Cell and Developmental Biology Commons^™

Modulation Of Kras Structure And Dynamics By Kras Ubiquitination And Membrane Depolarization, Vinay Nair

Dissertations and Theses (Open Access)

KRAS, a 21 kDa small GTPase protein, functions as a molecular switch playing a key role in regulating cell proliferation. Dysregulation of KRAS signaling by oncogenic mutations leads to uncontrolled cell proliferation, a hallmark of cancer cells. Attempts to therapeutically target oncogenic KRAS have led to limited success resulting in a need to identify new mechanisms to targeting KRAS. The interaction of KRAS with its regulators, effectors, and the membrane present one such avenue. In this study, we investigated how post-translational covalent and environmental modifications could modulate these interactions of KRAS. Using computational molecular dynamics simulations, nuclear magnetic resonance spectroscopy …

Integration Of Biomedical Imaging And Translational Approaches For Management Of Head And Neck Cancer, Abdallah Mohamed, Abdallah Mohamed

Dissertations and Theses (Open Access)

The aim of the clinical component of this work was to determine whether the currently available clinical imaging tools can be integrated with radiotherapy (RT) platforms for monitoring and adaptation of radiation dose, prediction of tumor response and disease outcomes, and characterization of patterns of failure and normal tissue toxicity in head and neck cancer (HNC) patients with potentially curable tumors. In Aim 1, we showed that the currently available clinical imaging modalities can be successfully used to adapt RT dose based-on dynamic tumor response, predict oncologic disease outcomes, characterize RT-induced toxicity, and identify the patterns of disease failure. We …

Development And Characterization Of B7-H3-Targeted Radioimmunotherapies For The Treatment Of Solid Tumors, Sarah Glazer

Dissertations and Theses (Open Access)

Radioimmunotherapy involves the selective targeting of therapeutic radionuclides to cancer-associated cell surface antigens using monoclonal antibodies. Radioimmunotherapy has been successful in treating hematologic cancers, such as non-Hodgkin’s Lymphoma. However, treatment of solid tumors by radioimmunotherapy remains a challenge, driven in part by a lack of high affinity antibodies against highly tumor-selective surface antigens. Herein, we characterize novel anti-B7-H3 immuno-conjugates for PET imaging and RIT of solid tumors in preclinical models.

Human B7-H3 (CD276) protein is highly expressed on solid tumor cell surfaces and endovascular surfaces in a wide variety of cancers (colon, breast, pancreatic, prostate, head and neck, glioblastoma, ovarian, …

Plant Homeodomain Finger Protein 20 (Phf20) And Its Homolog Phf20 Like 1 (Phf20l1) Define Two Distinct Non-Specific Lethal (Nsl) Complexes, Hieu Van, Hieu T. Van

Dissertations and Theses (Open Access)

Plant Homeodomain Finger Protein 20 (PHF20) and its homolog PHF20 Like 1 (PHF20L1) are known subunits of the Non-Specific Lethal (NSL) complex, which acetylates lysine residues on histone H4 and regulates gene expression. The current model assumes that PHF20 and PHF20L1 are present together in the NSL complex, although it has never been tested. Performing extensive biochemical analysis, we observed that PHF20 and PHF20L1 were exclusively and independently associated with the NSL complex. Our protein domain analysis showed that the C-termini of PHF20 and PHF20L1 are crucial for their interactions with the respective complexes. Furthermore, enrichment sites of PHF20 and …

4d Ex Vivo Crispr/Cas9 Whole-Genome Screen To Identify Genes Regulating Lung Cancer Metastasis, Alexandria Plumer

Dissertations and Theses (Open Access)

Metastatic lung cancer has a 5-year survival rate of 5%. Lung cancers tend to be asymptomatic until late stages, and almost 90% are not diagnosed until they are advanced. Metastases are very rare events, often initiated by a single cell from a primary tumor into a new niche at a distant location. Investigation of the early metastatic process is of urgent need for the development of early diagnostics and targeted therapeutics. We performed a proof-of-concept CRISPR/Cas9 whole genome knockout screen in the A549 lung adenocarcinoma cell line and utilized a novel ex vivo 4D lung metastasis model to find gene …

Lgr5 Regulation Of Stat3 Signaling And Drug Resistance In Colorectal Cancer, Tressie Posey, Tressie Alexandra Posey

Dissertations and Theses (Open Access)

LGR5 Regulation of STAT3 Signaling and Drug Resistance in Colorectal Cancer

Tressie Alexandra Capri Posey B.S.

