Theses and Dissertations from UMD
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Item Biomechanical regulation of T cells: The cytoskeleton at the nexus of force and function(2024) Pathni, Aashli; Upadhyaya, Arpita; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The adaptive immune response is a sophisticated and multi-pronged defense mechanism that provides specific and long-lasting protection against infections and cancer. Central to this response are T lymphocytes - immune cells that orchestrate the immune response and directly eliminate infected or malignant cells. T cell function is intricately linked to their cytoskeleton, a dynamic network of protein filaments, consisting of actin, microtubules, and intermediate filaments, which provides structure, facilitates movement, and regulates intracellular transport. While the biochemical aspects of T cell function have been well-studied, recent advances have highlighted how mechanical forces influence T cell behaviors such as activation, migration, and effector functions—all processes driven by dynamic cytoskeletal remodeling. However, the mechanisms by which cytoskeletal dynamics, forces and mechanical stimuli drive T cell function remain poorly understood. This dissertation investigates this interplay, focusing on cytotoxic T lymphocytes (CTLs), a subtype of T cells that directly kill infected or cancerous cells. To launch a killing response, naïve CD8+ T cells must be activated by antigen-presenting cells (APCs) in lymph nodes, following which they proliferate and differentiate into an effector CTL population. CTLs eliminate targets via a specialized interface called the immunological synapse (IS), where they release lytic granules containing cytotoxic molecules and exert cytoskeletal forces to induce target cell death. A key event in IS formation is polarization of the centrosome, or the microtubule-organizing center, facilitating directional release of lytic granules. We first examined how biochemical signals provided by APCs modulate the cellular cytoskeleton. APCs provide not only antigenic stimulation, but also co-stimulatory signals required for full activation. Inflammatory cytokines such as interleukin-12 (IL-12) act as a third signal, enhancing CTL proliferation and cytotoxicity. Our findings demonstrate that CTLs activated in the presence of IL-12 exhibit enhanced IS formation, altered actin dynamics and microtubule growth, and generate greater mechanical forces, thus highlighting how activation signals can shape T cell mechanics, dynamics and function. Next, we investigated how the mechanical properties of target cells influence CTL function. Employing a biomimetic hydrogel system that mimics the stiffness of target cells, we demonstrate that substrate stiffness modulates multiple aspects of CTL responses. CTLs interacting with stiffer substrates exhibit enhanced spreading, accelerated actin ring formation, increased contractile forces, and more efficient centrosome polarization. Mechanical cues also influence lytic granule release and the nuclear translocation of mechanosensitive transcription factors. This work underscores the importance of mechanical cues in regulating immune responses. Given that coordinated cytoskeletal interactions are crucial for T cells to effectively respond to environmental cues, we further examined this crosstalk with a focus on intermediate filaments, the third, often understudied component of the cytoskeleton. Our characterization of the vimentin intermediate filament network reveals an expansive structure complementary to and dependent on other cytoskeletal components. We study the dynamics and organization of the vimentin network and find a close association of this network with the centrosome. Our results suggest a structural role for vimentin in supporting IS formation. Throughout this work, we use advanced imaging techniques and analysis approaches to probe various facets of T cell function. By bridging immunology, cell biology, and biophysics, this research contributes to our understanding of how physical forces shape immune responses.Item Roles of Female Sex Hormones in Regulating Neisseria gonorrhoeae Colonization of the Human Cervix(2024) Di Benigno, Sofia; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) is a human-exclusive pathogen that infects the genital tract. Gonococcal infection may present with or without symptoms and can lead to a variety of serious sequelae if left untreated, especially in female patients. Despite this, there are few models that can effectively mimic GC infection in the female reproductive tract (FRT); of these, even fewer consider the impact of the menstrual cycle, an important feature of the FRT, on GC infection. I used the human cervical tissue explant model previously developed in our lab, which can recapitulate GC infection in vivo. Tissue explants were treated with