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Photobodies are plant subnuclear membraneless compartments, composed of the highly conserved red and far-red light receptor and temperature sensor, Phytochrome B (PHYB). Since the discovery of photobodies in 1999, many studies have tried to elucidate the exact function of photobodies in phytochrome B signaling, but function of photobodies has remained frustratingly elusive. Active PHYB interacts with a key family of bHLH transcription factors known as Phytochrome Interacting Factors (PIFs) which drive light and temperature signaling within Arabidopsis. The relationship of PIFs and photobodies suggest several possible mechanisms of regulation. My dissertation research investigates how the spatial compartmentalization of PHYB to photobodies regulates PHYB signaling outputs by using PIF5 as a model and how components like PIF5 are recruited and influence photobody formation. In the first chapter, I review what is known about Phytochrome B in its structure, signaling, composition, photobody formation and possible hypotheses of function. To better understand photobodies as biomolecular condensates and PIFs as components of the photobody, discusses concepts of formation such as liquid-liquid phase separation, multivalency, composition and functions of biomolecular condensates that have been applied to emerging plant biomolecular condensate studies. In the second chapter, we found that PHYB recruits PHYTOCHROME-INTERACTING FACTOR5 (PIF5) to photobodies. Surprisingly, PHYB exerts opposing roles in both degrading and stabilizing PIF5. Altering photobody size by overproducing PHYB created different responses. A moderate increase in PHYB enhanced PIF5 degradation, further elevating the PHYB level stabilized PIF5 by retaining more of it in enlarged PBs. Conversely, reducing PB size by dim light, which enhanced PB dynamics and nucleoplasmic PHYB and PIF5, switched the balance towards PIF5 degradation. Together, these results reveal a signaling mechanism where photobody formation

Open-Angle Glaucoma (OAG) is the leading cause of irreversible blindness. The cause of OAG is an increase in intraocular pressure, thought to be driven by fibrosis and other changes in the trabecular meshwork (TM). This accumulation is often the result of changes in the behavior of TM cells, which are responsible for maintaining the TM. In OAG, the cells are fewer in number, produce more extracellular matrix (ECM) proteins, and fail to remove ECM proteins and debris accumulation in the TM. Another change is mitochondrial damage, which takes the form of a decrease in mitochondrial DNA (mtDNA) per cell, an increase in the mitochondrial common deletion, and an increase in the damaged nucleotide 8-Oxo-2'-deoxyguanosine in mtDNA. This work sought to investigate the extent to which existing glaucoma models in TM cells affect mitochondrial performance and mtDNA damage. Further, this work also aimed to examine how mitochondrial DNA damage and depletion impacted levels of OAG-related transcripts. Dexamethasone (Dex) has been used to model glaucoma-like changes in TM cells. Specifically, it induces fibrotic changes in TM cells. In vivo, it can lead to glaucoma in humans and animal models. Our work shows that Dex leads to a decrease in ATP being produced by mitochondria in TM cells, as well as a decrease in mitochondrial performance. However, Dex treated TM cells did not show any evidence of the mtDNA damage found in OAG. For this reason, we next aimed to directly induce mtDNA damage and depletion in TM cells. Damage and depletion of mtDNA was induced by two methods, to account for potential off-target effects. One method was via treatment with ethidium bromide. In addition, another method involving doxycycline inducible expression of a mutant DNA repair enzyme was used as well. To allow for transduction and selection of cells with this construct, an immortalized version of TM cells was selected. Both of these methods yield a decrease in mtDNA per cell, as well a decrease in c

Genetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cell’s genome, thereby allowing the hidden forces that guide tissue development to be chronicled. After the developmental process of interest is complete, the cells’ experiences can be read out via high-throughput DNA sequencing. Two types of transient events are captured with DNA recorders—the existence of progenitor cells that give rise to daughter cells (which is recorded as lineage relationships among cells by generating lineage-specific DNA barcodes), and stimuli that trigger a molecular response in the cells (which is recorded in an analog fashion by linking the accumulation of mutations to the presence of inducers of interest). This dissertation focuses on the development of two distinct, but complementary DNA recorders. First, Cell History Recording by Ordered iNsertion (CHYRON), which uses a homing guide RNA (hgRNA), CRISPR-Cas9, and a template-independent polymerase (TdT) to generate ordered insertion mutations, was developed. A large portion of this technology was already established prior to my joining the project, so this dissertation focuses only on the developments that overlapped with my efforts. Specifically, alternative nuclease components (instead of Cas9) were explored for CHYRON recording, and dose- and duration-dependent hypoxia recording was accomplished. Also, the nucleotide insertion preferences of TdT were engineered in a novel, massively parallel screen, to achieve greater lineage barcode diversity and enable recording of two different stimuli with differently-biased TdTs. The other DNA recorder covered in this work is prime editing CHYRON (peCHYRON), which uses prime editor to generate iterative insertion mutations. Prime editor requires its edits to be precisely pre-defined in prime editing guide RNAs (pegRNAs), so pegRNA sequences that accomplish continuous insertion accumulation were carefully engineered via a combination of

Despite a global ban on their production, polychlorinated biphenyls (PCBs) remain widespread environmental contaminants and pose considerable risks to human health. Experimental findings demonstrate that PCBs have profound neurotoxic effects, with individual congeners varying in degrees of cytotoxicity and carcinogenicity. Exposure to PCBs is regarded as a probable risk factor for several neurological and psychiatric conditions of unknown etiology. Though more often investigated from a neurodevelopmental standpoint, PCB-induced neurotoxicity likely also has implications for adolescent- and adult-onset disease states. This review outlines significant findings of epidemiological studies that examined risks for neurodegenerative diseases and affective disorders in cases of adult PCB exposure. The biological plausibility of the observed associations between PCB exposure and increased risk for Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), and major depressive disorder (MDD) are discussed in the context of preclinical evidence supporting potential pathological mechanisms by which PCBs confer risk of neurodegenerative disease. Specifically, we focus on oxidative stress, altered neural connectivity, disrupted monoamine neurotransmitter homeostasis, and aggregated protein pathology. Lastly, key data gaps in the existing epidemiological literature are identified.

The constantly changing physical world subjects its inhabitants to variable abiotic conditions. Organisms function optimally within a range of abiotic parameters. Extreme divergence form that range due to conditions such as salinity fluctuations in some aquatic environments, imposes strain on their physiological systems such as hyper-osmotic (HO) stress. Persistence in such an environment is restricted to species capable of the necessary adaptive responses. The economically important Mozambique tilapia (Oreochromis mossambicus) is such a species, making it an ideal model for studying salinity stress tolerance. Previous work showed increased abundances of the myo-inositol biosynthesis (MIB) pathway enzymes myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1.1) in tissues of O. mossambicus subjected to elevated salinity. Previous work also suggested an involvement of the transcription factor NFAT5 (nuclear factor of activated T-cells 5) in the osmotic regulation of the MIB pathway. However, the causality of these relationships with salinity tolerance phenotypes had yet to be established. The objective of this research was to test the overall hypothesis that upregulation of the MIB pathway enzymes contributes to salinity tolerance and is mediated by NFAT5 using CRISPR/Cas9 gene editing and synthetic biology approaches in a simplified but representative tilapia cell line model (OmB) to manipulate the genetic loci encoding these proteins. Initial CRISPR/Cas9 editing attempts utilizing gRNA/Cas9 protein complexes and expression vectors optimized for zebrafish or mammalian cells failed to yield detectable gene edits. Therefore, a system customized for tilapia cells was developed using endogenous O. mossambicus EF1 alpha and U6 promoters to drive Cas9 and gRNA expression respectively. A version of the OmB cell line (Cas9-OmB1) was engineered to express the Cas9 protein by transposon mediated genomic integration of the Cas9 gene while different gRNAs

Neutrophils are the most abundant immune cell in the human body and are critically important in clearing infections and in wound healing. They rapidly migrate from circulation to sites of injury or infection by detecting chemicals that are released by the body or that emanate from degrading cells and bacteria. Upon reaching these sites, neutrophils engage pathogens with direct killing processes including pathogen engulfment by phagocytosis and by releasing pre-stored anti-microbial products from specialized vesicles called granules in a process called degranulation. While these destructive processes are effective at neutralizing pathogens, dysregulated activity is implicated in inflammatory diseases. Cleary defining the molecular mechanisms that regulate immune cell functions would present opportunities for new drug targets and therapeutics. However, achieving the comprehensive understanding necessary to derive novel therapies has been hampered by the complex interactions between molecules that regulate neutrophil functions. This dissertation is compilation of my contributions toward untangling these molecular pathways. Chapter 1 provides an introduction to neutrophils, their functions, and an overview of outstanding related questions. Chapter 2 describes discoveries about how neutrophils prioritize competing signals as they migrate, which helps them home in on the correct target. The work described in chapter 2 was a collaboration project with Briana Rocha-Gregg. Chapter 3 focuses on neutrophil function once they reach sites of infection. Specifically, it describes my findings about the molecular pathways that control degranulation, including the identification of components that may distinguish migration and degranulation behaviors. In Chapter 4 closing remarks and comments on future directions are made.

