Full text for dissertations and theses included in this collection dates back to 2011. For older dissertations, check the library’s catalog CatalogUSMAI or Dissertations and Theses database.

Recent Submissions

  • Characterizing enhancer-driven transcriptional networks in schizophrenia

    Casella, Alex; Ament, Seth A. (2021)
    Genetic studies of schizophrenia have demonstrated that more than 90% of genetic risk is confined to non-coding portions of the genome. Advances in chromatin state prediction and chromatin accessibility assays have enabled us to better characterize the genomic features making up these regions and annotate genetic risk to these elements. The focus of this dissertation is to understand the role that tissue- and cell type- specific regulatory elements and the transcription factors that bind them play in risk for schizophrenia. I hypothesized that enhancer-based transcription factor-target networks that direct neuronal development are disrupted in schizophrenia. To test this hypothesis, I used high-quality chromatin state predictions in both the developing and the adult brain to develop a framework for testing enhancer properties for association with genetic risk. Any enhancer-level annotation can be used in this type of test, including transcription factor binding counts and chromosomal contact information. I first described and validated an atlas of transcription factor binding sites across multiple human tissue, including the brain. I then used this atlas to show that neurodevelopmental transcription factors and target genes are most associated with risk for developing schizophrenia.
  • Genomic Medicine in Diabetes: Improving the Diagnostic Rate of Monogenic diabetes

    Zhang, Haichen; Pollin, Toni; 0000-0002-0615-2836 (2021)
    Monogenic diabetes is an uncommon type of diabetes caused by genetic defects in one of several genes, and it accounts for 1-2% of all diabetes. The primary subtypes are Maturity Onset Diabetes of the Young (MODY), neonatal diabetes, and syndromic diabetes. The correct treatment of each subtype of monogenic diabetes depends on the corresponding disease etiology that can only be confirmed by genetic testing. However, the diagnostic rate of monogenic diabetes is inadequate, mainly due to the overlapping phenotype of monogenic diabetes with type 1 diabetes and type 2 diabetes and lack of awareness among patients and physicians. To improve the diagnostic rate of monogenic diabetes, this project focuses on three aspects: 1) systematically screening of patients for genetic testing; 2) comprehensively re-analyzing next-generation sequencing (NGS) data from multiple diabetes cohorts; 3) assessing the ability of Direct-to-Consumer Genetic Testing (DTC-GT) raw data in detecting GCK-MODY variants. The Personalized Diabetes Medicine Program (PDMP) screened 2,522 patients with diabetes with a simple questionnaire, assigned patients to different algorithm criteria groups based on clinical features, and performed genetic testing on suspected patients. Overall, 38 of 313 patients suspected of monogenic diabetes were tested positive for causative variants. The group of patients diagnosed before age 30 who were not treated with insulin had the highest pick-up rate. The comprehensive re-analysis of NGS panel data in PDMP, including re-classification and updating variant calling algorithm, improved the diagnostic rate from 11.82% to 13.10%. Also, the comparison between exome sequencing (ES) and NGS panel or Sanger sequencing of the Progress for Diabetes Genetics in Youth samples showed ES failed to identify all MODY-causing variants, but re-analysis of ES unfiltered data discovered the missing variants. By analyzing the GCK variants in the 23andMe DTC-GT raw data from 3,044 anonymous volunteers and calculating the ancestry-specific allele frequency of GCK-MODY variants, some of the variants showed higher-than-expected minor allele frequency compared with the large population database. Such inconsistency suggests customers should not use DTC-GT as a supplementary method of clinical genetic testing for GCK-MODY. In a summary, these studies provide practical approaches to improve the diagnostic rate of monogenic diabetes.
  • Role of the Parabrachial Complex in Rodent Models of Pain

    Uddin, Olivia; Keller, Asaf; 0000-0001-7035-6836 (2021)
    The parabrachial complex (PB) is a midbrain structure that is vital to survival-related functions. This region receives and integrates incoming sensory information, communicating these signals to higher brain regions that are key in shaping behavior and affect. PB responds to painful somatosensory input, however, its most compelling role is in the development and maintenance of persistent pain. Pain persisting beyond the duration of a threat or tissue injury no longer serves a physiological purpose. Therefore, this type of maladaptive pain has a severe impact on health and quality of life. Current therapies for maladaptive pain are not optimal: it is necessary to better understand the neural circuitry underlying persistent pain so that we can design more effective therapies. The work presented here aims to outline PB’s role in four rodent models of pain. I approach this by combining anatomical tracing, electrophysiology, and behavioral studies. First, I show that in a model of orofacial neuropathic pain, PB neural activity is amplified. Next, I describe a novel and direct anatomical connection between the meninges and PB, via the trigeminal ganglion; this pathway is poised to underlie migraine headache-associated pain. Finally, I conduct behavioral studies aiming to hone rodent models of pain in the context of aging and amyloid accumulation, and pain during opioid withdrawal. These findings together confirm that PB is a crucial node in maladaptive pain processing and provide direction for further work clarifying PB’s role in new behavioral contexts.
  • Role of Adipose Lipolysis in Development of Alcoholic Liver Disease

