• T Cell Immune Response in Persistent Infection of Lymphocytic Choriomeningitis Virus (LCMV)

      Ou, Rong; Georgia Cancer Center (2004-07)
      The m urine LCMV system provides a ciassic model to study the mechanism of immunological tolerance, an efficient strategy used by virus to establish a persistent infection by selective down-regulation of virus-specific T lymphocytes. High viral burden in the onset o f infection drives responding cells into functional unresposiveness (anergy) that can, be followed by their physical elimination. In this study, the downregulation o f the virus-specific CD8^-T-ceil response was studied during a persistent infection o f adult mice, with particular emphasis on the contribution of the interferon response in promoting host defense, or perforin-, Fas/FasL-, or TN FR l-m ediated cytolysis in regulating T-cell homeostasis. Since LCMV infects a broad range o f host tissues, the functional properties o f virus-specific CD8'^ T cells in different tissues during LCMV infection were also evaluated. Infection of mice deficient in receptor for type I (IFN-a/p), type II (IFN-y), or both type I and II IFNs with LCMV isolates that vary in their capacity to induce T-celi exhaustion, revealed a critical role for IFN -a/p in restricting LCMV spread at the onset o f infection while IFN-y has impact on effector cells. The production o f IF N -a/p and/or IFN-y critically regulates the virus-host balance during the acute phase o f infection, such that a high viral burden drives responding cells into different programs o f exhaustion. Infection o f mice deficient in perferin, FasL or TNFRl with the Docile or Aggressive strains of LCMV revealed comparable kinetics of expansion and functional inactivation o f virusspecific C D ^ T cells in the early phase o f Infection in C57BL/6 controls. However, the data underscore a critical role for these molecules in the persistence o f the virus-specific CD8"‘-T-ceil population once it has become anergic. Study o f the functional properties of virus-specific CD8'^ T cells in different tissues during LCMV infections showed that a centra! role for the viral load in lymphoid tissue in the induction and maintenance of clonal exhaustion. The data strongly suggest that CD8^ T ceils may be differentially regulated in the environments o f lymphoid versus nonlymphoid tissues, and the pattern of T cell exhaustion observed with mice is likely a common feature o f the immune response during chronic infections in humans.
    • T-Type Calcium Current and Calcium-Induced Calcium-Release in Developing Chick Myocardium

      Kitchens, Susan A.; Department of Cellular Biology and Anatomy (2002-02)
      HYPOTHESES 1. The contribution of T-type calcium currents to the calcium transient are greater at young developmental ages, but decline with chick heart development. The decrease in contribution of T-type calcium current to the calcium transient mirrors the normal developmental reduction in magnitude of T-type current in the chick heart. 2. T-type calcium current plays a role in calcium-induced calcium-release during chick heart development. T-type current plays a significant role in the calcium-induced calcium-release process in younger embryos due to the greater magnitude of the current at earlier developmental stages. 3. More than one isoform of the T-type calcium channel is present in developing chick myocardium. The multiple isoforms will function concomitantly to provide sufficient T-type calcium current for proper development. 4. The expression of the T-type calcium channel in ventricle decreases with development. There is a concomitant decrease in T-type Ca2* current stimulation of CICR. SPECIFIC AIMS 1. To determine the contribution of T-type calcium current to the calcium transient during development in chick ventricular myocytes. The approach is to use a fluorescent calcium indicator to measure the transients from myocytes at embryonic day (ED) 5, EDI 1 andED15. 2. To determine the contribution of T-type calcium current to calcium-induced calciumrelease during chick heart development. The approach is to use pharmacological agents to quantify the contribution to the Ca3* transient from T-type Ca3* current stimulated CICR. 3. To determine which isoforms of the T-type calcium channel are likely to be present in chick myocardium. The approach is to use PCR methods to identify any T-type channel isoform mRNA expressed in chick ventricle. 4. To determine the level of expression of T-type calcium channel isoforms during the development of chick ventricle. The approach is to use molecular quantitation methods to examine the expression pattern of T-type channel isoforms in chick ventricle during development.
    • A Tailored Intervention Program for Overweight and Obese Vetrans: Who Benefits and When

