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Transcriptional Regulation of Neonatal Heart Regeneration and Direct Cardiac Reprogramming
No full textThe adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. Deciphering the molecular underpinnings of neonatal heart regeneration and the blockade to regeneration in later life may provide novel insights for heart repair. To elucidate the transcriptional responses of the different cellular components of the mouse heart following injury, we performed single cell RNA-sequencing on neonatal hearts at various timepoints following myocardial infarction, and coupled the results with bulk tissue RNA-sequencing and H3K27ac ChIP-sequencing data collected at the same timepoints. This approach provides detailed transcriptional dynamics of heterogeneous cardiac cell types during neonatal heart regeneration. Concomitant single cell ATAC-sequencing exposes underlying dynamics of open chromatin landscapes and regenerative gene regulatory networks of diverse cardiac cell types, and reveals previously unknown extracellular mediators of cardiomyocyte proliferation, angiogenesis, and fibroblast activation. Furthermore, using single-nucleus RNA sequencing, we mapped the dynamic transcriptional landscape of five distinct cardiomyocyte populations in healthy, injured and regenerating mouse hearts. We identified immature cardiomyocytes that enter cell-cycle following injury and disappear as the heart loses the ability to regenerate. These proliferative neonatal cardiomyocytes display a unique transcriptional program dependent on NFYa and NFE2L1 transcription factors, which exert proliferative and protective functions, respectively. Cardiac overexpression of these two factors conferred protection against ischemic injury in mature mouse hearts that were otherwise non-regenerative. Together, these findings provide mechanistic insights into the molecular basis of neonatal heart regeneration, and offer various pathways that can be manipulated to facilitate cardiac repair after injury.
Direct reprogramming of fibroblasts into induced cardiac-like myocytes using cardiac transcription factors offers another possible therapeutic approach for cardiac repair. To elucidate the gene regulatory network during direct cardiac reprogramming, we performed a genome-wide analysis of cardiac transcription factors binding sites and active enhancers during reprogramming. We found reprogramming factors cooperatively activate enhancers involved in cardiac development and maturation, and further delineated the regulatory relationships between reprogramming factors and cardiac gene expression. These findings reveal synergistic activation of the cardiac epigenetic landscape by cardiac transcription factors and key signaling pathways that govern direct cardiac reprogramming
Spooked
Get PDFThe author submitted this entry in the Fictional Short Story category (Amateur division) for the 2023 On My Own Time (OMOT) Art Show.There are always two sides to every story, and just because you hear someone doesn't mean you always hear everything. I wrote this story to show how easily miscommunication can happen, even with good intentions. When you love someone, never whisper, make it clear. Make it loud
Development of Applications and Quantitative Frameworks for Multispectral Optoacoustic Tomography
Get PDFPage xxxi is misnumbered as page ii.The file named "OKELLY-PRIMARY-2022-1.pdf" is the primary dissertation file. Four (4) supplemental files are also available; these files include 1 video file (MP4), 2 Microsoft Excel files (XLSX), and 1 Microsoft Word document (DOCX) and may be viewed individually.The tumor microenvironment is a highly complex system, with variations through space and time that are determined by the interplay of normal and cancerous cells, physiological phenomena, and treatments that can dramatically change the structural or biological dynamics underlying the emergent behaviors. Quantitatively and reliably imaging the microenvironment represents an opportunity to develop diagnostic and prognostic assessment of cancer patients, enabling a fuller understanding of the tumor's evolution, and response to treatments. Multispectral optoacoustic tomography (MSOT), a novel imaging modality, has the potential to reveal the spatiotemporal dynamics of oxygenation at high resolution through the use of multiplexed laser light and has shown promise in advancing both clinical and pre-clinical research. Nevertheless, current methods of analysis often fail to yield sensible data, and are prone to artifacts and quantitative errors that preclude the effective use of this imaging method for diagnostic or prognostic imaging and that add difficulties in downstream analyses. In this work, I developed a battery of methods and tools that bridge the gap of MSOT's theoretical capabilities and the practical realities of its usage. These include a transparent and open-source toolbox for image reconstruction and analysis along with its deployment to a cloud-based workflow service managed by the University of Texas Southwestern Medical Center at Dallas' BioHPC; a simple and scalable method to address spectral aliasing and improve the time resolution and signal-to-noise ratio of dynamic MSOT data; a method to extract quantitative breathing parameters from tomographic imaging data; and a model scheme of the systemic physiology that determines the response to gas-breathing challenges. These developments have laid the groundwork for more rigorous investigations using MSOT for preclinical imaging research
Spatiotemporal Regulation of the NADP(H) Phosphatase Nocturnin