Advisory Professor: Kendra Carmon, Ph.D.

The greatest difficulty in treating colorectal cancer (CRC) is the development of drug resistance which leads to relapse after treatment and progression to metastasis. Cancer stem cells (CSCs) are believed to drive relapse because of their capacity to self-renew, acquire resistance mechanisms, and differentiate promoting tumor growth and heterogeneity. Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), is a bona-fide marker of CSCs and has been considered a viable target for CSC specific therapeutic development. While we showed targeting LGR5 …

Investigating Therapeutic Strategies To Target Metabolic Vulnerabilities Of Nsclc Tumors With Mutant Keap1 Gene, Pranavi Koppula

Dissertations and Theses (Open Access)

The metabolic vulnerability of cancers has long been envisaged as an attractive window to develop novel therapeutic strategies. Metabolic flexibility at the cellular level encompasses the efficient rerouting of anabolic and catabolic pathways in response to varying environmental stimuli to maintain cellular homeostasis and sustain proliferation. The primary objective of this study is to identify metabolic vulnerabilities bestowed by KEAP1/NRF2 signaling axis through SLC7A11. SLC7A11 is a transcriptional target of NRF2, an essential regulator of cellular anti-oxidant response. Under unstressed basal conditions, NRF2 interacts with KEAP1, a tumor suppressor gene and a substrate adaptor protein of the Cullin3-dependent ubiquitin ligase …

Stat3 Inhibits Type I Interferon Signaling In Type I Conventional Dendritic Cells, Taylor Chrisikos

Dissertations and Theses (Open Access)

Conventional dendritic cells (cDCs) are an essential immune population, responsible for controlling adaptive immunity and tolerance. Recently, type I cDCs (cDC1s) have been delineated as a distinct cDC subset, uniquely responsible for coordinating T cell-mediated immunity against pathogens and tumors. Although the importance of cDC1s is now well established, the mechanisms that regulate cDC1 function remain largely unknown. Signal Transducer and Activator of Transcription 3 (STAT3) mediates the intracellular signaling of interleukin 10 (IL-10), an immunosuppressive cytokine. Therefore, we hypothesized that STAT3 and IL-10 inhibit cDC1 function and induction of T cell-mediated immunity. Herein, we show that IL-10 inhibits polyinosinic:polycytidylic …

Mutant Kras Alters Extracellular Vesicle Microrna Sorting In Pancreatic Cystic Neoplasms, Rachel L. Dittmar

Dissertations and Theses (Open Access)

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers by organ site with a 5-year survival rate of just 10.8%. This is largely because most patients do not experience symptoms until the disease has already metastasized. The best hope to cure PDAC is surgery, which can only be done with a curative intent at an early stage when the disease is localized. There are no reliable circulating, body-fluid-based biomarkers to detect early stage PDAC or its precursor lesions in a timely manner for effective surgical intervention. When potential PDAC precursor lesions, such as mucinous pancreatic cysts are found, there are …

Atrx Inactivation And Idh1-R132h Drive Preferential Sensitivity To Proton Vs. X-Ray Radiotherapy In Glioma Stem Cells, Ángel Adrián Garcés

Dissertations and Theses (Open Access)

Background: Glioma Stem Cells (GSCs) are self-renewable, treatment resistant cells in the glioma tumor mass known to promote tumor development. In contrast to traditional photon-based radiation therapy (XRT), proton radiation therapy (PRT) may induce more complex DNA damage and therefore might have the potential to eliminate GSCs. Although previous studies have individually linked IDH mutations, specifically IDH1^R132H, and ATRX inactivating mutations to improved patient outcomes and suppressed DNA damage repair compared to their respective wild-types, the mechanisms by which these two genetic alterations interact in GSCs treated with PRT compared to XRT are currently unknown. We hypothesize that …

