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THE ESSENTIAL DOESN’T CHANGE NOTHING TO BE DONE STORIES ABOUT LIFE AND DEATH AND RARE DISEASE
No full textWriting is exploration expressed in text. As Joan Didion once said, “I write entirely to find out what I’m thinking, what I’m looking at, what I see and what it means.” Life is learning and across these pages I learned some of my best and hardest lessons. Becoming a rare disease mom, adjusting to a new normal, having hope for a new treatment, looking ahead to the future, and living through the unique hell of parental grief. This is a collection of writing that exposes the fragility and resilience of humanity
Active transport phenomena in biology across different scales
Full text linkBiological systems function within an aqueous environment enriched with various solutes, including ions and proteins. The transport of these essential components—water and solutes—is an active process that operates across multiple scales, from single cells to multicellular structures and systemic circulation. Active transport plays a crucial role in cellular metabolism, growth, migration, morphogenesis, and systemic functions such as blood circulation. While many studies focus on transport driven by single environmental factors, such as hydrostatic pressure, it remains unclear how cells integrate multiple stimuli, including hydrostatic and osmotic pressures, to regulate active transport. Additionally, the interplay between transport processes across scales remains poorly understood, particularly in establishing stable metabolic activity and maintaining thermodynamic gradients out of equilibrium. In this thesis, we first provide a general framework for understanding active transport in biological systems, focusing on solute and water transport across different scales. For solute transport, we examine how the active intracellular transport of macromolecules, such as proteins, contributes to stable cell growth and nuclear size scaling. We then explore the coupling between solute and water transport, demonstrating how cells regulate their fluid environment by modulating membrane tension and ion transporter distribution. This regulation enables directed migration at both the single-cell and collective-cell levels. On a systemic scale, we investigate the role of active fluid pumping by renal epithelial and endothelial cells in maintaining sustained pressure and osmolarity gradients, which are essential for stable blood circulation in the human body. Finally, we discuss emergent behaviors driven by active transport in multicellular systems, using immune cell pattern formation as an example. Our work establishes a theoretical framework for understanding active transport in biological processes, providing new insights into cell migration, morphogenesis, and systemic fluid regulation
ROLE OF TEMPORALLY RESTRICTED TRANSCRIPTION FACTORS IN RETINAL TRANSCRIPTOMICS AND DEVELOPMENTAL CELL FATE SPECIFICATION
No full textThe mammalian central nervous system relies on complex signaling networks that drive cognition and problem-solving. Understanding how diverse cell populations arise and interact is crucial for assessing disease pathology. The retina serves as an ideal model for studying these processes and developing neuron-restorative therapies.
This thesis reviews recent advances in retinal neurogenesis, emphasizing insights from single-cell multiomic analyses. We explore how extrinsic signals drive transcriptional changes that pattern the optic cup and regulate neurogenesis. Additionally, we examine core, evolutionarily conserved gene regulatory networks (GRNs) that define early- and late-stage retinal progenitor cells (RPCs) and their roles in cell fate determination. Lastly, we discuss species-specific variations in retinal development and key unresolved questions.
To address these gaps, it is essential to understand molecular techniques available for studying developmental neuroscience. The Blackshaw lab has pioneered single-cell transcriptomics and genomics, contributing protocols on multiplexed single-cell RNA sequencing and fixed sample scRNA-seq and scATAC-seq. Multiplexing enables cost-effective phenotyping across genotypes or treatments early in investigations. Fixed sample sequencing provides insights into transcriptomic and genomic changes, revealing how gene expression is influenced by chromatin accessibility.
Our research focused on transcription factors regulating temporal identity. First, we examined the Kruppel-like factor family, which maintains late-stage progenitor identity. Overexpression during their temporal window expanded progenitor populations while repressing differentiation genes. Next, we studied MEINOX transcription factors, overexpressing them in late-stage development to assess their role in temporal identity shifts. While cell fate specification remained unchanged, we observed significant alterations in progenitor identity and maturation. Lastly, we analyzed NR2F transcription factors, identifying shifts in both cell fate specification and temporal identity through bioinformatics.
