• The c-MYC oncogene deregulates global DNA methylation and hydroxymethylation to control genome-wide gene expression for tumor maintenance in leukemia/lymphoma

      Poole, Candace Jean; Biomedical Sciences (Augusta University, 2019-05)
      Aberrant DNA methylation is a characteristic feature of tumor cells. However, our knowledge of how DNA methylation patterns are established and maintained to contribute to tumorigenesis is limited. Inactivation of the c-MYC oncogene triggers tumor regression in T-cell acute lymphoblastic leukemia (T-ALL) resulting in dramatic changes to the chromatin landscape including DNA methylation. In this study, I investigated how MYC regulates DNA methylation and hydroxymethylation patterns to contribute to gene expression programs important for tumor maintenance in T-ALL and Burkitt lymphoma. I report that MYC maintains 5-methylcytosine (5mC) and 5-hydroxy-methylcytosine (5hmC) patterns by regulating the DNA methylation machinery, which is important for gene expression in T-ALL. DNA methyltransferases (DNMTs) initiate 5mC marks, while Ten-eleven translocation methylcytosine dioxygenases (TETs) oxidize 5mC to produce 5hmC as an intermediate modification, ultimately leading to active DNA de-methylation. I demonstrated that DNMT1 and DNMT3B are MYC target genes and that their expression is dependent on high MYC levels. Knockdown of DNMT3B in T-ALL reduced cell proliferation through cell cycle arrest and caused the reactivation of gene transcription through reversing promoter/CpG island methylation. Furthermore, I demonstrated that TET1 and TET2 expression is MYC-dependent, as high TET1 and low TET2 levels depend on oncogenic MYC. Knockdown of TET1 in T-ALL reduced cell proliferation through cell cycle arrest and caused genome-wide changes in 5mC and 5hmC corresponding to changes in gene programs important for ribosomal biosynthesis and protein synthesis. In contrast, ectopic expression of TET2 reduced tumor cell proliferation through apoptosis/necrosis and caused genome-wide changes in 5mC and 5hmC corresponding to changes in transcriptional regulatory gene programs. My finding that a coordinated interplay between components of the DNA methylation machinery is necessary for MYC-driven tumor maintenance highlights the potential of targeting specific DNMT or TET proteins for therapeutic strategies.
    • 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.
    • CaMKIIβ association with F-actin in developing cortical neurons

      Lin, Yu-Chih; Department of Pharmacology and Toxicology (2008-08)
      Calcium/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase that is best known for its role in synaptic plasticity and memory. Although multiple roles of CaMKII have been identified in the hippocampus, its role in the developing cerebral cortex is less well understood. Immunostaining showed CaMKIIβ, but not CaMKIIα was expressed in embryonic day 18 (E18) cortical neurons at 4 days in vitro (DIV) and localized to a F-actin rich cytoskeletal structure we termed “microspike”. Further characterization of microspikes revealed that microspikes were composed of bundled actin, and were stable over time. Besides CaMKIIβ, several actin binding proteins, such as Arp3, cortactin and β1-integrin were also colocalized in microspikes. Fluorescence recovery after photobleaching (FRAP) analyses showed different dynamics of actin and CaMKIIβ in microspikes compared to dendrite spines. The colocalization of CaMKIIβ and F-actin in microspikes was dependent on the F-actin binding domain and the oligomerization domain. FRAP analyses confirmed the association of CaMKIIβ with F-actin in microspikes was via the F-actin binding domain. This association was altered by the co-expression of CaMKIIα. FRAP analyses with stabilized F-actin using jasplakinolide or cytochalasin-D further indicated CaMKIIβ, but not CaMKIIα, had a strong interaction with stable F-actin. Inhibiting calmodulin binding on CaMKII using a CaMKII inhibitor, KN93, dissociated CaMKIIβ from stable F-actin. Increasing CaMKIIβ activity with KCl or an active form of CaMKIIβ, CaMKIIβT287D, also dissociated CaMKIIβ from stable F-actin. A calmodulin binding mutant, CaMKIIβA303R, or a kinase dead mutant, CaMKIIβK43R, however, did not recover differently from wildtype CaMKIIβ. The differential binding of CaMKIIβ with F-actin shown in FRAP analyses correlated with CaMKIIβ enrichment in microspikes and the prominence of microspikes. While overexpressed CaMKIIβ increased the number of cells with microspikes, knockdown of CaMKIIβ with shRNA reduced it. Taken together, these data suggested that CaMKIIβ is associated with F-actin in cortical neurons, and this association is regulated by CaMKIIα and calcium signals contributing to the stability of microspikes.
    • Cancer Stressors and Protective Factors: Predictors of Stress Experienced During Treatment for Childhood Cancer

