• The Effect of Processing Techniques for rhBMP-2 Coated Titanium Implants on Alveolar Augmentation and Osseointegration in the Canine Supraalveolar Peri-Implant Defect Model

      Decker, John; Department of Oral Biology (9/5/2014)
      A current paradigm-shift in implant dentistry places restorative factors associated with esthetics and function in front of implant site selection based on bone quantity and quality. Marginal bone loss after implant placement, resorption of the edentulous alveolar ridge, bone defects from periodontal disease, and ridge aberrations due to trauma all challenge implant treatment driven by esthetics and function. Clinicians compensate for bone loss using bone augmentation procedures including bone grafts, bone materials, biologic mediators, barrier devices, or combinations thereof. The search for treatment modalities to address implant placement into compromised sites has lead to the development of a variety of products designed to replace or induce bone formation. Some believe an ideal material could be coated onto implants, to promote osseointegration, induce local bone formation, while not requiring adjunctive biomaterials, or procedures including placement of allogeneic and xenogeneic biomaterials, or autograft bone.
    • A MIXED METHODS EXAMINATION OF PRE-HOSPITAL TRAUMA TRIAGE DECISION MAKING

      Gollan, Srisuda Siera; Department of Physiological and Technological Nursing (9/15/2017)
      The objective of pre-hospital trauma care is ensuring that the most severely injured persons are transported to the facility best suited to meet their complex needs (Fitzharris, Stevenson, Middleton, & Sinclair, 2011; Hoff, Tinkoff, Lucke, & Lehr, 1992; Leach et al., 2008; Sasser et al., 2012). To support pre-hospital decision making regarding trauma triage destination determinations, the Guidelines for Field Triage of Injured Patients decision scheme (FTDS) was developed as an algorithmic decision tool (Sasser et al., 2012). The purpose of this study was to examine pre-hospital trauma triage transport decision making by EMS providers from multiple perspectives. This study used a concurrent mixed methods triangulation design (QUAL+QUANT). Mixed methods included: (1) Grounded theory methodology to describe a model of decision making used by EMS providers to make trauma triage determinations and (2) quantitative analysis of secondary data to determine how the FTDS criteria are utilized by EMS providers. The FTDS criteria were also examined relative to trauma outcomes: level of trauma team activation (TTA), patient disposition when leaving the emergency department (ED), and the injury severity score (ISS). A model of Interpreting Trauma into Action was elucidated to describe the processes used by EMS providers. Pre-hospital providers based their trauma transport decisions on the perceived patient level of injury severity. The FTDS criteria were not explicitly used in this study region, but were interwoven into practice through employer policies and other training. The convergence of these findings indicated congruence between the model and trauma outcomes. The quantitative data indicated relationships (p<.05) between 12 of the 29 FTDS criteria and trauma outcomes. Both sources of evidence supported the relationships between the model of Interpreting Trauma into Action, the FTDS criteria, and specific trauma outcomes.
    • Effect of chronic oral treatment with risperidone or quetiapine on cognitive performance and neurotrophin-related signaling molecules in rats

      Poddar, Indrani; Department of Pharmacology and Toxicology (8/7/2018)
      Antipsychotic (APs) drugs are among the top selling pharmaceuticals in the world and they have a variety of important therapeutic applications for neuropsychiatric disorders. However, there are a number of controversies related to this class of agents and many of the relevant questions are difficult to prospectively address in the clinical trial environment. For example, there have been multiple clinical trials for pro-cognitive agents in schizophrenia that have failed; however, the question of how chronic prior treatment with APs might influence the response to a pro-cognitive agent was not addressed. Moreover, there is clinical evidence that chronic treatment with some APs may lead to impairments in cognition, however, this issue and the potential molecular mechanisms of the deleterious effects have been not been prospectively addressed. Accordingly, the purpose of the work described in this dissertation was to prospectively address each of these issues in animals (specifically rats) were environmental conditions can be rigorously controlled. In each of the manuscripts included in this dissertation, two of the most commonly prescribed APs, risperidone and quetiapine were evaluated. In the work conducted in Manuscript 1, we established a therapeutic relevant dosing approach for rats (oral administration in drinking water) and reinforced the argument that these two APs are not pro-cognitive agents. Moreover, we determined that alpha-7 nicotinic acetylcholine receptor (nAChR) ligand like tropisetron has potential as an adjunctive medication in schizophrenia since the pro-cognitive effect was maintained in the presence of chronic AP treatment. In Manuscript 2, we concluded that chronic treatment with risperidone or quetiapine in rats can lead to impairments in a domain of cognition (recognition memory) that is commonly altered in neuropsychiatric disorders. Moreover, the negative effects of the APs appeared to be exacerbated over time. In Manuscript 3, we concluded that risperidone and quetiapine when administered chronically to rats have the potential to adversely affect neurotrophin-related signaling molecules that support synaptic plasticity and cognitive function. These data would suggest that the extensive prescribing of these APs across multiple conditions in patients ranging in age from the very young to the very old should be carefully reexamined. Key Words: antipsychotic, cognition, brain volume, schizophrenia, neurotrophin
    • ENCODING OF EPISODIC EVENTS BY THE RETROSPLENIAL CORTEX