This work enhances our understanding of temporal identity regulation. These findings provide critical insights into reprogramming strategies, offering a foundation for future therapeutic applications in neuroregeneration
INVESTIGATIONS ON HIGH VALENT IRON CORROLAZINE COMPLEXES: SYNTHESIS, CHARACTERISATION AND REACTIVITY
No full textHeme enzymes utilize a heme cofactor which contain iron bound porphyrin in the active site to facilitate several important chemical transformations in biology. Most common species that is responsible for enabling these enzymes to achieve their function is reactive high valent iron oxo porphyrin -radical cation. This intermediate is often fleeting requiring synthetic analogs necessary to study their spectroscopic and reactivity properties. This dissertation features synthesis, characterization and reactivity studies on iron corrolazine complexes to generate high valent iron oxo corrolazine species particularly providing insights on factors controlling their reactivity.
An overview to high-valent iron-oxo found in heme enzymes, as well as a background on the synthetic metalloporphyrinoid analogues using iron metal and the factors controlling their reactivity properties have been discussed in Chapter 1.
Chapter 2 describes the generation and characterization of a series of 6-coordinate iron (IV)-oxo porphyrinoid-π-cation-radical complexes, FeIV(O)(X)(TBP8Cz+•) using the ring contracted corrolazine ligand framework. This work demonstrate that the PCET reactivity of a Cpd-I analog is driven by the redox potential of Cpd-I rather than basicity and it provide the first evidence showing axial ligation inhibits the PCET reactivity of a Cpd-I analog.
Chapter 3 describes the isolation of first high valent Fe(IV)(X) (where X = Cl, triflate) corrolazine complexes. The reactivity of these complexes with O-atom donors were investigated. Utilizing an array of spectroscopic techniques (XAS, variable field Mossbauer, IR, LDI-MS), the new complex has been proposed to be hosting O-atom at one of the meso N atoms in the corrolazine ring. The OAT reactivity of this complex has been explored using PPh3 as the substrate.
Chapter 4 summarizes the optimized synthetic conditions to stabilize an Fe(IV)(OH) in corrolazine ligand. This involves characterization by oxidation and reduction titration UV-vis experiments, Mossbauer and DFT studies on the electronic structure of Fe(IV)(OH) corrolazine.
Chapter 5 reports the synthetic strategy to modify the remote ligand sites on iron corrolazine complex. Synthesis and isolation of monoprotonated and tri protonated iron corrolazine by crystal structure has been described in this chapter
ARACHNOBOT: DEVELOPING A ROBOT CAPABLE OF TRAVERSING NETTING
No full textRobots struggle to traverse netting upside down (e.g., fishing nets, climbing nets). Current literature does not have much information on robots walking on netting, such as an artificial web. On the other hand, spiders are incredible at maneuvering webs. We set an objective to create a spider-inspired robot able to traverse an artificial web (a form of netting) upside down, and explore what factors are important in traversing an artificial web. Our biological inspiration and reference was the spider Uloborus diversus. We tried a novel robot gait, which was using the limb, bearing the most body weight, to pull the body forward. Trials with our robot gait led us to realize an important guideline when traversing an artificial web upside down: only one limb should have its torque engaged when attempting to move the robot’s center of mass (CoM). An individual limb is not strong enough to move the robot’s center of mass. Coordinated movement amongst all the limbs is required, which involves releasing all limb torques except for the one limb pulling the robot’s body across the artificial web. In other words, our robot needs to swing its center of mass to traverse the web using the force of a single limb. In addition to the exploration of spider-inspired robophysics, we also developed a 3D visual analysis pipeline to process spider locomotion videos, generating tracked 3D locomotion
PHARMACOLOGICAL MODULATION OF GLUTAMINE METABOLISM ALTERS MACROPHAGE ACTIVITY AND TUMOR IMMUNE RESPONSES
