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The ability to learn and remember is crucial to our daily function, long-term survival, and personal identity. On a cellular level, this ability relies on the reshaping of synaptic connections between neurons. This means changing the cellular structure which houses most of the excitatory synapses in the brain- the dendritic spine. Dendritic spines are highly actin-rich structures, and their remodeling requires enlisting pathways which control actin dynamics, such as the Rho GTPases, and their activators, the Rho guanine nucleotide exchange factors (RhoGEFs). RhoGEFs are critical for regulating the development of dendritic spines, and facilitating their plasticity during the synaptic activation associated with learning. The RhoGEF Ephexin5 has been characterized as a negative regulator of spine outgrowth and is implicated in multiple diseases including Alzheimer’s. However, prior investigation has also revealed an additional for Ephexin5 in spine regulation: Ephexin5 is required for activity-dependent outgrowth. It is unclear what the underlying mechanisms of this role are, and it is unknown how the various roles of Ephexin5 are regulated. As activity-dependent spine remodeling is crucial for memory, uncovering the mechanisms of Ephexin5-mediated spine remodeling could lead to a deeper understanding of the fundamental processes which govern learning in the brain, as well as how those processes are disrupted in diseases such as Alzheimer’s.In Chapter 1 of this dissertation, I will review the current understanding of how RhoGEF signaling shapes dendritic spines, and how Ephexin5 fits into this framework. Chapter 2 of this work, which is adapted from a manuscript submitted for publication, explores the hypothesis that Ephexin5 carries out its role in facilitating activity-dependent spine remodeling by serving as a GEF for Cdc42. I demonstrate that during synaptic stimulation Ephexin5 is responsible for Cdc42 activation and spine growth. Further, I reveal an important activity-dependent de-phosphorylation mechanism for regulating Ephexin5-mediated Cdc42 activation. In Chapter 3, I present data which supplements my discoveries in the previous chapter, and adds further context to Ephexin5 signaling at the synapse by demonstrating a role in conformational NMDAR signaling and by providing a preliminary report of its hippocampal interactors. Finally, in Chapter 4 I will conclude by summarizing my contributions to the field and proposing important future directions for this research.

Metastasis is a major determinant of patient survival in cancer, yet the genetic and metabolic mechanisms underlying metastatic progression remain poorly understood. In our previous work, we identified PHLDA2, a gene not previously implicated in breast cancer metastasis, as a transcriptional marker of metastatic breast cancer. Analyzing patient datasets, we found this gene is more highly expressed in breast tumor tissue than normal tissue. This elevated expression is likely driven by methylation, as PHLDA2 is an imprinted gene, indicating a potential role for epigenetic regulation in its overactivation in cancer. Patient datasets revealed that high PHLDA2 expression is associated with reduced relapse-free and distal metastasis-free survival, underscoring its prognostic significance. Building on these findings, this thesis explores PHLDA2 as a driver of metastatic burden, demonstrating that it promotes breast cancer metastasis by restructuring the extracellular matrix and increasing vessel permeability, thereby reshaping the metastatic microenvironment. Additionally, to better study the metabolic processes driving aggressive cancer cell behavior at metastatic sites, I developed a single-cell metabolomics approach. This work identifies citrate as a potential metabolic drivers of cancer metastasis using a novel tool to study cancer metabolism at a single-cell resolution.

In nature, cephalopods’ astounding camouflage abilities are enabled by their sophisticated skin, that contains multiple types of cells acting as active optical elements, which can be actuated to enable the tunable optical properties of the skin, due to the presence of stimuli-responsive protein reflectin. First, we genetically engineer human cells to produce reflectin transiently and natively and extensively characterize those cells with three-dimensional holotomography, while demonstrating the potential of reflectin-based subcellular structures as high refractive index biomolecular reporters. Next, we establish chemical and electrical stimulation of our engineered human cells to alter the internal arrangements of reflectin-based structures and characterize such alterations with three-dimensional holotomography, while demonstrating the modeled optical properties of stimulated cells. Last, we extract single cells from squid skin tissue and characterize their subcellular structures with three-dimensional holotomography, while demonstrating the significance of subcellular morphological features for squid skin structural coloration. In summary, we enable a robust approach to systematically characterize engineered human single cells with three-dimensional holotomography, establish tunable optical properties of human cells and using three-dimensional data predict their optical properties, and determine the three-dimensional subcellular characteristics of squid skin cells that are responsible for their structural coloration.

Chronic pain is a hallmark of numerous disease conditions including nerve and spinal cord injury. Current mainstays of pain management include analgesics and anesthetics, treatments which are used despite their uncertain efficacy and known significant side effects. Safer and more productive approaches to pain management are urgently needed, but knowledge gaps in basic research have hampered the development and translation of novel treatments. The spinal cord is a crucial signaling hub involved in communicating pain-related signals between peripheral organs and the brain, serving as the first site of sensory integration within the central nervous system. Significant attention has focused on neuronal cell types and circuits perturbed during neuropathic pain. However, considerably less is known about the contributions of non-neuronal cells, such as astrocytes. Herein we investigate the role of astrocyte calcium signaling in the development of chronic neuropathic pain following peripheral nerve injury. We find by 1-Photon in vivo microscopy with wearable microscopes mounted to murine spinal cords that spinal cord dorsal horn astrocyte calcium signaling increases in frequency and intensity following peripheral nerve injury. Viral inhibition of Gq-GPCR dependent calcium signaling in dorsal horn astrocytes prevents the development of chronic mechanical and thermal hypersensitivity behavior after nerve injuryin mice of both sexes, driven by preventing pathological excitatory synaptogenesis as well as inhibitory synapse loss after nerve injury. This work unveils an essential role for spinal cord dorsal horn astrocytes and Gq-GPCR dependent calcium signaling in the development of chronic neuropathic pain following nerve injury, identifying astrocyte Gq-GPCRs as promising targets for next-generation pain therapeutics

Ras is a small GTPase that regulates cell growth and proliferation. Hyperactive Ras is prevalent in many cancers and has been a therapeutic target for decades. Given that functional roles of Ras are tightly coupled with its subcellular distribution and regulation, fluorescent Ras biosensors for tracking the spatiotemporal dynamics of Ras activity are instrumental for achieving a better understanding of both Ras signaling and Ras pathway inhibitors. However, current Ras biosensors are limited in their quantitative capacity. In this manuscript, we describe the development of two designs for endogenous Ras activity. FRET-based RasAR utilizes a pseudoligand design to modulate FRET between mCerulean3 and Ypet. RasAR is used measure the regulation of Src towards ectopically expressed HRas and has demonstrated capacity to measure Ras inhibition of overexpressed KRasG12C in response to novel Ras inhibitors Sotorasib and Adagrasib. To achieve endogenous Ras activity, we introduce a chemigenetic design for endogenous Ras engagement. HaloRasAR makes use of circularly permuted HaloTag covalently linked with chloroalkane-tethered far-red fluorescent JF635 dye. This HaloTag-based Ras Activity Reporter (HaloRasAR) reveals distinct temporal dynamics of endogenous Ras activity at plasma membrane and Golgi and uncovers two modes of regulation in the PKC-Ras signaling axis. In addition, live-cell characterization of RasG12C inhibitor RMC6291 and Ras-GEF inhibitor BAY293 reveals striking differences in their inhibitory effects on subcellular Ras activities, providing evidence for distinct pools of Ras that have differential susceptibility towards different inhibitors. HaloRasAR represents an important step forward in spatiotemporal interrogation of Ras signaling and live-cell pharmacology.