      Garvin, Jane; Department of Physiological & Technological Nursing (2012-06)
      This descriptive, exploratory study examined longitudinal clinical data for variables associated with weight reduction among veterans enrolled in a weight reduction intervention, the MOVE! Program. Variables of interest included background characteristics and exposure to components of the intervention. Background, intervention, and outcome variables were organized around the Interaction Model of Client Health Behavior. In addition to the outcomes related to weight, hemoglobin A1 Thirteen percent of participants (N = 53 of 404) achieved a 5% weight reduction. Overall, the sample was primarily non-Hispanic (96%), urban (83%), Black (58.4%), married (58.4%) and male (~80%). The mean age was 56 years. Common comorbidities associated with obesity were evident including diabetes (30.2%), hypertension (60.9%), and hyperlipidemia (54.0%). The average body mass index was ~35. All available data were collected from program entry to a designated stop date; therefore, participants had unequal and irregular data points. Participants were observed repeatedly over time with 51% having 10 or more observations. A little more than half of the participants were group attendees (~56%) rather than self-managed. The majority of the intervention exposures were group rather than individual or telephone visits with providers. C, blood pressure, and serum lipid levels were examined. Achieving a 5% weight reduction was significantly associated with age in years (OR 1.04), group attendance beyond the day of orientation (OR 6.61), attendance at the holiday eating class (OR 3.67), exposure over time (measured in weeks, OR 1.02), and the interaction between time and group (OR .97). Weight reduction in pounds was significantly associated with age, baseline body mass index, total number of group classes attended, and telephone contact with the registered nurse. Using repeated measures of weight, the trajectory of weight was significantly associated with gender, baseline body mass index, and exposure to the intervention over time. Examination of additional outcomes revealed that components of the intervention were associated with beneficial changes in hemoglobin A1C, blood pressure, and serum lipid levels. Further research is needed to more fully describe successful weight reducers and the identify best practices to convert unsuccessful weight reducers to successful ones.
    • Targeting cyclic GMP signaling for the treatment of gastrointestinal diseases

      Sharman, Sarah Kristen; Department of Biochemistry and Molecular Biology / Cancer Center (1/25/2018)
      Continual renewal of the luminal epithelium in the gut is essential for the maintenance of a healthy intestine as it sustains the barrier that protects underlying tissue from infiltration of material passing through the lumen. Dysregulation of homeostatic processes involved in maintenance of the barrier have been implicated in numerous gastrointestinal diseases. The cGMP signaling axis has emerged as an important regulator of homeostasis in the intestinal mucosa, and has been implicated in the suppression of visceral pain, colitis, and colon cancer. While there is considerable interest in exploiting this pathway, until recently the approaches used to increase cGMP have been limited. The present study sought to test the hypothesis that elevation of cGMP in the intestinal epithelium using PDE5 inhibitors will alter epithelial homeostasis and be therapeutic for constipation and preventative for colon cancer. Healthy mice treated with the PDE5 inhibitor sildenafil or the GC-C agonist linaclotide exhibited reduced proliferation and apoptosis, and increased numbers of differentiated secretory cells in the intestinal epithelium. In addition to these homeostatic effects, both drugs normalized intestinal transit and fecal water content in two mouse models of constipation. Furthermore, administration of sildenafil to mice treated with dextran sulfate sodium tightened the disrupted epithelial barrier. Treatment of ApcMin/+ mice with sildenafil or linaclotide significantly reduced the number of polyps per mouse (67% and 50%, respectively). The effect of these cGMP-elevating agents was not on the polyps themselves but was rather on the pre-neoplastic tissue, which was less proliferative and more apoptotic in the presence of the drugs. Taken together, the results of this study demonstrate that increasing cGMP with a pediatric dose of PDE5 inhibitors could be a potential alternative to GC-C agonists for the treatment of gastrointestinal diseases.
    • Temperament, childrearing practices and unintentional childhood injury