    Mathur, Mallika; Yu, Liqing (2021)
    Alcoholic liver disease (ALD) pathologies include hepatic steatosis, inflammation and liver injury. The liver receives ~60% of fatty acids from adipose tissue. We examined the role of adipose lipolysis in ALD pathogenesis using adipose-specific CGI-58 knockout (FAT-KO) mice, a model of impaired adipose lipolysis. FAT-KO versus control mice were almost completely protected against ethanol-induced hepatic steatosis and lipid peroxidation when subjected to the 15-day NIAAA chronic-binge ethanol diet. This was unlikely from reduced lipid synthesis because ethanol feeding downregulated hepatic expression of lipogenic genes similarly in both genotypes. On a control diet, FAT-KO relative to control mice had increased hepatocyte injury, neutrophil infiltration, and activation of transcription factor STAT3 in the liver, none exacerbated by ethanol. This was associated with increased hepatic leptin receptor mRNA and adipose inflammatory cell infiltration. These findings identify a critical role for adipose lipolysis in hepatic steatosis and oxidative stress during ALD development.
  • Neuronal Intrinsic Apoptosis Mechanisms and Their Modulation by Sp1 Inhibition

    Makarevich, Oleg; Stoica, Bogdan A.; 0000-0003-2061-2285 (2021)
    Drug-induced DNA damage, reactive oxygen species (ROS), inflammatory mediators and central neurotoxicity have all been shown to play a role in cognitive impairments after chemotherapy, after CNS injury or in neurodegenerative diseases. Intrinsic apoptosis is a regulated cell death pathway implicated in many of these conditions. This pathway proceeds sequentially through DNA damage responses, including phosphorylation of ATM, H2AX and Tumor Protein 53 (p53), transcriptional activation of pro-apoptotic BH3-only proteins, and mitochondrial outer membrane permeabilization (MOMP), activating caspase-dependent and caspase-independent apoptosis. Previous work has indicated that Sp1 may regulate p53’s transcriptional profile and ability to promote apoptosis after DNA damage. To examine the role of Sp1 in DNA-damage-induced apoptosis, my work has utilized Mithramycin, a drug that binds G-C rich DNA to compete with Sp1 chromatin binding. Our hypothesis is that Mithramycin competes with Sp1 chromatin binding and thus protects neurons from DNA damage-induced, p53-dependent intrinsic apoptosis. However, it is vital to recognize that neurons increase their resistance to cytotoxic stimuli as they mature, leading to decreased levels of apoptosis. Thus, we additionally characterized the changes in the intrinsic apoptosis pathway upon neuronal maturation. Despite significant attenuation of the intrinsic apoptosis pathway in mature neurons, we found evidence that Mithramycin can still attenuate DNA-damage dependent intrinsic apoptosis in this paradigm to the extent that it is induced by neurons. Additional experiments showed that mature animal cortices or hippocampi do not demonstrate evidence of end-stage apoptosis (caspase activity) in response to DNA damage, unlike those in immature animals. However, in vivo administration of Mithramycin was able to attenuate cortical expression of p53-dependent genes. Thus, our studies provide evidence that: 1) Mithramycin attenuates activation of the DNA damage-dependent intrinsic apoptosis pathway via indirect inhibition of p53-dependent transcription and 2) Mature neurons restrict intrinsic apoptosis pathway activation in response to DNA damage. Therefore, Sp1 is likely to play a significant role in DNA damage-dependent neuronal intrinsic apoptosis, and Mithramycin treatment is protective when this pathway is activated. However, mature neurons significantly downregulate this pathway, thus limiting the potential therapeutic effects of Mithramycin in adult animals.
  • Modulation of Inflammation and Stromal Remodeling Processes in Autoimmune Arthritis by Microbial Indole Derivatives