The Integrated Stress Response (ISR) is a conserved signaling network that detects aberrations and computes cellular responses. Dissecting these computations has been difficult because physical and chemical inducers of stress activate multiple parallel pathways. To overcome this challenge, we engineered photo-switchable control over the ISR sensor kinase PKR (opto-PKR), enabling virtual, on-target activation. Using light to control opto-PKR dynamics we traced information flow through the transcriptome and for key downstream ISR effectors. Our analyses revealed a biphasic, proportional transcriptional response with two dynamic modes, transient and gradual, that correspond to adaptive and terminal outcomes. We then constructed an ordinary differential equation (ODE) model of the ISR which demonstrated the dependance of future stress responses on past stress. Finally, we tested our model using high-throughput light-delivery to map the stress memory landscape. Our results demonstrate that cells encode information in stress levels, durations, and the timing between encounters. We expanded the single instance short-term activations to the long-term effects of consistent stress through dynamic conditioning regimes. We found that cellular stress response sensitivity is influenced by the duration of stress bursts during conditioning, with short bursts amplifying and long bursts muting the sensitivity. Additionally, we tested how virtual stress conditioning affects cellular fitness by challenging conditioned cells with toxins. Our findings indicate that continuous stress activation is detrimental and that proportional recovery periods are necessary to maintain cellular fitness, with minor benefits at consistent short bursts of activation. Moreover, we applied a similar methodology to opto-PKR for IRE1. We demonstrated that opto-IRE1 functions as intended, with light-enabled clustering and subsequent canonical IRE1 signaling, including IRE1 phosphorylation and the unconventional splicing of XBP1, serving as proof of concept for expanded studies in UPR.

Hedgehog (Hh) signaling is a critical pathway essential for embryonic development and maintaining adult tissue homeostasis. Dysregulation of this pathway is linked to numerous developmental disorders and various cancers. In vertebrates, Hh signal transduction is tightly dependent on the primary cilium, a small antenna-like organelle on the cell surface. Upon activation of the pathway, Smoothened (Smo), an atypical G protein-coupled receptor (GPCR), is released from the inhibitory control of Patched (Ptch) and translocates into the primary cilium, a pivotal step for Smo activation of downstream signaling, as discussed Chapter 1. Despite its importance, the molecular mechanisms governing Smo translocation and regulation remain inadequately understood.To address this, I leveraged the TurboID proximity labeling tool a method not previously used to study Smo signaling in the context of Hh pathway regulation. Chapter 2 details the development of Smo-TurboID system designed to label proteins that are proximal to Smo during its translocation into the primary cilium over the course of Hh signaling activation. Using Tandem Mass Tag (TMT)-mediated quantitative mass spectrometry, my work shows that Smo-TurboID system not only identified several well-established Hh signaling proteins, but also uncovered novel transient changes in the local micro-environment near Smo. In Chapter 3, my work focused on the newly identified Smo-interacting proteins, including the Catalytic subunit of Protein Kinase A (PKA-C) , which associates with Smo following Hh signaling activation, and G protein-coupled receptor kinase 2 (Grk2), a putative transient interactor. Notably, inhibition of Smo or Grk2 in Hh pathway diminishes the interaction between Smo and PKA-C, confirming the necessity of Smo-Grk2-PKA axis in Hh signaling.Chapter 4 extends this work by identifying GRK-interacting protein 1(Git1) as a positive regulator in Hh pathway. Using the Smo-TurboID system, Git1 emerged as a high-priority candidate, localizing at the base of primary cilium. CRIPSR/Cas9 knockout of Git1 in fibroblast markedly impaired Hh signaling and a reduction of phosphorylated Smo in the primary cilium. Importantly, I identified a critical link between Git1 and Grk2, a well-established activator that phosphorylates Smo within the cilia, offering fresh insight into the fine-tuning of Hh pathway regulation.Collectively, these findings provide a broader view of molecular regulation of Smo in Hh pathway at the primary cilium. By utilizing Smo-TurboID system, my work identified both known and novel Smo-associated proteins, revealing dynamic changes near Smo during it signaling transduction. Additionally, my work establishes the connection between Smo, Git1 and Grk2, offering valuable insights into potential therapeutic strategies targeting Smo interactors for Hh signaling-related cancers.

Protein Kinase A (PKA) is a highly conserved kinase that is involved with a myriad of cell signaling and physiological functions. It is interesting that PKA-null mouse embryos exhibit morphological phenotypes characteristic of Hedgehog (Hh) pathway mutants. Protein kinase A (PKA) has long been recognized as a potent inhibitor of the Hh pathway. However, how PKA specifically participates in Hh signaling has been an enigma. During embryonic development, the Hedgehog (Hh) pathway functions both as a crucial morphogenic signal that orchestrates tissue formation and as a potent mitogenic signal that drives cell proliferation. PKA and Hh signaling converge in the primary cilium, a specialized cell surface organelle viewed as the hub of cell signaling. In this study, we aim to identify the specific subcellular locations where PKA selectively regulates Hh signaling. Our findings suggest that two subcellular pools of PKA, one at the base of the cilium and one in the cilium, synergistically repress Hh signaling. Our results offer new insights into the spatial regulation of Hh signaling, and indicate that targeting PKA at specific subcellular location could be a potential therapeutic avenue for Hh-related diseases.

The underlying molecular mechanisms driving the wound response remain poorly understood. Thus, identifying the proper sequence of events responsible for tissue repair will be essential to clinical applications in regenerative medicine. Transcriptional and biochemical analyses of injured tissues have identified candidate genes and molecular pathways activated minutes to hours after injury. However, the upstream cues leading to genetic and biochemical changes remain elusive. Studies of the wound response in diverse organisms suggest that electrical changes at the site of tissue damage appear as one of the earliest cues. These studies imply that almost immediately after injury, ionic leakage and localized tissue depolarization result in lateral electric fields directed toward the injury site. Changes in endogenous potentials may also act as a first responder enabling tissues to sense the existence of injury. Here, we use the planarian flatworm Schmidtea mediterranea to test the role of tissue depolarization during early events of the wound response and try to identify which ions may be involved in this response. We developed a novel methodology to induce localized epithelial permeabilization without tissue damage. This is accomplished by brief exposure to amphipathic detergents. Our results show detergent-induced tissue permeabilization induces depolarization eliciting wound-related transcriptional changes, reactive oxygen species signaling, and biochemical activity consistent with the earliest stages of injury. Furthermore, we also noticed long-range spatiotemporal cell death and proliferation characteristics of tissue regeneration. Our experimental strategy revealed the possibility of dissociating molecular events of the injury response from physical tissue damage. We have also found that K+ may be the ion responsible for driving this process. We present comprehensive evidence about the activation of the regenerative response by a brief treatment to disrupt physical properties of the plasma membrane. These findings further support and give insight to the idea that it is possible to bypass the injury event to activate downstream components leading to tissue repair.