the sex hormones estradiol and progesterone to mimic various stages of the menstrual cycle and examine its impact on GC infectivity. Estradiol was used to mimic the late proliferative phase, and a combination of estradiol and progesterone was used to mimic the middle of the secretory phase. The effects of hormones on GC infectivity were examined after 72 total hours of hormone treatment and 24 hours of inoculation with GC of strain MS11. My results show that treatment with estradiol and with a combination of estradiol and progesterone both increase the level of GC colonization on the endocervix, but not on the ectocervix, compared to controls that were not treated with hormones. However, the hormone treatment did not affect GC penetration of the cervical epithelium. Both hormone treatments increased the number of GC colonies on the endocervical epithelium, and a combination of estradiol and progesterone produced an additional population of large GC colonies, leading to an increase in the average colony size. These increases in colony number and size were not associated with an increase in the expression of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which are the host receptors for GC Opa proteins. In contrast, treatment with estradiol induced a redistribution of CEACAMs from the luminal surface to the inside of epithelial cells. Additionally, estradiol altered the morphology of endocervical epithelial cells from columnar to cuboidal, but the integrity of cell-cell junctions was unchanged. The increase in colonization under high estradiol conditions was correlated with a decrease in levels of certain pro-inflammatory cytokines and chemokines, but this decrease was not sufficient to fully explain the increase in colonization. Next, I investigated the impact of cervical mucus on GC infectivity and interactions, as gel-forming mucin MUC5B but not MUC5AC increases with estradiol at the proliferation phase. Under both hormone treatment conditions, GC were able to establish close interaction with the luminal surface of the endocervical epithelial cells, displacing membrane-spanning mucin MUC1 in the membrane. Furthermore, GC were able to diffuse through an artificial mucin hydrogel and diffused more efficiently through a MUC5AC-dominant than a MUC5B-dominant hydrogel. Gel-forming mucins collected from cervical tissue explants enhanced GC aggregation in vitro, even at very low concentrations. However, mucins collected from estradiol-treated tissues showed less impact on GC aggregation than those collected from untreated tissues or tissues treated with both estradiol and progesterone. MUC5B and MUC5AC purified from cows and pigs also increase GC aggregation in vitro with GC aggregating more in a MUC5AC- than a MUC5B-dominant mucin mixture. Taken together, my research reveals for the first time that female sex hormones regulate GC colonization at the human cervix by changing the composition of the cervical mucus, providing a mechanism of hormonal regulation underlying the varying susceptibility of female patients to mucosal GC.Item INHIBITION OF TYPE ONE INTERFERON SIGNALING THROUGH CROSSTALK WITH TOLL-LIKE RECEPTOR SIGNALING(2024) Shuster, Michael; Briken, Volker V; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Interferons (IFNs) are a class of cytokines that play a prominent role in host immunity. Type I IFN is broadly associated with antiviral immunity and susceptibility to bacterial pathogens, but others have shown that type I IFN can be beneficial in some bacterial infections. Additionally, some bacterial infections such as Mycobacterium tuberculosis and Legionella pneumophila can inhibit type I IFN signaling. Questions remain such as how these bacteria inhibit type I IFN signaling as well as if other bacterial pathogens, such as Salmonella enterica, can also inhibit type I IFN signaling. Additionally, type III IFN is a relatively new class of IFN, providing antiviral protection similar to and at times redundant to type I IFN. There are some important non-redundant differences from type I IFN though, such as type III IFN’s broader activity at epithelial surfaces (like those in the lungs) and its reduced proinflammatory effects. The role of type III IFN in bacterial infections as well if bacteria can inhibit this signaling pathway remains poorly understood.Here, we examined if Salmonella enterica can inhibit type I IFN signaling, the specificities of the previously observed inhibition with Mtb infection, and how these bacterial infections are inhibiting this signaling. We demonstrate that Salmonella Typhimurium infection inhibits type I IFN signaling through crosstalk with TLR4 signaling. We establish that TLR4 signaling results in reduced surface level type I IFN receptor, which dampens cellular responsiveness to type I IFN. We show that Mtb does not inhibit type III IFN signaling and that it inhibits type I