Astrocytes are vital components in neuronal circuitry and there is increasing evidence linking the dysfunction of these cells to a number of central nervous system diseases. Studying the role of these cells in human brain function in the past has been difficult due to the limited access to human brain. Here, human-induced pluripotent stem cells (hiPSCs) were differentiated into astrospheres using a hybrid plating method, with or without dual-SMAD inhibition, and assessed in astrocytic markers, calcium-transient signaling, reactive oxygen species (ROS) production, and immune response. Neural degeneration was modeled via stimulation with amyloid-β (Aβ) 42 oligomers. Finally, co-culture was performed to for the derived astrospheres with isogenic neurospheres. Results show that the derived glial cells expressed astrocyte markers, secreted extracellular matrix (ECM) and capable of responding to with Aβ42 stimulation (higher oxidative stress, higher TNF-α and IL-6 expression, upon stimulation). In addition, the ECM remodeling proteins MMP2 and MMP3 were downregulated with Aβ42 stimulation. Co-culture experiments show the enhanced synaptic activities of neurons co-culture and neural protection ability of the glial cells. This study provides additional knowledge about the roles of brain glial cells, heterotypic cell-cell interactions, and the formation of engineered neuronal synapses in vitro. The implications lie in neurological disease modeling, drug screening, studying progression of neural degeneration and the role of stem cell microenvironment. / A Thesis submitted to the Department of Chemical and Biomedical Engineering in partial fulfillment of the requirements for the degree of Master of Science. / 2019 / August 28, 2019. / astrocytes, glial cells, human pluripotent stem cells, neural degeneration, spheroids / Includes bibliographical references. / Yan Li, Professor Directing Thesis; Sam C. Grant, Committee Member; Christina Holmes, Committee Member.

The Drosophila ovary is an important model system for studying oogenesis and has provided insight into broader, biological topics such as stem cell niche, differentiation, migration, morphogenesis, signaling, cell-size regulation, and tumorigenesis. The purpose of this dissertation was to build a comprehensive single-cell transcriptomic atlas of cells regulating oogenesis and ovulation and to use the ovarian follicle cells, as a model system to interrogate the role of ectopic Notch expression in development and during tumorigenesis. Single-cell RNA sequencing analysis revealed transcriptional signatures for each of the 28 expected ovarian cell types and developmental stages. Each stage of oogenesis was then separately analyzed with a special focus on the most diverse cell type, the follicle cells. This identified key cell-type specific expression patterns governing processes like differentiation, mitotic-to-endocycle switch, migration, morphogenesis, phagocytic removal of nurse cells, eggshell formation, and a newly identified shift of the pre-corpus lutuem cell expression from oogenesis-to-ovulation. Additional characterization of the interconnected tissues in the dataset identified novel cellular heterogeneity of the oviduct and a population of hemocytes associating closely with it. We further describe how this association occurs in a non-mating-dependent manner as early as the pupal stage (during oviduct development). Additional characterization of the hemocyte cluster reveals expression of many phagocytic genes including the newly identified enzymatic marker, Cp1. Additional validation of cell-type markers classifies these macrophage-like cells as plasmatocytes, one of the most common types of hemocytes in flies. Armed with a rich dataset describing proper signaling during development we turn to the follicle cells and switch focus to study dysregulated signaling of the Notch pathway and its involvement in tumor formation. First, we expressed ectopic Notch (NICD) in follicle cells and discovered an uneven pattern of nuclear NICD retention in a cell-cycle dependent manner. We find that the cell-cycle regulator, string (cdc25), can strongly impact the localization of NICD regardless of the cell-cycle status of the cell. We also identify that Vps proteins involved in forming the ESCRT complex can similarly regulate the nuclear NICD pattern. Next, we examined the role of ectopic Notch signaling in tumorigenesis and found that while not sufficient for tumor formation, Notch functions as a tumor promoter and leads to a more dysplastic tumor phenotype. Tumor cells with ectopic NICD gain a survival advantage which may be due to a bypass of the DNA damage sensing checkpoint. RNA sequencing revealed unique expression of these tumor cells with ectopic NICD in both pre- and post-tumor conditions. Specifically, we identified DNA stability related genes, RecQ4 and Xpd, which are upregulated in NICD-overexpressing pre-tumor cells. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / 2019 / November 8, 2019. / Drosophila, hemocyte, Notch, oogenesis, ovulation, tumor / Includes bibliographical references. / Wu-Min Deng, Professor Co-Directing Dissertation; Kathryn Jones, Professor Co-Directing Dissertation; Timothy Megraw, University Representative; Kimberly Hughes, Committee Member; Steven Lenhert, Committee Member.