Metabolic diseases, such as obesity or type 2 diabetes, are affecting millions of individuals globally and are projected to continue impacting sixty percent of the world’s population by 2050. Factors attributed to the development of metabolic diseases have often been identified as sedentary lifestyle and hypercaloric diets. In recent years the idea of the “exposome,” or the sum of an individual’s environmental exposures within their lifetime, has shed light on the importance of chemical exposure and incidence of metabolic disease. One environmental factor that can perturb metabolic function is the use of tobacco products. Specifically, exposure to nicotine can elicit metabolic disruption from direct, in utero, and ancestral exposures. Though global tobacco use rates among adults have declined, there are still communities that continue to use tobacco like adult men. Paternal smoking has also been associated with childhood overweight and obesity status in descendants and grandchildren. Paternal nicotine use has been shown to increase incidence of metabolic disruption in the next generation of male mice. However, further characterization of paternal nicotine exposure and metabolic outcomes in the next generation remains to be characterized. Investigation into the metabolic outcomes upon paternal nicotine exposure in females of the next generation remains to be elucidated. Also, investigation into dietary interventions upon paternal predisposition to nicotine exposure has not been previously explored. Referring to the idea of the exposome, we are exposed to multiple factors within our lifetime. It is important to understand the metabolic outcomes that arise upon paternal nicotine exposure and dietary intervention with a hypercaloric diet as we are exposed to multiple factors within ours and our ancestors’ lifetime. It is also important to understand the metabolic outcomes of direct exposure to nicotine. Direct nicotine exposure in the F0 generation has been associated with increased risk of metabolic disruption in the form of metabolic syndrome. Here we characterize the metabolic outcomes upon direct or ancestral exposure to nicotine in a rodent model.In Chapter 1 of this dissertation, I thoroughly detail relevant information on various fields that mesh into my novel research topic. Specifically, I outline the recent prevalence and projections of metabolic syndrome and its associated disorders at the global level. I discuss the factors that are associated with the development of metabolic diseases and summarize some of the recent investigation into chemical exposures and metabolic disruption. Specifically, exposure to endocrine-disrupting chemicals (EDCs) during critical windows of susceptibility during development can lead to adverse metabolic outcomes. One EDC, nicotine, found in tobacco, can elicit metabolic disruption from direct or developmental exposure. Paternal tobacco smoking has been associated with metabolic outcomes in unexposed grandchildren. Paternal nicotine exposure can also elicit metabolic disruption in males of the next generation in rodents. Mechanisms underlying these alterations that arise upon paternal nicotine exposure are still being elucidated; however, one hypothesis is the alteration of sperm small non-coding RNAs leads to metabolic outcomes observed. Investigation into paternal nicotine exposure and a secondary challenge with a different metabolic disease risk factor in the next generation, such as diet, has not been studied. The next two data chapters detail findings of nicotine exposure at two different moments in life, adulthood and after paternal preconception exposure, and the metabolic outcomes that arise at the physiological and transcriptomic levels.In Chapter 2 of this dissertation, I demonstrate that direct exposure of the F0 generation to nicotine leads to physiological and transcriptomic metabolic outcomes. Chronic nicotine exposure elicited cardiometabolic disruption at the physiological and molecular levels. Nicotine exposure elicited altered plasma metabolites, blood glucose levels during metabolic testing in both male and female rodents. Specifically, nicotine exposure in males was associated with impaired insulin tolerance and decreased body weights. Hepatic transcriptomics reveal alterations in gene expression of biological processes involved with cardiovascular disease upon nicotine or tributyltin exposures. These alterations suggest that direct exposure to endocrine disrupting chemicals like nicotine or tributyltin elicits cardiometabolic alterations in mice. Furthermore, exposures to these endocrine disrupting chemicals may be associated with increased risk of cardiometabolic disease in humans. In Chapter 3 of this dissertation, I demonstrate that paternal exposure to nicotine predisposes offspring to metabolic disruption that is further exacerbated in the presence of a hypercaloric diet. I also show that there is a sexually dimorphic phenotype observed in the F1 generation upon paternal preconception nicotine exposure. Specifically, there are different metabolic processes altered in the sexes upon paternal nicotine exposure, such as modifications to hepatic gene expression in gluconeogenesis in F1 females and glycogenolysis in F1 males. F1 males also had decreases plasma glucagon, an important metabolite involved in glycogenolysis. Although physiological outcomes were mild, hepatic transcriptomics reveal alterations in gene expression of biological processes involved with lipid and xenobiotic metabolism suggesting alterations in the fat metabolism. Transcriptomic alterations to the metabolically relevant liver tissue ultimately reveal that there was metabolic disruption that arises from paternal nicotine exposure and is further exacerbated by a hypercaloric diet.Finally, in Chapter 4 of this dissertation, I summarize the findings from both data chapters and discuss how these findings shed light into the effects of direct and ancestral exposure to nicotine on adverse metabolic outcomes. The findings here further provide compelling evidence that paternal nicotine exposure can elicit long-lasting metabolic effects that can be further exacerbated by a hypercaloric diet that represents the diet 50% of the American population follows. This dissertation demonstrates that nicotine and hypercaloric diet, two types of environmental factors, can elicit cardiometabolic alterations at the physiological and molecular levels. Future studies will investigate potential mechanisms that link paternal nicotine exposure and metabolic outcomes observed.

Small RNAs have been implicated in the intergenerational epigenetic inheritance of paternal environmental effects, with tRNA-derived fragments (tRFs) being one of the most abundant classes of small RNAs in mature mammalian sperm. These fragments are modulated by environmental conditions and can influence offspring phenotypes; however, their biogenesis and functions remain poorly understood. My research focused on uncovering the enrichment and functional roles of tRFs, particularly the 5′ fragment of tRNA-Valine-CAC-2 (tRFValCAC), which is highly abundant in mature mouse sperm. tRFValCAC is enriched during post-testicular maturation in the epididymis and delivered to sperm via extracellular vesicles secreted by epididymal epithelial cells.Mechanistically, I identified that tRFValCAC interacts with the RNA-binding protein heterogeneous nuclear ribonucleoprotein A/B (hnRNPAB) in the epididymis, which regulates tRFValCAC levels within these vesicles and further contributes to the enrichment of tRFValCAC in mature sperm. Functional analyses revealed that inhibiting tRFValCAC in preimplantation embryos altered the expression of genes involved in RNA splicing and mRNA processing, leading to delayed preimplantation development. Together, our results reveal a novel function of a sperm-enriched tRF in regulating alternating splicing and preimplantation embryonic development and shed light on the mechanism of sperm small RNA-mediated epigenetic inheritance.In a different project, I investigated the impact on diet on tRNAs on tissue level. Environmental factors play a crucial role in shaping tRNA levels and modifications, which in turn can influence protein synthesis and enable cells to adapt to various stresses. While much of our knowledge stems from studies in cell culture and yeast, evidence suggests that tRNA dynamics also occur at the tissue level. For instance, the microbiome has been shown to reprogram host tRNAs in a tissue-specific manner. Despite these insights, the connections between diet, tRNA expression, and tRNA modifications across different mammalian tissues remain largely unexplored. To investigate this, I collected tRNA from mouse tissues subjected to 10 weeks of low-protein and high-fat dietary challenges. RNA mass spectrometry analysis revealed tissue-specific changes in tRNA modifications, such as elevated galactosyl-queuosine levels in the epididymis compared to the liver, and demonstrated that diet influences bulk tRNA modifications across tissues. Using a modified-base-sensitive tRNA sequencing technique (OTTRseq), I identified that dietary challenges affect tRNA abundance in all tissues examined, with liver and heart showing more robust changes. Only liver and heart exhibit detectable mismatch rate changes at sites like 58A. This study could pave the way for exploring how tRNA dynamics in response to diet impact gene expression, uncovering tissue-specific RNA regulation and mechanisms of environmental tissue adaptation.

The localization of cellular components like organelles or protein complexes must be precisely controlled in living cells. Proper distribution is largely achieved by motor proteins that selectively bind and transport cargo along cytoskeletal tracks. The traditional view of motor-based transport is that cargo move by directly associating with a motor complex. However, a non-canonical form of motor-based transport termed “hitchhiking” was recently co-discovered by the Reck-Peterson lab, whereby “secondary” cargo move by tethering to motile “primary” cargo that are bound to a motor complex. Hitchhiking-like phenomena have been observed in diverse polarized cells such as filamentous fungi and neurons. The goal of my graduate work was to determine the function and mechanism of peroxisome hitchhiking using filamentous fungi as a model for studying this process in vivo. Peroxisomes are organelles ubiquitous to eukaryotic cells that exhibit different long-distance motility characteristics across diverse species and cell types. In the filamentous fungus Aspergillus nidulans, peroxisomes move long distances along microtubules by hitchhiking on motile early endosomes. This requires the fungal-specific protein PxdA. In my doctoral work, I helped identify the fungal-specific phosphatase DipA as an interaction partner of PxdA. I also explored the role of PxdA in different peroxisome functions, notably during the biogenesis and positioning of Woronin bodies and the compartmentalization of secondary metabolism. These findings highlight the molecular mechanisms underlying organelle hitchhiking in fungi and expand our understanding of dynamic peroxisome function.

Like most organisms, plants rely on a robust circadian clock to anticipate and adapt to periodic environmental changes. This clock-driven regulation enhances fitness by orchestrating 24-hour rhythms in essential growth and developmental processes. At its core, the circadian clock functions through a negative feedback loop involving the reciprocal regulation of morning-expressed transcription factors, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), and the evening-expressed TIMING OF CAB EXPRESSION 1 (TOC1). Beyond transcriptional regulation, the stability and activity of these core clock components are intricately controlled at the post translational level. This multilayered regulatory system enables the clock to maintain a precise 24-hour period, ensuring alignment between internal rhythms and environmental cycles—an essential feature for optimal plant fitness. Despite its importance, the molecular mechanisms governing the clock period, especially under environmental stress, remain poorly understood. This study investigates a novel layer of posttranslational regulation within the circadian system, shedding light on how plants sustain rhythmic precision in changing environments.