      Hockenberry-Eaton, Marilyn; Department of Physiological and Technological Nursing (1992-05)
      The purpose of this study was to evaluate cancer stressors and protective factors as predictors of stress experienced during treatment for childhood cancer. The conceptual framework evolved from the stress and coping literature and childhood cancer research. A convenience sample of 44 children between 6 ½ and 13 ½ years of age receiving treatment for cancer were evaluated during two clinic visits. Protective factors included the child’s self-perception, coping strategies, perceived social support, and family environment. Cancer stressors include acute stressors represented by the type of treatment received during two clinical visits. Chronic stressors were evaluated by the child’s perception of stressors related to the cancer experience. Responses to stressors were assessed by physiologic and psychologic indicators of stress. Physiologic measures include epinephrine, norepinephrine, and cortisol measures of urine and psychologic measures of state and trait anxiety. No significant differences were found in the physiologic or psychologic response to stressors in relation to the type of treatment received during either clinic visit. Epinephrine and norepinephrine were elevated for children during both clinic visits. Stepwise multiple regression analyses revealed that family expressiveness and the child’s perceived global self-worth were the best predictors of epinephrine levels. Family activities and recreation and family intellectual cultural orientation were the best predictors of state anxiety. The intensity of chronic cancer stressors, family activities and recreation, family intellectual cultural orientation, the child’s perception of physical appearance, and presence of family conflict had the greatest effect of trait anxiety. This study is the first to examine the child’s perception of chronic cancer stressors and protective factors associated with treatment for cancer. The findings provide insight into the importance of the interactions among the nature of the stressor, perceptual meaning of the stressor, and physiologic and psychologic responses to stressors that may affect long-term adjustment to childhood cancer.
    • 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.
    • Cardiovascular and behavioral changes associated with morphine abstinence in the physically dependent rat: a neuropharmacological study of both the spinal and supraspinal components of withdrawal

      Marshall, Dennis C; Department of Pharmacology and Toxicology (1984-10)
      ·.Physical depende.nce upon narcotics is, revealed in a characterfstic withdrawal syndrome of autonomic and behavioral changes evoked· by the administration of a narcotic antagonist. Although these changes have bee~ described, the mechanisms and locations within the central nervous system responsible for these effects are· unknown. My research was designed to develop an .objective animal model of dependency which could be used to identify mechanisms associated with narco.tic wi t.hdrawal. Cardiovascular and behavioral responses were ~valuat·ed following naloxone administration to·. freely moving rats made. physically dependent by a chronic intra-ar~ terial infusion of.morphine over 5 days. Naloxone evoked an elevation in mean arterial pressure (M·:AP) which· increased in a dose. and time dependent manner over the infusion schedule·whereas traditional behavioral signs of withdrawal were variable. Cardiovascular and behavioral signs of withdra~al also were e.lici ted by regional naloxone injection into various areas of the central nervo.us system including the spinal cord. Intrathecally administered, anticholinergic agents reduced the naloxone-induced increase in MAP along with some behavioral si.gns of wi thdr·awal. Spinal-t.ransected ( V.l), I dependent rats generated a profound hypertensive response to naloxone which was abolished·. by ganglionic or ct-adrenergic. blockade, spinal pithing or surgical dorsal root section. .Anticholinergic agents, intrathecally administered to spin~l-transected rats, resulted in an augmented hypertensive r~sponse to naloxone. These results indicate that 1) the naloxone-induced increase (MAP) in morphine dependent rats can serve as an objective and more sensitive measure of the degree of physical dependence than behavioral responses of withdrawal, 2) the spinal cord is capable of initiating cardiovascular and behavioral signs of withdrawal which can be modified by intrathecal administration of anticholinergic agents, 3) independent of supraspinal influences the spinal cord itself can generate a profound_ increase in mean arterial pressure which requires afferent information for its expression and, 4) at least two opposing cholinergic mechanisms which regulate cardiovascular activity exist with~n the spinal cord; one which is descending and facilitatory and another which is intrinsic and inhibitory.
    • Care-Cure Oreintations of Nurses Working in Critical Care and General Medical-Surgical Units