    Langan, David; Moudgil, Kamal; 0000-0002-4525-9767 (2021)
    Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the synovial tissue that can lead to joint damage and deformities. Inflammation, new blood vessel formation (angiogenesis), and bone resorption (osteoclastogenesis) are three key processes of the pathophysiology of RA. ‘Dysbiosis’ of the gut microbiota is implicated in RA pathogenesis because it can cause an imbalance in the microbial metabolites that regulate host health and disease. However, there is little information about the impact of two such indole derivatives, indole-3-aldehyde (IAld) and indole-3-acetic acid (I3AA), on arthritis-related processes. Using established cell-based models and the adjuvant-induced arthritis animal model, we conducted a comparative analysis of IAld and I3AA to understand how these metabolites might impact RA pathogenesis. To our surprise, despite their structural similarities, the bioactivities of these two metabolites were profoundly different. IAld, but not I3AA, altered the expression of genes encoding arthritis-associated cytokines (IL-1β, IL-6, VEGF) in RAW 264.7 (murine macrophage) cells stimulated with heat-killed M. tuberculosis. Further investigation of this anti-inflammatory activity of IAld suggested that inhibition of the MyD88-dependent activation of NF-κB and MAPK pathways was unlikely to be involved. IAld also exhibited pro-osteoclastogenic and pro-angiogenic activity. In contrast, I3AA exhibited only anti-angiogenic activity. Both IAld and I3AA are proposed agonists of the aryl hydrocarbon receptor (AhR). However, AhR inhibitor CH-223191 suppressed the anti-angiogenic activity of I3AA, but failed to mitigate any of the effects of IAld. There is a cross-talk between the AhR and Nrf2 pathways, and some plant-derived phytochemicals are multifunctional ligands of both pathways. Our findings show that IAld, unlike I3AA, can suppress the Nrf2-dependent antioxidant response of macrophages to an Nrf2 agonist, but that IAld also potentially reduces intracellular ROS levels during osteoclast differentiation. Furthermore, oral administration of IAld to rats resulted in the reduction of arthritis severity compared to the arthritic control group. Taken together, our findings suggest that the relative bioavailability of these microbial indole derivatives has the potential to influence their immunomodulatory effects in healthy individuals as well as patients with RA.
  • Monomeric IL-12p40 binds partner proteins to modulate immune cell function

    Gerber, Allison; Singh, Nevil; 0000-0001-9442-3255 (2021)
    Cytokines are critical mediators used by immune cells to communicate as well as protect. The IL-12 family of cytokines are made up of  and  subunits typically assembled within one cell and secreted as a heterodimer. IL-12p40 is the shared β-subunit for both IL-12 (paring with IL-12p35) and IL-23 (with IL-23p19). However, the IL-12p40 monomer is often secreted in excess during infections, but its biological role was not known. In this thesis we investigated the function of secreted IL-12p40 monomer in vivo with the central hypothesis that the monomer combines with multiple α-subunits in vivo to generate IL-12 as well as other heterodimeric cytokines. Consistent with this hypothesis, in chimeric mice containing mixtures of cells that can only express either IL-12p40 or IL-12p35, but not both together, we found that functionally active IL-12 was generated. This alternate two-cell pathway requires IL-12p40 from hematopoietic cells to extracellularly associate with IL-12p35 from radiation-resistant cells. The two-cell mechanism was sufficient to influence local T cell differentiation in sites distal to the initial infection and helped control systemic dissemination of a pathogen although not parasite burden at the site of infection. Broadly, this suggests that early secretion of IL-12p40 monomers by sentinel cells at the infection site may help prepare distal host tissues for potential pathogen arrival. In addition to this role in generating IL-12 through two-cell assembly, we found that IL-12p40 has a novel partner protein, CD5L. This novel heterodimer was present in the serum of uninfected mice, with differences in the basal levels between B6 and Balb/c animals, with Balb/c having higher amounts of p40-CD5L. Functionally, we found that treatment with p40-CD5L leads to IL-4 and IL-10 production by T cells. Taken together, this thesis offers at least two major fundamental advances in cytokine biology – one the concept of a two-cell assembled cytokine and second the identity of a novel TH2-promoting heterodimeric cytokine. The first has significance in immunotherapy and understanding immunity to tissue-specific modulation of immune responses. The second is expected to drive significant research on allergy, responses to parasites and immune deviation.
  • The Molecular Basis of IL-1 Family Signaling: Strategies to Modulate Inflammation

    Fields, James; Sundberg, Eric J.; 0000-0001-5923-108X (2021)
    Interleukin-1 (IL-1) family cytokines are potent signaling molecules that influence both innate and adaptive immune systems. The IL-1 family, composed of 11 cytokines and 10 receptors, mediate inflammation to a wide array of stimuli and act on myriad cell types for diverse immunological outcomes. Altogether, IL-1 family signaling is integral to a multitude of inflammatory responses and occurs in distinct steps. First, an agonist cytokine binds its cognate receptor at high affinity. Next, this cytokine-receptor complex recruits an often-shared co-receptor. As this cytokine/receptor/co-receptor complex forms, Toll/IL-1 Receptor (TIR) domains, residing cytoplasmically, oligomerize, initiating a potent signaling cascade that results in prototypical NF-κB signal transduction. Due to the strong nature of IL-1 family signaling, multiple physiological mechanisms exist to stem this inflammatory signal, including antagonist cytokines and decoy receptors. Within the IL-1 family, the cytokines and receptors can be further divided into four subfamilies dependent on their secondary receptors. The IL-1 subfamily contains IL-1, IL-33, and IL-36 as they all share IL-1RAcP as their secondary receptor; the IL-18 subfamily is distinct as it utilizes IL-18Rβ as its secondary receptor. Here, we describe how structural biology has guided our understanding of IL-1 family signaling and how that knowledge can be leveraged for the design of therapeutics to stem aberrant cytokine signaling. In our first study, we demonstrate the feasibility of targeting a shared co-receptor, IL-1RAcP, for selective cytokine inhibition. Indeed, dependent on the specific epitope targeted on IL-1RAcP, differential cytokine signaling inhibition can be achieved. In addition, we developed our own IL-33 therapeutics by leveraging the high affinity IL-33 has for its primary receptor, the stability imparted by the secondary receptor, and the extended half-life gained through an Fc-fused receptor. Two of these molecules inhibit IL-33 signaling better than the natural antagonist sST2. Altogether, structural biology has informed our understanding of IL-1 family signaling, generated approaches to improve existing therapeutics, namely antibody epitope targeting, and led to the creation of additional IL-33 target therapeutics in the form of our “cytokine traps.”
  • Mechanisms of Beta Cell Metabolic Coordination