No full textTumor-associated macrophages are the important components of the tumor immune repressive microenvironment, which present as the M2-like macrophage and have the capacity to repress anti-tumor immune response and mediate tumor cell immune escape. Targeting macrophages, especially macrophage metabolism, has become a promising direction to determine specific mechanisms as well as develop new therapy strategies for cancer immune therapy. Among them, glutamine metabolism is one of the vital metabolism pathways involved in macrophage polarization regulation. Previous studies have already shown evidence of the regulatory effects of glutamine metabolism during macrophage polarization. In the paper, we utilize broad pharmacological repression of glutamine metabolism to investigate the effects of glutamine metabolism in both M1 and M2 macrophages. Glutamine antagonist JHU083-treated iPAD mouse models exhibited reduced TAMs. Meanwhile, in vitro BMDM experiments showed repressive effects of DON (the active components of JHU083) on M2 macrophages while it also showed the beneficial effects of DON on M1 macrophage activation. Future research will focus on the roles of different metabolites of glutamine metabolism and oxidative stress in pharmacologically regulated macrophage polarization, thus providing deeper insight into specific mechanisms and benefiting new therapy development
DEVELOPING AND APPLYING SEQUENCING-BASED TOOLS TO UNDERSTAND ADAPTIVE IMMUNE RESPONSES IN MYOSITIS
No full textIdiopathic inflammatory myopathies (IIMs) are a group of heterogeneous, systemic diseases characterized by skeletal muscle pathologies, which most commonly result in chronic, progressive muscle weakness. Immune infiltrates, which includes B and T cells, are often found in the muscle of IIM patients. Currently, autoantibodies help stratify IIMs into clinical subtypes, however disease heterogeneity persists within subtypes of IIMs and the etiologies of IIMs remain unknown. Through the application of adaptive immune profiling methods: 1) Phage Immunoprecipitation Sequencing (PhIP-Seq) to understand antibody reactivities at cohort-scale and 2) B cell receptor sequencing, I have further characterized the antibody repertoires in IIMs. Using PhIP-Seq, I identified novel environmental and autoantibody reactivities in subtypes of IIMs. These findings may generate hypotheses about IIM etiologies, and improve IIM disease classifications. Using both PhIP-Seq and BCR sequencing, I characterized the diversity of the local antibody repertoire in muscle tissue of IIM patients, improving our understanding of the adaptive immune response within muscle. To improve our ability to perform immune receptor repertoire sequencing in a low-cost, high-throughput manner, I developed a method, INtraCEllular Reverse Transcription with Sorting and sequencing, or INCERTS, to efficiently link T cell phenotypes with T cell receptor sequencies. INCERTS is complementary to single-cell sequencing. Through the application of these sequencing- based methods, I have increased our understanding of the B cell immune response in IIMs. Through the development of INCERTS, I have improved our ability to perform immune receptor repertoire sequencing, which can be applied to further increase our understanding of the adaptive immune response in IIMs
BELOW COUNTRY: A COLLECTION OF SHORT STORIES
No full textThe South prides itself on being different, and these stories capture the Weird and quirkiness of the region, alongside its distinctive beauty, history and culture. Set in Dakker, a fictional small town along the South Carolina coast, this collection of short stories showcases a wide swath of the town’s people, from the elite to the fringes of society. In each piece, characters wrestle with demons; they struggle against who they are and who they want to be. Some are driven by the desire for money; some for respect and notoriety; and others grasp for a kind of divine ascendency
RMC-7977: A PROMISING INHIBITOR FOR THE TREATMENT OF NF1-RELATED TUMORS
Full text linkNeurofibromatosis type 1 (NF1) is a common genetic disorder characterized by mutations in the NF1 gene, leading to the loss of neurofibromin, a RAS GTPase-activating protein, which results in the persistent activation of the RAS-MAPK signaling pathway, facilitating tumorigenesis, such as cutaneous and plexiform neurofibromas, malignant peripheral nerve sheath tumors (MPNSTs), and gliomas. Despite advances in our understanding of NF1-associated tumor biology, current treatment options remain limited, especially for MPNSTs and high-grade gliomas. RMC-7977, a novel tri-complex pan-RAS inhibitor, selectively targets the active GTP-bound form of RAS by forming a stable complex with cyclophilin A (CYPA) and RAS, offering a new therapeutic approach.