      Fisher, Barbara; Department of Nursing (1980-11)
      The purpose of this study was to examine .the attitudes of hurses working in crtttca1 care and medical-surgical units toward patient care and cure. A total populatinn·and random sample of 167 subjects (77 nurses working tn critical· care untts, 3 males and 74 females and 90 nurses working on medical-surgi'cal units,. 3 males and 87, ·females) was selected from three participating acute care tertiary general hospitals in urban Georgia. Data wer~collected using a self-administered valid and reliable reseatch instrument consisting of Linn's (1974) C~re-Cure Scale as modified by - . . _the· investigator, and a -demographtc data questionhaire -also de~el_oped by the investigator. The primary finding of the study indicated that for the population studied medical-surgical nurses were more care oriented than were critical care nurses. Critical care and generai iried·ica·'i-surgi'cal nur-scis dif·t-ercd s·ignificantiy (iJ< 0.01) ii1 or·ientation toward patient care and cure.
    • Caring for Their Mothers: The Experiences, Self-Care Practices, and Cultural Influences of African American Women Caregivers

      Chappell, Harriet E. H.; Department of Physiological and Technological Nursing (2007-07)
      The growth of minority older populations has created an increased need for African American family caregivers. African American women are at increased risk when the demands of caregiving are added to their existing health disparities. The purpose of this study was to understand the caregiving experiences of African American women, their ability to provide selfcare, and the influences of the African American culture on caregiving. Qualitative description was used to study 12 African American women caring for their mothers. Themes extrapolated from the data were commitment to care, self-care by the daughter caregivers, and the difficult times. An integrated pattern was also identified: Influences of African American Culture on caregiving. African American daughter caregivers have a profound commitment to provide the care needed by their mothers. The daughter caregivers’ self-care was balanced within and impacted by the demands and stresses of caregiving. The African American culture embodied expectations for daughter caregivers to provide care but also facilitated sources of support and strength.
    • Catching the Asthma: Family Caring For School-Aged Asthmatic Children

      Horner, Sharon D; Catching the Asthma: Family Caring for School-Aged Asthmatic Children (1992-04)
    • Catching the Asthma: Family Caring for School-aged Asthmatic Children

      Horner, Sharon D; Department of Physiological and Technological Nursing (1992-04)
      The purpose of this study was to explore the process of family caring in families that had a school-aged child with asthma. One focus of this study was to explore the impact of chronicity on the family, specifically looking beyond illness-management issues. Another focus of this study was to uncover the evolution of family caring within the context of school-aged developmental changes. Grounded theory was the research methodology used to discover the strategies, goals, and dimensions of family caring. The research questions used to begin this exploration were: "What is (are) the experience(s) of family caring?" "How does chronicity impact family caring?" These questions presented a number of avenues for exploration. The impact of chronicity on family caring has not previously been explored in depth. Management of the needs of the family member who has a chronic illness (care-taking) has been studied extensively. Various studies have identified the problems families experience related to care-taking tasks, meeting family members' needs, financial burden, stress, role overload, as well as other dynamics of family functioning. None of these studies have explored the emotive and commitment dimensions of caring (caring for and caring about). An exploration of illness-management in day-to-day living was certainly included in the interviewing process; however, this study extended the exploration of chronicity into all aspects of family life to uncover the dimensions of family caring. Specifically, "How do family members care for and care about each other, while taking care of self and others?"
    • The CB1 Cannabinoid Receptor: Receptor States, Activity and G Protein Sequestration