      Markowitz, Monika S.; School of Nursing (1987-04)
      ~his study examined the rel.at~onsh.~p. a~()ng parents' . . . - ··- . . . . perceptions-of the~r c::hildren's temperament, self-reported ~hildrear:in~ practices and urililt·en_tional injury in children. ~he sample consisted of 23 injured children· • • ~ ' • • + between the ages of 1· an.d 7 Y~.CJ.rs who, wer~ _recr'lli~ed from the emergency rqoms, hospital·f~~ors and pedia~ric intensiv~ car~·units of two meiropolitan hospitals. . . . ' '. '. ' -. ~ ' ' . . . . . Parents completed·a temperament. questionnaire, a . . childrearing ~ractices questi6~naire and a demographic data . form. Inform·ation related to th~ injury was r~corded· and· the Physiologic Stability Index, which measured injury ' - ' ' ' . ' . . . . . . severity, was deter~ined by tne investigp.t_or afte:r a c}lar~ review. In this study, a.greater frequency of injury was rel.ated to a pa·re_nting style .of low warmth and a .greater injury severity was related to a childrearing practice of. low control, r=. 57 and ··51 respectiv.ely (p< ·• 01). Parents· of inj~red children reported themselves as practicing ' ' . ' . significantly more control and ·prot~ctiveness ·.than a sample ·of parents of non-injured children (n=20). The injured ·children were perceived l:?Y t~eir mothers as b.ein_g.slow in ·.adaptability, demonstrating low-rhythmicity, high withdrawal; and negative mood when compared to normative data bn other child subjects~
    • Temperamental Differences and Preschool Adjustment to Day Care

      Hatmaker, Debra; School of Nursing (1986-05)
      Adjustment of preschool children to group day care was studied in relation to their temperament characteristics. Temperament measurement, by means of the Toddler Temperament scale by Fullard, McDevitt, and carey, was completed at the beginning of group care on a sample of 18 children ages 23-42 months. Adjustment-to day care was measured at the.beginning of group care, at one week, and at four weeks after enrollment using the Preschool Behavior Questionnaiie by Behar and Stringfield. Three hypotheses were tested. The first hypothesis suggesting a positive correlation between children's average temperament scores and their negative behavior ratings was rejected after statistical testing by Pearson's Product Moment Correlation. Hypothesis II predicted a significant decline in negative behavior ratings from the first to the fourth week of day care enrollment. This second hypothesis was rejected after-results from paired t-tests failed to reach significance. Hypothesis III stated that there would be a positive-relationship between parents' and caregivers' ratings of ~hildren~ negative behaviors. Ratings from the first and fourth week were compared using the Pearson's Product Moment Correlation resulting in correlations of .61 and·.39, respedtiVely. These correlations were statistically signi~icant and the third hypothesis was accepted. These findings do suggest that parents and caregivers have similar perceptions in identifying certain negative behaviors for preschool children. As providers of anticipatory guidance, nurses must be well versed in the aspects of quality day care and be able to impart this imformation to families based

