials about mindfulness benefits; n=50). Upon arrival, women completed a PHQ-9, and baseline measurements of anxiety and pain using a visual analogue scale. They then completed the group specific activity and rated anxiety prior to examination. After examination participants rated exam pain, post-exam anxiety, and completed questionnaires assessing exam satisfaction and intervention acceptability. Physicians (n=2) rated ease of performing pelvic examination and ability to obtain clinical information. Results demonstrated clinically anxious individuals who completed the intervention reported significantly lower anxiety before pelvic examination (p=.000, partial η2=.262) and after (p= .010, partial η2=.126) compared to control group. No significant differences in pain, patient exam satisfaction, or physician perception of the exam were noted between groups. Acceptability and interest in the intervention was also high. This study demonstrates the ability to implement a brief mindful movement video into the flow of a medical clinic and reduce pelvic exam anxiety. In general, this broad investigation supports ongoing research exploring mindful movement from a mechanistic perspective, and the implementation of brief mindful movement interventions in medical settings. ItemThe Capabilities of Neural Systems Depend on a Hierarchically Structured World(August 2021) Blazek, Paul Joseph; Reynolds, Kimberly A.; Pfeiffer, Brad E.; Toprak, Erdal; Zinn, Andrew R.; Lin, MiloThe study of the human mind spans thousands of years, from the earliest philosophers to modern neuroscience. However, there still remains an incredible gap in our understanding of how cognitive functions arise from the biology of the brain. This has greatly hampered attempts to understand how the brain works, what its limitations are, and how to replicate it in artificial intelligence systems. Here I propose a general framework to understand how cognitive processes can be encoded by networks of interconnected neurons. I have taken a theoretical and computational approach, using artificial neural networks as a high-level quantitative model of basic neuroscientific principles. Neural networks are capable of reasoning by means of a series of specialized distinctions made by individual neurons that are integrated hierarchically. This framework enables the study of how the capabilities of neural systems are dependent on structural and functional constraints. Biological constraints on neural coding and network size and topology limit the complexity of stimuli that can be comprehended by the network. Surprisingly, though, neural networks are capable of comprehending much more complex stimuli than has been previously described, provided that the inherent distinctions between these stimuli are hierarchically structured. Functional constraints on neural networks require that they be able to perform cognitive processes and be able to reason in a way that can be communicated with other people. I have proposed a novel neurocognitive model which, when implemented in deep neural networks, is able to simulate a wide variety of cognitive processes. It is consistent with experimental evidence from neuroscience and theories from philosophy and psychology. By directly implementing symbolic reasoning within the structure and function of the network, it becomes possible to overcome many of the fundamental problems that face modern artificial intelligence systems, including their lack of explainability, robustness, and generalizability. This culminated in a novel algorithm that translates neural networks to human-understandable code, providing a complete picture of how neural networks can reason. All of these results suggest that neural systems require the world to be hierarchically structured in order to comprehend it, a direct reflection of their own hierarchical organization. ItemA Comparison of Post-Injury Symptomatology and Recovery Following Concussion Versus Orthopedic Injury(August 2021) Allen, Tahnae Tarkenton; Cullum, C. Munro; Didehbani, Nyaz; Hynan, Linda S.; Silver, Cheryl H.; Miller, ShaneOBJECTIVE: Concussion research has utilized orthopedic injury (OI) comparison groups to examine outcomes specific to concussion versus physical injury in general, due to the suggestion that preexisting, comorbid, and other injury-related factors influence post-concussion symptom reporting and recovery. The first aim of this dissertation (Study 1) was to conduct a review of the literature on post-injury symptoms and outcomes following concussion versus OI in children and adolescents, focusing on study design and synthesizing conclusions about concussion versus OI in youth populations. Incorporating findings from the review, the second objective of this dissertation (Study 2) was to collect original data from concussed youth and a carefully selected OI control