• B-Lymphoid Cells with Attributes of Dendritic Cells Regulate T Cells via Indoleamine 2,3 Dioxygenase

      Johnson, Burles Avner III; Cancer Research Center (2012-04)
      A rare subset of murine dendritic cells expressing the B cell marker CD19 are specialized to express the T cell regulatory enzyme indoleamine 2,3 dioxygenase (IDO-competent DCs). Here we show that IDO-competent DCs expressed Pax5, a transcription factor that maintains B cell lineage commitment, and drives expression of CD19 and surface immunoglobulin (slg). However IDO-competent DCs also exhibited multiple attributes of DCs including DC marker expression and potent T cell stimulatory properties when IDO was not induced. Unexpectedly, DCs expressing IDO were present in B cell deficient mice following TLR9 ligation, indication that B cell receptor (BCR) expression was not required for IDO function. Conversely, DCs from CD19 deficient mice did not express IDO after in vivo TLR9 ligation. This defect was not caused by blockade of IDO-competent DC development in CD19-deficient mice because IDO expression was incduced in these cells by in vitro interferon gamma treatment. Even though DCs from B cell deficient mice expressed IDO following TLR9 ligation, regulatory T cells (Tregs) from B cell deficient mice had impaired suppressor activity. IDO-competent DCs expressed high levels of CD1d-deficient mice. IDO-competent DCs also expressed IL-10 deficient mice to express IDO. Finally we demonstrated that DCs from draining lymph nodes (dLNs) of four week old prediabetic female non obese diabetic (NOD) mice expressed functional IDO following topical treatment with phorbol myristate acetate (PMA). However DCs from dLNs of six week old prediabetic NOD female mice did not express IDO following topical PMA treatment, indicating a critical defect in a specific immunosuppressive mechanism in NOD female mice that coincides with the appearance of insulitis. These data identify IDO competent DCs as a unique B lymphoid lineage cell type that has tightly controlled regulatory properties, and a DC subset whose acquired defect may contribute to autoimmune disease in NOD mice.
    • A Bayesian Framework To Detect Differentially Methylated Loci in Both Mean And Variability with Next Generation Sequencing

      Li, Shuang; Department of Biostatistics and Epidemiology (2015-07)
      DNA methylation at CpG loci is the best known epigenetic process involved in many complex diseases including cancer. In recent years, next-generation sequencing (NGS) has been widely used to generate genome-wide DNA methylation data. Although substantial evidence indicates that di erence in mean methylation proportion between normal and disease is meaningful, it has recently been proposed that it may be important to consider DNA methylation variability underlying common complex disease and cancer. We introduce a robust hierarchical Bayesian framework with a Latent Gaussian model which incorporates both mean and variance to detect di erentially methylated loci for NGS data. To identify methylation loci which are associated with disease, we consider Bayesian statistical hypotheses testing for methylation mean and methylation variance using a twodimensional highest posterior density region. To improve computational e ciency, we use Integrated Nested Laplace Approximation (INLA), which combines Laplace approximations and numerical integration in a very e cient manner for deriving marginal posterior distributions. We performed simulations to compare our proposed method to other alternative methods. The simulation results illustrate that our proposed approach is more powerful in that it detects less false positives and it has true positive rate comparable to the other methods.
    • Bayesian Functional Clustering and VMR Identification in Methylation Microarray Data