      Lynch, Thomas Joseph; Department of Physiology (1983-02)
      A finite epileptic focus was created in the brains of 25 Sprague-Dawley rats by kindling electrical stimuli delivered to the left amygdala. With the left amygdala thus defined to be the focus of epileptogenesis, changes in focal site temperature and p02 were monitored during progressively worsening convulsions. Convulsions were evoked by the daily administration of a kindling stimulus composed of a 3 second long, 60Hz, sinusoidal current of 150 microamperes. Two separate studies were conducted in which only those ictal temperature or ictal po2 transients occur~ing at the focus were monitored. In the main temperature study, the range of kindled seizure activity from simple electrographic afterdischarge (AD) through generalized convulsion caused a mean focal heating transient of .028°F with a range of from 0 to .738°F. Focal heating transients generally increased as the animals' convulsions progressed in severity from focal to generalized. In one supplementary temperature study using two rats, the contralateral unstimulated amygdala was found to undergo much the same progression of ictal heating transients as the stimulated amygdala. In a second supplementary study of four rats under neuromuscular block, the lack of convulsive motor activity seemed to be the cause of a significant reduction of the total, focal, ictal heating. In a third measurement between left and right amygdalae showed that their relative rates and directions of temperature change were different despite the gross similarity observed when left and right amygdala temperatures were recorded simultaneously. In the main po2 study, the standard ictal response was a 20- 50% increase in po2 over the baseline level. This increase occurred regardless of the severity of convulsion but- tended to become slightly enhanced as convulsions generalized. When p02 increased and peaked during a seizure, the p02 peak consistently occurred at the moment when the AD in the electroencephalogram ended. Among singly evoked kindled convulsions, significant decreases in focal p02 were rarely observed. Furthermore, in one rat evocation of 17 generalized convulsions, one every ten minutes, still caused a focal po2 increase during each convulsion. In another rat, six spontaneous seizures spaced about 2 minutes apart also caused only increased focal po2• These records are the first such reported for the kindling model of epilepsy and though the findings regarding temperature are similar to those in the available epilepsy literature, the findings regarding p02 are different in that no trend toward ictal reduction in brain po2 was found. That the overwhelming majority of kindled seizures cause focal brain p02 to increase is indication of a fundamental difference between kindled epileptic foci and epileptic tissue created by other models.
    • Tetrahydrobiopterin-Dependent Vasodilation is Impaired in Experimental Hypertension

      Mitchell, Brett M.; Department of Physiology (2003-05)
      Decreased nitric oxide (NO) bioavailability leads to decreased vasodilation and increased blood presssure. Adequate amounts of the NO synthase (NOS) cofactor, tetrahydrobiopterin (BH4), optimizes NO production. Reduced BH4 results in decreased NO and increased superoxide production. Therefore, we hypothesized that decreased GTP cyclohydrolase (GTPCH), the rate-limiting enzyme in BH4 production, decreases NO leading to impaired vasodilation and increased blood pressure. To examine the effect of in vivo GTPCH inhibition on vasodilation and blood pressure, we administered DAHP in the drinking water of rats. Systolic blood pressure increased significantly in DAHP-treated rats. Endothelium-dependent relaxation was decreased in aortas from DAHP-treated rats, but restored with superoxide dismutase or sepiapterin, which produces BH4 via a salvage pathway. In conclusion, in vivo GTPCH inhibition leads to decreased NO production resulting in decreased BH4-dependent vasodilation and increased blood pressure. Excess glucocorticoids (GC) cause hypertension. To assess the effect of GCs on BH4 biosynthesis and vasodilation, we implanted dexamethasone (DEX), a synthetic GC, in rats. Aortas were isolated after 12 hours, 4 days, or 15 days of DEX-treatment to examine the role of GTPCH in the onset, development, and maintenance of GC-induced hypertension, respectively. Aortic relaxation and GTPCH and eNOS mRNA levels were decreased significantly in aortas from 4- and 15-day DEX-treated rats and restored with sepiapterin. In conclusion, excess GCs down-regulate GTPCH leading to decreased BH4-dependent vasodilation, which contributes to GC-induced hypertension. To assess if GCs act directly on the blood vessel to down-regulate GTPCH and decrease vasodilation, we incubated rat aortic rings with various compounds and found endothelium-dependent relaxation and GTPCH mRNA decreased significantly following incubation with DEX for 6 hours. This effect was blocked by a GC receptor antagonist and reversed by sepiapterin. In conclusion, GCs act through the GC receptor to down-regulate GTPCH leading to decreased BH4- dependent vasodilation. The results support the overall hypothesis that decreased BH4 biosynthesis, either by GCs or pharmacological inhibition, results in decreased NO production leading to reduced vasodilation and increased blood pressure.
    • The Influence of Porphyromonas gingivalis Fimbrial Expression on Human Dendritic Cell Signaling and Innate Immune Homeostatic Functions