group to compare symptomatology and recovery at initial presentation and 3-months post-injury to examine whether concussion outcomes are unique to a brain injury or more related to response to injury in general. METHODS: A scoping review using MEDLINE and PubMed to query databases from 2000 to 2020 was performed. Studies were included if they reported children, adolescents, or young adults with mild traumatic brain injury (mTBI)/concussion, used an orthopedic control group, and compared post-injury outcomes, which resulted in a total of 52 articles out of the 526 initially identified. For the second study, participants age 12-18 who sustained a concussion (n = 50) were matched by sex, age, and days since injury to an OI group (n = 50). Repeated measure analyses of covariance (ANCOVAs) were used to compare post-concussion symptoms, emotional symptoms, and recovery outcomes between injury groups at initial and 3 months post-injury. Binary logistic regression analyses were used to determine predictors of prolonged recovery separately in concussion and OI groups. RESULTS: Study 1: sixty-nine percent of the studies included in the scoping review reported differences between concussion and OI outcomes during at least one assessment time point during the recovery period, with higher and more persistent symptomatology in the concussion group. Study 2: the repeated measure ANCOVAs indicated that concussion participants reported significantly higher post-injury symptomatology and psychological sequelae within the first week of injury compared to OI subjects, but by 3 months, the groups showed no differences. Within the concussion group, females reported significantly higher symptoms compared to males, but this pattern was not observed in the OI group. Significant predictors for prolonged recovery also differed between injury groups. For the concussion group, previous concussion was the only significant predictor in our model for prolonged recovery. In the OI group, time since injury and functional impairment rating scores predicted prolonged recovery. CONCLUSIONS: Overall, findings suggest concussion results in a unique expression of symptoms, and recovery following concussion is influenced by a specific set of concussion-related factors that are not commonly seen in OI. Clinically, a unique presentation and recovery course following concussion versus OI supports the utility of specialized concussion treatment and clinical protocols, and may help identify individuals at greater risk of prolonged recovery. ItemInteraction of Quantal Inhibitory and Excitatory Neurotransmission in Regulation of Synaptic Homeostasis via Molecular Signaling(August 2021) Horvath, Patricia Mary; Chahrour, Maria; Monteggia, Lisa; Cobb, Melanie H.; Kavalali, Ege T.; Konopka, GenevieveThe processing and flow of information through brain circuits is undeniably shaped by individual neurons and synapses. The type of synapse between any two given neurons determines the reliability, speed, and strength of signal transfer. Therefore, studying the organization and signaling capacity of the variety of synapses present in our nervous system is critical to understanding how information is shaped and communicated between neurons. Neurotransmission can be classified into two broad types: spontaneous and evoked. Much of the work examining these types of neurotransmission and their properties has been done in excitatory synapses. Yet the variety of inhibitory neurons is so vast that Ramon y Cajal referred to them as the "Butterflies of the Soul." In order to help paint a more complete picture of the brain, this PhD thesis is focused on the structure, function, and regulation of synapses and neurotransmission, with a focus on inhibitory synapses. We first expanded the tools available to study synapses by demonstrating that lentiviral CRISPR could be used in postmitotic cells to knockout synaptic proteins and study non-cell autonomous phenotypes. Next, we took advantage of the use-dependent properties of the GABAA receptor antagonist picrotoxin, demonstrating that this drug could be used to interrogate inhibitory synapse parameters, such as presynaptic release probability. By utilizing picrotoxin, we were able to examine the postsynaptic organization of spontaneous and evoked neurotransmission at inhibitory synapses, and discovered a partial segregation of the postsynaptic receptors activated by evoked and spontaneous neurotransmission. This result implied that spontaneous and evoked neurotransmission at inhibitory synapses may have partially non-overlapping functions. Because the function of inhibitory spontaneous neurotransmission is largely unknown, we next examined the signaling capacity of inhibitory spontaneous neurotransmission. We discovered that modulation of inhibitory, but not excitatory, spontaneous neurotransmission alters