      Fox, Grace Ellen; Department of Neuroscience and Regenerative Medicine (8/7/2018)
      Fear is an adaptive response that permits organisms to reduce or avoid danger. In many animal species, behavioral correlates of fear can be seen, which highlights its essential role in survival; conversely, inappropriate and exaggerated fears are a hallmark of psychiatric disorders, such as anxiety-related disorders. Recently, the retrosplenial cortex (RSC) has been implicated for long-term storage of fear-related memories. However, the basic physiological properties of RSC cells as well as their response to fearful events remained largely unexplored. Our experiments demonstrate that excitatory principal cells within RSC layers 2&3 and 5&6 contain multiple physiologically distinct sub-populations that are differentially affected by ketamine. We also demonstrate the novel finding that the RSC utilizes the specific-to-general cell-assembly logic for processing neural information about fearful experiences. Taken together, these results illustrate that the RSC generates a cell assembly-level representation of fear memory engrams and supports the pursuit of the RSC as a therapeutic target for anxiety-related disorders.
    • THE TUMOR SECRETORY FACTOR ZAG PROMOTES WHITE ADIPOSE TISSUE BROWNING AND ENERGY WASTING IN CACHEXIA

      Elattar, Sawsan; Department of Biochemistry and Molecular Biology / Cancer Center (8/7/2018)
      SAWSAN ELATTAR The Tumor Secretory Factor ZAG Promotes White Adipose Tissue Browning and Energy Wasting in Cachexia (Under the direction of SATYANARAYANA ANDE) Cachexia is a complex tissue-wasting syndrome characterized by inflammation, hyper-metabolism, increased energy expenditure and anorexia. Browning of white adipose tissue (WAT) is one of the significant factors that contribute to energy wasting in cachexia. Tumors secrete an array of secretory factors, such as tumor necrosis factor α (TNFα), interleukin-1 (IL-1), interleukin-6 (IL-6), interferon γ (IFNγ) and zinc-α2-glycoprotein (ZAG), that have been implicated in altering metabolism and promoting cachexia. Previous studies have demonstrated that ZAG can induce lipolysis; however, whether ZAG plays a role beyond lipolysis remains unclear. Here, by utilizing a cell implantation model, we demonstrate that the lipid-mobilizing factor, ZAG, induces WAT browning in mice. Increased circulating levels of ZAG not only induced lipolysis in the adipose tissues, but also caused robust browning in the WAT. Stimulating white adipose progenitors with ZAG recombinant protein or expression of ZAG in mouse embryonic fibroblasts (MEFs) strongly enhanced brown-like differentiation. At the molecular level, ZAG stimulated peroxisome proliferator-activated receptor gamma (PPARƔ) and early B cell factor 2 (Ebf2) expression and promoted their recruitment to the PR/SET domain 16 (Prdm16) promoter, leading to enhanced expression of Prdm16, which determines brown cell fate. In the brown adipose tissue (BAT), ZAG stimulated the expression of PPARƔand peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and promoted recruitment of PPARƔ to the uncoupling protein 1 (UCP1) promoter, leading to increased expression of UCP1. Collectively, by promoting WAT browning and by activating thermogenesis in the BAT, ZAG increased body energy expenditure. Overall, our results revealed a novel function of ZAG in WAT browning and highlight that targeting ZAG may have therapeutic applications in humans with cachexia. KEYWORDS: (Cachexia, beige adipocyte, brown fat, adipose atrophy, Zinc-α2-glycoprotein, Ebf2, Prdm16, PPARƔ, UCP1)
    • Canonical Wnt Signaling in Antigen Presenting Cells Regulates Microbiota-Induced Inflammation and Immune Cell Homeostasis in the Colon