Neuroendocrine prostate cancer (NEPC) represents an aggressive subtype of prostate cancer, which may either manifest de novo or emerge because of anti-androgen receptor (AR) drug treatment. Unfortunately, there is currently no established effective treatment for NEPC. Alectinib and entrectinib are FDA-approved receptor tyrosine kinase inhibitors designed to target the anaplastic lymphoma kinase receptor (ALK) initially developed for NSCLC patients who developed resistance to the receptor tyrosine kinase (RTK) inhibitor crizotinib. In this study, we sought to assess the potential of alectinib and entrectinib as therapeutic agents for NEPC. We conducted experiments using various NEPC cell lines and castration-resistant prostate cancer (CRPC) cells, subjecting them to treatment with alectinib and entrectinib. Notably, treatment with these drugs, particularly in cell lines 42D, NCI-H660, C42B-entry, and LuCaP, significantly inhibited cell growth and proliferation. Further investigation revealed that alectinib and entrectinib exerted their effects by impacting the ALK signaling pathway. Specifically, downstream targets of ALK signaling, such as STAT3 and AKT phosphorylation, exhibited reductions in treated cells. Additionally, the expression of NEPC cellular markers, including enolase2, BRN2, and ASCL1, decreased upon alectinib treatment. Notably, NEPC tumors treated with alectinib or entrectinib in combination with CM 272, a G9a/DMNT1 inhibitor, exhibited a notable decrease in tumor volume. Both alectinib and entrectinib demonstrated their efficacy in inhibiting various cellular pathways, including the Neuroactive ligand-receptor and cytokine-to-cytokine receptor interaction pathways. The findings of our current study provide compelling evidence that alectinib and entrectinib have the potential to effectively inhibit NEPC cell and tumor growth. Consequently, these drugs hold promise as therapeutics for patients grappling with NEPC, offering a potential avenue for improv

Tissue regeneration, once a dream or a fantasy, is now becoming a reality. Science has advanced so much that cells can be cryopreserved and expanded into millions under laboratory conditions. Cells can be manipulated to become something they were not before and can be coerced into forming actual tissues. The discovery of powerful growth factors and the ability to synthesize them, helps to achieve these goals. Our understanding of pathologic processes and aging also plays an essential role in our ability to regenerate tissues. Laboratory methods have advanced immensely, allowing more efficient and rapid progress in regenerative medicine. This thesis addresses the regenerative efforts of two types of tissues: cartilage and skin. These tissues have an essential function in our bodies, but their structure and biology are dramatically different. Yet the common denominator is that people can benefit greatly from learning more about the regeneration of these tissues.Cartilage is a shock-absorbing tissue lining the ends of diarthrodial joints that aims to provide a frictionless surface for joint articulation. In disease, damage of articular cartilage is often accompanied by tissue thinning and an irreversible yet progressive degeneration. Cartilage’s innately poor regenerative capacity and progressively increasing demand for its regeneration have resulted in astonishing developments and achievements in the field of tissue engineering (TE) and regenerative therapies. However, despite advancements in cartilage tissue engineering and current clinical transplantation practices, the ability to replace deficient or damaged cartilage remains limited. In response, researchers are urgently trying to develop novel and clinically applicable solutions for the treatment of cartilage degenerative disorders.The translation of cartilage tissue engineering practices is hindered by the lack of suitable cell sources and implant integration with surrounding native tissue. The lack of implant i

The precise spatial control of the division plane is crucial for tissue and organpatterning in plants, and the preprophase band (PPB) is a cytoskeletal structure that regulates division plane orientation in somatic cells. The PPB is primarily composed of bundled cortical microtubules (MTs) with actin and protein components and functions to organize a cortex region that becomes the future division site. The PPB-dependent cue is a positional landmark that guides centrifugal phragmoplast expansion during cytokinesis. Chapter 1 of this thesis offers a comprehensive literature review on the PPB, delving into our current understanding of the molecular mechanisms controlling its formation and function and tracing its evolutionary origins. In Chapter 2, we identify and characterize the IQ Domain (IQD) containing proteins 6/7/8 as microtubule-binding proteins associated with PPB MTs. Triple mutant plants of these IQD genes exhibit oblique cell division planes caused by disorganized PPBs that led to the misspecification of the division plane. Our studies show that robust and solid PPBs are crucial for precise division plane control and that IQD6/7/8 are necessary for properly organizing PPB MTs. Another discovery highlights the cooperation between the MTpolymerase CLIP Associated Protein (CLASP) and IQD6/7/8 in maintaining PPB MT organization and stability. Considering the conserved IQ67 domain in IQD proteins is known to bind calmodulin (CaM) in the presence of calcium (Ca2+), it raises intriguing questions about the role of Ca2+/CaM in division plane regulation. Chapter 3 focuses on the regulation of PPB formation by cyclin and cyclin-dependent kinase (CDK). We confirm that the cyclin A1;1 (CYCA1;1) and CDKA;1 localize at the PPB when expressed under native promoters, revealing an interdependence between CDKA;1 and CYCA1;1 for their PPB localization. However, the specific functions of CDKA;1 and CYCA1;1 at the PPB still need to be clarified. Our findings introduce a connect

Well-differentiated/dedifferentiated liposarcoma (WD/DD LPS) is a common subtype of soft tissuesarcoma in adults. Our understanding of this disease is lacking and treatment options are limited. First, by investigating tumor metabolomics, we identify asparagine (Asn) as a fundamental purpose for mitochondrial respiration in WD/DD LPS. Asn promotes mTORC1 activity to support tumor growth. Depleting Asn by combining mitochondrial complex I inhibition with asparaginase holds therapeutic potential. Next, we characterize WD/DD LPS beyond histological classification by defining mechanisms of dysregulation in mitochondrial dynamics. WD LPS displays low MAPK signaling activity, elongated mitochondrial morphology, and high mitochondrial respiration. Whereas DD LPS displays high activation of MAPK signaling, which promotes activation of DRP1, fragmentation of mitochondria and a glycolytic phenotype. This shift in mitochondrial bioenergetics promotes tumor growth. Trametinib represents a novel therapeutic approach to target this adaptation. Moreover, Trametinib synergizes with chemotherapy, including agents Pazopanib and Palbociclib, further expanding its clinical potential. DRP1 expression may also serve as a prognostic biomarker, associating with worse survival. Altogether, these findings reveal several adaptations in mitochondrial bioenergetics that provide opportunities for targeted therapy in patients with WD/DD LPS.

The Histone lysine demethylase 5 (KDM5) is recognized as a potential therapeutic target due to its involvement in several diseases, including cancer. While recent studies have advanced our understanding of its catalytic functions, KDM5’s scaffolding functions to recruit other factors to chromatin remain underexplored. This dissertation serves to investigate the non-catalytic roles of KDM5 in chromatin regulation, particularly in modulating immune responses. Using a multiomics approach, we have characterized KDM5A and KDM5B as regulators of the KRAB-ZNF (Krüppel-associated box domain zinc finger) genes. The loss of KDM5A and KDM5B are associated with increased expression of ERV genes and upregulation of immune response markers. Additionally, inhibition of KDM5 catalytic activity did not phenocopy genetic ablation. In contrast, acute degradation of KDM5A resulted in increased ERVs expression. Additionally, this dissertation details efforts to develop targeted protein degradation strategies that address both the catalytic and scaffolding functions of KDM5.Chapter 1 provides an introduction to KDM5 demethylases and their roles in disease, discussing recent advancements in catalysis and therapeutic efforts for KDM5 and other JMJC KDM demethylases. Chapter 2 employs a mult-iomics approach to characterize KDM5 role in regulating the chromatin landscape, highlighting the significant roles of KDM5A and KDM5B in modulating immune responses. Chapter 3 outlines the development of small molecule degraders targeting KDM5

To orchestrate the complex events of craniofacial morphogenesis requires a concerted effort between gene expression and tissue specific events. In the embryonic midface, facial prominences must execute a coordinated fusion between maxillary, medial and lateral nasal prominences at the lambdoidal junction, requiring breakdown and removal of epithelium to permit coalescence of underlying mesenchyme. When this process is disrupted by genetic mutations, cleft lip, the most common craniofacial birth defect in humans, is created. Key to understanding cleft lip pathogenesis is the reliance on mouse models. Models of cleft lip and studies of the role of epithelium are rare in comparison to cleft palate and research on underlying mesenchyme. The epithelium possesses discrete roles that facilitate fusion at the lambdoidal junction, including apoptosis and epithelial to mesenchymal transitions. To better understand its heterogeneity, we sought to enrich for the midface epithelium and isolate it from mesenchyme to gain fuller resolution of the lambdoidal junction transcriptome. This dissertation contains the product of that endeavor to focus on the role of the epithelium alone both in normal conditions and in cleft lip pathogenesis. In doing so, an atlas of lambdoidal junction epithelium at timepoints before, during and after fusion of the midface was created and validated. Through these methods, a deep analysis of each cluster resulted in the localization of a cluster undergoing cell cycle arrest and located at the fusion site of each prominence. A series of experiments were conducted which demonstrated that cell cycle arrest is characteristic of this population that is altered in cleft lip models. Additional research yielded ties to normal midface morphogenesis as well as novel orofacial cleft risk candidates, including Zfhx3 a gene with ties to cell cycle arrest. This thesis also expands upon the phenotypic characterization of a novel mouse cross which conditionally removes