Epithelial Memory Of Resolved Inflammation Limits Tissue Damage While Promoting Pancreatic Tumorigenesis, I-Lin Ho

Dissertations and Theses (Open Access)

Inflammation is a major risk factor for pancreatic ductal adenocarcinoma. When occurring in the context of pancreatitis, mutations of KRAS accelerate tumor development. We discovered that long after its complete resolution, a transient inflammatory event primes pancreatic epithelial cells to subsequent transformation by oncogenic KRAS. Upon recovery from acute inflammation, epithelial cells of the pancreas display an enduring adaptive response associated with sustained transcriptional and epigenetic reprogramming. Such adaptation enables the prompt reactivation of acinar-to-ductal metaplasia (ADM) upon subsequent inflammatory events, thus efficiently limiting tissue damage via rapid decrease of zymogen production. We propose that since activating mutations of KRAS …

Npsd4: A New Player In Sumo-Dependent Dna Repair, Erin Atkinson

Dissertations and Theses (Open Access)

The human genome is under constant threat from sources of damage and stress. Improper resolution of DNA damage lesions can lead to mutations, oncogene activation, and genomic instability. Difficult-to-replicate-loci present barriers to DNA replication that, when not properly resolved, lead to replication fork stalling and collapse and genomic instability.

DNA damage and replication stress trigger signaling cascades potentiated by multiple types of post-translational modifications, including SUMOylation. Through proteomic analysis of proteins involved in SUMOylation following DNA damage, our lab identified an uncharacterized protein that we named New Player in SUMO-dependent DNA damage repair 4 (NPSD4). Through an additional proteomic screen, …

Targeting Plasma Membrane Phosphatidylserine Content To Inhibit Oncogenic Kras Function, Walaa E. Kattan

Dissertations and Theses (Open Access)

The small GTPase KRAS, which is frequently mutated in human cancers, must be localized to the plasma membrane (PM) for biological activity. We recently showed that the KRAS C-terminal membrane anchor exhibits exquisite lipid-binding specificity for select species of phosphatidylserine (PtdSer). We therefore investigated whether reducing PM PtdSer content is sufficient to abrogate KRAS oncogenesis. Oxysterol-related binding proteins ORP5 and ORP8 exchange PtdSer synthesized in the ER for phosphatidylinositol-4-phosphate (PI4P) synthesized in the PM. We show that depletion of ORP5 or ORP8 reduced PM PtdSer levels, resulting in extensive mislocalization of KRAS from the PM. Concordantly, ORP5 or ORP8 depletion …

Combination Of Oncolytic Adenoviruses, T-Cell Activation, And Blockade Of Ido Metabolic Circuitry For The Treatment Of Glioma, Teresa Nguyen

Dissertations and Theses (Open Access)

Glioblastoma is the most common malignant primary brain tumor in adults; the current aggressive treatment results in a 5% five-year survival rate. More effective therapies should be developed. One promising alternative is oncolytic adenovirus, Delta-24-RGD, which elicits cancer cell lysis and immunogenic cell death. In fact, Delta-24-RGD produced complete responses in 20% of recurrent glioblastoma patients through immune mechanisms that activate anti-tumor cytotoxic properties of T-cells. This cytolytic effect can further be enhanced by adding immune agonists, namely OX40L, which engages the OX40 receptor to co-stimulate activated T cells for enhanced proliferation. Hence, we produced the next generation of Delta-24-RGD, …

The Functional Analysis Of A Major Tyrosine Phosphorylation Site On Actin, Amelie Simone Cordelia Albrecht

Dissertations and Theses (Open Access)