      Nie, Jingjiang; Department of Pharmacology and Toxicology (2001)
      The human CB1 cannabinoid receptor is a member o f the G protein coupled receptor family. The CB1 cannabinoid receptor couples to pertussis toxin sensitive Gi/o proteins and inhibits neuronal voltage-gated Ca2+ channels. The hCBl receptor has two unusual properties: 1) it is constitutively active in the absence o f agonist and 2) it can prevent other G protein coupled receptors from signaling by sequestering a common pool of Gi/o proteins. The mechanism o f constitutive activity and G protein sequestration by the hCB 1 receptor is unknown. In this study, two carboxyl terminal truncation mutants (hCB 1-417 and hCBl-400) were used to test the hypothesis that the proximal carboxyl terminal couples to G proteins while the distal carboxyl terminal modulates G protein sequestration and constitutive activity. Additionally, mutation o f a single amino acid in the second transmembrane domain (rCBl-D164N) was used to test the hypothesis that this amino acid plays a critical role in the structural basis o f G protein coupling and constitutive activity. Receptor cDNA constructs were injected into the nucleus of superior cervical ganglion neurons. After an overnight incubation to allow for receptor expression, neurons were voltage clamped and Ca2+ current were recorded. Inhibition of the Ca2r current by the cannabinoid agonist WIN 55,212-2 was used as an index o f CB1 cannabinoid receptor G protein coupling and activation. Ca2' channels are inhibited by G{3y subunits released from activated Gi/o proteins. In contrast to the wild type CB1 cannabinoid receptor, the mutant receptor in which the entire carboxyl terminal (amino acids 401-472) was deleted (hCB 1-400) failed to inhibit the Ca2+ current. Deletion o f only the distal carboxyl terminal (amino acids 418-472; hCB 1-417) restored Ca2+ current inhibition. These results demonstrate the critical role o f the proximal domain (amino acids 401-417) o f the carboxyl terminal of the hCB 1 receptor in coupling to G proteins. Truncation o f the distal carboxyl terminal domain, however, changed the magnitude o f Ca2+ current inhibition. The hCBl-417 receptor produced significantly less inhibition of the Ca2+ current in the presence o f WIN 55,212-2 compared to the wild type receptor (22.6±3.0% vs 43.7±6.5%, respectively). Thus, the distal carboxyl terminal domain is important in modulating the magnitude of Ca2* current inhibition. In addition to the change in the magnitude o f Ca2+ current inhibition, deletion o f the distal carboxyl terminal significantly slowed the kinetics o f Ca2l~ current inhibition by WIN 55,212-2 (time to peak o f effect: 146.0+8.7 second). The distal carboxyl terminal tail of the CB1 cannabinoid receptor also played a role in constitutive activity and G protein sequestration. The hCB 1-417 receptor displayed enhanced constitutive activity. In neurons injected with 50 ng/pl hCBl-417 cDNA the inverse agonist SR141716A increased the Ca2+ current 101.1±21.3%. The inverse agonist acts to reverse the constitutive activity o f the