Get PDFPeriodic changes in the environment are ubiquitous in the natural world. Among these, the most biologically relevant rhythm is the 24-hour geophysical day/night cycle. As an adaptive strategy, many organisms have evolved an endogenous biological clock to temporally organize their physiology and anticipate daily changes in the environment. At its core, the mammalian "circadian clock" is a molecular oscillator driven by a genetic transcription/translation feedback loop, which orchestrates the rhythmic expression of thousands of genes. An intimate link between circadian clocks and metabolism is established by the rhythmic transcription of output genes involved in almost every metabolic pathway. Among these oscillating genes, Nocturnin (also Noct; protein name: NOC) has one of the highest amplitude rhythms at the mRNA level. Mice with a loss-of-function in Noct possess metabolic phenotypes, where they are protected from high-fat diet-induced obesity and LPS-induced septic shock. However, the mechanism by which this occurs is not well-understood. Here, in collaboration with Green lab members and the Liou lab, I used both in vitro biochemical and in vivo cellular and mouse models to elucidate the molecular and physiological function of NOC. Even though NOC is highly-conserved with the endonuclease/exonuclease/phosphatase (EEP) domain-containing CCR4 family of deadenylases, we show that highly-purified recombinant NOC lacks ribonuclease activity. Instead, NOC catalyzes the dephosphorylation of NADP(H), and its activity level is associated with the cellular response to oxidative stress. Furthermore, we describe two isoforms of NOC and their spatiotemporal regulation in the mouse liver. Cytoplasmic NOC is constitutively-expressed throughout the day and associates externally with the endoplasmic reticulum and other membranes via N-terminal glycine myristoylation. In contrast, mitochondrial NOC levels are highly circadian with peak expression during the early dark phase. Overall, our work suggests that NOC links circadian clocks to metabolism by regulating local intracellular concentrations of NADP(H) in a manner that changes throughout the day
Alternating Sequences of Future and Past Behavior Encoded Within Hippocampal Theta Oscillations
No full textExperience necessarily occurs in a sequence, and adaptive behavior requires the ability to analyze experience, both prospectively and retrospectively. It remains unclear how forward-ordered neural activity can facilitate storage or expression of reverse-ordered sequences, which are observed in ripple-based reverse replay and may underlie human episodic memory retrieval. Specifically, hippocampal function is critical for representation and storage of sequential information during spatial navigation of animals. Pyramidal neurons in the hippocampus have been found to intuitively encode locations in space, and are thus termed "place cells". During active exploration, place cells display "phase precession" relative to the ongoing 4-12 Hz theta rhythm, firing at progressively earlier phases of theta as the rat traverses a cell's place field. Across large populations of place cells, phase precession is hypothesized to produce theta sequences, temporally organized sequences of neural firing which encode short virtual trajectories ahead of the animal. While forward theta sequences during experience help explain prospective memory formation, it is unclear how retrospective evaluation of prior behavior can be consolidated.
To understand how reverse-ordered memory arises from forward-ordered behavior, I analyzed an in-vivo electrophysiological dataset in which the simultaneous activity of hundreds of neurons was recorded while rats were engaged in navigational tasks in multiple environments. I observe that during active navigation, hippocampal CA1 place cell ensembles are inherently organized to produce independent forward- and reverse-ordered sequences within each theta oscillation, providing a circuit-level basis for retrospective evaluation and storage during ongoing behavior. The cellular mechanisms underlying the reverse-ordered sweep of the theta sequences is theta phase procession arising in a minority of place cells (bimodal cells) which display two preferred firing phases in theta and preferentially participate in reverse replay during subsequent rest. Independent modulation of the reverse and forward theta sweeps suggests separate upstream circuit inputs, with the reverse theta sequence likely driven by layer III entorhinal cortex. These findings reveal a novel and unexpected aspect of theta-based hippocampal encoding and provide a biological mechanism supporting the expression of reverse-ordered memory
(A)I Made a Promise
Get PDFThe author submitted this entry in the AI Literature category (Amateur division) for the 2023 On My Own Time (OMOT) Art Show.Written with the feedback and revisions of an AI ChatBot, this piece is a reflection on the promise I made to a brother taken by cancer
Quantitative Studies of Composition and Formation of Yeast P Bodies
Get PDFEukaryote cells organize their internal spaces into distinct compartments to achieve precise spatiotemporal regulation of biochemical reactions. One level of organization is achieved through membrane borders that form classical organelles such as nuclei and mitochondria. However, another widespread type of structures concentrates distinct molecular components without being enclosed by membranes--these are termed biomolecular condensates. Quantitative studies are lacking to mechanistically understand condensates within the complicated cellular environment. Toward this aim, I developed live cell imaging methods to quantitatively measure protein partitioning into condensates. Using P bodies, an archetypal biomolecular condensate that concentrate proteins and RNA, I first generated a quantitative inventory of the major proteins in yeast P bodies. I found that only 7 proteins are highly concentrated in P bodies while the 24 others examined are appreciably lower. P body concentration correlates inversely with cytoplasmic exchange rate. Based on the results, I proposed that the compositions of natural condensates can be classified into scaffold-like and client-like components based on their distinct partitioning and interaction network.