Actin is an abundant and evolutionarily conserved protein and a key component of the cytoskeleton. Post-translational modifications of actin are emerging as an important mechanism for regulating actin functions, and may form an ‘Actin Code’. In this work, I investigate the role of actin phosphorylation at tyrosine 53 (pY53), one of the most frequently detected actin PTMs, through identifying interaction partners, or ‘readers’, for this modification. Using an SH2 (Src Homology 2) protein domain array, we identify N-terminal SH2 domains of p85, regulatory subunits of Phosphatidylinositol 3-kinase (PI3K), and VAV2, a Rho GTPase guanine nucleotide exchange factor, as phosphorylation-dependent binding …

Understanding The Pathogenesis Of Renal Medullary Carcinoma, Melinda Soeung

Dissertations and Theses (Open Access)

Renal medullary carcinoma (RMC) is a lethal cancer that predominantly affects young individuals with sickle cell trait (SCT). It is not currently understood why RMC only affects certain individuals with SCT. We found that patients with RMC more frequently participated in high-intensity exercise than matched controls. Using mouse models of SCT, we demonstrated the significant increase of renal hypoxia in the right kidney following high- but not moderate-intensity exercise. We also demonstrated in cell culture studies that SMARCB1 is ubiquitinated for proteasome-mediated degradation in hypoxia, and the re-expression of SMARCB1 leads to compromised proliferation in renal cells specifically in the …

Regulation Of Lung Mesenchymal Cells By Epithelial Wnt Ligands, Odemaris Narvaez Del Pilar

Dissertations and Theses (Open Access)

The mesenchyme comprises of heterogeneous cell populations that support neighboring structures and are integral to intercellular signaling. Despite such importance, mesenchymal cells are poorly defined morphologically and molecularly. Their signaling regulation is not well understood, lagging their counterparts in the epithelial, endothelial, and immune lineages. Leveraging single-cell RNA-seq, three-dimensional imaging, and lineage tracing, first, we delineated different lung mesenchymal cell types and classified them into three-proximal-distal axes in the neonatal and adult murine lung. Each axis is associated to the structure they support – the endothelium, epithelium and interstitium. We also identified two subpopulations of neonatal lung myofibroblasts – ductal …

The Role Of Pag1 In The Activated B-Cell Subtype Of Diffuse Large B-Cell Lymphoma, Jared Henderson

Dissertations and Theses (Open Access)

Diffuse Large B Cell Lymphoma (DLBCL) is the most common aggressive hematologic malignancy in adults, but despite recent advances in treatment, many patients today still face poor outcomes. Of the two types of DLBCL, activated B-cell (ABC) and germinal center B-cell (GCB), patients diagnosed with the ABC subtype especially face a lack of effective treatment options. Unlike GCB-DLBCL, ABC-DLBCL is characterized by chronic antigen-driven signaling by the B-cell receptor (BCR), indicating an escape from immunologic tolerance mechanisms that normally regulate self-reactive B cells. While recent therapies have focused on inhibiting the kinases that propagate BCR signaling with mixed success, we …

Genetic Pathway Analysis Of Abnormal Facial Development In Nonsyndromic Cleft Lip And Palate, Lorena Maili

Dissertations and Theses (Open Access)

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is the most common craniofacial birth defect resulting from incomplete fusion of the facial prominences during development, which leaves a gap in the lip, primary palate and/or the secondary palate. NSCLP affects 135,000 NSCLP newborns worldwide each year based on a birth prevalence of 1 per 700 live births. While surgical treatments have dramatically improved, many long-term health issues persist, imposing significant medical, psychosocial and economic burdens. Familial aggregation and segregation analyses suggest genetic contributions underlie NSCLP, but despite decades of study, only a small portion of the NSCLP genetic liability …

Assessing The Outcomes Of Blocking Ccl2-Ccr2 Signaling Axis On Breast Cancer Brain Metastasis, Yutao Qi

Dissertations and Theses (Open Access)