    Rao, Vishnu; Rizzo, Megan A.; 0000-0002-4396-1579 (2021)
    Glucose homeostasis is predominantly regulated by pancreatic hormones. Insulin, which is secreted by pancreatic beta cells, is vital for maintaining normoglycemia; insulin secretory failure is a prime contributor to diabetes progression. The insulin secreting beta cells are a heterogeneous population that are organized into islets, which display coordinated responses to glucose. Although the electrical coupling of beta cells is well described, upstream metabolic coupling has not been sufficiently explored. The goal of this research was to (1) determine if beta cells are metabolically coordinated prior to reaching electrical threshold potential, (2) confirm evidence of metabolic heterogeneity, and (3) examine the importance of this heterogeneity. To accomplish this goal, we used a variety of novel techniques and models. We restricted application of glucose to portions of the islet to show beta cells are metabolically coordinated by gap junctional diffusion of metabolites. Then, using mice expressing a plasma membrane ChR2, we demonstrated that beta cell metabolism is differentially regulated by calcium. Finally, we generated a novel mouse model expressing an anisotropic reporter of glucokinase activity to illustrate that differences in glucokinase activity are averaged by metabolic coupling. These results highlight the importance of metabolic coordination in evening out differences between cells. Our findings may explain how insulin secretion is enhanced in the presence of glucose but contained in its absence.
  • The Role of Testisin in Endothelial Cells and Angiogenesis

    Peroutka, Raymond; Antalis, Toni M.; 0000-0002-8639-5773 (2021)
    Testisin (PRSS21) is a membrane anchored serine protease, which is tethered to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor. Testisin expression has been documented in eosinophiles, ovarian cancers, endothelial cells, and spermatozoa where its expression is highest. Although two substrates of testisin have so far been identified, protease activated receptor 2 (PAR2) and protein inhibitor C (PCI), little is known of the biological, physiological, and pathophysiologic characteristics of testisin. Thus far, there are no published data identifying an activator or inhibitor of testisin. To better characterize the biochemistry of testisin we produced the zymogen as inclusion bodies in E. coli and refolded using the insoluble cellular fraction. To better characterize the cellular functions of testisin, hybridomas producing anti-testisin monoclonal antibodies were acquired, antibodies purified, and then characterized. In an investigation of testisin’s function in endothelial cells we identified testisin as a novel regulator of physiological hormone-induced angiogenesis and microvascular endothelial permeability. Using a murine model of rapid physiological angiogenesis during corpus luteal development in the ovary, we found that mice genetically deficient in testisin (Prss21-/-) show a substantially increased incidence of hemorrhages which are significantly more severe than in littermate control Prss21+/+ mice. This phenotype was associated with increased vascular leakiness, demonstrated by a greater accumulation of extravasated Evans blue dye in Prss21-/- ovaries. Live cell imaging of in vitro cultured microvascular endothelial cells depleted of testisin by siRNA knockdown revealed that loss of testisin markedly impaired reorganization and tubule-like formation on Matrigel. Moreover, testisin siRNA knockdown increased the paracellular permeability to FITC-albumin across endothelial cell monolayers, which was associated with decreased expression of the adherens junction protein VE-cadherin and increased levels of phospho-(Tyr658)-VE-cadherin, without affecting the levels of the tight junction proteins occludin, claudin-5, or ZO-1. Decreased expression of VE-cadherin in the neovasculature of Prss21-/- ovaries was also observed without marked differences in endothelial cell content, vascular claudin-5 expression or pericyte recruitment. Together, these data identify testisin as a novel regulator of VE-cadherin adhesions during angiogenesis and indicate a potential new target for regulating neovascular integrity and associated pathologies.
  • Mitochondrial Dysfunction is Linked to Pathogenesis in the P497S UBQLN2 Mouse Model of ALS/FTD