Nanoparticle based cellular therapies hold great promise for clinical applications and medicinal use in human patients by allowing for targeted delivery of a personalized medicine payload to specific cells and tissues in a variety of disease states. The large surface to volume ratio of solid inorganic nanoparticles and the availability of facile surface functionalization chemistries with gold, gold-coated, and semi-conductor nanoparticles enables the design of delivery agents which can simultaneously carry a targeting molecule, such as an antibody or cell penetrating peptide (CPP), a short oligonucleotide for RNA interference or intracellular sensing, and a full gene for genetic therapy to correct aberrant protein function or cause apoptosis of cancer cells. The scope of applicability for nano-therapy technology is incalculable, however current understanding of the intracellular uptake and processing of nanomaterials-based therapeutics is limited and many facets of the cellular response to nanoparticle therapy are still in need of investigation. The goal of this dissertation work has been to elucidate the effects of nanoparticle-based therapeutics to ascertain the intracellular fate and processing of model nanomedicines by designing nanoparticle-bioconjugates capable of spatiotemporally reporting live intracellular uptake and processing events using fluorescence microscopy, and magnetic detection. The first chapter gives an introduction to nanoparticles and their use in biological applications, as well as detailing how they can be used as intracellular sensors. The second chapter investigates the ability to control therapeutic DNA cargo release from a gold nanoparticle in live cells using different appendage chemistries. The third chapter probes the intracellular environment experienced by the nanotherapeutic and discusses nano-induced effects to the intracellular environment. The fourth chapter investigates nanotherapy cellular uptake targeting using cell penetrating peptides, as probed by fluorescent quantum dots, to determine outcomes for a variety of naïve and drug resistant mammalian cell lines, include human lung, skin and brain cancers as well as rodent cancer model cell lines. The fifth chapter combines optical and magnetic analysis techniques to investigate multiplexed sensing with gold shelled iron oxide nanoparticles to investigate cellular uptake. And the last chapter summarizes the work and provides a discussion of the outlook for the work. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester 2018. / July 11, 2018. / cell penetrating peptide, DNA, gold nanoparticle, nano-probe, nano-therapeutic, surface energy transfer / Includes bibliographical references. / Geoffrey F. Strouse, Professor Directing Dissertation; Cathy W. Levenson, University Representative; Joseph Schlenoff, Committee Member; Wei Yang, Committee Member.

Specific mutations in the replacement histone variant H3.3 are known to drive cancers such as glioblastomas, chondroblastomas and large cell tumors of the bone primarily in children and young adults. Several recent studies have suggested that transcriptional defects associated with H3.3 mutations are likely to be involved in tumor formation. However, transcription independent roles of H3.3 and their potential contribution to cancer have not been investigated. Here we report that histone H3.3 (but not the related replication dependent H3.1 or H3.2 variants) is recruited within ~60 seconds to sites of laser induced DNA damage. This recruitment is dependent on acetylation at the N terminus of the protein. A stable depletion of this protein in human cells leads to high levels of endogenous DNA damage and an impaired ability to repair this damage, leading to genomic instability and sensitivity to DNA damaging agents. Flies with reduced levels of H3.3 are also sensitive to damaging agents and accumulate spontaneous damage. H3.3 depletion alters the recruitment of double strand break (DSB) repair factors, with defects in the recruitment of proteins involved in homologous recombination (HR) repair and faster recruitment of some proteins involved in non-homologous end joining (NHEJ). We also find that the cancer associated mutants are defective in localizing to damage sites and their expression leads to an accumulation of high levels of endogenous damage. Overall, our data from evolutionarily distant species suggest that histone H3.3 plays a crucial role in HR-mediated DNA repair which is conserved across eukaryotes. Hence, based on the strong links between defective DNA repair and cancer, we propose that the DNA repair defects associated with H3.3 mutations are likely to contribute to genomic instability, and thereby to carcinogenesis, independent of the known transcriptional roles of histone H3.3. Based on these findings, we propose a potential therapeutic strategy that combines the use of inhibitors along with a DSB inducing agent to selectively target cells that are defective in HR repair due to H3.3 depletion or mutation. Our promising preliminary data show that this is effective in H3.3 knockdown cells and may also be useful in killing H3.3 cancer mutants. This knowledge helps us to understand the complex details of these deadly mutations and hopefully brings us closer to a treatment. / A Dissertation submitted to the Department of Biomedical Sciences in partial fulfillment of the Doctor of Philosophy. / Fall Semester 2015. / October 29, 2015. / cancer, chromatin, DNA repair, H3.3, histone / Includes bibliographical references. / Akash Gunjan, Professor Directing Dissertation; Hong-Guo Yu, University Representative; Yanchang Wang, Committee Member; Jonathan Dennis, Committee Member.

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