How nature builds complex and beautiful architecture from very simple building rules and building blocks is a fascinating biological question. During every cell division, nanometer-scale components self-organize to build a micron-scale spindle. In mammalian spindles, microtubule bundles called kinetochore-fibers attach to chromosomes and focus into spindle poles. Despite evidence suggesting that poles can set spindle length, their role remains poorly understood. In fact, many species do not have spindle poles. Here, we probe the pole’s contribution to mammalian spindle length, dynamics, organization, and function by inhibiting dynein to generate spindles whose kinetochore-fibers do not focus into poles, yet maintain a metaphase steady-state length. We find that unfocused kinetochore-fibers have a mean length indistinguishable from control, but a broader length distribution, and reduced length coordination between sisters and neighbors. Further, we show that unfocused kinetochore-fibers, like control, can grow back to their steady-state length if acutely shortened by drug treatment or laser ablation: they recover their length by tuning their end dynamics, albeit slower due to their reduced baseline dynamics. Thus, kinetochore-fiber dynamics are regulated by their length, not just pole-focusing forces. Next, we probe force-induced displacement of kinetochore-fibers lacking pole-focusing forces to reveal hints about mechanisms underlying kinetochore-fiber anchorage and organization. Finally, we show that spindles with unfocused kinetochore-fibers can segregate chromosomes but fail to correctly do so. We propose that mammalian spindle length emerges locally from individual k-fibers while spindle poles globally coordinate k-fibers across space and time.

G protein-coupled receptor (GPCR) signal transduction is a fundamental building block of cell biology, yet how different ligands such as peptide agonists and small molecules can act at the same receptor to produce different functional outcomes is not fully appreciated. A proliferation of biochemical, biophysical, and pharmacological studies led to an abundance of receptor active-state structures and candidate molecules selective for desired outcomes while avoiding side effects. With preponderance of data, however, also came contradictions and challenges in interpretations. This thesis returns to the basic step of ligand-receptor-effector interaction, and explores two fundamental questions: 1) can different agonists selectively couple the receptor to different cytoplasmic proteins in intact cells? and 2) in the agonist-selective receptor endocytosis pathway, how do GPCR kinases (GRKs) recognize different agonist-bound receptors? Using two engineered protein biosensors as discreet intracellular proteins, two agonists showed distinctly different profiles in recruiting the protein biosensors. This result provides a proof of concept of a potential answer to the first question, that in a ‘clean’ cellular environment without native signaling interference, agonists can indeed engage two different biosensors with selective preferences. Similarly for the second question, using an engineered GRK-free cellular background, a single GRK subtype was shown to produce agonist-selective functional outcome of receptor internalization. This GRK was also recruited to the receptor in an agonist-selective manner, supporting the hypothesis that selective functional outcomes begin upstream at selective receptor-GRK interactions. Furthermore, a single domain was shown to be the key selectivity determinant for GRK recruitment to the receptor and internalization function.

Intracellular bacterial pathogens have evolved to survive in a distinctly hostile environment. Intravacuolar pathogens such as Legionella pneumophila (L.p.) reside within a membrane bound compartment in their host cell, reshaping host materials into a new, protected organelle. The molecular mechanisms employed by L.p. during early infection to defend its vacuole are not entirely understood. As host cells uptake the bacterium by phagocytosis, L.p. must avoid classical trafficking of its phagosome through the endolysosomal pathway or attack by autophagy, which would ultimately lead to destruction of the bacterium in the lysosome. In this work, we set out to define how L.p. manipulates host regulatory proteins to defend its vacuole, termed the Legionella-containing vacuole (LCV), from these threats during early infection. First, we explore the interaction of Rab5, the master regulator of the early endosomal compartment which is required for phagosome maturation, with the LCV. We determine that Rab5 associates with the LCV and is both mono- and polyubiquitinated during infection. This ubiquitination is dependent on two families of secreted bacterial proteins, SidC/SdcA and the SidE family of effectors. Finally, we find that SidE family dependent polyubiquitination is necessary for retention of Rab5 at the LCV membrane. Next, we recognized the unique opportunity presented by Rab5, as this host protein localizes to both the WT and avirulent strain LCV. We determine that Rab5 associated with the WT LCV has a distinctive morphology and stability that is dependent on the SidE family of effectors rather than host regulators. Finally, we discover that L.p. infection induces a burst of ubiquitination of both host and bacterial proteins, and that SidC/SdcA play a central role in this burst. We propose a new role of SidC/SdcA as a metaeffector, which regulates the activity of other as yet unknown bacterial proteins. Taken together, our findings suggest a model in which ubiquitin

Stress granules (SGs) are macromolecular assemblies that form under cellular stress. Formation of these condensates is driven by the condensation of RNA and RNA-binding proteins such as G3BPs. G3BPs condense into SGs following stress-induced translational arrest. Three G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) have been identified in vertebrates. However, the contribution of different G3BP paralogs to stress granule formation and stress-induced gene expression changes is incompletely understood. Here, we identified key residues for G3BP condensation such as V11. This conserved amino acid is required for formation of the G3BP-Caprin-1 complex, hence promoting SG assembly. Total RNA sequencing and ribosome profiling revealed that disruption of G3BP condensation corresponds to changes in mRNA levels and ribosome engagement during the integrated stress response (ISR). Moreover, we found that G3BP2B preferentially condenses and promotes changes in mRNA expression under endoplasmic reticulum (ER) stress. Together, this work suggests that stress granule assembly promotes changes in gene expression under cellular stress, which is differentially regulated by G3BP paralogs.

Neutrophils collectively migrate to sites of injury and infection. How these swarms are coordinated to ensure the proper level of recruitment is unknown. Using an ex vivo model of infection, we show that human neutrophil swarming is organized by multiple pulsatile chemoattractant waves. These waves propagate through active relay in which stimulated neutrophils trigger their neighbors to release additional swarming cues. Unlike canonical active relays, we find these waves to be self-terminating, limiting the spatial range of cell recruitment. We identify an NADPH-oxidase-based negative feedback loop that is needed for this self-terminating behavior. We observe near-constant levels of neutrophil recruitment over a wide range of starting conditions, revealing surprising robustness in the swarming process. This homeostatic control is achieved by larger and more numerous swarming waves at lower cell densities. We link defective wave termination to a broken recruitment homeostat in the context of human chronic granulomatous disease.

In the developing vertebrate brain, Radial Glia Progenitor (RGP), the principal neural stem cells, undergo symmetric and asymmetric cell division (ACD), giving rise to both RGPs (self-renewal) and differentiated cell types (e.g., neurons, oligodendrocytes, and astrocytes) that will ultimately form the central nervous system. Perturbations of RGPs divisions will result in neurodevelopmental disorders and brain tumors. Understanding the basic biology of RGPs divisions will potentially have a significant impact on understanding brain development and may provide insights into treatments for developmental disorders or brain tumors. During ACD, the mother cell must establish the proper axis of polarity with respect to asymmetrically localized cell fate determinants. These processes are regulated by the evolutionarily conserved Partition defective protein (Par) complexes. The Par complexes have been extensively characterized in invertebrates, but much remains to be understood in vertebrates, especially in the context of RGPs ACD. Previous studies in invertebrates and vertebrates have demonstrated that Par-3 is essential for regulating ACD by localizing asymmetrically along the division axis. Par-3 has long been thought to function exclusively at the cell cortex. Recent findings have led us to investigate the role of Par-3 during ACD in RGPs. Therefore, one approach to gain insights into this question is the utilization of zebrafish (Danio rerio), a powerful model organism used in biological research. Proteins of interest can be easily targeted by both pharmacological agents and genetic alterations within zebrafish, allowing for examination of complex phenotypes resulting from desired perturbations. Unlike mammalian models, development is relatively fast, and breeding produces a large amount of progeny, allowing for quick generation progression and large sample sizes. Also, unlike mammals, development occurs externally, and the brain is transparent, allowing for accessible

Muscle contractions, among other cellular functions, are driven by highly ordered structures composed of actin and myosin. For cells to properly execute these functions, actin filaments must be specifically organized, assembled, and maintained throughout the cell. The precise timing of actin assembly required for these functions is helped by actin nucleators, such as formins. Formins assemble many actin-based structures through their formin homology (FH) domains, FH1 and FH2. The FH2 domain helps form new filaments in a process called nucleation, while the FH1 domain helps to elongate actin filaments in a processive manner while the FH2 domain “walks” along the growing barbed ends of filaments. The formin homology domain-containing protein (Fhod) family of formins are important for building several contractile actin structures, including sarcomeres in muscle cells and stress fibers in various non-muscle cells. Despite Fhod family formins being required for structures in vivo, mammalian Fhods were initially reported to instead inhibit actin assembly in vitro. Here, we establish that mammalian formin FHOD3 (both isoforms FHOD3S and FHOD3L) nucleate and elongate actin filaments in vitro. Human FHOD3S/L elongate actin filaments quite differently than other formins, where we observe brief, rapid moments of elongation after elongation is paused. We performed rescue experiments for FHOD3L in neonatal rat ventricular myocytes (NRVMs) with mutants that separated its actin assembly activities to better understand whether nucleation or elongation is more important for sarcomere formation. We found that elongation activity by FHOD3L is necessary and sufficient for proper sarcomere formation and maintenance, whereas reducing its nucleation or bundling activity is tolerated in NRVMs. Further, mutations in FHOD3 have been implicated in 1-2% of cases of hypertrophic cardiomyopathy (HCM), a heart disease which results in the thickening of the septal muscle, eventually leading to arr