    Lin, Brian; Monteiro, Mervyn J.; 0000-0003-2944-3051 (2021)
    Ubiquilin-2 (UBQLN2) mutations cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD), but the mechanisms that drive disease pathogenesis remain unclear. Neurons have especially high energy requirements and consume copious amounts of ATP to support synaptic transmission and other complex processes. As such, mitochondrial dysfunction has been thought to play a pathogenic role in ALS. Recently, UBQLNs have been implicated in mitochondrial protein quality control whereby their inactivation in cells leads to the accumulation of cytostatic mitochondrial precursors. However, it is unclear what specific role UBQLN2 plays in maintaining mitochondrial proteostasis and how UBQLN2 mutations impact mitochondria physiology. In this thesis, I tested my hypothesis that the ALS-linked UBQLN2 P497S mutation causes mitochondrial dysfunction through loss of UBQLN2 chaperone function and impaired mitochondrial import. Our lab previously generated proteomic profiles of early-stage (8 weeks) hippocampal and spinal cord (SC) tissues isolated from non-transgenic (Non-Tg), wild-type (WT356), and P497S UBQLN2 mutant mice, whereby the mutant animal closely models human ALS/FTD. Gene ontology analysis revealed “mitochondrial proteins” as a major category altered in P497S animals. I immunoblotted SC lysates of Non-Tg, WT356 and P497S UBQLN2 animals for various mitochondrial proteins, and found decreased levels of many mitochondrial proteins, including those involved in oxidative phosphorylation (OXPHOS), network dynamics and import. I discovered through Seahorse respiration assays that mitochondria purified from the SC of P497S mice have age-dependent respiration deficits unlike those of age-matched Non-Tg and WT356 animals. Electron microscopy of spinal motor neurons in the P497S animals revealed distortions to mitochondria cristae. I demonstrated that mitochondrial alterations found in P497S mutant animals are recapitulated in UBQLN2 knock-out cells, suggesting loss of UBQLN2 function may underlie the mutation’s effects. Additionally, inactivation of UBQLN2 compromised proper targeting and processing of the mitochondrial import factor, TIMM44, which subsequently could be rescued by reexpression of WT UBQLN2, but not by mutant UBQLN2 proteins. ALS/FTD UBQLN2 mutants bind weaker to TIMM44 compared to WT UBQLN2, providing a possible mechanism for the mitochondrial import defects. Overall, these studies highlight a potential key role of UBQLN2 in maintaining mitochondrial health, and how its function is impaired by mutations in UBQLN2.
  • BECC Adjuvanted Vaccine Provides Broad Protection from Homologous and Heterologous Influenza A Virus Infections

    Haupt, Robert; Frieman, Matthew B.; 0000-0001-8300-7184 (2021)
    Influenza A virus (IAV) is a leading cause of mild to severe respiratory disease worldwide with significant infections resulting in hospitalization or death, especially in older individuals, young children, and people with comorbidities. Because the influenza virus has a high rate of mutation, this allows the virus to evade the host immune system against new variants, new vaccines are produced annually to match circulating strains. FluAd, is currently the only approved influenza vaccine formulated with the adjuvant MF59, a squalene oil emulsion, to create a stronger immune response to the vaccination and is limited for use in people 65 years and older. Classic adjuvants such as oil emulsions and alum are not universally effective and potentiate toward a T helper 2 (Th2) response (effective versus extracellular pathogens), while another often-used vaccine adjuvant, monophosphoryl lipid A, skews toward a Th1 response (effective versus intracellular pathogens). Unique adjuvants have been created using the Bacterial Enzymatic Combinatorial Chemistry (BECC) system to produce novel Toll like receptor 4 (TLR4) immunostimulatory molecules. These molecules drive a balanced Th1/Th2 response and enhanced immunogenicity in the context of an influenza virus vaccine candidate as well as other vaccine platforms. These BECC adjuvanted vaccines display superior protection to challenge from a homologous IAV strain in a BALB/c mouse model, even in aged mice with a single prime vaccination. More important, these vaccines stimulate the production of broadly reactive antibody to the hemagglutinin stem and protection against heterologous IAV strain infection, possibly skirting the need to formulate new influenza vaccine annually.
  • Immunologic and pathogenic interactions between burned mice and Pseudomonas aeruginosa