Human stem cell-derived in vitro models of various cell types and tissues act as useful tools for the study of human biology and disease in a genetic editable, scalable setting. Years of studying in vivo pancreatic development has facilitated the development of in vitro protocols. Stem cell-derived islets (SC-islets) provides an alternative source of cells to study β-cell development, function, and dysfunction. This is particularly useful for research regarding the human pancreatic islet, which is difficult to access in vivo and difficult to obtain for experiments ex vivo. Despite successful induction of pancreatic β-cells which produce and secrete insulin in a glucose-responsive manner, stem cell-derived β-cells (SC-β-cells) still fail to recapitulate primary human β-cell gene expression and functional maturity. We developed a genome-wide screening platform using CRISPR-Cas9 technology to identify genes regulating the mitochondrial glucose response, an important aspect of insulin secretion. Our results identified new avenues of study to understand the genetic underpinnings of β-cell functional acquisition. Among these is the folate cycle, an important driver of cellular proliferation. The folate cycle has been implicated in metabolic function and β-cell development in studies of maternal dietary folate. We assayed each stage of development for folate cycle impacts on β-cell development. We discovered the folate cycle primarily influences β-cell development during foregut specification. This novel finding improves our understanding of the in vivo studies on maternal folate supplementation. Furthermore, we find inhibition of the cycle component SHMT2 is necessary to induce a crucial pancreatic transcription factor. These findings, together with literature evidence and qualitative observations, hint at the importance of cell density for pancreatic differentiation, possibly through mechanotransducive signals. In our investigation of the folate cycle impact on mature β-cell function and metabolism, we report antifolate drugs promote oxygen consumption in SC- and primary human islets. Furthermore, potent folate cycle inhibition augments SC-islet insulin, and elevates AICAR, which amplifies insulin secretion through AMPK activation. Together, this paints a picture of folate cycle inhibition amplifying insulin secretion through AICAR accumulation.Altogether, this dissertation details folate cycle regulation of β-cell development and function, identifying a new target for improving SC-islet differentiation protocols.

Defects in telomerase and telomere length maintenance have been implicated in difficult to treat genetic disorders, premature senescence underlying aging disorders, and chromosomal instability contributing to increased risk of cancer development. It has relatively recently been noted that patients with congenitally short telomeres and telomerase dysfunction diseases present with microcephaly and associated neurological symptoms. However, few studies have explored the role of telomere dysfunction on neurodevelopment.This proposal aims to study the role of shortened telomeres and telomerase dysfunction using human induced pluripotent stem cells (hiPSCs) to model neurodevelopmental defects. In previous studies, hiPSCs with measurably shortened telomere length, as well as hiPSCs that have been CRISPR engineered with defects in the telomerase reverse transcriptase (TERT) component of the telomerase complex that is important for end replication function, have elucidated that bone marrow failure associated with the disease can be attributed to cellular dysfunction that underlies failure of hematopoietic stem cell development. Using hiPSCs that have similar defects in telomere length and in TERT (the enzymatic component of telomerase), this proposal will utilize a directed differentiation approach to study early neurodevelopment and neuronal maturation to measure critical points of cellular development that potentially give rise to the reduced neuronal volume that characterizes microcephaly. Furthermore, this proposal will utilize a previously published three-dimensional cellular protocol for studying the microcephaly phenotype using cortical organoid culture, which will allow for visualization and measurement of neuronal development related to spatial organization. Finally, the cellular basis of associated neurodevelopmental aberrations will be measured via bioinformatics analysis and functional activity assays, to elucidate the underlying contribution of prematurely shortened telomeres to neuronal activity and maturation.

RNA-binding proteins (RBPs) regulate post-transcriptional gene expression, influencing key cancer pathways. Understanding how RBPs control these processes can uncover new treatment strategies, which is crucial for cancers that lack effective targeted therapies such as triple negative breast cancer (TNBC). Here, we employ dual in vitro and in vivo CRISPR/Cas9 screens to investigate the role of RBPs in TNBC, revealing the poly(U)-binding splicing factor 60 (PUF60) as a key modulator of TNBC cell survival. Integrated eCLIP and RNA-sequencing identifies that PUF60 drives exon inclusion within proliferation-associated transcripts that, when mis-spliced, induce cell cycle arrest and DNA damage. Furthermore, disrupting PUF60 splice site activity via a substitution in its RNA-binding domain causes widespread exon skipping, leading to the downregulation of proliferation-associated mRNAs and inducing apoptosis in TNBC cells. We demonstrate that this RNA-binding disruption inhibits TNBC cell proliferation and shrinks tumor xenografts, highlighting the molecular mechanism through which PUF60 supports cancer progression. Our work demonstrates functional in vivo screening of RBPs as an effective strategy for identifying unexpected cancer regulators. Here, we reveal a crucial role for PUF60-mediated splicing activity in supporting oncogenic proliferation rates and highlight its potential as a therapeutic target in triple negative breast cancer.

Cancer is a complex disease broadly characterized by disruption of cellular homeostatic mechanisms. Tumors exhibit a high degree of cellular heterogeneity, comprised of polyclonal populations that interact and elicit coordinated responses. Such complexity presents a significant challenge for the development of disease-state models and effective therapies that accurately capture intricate cellular interactions within solid tumors. This dissertation presents two studies examining alternative methods for in vitro exploration of tumor cell growth focusing on 1) mechanisms of drug resistance and; 2) novel cell-cell interactions in three-dimensional space. In Chapter 2, we first interrogate acute non-small cell lung cancer (NSCLC) tumor cell resistance to targeted EGFR/RAS/RAF- pathway inhibition. Strikingly, we find that acute response to EGFR/RAS/RAF pathway inhibitors is culture context-specific, demonstrating that spheroid culture better recapitulates EGFRi-mediated apoptosis observed with in vivo modeling of EGFR-mutated NSCLC, whereas monolayer culture intrinsically mimics a drug tolerant persister (DTP) state. We identify differential YAP activity between monolayer and spheroid culture as a common, non-genetic mechanism of resistance to acute EGFR/RAS/RAF-pathway inhibition. In line with exploring methods to model intratumoral cell-cell interactions, in Chapter 3 we focus on generating models of cancer heterogeneity by constructing complex tumors with engineered tumor subclones driven by distinct oncogenes. We hypothesize tumor subclones cooperate to elicit emergent tumor properties, promoting tumor growth and, potentially, metastasis and drug resistance. Both studies emphasize that distinct cell culture contexts can significantly impact outcomes in drug discovery and resistance research and should be considered for future work exploring these topics.

Tumors display altered metabolism, often reflective of their microenvironment. In many cases including breast cancer, the invasive tumor front borders adipocytes. Reliance on mitochondrial fatty acid oxidation (FAO) has been observed in aggressive receptor ‘triple-negative’ breast cancers (TNBC), but the adaptations that permit elevated FAO, and the molecular mechanisms by which tumors coopt adjacent adipocytes for growth, remain elusive. The oncogene MYC dysregulates a range of cellular programs, including metabolism; MYC expression is elevated in most TNBC, and linked to increased FAO. To delineate alterations facilitating FAO in TNBC, I examined fatty acid binding proteins (FABP) thought to traffic FA to the mitochondria. Prior work identified FABP5 elevation in patient TNBC and a model of MYC-driven breast cancer. I observed increased FABP5 transcription in TN compared to ‘receptor-positive’ patient-derived cell lines, yet elevation across MYC-low and -high TNBC suggested levels are not solely MYC-regulated. Treating MYC-driven breast cancer cells with an FABP5/7 inhibitor caused lipid accumulation and impaired proliferation, but I found no growth defect after FABP5 knockout. While compensation by other FABPs may complicate knockout studies, specific inhibitors for FABP5 and other FAM targets are in clinical development. Despite evidence indicating reliance on FAO in TNBC, the source of FA fueling aggressive tumor growth is unclear. I next described a direct interaction linking cancer cell-adipocyte contact to tumor progression. Examining breast tumors and normal adjacent tissue from patient cohorts, patient-derived xenografts and mouse models, we observed activation of lipolysis and lipolytic signaling in neighboring adipose tissue. Using cancer cell adipocyte co-cultures, I found that functional gap junctions (GJ), small intercellular channels, form and permit cAMP transfer from breast cancer cells to adipocytes, activating lipolysis in a GJ-dependent manner. We identified connexin 31 (GJB3) as a promoter of in vivo TNBC growth and activation of adjacent lipolysis, or FA release. Our findings indicate a pro-tumorigenic role for direct tumor cell-adipocyte interactions. These studies reveal that TNBC dysregulate tumoral lipid metabolism and stimulate altered FAM in surrounding tissue.