transcription of certain activity-induced genes, including Bdnf. Furthermore, blockade of inhibitory spontaneous neurotransmission leads to downscaling of excitatory synapses through a BDNF-dependent mechanism. Finally, we examined the regulation of synapse function by miRNAs regulated by MeCP2, a gene in which loss of function mutations are the primary cause of Rett Syndrome. We characterized the effects of two candidate miRNAs on synapse function. One of these miRNAs, miR-101a, has opposing impacts on excitatory and inhibitory synapses, suggesting a role in regulating excitatory/inhibitory balance, a feature that is often altered in ASD and ASD-like disorders such as Rett Syndrome. In summary, we have contributed to the study of synapses by expanding the tools available, improving understanding of inhibitory synapse structure and function, and examining the broad regulatory capacity of certain miRNAs over synaptic function. ItemBiochemical Characterization of IpaH E3 Ubiquitin Ligase Effector Proteins and Their Host Substrates(August 2021) Hansen, Justin Mark; Orth, Kim; Sperandio, Vanessa; Reese, Michael L.; Alto, NealShigella flexneri is a gram negative pathogen that utilizes its type 3 secretion system (T3SS) to inject effector proteins in the cytoplasm of host cells to manipulate host cells processes. T3SS effectors are able to post translationally modify host proteins to reprogram intracellular signaling pathways, actin dynamics, membrane trafficking, and innate immune pathways. This allows Shigella to modify the intracellular environment to be conducive to bacterial replication and dissemination to neighboring cells. Shigella flexneri and other bacteria including Salmonella and Yersinia secreted E3 ubiquitin ligases into the host cell cytoplasm via the Type III secretion system (T3SS) apparatus. The invasion plasmid antigen Hs (IpaHs) are a novel family of bacterial E3 ubiquitin ligases that are secreted by Shigella, Salmonella, and Yersinia. These bacterial enzymes highjack the host ubiquitin conjugation machinery by binding to ubiquitin-charged E2 conjugating enzymes and facilitating direct transfer of ubiquitin onto host substrates. IpaH effectors induce polyubiquitination and subsequent proteasomal degradation of their substrates during bacterial infection. The effector substrate interaction of IpaH1.4/2/5 and HOIP was previously characterized. I went on to identify that IpaH2.5 is able to inhibit the in vitro catalytic activity of HOIP via mono-ubiquitination of catalytic lysine residues in the HOIP ring-between-ring domain (RBR-C). Subsequent to this Ubiquitin activated interactive trapping (UBAIT) screening was then utilized to identify the host substrate of IpaH7.8, Gasdermin B (GSDMB). GSDMB belongs to a large family of pore forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to inflammatory disease, its function in human physiology remains poorly understood. I investigated a previously unrecognized host-pathogen conflict between GSDMB and the IpaH7.8 effector protein encoded by Shigella flexneri. Through extensive biochemical and cellular characterization, I show that IpaH7.8 ubiquitinates and targets GSDMB for proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of Natural Killer cells by suppressing Granzyme-A mediated activation of GSDMB. In contrast to the canonical function of most Gasdermin-family members, GSDMB does not inhibit Shigella by lysing infected cells. Rather, GSDMB exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveals a mechanism employed by pathogens to counteract this host defense system. ItemSAM Homeostasis Is Regulated by CFIm-Mediated Splicing of MAT2A(August 2021) Scarborough, Anna Maurine; Tu, Benjamin; Mendell, Joshua T.; Green, Carla B.; Conrad, NicholasS-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of MAT2A, the only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) as a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in the detained intron and 3´ UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal a previously undescribed role for CFIm in splicing and SAM metabolism. ItemNeuropsychological Predictors of Time to Conversion from Mild Cognitive Impairment to Alzheimer's Disease(August 2021) Parker, Allison Nicol; Cullum, C. Munro; McClintock, Shawn Michael; Rossetti, Heidi; Hynan, Linda S.; Nguyen, TrungMild Cognitive Impairment (MCI) is a risk state for the development of Alzheimer's disease (AD), though individual outcomes vary. Accurately predicting which MCI patients are likely to develop AD and how long they have until the onset of dementia could provide both patients and their families sufficient time to prepare. Neuropsychological tests have the advantage of objectively quantifying cognitive impairments, and may be useful in predicting time to conversion. The present project aimed to 1) compile the