      Swafford, Daniel Joseph; Department of Biochemistry and Molecular Biology / Cancer Center (8/3/2018)
      Aberrant Wnt/β-catenin-signaling occurs in several inflammatory diseases including inflammatory bowel disease (IBD) and IBD-associated colon carcinogenesis. However, its role in shaping mucosal immune responses to commensals in the gut remains unknown. Here, we investigated the importance of canonical Wnt signaling in CD11c+ antigen presenting cells (APCs) in controlling intestinal inflammation. Using a mouse model of ulcerative colitis, we demonstrated that canonical Wnt-signaling in intestinal CD11c+ antigen presenting cells (APCs) controls intestinal inflammation by imparting an anti-inflammatory phenotype. Genetic deletion of Wnt co-receptors, low-density lipoprotein receptor-related protein 5 and 6 (LRP5/6) in CD11c+ APCs in mice (LRP5/6ΔCD11c mice) resulted in enhanced intestinal inflammation with increased histopathological severity of colonic tissue. This was due to microbiota-dependent increased production of pro-inflammatory cytokines and decreased expression of immune regulatory factors such as IL-10, retinoic acid (RA), and IDO. In addition, loss of LRP5/6-mediated signaling in CD11c+ APCs resulted in altered microflora and T cell homeostasis, which led to a loss of systemic tolerance to oral antigen. Furthermore, our study demonstrates that conditional activation of β-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice resulted in reduced acute intestinal inflammation with decreased histopathological severity of colonic tissue. Loss of canonical Wnt signaling in CD11c+ APCs also results in an increase in colonic polyp formation and exacerbation of chronic inflammation/injury. This was also heavily dependent on the presence and composition of the gut microbiota, as fecal transfers from LRP5/6ΔCD11c mice to floxed control (LRP5/6FL/FL) mice that were administered an antibiotic cocktail produces a polyp load and weight loss similar to that of LRP5/6ΔCD11c mice without treatment. Additionally, our study demonstrates that conditional activation of β-catenin in CD11c+ APCs in LRP5/6ΔCD11c mice reduces severity of inflammation-associated colon carcinogenesis in these mice. Furthermore, we show that treatment of LRP5/6ΔCD11c mice with either RA or IL-10 reduces severity of inflammation-associated colon carcinogenesis. Mechanistically, RA and IL-10 may independently reduce key inflammatory factors at the acute phase of colitis. These results ultimately reveal a mechanism by which intestinal APCs control intestinal inflammation and immune homeostasis via the canonical Wnt signaling pathway, which may serve as a promising target for chronic inflammatory disorders.
    • Mathematical and Stochastic Modeling of HIV Immunology and Epidemiology

      Lee, Tae Jin; Department of Biostatistics and Epidemiology (8/3/2017)
      In HIV virus dynamics, controlling of viral load and maintaining of CD4 value at a higher level are always primary goals for the providers. In recent years, a new molecule was discovered, namely, eCD4-Ig, which mimics CD4 if introduced into the human body and has potential to change existing HIV virus dynamics. Thus, to understand dynamics of viral load, eCD4-Ig, CD4 cells, we have developed mathematical models by incorporating interactions between this new molecule and other known immunological, virological information. We further investigated model based speculations for management, and obtained the level of eCD4-Ig required for elimination of virus. Next, we built epidemiological model for HIV spread and control among discordant couple through dynamics of PrEP (Pre-exposure prophylaxis). For this, an actuarial assumptions based stochastic model is used to obtain the mean remaining time of couple to stay as discordant. We generalized single hook-up/marriage stochastic model to multiple hook-up/marriage model.
    • Polyfunctional CD4+ T cells synergize with chemotherapy to reprogram tumor metabolism towards a curative outcome