receptor. The effect o f SRI 41716 A on the hCBl-417 receptor was significantly greater than the 42.9±7.6% Ca2+ current increase in neurons expressing the wild type hCB 1 receptor. G protein sequestration was also enhanced in neurons expressing the truncated hCBl-417 receptor. Wild type hCBl cannabinoid receptors when expressed by injecting 100 ng/p.1 cDNA completely abolish signaling by other G protein coupled receptors including a 2 -adrenergic receptors. Normally activation o f a 2 -adrenergic receptors inhibits the Ca2+ current 44.5±5.7%. In the presence o f hCBl receptors activation of the a 2- adrenergic receptors by UK14304 inhibited the Ca2+ current only 1.5±4.2%. Signaling by a 2-adrenergic receptors can be partially restored by injecting a lower concentration o f hCBl cDNA. In neurons injected with 50 ng/pl hCBl cD N A the ct2 -adrenergic agonist UK14304 inhibited the Ca2+ current 20.0±3.7%. In neurons injected with 50 ng/pl hCBl- 417 cDNA UK14304 inhibited the Ca2+ current 7.0±1.2%. Thus, signaling by the a 2- adrenergic receptor was abolished by the carboxyl terminal truncated hCBl-417 receptor. These results indicate that deletion of the distal carboxyl terminal enhances the ability o f the receptor to sequester G proteins. The aspartic acid residue in the second transmembrane domain of G protein coupled receptors is highly conserved. Mutation o f this aspartic acid (rCBl-D164N) had profound effects on the constitutive activity o f the C B 1 receptor as well as on the ability of the receptor to sequester G proteins. Both the constitutive activity and the ability to sequester G proteins were abolished by the rCBl-D164N receptor. The inverse agonist increased the Ca2+ current only 11.6+6.9% in neurons expressing the mutant rC Bl- D164N receptors. The mutant rCBl-D164N receptors failed to block signaling by the a 2- adrengic receptor. UK14304 inhibited the Ca2+ current 35.8+6.8% in neurons expressing the rCBl-D164N receptors and was not different from control neurons. Additionally, the D164N mutation in the second transmembrane domain decreased the time to peak o f the WIN 55,212-2 inhibition o f the Ca2+ current to 24+4 seconds. These results demonstrate that 1) the proximal carboxyl terminal domain o f the hCB 1 receptor is critical for G protein coupling, 2) the distal carboxyl terminal domain regulates constitutive activity, G protein coupling kinetics and G protein sequestration and 3) the aspartic acid in the second transmembrane domain plays a critical role in G protein sequestration, G protein coupling kinetics and constitutive activity o f the hCBl receptor. Taken together, the mutant receptors shift the CB1 receptor into different receptor states. The D164N-CB1 receptor exists primarily in an inactive state uncoupled from G proteins, the wild type CB1 receptor exists in both active and inactive G protein coupled states and the carboxyl terminal truncated CB 1-417 receptor exists primarily in an active G protein coupled state.
    • Cellular and Immunocytochemical Response to Mandibular Distraction Using an Implanted Lengthening Device