To understand compositional specificity, I showed that sequence elements driving Dcp2 enrichment into P bodies are distributed across the protein, and that these elements act cooperatively. Multiple distributed enrichment elements provide a thermodynamic framework for regulating compositional specificity of P bodies. I further illustrated that changing the molecular interactions could shift phase boundaries, suggesting that behaviors of biomolecular condensates are dictated by molecular interactions. Taken together, my work provides a quantitative view of compositions and formation of natural biomolecular condensates
Discovery, Biological Profiling and Mechanistic Studies of Three Novel Antimalarials
No full textEmerging resistance of the malaria causing Plasmodium parasite to current first-line therapies underscores the need for new antimalarial agents with broad ranging activity against multiple stages of the parasite. No new chemical class of antimalarials has been introduced into clinical practice since 1996. Overcoming emerging drug resistance requires new drugs with novel modes of action. With the aim of identifying new classes of antimalarials, I completed a phenotypic high throughput screen of two synthetic chemistry libraries against erythrocytic stage P. falciparum and compiled a portfolio of chemically novel validated antiplasmodials (ALCHM1 - 18). Herein I describe the discovery and characterization of three prioritized scaffolds: a tetrazole-based ALCHM3 series, an azetidine amide ALCHM17 series and a piperidine carboxamide ALCHM18 series. I report here on the biological profiling, mechanistic characterization and potential as next-generation anti-malarial agents of these three previously unreported scaffolds. ALCHM3 is a novel chemical series, with fast kill kinetics that targets the historically druggable heme polymerization pathway. The fast kill azetidine amide ALCHM17 series, is a novel scaffold with inhibitory activity in the pre-erythrocytic and erythrocytic stages, and the first azetidine scaffold with Pfcarl associated resistance. The piperidine carboxamide ALCHM18 is a proteasome β5 subunit-selective inhibitor, with a moderate rate of kill, species selectivity, strong starting in vitro and in vivo ADME properties, and potential to become the first proteasome inhibiting preclinical candidate for malaria treatment
Space I
Get PDFThe author submitted this entry in the Open Verse Poetry category (Amateur division) for the 2023 On My Own Time (OMOT) Art Show.I struggle to articulate myself aloud and, in the moment, but writing gives me the ability to stop and really think of how I am feeling. The ability to reflect my emotions and thoughts in an abstract manner is a great way of relieving stress, and I think this piece gives insight into a time when there was immense pressure I was facing
Ethics in academia and the pharmaceutical industry: personal experience
Get PDF[Note: The slide presentation is not available from this event.] Tuesday, March 14, 2023; noon to 1 p.m. (Central Time); via Zoom. "Ethics in Academia and the Pharmaceutical Industry: Personal Experience". Nassir Ghaemi, M.D., M.P.H., Professor of Psychiatry and Pharmacology at Tufts University School of Medicine School and Director of the Mood Disorder Clinic and the Psychopharmacology Consultation Clinic at Tufts Medical Center, Boston, Massachusetts.[Note: The slide presentation is not available from this event.] Many academics in medicine assume that they hold a higher ethical standard than the pharmaceutical industry. Some of this assumption has to do with the presumed not-for-profit nature of academia and the for-profit nature of the pharmaceutical industry. In this discussion, based on my personal experience in both settings, I will contrast and compare the ethical standards of the two groups. I will conclude that they are both ethically challenged with different drawbacks. In academia, the usual ethical problem is an excessive focus on power and prestige; in the pharmaceutical industry, as is well known, the ethical problem is the profit motive. Both are highly problematic, with different causes producing the same effects: the interests of the public are sacrificed for the desires of academics and the pharmaceutical industry. Different strengths also exist: Academic freedom of thought allows for free public expression of ideas, but, combined with tenure and the wish for inclusion in the status quo power structure, it leads to self-censorship and a conservatism of thinking. Academic life begins with freethinking and ends in group think. The pharmaceutical industry is much more open to new ideas because it is not attached to any idea; its main test is practical utility in the marketplace. Hence, it is, paradoxically, much more innovative than academia, but this innovation is only tied to commercial results. Many public health needs are ignored due to non-commercial potential.UT Southwestern--Program in Ethic