With the global population projected to reach 9.8 billion by 2050, there is a critical need for sustainable and innovative food sources. Cultured meat, an innovative and sustainable approach to food production that allows the production of meat by cultivating animal cells in vitro, exhibits the potential to circumvent the challenges associated with traditional animal agriculture. A key to the success of cultured meat is developing adipose tissue, crucial for achieving the desired taste, texture, and nutritional quality. Accelerating adipogenesis has the potential to improve production efficiency, making large-scale cultured meat manufacturing more viable and cost-effective. This study aims to identify small molecules that accelerate adipogenesis for edible fat tissue production using a high-throughput screen. Our findings identified magnolol and dicoumarol as effective natural compounds that promote lipid accumulation in differentiating mouse, rabbit, and porcine preadipocytes. These compounds have also demonstrated a synergetic effect in accelerating adipogenesis, which highlights the potential of novel treatments in effectively promoting adipocyte differentiation across various species.

Adult stem cells are crucial for tissue homeostasis and repair. Their fate is tightly regulated by intrinsic genetic factors and extrinsic cues within the stem cell niche. These niches are adaptable, responding to stimuli to maintain homeostasis, but the molecular details of this regulation are not fully understood. This study explores a mechanism that regulates the fate of somatic support cells in the Drosophila testis to maintain tissue homeostasis.The hub in the Drosophila testis niche secretes factors that regulate germline stem cells (GSCs) and cyst stem cells (CySCs). Our lab previously reported that the Snail family transcriptional regulator Escargot (Esg) is necessary for maintaining the identity of both CySCs and hub cells. Depleting esg from hub cells leads to their conversion into CySCs, resulting in the loss of both GSC and CySC populations. Similarly, esg depletion from early somatic cells causes CySCs to differentiate prematurely into daughter cyst cells. These findings indicate that Esg is central to determining somatic cell fates, although the mechanisms are not fully understood. In the adult testis, Egfr activity is low in hub cells under homeostatic conditions but active in CySCs and early cyst cells, regulating their differentiation. Interestingly, Esg protein levels are significantly higher in hub cells than in cyst cells. We hypothesized that Esg influences somatic cell fate by repressing the Egfr signaling cascade. Our results show that repressing Egfr signaling suppresses hub-to-CySC conversion upon esg depletion. Conversely, overexpressing esg in CySCs increases the number of early cyst cells and induces CySC-to-hub cell conversion, which depends on Egfr activity. Constitutive Egfr activation in CySCs prevents this conversion upon esg overexpression. These findings support the hypothesis that Escargot acts upstream of Egfr to regulate somatic cell identity and maintain tissue homeostasis in the testis.

Mucosal-associated invariant T (MAIT) cells are innate T cells that were first believed to specialize in anti-bacterial responses, however, have shown to be involved in other immune responses, for example, against viruses, as well as processes of tissue-repair. After a lung infection with bacteria containing riboflavin metabolic pathways, Mucosal-associated invariant T (MAIT) cells expand and remained expanded in the tissue for long-term. Subpopulations of KLRG1+ and CD127+ MAIT cells are observable after infection, but only CD127+ population is detectable at steady state. Some of the transcriptional changes observable long-term after exposure are also found in canonical T cell memory populations when compared to naïve cells. We plan to use these conventional immunological memory overlapping genes and MAIT-exclusive gene hits to perform a in vivo CRISPR screen that would help us determine which genes enable the generation of these long-term expanded MAIT cells.

Cardiovascular disease (CVD) is a leading cause of deaths in the United States, however the underlying inflammatory pathways that contribute to CVD are poorly understood. Endothelial dysfunction is a hallmark of CVD and induced by various inflammatory mediators, many of which signal through G-protein coupled receptor (GPCRs). Thrombin, an inflammatory mediator, is generated in response to vascular injury and disrupts endothelial barrier integrity and promotes vascular leakage, a hallmark of inflammation. Thrombin activates endothelial protease-activated receptor-1 (PAR1) to disrupt endothelial barrier through the RhoA/myosin light chain (MLC) pathway. However, thrombin/PAR1-stimulated p38 mitogen-activated protein kinase (MAPK) signaling also promotes barrier permeability through a pathway that does not integrate with the RhoA/MLC signaling, suggesting that p38 acts through an uncharacterized pathway to promote inflammatory responses in endothelial cells. Our previous work showed that thrombin-activated PAR1 ubiquitination drives the recruitment of TAB2-TAB1 protein complexes on endosomes to trigger non-canonical p38 activation and endothelial barrier disruption. The mechanisms that regulate PAR1 ubiquitination/de-ubiquitination are not known. This proposal aims to understand the molecular processes that regulate PAR1-ubiquitin-driven p38 signaling and most importantly its impact on endothelial barrier integrity. The central hypothesis is that de-ubiquitination of PAR1 leads to termination of p38-mediated pro-inflammatory signaling. Using advanced biochemical, microscopy and genome-wide RNAi screening methods, we for the first time investigate molecular determinants that regulate the dynamics of PAR1-ubiquitination and the impact on endothelial proinflammatory responses in vitro using human endothelial cells. We reveal new information regarding the function of PAR1 ubiquitination in driving non-canonical endosomal p38 MAPK signaling in endothelial barrier disruption

Nematodes and their naturally associated pathogens are useful systems for dissecting host-pathogen interactions in vivo. Understanding the mechanisms of pathogenesis and innate immunity from these models could draw parallels to relevant human diseases and open new potentials for therapeutic interventions. This dissertation focuses on a new system, the free-living soil nematode Oscheius tipulae and its intracellular bacterial pathogen Bordetella atropi. To understand what nematodes, representative by Caenorhabditis elegans, can offer in terms of gaining relevant insights into the biology of immunity, the first chapter will review current knowledge on nematode immune responses against a wide variety of pathogens and how they are functionally and conceptually similar to those in vertebrate immunity. Chapter two will then describe our discovery that B. atropi employs filamentation – a morphological change from the normal coccobacillus to filamentous shape – for spreading from the initially infected cell to neighboring cells. We showed that this filamentation process is under control of a metabolic pathway highly conserved among bacterial species, which has been shown to regulate bacterial cell sizes under rich conditions. From the current available data, we proposed a model in which B. atropi, upon entering host cell, detects the rich environment of host cytoplasm and initiates filamentation to quickly spread to other cells, maximizing host cell space and resource utilization. Furthermore, we posited that spreading by means of filamentation represents a new paradigm of intracellular dissemination compared to that of more well-studied mammalian intracellular bacteria, including Rickettsia spp., Shigella spp., Burkholderia spp., and Listeria monocytogenes, where the pathogens often hijack host actin polymerization to form actin tails for the same purpose. Chapter three will present the mechanism that B. atropi uses to invade intestinal cells, helping it establish a replic

Retinogenesis, the development of the retina, is regulated by dynamic changes in transcriptional expression and the chromatin landscape. While model organisms have contributed significantly to understanding retinal development, they do not allow studies of human-specific conditions. We utilized a SIX6-eGFP/VSX2-tdTomato dual fluorescent pluripotent stem-cell (PSC) line and grew retinal organoids (ROs) through 45 days. RNA- and ATAC-seq analyses through 45 days revealed transcriptomic and chromatin-landscape regulatory changes that drive retinal development and the activation of retinal specific pathways. To study an early modifier of retinal development we also investigated the Sonic Hedgehog (Shh) signaling pathway by modulating the timing and concentration of a smoothened agonist (SAG) in SIX6-eGFP/POU4F2-tdTomato dual reporter ROs. Early SAG treatment led to the development of non-retinal tissue while later treatment favored retinal development, indicated by robust POU4F2-tdTomato expression. To investigate factors governing the formation of retinal ganglion cells (RGCs; the first cell born in the eye), we developed an inducible transgene cassette expressing the transcription factors (TFs) NEUROG2-ATOH7-ISL1-POU4F2 which, in combination with Bone Morphogenic Protein (BMP) inhibition, led to robust formation of RGC-like neurons in as little as one week. An RGC-like identity was validated via fluorescent reporter expression, transcriptomic analysis, and electrophysiology. Our findings provide a better understanding of the complex interplay of transcriptional regulation, chromatin dynamics, and microenvironmental cues in RO development and advancing our understanding of retinal biology. Generating RGC-iNs illuminates factors governing RGC formation and provides insight into the early formation of the eye.