    brammer, jerod; Cross, Alan S.; 0000-0003-0368-0745 (2021)
    Fire is a pinnacle staple of human life. Consequently, burn injuries are inevitable. People that survive the initial burn trauma are at higher risk of severe complications due to secondary bacterial infections from either environmental exposure or hospital-acquired infections. The most common Gram negative bacterium found in burn wounds worldwide is Pseudomonas aeruginosa (PA). Here we employ a non-lethal 10% total body surface area flame-burn. A superimposed infection with PA strain M2 resulted in 100% mortality post-burn with a reduction in the lethal dose from >106 to <102 CFU when administered in the burn site immediately after the burn. This reduction in LD50 only lasts for 72 hours post-burn, suggesting the burn caused a transient reduction in host defenses that reduced the ability to fight infection. This model allowed for the discrimination between immunological events caused by the burn itself and subsequent bacterial infection. We determined that a high concentration of High Mobility Group Box 1 (HMGB1), a danger-associated molecular pattern, was released into the circulation directly after the burn. This release of HMGB1 into the circulation was independent of infection and preceded detectable cytokine responses. With infection, there was a 10-fold increase in circulating HMGB1 that continued until death. The inhibition of circulating HMGB1’s ability to cause inflammatory signaling through the TLR4-signaling pathway with a small molecule inhibitor, P5779, almost doubled the mean time to death and even resulted in a group of survivors. During routine necropsy post-burn, we identified a previously undescribed seroma. The seroma fluid supported the robust growth of PA and recruited neutrophils from the circulation, possibly sequestering them from vital organs at a critical time, thus facilitating burn wound sepsis. This sublethal mouse burn model enabled the study of effects from the burn injury on both the innate response to the burn and the pathogen.
  • Development of Heterogeneous Nuclear Ribonuclear Protein A18 (hnRNP A18) Small Molecule Inhibitors and Phosphorylation of hnRNP A18 by Casein Kinase-2

    Coburn, Katherine; Weber, David J., Ph.D.; 0000-0001-8325-9437 (2021)
    Heterogeneous nuclear ribonuclear protein A18 (hnRNP A18) is an RNA binding protein (RBP) is upregulated in response to cellular stressors such as cold, UV, and hypoxia. Upon cellular stress, hnRNP A18 is phosphorylated in the nucleus by casein kinase-2 (CK-2) and glycogen synthase kinase-3β (GSK-3β). Upon phosphorylation, hnRNP A18 translocates to the cytosol where it interacts with pro-survival mRNA transcripts and stabilizes them to increase their translation. hnRNP A18 is differentially upregulated in solid tumors in response to low oxygen tension where it stabilizes pro-survival mRNA transcripts, such as hypoxia inducible factor-1α (HIF-1α) and Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Chemotherapeutics that target HIF-1α and CTLA-4 have demonstrated increased patient progression-free survival. hnRNP A18 presents a unique advantage in the ability to target both the hypoxic cellular responses and immune checkpoints in cancer. Toward the development of hnRNP A18 inhibitors, the X-ray crystal structure of the hnRNP A18 RNA recognition motif (RRM) was solved and used in the design of inhibitors for hnRNP A18 that disrupt RNA binding with the RRM. Initial inhibitors were identified through the Site Identified Ligand Competitive Saturation (SILCS) computational screening method. Nuclear magnetic resonance (NMR) screening of these compounds produced a lead compound that was further refined. Subsequently identified compounds were analyzed for hnRNP A18 binding, specificity, membrane permeability, and binding affinity. This work serves as a foundation for investigation of hnRNP A18 inhibitors in vivo and toward the development of an hnRNP A18 therapeutic approach. Phosphorylation by CK-2 and GSK-3β is required for nuclear to cytosolic translocation upon cellular stressors. CK-2 phosphorylation primes hnRNP A18 for GSK-3β phosphorylation, which subsequently increases hnRNP A18 affinity for target mRNA transcripts. To understand the structural and functional impact of such post translational modifications, hnRNP A18 was phosphorylated by CK-2 and the structural impacts of the phosphorylation were analyzed by NMR. RBPs, like hnRNP A18, are involved in a variety of cellular processes and disease pathologies, such as carcinogenesis and neurodegenerative disorders. Insight into the biological and structural mechanisms of RBP action can aid in the development of therapeutic strategies important for the treatment of such pathologies.
  • Genetic and Functional Studies of the Evolutionarily Oldest Natural Killer Receptor, NKp30

    Kinlein, Allison; Ohta, Yuko; 0000-0002-3944-2530 (2021)
    NKp30 is a Natural Cytotoxicity Receptor (NCR) expressed by Natural Killer (NK) cells and other lymphocytes. NKp30 is the only evolutionarily conserved NCR found in all jawed vertebrates and it coevolves with its ligand B7-H6. Using sharks as a model, we found NKp30 gene expression in subsets of mature and immature T cells, suggesting that T cells and NK cells share common features in these primitive vertebrates. To understand NKp30’s evolutionary origin, we examined genomic regions containing NKp30 and its homologs in vertebrates in different Classes. We observed that loci in paralogous regions containing NKp30 homologs are well conserved, suggesting the presence of NKp30 ancestors and other linked immune genes before the emergence of vertebrates 550 million years ago. Indeed, the corresponding region is also present in invertebrates. We hypothesize that this genomic region encompassed the “Primordial Immune Complex,” containing genes playing roles in immunity at the origin of vertebrates.
  • Inhibition of GPR68 Sensitizes GBM to Temozolomide Treatment via the NF-kB Pathway