      Habtetsion, Tsadik Ghebreamlak; Department of Biochemistry and Molecular Biology (8/3/2017)
      CD4+ T cells are critical mediators of anti-tumor immunity. Accumulating evidence from preclinical and clinical studies suggests that tumor-reactive CD4+ T cells in adoptive T cell therapy (ACT) have the potential to effectively control tumor growth. In most ACT clinical settings, chemotherapeutic agents are used to induce an immunostimulatory milieu which facilitates the effector function of donor T cells. Although the efficacy of ACT has been well-established, currently only a fraction of patients with certain types of malignancy have benefited, highlighting the need for improved ACT strategies. Recent studies have revealed that the metabolic reprogramming by cancer cells attenuate antitumor immune response by imposing nutrient restrictions in the tumor microenvironment, which leads to defective T cell responses. In the current study, we set out to explore how Cyclophosphamide (CTX) and tumor reactive CD4+ T cells alter the metabolic features of cancer cells. By comparing the global metabolic profiling of tumors pre and post-treatment, we found that CXT+CD4 ACT elicited a metabolic catastrophe affecting multiple pathways critical for cancer progression. Particularly, CTX+CD4 ACT led to marked reduction in glutathione (GSH) levels, increased accumulation of reactive oxygen species (ROS) and oxidative DNA damage product in tumors. Importantly, administration of N-acetyl-L-cysteine diminished the curative effect of CTX+CD4 ACT. Moreover, pharmacological inhibition of GSH using Buthionine Sulfoximine (BSO) following CTX significantly delayed tumor growth in mice. Mechanistically, we found that TNFα synergized with chemotherapy to reduce intracellular GSH levels and promote ROS induction. TNFα enhanced cell death in chemotherapy pre-treated tumor cells and the cytotoxic effect was reversed by adding GSH exogenously. Importantly, the curative effect of CTX+CD4 ACT was abrogated after TNFα neutralization. Additionally, we found that CTX+CD4 ACT led to tumor vascular disruption causing hemorrhagic necrosis of tumors. IFNγR-deficient mice failed to reject tumor after CTX+CD4 ACT and had intact tumor vasculature. Collectively, our data reveal that tumor reactive CD4+T cells disrupt the redox homeostasis of cancer cells in TNFα-dependent manner. Whereas CD4+T cells derived IFNγ targeted tumor endothelial cells to cause vascular disruption and tissue ischemia. The combined action of these two cytokines leads to eventual eradication of established tumor.
    • Dopamine Regulation of Fear Processing and Social Motivation: Implication for Common Psychiatric Comorbidities

      Lee, Jason ChiaTse; Brain and Behavior Discovery Institute (8/3/2017)
      Psychiatric disorders such as post-traumatic disorders and schizophrenia often present with common comorbidities such as increased depression, anxiety, and decreased social motivations. However, the underlying neural circuit that may account for occurrence of multiple psychiatric comorbidities remained unidentified. The dopamine system has been known to play prominent roles regulating emotional states and motivations. We therefore hypothesized that alteration in the dopamine system may lead to comorbidities such as negative mood and social isolation commonly observed in many psychiatric disorders. In this thesis work, we first examined how the dopamine system processes known triggers of psychiatric disorders, such as fear-charged stimuli. We then examined how the dopamine system regulates normal social interactions as well as how an altered dopamine system affects social interactions
    • A modified bump hunting approach with correlation-adjusted kernel weight for detecting differentially methylated regions on the 450K array