IFN signaling independently of virulence, specifically EsxA and ESX-5. Additionally, this inhibition of type I IFN signaling seems specific to mouse cells as Mtb-infected human macrophages and dendritic cells did not have inhibited type I IFN signaling. We observed that other TLR signaling pathways result in specifically inhibited type I IFN signaling. Synthesizing a model from our results, there appears to be a mouse-specific crosstalk pathway between TLR signaling and type I IFN signaling, resulting in dampened responsiveness to type I IFN through downregulation of cell surface type I IFN receptor.Item Characterization of the GBF1-Arf1 axis in enterovirus RNA replication(2024) Gabaglio Velazquez, Samuel Maria; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Enterovirus genus includes many known and emerging pathogens, such as poliovirus, enteroviruses A71 and D68, rhinoviruses, and others. Enterovirus infection induces the massive remodeling of intracellular membranes and the development of specialized domains harboring viral replication complexes, called replication organelles. The cellular protein Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) is essential for the replication of enteroviruses, but its molecular role in the replication process is unclear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including the functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and other enteroviruses directly interact with and recruits GBF1 to the replication organelles. Moreover, enterovirus infection induces the massive recruitment of all isoforms of the small cellular Arf GTPases to the replication organelles, but the mechanistic role of these proteins in the replication process is not understood either. Here, we sought to characterize the role of the GBF1-Arf1 axis in enterovirus replication. First, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrated that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results showed that the Arf-activating property, but not the primary structure of the Sec7 catalytic domain is essential for viral replication. They also suggest a redundant mechanism for recruiting GBF1 to the replication sites. This mechanism depends not only on the direct interaction of the protein with the viral protein 3A but also on elements located in the noncatalytic C-terminal domains of GBF1. Next, we investigated the distribution of viral proteins and Arf1 on the replication organelles and their biochemical environment. Pulse-labeling of viral RNA with 5-ethynyl uridine showed that active RNA replication is associated with Arf1-enriched membranes. We observed that Arf1 forms isolated microdomains in the replication organelles and that viral antigens are localized in both Arf1-depleted and Arf1-enriched microdomains. We investigated the viral protein composition of the Arf1-enriched membranes using peroxidase-based proximity biotinylation. Viral protein biotinylation was detected as early as 3 h.p.i., and the non-cleaved fragments of the viral polyprotein were overrepresented in the Arf1-enriched domains. Furthermore, we show that after 4 h.p.i. viral proteins could be efficiently biotinylated only upon digitonin permeabilization of the replication organelle membranes, while such permeabilization inhibited the Arf1 biotinylation signal at the Golgi in non-infected cells. Together, these data support a model that recruitment of GBF1 to the replication organelles generates foci of activated Arfs on the membranes, which further differentiate into specific microdomains through the recruitment of a specific complex of viral proteins and cellular Arf effectors likely needed to establish the lipid and protein composition required for viral replication.Item COORDINATED TRAFFICKING OF HEME TRANSPORTERS BY CARGO SORTING COMPLEXES IS ESSENTIAL FOR ORGANISMAL HEME HOMEOSTASIS(2025) Dutt, Sohini; Hamza, Iqbal IH; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme, an iron-containing organic ring, is a vital cofactor responsible for diverse biologicalfunctions and is the major source of bioavailable iron in the human diet. As a hydrophobic and cytotoxic cofactor, heme must be transported in a highly controlled manner through membranes via specific intra- and inter-cellular pathways. However, the genes and pathways responsible for heme trafficking remain poorly understood. Unlike other metazoans, Caenorhabditis elegans cannot synthesize heme but requires heme for sustenance. Thus, C. elegans is an ideal animal model to identify heme trafficking pathways as it permits organismal heme homeostasis to be directly manipulated by controlling environmental heme. Heme is imported apically into the intestine by HRG-1-related permeases and exported basolaterally by MRP-5/ABCC5 to extra- intestinal tissues. Loss of mrp-5 causes embryonic lethality that can be suppressed by dietary heme supplementation