Appropriate 3D tissue structure supports cell and tissue function; inappropriate tissue structure correlates with cell and tissue dysfunction. Quantitative studies of this structure-function relationship are hampered by a paucity of techniques for tissue structure analysis. Using pancreatic islets as a model system, I have developed a novel method for high-resolution quantitative analysis of 3D tissue structure. Here, I summarize what is known about tissue structure in pancreatic islets, outline the processes of collecting high-resolution spatially quantitative 3D images of tissue and of extracting spatial data, detail qualitative observations of structural phenomena gleaned from observation of hundreds of 3D images of adult human and mouse pancreatic islets, and provide a proof-of-concept quantitative analysis of adult mouse and human islet tissue structure. My dissertation work sets the stage for quantitative study of tissue structure and function not just in healthy adult islets but also in other tissue types, and in unhealthy, developing, or engineered tissue. The rich image datasets acquired are also a valuable resource for both pancreatic biologists and those developing tools for image processing and image analysis.

Activating mutations in the Telomerase Reverse Transcriptase (TERT) promoter are prevalent in cancer and enable limitless cell division characteristic of immortal cells1-12. Solving the immortality mechanism represents a major step towards selective reversal in cancer cells. TERT promoter mutations create a de novo E26 transformation specific (ETS) transcription factor binding motif. Here, we analyzed fifty-three cell lines representing sixteen cancer types and six recurrent TERTp mutations and found that the GA-binding protein (GABP) tetramer is responsible for promoter activation in all cases, extending prior studies on a few cancers and two hotspot mutations. Surprisingly, TERT expression is maintained after tetramer depletion. Further investigation revealed an underlying network of auto-suppression, the release from which drives TERT maintenance via upregulated GABP dimers or a paralogous tetramer. To target all three complexes, we used AlphaFold2 to design a biological proteolysis-targeting chimera to specifically degrade GABPA protein. TERT expression was abolished in a promoter mutation-specific manner, shortening telomeres and improving survival in a GBM xenograft model. The GABPB1L tetramer is, therefore, a pan-cancer, pan-mutation activator of the mutant TERT promoter, but it is replaceable. Domains shared by the three GABP complexes, rather than solely the B1L tetramer, are mutation-specific vulnerabilities.

The cytoplasm is a dynamic fluid where cellular building blocks and components are continuously mixing. Cytoplasmic mixing is important for transporting intracellular material ranging from nucleotides and proteins to full organelles. In large cell types, such as ameboid and neutrophil cells, cytoplasmic streaming is coupled to membrane deformations that accompany in motility. How well or how long it takes for material to mix in cytoplasmic streams during motility remains an open question. In relatively small volumes of viscous fluids at very low Reynolds numbers, such as the cytoplasm, it should be difficult to obtain significant mixing for structures in the size range of organelles simply from streaming laminar flows. Although small things like proteins can eventually mix through diffusion, microscale structures are expected to mix on a significantly slower time frame or not at all. In this work we discovered how the giant amoeba Chaos carolinensis overcomes these limitations using a novel cytoplasmic gel state capture and release strategy to facilitate the extremely efficient mixing of its cytoplasm. While it was previously thought that the amoeba solution to gel state transitions only occurs at the trailing and leading edge of the cell body, our work indicates that these transitions occur frequently throughout the mid-cell region, driving the cytoplasmic mixing of beads and organelles. These results indicate that amoeba reaches a stable mixed state during motility in as little as one cytoplasmic stream/flow cycle, effectively making it a Bernoulli system and thus one of the fastest possible known intracellular mixers. To study cytoplasmic streams, we microinjected fluorescent beads into amoeba and recorded their movement using time-lapse microscopy for up to 12 hours. In addition to bead trajectories, we microinject histone H1 labeled with alexa 488 and tracked nuclei movement. To separate the movements of beads and nuclei from overall cell motility, we employed automated image processing to stabilize the videos, making the amoeba appear to crawl in place. Power law fits of interparticle distance over time allowed us to classify bead pair separation events into sub-diffusive, diffusive, super-diffusive, and ballistic transport regimes. Using mean squared displacement analysis, we also distinguished gel-state from liquid-state flows. This classification enabled us to calculate diffusion coefficients, mean velocities, and dwell times for each state, which we used to simulate the time required for two particle populations to achieve stable mixing. To validate these simulations, we developed a method to computationally label and track two distinct bead populations from a single-color bead injection. This approach allowed us to observe new mixing events frame by frame and showed strong agreement between simulated and experimental results.Here we show the innovative strategy through which the giant amoeba Chaos carolinensis achieves efficient cytoplasmic mixing despite its reliance on low-Reynolds-number, laminar flow. Our results highlight the role of mid-cell sol-to-gel transitions in facilitating efficient mixing, enabling material exchange between cytoplasmic layers and allowing complete intracellular mixing within a single flow cycle. Novel computational approaches validated the robustness of these dynamics through simulation, showing alignment with experimental data for both bead and nuclear trajectories. Moreover, pseudopod modulation was found to have minimal impact on mixing efficiency, underscoring the unique contribution of sol-to-gel state dynamics. This work establishes a robust framework for studying intracellular mixing, with potential applications across cellular systems characterized by cytoplasmic streaming.

Obesity in the United States is a problem of epidemic proportions. The CDC suggests that around 40% of adults and 20% of children are obese, and these numbers show no signs of tapering off. In the last few decades, it has been established that in addition to many well-known cardiometabolic complications, increased adiposity also plays a role in cancer—forming a synergistic relationship with cancer cells resulting in more aggressive, difficult to treat disease. Compelling evidence has been presented supporting the negative impact of obesity in numerous cancers, namely pointing toward mechanisms related to lipid metabolism and increased inflammation resulting from aberrations in adipose tissue. One caveat to the bulk of obesity and cancer research is the focus on solid tumor cancers occurring in adults, thus failing to account for juvenile and hematological malignancies such as pediatric acute lymphoblastic leukemia (ALL)–the most common childhood cancer. Additionally, adipose tissue is notoriously difficult to culture ex vivo, resulting in frequent use of artificial in vitro adipocyte models and animal models that limit translational application. To address shortcomings in adipose culture methods, we developed a novel culture method using polydimethylsiloxane (PDMS) flow chambers, allowing us to more effectively culture whole patient adipose tissue ex vivo. To investigate the crosstalk between adipose tissue and acute lymphoblastic leukemia, we conducted single-cell RNA sequencing of epididymal adipose tissue in a syngeneic obese mouse model of juvenile ALL. Results show that PDMS flow chambers are a promising alternative to current methods used to study adipose tissue, as they maintain tissue viability, functionality, and better simulate adipose tissue in vivo since 3D structure and stromal cell network are retained. Our exploration of adipose tissue-ALL crosstalk at the single-cell resolution revealed a novel mechanism by which Prostaglandin E2 released from adipose tissue binds leukemia cells and increases expression of Receptor Activator of Nuclear Factor Kappa- beta Ligand (RANKL), which is associated with bone breakdown and central nervous system metastasis. Collectively, our findings help improve on current adipose tissue culture methods and provide novel insight into how the obese adipose tissue microenvironment plays a role in promoting cancer progression.

The L-type Ca2+ channel (CaV1.2) is essential for cardiac excitation-contraction coupling. Alterations in CaV1.2 expression, localization, or function can underlie cardiac dysfunction found in aging and heart disease; thus the regulatory mechanisms that tune CaV1.2 expression to meet contractile demand are a crucial area of research. This dissertation work highlights the function of two proteins that regulate CaV1.2 trafficking: 14-3-3 and BIN1. We used a range of techniques including confocal imaging, proximity ligation assays, super-resolution imaging, In vivo echocardiography, and co-immunoprecipitation to explore the dynamics of this regulation. Our work elucidates a novel mechanism where 14-3-3 interacts with CaV1.2 in a phosphorylation-dependent manner to promote enhanced trafficking/recycling, clustering, and activity during β-adrenergic stimulation. We also report that a two-week treatment to restore youthful Bridging Integrator 1 (BIN1) levels in the hearts of 24-month-old mice rejuvenates cardiac function and substantially reverses the dysregulated endosomal recycling and disrupted CaV1.2 trafficking phenotype found in aging. These results increase our understanding of CaV1.2 regulation and provide a foundation for future work establishing a complete model of CaV1.2 trafficking.