      Daniel, Jeannie T; Department of Biostatistics and Epidemiology (8/3/2017)
      DNA methylation plays an important role in the regulation of gene expression, as hypermethylation is associated with gene silencing. The general purpose of this dissertation is the development of a statistical method, called DMR Detector, for detecting differentially methylated regions (DMRs) on the 450K array. DMR Detector makes three key modifications to an existing method called Bumphunter. The first is what statistic to collect from the initial fitting for further analysis. The second is to perform kernel smoothing under the assumption of correlated errors using a newly proposed correlation-adjusted kernel weight. The third is how to define regions of interest. In simulation, the method was shown to have high power comparable to Bumphunter, with consistently lower family-wise type I error rate, controlled well below the 0.1 FDR. DMR Detector was applied to real data and was able to detect one DMR that was not detected by Bumphunter.
    • A Modified Information Criterion in the 1d Fused Lasso for DNA Copy Number Variant Detection using Next Generation Sequencing Data

      Lee, Jaeeun; Department of Biostatistics and Epidemiology (8/3/2017)
      DNA Copy Number Variations (CNVs) are associated with many human diseases. Recently, CNV studies have been carried out using Next Generation Sequencing (NGS) technology that produces millions of short reads. With NGS reads ratio data, we use the 1d fused lasso regression for CNV detection. Given the number of copy number changes, the corresponding genomic locations are estimated by fitting the 1d fused lasso. Estimation of the number of copy number changes depends on a tuning parameter in the 1d fused lasso. In this dissertation, we propose a new modified Bayesian information criterion, called JMIC, to estimate the optimal tuning parameter in the 1d fused lasso. In theoretical studies, we prove that the number of change points estimated by JMIC converges the true number of changes. Also, our simulation studies show that JMIC outperforms the other criteria considered. Finally, we apply our proposed method to the reads ratio data from the breast tumor cell HCC1954 and its matched cell line provided by Chiang et al. (2009).
    • A Novel Function of ADP-Ribosylation Factor 1 in Prostate Cancer Cell Proliferation through Activating the Mitogen-Activated Protein Kinase Pathway

      Davis, Jason E.; Department of Pharmacology and Toxicology (8/23/2016)
      The enhanced activation of the mitogen-activated protein kinase (MAPK) Raf-MEK-ERK1/2 pathway directly correlates with the growth, androgen-independence, and poor prognosis of prostate cancer. However, the underlying molecular mechanisms remain poorly understood. Here, we have demonstrated that ADP-ribosylation factor 1 (ARF1), a Ras-like small GTPase, was highly expressed in human prostate cancer cells and tissues. In addition, ARF1 was markedly activated in prostate cancer cells. More interestingly, oncogenic G protein-coupled receptors (GPCRs) strongly activated ARF1 and the activation was mediated through Gβγ subunits. These data indicate that GPCRs and heterotrimeric G proteins are the upstream activators of ARF1 in prostate cancer cells. Next, we determined the role of ARF1 in the MAPK activation and proliferation in prostate cancer cells. Lentiviral-mediated overexpression of ARF1 remarkably enhanced, whereas shRNA-mediated depletion of ARF1 dramatically reduced ERK1/2 activation in prostate cancer cells. In addition, disruption of both the Golgi localization of ARF1 and the Golgi structure substantially attenuated ERK1/2 activation in prostate cancer cells. In parallel with their effects on the MAPK activation, ARF1 overexpression greatly enhanced and ARF1 knockdown inhibited the proliferation of prostate cancer cells. These data suggest that ARF1, by its ability to activate the MAPK pathway likely at the Golgi, controls prostate cancer cell proliferation. We then investigated the consequence of pharmacologically inhibiting ARF1 activation. Small molecule inhibitors including brefeldin A, golgicide A, and Exo2 that specifically target Golgi-localized ARF1 markedly reduced both ERK1/2 activation and proliferation in prostate cancer cells. These results further indicate an important role of ARF1 activation in regulating the MAPK pathway and prostate cancer cell proliferation. Altogether, our data suggest a possible GPCR-G-ARF1-MAPK signaling pathway, which may be responsible for the hyperactivation of the MAPK ERK1/2 in prostate cancer and contributes to prostate cancer progression. Our results also imply a novel approach for prostate cancer therapy by targeting ARF1 activation
    • Calpain-2 Activates Akt via the TGF~ 1-mTORC2 Pathway in Pulmonary Artery Smooth Muscle Cells