    Ahmad, Jovanni; Hong, Charles C., 1967-; 0000-0001-9550-8090 (2021)
    Glioblastoma Multiforme (GBM) remains as one of the most aggressive and lethal cancer types, often resulting in poor prognosis. Currently, Temozolomide (TMZ) is the standard chemotherapy for combating GBM. However, GBM’s upregulation of O-6-Methylguanine-DNA Methyltransferase (MGMT) mitigates the alkylating effects of TMZ treatment, generating a dire new need for novel or adjuvant therapy. U138MG is a TMZ-resistant GBM cell line used for the development of new chemotherapy. Here, we investigated whether inhibition of proton sensing GPR68 would decrease MGMT expression and sensitize U138MG cells to TMZ treatment. Using various genetic, protein, and cell-based assays we determined that inhibition of GPR68 may be decreasing MGMT protein expression and sensitizing U138MG to TMZ via the Gq/NFkB pathway. Furthermore, we identified that co-administration of TMZ and OGM resulted in a synergistic decrease in cell growth compared to OGM treatment alone.
  • Actin-Like Protein 6A (ACTL6A) Suppresses p21Cip1 Expression to Maintain an Aggressive Cancer Phenotype

    Shrestha, Suruchi; Eckert, Richard (Richard L.); 0000-0002-9550-816X (2021)
    Epidermal squamous cell carcinoma (SCC) and mesothelioma are two distinct but highly aggressive forms of cancer. SCC is a common and highly invasive cancer that arises from the epidermis. The major cause of epidermal SCC is repeated exposure to ultraviolet light and other DNA damaging agents such as oxidative stress which causes mutations eventually leading to increased expression of pro-tumor genes and reduced expression of tumor suppressors. Mesothelioma is highly invasive and lethal cancer that arises from the mesothelial lining and is linked to exposure to asbestos and other toxic agents. Actin-like protein 6A (ACTL6A, BAF53A) is a member of SWI/SNF chromatin remodeling complex that has been implicated in many cancers as a driver of cancer survival and tumor formation. We show that ACTL6A functions to maintain an aggressive cancer phenotype in both SCC and mesothelioma. We further show that ACTL6A reduces expression of the p21Cip1 cyclin-dependent kinase inhibitor and tumor suppressor protein. Biochemical studies reveal that loss of ACTL6A leads to increased p21Cip1 promoter activity, and mRNA and protein expression suggesting transcriptional regulation of p21Cip1 gene. Moreover, chromatin immunoprecipitation studies show that ACTL6A interacts at the p21Cip1 promoter proximal Sp1 site and distal p53-responsive enhancer sites to suppress transcription. We further report that the increase in p21Cip1 upon ACTL6A knockdown is required to suppress the SCC and mesothelioma cancer phenotypes. This suggests that p21Cip1 is the key mediator of ACTL6A function in SCC and mesothelioma. p53 is a key tumor suppressor that interacts with the p21Cip1 promoter to increase expression; however, we show that it may not play a regulatory role in these cancers. These findings suggest that ACTL6A suppresses p21Cip1 transcription to reduce p21Cip1 function as a mechanism to maintain an aggressive cancer phenotype in SCC and mesothelioma.
  • Calcium and BK Potassium Channel Regulation of Circadian Rhythms in the Suprachiasmatic Nucleus

    Plante, Amber; Meredith, Andrea L.; 0000-0002-1010-2348 (2021)
    Mammalian circadian rhythms are driven by a network of neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN exhibits daily (24-hour) rhythms in spontaneous action potential (AP) firing rate that encodes a time-of-day signal that coordinates the timing of circadian physiological and behavioral processes. Large-conductance Ca2+-activated K+ (BK) channels have a major role in driving the diurnal patterns of spontaneous firing in SCN neurons. BK K+ currents are larger at night, correlating with reduced neuronal excitability. The diurnal variation in BK current in the SCN is required for setting the day-night difference in firing frequency. BK currents undergo multi-level regulation by genetic and posttranslational mechanisms as well as functional coupling to Ca2+ channels. Intracellular Ca2+ (Ca2+i) is required for BK channel activation and previous studies have shown BK current is predominantly coupled to two types of Ca2+ sources in the SCN: L-type Ca2+ channels (LTCCs), and ryanodine receptors (RyRs). Circadian rhythms in Ca2+i have also been identified in SCN neurons. However, the Ca2+ channels involved in generating both AP and Ca2+i rhythms have not been clearly identified. First, to determine which Ca2+ channels are involved in AP rhythms, this study measured the impact of Ca2+ channel agonists and antagonists on the circadian parameters of spontaneous AP activity from organotypic SCN slice cultures grown on multi-electrode arrays. Next, to determine which Ca2+ channels are involved in Ca2+i rhythms, this study tested the effects of the same Ca2+ channel pharmacology on the circadian parameters of Ca2+i measured from SCN slice cultures transfected with a fluorescent Ca2+ sensor. Lastly, this investigated a potential mechanism by which LTCCs contribute to firing rate in SCN neurons by examining their ability to activate BK channels under controlled conditions. This study provides insight into the roles of specific Ca2+ sources in neural coding of the circadian time signal in the SCN.
  • Theileria infections in African cattle and buffalo: understanding genetic variation and speciation