raising the possibility that MRP-5-independent heme export pathways must exist. Here we show, by performing a forward genetic screen in mrp-5 null mutants, that loss of the vesicular cargo sorting Adaptor Protein complexes (AP-3) fully rescues mrp-5 lethality and restores heme homeostasis. Remarkably, intestinal heme accumulation due to mrp-5-deficiency causes a concomitant deficit in the lysosomal heme importer HRG-1 abundance and localization. Loss of both MRP-5 and AP-3 subunits resurrects HRG-1 levels and localization, thus underscoring the crucial role of HRG-1 in dictating mrp-5 mutant phenotypes. In the absence of MRP-5, heme is exported by SLC49A3 homolog, a previously uncharacterized transporter. Live- cell imaging reveals vesicular coalescence that facilitates heme transfer between the importers and exporters at the interface of lysosomal-related organelle. These results define a mechanistic model for metazoan heme trafficking and identifies SLC49A3 as a promising candidate for heme export in mammals.Item INVESTIGATION OF A NOVEL O-GLCNAC MODIFICATION OF A VACCINIA VIRUS CORE PROTEIN(2024) Zhang, Yunliang; Scull, Margaret; Moss, Bernard; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Vaccinia virus (VACV) is a large, complex, enveloped virus that is the prototypic member of the genus Orthopoxvirus of the Poxviridae family and is well known as the live-virus vaccine that eradicated smallpox. It has a linear, double-stranded DNA genome of approximately 190 kbp that encodes about 200 proteins some of which undergo various post-translational modifications. These modifications are crucial for regulating protein function and influencing the virus behavior within the vertebrate and insect cells. Among these, O-GlcNAcylation is notable for its reversible modulation of protein function, like phosphorylation. Although over 5,000 human proteins have been documented as O-GlcNAcylated, the prevalence and function of this modification in viral proteins remain underexplored.Early studies from the Moss laboratory demonstrated the presence of a 40-kDa protein that contained N-acetylglucosamine in purified virions. The small size of the pronase-digestion product and the absence of other sugars suggested one or few glucosamines. The current study advances this understanding by pinpointing the novel O-linked β-N-acetylglucosamine (O-GlcNAc)-modified protein in VACV infectious particles. Enzymatic labeling of purified virions was performed using the mutant β-1,4-galactosyltransferase (GalT1 (Y289L)) to specifically transfer azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAc residues. Following copper catalyzed azide-alkyne cycloaddition (CuAAC) of biotin or an infrared dye, the candidate O-GlcNAc proteins were detected by SDS-polyacrylamide gel electrophoresis and identified by mass spectrometry (MS). Then using strain-promoted cycloaddition (SPACC) chemistry to attach a polyethylene glycol mass tag of 10 kDa to the O-GlcNAc protein, a significant shift in the electrophoretic mobility of the VACV A4 protein was documented by western blotting. The presence of O-GlcNAc in A4 was confirmed by MS and by binding to specific antibodies. Multiple modification sites were pinpointed using higher-energy collisional dissociation induced electron-transfer dissociation in MS. Further evidence linking cellular protein O-GlcNAc transferase (OGT) to the modification of A4 was derived from experiments conducted with an A4-expressing cell line. Disruption of OGT activity, either through chemical inhibition or knock-down techniques, reduced A4 O-GlcNAc modification without impairing VACV infectivity. This finding suggests that the O-GlcNAc modification of A4 does not play an essential role in VACV infectivity, which is not correlated with the A4 deletion phenotype. Therefore, the specific effects of O-GlcNAc modification on the VACV lifecycle remain elusive, indicating further studies are required to determine the potentially subtle effects of O-GlcNAcylated A4 on the VACV life cycle.Item CHARACTERIZING THE ROLES AND MECHANISMS OF CYTONEMES IN ASYMMETRIC SIGNALING AND ORGANIZATIONS IN THE DROSOPHILA MUSCLE PROGENITOR NICHE.