      Abeyrathna, Prasanna; Deparment of Pharmacology and Toxicology (8/23/2016)
      Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to the AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibition of calpain-2 using the calpain inhibitor MDL28170 and calpain-2 siRNA attenuated Akt phosphorylation at serine-473 (S473) and threonine-308 (T308) as well as collagen synthesis and cell proliferation ofPASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylation at S4 73 and T308. Moreover, knockout of calpain attenuated Akt phosphorylation at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable specific TGF~ receptor inhibitor SB431542 attenuated Akt phosphorylation at both S473 and T308 induced by PDGF and overexpressed calpain- 2 in PASMCs. Moreover, SB-431452 and knock down of ALK5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGF~l using a cell-impermeable TGF~l neutralizing antibody did not affect PDGF-induced Akt phosphorylation at S473 and T308. Further, inhibition of mTORC2 by knocking down its component protein Rictor prevented Akt phosphorylation at S473 and T308 induced by PDGF and overexpressed calpain-2. These data provide the first evidence supporting that calpain-2 up-regulates PDGF-induced Akt phosphorylation via an intracrine TGF~ 1/mTORC2 mechanism.
    • The Role of Toll-Like Receptor 4 in the Secondary Pathogenesis of Spinal Cord Injury

      Fessler, David R.; Department of Neuroscience and Regenerative Medicine (7/31/2016)
      Spinal cord injury (SCI) is a leading cause of paralysis and disability worldwide, however no successful treatments have been developed partly due to a lack of understanding of SCI pathophysiology. This study established a model of compression SCI for investigation of the proinflammatory receptor, toll-like receptor 4. The model was validated measuring motor function, lesion size, Fluoro-Jade B staining, and western blots of PARP cleavage and 3-nitrotyrosine. Western blots and immunohistochemistry showed a biphasic upregulation of TLR4 in neurons after injury with peaks at 6 and 48 hours post-SCI. TLR4 mutant mice showed diminished lesion size, Fluoro-Jade B and hydroethidine staining, as well as improved motor function; and pre-treatment with TLR4 antagonist, VGX-1027, in wild type (WT) mice improved motor function as well. GP91 expression was unchanged in TLR4 mutants, suggesting changes in superoxide production may be NOX2 independent. This study suggests that targeting TLR4 may be viable therapeutic strategy for the repair/treatment of SCI.
    • Identifying the Function of Nasal Embryonic LHRH factor (NELF) in Immortalized GnRH Neurons

      Ko, Eun Kyung; Department of Neuroscience and Regenerative Medicine (7/30/2018)
      Hypothalamic gonadotropin releasing hormone (GnRH) is crucial for the proper function of the hypothalamic-pituitary-gonadal (HPG) axis, puberty, and reproduction. When GnRH neuron migration or GnRH regulation is impaired, hypogonadotropic hypogonadism results. Mutations in the gene for nasal embryonic LHRH factor (NELF) have been identified in GnRH-deficient humans. NELF is a predominantly nuclear protein that may participate in gene transcription, but it is unlikely to be a transcription factor. Thus, our hypothesis is that NELF may indirectly regulate transcription via protein-protein interaction within a transcription complex. To address this question, RNA was extracted from NLT GnRH neuronal cells following either stable Nelf knockdown or scrambled control and subjected to cDNA arrays. Expression of transcription factors and cell migration gene expression was most commonly altered. Members of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, including Stat1, Stat2, Stat5a, Jak2, Irf7 and Irf9, were significantly down-regulated as assessed by RT-qPCR. Protein levels of STAT1, phospho-STAT1, and JAK2 were reduced, but the levels of phospho-JAK2 were not. These findings suggest a role for NELF in the regulation of the JAK/STAT signaling pathway, which has important functions in GnRH neurons. Jacob, the rat orthologue of NELF, is phosphorylated at the serine 180 residue by phosphorylated Erk1/2 which is activated by synaptic NMDA receptor activation and then translocates to the nucleus. Phosphorylated Jacob in the nucleus interacts with CREB and regulates the expression of BDNF, which is associated with synaptic plasticity in the brain. Proteins, such as caldendrin, importin-α and α-interxin, have been identified as binding proteins with Jacob. However, binding proteins, phosphorylation sites and/or kinases for NELF have not yet been reported. To demonstrate novel and putative functions of NELF in the nucleus, identification of binding proteins and phosphorylation sites is required. To address this question, co-immunoprecipitation (Co-IP), mass spectrometry and in vitro kinase assays were performed. We found six putative binding proteins that could interact with NELF, including 14-3-3ε. We also identified phosphorylation sites on NELF, including serine 288, which could be phosphorylated by AKT1 kinase. These new findings will be helpful to understand the function of NELF in the nucleus
    • NAE1-MEDIATED NEDDYLATION IS REQUIRED FOR POSTNATAL LIVER DEVELOPMENT AND FUNCTION