      Campbell, Jeff; Department of Biostatistics and Epidemiology (2015-07)
      The study of the relation between DNA and health and disease has had a lot of time, energy, and money invested in it over the years. As more scientific knowledge has accumulated, it has become clear that the relations between DNA and health isn’t just a function of the sequence of nucleotide bases, but also on permanent modifications of DNA that affect DNA transcriptions and thus have a macroscopic effect on an individual. The study of modifications to DNA is known as epigenetics.Epigenetic changes have been shown to play a role in certain diseases, including cancer (Novak 2004). Finding locations of differential methylation in two groups of cells is an ongoing area of research in both science and bioinformatics. The number of developed statistical methods for establishing differential DNA methylation between two groups is limited (Bock 2012). Many developed methods are developed for nextgeneration sequencing data and may not work for microarray data, and vice versa. Bisulfite sequencing, the next-generation sequencing technique for attaining methylation data, often comes with limited sample size and considerations must be made for low and variable coverage, and smoothing the methylation values. The analysis of nextgeneration sequencing data also involves small sample sizes.In addition, these methods can be sensitive to how individual CpG regions are grouped together as a region for analysis. If the DMRs are small relative to the sizes of 5 established regions, then the method may not detect a region as having differential methylation. Robust methods for clustering microarray data have also been an ongoing area of research. It is desirable to have a method that could be applied to microarray data could increase the sample size and mitigate the previous problems if the method used is robust to missing values, outliers, and microarray data noise. Functional clustering has shown to be effective when properly conducted on gene expression data. It can be used when the data have temporal measurements to identify genes that are possibly co-expressed. The clustering of methylation data can also be shown to identify epigenetic subgroups that can potentially be very useful (Wang, 2011). [introduction]
    • Being Homeless: An Ethnographic Study of Women's Experiences

      Hdnicki, Donna R; Department of Physiological and Technological Nursing (1992-04)
      This field research used ethnographic techniques to study women's experiences of homelessness while living in a shelter. A feminist approach which values women and the knowledge that women can share provided an orientating framework for this study. Data were collected by means of participant observation and in-depth, semi-structured interviews with 23 homeless women living in a shelter. A constant comparative analysis of the data yielded two major domains of the experiences of homelessness: Disconnected— Loss of Major Support and Rebuilding— The Regrouping of Assets. Themes within the first domain included disaffiliation, significant loss, homelessness hurts, facing uncertainty, and being pressured. Themes within the second domain were heightened awareness, making adjustments, living with limitations, a period of growth, and taking a proactive stance. A model of the experiences of womens' homelessness in a shelter was developed. Women experience vulnerability throughout the homeless experience, but it is most intense when the women are disconnected from major sources of support. Vulnerability lessens as the women begin to rebuild their lives. The women in this study exhibited a proactive behavior during Rebuilding which has not previously been described in the literature. The shelter used by the women in this study provided a "resource rich" environment that undoubtedly contributed to the women's proactivity and to Rebuilding.
    • Bioactivity and Mechanism of Action of Resveratrol, A Polyhenolic Phytoalexin, in Sickle Cell Disease

      Agyekum, Davies G.; Department of Anesthesiology and Perioperative Medicine (2013-07)
      The primary goal of this research was to evaluate the fetal hemoglobin (HbF)- inducing efficacy of resveratrol (RSV), a plant polyphenol, in relevant in vitro systems, a preclinical mouse model of sickle cell disease (SCD) and to determ ine its mechanism of action. An overview of the public health impact of SCD, the molecular pathogenesis of the disease and our current understanding of the fetal-to-adult hemoglobin switch are discussed in detail below. Additionally, the roles of erythroid Kruppel-like factor 1 (KLF1) and B-cell lymphoma 11A (BCL11A) in silencing the gene responsible for fetal hemoglobin are discussed, followed by a description of experimental models used to study sickle cell disease. Lastly, the properties and applications of RSV in relevant disease models are discussed, followed by a description of the objectives and specific aims of this study.
    • Biosynthesis and Modification of Helicobacter pylori Lipid A