(2024) Patel, Akshay Jitendrakumar; Roy, Sougata; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tissue development and homeostasis rely on the ability of embryonic or stem cells to efficiently determine whether to multiply for self-renewal or differentiate to generate a wide range of cell types that constitute an adult body. Stem cells determine these fates in the context of a specialized microenvironment or the niche that they occupy. All stem cell niches characterized to date are known to function using two key processes - adhesive interactions and asymmetric growth factor signaling between the niche and stem cells. While adhesion to the niche maintains niche occupancy and stemness, the loss of niche adhesion and occupancy initiates stem cell differentiation. Moreover, niche cells produce secreted growth factors to support stem cell self-renewal. Despite the ability of secreted growth factors to disperse across tissues over a long range, only the niche-adhering stem cells receive the self-renewal signals. The genetically identical daughter cells that lack adhesion to the niche fail to receive self-renewal signals, even when located within one or two cell diameters away, leading to the activation of their post-mitotic fates. Therefore, understanding how asymmetric signal distribution and adhesive interactions are produced and coordinated within the niche is critical to understanding how stem cells determine their identity and prime differentiation to generate or regenerate tissues. This thesis investigated and characterized a new mechanism of asymmetric signaling and cell organization in the Drosophila Adult Muscle Progenitor (AMP) niche. By employing genetic, cell-biological, and high-resolution microscopy techniques, this work discovered that AMPs extend thin polarized actin-based filopodia, called cytonemes, by orienting toward the wing disc niche. Cytonemes play a dual role. Cytonemes help AMPs to physically adhere to the wing disc niche and also directly receive a self-renewal Fibroblast Growth Factor (FGF) through the cytoneme-niche contact sites. AMP cytonemes localize the FGF-receptor (FGFR), called Heartless (Htl), and selectively adhere to the wing disc areas that express two different Htl ligands, Pyramus and Thisbe, both mammalian FGF8 homologs. Htl on these cytonemes directly receives Pyramus and Thisbe through the cytoneme-niche contact sites. Although FGFs are long-range secreted paracrine signals and Htl is the only receptor shared by Pyramus and Thisbe, these FGFs are received and restricted only to the niche-adhering AMPs due to the contact-dependent cytoneme-mediated asymmetric delivery of the signals. Moreover, despite employing a common FGF signal transduction pathway, Thisbe- and Pyramus-signaling initiates divergence of AMP fates into two distinct muscle-specific lineages. These experiments showed that cytoneme-mediated signal communication forms the basis of asymmetric signaling and organization within the AMP niche. We next asked how AMPs determine the niche-specific polarity and affinity of cytonemes. This research discovered that FGF reception and signaling activation in AMPs are required to activate polarized cytoneme formation orienting toward the wing disc niche. Without FGF signaling, AMPs cytonemes fail to polarize and adhere to the FGF-producing niche, causing them to exit the niche and start to differentiate. Thus, while target-specific asymmetric FGF distribution relies on cytonemes, activation of FGF signaling feedback maintains the polarity and adhesion of the signaling cytonemes toward the FGF-producing niche. A consequence of this interdependent relationship between niche adhesion, polarized FGF-reception, and stimulation of FGF signaling feedback is the maintenance of the self-organized niche-specific asymmetric signaling and organization via cytonemes. We next investigated whether the niche-adhering cytonemes receive additional fate-specifying cues, particularly the mechanical cues from the niche. Recent evidence suggests a critical role of mechanical and physical cues in determining stem cell fates. This work discovered that the AMP cytonemes are enriched with a common mechano-transducer, named Talin. AMP-specific genetic manipulation of talin indicates that Talin is critical for cytoneme-mediated niche occupancy and FGF signaling. Using a Talin-based force probe expressed at the physiological levels and FLIM-FRET microscopy, we discovered that Talin experiences pN level force within the cytonemes. These findings suggest that AMPs employ cytonemes not only for receiving FGFs in a restricted polarized manner but also for a mechanosensory function. In conclusion, these results strongly suggest a critical role of cytonemes in coordinating asymmetric signaling and organization in the stem cell niche. In addition, the work provides evidence that the stem cell cytonemes are critical organelles for integrating the inputs and outputs of both growth factor signaling and mechanical cues to sculpt tissues.Item AN INTERSECTING NETWORK OF REGULATORS IS REQUIRED FOR RNA SILENCING AND NUCLEAR INTEGRITY IN C. ELEGANS(2024) Knudsen-Palmer, Daphne R; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Regulation of gene expression is required for an organism to develop, maintain homeostasis, and respond to environmental stimuli. While each cell in a multicellular organism contains the same genetic information, the epigenetic control of the expression of genes at different times is crucial for processes such as cell differentiation, division, and for allowing for cells to carry out different functions