Proper regulation of microtubule dynamics is critical for multiple events in embryogenesis, as microtubules are key cellular components that contribute to cell division, polarity, and migration. In C. elegans, the precise involvement and regulation of microtubules during embryonic development remains unclear. Our lab previously identified a gain-of-function (gf) mutation in an alpha-tubulin tba-1, which causes embryonic lethality at the restrictive temperature of 25°C. To identify the interactors of TBA-1 during embryo development, an EMS mutagenesis screen was performed and several lines that show suppression of tba-1(gf) embryonic lethality at 25°C were isolated. In this thesis, I analyzed the WGS data for all the obtained tba-1(gf) suppressor lines and focused on mapping the causative mutation for the ju1911 suppressor allele. By generating ju1911 recombinants, the genetic position of ju1911 is narrowed down to between -2.95 and +5.3 on chromosome III, containing function-altering mutations in two genes. I checked the candidate gene mapk-15 by introducing a loss-of-function allele in the tba-1(gf) background and CRISPR-Cas9 mediated gene knockout. I found that mapk-15(III, -1.42) is not responsible for the suppression. Overall, this study shows the disruptive effect of TBA-1(gf) in embryo development and narrows down the genetic locus of one of the tba-1(gf) suppressor alleles.

Iron is found within the cell as Fe3+, ferritin-bound iron, and Fe2+, intracellular labile iron, which can be readily used for a variety of cellular functions, especially in the mitochondria. Macrophages, a major site for iron storage and recycling, undergo significant metabolic changes upon activation. Here, we demonstrate that sequestration of the labile iron pool to the mitochondria is characteristic of the macrophage proinflammatory response. Using LPS to stimulate proinflammatory activation in bone marrow derived macrophage (BMDMs) and embryonic Kupffer cells (emKCs), we measured labile iron content and mitochondrial iron with specific probes detected by flow cytometry. The expression of key iron metabolism genes and proinflammatory cytokines were measured to assess the iron metabolism and proinflammatory responses of macrophages. Treatment with LPS in vitro and in vivo demonstrated increased the mitochondrial Fe2+ content, while the intracellular labile iron pool was decreased. The emKC population was decreased after LPS stimulation, but this effect was attenuated by depletion of ferritin. Other hepatic macrophage populations, Kupffer cell-like (KC-like) cells and recruited hepatic macrophages (RHMs), increased in response to LPS. Nrf2, a master regulator of redox homeostasis, which was repressed in emKCs but upregulated in KC-like cells and RHMs, may be key to understanding the differences in responses to LPS exhibited by these macrophage populations. Our results expand upon the interplay between iron homeostasis and the metabolic changes required for macrophage activation.

Transposable elements (TE) are mobile DNA elements that can replicate and re-insert themselves into different locations within the host genome. This retrotransposition has resulted in TEs accounting for more than 50% of the human genome. Much like other non-coding RNAs, TE insertions with multiple copies were once considered non-functional. But recently it has been shown to have much more regulatory role in different cellular pathways. The first part of this thesis delves in describing the different types of TE and other non-coding RNAs like non-coding RNA and their significance in inflammation and cancer. Noncoding and repeat-derived cell-free RNA transcriptome (TE) is a rich source of novel, abundant, and disease-specific RNA biomarkers. We show that repeat-derived cell-free RNAs, including simple repeat RNAs and TE RNAs transcribed from LINE, SINE, and LTR elements, are cancer-specific RNAs that are normally present at low or undetectable levels in healthy individuals. The second part explains about the role of RAS in regulating TE in the context of cancer. Here I describe about RAS effector transcripts give rise to a diverse array of non-coding RNAs that contribute significantly to cancer initiation and progression. Our study also reveals the transcriptomic and epigenomic impact of oncogenic KRAS signaling on TE RNAs and ISGs. Our study suggests that KZNF repression by mutant KRAS signaling leads to de-repression of TE RNAs, triggering an intrinsic ISG response. This model is supported by broad and significant downregulation of these same KNZFs in mutant KRAS-driven lung adenocarcinomas in vivo. We also identified mutant KRAS(G12C)-specific exRNA signatures secreted from LUAD cells by comprehensively analyzing the coding and noncoding RNAs secreted in extracellular vesicles (EV). The final section addresses the regulation of transposable elements (TEs) by KRAS during the pluripotency and differentiation of human induced pluripotent stem cells (hiPSCs). My resear

This dissertation investigates the multifaceted roles of transfer RNAs (tRNAs) in gene expression regulation, extending beyond their traditional roles in protein translation. Chapter I explores tRNAs in human brain development, utilizing cerebral cortical organoids and specialized tRNA sequencing to reveal dynamic expression patterns of tRNAs and tRNA-derived small RNAs (tDRs) in the early human cerebral cortex. Notably, it identifies a variety of upregulated tDRs from diverse isodecoders, with sequence-specific conservation among neural-specific groups, suggesting a pivotal role in neural development. Chapter II examines the impact of complete telomere-to-telomere (T2T) assemblies of great ape (and human) genomes on tRNA gene discovery. This has led to the identification of nearly 100 new human cytosolic-tRNA gene loci, particularly in regions of chromosome 1 associated with neural gene regulation and implicated in the neural expansion of humans and primates. The chapter also discusses the dynamic nature of tRNA loci copy number variation across tandem repeats and their roles on adjacent neural genes. Chapter III introduces two novel bioinformatics tools, tRNAgraph, and tRNAmap, designed to address the challenges of analyzing tRNA sequencing data across multiple species and experimental conditions. tRNAgraph offers multi-variate analysis, automated clustering, and classification of tRNAseq data, while tRNAmap aligns the ‘tRNAnome’ of eukaryotic species for cross-species comparison. These tools enable advanced analysis of the increasingly common tRNA sequencing data, contributing significantly to our understanding of tRNAs in neural development and evolution. This dissertation’s exploration into the complex roles of tRNAs and the innovative tools developed for their analysis not only advances our understanding of neural development and evolution but also paves the way for future genomic research to uncover the intricate mechanisms of gene regulation.

Technological and computational advances in analysis of cell size have led to new insights into mammalian size control. These new techniques have allowed high-resolution, single-cell measurement of size in large numbers of proliferating cells, which allowed testing of models of cell size control. Here, I will describe the techniques used, and put the data generated into context for the established models of size control that are currently under debate. After a comprehensive study of the literature these techniques support an adder model of growth control in mammalian cells, in which there is a constant amount of growth generated per cell cycle, regardless of starting cell size.

Nucleus-forming bacteriophages are a biologically unique and functionally fascinating clade of viruses. These recently characterized phages orchestrate massive and complex changes in their host bacteria by remodeling the cell wall, constructing a nucleus-like replication compartment, and using tubulin-based filaments to organize and facilitate the efficiency of the infection. Nucleus-forming phages are able to accomplish this level of organization in part due to the large sizes of their genomes, which are mostly comprised of genes of unknown function. Here I characterize divergent nucleus-forming phages in order to understand the mechanisms they share. From a phylogenetic perspective, I show that all phages that encode the major nuclear shell protein form one monophyletic clade: the chimalliviruses. Coupled with cell biology studies to characterize the replication cycles of divergent nucleus-forming phages in vivo, I propose that all chimalliviruses share the nucleus-based phage replication strategy, but that subgroups within this family each have their own variations on this theme.

Proper cell division orientation is an important component to plant and animal growth and development. In contrast to animal cells which divide through the constriction of the contractile ring, plant cells divide through the expansion of a microtubule and actin structure, the phragmoplast, to a division site specified at the cell cortex. Plant cells are encased in a semi rigid cell wall and do not rotate or migrate, thus coordination of division site structures with the division site is very important. In Chapter 1, I discuss temporal, genetic, and synthetic redundancies that ensure plant cell division planes are positioned correctly. In Chapter 2, I examine and characterize the role of cortical telophase microtubules in positioning the phragmoplast through interactions with the division site protein TANGLED1 (TAN1). In Chapter 3, I demonstrate that TAN1 is also recruited independently of the preprophase band by the phragmoplast. Finally, in Chapter 4, I use bioinformatics and whole genome resequencing to identify new molecular players important to division plane orientation or cytokinesis in maize. Altogether these chapters provide new perspectives into the function of TAN1 in maize.