      Sahay, Khushboo; Department of Physiology (7/26/2018)
      Liver disease is an important health concern and a significant source of morbidity and mortality in the United States and worldwide. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is a novel ubiquitin-like protein modifier. The conjugation of NEDD8 to target proteins, termed neddylation, requires NEDD8 specific E1, E2 and E3 ligases. Neddylation participates in various cellular processes. However, whether neddylation regulates liver development and function is completely unknown. We created mice with hepatocyte specific deletion of NAE1, a subunit of the only NEDD8 E1 enzyme, and identified that they display severe hepatomegaly, hypertriglyceridemia, and hypercholesterolemia from 10 days after birth. By postnatal 14 days, their liver cytoarchitecture is completely disrupted, along with formation of numerous biliary cysts, fibrosis and hypoglycemia, which ultimately result in liver failure and premature death by 6 weeks. Mechanistically, NAE1 deficiency in hepatocytes caused reduced hepatocytespecific gene expression but increased biliary/oval cell gene expression in liver. In vitro, NAE1 inhibition by MLN4924 and CRISPR/Cas9-mediated NAE1 deletion in HepG2 cells recapitulated in vivo findings with repressed expression of hepatocyte specific genes but elevated biliary/oval cell gene expression. Together, these data highlight an essential role for neddylation in regulating hepatocyte lineage commitment and function as well as polycyst formation through trans/de-differentiation of hepatocytes.
    • CXCR2 EXPRESSING TUMOR CELLS DRIVE VASCULAR MIMICRY IN ANTI-ANGIOGENIC THERAPY RESISTANT GLIOBLASTOMA

      Angara, Kartik Prasad; Department of Biochemistry and Molecular Biology (Augusta University, 7/20/2018)
      Glioblastoma (GBM) is a hypervascular and hypoxic neoplasia of the central nervous system with an extremely high rate of mortality. Owing to its hypervascularity, anti-angiogenic therapies (AAT) have been used as an adjuvant to the traditional surgical resection, chemotherapy, and radiation to normalize blood vessels, control abnormal vasculatures and prevent recurrence. The benefits of AAT have been transient and the tumors were shown to relapse faster and demonstrated particularly high rates of AAT-induced therapy resistance due to activation of alternative neovascularization mechanisms. Vascular Mimicry (VM) is the uncanny ability of tumor cells to acquire endothelial-like properties, lay down vascular patterned networks reminiscent of host endothelial blood vessels and served as an irrigation system for the tumors to meet with the increasing metabolic and nutrient demands in the event of the ensuing hypoxia resulting from AAT. In our studies, we have demonstrated that AAT accelerates VM. We observed that Vatalanib (a VEGFR2 tyrosine kinase inhibitor) induced VM vessels are positive for periodic acid-Schiff (PAS) matrix but devoid of any endothelium on the inner side and lined by tumor cells on the outer side. Interestingly, 20-HETE synthesis inhibitor HET0016 significantly decreased GBM tumors through decreasing VM structures both at the core and at the periphery of the tumors. During our extensive studies to understand the tumor-inherent mechanisms of AAT-induced resistance, we identified a crucial chemokine, CXCL8 or IL-8, to be highly upregulated in the GBM tumors treated with AAT. IL-8 has been well established as a highly prevalent cytokine in GBM with potent pro-migratory and pro-angiogenic functions. AAT-treated groups had significantly higher populations of CXCR2+ glioma stem cells and endothelial-like subpopulations and these populations were decreased following treatment with HET0016 and SB225002 (a CXCR2 antagonist). CXCR2+ GBM tumor cells were shown to form VM-like vascular channels carrying functional RBCs. Knocking down CXCR2 led to smaller tumor size in the animals and improperly developed vascular structures without CXCR2+ GBM cells lining them. This confirms our hypothesis that CXCR2+ GBM cells initiate VM and contribute to AAT resistance in GBM. Our present study suggests that HET0016 and SB225002 have potential to target therapeutic resistance and can be combined with other antitumor agents in preclinical and clinical trials.
    • Super enhancer-associated molecular signatures reveal a dependency on immune and metabolic mechanisms in chronic lymphatic leukemia