available literature concerning neuropsychological predictors of conversion from MCI to AD using systematic review and meta-analytic techniques and 2) to determine if neuropsychological profiles differentiate MCI patients who convert to AD sooner, those who convert later, and those who do not convert utilizing a statistical technique known as profile analysis. Findings from the systematic review illuminated several gaps in the literature such as the small number of studies that follow patients over longer periods of time. Results from the meta-analysis suggested that word recall and recognition tasks, complex figure recall tasks, simple shape recall tasks, Trail Making Task B (TMT-B), semantic fluency, and the Mini Mental State Examination (MMSE) differentiated between MCI patients who convert sooner (within three years) from those who maintain an MCI diagnosis over three years. Results from the second part of the project found that verbal memory measures best distinguished those who converted sooner from those who converter later. In comparison with those who convert within three years, the group that maintained an MCI diagnosis completed TMT-B more quickly and performed better on memory measures. Neuropsychological measures did not distinguish between those who maintained an MCI diagnosis and those who converted after three years. Taken together, results from the two studies suggest that clinicians may wish to rely upon memory measures and TMT-B performance when considering recommendations regarding length of follow-up and planning for the onset of dementia in patients with MCI. In order to better understand predictors of time to conversion from MCI to AD, future studies should follow participants over several years and make direct comparisons between those who convert sooner and those who convert later. ItemTransplant as a Medical Trauma: Posttraumatic Stress Symptoms in Pediatric Solid Organ Transplant Patients, a Mixed Methods Approach(August 2021) Masood, Saba Saleem; Triplett, Kelli; Schuster, Lisa; Lippe, Ben; Killian, Michael; Desai, Dev; Stewart, Sunita M.Posttraumatic stress symptoms (PTSS) have been reported by pediatric solid organ transplant (SOT) patients well after transplantation. Despite the elevated levels of PTSS in this population, subjective factors associated with PTSS and patients' perception of transplant as traumatic and symptom manifestation of PTSS remains unclear. This study aimed to examine the prevalence of PTSS in pediatric SOT utilizing the DSM-5 PTSD criteria and compare the rate to existing rates in pediatric populations, assess variables associated with PTSS, and explore patients' perception of trauma in SOT and clinical presentation of PTSS. Forty patients participated in the study with twenty-six completing qualitative interviews. One participant (2.5%) met criteria for PTSD with a slightly larger proportion (n=5;12.5%) being characterized as "partial PTSD likely." Clinical elevated symptoms of hyperarousal were most commonly endorsed. The majority of the sample endorsed functional impairment/distress associated with the PTSS. Patients with lifetime trauma exposure and a post-transplant psychiatric diagnosis reported significantly higher PTSS. Greater perceived life threat, treatment intensity, and maladaptive coping strategies were associated with higher PTSS. Post-transplant medical complications was associated with lower post-transplant PTSS. Lastly, patients described various aspects of SOT as traumatic with symptoms being pervasive across life domains. Results indicate subclinical levels of PTSS are prevalent in pediatric SOT patients with some experiencing clinically elevated PTSS. Patients with a post-transplant comorbid psychiatric diagnosis are at-risk and would benefit from transdiagnostic assessment and intervention to address symptoms overlapping across disorders. Post-transplant subjective variables and maladaptive coping strategies appear to be an important component to assess, as it relates to PTSS. Objective variables, such as frequent hospitalizations, may be less suggestive of post-transplant PTSS. In-depth examination of the SOT experience and presentation of PTSS via qualitative methods provides insight into how disruption in adolescents' developmental trajectory due to medical trauma is experienced and manifested. ItemBuilding a Methodological Framework for Cell Fate Engineering(August 2021) Li, Boxun; Xu, Jian; Hon, Gary C.; Banaszynski, Laura; Cleaver, Ondine; Munshi, NikhilCell fate engineering has become an area of intense research in the last fifteen years. A useful framework of cell fate engineering should include three pillars: the discovery of new cell fate-reprogramming cocktails of factors, the evaluation of engineered cells, and the revelation of underlying molecular mechanisms. One major challenge has been the lack