from one another. One type of such epigenetic regulation is mediated by small non-coding RNAs. Introduction of double-stranded RNA (dsRNA) and subsequent production of small interfering RNAs can result in sequence-specific mRNA silencing, creating the potential for highly specific therapeutics and pesticides. However, some targets are more easily silenced than others, and the mechanisms of silencing are not fully understood. Here we investigate regulators of small RNA-mediated silencing in the nematode C. elegans and find that they function in an intersecting network, allowing the potential for regulators to contribute to the silencing of any target. Quantitative modeling suggests that the production and turnover rates of a target at steady-state can affect the ease with which a target can be knocked down, and experimentally we found that changing the cis-regulatory sequences of a target can make it more susceptible to silencing. We found restricted production of RNA silencing intermediates, allowing for the recovery of a target in response to dsRNA, which we observed experimentally in non-dividing cells. In addition to roles in response to dsRNA, we report that disruption of small RNA-based regulation can result in germline nuclear defects. In the absence of the intrinsically disordered and perinuclear granule-forming protein MUT-16, some of the nuclei in the syncytial germline appear enlarged, suggesting that small RNA-based regulation may be playing an active role in maintaining nuclear size. Taken together, these findings suggest that (1) regulators of small RNA silencing can contribute to the silencing of all targets as part of an intersecting network, as opposed to operating in specialized pathways and (2) small RNA-based regulation is required for nuclear integrity, providing a paradigm for studying control of nuclear size, where enlarged nuclei can be compared with wildtype nuclei in a shared syncytium. We speculate that these findings will improve understanding of RNA silencing across species and provide insight into understanding how nuclear size is controlled, a fundamental ability of all eukaryotes.Item Functionalized Nanoparticles for the Controlled Modulation of Cellular Behavior(2023) Pendragon, Katherine Evelyn; Fisher, John; Delehanty, James; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The ability to control cellular behavior at the single-cell level is of great importance for gaining a nuanced understanding of cellular machinery. This dissertation focuses on the development of novel hard nanoparticle (NP) bioconjugate materials, specifically gold nanoparticles (AuNPs) and quantum dots (QDs), for the controlled modulation of cellular behavior. These hard NPs offer advantages such as small size on the order of 1 – 100 nm, high stability, unique optical properties, and the ability to load cargo on a large surface area to volume ratio, making them ideal tools for understanding and controlling cell behavior. In Aim 1, we demonstrate the use of AuNPs to manipulate cellular biological functions, specifically the modulation of membrane potential. We present the conception of anisotropic-shaped AuNPs, known as gold nanoflowers (AuNFs), which exhibit broad absorption extending into the near-infrared region of the spectrum. We demonstrate the effectiveness of utilizing the plasmonic properties AuNFs for inducing plasma membrane depolarization in rat adrenal medulla pheochromocytoma (PC-12) neuron-like cells. Importantly, this is achieved with temporal control and without negatively impacting cellular viability. Aim 2 explores the use of QDs as an optical, trackable scaffold for the multivalent display of growth factors, specifically erythropoietin (EPO), for the enhanced induction of protein expression of aquaporin-4 (AQPN-4) within human astrocytes. This results in enhanced cellular water transport within human astrocytes, a critical function in the brain's glymphatic system. We show that EPO-QD-induced augmented AQPN-4 expression does not negatively impact astrocyte viability and augments the rate of water efflux from astrocytes by approximately two-fold compared to cells treated with monomeric EPO, demonstrating the potential of EPO-NP conjugates as research tools and prospective therapeutics for modulating glymphatic system function. Overall, the body of work presented in this dissertation develops new NP tools, namely solid anisotropic AuNFs and growth factor-delivering QDs, for the understanding and control of cell function. These new functional nanomaterials pave the way for the continued development of novel NP-based tools for the precise modulation of cellular physiology.Item CHARACTERIZATION OF THE SHEDDASE-MEDIATED RELEASE OF A DROSOPHILA MEMBRANE-ANCHORED FGF THROUGH CYTONEMES(2023) Li, Yujia; Roy, Sougata; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)During tissue morphogenesis, cells