      Shull, Austin Y.; Department of Biochemistry and Molecular Biology (7/12/2016)
      Chronic lymphocytic leukemia (CLL), characterized by the progressive and uncontrolled accumulation of CD19+ B cells, currently remains as an incurable malignancy despite recent advancements in treatment options. The difficulties of eliciting curative measures in CLL are partly driven by the adaptability of the transcriptional response in CLL cells. In this study, we sought to better understand the complexities of the CLL transcriptional profile by defining the large histone H3 lysine-27 acetylation regions known as “super enhancers” within B cells and determining which genes overexpressed in CLL overlapped with super enhancers. From this analysis, we identified 190 super enhancer-associated genes overexpressed in CLL and determined that many of the genes identified were either involved in immune signaling cascades (e.g. LCK, FCER2) or metabolic regulation (e.g. LSR, ENO2). These processes corresponded with our reverse phase protein array (RPPA) profile of CLL patients, which shows overexpression of immune signaling kinases (e.g. LCK) as well as alteration of metabolically sensitive translation regulators (e.g. 4E-BP1 phosphorylation). Additionally, we determined that CLL cells are apoptotically sensitive to dual PI3K/mTOR inhibition when compared to upstream B cell receptor pathway inhibition due to their differential effects on 4E-BP1 phosphorylation. Based on the derived information from our super enhancer expression signature, we then compared the effects of preferentially targeting super enhancers with either the BET bromodomain inhibitor JQ1 or the cyclin dependent kinase-7 (CDK7) inhibitor THZ1. From this comparison, we saw that JQ1 could inhibit cell cycle progression in CLL cell lines as well as differentially disrupt transcription of genes involved in immune signaling. Contrastingly, we saw that THZ1 elicited a different response in CLL cells by inducing apoptosis and differentially downregulating genes involved in metabolism. The specific super enhancer-associated genes disrupted by the respective treatments further highlighted the dichotomy of JQ1 and THZ1-mediated effects, as JQ1 suppressed the B cell activation marker gene FCER2 whereas THZ1 suppressed the glycolytic enolase gene ENO2. Collectively, these results reveal that super enhancers play a role in mediating both immune signaling and metabolic expression signatures in CLL and that super enhancers can be differentially disrupted by BET bromodomain or CDK7 inhibition.
    • Evaluation of a Novel Compression Resistant Matrix for Recombinant Human Bone Morphogenetic Protein-2 (RHBMO-2) for Onlay Graft Indications

      Lu, Sheldon; Department of Oral Biology (6/4/2014)
      The discovery and subsequent characterization of endogenous signaling peptides known as bone morphogenetic proteins (BMPs) capable of inducing de novo bone formation in postfetal life represents a critical advancement in the understanding of tissue morphogenesis and has become an incentive to develop additional growth factor based tissue engineering strategies (Wozney & Seeherman 2004). Because BMPs act locally, a suitable carrier system must be used to ensure effective presentation of an adequate dose to a target site (Mont et al. 2004). A number of candidate biomaterials have thus been tested as potential carrier technologies (Huang et al. 2008). Currently, recombinant human BMP-2 (rhBMP-2) coupled with an absorbable collagen sponge (ACS) manufactured from bovine Achilles tendon Type 1 collagen is the only FDA approved device for orthopedic and craniofacial indications. Although the rhBMP-2/ACS construct has demonstrated clinical efficacy for indications including spine fusion, long bone fracture healing, sinus and alveolar augmentation, the ACS’s inability to resist tissue compression limits its use for onlay indications (Wikesjö et al. 2007).