of a scalable screening approach in vitro for the performance of reprogramming cocktails. This limits the speed of discovering new cocktails that can efficiently reprogram diverse cell types. Such new cocktails are needed to unleash the full applicational potential of engineered cells in regenerative medicine, disease modeling, and drug discovery. Another challenge is that despite the advantages of in vivo reprogramming, such as more efficient and mature fate conversion, the underlying gene programs, and thereby the molecular mechanisms, have been largely unknown. This is in large part due to the difficulty of specifically isolating and analyzing reprogrammed cells, without contamination from their endogenous counterparts. To address these, in this thesis, I first develop Reprogram-seq, a method that screens thousands of transcription factor cocktails for their reprogramming performance by single-cell perturbation screens. Reprogram-seq found a cocktail of three factors that efficiently and functionally reprograms fibroblasts to epicardial-like cells. Thus, Reprogram-seq accelerates rational cell fate engineering. Next, I performed single-cell transcriptomic analysis of in vivo neurogenesis induced in astrocytes by a novel reprogramming factor, DLX2. This is enabled by a lineage tracer that highly specifically tracks all cells reprogrammed from astrocytes. My analysis reveals that DLX2 induces a neural stem cell-like behavior, transitioning from quiescence to activation, proliferation, and neurogenesis. Gene regulatory network analysis and mouse genetics identify and confirm key nodes mediating DLX2-dependent fate reprogramming. Therefore, this study dissects the gene programs of in vivo reprogramming with single-cell transcriptomics and paves the way for applying Reprogram-seq in vivo. Together, my thesis research has demonstrated that single-cell omic technologies accelerate the discovery of new reprogramming cocktails, streamline the transcriptional evaluation of engineered cells, and dissect gene programs that underlie reprogramming, contributing to all three pillars of the framework. I expect these methodologies to be generalizable to and useful for other cell fate engineering scenarios. ItemNuclear Receptor FTZ-F1 and Downstream Target Gene Meg-8.3 Control Esophageal Gland Function in S. Mansoni(August 2021) Romero, Aracely Alicia; Reese, Michael L.; Collins, James J.; Mangelsdorf, David J.; Phillips, Margaret A.; Kliewer, Steven A.Schistosomes infect over 200 million of the world's poorest people, yet treatment relies on a single drug. Nuclear hormone receptors are ligand-activated transcription factors that regulate diverse processes in metazoans and are known drug targets in humans. However, few nuclear hormone receptors have been characterized functionally in the parasite. During a systematic analysis of nuclear receptor function, we have identified one receptor, an FTZ-F1-like receptor that is essential for parasite survival. Using a combination of transcriptional profiling and chromatin immunoprecipitation we identify the micro-exon gene, meg-8.3, a direct transcriptional target of FTZ-F1 which is also essential for parasite survival. We find that RNAi of either ftz-f1 or meg-8.3 are required for esophageal gland function and maintenance of tissue in the worm head. Together, our data suggest that FTZ-F1 regulates meg-8.3 transcription, and this regulation is essential to esophageal gland function. These data suggest that drugs that alter the activities of proteins in the parasite's esophageal gland could have therapeutic potential. ItemMetabolic Reprogramming in Response to Mitochondrial Electron Transport Chain Dysfunction(August 2021) Lesner, Nicholas Paul; Reynolds, Kimberly A.; DeBerardinis, Ralph J.; Malloy, Craig R.; Zhu, Hao; Mishra, PrashantMitochondrial electron transport chain (ETC) diseases are genetic disorders of energy production with an occurrence rate of approximately 1:4300 and no effective treatment options. Here we show in vitro models of mitochondrial ETC dysfunction display shunting of major carbon sources (glucose and glutamine) from the TCA cycle. Additionally, SLC7A11 (a plasma membrane glutamate/cystine antiporter) promotes pyruvate oxidation in the mitochondria via generation of an electron acceptor, a-ketobutyrate. Furthermore, in the liver, loss of ETC complex I results in no observable clinical or biochemical phenotype, while complex IV deficiency results in steatosis, liver damage, and significant biochemical alterations. The studies herein demonstrate metabolic reprogramming is important in mitochondrial dysfunction; however, the reprogramming is ETC complex and tissue specific. ItemSensory Neurogenesis and the Role of PRDM12 in Nociceptor Development and Function(August 2021) Landy, Mark Andrew; Johnson, Jane E.; Henkemeyer, Mark; Price, Theodore; Lai, HelenNociceptors