      Meghil, Mohamed; Biomedical Sciences
      Perturbation of fundamental processes of immune homeostasis, such as apoptosis and autophagy, can result in dire consequences such as autoimmune diseases. Periodontitis is an inflammatory oral disease that is characterized by oral microbial dysbiosis, and deregulation of the host immune response. The aim of this study was to elucidate the underlying mechanisms by which Porphyromonas gingivalis (P. gingivalis) manipulates dendritic cell (DC) signaling to perturb immune homeostasis. Using a combination of Western blotting, flow cytometry, qRT-PCR and immunofluorescence analysis, we show a pivotal role for the minor (Mfa1) fimbriae of P. gingivalis in nuclear/cytoplasmic shuttling of Akt and FOXO1. Mfa1-induced Akt nuclear localization and activation ultimately induced mTOR. The upregulated Akt/mTOR axis was shown to downregulate the intracellular levels of LC3II, an autophagy protein also designated Atg8, required for autophagosome formation and maturation. Further studies utilizing the allosteric panAkt inhibitor MK2206 and rapamycin an mTOR inhibitor confirmed that the activation of Akt/mTOR signaling by P. gingivalis inhibited autophagy in DCs. Concomitant with inhibiting autophagy, we show a pivotal role for Mfa1 fimbriated P. gingivalis in induction of the antiapoptotic protein BCL2, decreased caspase-3 cleavage and decreased expression of pro-apoptotic proteins Bax and Bim in infected DCs, ultimately blocking programmed death of infected DC cells. Importantly, we show that, by using ABT-199 peptide to disrupt interaction of antiapoptotic BCL2 and its proapoptotic partners BAK/BAX, we can restore programmed cell death to P. gingivalis-infected DC cells. In summary, we have identified the underlying mechanism utilized by P. gingivalis to promote the survival of its host immune cells while preventing its own autophagic elimination.
    • Therapeutic induction of fetal hemoglobin in sickle cell disease: development of a novel prodrug AN-233

      Oseghale, Aluya; Biomedical Sciences (Augusta University, 2019-05)
      The reactivation of fetal hemoglobin (HbF) in sickle cell disease (SCD) ameliorates the clinical severity of the illness and improves patient's survival. Pharmacological induction of HbF has been a major strategy for SCD treatment and several research studies have focused on a wide variety of agents for their potential to induce HbF. However, hydroxyurea (HU) remains the only Food and Drug Administration (FDA)-approved drug proven to elevate HbF in about 50% of adults with SCD. Efficacy of HU has been very limited due to many side effects including bone marrow suppression, susceptibility to infections and long-term infertility. Our group reported potent HbF induction by sodium butyrate in erythroid cells through p38 MAPK activation. Nevertheless, oral administration of a butyrate (BA) derivative to SCD patients was ineffective due to rapid metabolic inactivation by the liver. Therefore, the need for better therapies exists. This project investigated a novel prodrug conjugate of BA and δ-aminolevulinate (ALA) denoted as AN-233. As an ester, AN-233 [1-(butyryloxy) ethyl-5-amino-4-oxopentanoate] undergoes cellular hydrolysis in an esterase dependent manner to yield two active drugs BA and ALA. In prior studies, oral administration of AN-233 to mice increased total hemoglobin but the effect on HbF was unknown. We proposed the hypothesis; that AN-233 upregulates γ-globin gene expression and elevates HbF synthesis via transcriptional and post-transcriptional mechanisms. We investigated AN-233 using in-vitro, in-vivo and ex-vivo model systems including K562 cell lines, sickle progenitors and β-YAC mice. Treatment of K562 cells showed AN-233 significantly increased mRNA levels. Flow cytometry analyses show the prodrug significantly increased HbF protein expression and Western blotting of whole cell lysates confirmed increased synthesis of HbF. Treatment of CD34+ stem cell-derived primary erythroid cells increased early stage (basophilic) erythroblasts by 2.4fold and decreased late stage (orthochromatophilic) erythroblasts by 2.5 fold. In sickle progenitors, AN-233 again elevated F-cell% by 1.5fold and HbF protein by over 2.6fold. Mechanistic studies in K562 cells show AN-233 significantly elevated heme biosynthesis, decreased phosphorylation of HRI and eIF2α thereby promoting the protein synthesis of globin chains. Additionally, AN-233 enhanced histone acetylation at the γ-globin promoter and LCR DNase hypersensitive site 2 (LCR HS2). Treatment of sickle progenitors with AN-233 decreased %sickled cells by up to 50%. The transcription factor BACH1 was reduced while NRF2 was increased in AN-233 treated K562 cells. In vivo, AN-233 increased F-cell% and F-cell MFI of treated β-YAC mice within 4-weeks. Our data support AN-233 as a potent HbF inducer in erythroid progenitors and in mice. The prodrug represents a drug candidate, which can be developed for the treatment of SCD patients.
    • Therapeutic Targeting of P2X7 After Traumatic Brain Injury