      Elbokle, Nadar N; Department of Oral Biology (2004)
      Distraction osteogenesis (DO) is a biologic process that generates new bone between surfaces of bone segments, which are gradually separated by traction forces. It is a uniquely effective method with multiple applications in the craniofacial region. This concept has been the basis of all bone-lengthening operations; it involved an osteotomy of the shortened bone and an external/internal fixator device, which slowly elongates the bone to its new dimension while a bony callus is being formed at the side to distraction. The biology of DO is similar to callus fracture healing. The bony regenerate passes through the same phases: formation of a collagen fibril template, mineralization, bony union and finally remodeling. The mechanisms by which the mechanical stresses applied to the bone tissue cause the cells to proliferate and form new bone are not well understood. More studies are needed to understand the cellular events underlying DO and the effects of the strains applied during DO on cellular proliferation and mineral apposition.
    • Cellular and Molecular Mechanisms of Retinal Bipolar Regeneration in Zebrafish

      Ariga, Junko; Department of Pediatrics (2012-03)
      Human retinal degenerative diseases are characterized by slow progressive loss of retinal cells which induces reactive gliosis in Muller glia cells. In mammalian systems, this results in scar tissue formation which exacerbates loss of vision. Similar initial responses are observed following injury in highly regenerative species, such as zebrafish. However, Muller glia cells in these systems are capable of regenerating a functional retina. We are interested in determining how the regenerative potential of Muller glia cells is triggered and controlled. Thus, we are studying the cellular and molecular mechanisms governing how zebrafish regenerate specific retinal cell types. Ultimately, we seek to identify factors that could be harnessed to redirect mammalian Muller glia cells into regenerative pathways. Such insights could aid the development of regenerative therapies for degenerative diseases. Despite the relevance to disease, little is known about how the retina responds to loss of discrete cell types. Here, we focused on characterizing this paradigm to study four principle aspects of the regenerative process: 1) endogenous stem cell activation, 2) stem and progenitor cell proliferation, 3) progenitor cell differentiation, and 4) functional recovery. By studying all four of these aspects in relation to each other we were able to reveal fundamental insights into how retinal regeneration is governed. Specifically in Aim 1, we used transgenic and pharmacological techniques to induce ablation of /^-expressing retinal bipolar cell subtypes and asked whether the lost cells were subsequently regenerated in zebrafish larvae. We then sought to identify potential stem cell sources. In Aim 2, lineage tracing of retinal stem cell populations was used to ask whether the extent of bipolar cell loss altered the specificity of the regenerative response. In Aim 3, we manipulated the Wnt pathway to investigate the role of Wnt signaling in bipolar cell regeneration. Finally, in Aim 4 we used visual behavior assays to determine if functional deficits attend the loss of nyx-expressing bipolar cells and, if so, whether functional recovery was evident following their regeneration. In particular, our observations demonstrating opposing roles of the Wnt pathway in regeneration have implications regarding the development of age-appropriate and/or cell-specific regenerative therapies.
    • Cellular and Molecular Players in Neuromuscular Junction (NMJ) Formation and Function

      Barik, Arnab; Institute of Molecular Medicine and Genetics (2014-04)
      There are three distinct segments in this dissertation. First, I attempted to address the role of Schwann cells in mammalian neuromuscular junction (NMJ) development and function. Schwann cells at the NMJs do not form myelin sheaths and are known as terminal Schwann cells. Terminal Schwann cells are thought to be analogous to astrocytes in the central nervous system. Schwann cells (as described in details in the next section) provide trophic support to motor axons and modulate synaptic activity by sensing neurotransmitter release at the nerve terminal. However, the role of Schwann cells in synapse formation and maintenance remains unknown. Second, during NMJ formation, anterograde signals from nerve to muscle, and retrograde signals from muscle to nerve are critical for the establishment of a functional synapse. Research over the last three decades has contributed to our understanding of the role of the anterograde signaling at NMJ. However, identification of muscle-derived retrograde signals involved in motoneuron terminal differentiation remains scarce. Recent work from our laboratory suggests that genes that are transcriptionally regulated by p-catenin in muscles might play a crucial role in pre-synaptic differentiation at the NMJ.2 Third, Agrin-LRP4-MuSK signaling is critical for NMJ formation. At the NMJ, LRP4-mediated activation of MuSK by neural Agrin is required for post-synaptic differentiation. Mice that lack any one of the three genes fail to form NMJs and die at birth. Due to perinatal lethality of these null mice, less is known about how Agrin-LRP4-MuSK might regulate NMJ maintenance. Moreover, mutations in Agrin, LRP4, and MuSK have been reported in patients diagnosed with congenital myasthenic syndrome (CMS), and autoantibodies against MuSK and LRP4 have been detected in patients with myasthenia gravis (MG). However, the role of Agrin-LRP4-MuSK in the etiology of these neuromuscular disorders is not clear.
    • Ceramide Compartments and Protein Interaction: Structure Meets Function