are a set of peripheral neurons responsible for the detection of noxious stimuli. They function to alert us to the presence of potentially damaging internal and environmental threats, thereby playing an important role in allowing us to react and avoid danger. Unfortunately, for over 30% of the population, the sense of pain derived from nociception becomes maladaptive, leading to chronic painful conditions which carry an estimated economic burden of over USD 600 billion. While opioids are the current mainstay analgesic medication, they have a vast array of side effects, including risk of overdose and death, which makes their use in treatment of chronic conditions far from ideal. Recently, studies of genetic mutations leading to congenital insensitivity to pain (CIP) have provided a springboard for the development of novel analgesics targeting nociceptor function in the periphery. The identification of additional such mutations in the gene Prdm12 raised questions about processes inherent to nociceptor development and sensory neurogenesis as a whole, and what function this gene plays in mature primary afferent neurons. Over the course of my dissertation work I sought to address some of these questions by studying the development of nociceptive neurons in mice, and the effect of Prdm12-knockout on pain sensation. To establish a framework for sensory neuronal development, I first completed a birthdating study using a thymidine analog to permanently label cells undergoing their final mitotic divisions and identify them at later timepoints. With this, I show that there is no temporal difference in the birth rates of different nociceptor subtypes, but that most are born after touch and proprioceptive neurons. Next, using multiple models to knockout Prdm12 at various timepoints ranging from conception to adulthood, I then provide evidence that this is a key transcription factor for specification of the nociceptor population, but that its function likely changes over time. Loss of Prdm12 during development causes defects in nociception, but no behavioral phenotype was readily discernible following adult knockout. Instead, I show that Prdm12 appears to regulate a population of stem cell-like neurons, with an as-yet unknown role in dorsal root ganglia. Altogether my work highlights the role of Prdm12 in nociceptor development and lays the groundwork for additional studies to investigate the clinical relevance of this gene. ItemInhibitory Control of Contextual Fear Memory and Memory Specificity(August 2021) Guo, Jun; Pfeiffer, Brad E.; Roberts, Todd; Takahashi, Joseph; Xu, WeiThe brain functions are supported by close interactions between excitation and inhibition. Inhibition contributes to neural computation by gating information flow, tuning the gain of the network, and modulating the output strength of the system. The inhibition in the brain is mainly achieved by GABAergic inhibitory neurons which exert their effect by acting on multiple types of GABA receptors. In this dissertation, I examined the roles of specific types of GABAergic neurons and GABA receptors in learning and memory. The study consists of three major components. In the first component (Chapter 2), I developed a novel technique to selectively target and control GABAergic interneurons (NDNF cells) distributed at the SLM of the hippocampus. With this technique, I found that the activities of NDNF cells increased during memory encoding and decreased during retrieval. Enhancing their activity improved memory encoding but impaired memory retrieval. I further discovered that NDNF cells coordinate memory encoding and retrieval by differentially regulating the two major excitatory inputs to the CA1 region of the hippocampus. In the second component (Chapter 3), I identified a single nucleotide deletion in the gene of a subunit of GABA receptors - Gabra2 that reduces contextual fear memory in C57BL/6J by using quantitative trait locus analysis. In the third component (Chapter 4), I found a change in GABAB receptor-mediated feedforward inhibition led to distinct hippocampal responses to environmental stimuli. This difference further led to distinct hippocampal representation and generalization of contextual fear memory. These studies were carried out at genetic, molecular, circuit, and behavioral levels, and involved a combination of techniques including genetic mapping, in vivo recording, circuit manipulation, and behavior analysis. They exemplify how inhibition shapes neuronal activity and animal behavior. They also provide valuable tools and ideas for future research on the function of inhibition in the brain. The knowledge gained through these studies on how the brain inhibitory network interacts with excitatory neurons to regulate memory facilitates our understanding of the cognitive processes in the brain.