      Stead, Christopher Michael; Department of Biochemistry and Molecular Biology (2010-05)
      The secondary acylation steps of Helicobacter pylori lipid A biosynthesis are poorly understood because H. pylori only has one homolog (Jhp0265) to the Escherichia coli secondary acyl transferases LpxL and LpxM. Jhp0265 was shown to be responsible for the transfer of a secondary C18 acyl chain to the 2′-linked acyl chain of lipid A, making Jhp0265 homologous to LpxL. An activity was also demonstrated for the addition of a secondary acyl chain to the 3′-linked acyl chain of H. pylori lipid A, although the enzyme responsible for the transfer remains unknown. After synthesis, H. pylori lipid A is modified by the action of five enzymes. Mutation of the candidate modification enzyme Jhp0634 demonstrated that the enzyme catalyzes the removal of the 3′-linked acyl chains of H. pylori lipid A, producing a tetra-acylated lipid A species. Continuing with the characterization of H. pylori lipid A modification enzymes, we were also able to demonstrate an activity for a Kdo trimming enzyme in vitro. Requirement for a Kdo hydrolase in vivo was confirmed after the Kdo transferase of H. pylori was shown to be bifunctional despite the presence of only one Kdo sugar in H. pylori lipopolysaccharide. Attempted identification of the Kdo hydrolase revealed that both Hp0579 and Hp0580 were required for the removal of the Kdo sugar, which occurred in the periplasm. A Kdo hydrolase mutant revealed two unexpected phenotypes related to interaction with the innate immune system. The first was an increased sensitivity to cationic antimicrobial peptides, which was explained by a downstream effect on modification to the 4′- phosphate group of lipid A. The second phenotype related to the expression of Oantigen on the bacterial cell surface. The Kdo hydrolase mutants produced a reduced amount of fully extended lipopolysaccharide and conversely, an increased amount of core-lipid A. The type of O-antigen epitope displayed was also affected by a Kdo hydrolase mutation, in a strain specific manner.
    • Biosynthesis of the Vibrio cholerae Kdo-lipid A Domain and its Role in Pathogenesis

      Hankins, Jessica V.; Department of Biochemistry and Molecular Biology (2011-05)
      Bacteria assemble remarkable surface structures that interface with their surrounding environment. One such structure is the glycolipid lipopolysaccharide (LPS) that covers the surface of Gram-negative bacteria. LPS is anchored to the bacterial cell by its lipid anchor known as lipid A. Since lipid A is the bioactive component of LPS, modulation of its structure can have a profound impact on disease by altering the host immune response. Additionally, LPS structure directly impacts the outer membrane permeability barrier and bacterial resistance to host antimicrobial peptides. Although the lipid A domain of Escherichia coli has been well characterized, the Vibrio cholerae lipid A biosynthetic pathway has received little attention. The late stages of lipid A biosynthesis include the transfer of the 3-deoxy-Dmanno- octulosonic acid (Kdo) sugars and the secondary acyl chains to the lipid A backbone. Here, the V. cholerae Kdo transferase (Vc0233) was shown to be monofunctional, transferring one Kdo residue to the lipid A precursor, lipid IVA. V. cholerae encode a Kdo kinase (Vc0227) responsible for the phosphorylation of the Kdo residue. The functionality of Vc0227 was shown to be required for the activity of the V. cholerae lipid A LpxL homologue, Vc0213. Interestingly, the addition of the phosphate group on the Kdo sugar was shown to be essential for lipid A secondary acylation in Haemophilus influenzae and Bordetella pertussis. Vc0213 was shown to catalyze the transfer of a myristate (C14:0) to the 2′-position of the V. cholerae phosphorylated Kdolipid A domain. A second protein, Vc0212, acts as an LpxM homologue and transfers 3- hydroxylaurate (3-OH C12:0) to the 3′-position creating hexa-acylated V. cholerae lipid A domain. Although lipid A secondary acyltransferases have been characterized among various Gram-negative bacteria, this is the first report of a lipid A secondary hydroxyacyltransferase. Further, the transfer of 3-hydroxylaurate (3-OH C12:0) was demonstrated to be essential for antimicrobial peptide resistance in V. cholerae and required for activation of the innate immune receptor TLR4.
    • Bisphosphonate-Related Osteonecrosis of the Jaw: From Mechanism to Treatment

