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Get PDFThe author submitted this entry in the Open Verse Poetry category (Amateur division) for the 2023 On My Own Time (OMOT) Art Show.The definition of time is unclear, to say the least. However, it is finite and managing it is paramount in our lives. Often, we don't notice how rapidly it passes in our day-to-day living. Other times, it can seem to crawl during difficult periods that we endure. It is a complex, abstract, and paradoxical concept all rolled into one
Endoscopic therapies for weight loss: current and future paradigms
Get PDFDetailed formal protocol with illustrations and extensive bibliography.A recording of the protocol presentation is available on UT Southwestern's Mediasite. Note: Access to the video is restricted to authorized UT Southwestern users only.UT Southwestern--Internal Medicin
Congenital Heart Defect-Associated Enhancers Shape Human Cardiomyocyte Lineage Commitment
Get PDFPages vii-xiv are misnumbered as pages vi-xiii.Advancements 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
Re-engineering of Dendrimer-Based Lipid Nanoparticles for Efficient and Precise HDR-Mediated Gene Editing
Get PDFCRISPR/Cas gene editing is poised to transform the treatment of genetic diseases. However, limited progress has been made toward precise editing of DNA via Homology Directed Repair (HDR) that requires careful orchestration of complex steps. Rather, many reports of in vivo gene editing rely on an error-prone mechanism called Non-Homologous End Joining (NHEJ). While this pathway is effective for elimination of protein function via the introduction of insertions and deletions (Indels) into the genome, it presents little to no utility for correcting disease-causing mutations in DNA. As such, there is a pressing need to develop effective non-viral carriers capable of replacing the mutated DNA sequence with the corrected sequence via HDR.
Currently, non-viral, in vivo gene editing techniques have been limited in their capacity to precisely correct mutations in DNA, with most examples yielding correction rates of less than 1%. Additionally, many delivery systems aimed at inducing HDR have consisted of multiple transfection vehicles, including virus, due to the diverse set of nucleic acid cargoes required for this process. However, techniques relying on viral vectors and/or separate carriers are nonoptimal due to potential immunogenicity and process dependence all three nucleic acids.
This dissertation details the development of dendrimer-based lipid nanoparticles (dLNPs) for the encapsulation and delivery of multiple nucleic acids necessary for HDR: Cas9 mRNA, sgRNA, and a donor DNA template containing the correct nucleic acid sequence, as well as the optimization of intra-particle nucleic acid ratios for efficient induction of HDR in vivo. To assess in vivo HDR efficiency, we employed xenograft tumors consisting of BFP/GFP switchable HEK293 cells with a single Y66H amino acid mutation. Through systematically adjusting the individual internal ratios of Cas9 mRNA, sgRNA, and donor ssDNA, an optimal balance of components resulted in a HDR rate of greater than 20% in vivo. This is the first report of a completely non-viral, LNP-based, fully nucleic acid-mediated delivery system capable of inducing HDR. Due to the all-in-one simplicity and high efficacy, HDR dLNPs provide a route forward towards correcting DNA mutations responsible for genetic disease
Recruitment of Enzyme Cascade to Phase-Separated Biomolecular Condensates Accelerates Reactions via Concentration-Dependent and Concentration-Independent Mechanisms
Get PDFBiomolecular condensates are ubiquitous throughout biology, but their functions remain largely poorly understood. Biomolecular condensates concentrate biomolecules relative to the surrounding medium. For biomolecular condensates that concentrate enzymes and their substrates, classic enzyme kinetics predicts an acceleration of the reaction rate within the condensates, but the effect of condensates on enzymatic activity both within and outside condensates has not been widely investigated. In order to understand these effects in more detail, we developed an in vitro model system consisting of multivalent protein scaffolds and a minimal enzyme system-the SUMOylation cascade. By inducibly recruiting various combinations of components of the SUMOylation cascade to condensates, we are able to uncouple the contributions of individual components and phases to enzymatic activity. We find that the reaction is accelerated when all SUMOylation components are recruited to condensates, and this acceleration