Stem cell biology has the potential to solve many problems related to disease modeling, and personalized regenerative medicine. Experimental research involving stem cells is often quantified via non-invasive microscopy imaging to observe morphological behavior and determine the underlying mechanisms of observed patterns. The image data collected from these experiments are very large and require extensive by-hand analysis by a skilled biologist to evaluate experimental outcomes. To this end, computer vision programs aimed at feature extraction and classification have become an indispensable tool to accelerate and standardize the analytical pipeline. Furthermore, deep learning has automated these programs to improve the accuracy and reliability of the results obtained from analysis. There remain limitations imposed on deep learning that are a consequence of the unique circumstances under which the data are collected. For example, class imbalances, lack of data, ground-truth annotation, contiguous class boundaries, and multi-label images, to name a few. The goal of this thesis is to overcome these limitations through the novel use of biological features which inform and guide model design and implementation. The major works comprising this dissertation involve deep neural network-based methods for stem cell microscopy image classification. Specifically, these methods address complex biological dataset problems including contrastive learning for improving discrimination of overlapping features, unsupervised generative adversarial networks for supplementation of limited datasets, and semi-supervised, pseudo-labeling to overcome costly manual annotations. Paramount to the success of these projects is the use of domain knowledge in the form of biological relationships between image classes, temporal and dynamic features, and microscopy scale-space, which are exploited to improve classification performance and are shown to be indispensable in designing efficient and effecti

Bone is one of the most complex tissues due to the numerous embryonic origins it is derived from to its ossification processes. The transcriptional network that regulates bone development has been extensively studied. However, the insight pertaining to microRNAs and osteogenesis is either limited or seen at later stages of development. MicroRNAs are small non-coding RNAs that are involved in RNA silencing and post-transcriptional regulation of gene expression. These negative epigenetic modulators bind to the 3’Untranslated Region of their target mRNAs to initiate mRNA degradation or translational inhibition. They have been linked to numerous biological processes ranging from maintenance of pluripotency in embryonic stem cells, to differentiation, and later stages of osteogenesis. We have identified a novel microRNA, miR361, that impacts early osteoprogenitors and promotes osteogenesis. MiR361 accomplishes this role by directly targeting negative modulators of non-canonical Wnt, Planar Cell Polarity (PCP) pathway, Prickle 1, and Prickle 2. PCP signaling, known for establishing polarity and directing cell locomotion, is a crucial aspect of proper bone development, as mutations have led to severe skeletal deformities and disorders such as Robinow Syndrome. Studies on both Prickles are typically centered around their responsibilities in neurogenesis. Recently, research has shown that Prickle 1 plays a necessary role in bone development and is a novel marker for Robinow Syndrome. Furthermore, Prickle 1 has been shown to act as a nuclear translocator for Re1-silencing transcription factor (Rest). Recently, Rest has been linked to osteoblast differentiation. Prickle 2’s osteogenic function has not been established. Therefore, the objective of this dissertation is to (i) determine the role of miR361 during early osteogenesis, (ii) delineate the individual functions of Prickle 1 and Prickle 2 in bone development. This will provide the first evidence for Prickle 2. (iii) Unco

Over the last few decades, the development and application of advanced fluorescent probes have revolutionized our ability to assess complex biological processes visually and quantitatively with unprecedented specificity. Employing these probes alongside sophisticated microscopy techniques, this dissertation explores the development and application of diverse technologies within biological research to enhance our understanding of RNA biology, drug interactions, and cellular mechanisms.The use of fluorescent probes in real-time metabolic labeling of RNA, both in cellulo and in vivo, is explored. This approach provides critical insights into RNA dynamics, enabling the precise measurement of RNA expression patterns that are pivotal for understanding cellular function in health and disease.The development of a fluorescent reverse-transcription assay for detecting covalent interactions between small molecules and RNA is detailed. This method offers valuable perspectives on RNA modifications and their potential implications in therapeutic development, particularly through the identification of small molecule binders that could lead to innovative treatment strategies. Novel photocrosslinking fluorescent probes were developed for a new spatial transcriptomics technology employing spatially restricted cell tagging by confocal microscopy and cellular isolation with fluorescence-activated cell sorting. This technique allows for high- resolution mapping of gene expression within specific cellular contexts, thus deepening our understanding of the spatial and temporal dynamics of gene activity crucial for comprehending complex biological phenomena.The exploration of drug permeability through the development of a new fluorescence cell-based assay highlights how this technology can assess the bioavailability of pharmacologically relevant molecules, particularly those that challenge the conventional boundaries of drug design. By enabling the direct measurement of compound accumulatio

Ketogenesis requires fatty acid influx from stored lipid droplets or extrahepatic tissues to hepatic mitochondria. However, whether inter-organelle contact sites can regulate this process is unknown. Recent studies have revealed a role for Calsyntenin-3b (Clstn3b), an endoplasmic reticulum-lipid droplet contact site protein, in control of lipid utilization in adipose tissue. Here we show that Clstn3b transcription is induced in liver by PPARa in settings of high lipid utilization, including fasting and ketogenic diet feeding. Hepatocyte-specific loss of Clstn3b in mice inhibits ketogenesis independent of changes in PPARa activation. Conversely, hepatic overexpression of Clstn3b promotes ketogenesis in mice. Furthermore, the absence of Clstn3b alters LD- mitochondrial crosstalk, as revealed by impaired fatty acid oxidation and mitochondrial respiration, and a decreased mitochondrial integrated stress response. These findings define an important function for Clstn3b in ketogenesis and lipid utilization in hepatocytes.

The human mammary gland is a pivotal organ within the human anatomy, serving as a paramount role in sustaining progeny through the synthesis of milk for nourishment during infancy and early childhood. However, this lactation system of the human mammary gland remains inadequately explored due to the challenge of replicating this process in vitro. Although several studies have utilized the human mammary gland for breast cancer research, the lactation function of the human mammary gland is understudied. Additionally, existing lactation studies predominantly rely on other cell lines such as mouse or bovine sources. The rise of developing lab-engineered meat and fish caused by up-and-coming food industry companies caused quite a stir in the cellular agriculture industry. At around the same time, the baby formula shortage revealed a need for an alternative source of breast milk, akin to the recent achievements in the cellular agriculture industry. Based on this background, my thesis aims to address these needs by using the human mammary cell line MCF10A, along with our novel 3D bioprinting system and developmental stage-specific culture media, to recapitulate the physiological environment of the human mammary gland in vivo. To this end, we confirm that our bioprinting setup consistently produces long, connected mammary mini-tissues from MCF10A which express differentiated luminal markers EpCAM and MUC-1, with evidence of milk proteins detected. These MCF10A mini-tissues also exhibit an appearance not documented before; cell swelling and cell transparency reveal the surface monolayer and interior tubular structures at around 25 days of culture.

Generally, the biology field has treated cell signaling as if it operates in steady-state conditions, collecting data in bulk measurement assays or at single time points. However, this assumption is now being challenged with the advancement of live-cell imaging tools. Many cellular processes are highly dynamic, for example, the cell cycle, or heterogeneous, like cell signaling. These differences in individual cells play a role in cell fate decisions and behavior, such as proliferation, differentiation, or immune response. Cell signaling is also complex in its spatial organization, whereby information diffuses throughout a monolayer and creates an intricate paracrine network influencing gene expression throughout the tissue. Recently cell signaling dynamics have been shown to drive cellular behavior, yet often a stimulus activates more than a single pathway. This raises the question of which information is most critical in deciding cell fate. In the first part of the dissertation, I explore the current state of live-cell biosensors available, the mechanisms behind signaling dynamics, and how temporal information codes cellular outcomes. In the second section, I specifically focus on one of the most well-studied dynamic systems, the MAPK-ERK pathway. In the second part of the dissertation, I expand my study to include other biosensors including NF-kB, AMPK, and JNK as well. I developed a model of lung epithelium that expresses three live-cell reporter constructs simultaneously to measure their role in determining cytokine production, specifically of interleukin-8 (IL-8). I show that multiple signaling pathways are responsible for regulating IL-8 in a time-dependent manner. This study indicates multifactorial regulation of the cellular immune response.

Despite advances in cancer treatment, relapse remains a critical factor in the overall mortality of patients. Relapse is often driven by a rare population of cells within the tumor that have the ability to evade treatment, self-renew, and repopulate a tumor. The initiating cell in leukemia is often a normal hematopoietic stem cell that accumulates mutations over time, causing aberrant function. These cells, often referred to as cancer stem cells, co-opt stem cell programs. These cancer stem cells maintain their ability to interact with and manipulate the stem cell niches within the bone marrow microenvironment to further propagate disease. To understand the programs that are necessary for successful leukemia engraftment and progression, the Reya lab conducted an in vivo CRISPR screen. From the dropouts of this screen, we targeted GFER. GFER, a secreted protein, could potentially influence the surrounding niche to make it more permissive to the cancer stem cell. We found that the inhibition of GFER led to mislocalization within the bone marrow as well as a decrease in leukemia blasts in the bone marrow and spleen. This could be evidence that the cancer stem cell relies on the niche and, without it, they are left vulnerable. To further target the cancer stem cell and its relationship with the niche, we developed a monoclonal blocking antibody that binds to Tspan3. This anti-Tspan3 antibody reduced colony forming ability of myeloid leukemia patient samples, both in vitro and in vivo. We found that inhibition of Tspan3, by either blocking antibody or shRNA, led to alterations in stem programs that could push the cancer stem cell to differentiate. This would leave the cancer stem cell more susceptible to traditional cancer treatments. Indeed, we found that the anti-Tspan3 antibody synergized with imatinib, a front-line tyrosine kinase inhibitor for the treatment of CML. To further develop the antibody, we used humanized variants to test against myeloid leukemia cells. Th