Breast cancer brain metastases have remained one of the most intense challenges for precision cancer therapeutics, but current treatment options are limited and not curative. Recently, our lab reported that adoptive PTEN downregulation in metastatic breast tumor cells activates PI3K/NF-ƙB signaling and increases the secretion of the chemokine CCL2, which enhances the chemotaxis of CCR2⁺ myeloid cells, a major subpopulation of bone marrow-derived myeloid cells (BMDMs), from peripheral blood into the brain tumor microenvironment (TME), eventually promoting brain metastasis outgrowth by driving immune suppression. Here, in this project we have been aiming to develop effective therapies by immune-modulating the …

Disruption Of Dna Polymerase Zeta Promotes An Innate Immune Response, Sara Martin

Dissertations and Theses (Open Access)

DNA polymerase ζ (pol ζ) is a specialized polymerase that protects cells from the consequences of DNA damage and stress. Without pol ζ function mammalian cells and yeast are exquisitely sensitive to genotoxic stresses including ultraviolet irradiation and platinum compounds such as the chemotherapeutic agent cisplatin. This elevated sensitivity arises from the central role of pol ζ in lesion bypass. Pol ζ is also required to protect cells from endogenous genomic damage or stress. In primary mammalian cells, the function of pol ζ is required for genome protection and normal proliferation. Immortalized Rev3lknockout (KO) MEFs are under considerable constitutive …

Calcium Dyshomeostasis In Neurodegeneration, Nicholas Emanuel Karagas

Dissertations and Theses (Open Access)

Neurodegenerative diseases, despite constituting a major and growing cause of mortality globally, have few effective treatments. In order to develop novel therapeutics to combat neurodegeneration, a better understanding of the molecular mechanisms underlying these diseases is needed. Neurons rely on Ca²⁺ to mediate many of their unique functions, and aberrant Ca²⁺ signaling has been broadly implicated in neurodegeneration. The goal of this dissertation is to delineate specific examples of Ca²⁺ dyshomeostasis that I have uncovered in Drosophila models of neurodegeneration.

I first define the role a neurodegeneration-associated mutation plays in perturbing presynaptic [Ca²⁺], which is …

Subclonal Evolution Of Chronic Lymphocytic Leukemia After Allogeneic T Cell Therapies, Haven Garber

Dissertations and Theses (Open Access)

Subclonal evolution of chronic lymphocytic leukemia after allogeneic T-cell therapies

Haven Garber, MD

Advisory Professor: Jeffrey Molldrem, MD

Intratumoral genetic heterogeneity describes the molecular differences among subclones within a tumor and is a major barrier to effective therapy in many solid and liquid cancers, including chronic lymphocytic leukemia (CLL). Rare, treatment-resistant subclones can expand to compose relapsed disease during tumor evolution. Examination of malignant evolution in the context of specific treatment provides insight into the molecular lesions that mediate therapeutic response and resistance. Both chemotherapy and targeted therapy were shown to precipitate CLL subclonal evolution. We hypothesized that allogeneic T-cell …

P53 Drives A Transcriptional Program That Elicits A Non-Cell-Autonomous Response And Alters Cell State In Vivo, Sydney Moyer

Dissertations and Theses (Open Access)

Cell stress and DNA damage activate the tumor suppressor p53, triggering transcriptional activation of a myriad of target genes. The molecular, morphological, and physiological consequences of this activation remain poorly understood in vivo. We activated a p53 transcriptional program in mice by deletion of Mdm2, a gene which encodes the major p53 inhibitor. By overlaying tissue-specific RNA-sequencing data from pancreas, small intestine, ovary, kidney, and heart with existing p53 ChIP-sequencing, we identified a large repertoire of tissue-specific p53 genes and a common p53 transcriptional signature of seven genes which included Mdm2 but not p21. Global p53 activation …

Sine Oculis Homeobox Homolog 1 (Six1) Plays A Critical Role In The Progression Of Pulmonary Fibrosis., Cory Wilson

Dissertations and Theses (Open Access)