      Howie, Rebecca; Department of Cellular Biology and Anatomy (2015-04-20)
      With 55 million prescriptions issued each year, bisphosphonates are the second most prescribed class of drug in the United States. They are widely used to treat diseases with excessive osteoclastic resorption, including post-menopausal osteoporosis, Paget’s disease, and tumor metastasis to bone. Unfortunately, with long term intravenous administration of nitrogen-containing bisphosphonates some patients develop bisphosphonate-related osteonecrosis of the jaw (BRONJ). This debilitating disease has limited treatment options once it has manifested and no mechanism for its development has been elucidated. This dissertation explores the novel concept that bisphosphonates cause osteonecrosis of the jaw by impairing osteocyte-induced osteoclastogenesis and, through the physical accumulation of bisphosphonates in bone, impairing the ability of recruited osteoclasts to attach thereby arresting bone healing. Furthermore, it explores the possibility that chelating agents can be used for the removal of bisphosphonate attachment from bone systemically and locally during extractions, potentially leading to a future preventive treatment. It was found that 13 weeks of 80µg/kg intravenous tail vein injections of Zoledronate followed by two mandibular molar extractions caused the clinical presentation of BRONJ as analyzed by the gross, radiographic, and histological methods. Bone dynamic parameters and TRAP staining suggested an impaired ability for the bone to remodel and defective osteoclast attachment in treated groups that persisted eight weeks after the cessation of treatment. Additionally, it was found through the use of a fluorescently tagged bisphosphonate, that the decalcifying agents cadmium, EDTA, and citric acid all had the ability to cause the significant release of bound bisphosphonate from bone. Finally, this dissertation showed that the migration of monocytes treated with low doses of Zoledronate had increased migration, while their migration to conditioned media of osteocytes treated with Zoledronate was impaired. Collectively, these data suggest that invasive trauma by itself consistently precipitated massive bone necrosis in Zoledronate treated animals, possibly through a bisphosphonate driven alteration of monocyte migration and that the use of decalcifying agents could acutely remove bisphosphonate from bone both systemically and locally. This study establishes and effective rodent model for BRONJ and a possible preventive strategy for the side-effects of bisphosphonates during high-risk situations.
    • Blood pressure impacts the renal T cell profile of male and female spontaneously hypertensive rats

      Tipton, Ashlee J.; Department of Physiology (2014-03)
      Of the 68 million Americans with hypertension, fewer than 46% have their blood pressure (BP) adequately controlled and women are more likely than men to have uncontrolled hypertension. This underscores the critical need for new treatment options; however, this is a challenge due to our lack of knowledge regarding the mechanism(s) driving essential hypertension. T cells have been implicated in hypertension in males. Prior to our work, the role of T cells in hypertensive females had been unexplored. We demonstrate that female spontaneously hypertensive rats (SHR) have a decrease in BP in response to an immunosuppressant, supporting an immune component to their hypertension. We further defined a sex difference in the renal T cell and cytokine profile in SHR. Female SHR have a more anti-inflammatory immune profile in their kidneys than males. To gain insight into the mechanisms mediating sex differences in the immune profile, male and female SHR were gonadectomized. Gonadectomy increased pro-inflammatory markers in both sexes and attenuated anti-inflammatory markers particularly in females. Therefore, while both male and female sex hormones promote an anti-inflammatory immune profile, female ii sex hormones contribute greater to their more anti-inflammatory profile, but do not explain the sex difference. To determine the impact of hypertension on the renal immune profile, experiments measured renal T cells and cytokines in hypertensive male and female SHR, normotensive Wistar Kyoto rats (WKY), and SHR treated with antihypertensive therapy. All T cells and cytokines measured were higher in SHR compared to the same sex WKY. Moreover, antihypertensive therapy decreased renal Tregs only in female SHR. These data suggest that increased BP in both sexes is associated with an increase in renal inflammation; however female SHR have a compensatory increase in renal Tregs in response to increases in BP. TGF-β is a key cytokine regulating Treg and Th17 differentiation and we found that female SHR express more TGF-β than males. Experiments assessed if female SHR possessed a sex hormone or BP-mediated increase in renal TGF- β corresponding with increases in Tregs. We determined that loss of female sex hormones and increased BP in female SHR increase renal TGF-β expression. We conclude that BP status drive sex differences in the renal T cell and cytokine profile of SHR.
    • Bone and soft tissue regenerative response following alveolar ridge augmentation using polysulfone implants with and without demineralized bone powder in macaca fascicularis