La ricerca verte sulla comprensione del ruolo del ferro e della ferroptosi in diversi contesti patologici. La ferroptosi è una forma di morte cellulare non apoptotica, ferro-dipendente, causata dai ROS. Ci siamo focalizzati sulla leucemia linfoblastica acuta di tipo B (LLA-B), uno dei tumori ematologici più frequenti nei bambini. Per avere una prima idea sul ruolo del ferro LLA-B, si è valutata l'espressione di geni ferro in campioni di midollo osseo. Si è lavorato principalmente su modelli cellulari di LLA-B, i quali rappresentano diversi sottotipi di LLA. Le cellule sono esposte a concentrazioni crescenti di ferro: a dosi basse il ferro promuove la proliferazione cellulare, a livelli elevati si dimostra citotossico. È interessante notare che solamente una delle quattro linee cellulari mostra una risposta favorevole ad alte concentrazioni di ferro. Questa linea cellulare, RS4;11, è caratterizzata dalla traslocazione t(4;11), tipica della leucemia a linea cellulare mista (MLL). MLL è associata a un'insorgenza improvvisa, è molto aggressiva e a una prognosi sfavorevole. Utilizzando diversi approcci sperimentali, si sono studiate le risposte cellulari post-somministrazione di ferro e RSL3, induttore di ferroptosi. I risultati dimostrano che le cellule dipendono fortemente dal ferro per la loro crescita. Inoltre, si è dimostrato che RSL3 innesca perossidazione lipidica, produzione di ROS e morte cellulare. Tutti questi processi vengono bloccati in presenza di Liproxstatin-1, un inibitore di ferroptosi. Confermato che i modelli in vitro mostrano risposte diverse all'induzione della ferroptosi, si è ipotizzato che questa diversa sensibilità possa essere attribuita all'attivazione di diversi set di geni che conferiscono resistenza all'induzione della ferroptosi. La comprensione del ruolo del metabolismo del ferro e dell'impatto della ferroptosi in varie patologie rappresenta il fulcro del progetto di dottorato. Studiando il metabolismo del ferro nel LLA-B, il g, The research was focused on understanding the role of iron and ferroptosis in different pathological contexts. Ferroptosis is a form of non-apoptotic, iron-dependent cell death caused by lipid-based reactive oxygen species (ROS). Firstly, we focused on B-acute lymphoblastic leukaemia (B-ALL), the most diagnosed cancer in children. To obtain an initial understanding of iron's role in B-ALL, we assessed the expression of iron-related genes in patients’ bone marrow samples. Then, we worked on four B-ALL in vitro models, which represented different B-ALL subtypes. We exposed cells to varying iron concentrations, observing that lower doses promoted cell proliferation, while higher levels proved cytotoxic. Interestingly, one (RS4;11) out of the four cell lines exhibited a favourable response to high iron concentrations. RS4;11 is characterized by translocation t(4;11), which is typical of MLL (Mixed-Lineage Leukaemia) – rearranged leukaemia. MLL-r leukaemia is associated with a sudden onset, aggressive progression, and notoriously poor. Using different experimental approaches, we studied cellular responses in two MLL cell lines after administrations of iron and RSL3, ferroptosis inducer. Our results demonstrated that cell lines strongly depend on iron for growth and expansion. Moreover, we showed that the RSL3 triggers lipid peroxidation, production of ROS and cell death. All these processes are arrested in the presence of Liproxstatin-1, a small-molecule inhibitor that prevents lipid peroxidation. Moreover, we confirmed that in vitro MLL models exhibited distinct responses to ferroptosis induction. We postulated that this varying sensitivity might be attributed to the activation of different gene sets capable of conferring resistance to ferroptosis. Understanding the role of iron metabolism and the impact of ferroptosis in various diseases settings represented the core of the PhD project. While studying iron metabolism in B-ALL, Ceruloplasmin protein resulted as one of t

The rhomboid protein superfamily has been implicated in many important cellular functions including growth factor signaling, protein quality control, mitochondrial homeostasis, and parasite invasion. There are two primary subclasses of the rhomboid superfamily: rhomboid proteases, intramembrane serine proteases that cleave misfolded membrane proteins, and rhomboid pseudoproteases, catalytically inactive proteases. The disposal of misfolded proteins is essential as the accumulation of misfolded proteins causes aggregation, which can become precursors for many neurodegenerative diseases and cancer. Two protein quality control pathways associated with the rhomboid proteins help alleviate this cellular stress, 1) Endoplasmic Reticulum Associated Degradation (ERAD) degrades misfolded proteins while, 2) chaperones reconfigure the folding of misfolded proteins. Dfm1, a yeast rhomboid pseudoprotease, is essential in the degradation of misfolded membrane proteins through ERAD in yeast and regulates sphingolipid homeostasis. Additionally, RHBDL4, a mammalian rhomboid protease, is implicated in breast cancer progression with increased RHBDL4 activity resulting in decreased apoptosis and increased proliferation of breast cancer cells. This thesis describes our original research in further characterizing the function of the rhomboid proteins Dfm1 and RHBDL4. Dfm1, a yeast rhomboid pseudoprotease, is essential in the degradation of misfolded membrane proteins through ERAD in yeast. We establish that Dfm1 has a chaperone-like activity independent from Dfm1’s retrotranslocation role in ERAD. Additionally, we establish another independent and novel role of Dfm1 in the sphingolipid biosynthesis pathway. Here, Dfm1 facilitates sphingolipid homeostasis by exporting phosphorylated Orm2 from the ER which results in the degradation of Orm2 through Endosome and Golgi-Associated Degradation (EGAD). We have also begun to establish a novel in vivo split nano-luciferase assay to interrogate th

Transport proteins are crucial to essential cellular processes. Databases such as the Transporter Classification Database (TCDB) hold a wealth of hypothetical transport proteins in need of adequate characterization. However, there are significant challenges to transport protein experimental research in vitro and in vivo due to the physico-chemical properties of transport proteins linked to their membrane-bound nature. Instead, bioinformatic methods may be utilized to elucidate structural features, which facilitate the identification of homology between families. If one of the families is well characterized, it may provide clues to the potential functions and mechanisms of action of the poorly understood family.In this project, potential homologous relationships were explored between the well-characterized Major Facilitator Superfamily (MFS) and the uncharacterized Sar TMS10 Membrane Protein (S10MP) Family. To infer homology between the families, we searched for seven types of bioinformatic evidence: 1) sequence similarity, 2) topology and hydropathy profile compatibility, 3) shared domains, 4) conserved motifs, 5) similarity of hidden Markov model-profiles at the family level, 6) common 3D structural folds, and 7) a superfamily tree supporting the proposed relationships. Our results showed that one established MFS family, The Anion: Cation Symporter (ACS) Family, generated reliable signals of homology with the S10MP family. Therefore, we concluded that the S10MP family is a distant member of the MFS. Characteristics of the MFS proteins may extend to S10MP members, warranting further research.

Due to the ongoing rise in atmospheric carbon dioxide (CO2) concentrations and other downstream effects of climate change, global crop agricultural yields are projected to be severely impacted. To combat this issue, a clear understanding of the molecular mechanisms by which plants are able to respond to increasing CO2 levels is imperative. This study serves to characterize and investigate the roles of several proteins suspected to function in CO2-induced signal transduction in Arabidopsis thaliana and Solanum lycopersicum guard cells. This research was conducted through the construction and screening of multiple mutant plant lines as well as analysis of their stomatal gas exchange responses to imposed shifts in [CO2]. Previous research has revealed the importance of the MAP kinase MPK12 in its role as an inhibitor of the Raf-like kinase HT1, a principal negative regulator of high CO2-induced stomatal closure. However, the mechanism by which this inhibitory interaction occurs is not yet confirmed. Results obtained from this study support previous findings suggesting that the phosphorylation activity of MPK12 is not required for its function in CO2 signaling through gas exchange analysis of a kinase inactive isoform. Additionally, another protein named AtMRK1 is suspected to function in guard cell CO2 signaling in tandem with two previously identified MAPKKK proteins of the same clade, named CBC1 and CBC2. Thus, a cbc1/cbc2/atmrk1 allelic triple mutant for all three genes was developed and screened for in A. thaliana to potentially observe whether AtMRK1 partially complements the activity of CBC1 and CBC2 in future analyses. Lastly, preliminary gas exchange experiments were pursued on mutant alleles of the SlROP9 protein in S. lycopersicum. The preliminary data obtained here suggest that rop9 mutants experience constitutively reduced levels of stomatal conductance with no impairment to CO2 signal transduction, and thus are likely to exhibit increased water use efficie

This study focused on understanding the subcellular distribution of the oncogenic TRK-T3 fusion protein. TRK-T3 results from a chromosomal translocation event, where the gene encoding for TFG is spliced onto the NTRK1 locus. The resulting TFG-NTRK1 fusion induces growth factor signaling and cell transformation. To investigate the molecular mechanism and how this occurs, recombinant TRK-T3-SNAP constructs were cloned. The subcellular distribution of the fusion protein was investigated by transfection and overexpression of TRK-T3-SNAP at varying levels, and robustly resulted in the formation of nanoscale foci, reminiscent of protein aggregates. Live-cell microscopy revealed that these foci are not aggregates, but rather dynamic structures that resemble biomolecular condensates. Fluorescence Recovery After Photobleaching (FRAP) approaches support the fluidity of TRK-T3 foci, prompting the hypothesis that aggregation of TRK-T3 results in ligand-free activation of the Receptor Tyrosine Kinase pathway (RTK) via the close juxtaposition of kinase domains and induction of autophosphorylation reactions within the putative condensate. Finally, TRK-T3 condensates did not colocalize with endogenous markers of the early secretory pathway, suggesting that the oncoprotein may form distinct signaling compartments in the cytosol of cells expressing the fusion protein.