Osteosarcoma (OS) is the most common primary bone malignancy in humans and canines, and in humans primarily affects younger patients 10-14 years of age. While considerable efforts have been put forth in new therapeutic approaches to this disease, the treatment and prognosis for OS has changed very little since the 1980s. Targeted therapeutics have made considerable progress in other cancer types, leveraging characteristics of cancer cells which differentiate them from that of normal healthy cells. In comparison to other cancers, OS is highly heterogeneic, and no single unifying driver mutation has yet been found. Development of a therapy which could overcome the high degree of heterogeneity amongst OS tumors could go a long way in improving the lives of OS patients. Targeting protein translation has been proposed as one mechanism by which to overcome tumor heterogeneity, and this dissertation focuses on studying a noncanonical role of core binding factor beta (CBFβ) as a regulator of protein translation, and elucidating whether this could represent a potential therapeutic target for OS. Utilizing a wide array of in vitro assays, we have been able to demonstrate that loss of CBFβ reduces protein expression of RUNX2 in a post-transcriptional manner, and this decrease in RUNX2 protein level is not fully explained by alterations in RUNX2 stability brought about by loss of its binding partner CBFβ. Additionally, we demonstrate that loss of CBFβ also causes a decrease in global protein translation, and confirmed an interaction between CBFβ and hnRNPK which has thus far only been observed in breast cancer cells. Importantly, this interaction with hnRNPK is said to be the mechanism by which CBFβ influences protein translation, and our results corroborate those observed in breast cancer cells and suggest CBFβ may also perform this role in OS. Reports of the interactions between CBFβ and hnRNPK or RUNX2 allude to mutual exclusivity in interaction, and with the transcriptional role of CBFβ accomplished via binding to RUNX proteins, and the translational role of CBFβ accomplished via binding to hnRNPK, it is entirely possible these two roles are antagonistic in some fashion. To investigate the relevance of certain CBFβ residues in terms of this translational role of CBFβ, and avoid confounding variables from the transcriptional role of CBFβ, we utilized point mutations to interrupt CBFβ-RUNX2 interaction. Using various in vitro assays, we validated key residues of CBFβ which are involved in its interaction with RUNX2, re-introduced this mutant form into CBFβ knockout cells, and measured alterations to RUNX2 interaction and nuclear shuttling. We confirmed that our mutant displays reduced binding to RUNX2, and drastically reduced nuclear shuttling. Lastly, we expanded our studies from RUNX2 to the entire genome and proteome. Encouraging data thus far had suggested CBFβ may play a role in protein translation, and necessary next steps were to assess which proteins CBFβ may be interacting with in performance of this role, and elucidate which proteins may be under the translational purview of CBFβ. Using immunoprecipitation mass spectrometry we identified numerous specific interactors of CBFβ, with high enrichment in pathway analysis terms associated with protein translation. Additionally, using two different methods we generated a list of proteins which may be under the translational purview of CBFβ, and found strong enrichment of numerous cancer-associated terms among this list. These studies establish that CBFβ participates in protein translation in OS, with many genes under its purview associated strongly with cancer in general, and OS specifically. This provides justification for future studies delving deeper into this novel role of CBFβ, and opens up another mechanism by which protein translation could be targeted therapeutically in OS.

AbstractThis dissertation investigates the role of the translation initiation machinery in the dynamics of stress granules (SGs). Stress granules are cytoplasmic, non-membrane bound organelles composed of mRNA transcripts, RNA-binding proteins, 40S ribosomal subunits, and eukaryotic initiation factors (eIFs). They form in response to environmental stress to promote cell survival and recovery. A major gap in knowledge addressed in this work is the need to measure real- time kinetics of SG assembly and disassembly. Furthermore, the involvement of translation initiation factors in SG assembly remains unclear. My research addresses these gaps by developing a system to monitor SG dynamics using a stable inducible Flp-In™ T-REx™-HeLa Cell Line system, enabling real-time tracking and modeling.Chapter 1 reviews the mechanisms of SG formation, their interplay with the translation initiation machinery, and their regulation under stress conditions. In Chapter 2, I describe the protocols developed to characterize the HeLa Fl-In cell line used throughout this study. Chapter 3 focuses on the role of the RNA Recognition Motif (RRM) of the initiation factor eIF4B in regulating SG assembly. Using single-cell live-imaging, I quantified the kinetics of SG formation and discovered that overexpression of GFP-tagged eIF4B with a point mutation in the RRM domain results in a decreased rate of SG formation, fewer total SGs, and a delayed stress response compared to wild type eIF4B. Furthermore, I demonstrated that eIF4B directly binds to G3BP1, a known SG nucleator, suggesting a mechanism by which eIF4B influences SG assembly. Chapter 4 examines the mechanisms of SG disassembly, a process poorly understood but linked to neurodegenerative disorders due to the inability to disassemble SGs, leaving cells in aconstant state of stress. I developed a method to measure the rate of SG disassemblyaccurately, addressing the challenge of focal drift in time-lapse imaging. Future work will explore overcoming focal drift using microfluidics devices or environmental chambers. Chapter 5 presents preliminary data on the interaction of GFP-tagged eIF4G with SGs. Unexpectedly, modest overexpression of eIF4G in HeLa cells results in hypersensitivity to oxidative stress, even though SGs still form. Proposed future experiments aim to confirm and extend understanding of this phenomenon.Overall, this dissertation provides novel insights into the dynamic interplay between translation initiation machinery and SGs, with potential therapeutic implications for targeting SG dynamics in diseases such as cancer and neurodegeneration.

During my dissertation in the lab of John Albeck, I have worked on various projects involving measurement of single-cell kinase signaling activity in response to stress using live-cell biosensors. This included optimizing and publishing methodology, the use of live-cell biosensors to measure cellular response to pro-inflammatory cytokines and oxidative stress, and the development of new biosensors. In Chapter 1, I introduce the methods we use to measure signal transduction in real time using fluorescent biosensors, which includes optimizations that I contributed to the lab. This protocol was accepted for publication as a chapter in Methods of Molecular Biology. In Chapter 2, with the help of my co-mentor Amir Zeki, I delve into the application of biosensors and show my finding that spatially coordinated ERK signaling (SPREADs) increased when airway epithelial cells are exposed to pro-inflammatory stimuli like those seen in common airway diseases. I then investigated the potential mechanisms of SPREADs and their suppression by hydrocortisone and metabolic stress that coincides with increased AMPK activity. Chapter 3 focuses on using live-cell microscopy to understand how the RNA-recognition motif of the translation protein eIF4B regulates the cell’s ability to form stress granules and suppress protein synthesis following treatment with sodium arsenate. Finally, in Chapter 4, I discuss my work developing, validating, and optimizing Far-Red FRET biosensors for ERK (REKAR67 and REKAR76) and AMPK (RAMPKAR2). Including the use of RAMPKAR2 to understand the single-cell dynamics of AMPK, ATP:ADP ratio, and glycolysis. This last chapter includes work from two different publications in preparation for submission.

Per- and polyfluoroalkyl substances (PFAS) are a class of compounds commonly used as surfactants in food contact materials and household items due to their hydrophobicity. However, their physicochemical properties render them highly resistant to degradation in the environment and human body. Thus, these chemicals are environmentally ubiquitous and have been found in the serum of 98% of U.S. citizens. Evidence implicates some well characterized PFAS in breast cell carcinogenesis and cancer progression. While PFAS-induced receptor-positive breast cancer progression has been widely investigated, PFAS-induced receptor-negative breast cancer has not, and molecular mechanisms remain unclear. Additionally, Generalized Read-Across of PFAS using cancer progression endpoints are lacking. This research analyzes and compares the effects of five PFAS of increasing alkyl chain length on triple negative breast cancer progression in vitro. Using the aggressive human cell line Hs578T the effects of five PFAS were analyzed across environmentally relevant doses. Progression was assessed through 2D cell proliferation assays and “2.5D” Matrigel migration and invasion assays. Results showed significant decreases in mean number of protrusions per cell and total colony formation per well with PFBA and PFDoA treatment. Significant decreases in colony formation were observed with PFDA treatment at all doses. The mean length of the longest protrusion per cell was largely unimpacted by PFAS, except for decreases by PFDoA. When viewing the data though read-across lenses, near significant trends were seen in protrusion counts with increasing alkyl chain length at 1 nM and 10 nM PFAS. The highest alkyl length significantly decreased colony formation in a pattern seen across 10 nM, 100 nM and 1000 nM doses. Cell proliferation was not impacted by PFAS treatment. These results will help elucidate whether triple negative breast cancer progression is related to dose and alkyl chain length, and whether short-chain PFAS exhibit increased progression as longer PFAS have been shown to promote.