      Fouad, Salama S; Department of Oral Biology (1987-06)
      Successful augmentation of bone surfaces has great clinical application, particularly to the face and oral cavity regionso More than 24 mi 11 ion Americans are edentulous and must depend upon dentures to eat and to restore their norma 1 speech and appearance o Porous po lysul fane (PPSF) is frequently used to fill osseous voidso The purpose of this study was to· test tooth soft, ti.ssue and bone response to porous po lysul fane (PPSF), with and without demineralized bone powder (DBP) in Macaca fascicularise Six adult female monkeys, 12-15 year~ of ~ge, were used in this study. One animal was sacrificed and used as a bone donor and the other five were recipientso All mandibular molar teeth were extracted and masstve alveolectomies were performedo The wounds were left to heal for 5 to 8 1/2 months postoperativelyo At the time of implantation~ PPSF with DBP was inserted subperiosteally into the left mandibular edentulous areas while PPSF alone was inserted into the right sideso The. animals were sacrificed at 42, .60, or 90 days following implantationo Each mandible was cut- into 3mm thick coronal sections which were then examined and photographed with a dissecting microscopeo Some specimens were then decalcified, embedded in paraffin and sect i a ned and stained With. H & Eo Other specimens were processed undecalcified in glycol and methylmethacrylate for histomorphometric measurements and tetracycline labe1ling·., Also, some specimens were processed for scanning electron microscopyo No inflammation or untoward reaction of the 'implantation sites were noted at the time of sacrifice. Histologically, the 42 day specimens of the' DBP-PPSF side· (experimental side) revealed penetration of fibrous tissue rich in fibroblasts and vessels into the pores of PPSF comparirig to the PPSF side (control side)., The fibrous tissue also surrounded the implant., Some multinucleated giant cells and macrophages were present., At 60 days, the PPSF side showed more organized fibrous tissue and bone grew only for a. short distance into the polysulfone. In contrast, the PPSF-DBP side showed large amounts of .bone formation within the pores of the polysulfone and almost covered the implant., The newly formed bone contained osteocytes and was -surrounded by osteob 1 asts. At 90 days, the PPSF side showed more bone tormation on the lower half of the. implant" These res~lts suggested that PPSF is a suitable non-resorbable material that accommodates bone and soft tissue formation. A 1 so :p the use of DBP enhanced both rate and amount of the new bone., In· conclusion; PPSF with and without DBP is a suitable material that can be used successfully for alveolar ridge augmentation.
    • Burnout and Job Satisfaction of Medical-Surgical Nurses

      Garbutt, Susan J.; School of Nursing (1981-12)
      Job satisfaction and burnout of medical-surgical nurses a n d t h e i r . r e 1 at i_ o n s h i p to . t h. e ~ ri u· r s .. e ' s o p t i. on t o w or k h e r I h i s s hi f t ·of choice was stud i e d . The Mas 1 a c h Burnout Inventory (Maslach and Jackson, 1981-), the Job Satisfaction-Instrument (Wall, ·1980; and Sorrow, 1980) and a Demographic Data Form were used to survey t~enty randomly selected registe~ed nurses f r o m e a c h . o f t h r e e s o u t h e a s t e r n h o s p l t a 1 s . ( t o t-a 1 N = 6 0 ) .. Result~ of this study indicated that as job satisfact}on increased, the intensity of burnout reported by medical-surgical nurses decreased. Frequency and intensity of burnout were positively correlated. Burnout occorred among n~rses who worked their choice of shift and among nurses who did not. However, nurses who worked their choice of shift reported a significantly lower intensity of burnout than nurses who did not work their choice of shift. This study prov.ides some interest-· ing data to be ~onside~ed by nurse administrators in working .with medical-sur9ical nurses. Replication and extension of this study is recommended.
    • 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.