    Palmateer, Nicholas; Carneiro da Silva, Joana; 0000-0002-4307-8049 (2021)
    East Coast fever, caused by the apicomplexan parasite Theileria parva, has an estimated annual death toll of over a million cattle in endemic sub-Saharan regions. The African Cape buffalo is the natural reservoir of T. parva and rarely exhibits clinical symptoms when infected, but transmits the parasite to cattle via a tick vector. Previous studies based on a few genetic markers showed that buffalo-derived T. parva subpopulations contain greater antigenic diversity than those from cattle. Interestingly, cattle are infected and killed by T. parva of buffalo origin, but cannot transmit those parasites, suggesting that a degree of host specificity exists. The characterization of genetic variation within and between cattle- and buffalo-derived T. parva is critical to understand the molecular mechanism(s) of host specificity. To overcome obstacles in T. parva biology that prevent the straightforward acquisition of sufficient DNA for whole genome sequencing (WGS), we adapted a DNA capture approach to select T. parva from a mix of parasite and bovine DNA obtained from T. parva-infected bovine lymphocyte cultures. To gain access to variable genomic regions that cannot be characterized through read mapping approaches, we assembled the captured reads de novo. From starting material of <1%-4% parasite DNA in a mixed sample from host and parasite, >98% of sequence reads post-capture are of parasite origin and >97% of the genome is recovered, reflecting the method’s high specificity and sensitivity. We used this whole genome DNA capture followed by sequencing to generate WGS data from 15 cattle- and 24 buffalo-derived T. parva isolates. This resulted in the generation of the first assembly of a buffalo-derived T. parva isolate. Furthermore, we determined that cattle- and buffalo-derived T. parva isolates differ in various measures at levels consistent with speciation. Finally, capture and analysis of members of the T. parva repeat (Tpr) multigene family, which encode some of the most variable antigen families in the species, enabled the study of Tpr evolution and initial inferences of its possible involvement in parasite-host interactions. These results have greatly advanced the study of the T. parva genome and improved our understanding of the evolution of this parasite population.
  • Cancer Mortality among US Solid Organ Transplant Recipients: Novel Methodologies to Estimate Cancer Burden using Linked Population-Based Registries

    Noone, Anne-Michelle; Dorgan, Joanne; Engels, Eric A.; 0000-0001-6997-4004 (2021)
    Background: The solid organ transplant population has an elevated risk of cancer compared with the general population. Excess risk is largely due to immunosuppression. As this population grows, understanding long-term health risks such as cancer is critical. Population-based estimates of cancer mortality are needed since they measure the downstream outcome following a cancer diagnosis. Furthermore, quantifying deaths attributable to cancer can inform priorities to reduce the cancer burden. Methods: Linked transplant and cancer registry data were used to identify incident cancers and deaths among solid organ transplant recipients in the United States (1987-2014). Population-attributable fractions (PAFs) of deaths due to cancer and corresponding cancer-attributable mortality rates were estimated. Cancer-attributable mortality rates computed using the PAF were compared to cancer-specific mortality rates computing using cause of death (COD). The life-years lost (LYL) to cancer were estimated using two methods: an approach using matching to construct a cancer-free cohort and an approach using Cox proportional hazards regression models. Results: Among 221,962 transplant recipients, 15,012 developed cancer. Thirteen percent of deaths (PAF=13.2%) were attributable to cancer, corresponding to a cancer-attributable mortality rate of 516 per 100,000 person-years. Lung cancer was the largest contributor to mortality (PAF=3.1%), followed by non-Hodgkin lymphoma (NHL, PAF=1.9%), colorectal cancer (PAF=0.7%), and kidney cancer (PAF=0.5%). Overall, the cancer-specific mortality rate lower, 368 per 100,000 person-years. Within 10 years post-transplant, the mean LYL was 0.16 years per transplant recipient and 2.7 years per cancer. Cancer accounted for 1.9% of the total LYL expected in this population. Lung cancer was the largest contributor, accounting for 24% of all LYL, and NHL had the next highest contribution (15%). Conclusions: Cancer is a substantial cause of mortality among solid organ transplant recipients resulting in excess deaths and a shortened lifespan. Lung cancer and NHL are major contributors to the cancer burden including LYL to cancer, highlighting opportunities to reduce cancer mortality through prevention and screening.

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