      Kimbler, Donald E.; Department of Neurosurgery (2012-02)
      Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Cerebral edema, the abnormal accumulation of fluid within the brain parenchyma, contributes to elevated intracranial pressure (ICP) and is a common life-threatening neurological complication following TBI. Unfortunately, neurosurgical approaches to alleviate increased ICP remain controversial and medical therapies are lacking due in part, to the absence of viable drug targets. In the present study, genetic inhibition (P2X7-/- mice) of the purinergic P2X7 receptor attenuated the expression of the pro-inflammatory cytokine, interleukin-iP (IL-ip) and reduced cerebral edema following controlled cortical impact, as compared to wild-type mice. Similarly, the clinically useful P2X7 inhibitor, brilliant blue G (BBG), inhibited the expression of IL-ip, limited edemic development and prevented the development of post-traumatic depression and anxiety. The beneficial effects of BBG were observed following either prophylactic administration via the drinking water for one week prior to injury or via an intravenous bolus administration up to four hours after TBI, suggesting a clinically-implementable therapeutic window. Notably, P2X7 localized within astrocytic end feet and administration of BBG decreased the expression of glial fibrillary acidic protein (GFAP), a reactive astrocyte marker, and reduced the expression of aquaporin-4 (AQP4), an astrocytic water channel that promotes cellular edema. Together, these data implicate P2X7 as a novel therapeutic target to prevent secondary neurological injury after TBI, a finding that warrants further investigation.
    • Toll-like receptor 2 contributes to cerebrovascular dysfunction and cognitive impairment in diabetes

      Hardigan, Trevor; Department of Physiology (2016-03)
      The risk of cognitive decline in diabetes (Type 1 and Type 2) is significantly greater compared to normoglycemic patients, and the risk of developing dementia in diabetic patients is doubled. The etiology for this is likely multifactorial, but one mechanism that has gained increasing attention is decreased cerebral blood flow (CBF) as a result of cerebrovascular dysfunction. The innate immune system has been shown to play a role in diabetic vascular complications, notably through Toll-like receptor (TLR) stimulated release of proinflammatory cytokines and chemokines that leads to vascular damage. TLR2 has been implicated in the development of diabetic microvascular complications such as nephropathy, and thus we hypothesized that TLR2-mediated cerebrovascular dysfunction leads to decreased CBF and cognitive impairment in diabetes. Vascular TLR2 expression was increased and local TLR2 antagonism improved cerebrovascular function in diabetes. While the anti-hyperglycemic dipeptidylpeptidase-IV (DPP-IV) inhibitor linagliptin prevented TLR2 expression in brain microvascular endothelial cells (BMVEC) when applied locally, chronic in vivo treatment did not decrease vascular smooth muscle TLR2 expression. Treatment with linagliptin restored CBF in diabetes independent of effects on blood glucose levels, and this increase in CBF was correlated with decreased endothelin-1 (ET-1)-mediated vasoconstriction, decreased pathological remodeling, and increased endothelium-dependent relaxation. Knockout of TLR2 conferred protection from impaired CBF in early-stage diabetes and from hyperperfusion in long-term diabetes, prevented the development of endothelium dependent vascular dysfunction in diabetes, created a hyperactive and anxiolytic phenotype, and protected against diabetes induced impairment of long term hippocampal- and prefrontal cortex- mediated fear learning. In conclusion, these findings support the involvement of TLR2 in the pathogenesis of diabetic vascular disease and cognitive impairment.
    • Toll-like receptor 9 contributes to vascular dysfunction in hypertension

