• 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 bacteriophage P22 gene necessary for DNA synthesis which maps in the late region

      Ruddel, Mark Eric; Department of Cell and Molecular Biology (1977-06)
    • 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]
    • A behavior-oriented approach to measuring the power motive

      Welcher, Portia G.; School of Nursing (1983-05)
      Based on McClelland's need theory, thij investigation represents an attempt to develop and test a research jnstrument for measuring power, achievement, and-affiliation motive,. The primary study variable is the power motive. The significance of the study is supported by· literature citing the need for instruments to ~nhance power research in nursing. A 32-item instrument, the Behavior-Oriented Motive Measurement (BOMM), was designed by the investigajor as an alternative to . existing more complex and lengthy motive mlasurements·. The design of the BOMM is·intended to minimize the time ~equired for completion and to enhance feasibility for use in nursing 1rganizational settings. This investigation included two BOMM tlst administrations. Subjects I . . consisted of a. convenience sample of 26 graduate nursing students,: .16 of · whom repeated testing three weeks later.to provide data for testretest reliability estimates. Reliabilities for internal consistency, content validity and convergent validity wrre also estimated. The BOMM was judged to have content va~idity ·on the basis of expert judges' assessment. In this measur~, the power and achievement scales demonstrated acceptable levels of internal consistency with standardized alphas of .71 and .56, respeclively in Test 1 and alphas of .78 and .73 in Test 2. The powjr motive scale demonstrated an acceptable test-retest reliability: r ·~·.50. This is somewhat low, but comparable to that of other motile measures. Conceptual evidence is cited to support the notion t~at there may be no need to expect high test;_,retest reliabilities for motive measurements. Convergent validity of the power and achievement scales was evidenced by relatively high correlations with the-Manifest Needs Questionnaire( MNQ) (r = .76, p = .001) and (r = .72, p = :001). Acceptable levels of reliability and validity were not obtained for the affiliation scale. On the basis of reliability and val-idity coefficients obtained, the conclusion is drawn ~hat the power and achievement- scales of the BOMM may be useful tools for further research on the power motive.·
    • Being homeless : an ethnographic study of women's experiences in a shelter

      Hodnicki, Donna R.; School of Nursing (1992-04)
      This field research used ethnographic techniques to study women's experiences ~f 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: Disc.onnected-Loss of Major Support and Rebuilding--The Regrouping of Assets. Themes within the first domain 1 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 whe11 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.
    • Being Homeless: An Ethnographic Study of Women's Experiences

      Hodnicki, 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.
    • Beta-arrestin1 and G protein-coupled receptor Kinase 5 regulate cancer progression

      Kim, Jae II; School of Graduate Studies (2010-12)
      Cancer is a leading cause of death worldwide, accounting for nearly seven million deaths per year. Available clinical data establish a· protective effect of COX-2 inhibition on human cancer progression, but the appearance of unwanted side effects remains a major hurdle for the general application of COX-2 inhibitors as effective cancer therapeutics. Major COX-2 effectors are prostaglandins and we explored the idea that PGE2 promotes mitogenic signals that could be exploited for targeted therapy of cancer. PGE2 signals through EP1, EP2, EP3, and EP4 that belong to the superfamily of GPCR that have been demonstrated to signal through G proteins and r3Arrestins. In the first part of this . thesis project we determined the role of r3Arrestins in PGE2-regulated cancer cell migration. We report that the COX-2 effector PGE2 signals selectively via EP4 to enhance A549 lung cancer cell migration. We further find that this mode of ·.signaJing requires the presence of r3Arrestin1 and tyrosine kinase c-Src activity. Hehce, this study provides preclinical-based rationale for the selective targeting of EP4 to inhibit PGE2-induced lung cancer cell migration. In the second part of this thesis project we determined the role of G protein-coupled receptor kinase 5 (GRK5) in cancer cell proliferation and tumor growth. Recent studies have implicated distinct GRK roles in the regulation of non-GPCR substrates, some of which have well-defined roles in cancer progression such as tumor suppressor p53. Here we report that GRK5 is required for prostate cancer cell cycle progression, cell proliferation, and prostate tumor growth. We identified HDAC4 as a novel GRK5 substrate, whose gene and protein expression is regulated by its kinase activity. In addition, we found that serine 246 residue of HDAC4 is phosphorylated by GRK5, a site known to regulate HDAC4 activity and subcellular localization. GRK5 can also phosphorylate an HDAC4 fragment (419-670 amino acid residues) that also contains two important regulatory serine phosphorylation sites. As many studies have shown HDAC4 involvement in cancer, our findings may provide a possible mechanism of HDAC4 regulation by GRK5
    • Binding of 2, 3-DPG to high molecular weight substances in the red cell