      Kong, JiNa; Department of Neuroscience and Regenerative Medicine (12/27/2016)
      Ceramide is a key sphingolipid, regulating a variety of critical cellular processes. Although exosomes and cilia are derivatives of the membrane, little is known about the role of lipids in their formation. Here we examined the novel role of ceramide in two ceramide-enriched, subcellular compartments: 1) secreted, extracellular vesicles (EVs) termed exosomes, and 2) cell membrane protrusions termed cilia. Firstly, we attempted to address the role of ceramide in exosome secretion and breast cancer. Breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP). We show that incubation of human breast cancer MDA-MB-231 cells with the farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, which is known to induce exosome secretion. Ceramide elevation by combined treatment with gug and bex induced BCRP secretion in exosomes and reduced cellular BCRP in cancer and cancer stem-like cells. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex treatment increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death. Our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy. Secondly, to investigate the role of ceramide in ciliogenesis, in particular motile cilia, we used Chlamydomonas reinhardtii (Chlamydomonas) and murine ependymal cells as models. Motile cilia are specialized organelles formed by cell membrane protrusions to function in movement of body fluids. We show for the first time that Chlamydomonas expresses serine palmitoyl transferase (SPT), the first enzyme in the sphingolipid biosynthetic pathway. Ceramide depletion, by the SPT inhibitor myriocin and a neutral sphingomyelinase deficiency (fro/fro mouse), led to glycogen synthase kinase-3 (GSK3) dephosphorylation and defective flagella and cilia, respectively. A novel activation mechanism for GSK3 by the sphingolipids phytoceramide and ceramide is shown to be critical for ciliogenesis in Chlamydomonas and ependymal cells, respectively. We conclude that ceramide promotes exosome secretion to reduce MDR in MDA-MB-231 cells and regulates GSK3-mediated ciliogenesis in Chlamydomonas and murine ependymal cells.
    • Ceramide-mediated Regulation of Cell Polarity in Primitive Ectoderm Cells: A novel role for sphingolipids in morphogenesis

      Krishnamurthy, Kannan; Institute of Molecular Medicine and Genetics (2009-01)
      Ceramide is considered a key sphingolipid, regulating a variety of critical cellular processes. To facilitate the study of ceramide localization and its interaction with cellular proteins, we have developed a novel antibody against ceramide, raised in rabbit (rabbit IgG). The novel antibody specifically recognizes ceramide in lipid overlay assays and detects ceramide containing different fatty acid chain lengths (i.e. C2-, C16-, C18-, C20- and C24 ceramide). The new antibody was compared with the commercially available anti-ceramide mouse IgM antibody in immunocytochemistry experiments to study the localization of ceramide. Although both antibodies stain similar regions on the cell membrane, the rabbit IgG reveals the distribution of ceramide in intracellular compartments that are not well identified with the commercially available antibody. Pharmacological depletion or increase of ceramide levels results in a corresponding change in staining intensity, confirming the specificity of the antibody. These results indicate that the rabbit IgG is a suitable antibody to determine both the localization of ceramide, and its interaction with proteins by immunocytochemistry. To investigate the role of ceramide in early embryonic development, we used embryoid bodies (EBs) differentiated from mouse embryonic stem cells as a model. The primitive ectoderm cell layer of EBs represents the primitive ectoderm of the early embryo. In mammals, the primitive ectoderm is an epithelium of polarized cells that undergoes gastrulation and differentiates into all embryonic tissues. We find that in primitive ectoderm cells, ceramide was elevated and asymmetrically distributed to the apico-lateral cell membrane, where it was co-distributed with Cdc42 and F-actin. Pharmacological or siRNAmediated inhibition of ceramide biosynthesis impaired primitive ectoderm formation and concomitantly increased apoptosis in EBs. Primitive ectoderm formation was restored by incubation with ceramide or a ceramide analog, indicating that the observed defect was due to loss of ceramide. Ceramide depletion also prevented membrane translocation of atypical PKC (aPKC), interaction of aPKC with Cdc42, and phosphorylation of GSK-3|3. Recombinant aPKC, when bound to ceramide-containing lipid vesicles, formed a complex with the polarity protein Par6 and Cdc42. Taken together, our data suggest a novel mechanism by which a ceramide-induced, apico-lateral polarity complex with aPKC regulates primitive ectoderm cell polarity and morphogenesis.
    • Changes in parental perceptions of stress in a pediatric intensive care units