Feline Chronic Gingivostomatitis (FCGS) is a common oral inflammatory disease of cats. FCGS is associated with chronic viral infections, such as feline calicivirus (FCV) and feline foamy virus (FFV) and can be treated by full mouth extraction (FME) or mesenchymal stromal cell (MSC) therapy in cases where FME fails. We hypothesized that FCGS is characterized by a dysfunctional, exhausted, CD8+ T cells state that is characterized by decreased cytokine production, decreased proliferative potential, decreased cytotoxic activity and increased expression of inhibitory receptors. We further hypothesized that MSC therapy helps to reinvigorate exhausted CD8+ T cells. Transcriptomic analysis of peripheral blood CD8+ T cells indicate upregulation of genes and pathways associated with exhaustion in peripheral blood CD8+ T cells in cats with FCGS. Moreover, confocal immunofluorescence indicated a marked expansion of CTLA-4 positive T cells in the mandibular lymph nodes of cats with FCGS. Finally, MSC therapy suppressed CD8+ T cell activation and proliferation. By comparing the pre-treatment pathways of CD8+ T cell effector activity in successful and failed FME therapy, we identified IL12R, IL18R, and AKT1S1 as crucial for successful FME therapy, potentially linked with the Ras-MEK-ERK pathway, reflecting enhanced T cell signaling pathway activation. In summary, our study found CD8 T cell exhaustion in FCGS and identifies key molecular targets for FME and MSCs treatment.

Our immune system responds to extracellular cues to protect our health by coordinatingnecessary cell functions from cell motility to apoptosis. Engineering signaling pathwayscontrolling these processes is rapidly becoming a powerful approach for developing newbiomedical technology, with cancer immunotherapies serving as a prominent example. However,most current strategies rely on modifying or repurposing endogenous human signaling cascades.This can result in unintended crosstalk between the synthetic signaling pathways and thecomplex native signaling of human cells. Two-component systems (TCS) could address thischallenge and establish a scientific foundation for systematically improving and adding to thenatural abilities of cells to sense and respond to their environment in a robust yet flexiblemanner. With a newly paved avenue for engineering cell communication, researchers coulddevelop strategic signaling cascades in any human cell for their specific research interests.Importantly, TCS are absent in human cells, but present in all three domains of life, and offerdiverse yet simple and linear signaling pathways. In TCS, ligand binding modulates a histidinekinase (HK) sensor, a protein characteristically containing separate ligand binding and HKdomains. When activated, the HK transfers a phosphoryl group to the response regulator (RR),and the phosphorylated RR activates a specific downstream response through mechanismsranging from protein-to-protein interactions to transcription. In its inactive state, the HK acts as aphosphatase. These studies focus on a light-sensitive LOV TCS and a nitric oxide-sensitive HnoTCS as candidate model pathways for modifying mammalian cells. We demonstratedconstitutive activity with the LOV pathway and tested several modifications to achieve lightsensitivity. While some modifications affected pathway output, all tested pathways appeared tohave light-independent constitutive activity.

Recent reports of bovine embryonic stem cell (bESC) establishment and in vitro gametogenesis (IVG) in the mouse open the door to recapitulate oogenesis from bESCs in vitro, however little progress has been made toward this feat. Critical gaps remain in our understanding of bovine germ cell development, which will be necessary to establish a system that supports efficient in vitro germline formation as well as to assess the resulting cells once obtained. Additionally, progress in other models has shown the importance of pluripotent state on cellular capacity for in vitro germline induction. Therefore, the goal of this dissertation was to shed light on the requirements of livestock pluripotency and germline development to set the basis to establish reliable bovine in vitro oogenesis systems.We first examined the pluripotent state of bESC lines and explored the potential to stabilize these cells in intermediate states of pluripotency, due to the limitations of primed ESCs for germline induction. We found that regardless of the initial blastocyst stage that bESCs were derived from, cell lines exhibited similar transcriptional and functional characteristics of primed pluripotency. However, the demonstration that bESCs could be efficiently established from early blastocysts increases the pool of embryos that may be targeted for bESC derivation approaches. We also investigated alternative formative media for bESC establishment, but found that conditions that supported this state in other mammalian species did not translate to cattle. This highlights the unique signaling requirements of bovine pluripotency and the necessity of further studies to establish alternative conditions for bESCs with high germline competence.Specification of primordial germ cells (PGCs), the precursors of oocytes and sperm, occurs during early embryonic development. The first step of IVG is induction of ESCs into PGC-like cells. Despite wide interest in replicating this event in vitro, little was known about PGC specification in the cow. We identified founder PGCs in the posterior epiblast of bovine embryos at day 16, by unique co-expression of conserved early germline proteins SOX17, PRDM1, and TFAP2C, with absent SOX2. We found that PGCs had initiated migration by day 20, at which point they were distributed through the allantois and hindgut endoderm, but were not yet highly proliferative. Single-cell transcriptome analysis of bovine embryos at day 20-22 allowed us to capture a small population of bovine PGCs, and gave insights into early germline development, including acquisition of the germ cell program, repression of somatic marks, and initiation of epigenetic remodeling. Further, we identified candidate cell surface markers for sorting PGCs – or in vitro-induced counterparts – for future experiments. This work provides the earliest observation of PGCs in cattle and demonstrates a conserved program in bovine germline emergence with other species that form as an embryonic disc.We continued from studying PGC specification and migration onset in the embryo through the advanced development of these cells into oocytes in the fetal ovary. Expanding upon a report from day 50, we traced the bovine germline by single-cell RNA-sequencing at days 70, 90, and 120 of fetal ovarian development. This revealed the transcriptional profile of bovine germ cells from gonadal PGCs through germline commitment into oogonia, initiation of meiosis, arrest at prophase I, and development of primordial oocytes within follicles, as well as cell surface marker candidates for each of these stages. Further, we explored the molecular profile of the somatic niche of the ovary and the interactions of these cells with the germline, which gives evidence for signals that could support germ cell development in a culture dish.In conclusion, this dissertation provides detailed and novel insights into bovine pluripotency and germline establishment. The knowledge gained from these studies may help in the development of assisted reproductive technologies of livestock, humans, and endangered ungulates. Moreover, these studies highlight the cow as a valuable model of human reproductive development and offer important context of embryonic/fetal events that cannot be directly studied in humans.