must precisely coordinate their functions over distance by communicating with secreted paracrine signaling proteins, such as the fibroblast growth factors (FGFs). The spatiotemporal regulations of FGF signaling are critical for development and homeostasis in all organisms. However, the mechanisms that control the location, timing, and level of FGF secretion and ensure its target-specific delivery over distance are poorly understood. This thesis has addressed this fundamental question by using a Drosophila FGF Branchless (Bnl) during tracheal development. Bnl is produced in a small group of wing-disc cells. Bnl regulates the development of a disc-associated tracheal branch, the air-sac-primordium (ASP), a vertebrate lung bud analog. Although FGFs are secreted diffusible signals, previous work has shown that the inter-organ dispersion of Bnl from the source to the ASP is highly polarized, target-specific, and is mediated over distance via long polarized actin-based filopodia, named cytonemes. The contact-dependent cytoneme-mediated Bnl delivery is programmed by the glycosylphosphatidylinositol (GPI) anchoring of Bnl. A GPI anchor tethers Bnl to the outer leaflet of the source cell membrane and inhibits free/random Bnl secretion. Surprisingly, GPI anchoring also facilitates Bnl’s release, but only through the cytoneme contact sites, thereby activating MAPK signaling exclusively in those recipient cells that establish contact with its source. However, the molecular mechanisms that might spatiotemporally activate the release of the membrane-anchored Bnl at the cytoneme contact sites remained unknown. This work addresses this knowledge gap by using a combination of cell biological, biochemical, and genetic analyses. Visualization of the distribution of a fluorescently-tagged Bnl revealed that a proteolytic cleavage within the Bnl backbone is essential for its target-specific release. Phenotypic analyses of an uncleavable Bnl mutant generated by genome editing suggested that the proteolytic event that releases Bnl is essential for Bnl signaling and morphogenesis. An in vitro cell-culture-based screening for candidate sheddases identified five membrane-localized enzymes that specifically shed Bnl from the cell surface. These enzymes include two matrix metalloproteinases, MMP1 and MMP2, a pro-protein convertase, Furin1, and two ADAM family metalloproteases, Meltrin and Tace. An inherently disordered region within the Bnl backbone was identified as the substrate for the proteolytic cleavage. Serial mutagenesis in this region revealed four minimal zones required for enzymes to act on Bnl. Interestingly, one of the minimal regions at the juxta-membrane position harbors the Bnl cleavage site, and the other three zones apparently play regulatory roles. Further work on MMPs using mosaic gain-of-function analyses confirmed MMP-mediated Bnl shedding in vivo. However, MMPs are not expressed in the Bnl source. Consequently, Bnl is not freely released. In contrast, MMPs are expressed in the ASP and are localized on the recipient ASP cytonemes. in vitro cell culture experiments that reconstituted contact-dependent Btl-Bnl interactions between Bnl-source and recipient cells confirmed that MMPs are selectively recruited by the recipient cells to the signaling contact sites and activate Bnl shedding in trans. These results explained why and how Bnl is released only via the cytoneme contact sites. Notably, MMP1 and MMP2 are GPI-anchored proteins but are also catalytically active in secreted soluble forms. Therefore, how is Bnl prevented from non-specific shedding by the extracellular soluble MMPs? in vitro and in vivo experiments using GPI-modified and non-GPI-modified MMPs revealed that only the membrane-tethered MMPs that are retained on the cell surface could efficiently shed cell surface Bnl. These findings supported a working model suggesting that recipient ASP cells extend receptor-containing cytonemes and establish contact with the Bnl-source via the receptor-ligand interaction. Due to the GPI-anchoring, MMPs are presented on the surface of ASP cytonemes and are delivered via these cytonemes to their contact sites, leading to the contact-dependent target-specific Bnl trans-shedding. These results provide new insights into how contact-mediated signal dispersion via cytonemes can be modulated by cell surface sheddases. Additional work on Furin1 and ADAM proteases showed that Tace and Meltrin shed Bnl in a cell-non-autonomous manner, like MMPs. In contrast, Furin1 is required only in the source cells for Bnl shedding. How and under what contexts Furin1 and ADAM proteases are activated to shed Bnl needs to be explored in the future. Collectively, this work characterized a novel enzymatic Bnl shedding mechanism and provided insights into how Bnl sheddases might be controlled in space and time to ensure cytoneme-mediated Bnl exchange.