Here, we evaluated the efficacy of RMC-7977 in NF1 -mutated tumor models. IC50 assays were performed on multiple NF1 cell lines, identifying sensitive lines for in vivo investigation. Xenograft mouse models were then established via orthotopic implantation of luciferase-tagged cells into the sciatic nerve, brain, or subcutaneously of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. We found that RMC-7977 significantly suppressed tumor progression in ST8814, S462, and JH-2-002 models, and improved survival in intracranial LN229 tumor-bearing mice, with delayed but statistically significant tumor growth inhibition and prolonged survival. Interestingly, despite its high molecular weight of 865.1 Da, RMC-7977 demonstrated the ability to penetrate the Blood-Brain Tumor Barrier (BBTB) and exert antitumor activity within the central nervous system. Overall, these findings provide foundational evidence for RMC-7977 as a promising therapeutic strategy for targeting RAS-driven NF1 malignancies
Evaluating the effect of osteoarthritis on the distant tumor microenvironment and response to immunotherapy
No full textDespite unprecedented outcomes associated with immune checkpoint blockade (ICB), most cancer patients either do not respond or relapse after a partial response. The mechanisms responsible for heterogeneous ICB responses are poorly understood. Acute and chronic stressors are known to influence cancer development. The induction of senescent tumor cells and their senescence-associated secretory phenotype (SASP) has been shown to remodel tumor vasculature, increasing ICB responsiveness. Further, clinical studies have reported patients with osteoarthritis (OA), a disease associated with senescent cell (SnC) accumulation, have slower solid tumor progression and a lower risk of cancer-related mortality. Thus, by understanding the impact of cellular senescence and other systemic responses induced by OA on tumor growth, we sought to elucidate the characteristics of therapy resistance.
Anterior cruciate ligament transection (ACLT), a model of post-traumatic OA, leads to SnC accumulation in the joint and systemic SASP-mediated effects. Here, we investigated the immunological and stromal changes arising with distant ACLT-induced OA (ACLT-OA) and ICB within various murine syngeneic tumor models. ACLT, performed prior to tumor injection, significantly delayed tumor growth and increased ICB responsiveness. Additionally, distant ACLT-OA induced an increase in T cell, especially CD8+ tumor-specific, infiltration into the tumor. Proteomic profiling revealed elevated serum-levels of pro-inflammatory cytokines and factors associated with vessel maturation in ACLT-OA mice. scRNAseq identified transcriptional changes in endothelial cells associated with vascular remodeling. Immunostaining validated the predicted tumor vascular maturation effects; ACLT-OA increased tumor vascular integrity to support anti-tumor immune infiltration and induced the accumulation of vascular-associated SnCs. SnC deletion, using the transgenic INK-ATTAC model, abrogated all of the ACLT-OA-induced effects on the tumor microenvironment, highlighting the critical role of senescence in this interaction. Our findings suggest that the response to OA induces SnC accumulation within the tumor, remodeling the tumor vasculature to promote immune cell infiltration and overcome ICB resistance.
In summary, this work explores the senescent-vascular-immune axis to reveal factors that influence favorable responses to ICB to help predict non-responders and develop sensitizing therapies. Future work seeks to identify the systemic factors of OA that promote ICB sensitivity and design combination therapies to overcome ICB resistance