      Chen, Clara Johsien; Department of Cell and Molecular Biology (1974-06)
    • 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.
    • Biocompatibility and mechanical/physical properties of 3D printed, milled, and conventionally processed denture base materials

      Ulmer, Mallory; Biomedical Sciences (Augusta University, 2019-12)
      According to the American College of Prosthodontists, over 36 million people in the USA are edentulous with a 2:1 predilection for geriatric patients1. Each year, an estimated 15% of edentulous Americans will seek denture treatment1. Conventional dentures require multiple visits and lab processing time. 3D printing technology offers the potential to reduce the number of appointments and speed up the time until patient rehabilitation. However, the newly FDA-certified 3D printer denture resins, featuring secretive and proprietary formulae, lack studies concerning their biocompatibility/safety and mechanical strength. This study aims to investigate the biocompatibility and physical properties of one such 3D printer resin, NextDent® Base (Vertex, Soesterberg, The Netherlands), and compare it to pre-existing conventional polymethyl methacrylate (PMMA) denture base (Lucitone 199, Dentsply Sirona, York, Pennsylvania) and milled PMMA denture base (IvoBase CAD®, Ivoclar Vivadent AG, Schaan, Liechtenstein). The cytotoxicity was examined using of 12 discs: conventional PMMA, milled PMMA, as-printed 3D printer resin, post-cured 3D printer resin, and Teflon controls. An MTT assay using human periodontal ligament (900L) cells was employed, and specimens were aged for 1, 3, 7, 10, and 14 days. After day 7, there were no statistically significant differences among the groups, excluding the Teflon control, which showed significantly less cell viability on day 14. Bars of conventional PMMA, milled PMMA, as-printed 3D printer resin, and post-cured 3D printer resin were subjected to a 3-point bend test to examine flexural strength and moduli differences. The mean flexural strength was 63.8 ± 3.06, 82.6 ± 1.9, 5.1 ± 0.4, and 22.1 ± 6.4 MPa, respectively, while the flexural moduli were 1757.3 ± 109.5, 2226.7 ± 76.3, 110.3 ± 20.3, and 537.0 ± 210.6 MPa, respectively. The flexural strength and modulus were significantly different among all groups. Weibull analyses for conventional PMMA, milled PMMA, as-printed 3D printer resin, and post-cured 3D printer resin revealed a Weibull modulus of 23.5, 42.8, 16.6, and 3.7, respectively, and a characteristic strength of 65.2, 83.5, 5.3, and 24.5 MPa, respectively. The characteristic strength was significantly different among all groups as well. The Weibull modulus was significantly different between all groups, except for conventional vs. as-printed, which were not significantly different. In summary, milled PMMA featured significantly greater mechanical properties. Both 3D printed groups proved to be very weak, with the as-printed group being the weakest of all. The differences between the as-printed and post-cured groups highlight the importance of properly post-curing the resin. While the biocompatibility results showed promise, the mechanical testing results were disappointing. Unfortunately, the findings suggest that 3D-printed denture base resin is not yet ready for clinical use.
    • 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.