requires recruitment of both enzyme and substrate. This is despite condensates representing only 1 % of total solution volume. This enhancement is limited to substrates whose KM is well above total substrate concentration. This selective enhancement is further demonstrated with simple modeling to show that substrate concentration relative to KM is a key factor in understanding the degree to which different substrates are likely to be influenced through condensate recruitment. Recruitment accelerates not only the reaction within the condensate but also the reaction outside the condensates. To understand what fraction of this increased activity within condensates is attributable to increased concentration of enzyme and substrate, we measured activity at identical concentrations of enzyme and substrate but lacking the scaffolds. We find that condensate activity exceeds the concentration-matched reaction, suggesting there is concentration-independent activity enhancement. Further investigation found that this excess enhancement is likely due to a scaffold-induced reduction in apparent KM. These results suggest that condensates can accelerate enzymatic activity through multiple mechanisms, including concentration and molecular organization of enzyme and substrate. Condensates selectively accelerate substrates whose total concentration is low relative to KM. Together these effects demonstrate the capacity of condensates to impart activity enhancement, specificity, and potentially sequestration through regulated enzyme and substrate recruitment
The PNUTS-PP1 Complex Acts as an Intrinsic Barrier to Kaposi's Sarcoma Associated Herpesvirus Gene Expression and Replication
Get PDFControl of RNA Polymerase II (pol II) elongation is a critical component of gene expression in mammalian cells. The PNUTS-PP1 complex controls elongation rates, slowing pol II after polyadenylation sites to promote termination. The Kaposi's sarcoma-associated herpesvirus (KSHV) co-opts pol II to express its genes, but little is known about its regulation of pol II elongation. I identified PNUTS as a suppressor of a KSHV reporter gene in a genome-wide CRISPR screen. PNUTS depletion enhances global KSHV gene expression and overall viral replication. Mechanistically, PNUTS requires PP1 interaction, binds viral RNAs downstream of polyadenylation sites, and restricts transcription readthrough of viral genes. Surprisingly, PNUTS also represses productive elongation at the 5´ ends of the KSHV reporter and the KSHV T1.4 RNA. From these data, I conclude that PNUTS' activity constitutes an intrinsic barrier to KSHV replication likely by suppressing pol II elongation at promoter-proximal regions
NAFLD and cardiovascular risk: translational implications for clinical practice
Get PDFDetailed formal protocol with illustrations and extensive bibliography.A recording of the protocol presentation is available on UT Southwestern's Mediasite. Note: Access to the video is restricted to authorized UT Southwestern users only.UT Southwestern--Internal Medicin
Uncovering Molecular Determinants of Pathogenic Non-Coding Structural Variation in Leukemia Genomes
No full textNon-coding structural variants (SVs) can rewire chromatin topologies to cause oncogene activation in cancer genomes, yet the molecular determinants for the transcriptional output mediated by SVs remain poorly understood. Advances in sequencing methodologies are increasing the number of SVs identified yet challenges remain to distinguish non-coding SV cancer drivers from non-functional passenger mutations. Hence, deciphering the regulatory principles governing how SVs, epigenetic landscapes, and 3D genome structure cooperate to control oncogenic transcription will be crucial for understanding the pathogenic potential of non-coding SVs. This dissertation outlines the development and implementation of a novel multimodal strategy to detect non-coding SVs with potential functional consequence and the characterization of an interdependency between genetic and chromatin variation for SV-mediated transcriptional effects. Together, these findings broaden our understanding of the features controlling the transcriptional output of pathogenic non-coding SVs and highlight new opportunities to reprogram gene regulation as epigenetic therapies in human disease
Liquid-Liquid Phase Separations in Innate Immune DNA Sensing and NF-κB Signaling Pathways
Get PDFThe 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
Neuropsychological Predictors of Time to Conversion from Mild Cognitive Impairment to Alzheimer's Disease
Get PDFMild 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