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia with a median survival time of 2-4 years after diagnosis. The alarming mortality rate is due to the lack of effective treatments. IPF is a chronic disease that is characterized by alveolar destruction due to increasing extracellular matrix deposition that leads to poor lung compliance, impaired gas exchange, and ultimately respiratory failure. Repetitive alveolar epithelial injury is a central process to the underlying pathology with injury to the type II alveolar epithelial cells (AT2) specifically being a key player in the pathogenesis of IPF. Recent studies have shown that …

The Genome-Wide Roles Of The Lung Lineage Transcription Factor Nkx2-1 In The Regulation Of Opposing Cell Fates In Vivo, Danielle Renae Little

Dissertations and Theses (Open Access)

Lineage transcription factors mark, promote, and maintain multiple distinct cell types originating from a common progenitor. Despite their essential role, how such factors function and bind genome wide to orchestrate the epigenetic changes necessary to form and maintain these identities in vivo is unclear. One lineage transcription factor NK Homeobox 2-1 (NKX2-1) is expressed throughout the lung epithelium during development and was thought to be lost in the extraordinarily thin cell type required for gas exchange– the alveolar type 1 (AT1) cell. Complementing precise genetic knockouts with cell type-specific ChIP-seq, ATAC-seq, and scRNA-seq, our study shows that AT1 and AT2 …

The Role Of Daam1 In Kidney Development, Vanja Krneta-Stankic

Dissertations and Theses (Open Access)

Kidneys, like most organs in our bodies, consist of a network of epithelial tubules. Kidney tubules are called nephrons, and their morphology is important for kidney function. Nephrons develop from mesodermally derived aggregates of progenitor cells. The nephric progenitors organize into nephric tubules lined with hair-like sensory projections called cilia. Many diseases of the kidney are characterized by abnormal nephron morphology with current treatment aimed at symptom control. To understand the mechanisms underlying kidney diseases and achieve the development of novel therapies, a better understanding of how nephrons develop is needed. Although the actin cytoskeleton is critical for cell behaviors …

Micrornas Associated With Melanoma Inflammation And Response To Pd-1 Inhibition, Robert Szczepaniak Sloane

Dissertations and Theses (Open Access)

Melanoma is an aggressive malignancy of melanocytes with historically poor outcomes. Melanoma therapy has improved markedly over the past decade with advances in molecularly targeted agents and immunotherapies. Immune checkpoint inhibitors achieve T-cell mediated anti-tumor efficacy by blocking engagement of inhibitory checkpoints on T-cells to overcome immunosuppressive signals from tumor cells and the broader microenvironment. Despite these advances, there are a significant proportion of patients who do not benefit from existing immunotherapy strategies making it a priority to identify and target the mechanisms that confer resistance to therapy. We demonstrate that microRNAs are accurate markers of microenvironment composition with prognostic …

The Role Of Daam1 In Kidney Development, Vanja Krneta-Stankic

Dissertations and Theses (Open Access)

Kidneys, like most organs in our bodies, consist of a network of epithelial tubules. Kidney tubules are called nephrons, and their morphology is important for kidney function. Nephrons develop from mesodermally derived aggregates of progenitor cells. The nephric progenitors organize into nephric tubules lined with hair-like sensory projections called cilia. Many diseases of the kidney are characterized by abnormal nephron morphology with current treatment aimed at symptom control. To understand the mechanisms underlying kidney diseases and achieve the development of novel therapies, a better understanding of how nephrons develop is needed. Although the actin cytoskeleton is critical for cell behaviors …

Packaging And Secretion Of Airway Mucins, Oanh Ngoc Hoang

Dissertations and Theses (Open Access)

In the conducting airways of the lungs, the mobile mucus gel layer sits on top of the airway epithelium, and through ciliary beating it is propelled up the trachea into the pharynx where it is swallowed and cleared. This gel layer is composed of secreted mucins that become hydrated once reaching the airway lumen. Mucins are secreted at a baseline rate and at a stimulated rate, dependent on the amount of agonist. Baseline secretion is responsible for steady clearance of inhaled particles and pathogens. Stimulated secretion is thought to play a role in trapping helminths though mucus occlusion in small …