Photosymbioses between cnidarian hosts and Symbiodiniaceae algae enable the success of coral reef ecosystems. Algae remain photosynthetically active in symbiosis capturing light energy to fix CO2, evolve O2, and produce photosynthates which are transferred to hosts as substrate for growth and metabolism. Host cells phagocytose free-living algae and sequester them in arrested phagosomes, termed “symbiosomes,” enabling control over symbionts’ metabolisms promoting the production of photosynthates while regulating symbiont population growth. The underlying cellular mechanisms are mostly unknown and only a V-type H+-ATPase (VHA)-dependent carbon-concentrating mechanism (CCM) is documented in corals. My thesis sought to identify whether the CCM is widespread in cnidarian photosymbioses, establish the CCM’s importance under various environmental conditions, and identify nitrogen transport mechanisms. Chapter 2 describes the cloning of a candidate coral NH3/NH4+ (total ammonia, Tamm) channel (ayRhp1) from Acropora yongei. Heterologous expression in Xenopus oocytes and flux assays determined ayRhp1 is a dual NH3/CO2 gas channel. Immunofluoresence Airyscan confocal microscopy (IACM) revealed ayRhp1 is present in the symbiosome membrane where, together with VHA, it concentrates Tamm in the symbiont’s microenvironment. Furthermore, ayRhp1 is preferentially present in the symbiosome membrane during the day; presumably to provide algae with Tamm to ensure photosynthate production during photosynthesis. Chapter 3 tested whether Cassiopea jellyfish, which host their symbiotic algae in motile cells called amebocytes, employ a CCM like that from coral host cells. Western blotting and AICM revealed that Cassiopea amebocytes express both VHA and carbonic anhydrase (CA) and that both proteins localized to the symbiosome membrane. Respirometry using VHA and CA inhibitors revealed significant reductions in O2 evolution rate, a proxy for photosynthetic activity, suggesting that host VHA and CA are functionally coupled in the CCM. Chapter 4 explored the prevalence of the CCM in the coral Stylophora pistillata after acclimation to various environmental light conditions. While VHA protein abundance correlated with increased irradiance, AICM found limited symbiosomal VHA and respirometry experiments did not detect functional evidence for CCMs. Instead, VHA was prominently observed in calcifying cells, where it may contribute to pH regulation for biomineralization. Together, my thesis advances our mechanistic understanding of nutrient exchange in cnidarian photosymbioses.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer, with von Hippel-Lindau (VHL) gene mutations prevalent in approximately 90% of cases. We have previously demonstrated synthetic lethality between VHL loss and the inhibition of Cyclin-Dependent Kinases (CDKs) in ccRCC, using the multi-CDK inhibitor Dinaciclib. Here, we extend these findings by investigating the specific role of CDK9 in synthetic lethality with VHL loss. CDK9 is a transcriptional CDK involved in the process of transcription elongation and splicing. Using three distinct and specific CDK9 inhibitors (AZD45753, KB0742 and NVP-2), we confirmed preferential toxicity towards VHL-deficient ccRCC cells that acted independently of the hypoxia-inducible factor (HIF) pathway, the main pathway regulated by VHL. An analysis of the current literature connecting inhibition of CDK9 to cancer cell death suggests that the prime mechanism of action is the reduction of CDK9’s target pro-survival proteins, MCL-1 or BFL-1. Interestingly, we found that BFL-1, not MCL-1, is the critical anti-apoptotic protein involved in the mechanism of synthetic lethality. The literature further suggested that CDK9 inhibition might cause immunogenic cell death (ICD). In vivo, immunocompetent and immunodeficient mouse studies using AZD4573 showed that CDK9 inhibition suppressed ccRCC tumor growth, although we did not observe significant changes in immune cell populations upon treatment in an immunocompetent mouse model. At the same time, CDK9 inhibition did not compromise immune system. Our results offer novel insights into the mechanism of CDK9 inhibition / VHL loss synthetic lethality in ccRCC and provide a compelling rationale for further development of CDK9 inhibitors as potential therapeutics in VHL-deficient ccRCC. The BFL-1 pathway, which is a part of the mechanism of AZD4573 action, represents a biomarker for drug sensitivity, paving the way for improved patient stratification and treatment outcomes.

The broader theme of this dissertation is concerned with the molecular mechanismsemployed by neurons to regenerate dendrites after injury, with a special emphasis ondendrite injury detection. Much of the work provides evidence for a fundamental, yetnecessary, question: Does dendrite injury trigger calcium influx and, if so, how necessary isthis early calcium influx for repair? To answer this, I used Drosophila dendritic arborizationneurons of the larval peripheral nervous system as a model. Injuries were performed usingprecise two-photon laser-mediated removal of dendrites in live, intact larvae. Elevations incalcium were measured using the most advanced version of the genetically encoded calciumindicator GCaMP7f and morphological regeneration was assessed using membrane-boundfluorophores. The findings indicate that dendrite injury-induced elevations in calcium maybe cell type- and neurite type-specific. Importantly, shunting injury-induced calcium influxresulted in less, but not abolished, regeneration, suggesting that this early calcium plays asupportive role in dendrite regeneration. Mechanistically, I provided evidence for thepositive role of L-type VGCCs, IP3 signaling, and PKD activity on dendrite regeneration. Inaddition to my research endeavors, this thesis also describes my experience completing agraduate research internship with Eli Lilly and Company in Boston, MA. I then reflect on howthat opportunity made me a better scientist, and briefly discuss the literature on graduatestudent internships. Lastly, I detail the results of some final projects regarding the roles ofhistone deacetylases, protein kinase D, and the neuronal cytoskeleton, in addition to the useof hippocampal neuron culture to study dendrite regeneration in a system that providesunique characteristics distinct from fly sensory neurons.

Autophagy is a fundamental process of cellular quality control and maintenance that is used to protect cells during natural, healthy aging. Defects in autophagy are implicated in a host of pathologies of aging, notably neurodegenerative diseases. The Rubicon Homology (RH) proteins are a class of autophagy-regulating proteins defined by their RAB7A binding RH domains, consisting of the proteins Rubicon, Pacer, and PLEKHM1. These proteins bidirectionally regulate autophagy, as determined by the phosphorylation state of RAB7A.Here, I demonstrate a mitochondrial autophagy (mitophagy) specific form of regulation for RH family proteins. Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A–positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.The stability of the Rubicon protein has recently been established as a natural regulator of autophagy flux in a variety of tissues (adipose, hepatic, cardiac, and neuronal), where cells will stabilize or degrade Rubicon as a means of inhibiting or promoting autophagy flux respectively. To develop a novel pharmacological inducer of autophagy, I sought to mimic this form of regulation by creating a small molecule heterobifunctional degrader of Rubicon that colocalizes Rubicon with the E3 ligase Cereblon, inducing ubiquitinylation and subsequent degradation.

In order to promote sustained growth and health, the cell must carefully balance its metabolic state, responding to nutrient shortages, acute injury, and altered environment to maintain energetic homeostasis. The machinations of this task are extremely complex, and require extensive sensors and signals to diagnose cellular nutrient state, identify specific issues, and maintain functional balance. Two organelles, the lysosome and the mitochondrion, are central to cellular metabolic processes and host a wide variety of signaling pathways that are used to assess energetic health and capacity. Their coregulation is necessary to support cellular health in instances of energetic stress, and the breakdown of their communication often leads to debilitating degenerative diseases. While much work has been done to understand how the signals and functions of these two organelles overlap during stress, it is less clear what functional changes are initiated by lysosomal signaling itself, and how these may alter mitochondrial function to promote survivability.Lysosomes are the primary catabolic site of the cell, where the autophagic and endocytic pathways converge to deliver cargo for degradation. By this mechanism, the lysosomal lumen reflects the nutrient status of the cellular environment and can use this information to influence the metabolic state of the cell. To this purpose, the lysosomal surface hosts the active state of the kinase mechanistic Target of Rapamycin (mTOR) as part of mTOR Complex I (mTORC1). Here, mTORC1 responds to the presence of insulin, growth factors, and nutrients to promote anabolic processes for cell growth. If any of these inputs are depleted, mTORC1 is rendered inactive and removed from the lysosome, switching the cell to catabolic programming. With this process, mTORC1 is central to maintaining metabolic and energetic homeostasis in the cell.Meanwhile, the mitochondria is the cellular site of bulk metabolite and energetic production. Robust mitochondrial function is required for the survival of high-energy tissues, such as the heart and brain, and is necessary for cellular growth and differentiation. Mitochondrial stress results in depleted energetic supply, toxic redox species, and in some cases, the trigger of apoptotic cell death. Defects in mitochondrial function are thus detrimental to cellular and organismal health. Previous work has established that many signals that reflect mitochondrial stress interact with mTORC1, either directly or by pathway convergence, to coordinate energetic status with whole-cell programming. In many models of mitochondrial disease, coordinated mTORC1 function is required to allow survivability.While we generally understand how mTORC1 interacts with and supports the objectives of mitochondrial stress pathways, what is less clear is how mTORC1 signaling influences mitochondrial function at baseline. Here, I study how acute inhibition of mTORC1 by pharmacological intervention alters mitochondrial function in the absence of induced stress, thereby shedding light on mitochondrial pathways that may be influenced by mTORC1 status. Then, I study how these alterations map to mitochondria who have been injured by depletion of the iron-sulfur cluster biogenesis protein Frataxin (FXN) to identify which pathways may be valuable for mTORC1-supported mitochondrial survival under stress.With functional assays, we identify impaired function of Complex III of the mitochondrial electron transport chain as a result of FXN loss, with a converse restoration when mTORC1 is inhibited. Proteomic analysis of mitochondria in these conditions reveal an alteration of redox homeostasis upon mTORC1 inhibition, irrespective of FXN loss. Further exacerbation of the system by FXN loss then exposes a potential role of mTORC1 signaling for modulation of CoQ biosynthesis machinery, which may represent a newly discovered branch of mTORC1 influence.