      Sharp, Emily A.; School of Nursing (1985-05)
      . Pare~tal perceptions of stress associated wfth having a thild hospitalized in a Pediatric Intensive Care Unit {PICU) were studied. The Parental Stressor Scale: Pediatric Intensive Care Unit .{Miles & Carter, 1983) and a Demographic Data Sheet were used to assess nineteen parents• stress who had a child hospitalized in a PICU of a southwestern hospital {n = 19). Results of. the study indicated that parental perceptions of stress did not significaritly differ at three different times during their child's hospitalization in a PICU. The parental stress was assessed at the beginning, at the midpoint, and ·at the end of their child's hospitalization in the PICU. The parental stress was also analyzed for correlation with the child's age, admi~sion status, and parent's educational level. The parental stress was significantly greater if the. child's admission to-the PICU was unplanned., The study provides I some interesting data to be considered by profess-ionals caring for parents with a child hospitalized in a PICU.
    • Changes in the RANK/RANKL/OPG Signaling System as a Mechanism of Zoledronate-Induced Osteonecrosis of the Jaw

      Lane, Jonathan; Department of Oral Biology (3/22/2016)
      Bisphosphonates (BPs) are widely used for the treatment of osteoporosis, hypercalcemia of malignancy, skeletal-related events associated with bone metastases, and for managing lytic lesions of multiple myeloma. A serious risk associated with the use of BPs is the development of Bisphosphonate Related Osteonecrosis of the Jaw (BRONJ), a painful and inflamed area of exposed bone in the oral cavity that fails to heal after 6-8 weeks. The cause of BRONJ is unknown, but it is believed to be due primarily to a longterm suppression of bone remodeling, caused by BP’s potent inhibition of osteoclastic activity. At the cellular level, it is generally accepted that bisphosphonates are taken in by osteoclasts at sites of relatively greater bone remodeling, owing to the strong affinity of bisphosphonates for the mineralized matrix and the increased activity of osteoclasts at active sites of resorption. The accumulation of intracellular bisphosphonates ultimately leads to osteoclast dysfunction or apoptosis through the formation of nonhydrolyzable ATP-analogues, or due to inhibition of the mevalonate pathway responsible for synthesis of sterols and lipids necessary for proper cellular membrane structure. However, the refined details of the pathophysiology of BRONJ remain elusive. The RANK/RANKL/OPG system is a well-known signaling pathway for the recruitment and differentiation of osteoclasts. RANK is a surface-bound receptor on osteoclasts, and requires binding of its ligand, RANKL, for cell activation and ultimately resorption of bone. On the other hand, OPG is a soluble decoy receptor for RANKL. Therefore, osteoclastic activity is effectively regulated by the ratio of RANKL to OPG. For years, it has been generally accepted that osteoblasts are the primary source of both RANKL and OPG. However, it is now recognized that the master orchestrator of bone activity, the osteocyte, contributes to the pathway. Furthermore, it has been shown that in localized tissue damage or hypoxia, such as in a dental extraction, immediately adjacent surviving nonapoptotic osteocytes upregulate RANKL and downregulate OPG. It is unknown to what extent BPs may alter the normal osteocyte response to injury and hypoxia or, ultimately, the dynamics of the RANK/RANKL/OPG system. Furthermore, the extent to which this could contribute to the development of BRONJ is unexplored.There is a paucity of studies concerning how the fundamental system responsible for bone remodeling, RANK/RANKL/OPG, is effected by BPs. It may be that changes in this system, especially in signals derived from the osteocyte, contribute to the pathophysiology of BRONJ.