Filarial nematodes are human parasites that infect millions of people across the globe and cause debilitating diseases such as Elephantiasis and African River Blindness. These worms share a symbiotic relationship with Wolbachia, an obligate, alpha-proteobacteria endosymbiont, and rely on these bacteria for survival and proper embryogenesis. Taking advantage of this crucial symbiosis, efforts to identify drugs that kill the adult worm by targeting Wolbachia have proven to be promising. Here, I describe the discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination of Wolbachia in the in vivo Litomosoides sigmodontis filarial infection model. These potent quinazolines were identified by pairing a primary cell-based high-throughput imaging screen with a secondary ex vivo validation assay to rapidly quantify Wolbachia elimination in Brugia pahangi filarial ovaries. To better understand the relationship between Wolbachia and its worm host, adult Brugia pahangi were exposed to varying concentrations of common antibiotics in vitro and assessed for Wolbachia numbers in the germline tissue. Surprisingly, we found that worms treated with higher concentrations of antibiotics had higher Wolbachia titers, and antibiotics given at low concentrations reduced Wolbachia titers. This counterintuitive dose response is known as the “Eagle effect” and the presence of this effect in Wolbachia suggests a common underlying mechanism that allows diverse bacterial and fungal species to persist despite exposure to high concentrations of antimicrobial compounds. To our knowledge this is the first report of this phenomenon occurring in an intracellular endosymbiont, Wolbachia, in its filarial host. While several studies have shown that novel and FDA-approved antibiotics are efficacious at depleting the filarial nematodes of their endosymbiont, thus reducing female fecundity, it remains unclear if antibiotics can permanently deplete Wolbach

The rhythmic contraction of the heart depends upon the conductive properties of a small population of highly specialized cardiomyocytes, known as pacemaker cells. In the early embryonic heart, pacemaker cells reside in a ring-like pattern at the venous pole of the atrial chamber, in a region known as the inflow tract (IFT). While the gene regulatory networks that contribute to pacemaker cell differentiation have been previously investigated, we do not fully understand the upstream signaling landscape that defines the dimensions of the IFT. Our analyses in zebrafish suggest an essential role for Bmp signaling in promoting the formation of an appropriate number of IFT cardiomyocytes. Diminished Bmp signaling activity in alk8 mutants results in a substantial reduction of the number of IFT cells. Oppositely, heightened levels of Bmp signaling in chordin mutants leads to an increased number of IFT cells. In both alk8 and chordin mutants, altered numbers of IFT cells are evident within the primitive heart tube, indicating that Bmp signaling impacts IFT development prior to heart tube formation. Indeed, treatment with DMH1, a pharmacological antagonist of Bmp signaling, during gastrulation results in a significant reduction of IFT cell number, but treatment with DMH1 during early somitogenesis or during heart tube formation does not alter the number of IFT cells. Intriguingly, pharmacological inhibition of Wnt signaling during IFT cell differentiation suppresses the enlargement of the IFT in chordin mutants, implying that Wnt signaling acts downstream of Bmp signaling during IFT development. Together, these data suggest a model in which Bmp signaling acts during IFT progenitor specification, upstream of Wnt-directed IFT differentiation, to produce an appropriate number of IFT cardiomyocytes.

Base editors are an innovative addition to the genome editing toolbox that introduced a new genome editing strategy to the field. Instead of using double-stranded DNA breaks, base editors utilize unique types of DNA damage products featuring nucleobase mismatches with a DNA backbone nick to facilitate the efficient incorporation of single nucleotide variants into the genome of living cells. Two classes of DNA base editors current exist: cytosine deamination-derived editors and adenine deamination-derived base editors. Despite their widespread use in both basic research and therapeutic settings, the biological pathways processing base editing intermediates remain unclear. Chapter 1 provides a general introduction to base editors. Chapter 2 presents the elucidation of the genetic mechanisms governing cytosine base editing outcomes through CRISPRi screens. This work demonstrates that competition and collaboration among various DNA repair proteins from different pathways shape cytosine base editing outcomes. It provides a deeper understanding of the mechanisms of cytosine base editing and sets a foundation for understanding the genetic mechanisms of other base editors. All current base editors rely on deamination chemistry to introduce DNA damage products that serve as their mutagenic intermediates. In chapter 3, the development of a G•C to T•A base editor utilizing methylation chemistry to introduce N1-methylguanine as the mutagenic intermediate was described. This project sets a precedent for harnessing non-deamination chemistry to perform genome editing and demonstrates a novel strategy for the development of new base editors.

Organ boundaries serve several important roles in development, but the mechanisms downstream of boundary-defining factors are not well understood. In Chapter 1 of my dissertation, I show the Arabidopsis transcription factor LOB regulates leaf angle via a phototropism-dependent mechanism, reducing phototropic responses at the base of the leaf. LOB and other boundary-defining transcription factors also regulate numerous cell wall genes, implying a biophysical mechanism is an important aspect of boundary function. Given the inherent challenges of measuring cell wall mechanics in the Arabidopsis leaf-meristem boundary, I used a different system, the maize ligular region, which is highly amenable to AFM experiments. In Chapter 2, I characterized mechanical patterns during the development of the ligular region. My findings are consistent with the existence of a cell wall rigidification program in the boundary, which correlates with patterns of growth, cell division, and auxin dynamics. In Chapter 3, I measured epidermal cell geometry and cell wall stiffness over a wide area of the maize leaf. I found that the establishment of the boundary in the maize leaf was linked not just to local changes, but to broader growth and mechanical patterns outside the boundary. This suggests that coordination between the boundary and adjacent leaf zones is involved in the morphogenesis of the maize leaf. My findings further our understanding of how plant boundaries regulate growth.

Musculoskeletal injuries account for the highest percent of time away from work and affect over 100 million people in the United States [1,2]. Ligament and tendon injuries account for approximately 50% of all musculoskeletal injuries [3]. Despite the prevalence of these injuries, current tendon and ligament research is lacking, with little advancement in the treatment of tendinopathies for decades. Tendinopathy describes an injured or diseased tendon/ligament. Underneath this umbrella term, tendinitis is used to describe an injured tendon/ligament that has signs of inflammation. There are many molecular pathways which have been explored in great lengths in muscle and bone; however, the same signals in tendon/ligament have significantly less research devoted to them – including the pro-inflammatory pathways upregulated in tendinitis. In this dissertation, we characterize an engineered ligament model, determine the effects of pro-inflammatory cytokines on engineered ligaments, and explore possible interventions to treat tendinitis such as anti-inflammatories and isometric loading.The in vitro engineered ligament model used throughout this dissertation was first developed by the Baar lab group [4]. In order to further validate the use of this model, we characterized the effect of passage number on cell gene expression and ligament function, as well as the ligament development over time. Despite previous studies suggesting multiple passages of fibroblasts led to decreased gene expression of typical tenoblast markers, our results found that there was no significant change in gene expression across multiple passages. Furthermore, from passage 4 to passage 11 there was no observed change in mechanical function or matrix composition of engineered ligaments. These results validate the use of this in vitro engineered ligament model using both early and later passage cells. We then characterized the development of engineered ligaments over the course of 5 weeks. The results sh

Aberrations in signal transduction pathways are major drivers of human cancers. Thefirst oncogene discovered, encoded by the Rous sarcoma virus, is a mutant form of a cellular kinase capable of activating multiple signaling pathways, transforming healthy cells into cancerous ones. The characterization of this kinase, viral-Src, and the protooncogenic endogenous kinase cellular-Src, has illuminated the mechanisms underlying the activation of this kinase. Many other oncogenes also encode hyperactive kinases that dysregulate cell signaling. In this study, I investigated the mechanism of activation of the proto-oncogenic kinase BRAF. Mutations that activate BRAF, in part by promoting the constitutive dimerization of BRAF kinase domains, are associated with human cancers. In structures of BRAF dimers bound to the scaffold protein 14-3-3, the distal carboxy-terminal tail of one kinase directly interacts with the active site of the cognate kinase. I examined the role of this element in BRAF activation, finding that extensive mutation and truncation of this segment do not compromise the activity of BRAF dimers. These modifications also do not disrupt the binding of BRAF dimers to 14-3-3. These data suggest that the conformation of the BRAF tail segment in the dimeric structure could reflect an autoinhibitory instead of an activating function. Oncogenic mutations can destabilize signaling proteins, paradoxically resulting in increased or unregulated activity in these molecules. In this work, I also developed a tool to study protein-kinase stability in live mammalian cells and investigated the effects of the HSP90 chaperone system on the stability of these kinases. I utilized this tool to study the dependence of Src- and Raf-family kinases on the HSP90 system, demonstrating that this sensor reports on destabilization induced by oncogenic mutations in these kinases. I also show that domains required for the autoinhibition of c-Src and related kinases stabilize these kinase dom