      McCarthy, Cameron; Department of Physiology (2016-03)
      Inappropriate immune system activation is common in hypertension; however, the exact mechanisms by which this occurs are not well understood. Innate immune system recognition and response to damage-associated molecular patterns (DAMPs) is becoming an increasingly accepted mechanism. Mitochondrial DNA (mtDNA) is a DAMP that is recognized by Toll-like receptor (TLR)9, and it is elevated in the circulation of spontaneously hypertensive rats (SHR). Therefore, we hypothesized that (1) inhibition of TLR9 in SHR with a TLR9 antagonist (ODN2088) or TLR9 inhibitor (chloroquine) would lower blood pressure and improve vascular function and that (2) treatment of normotensive rats with a TLR9 agonist (ODN2395) would cause vascular dysfunction and increase blood pressure. Both ODN2088 and chloroquine lowered high blood pressure in SHR and treatment with chloroquine also improved cyclooxygenase-dependent endothelial function and prevented the full recruitment of the adaptive immune system in SHR. On the other hand, treatment of normotensive rats with ODN2395 increased blood pressure and rendered their arteries less sensitive to acetylcholine-induced relaxation and more sensitive to norepinephrine-induced contraction. This dysfunctional vasoreactivity was due to cyclooxygenase activation, increased reactive oxygen species generation, and reduced nitric oxide bioavailability. In conclusion, these findings support the involvement of the innate immune system pattern recognition receptor TLR9 in the pathogenesis and maintenance of hypertension. Specifically, circulating mtDNA may activate TLR9 and contribute to high blood pressure and endothelial dysfunction in SHR.
    • Transcriptional Coactivator and Oncoprotein CoAA

      Brooks, Yang Sui; Department of Pathology (2008)
      CoAA contains two copies of RNA recognition motifs (RRM) and an intrinsic transactivation domain rich in repetitive tyrosines and glutamines (YxxQ domain). Previously, CoAA has been shown to be a transcriptional coactivator that stimulates transcriptional activation and regulates alternative splicing. A pattern and profile search revealed that the YxxQ domain in CoAA shared significant pattern homology with the oncogenic EWS activation domains (EAD) in TET family proteins, including, TLS/FUS, EWS and TAFII 68. It was further demonstrated that CoAA’s YxxQ domain and EWS’ EAD also shared functional similarities. Based on these findings, this work investigated the aberration of CoAA in cancers and its pathophysiological significance. The results showed that the CoAA gene was amplified in a high percentage of inflammation-related human cancers with recurrent loss of the 5’ regulatory element upstream of its promoter. This genomic aberration resulted in CoAA protein overexpression, which in turn, induced the transformation of NIH3T3 cells. Subsequently, it was shown that the lost 5’ regulatory element could modulate the alternative splicing of the CoAA gene during stem cell differentiation and that the unbalanced expression of CoAA and its splice variant, CoAM could potentially impact the cell differentiation process. To further characterize the regulation of CoAA alternative splicing, two conserved trans-splicing events between CoAA and its downstream RBM4 were identified. These events yield a novel zinc finger- containing coactivator, CoAZ, and a non-coding splice variant, ncCoAZ. Both variants regulated their parental genes’ mRNA expression as well as activities, suggesting a linked control between CoAA and RBM4. Moreover, the expression patterns of CoAA, RBM4 and their trans-splicing variants switched during neural stem cell differentiation, resulting in lineage-specific expression of each variant. Our phylogenetic analysis suggests that mammalian CoAA and RBM4 share a common ancestor with the Drosophila melanogaster gene, Lark. In this regard, the trans-splicing events between CoAA and RBM4 represent a functional regulation preserved during evolution. This study established the connection between CoAA and human cancer and provides evidence for CoAA’s involvement in the regulation of cell differentiation. Moreover, this study is the first to report a functional trans-splicing variant in mammalian cells.
    • Transcriptional Regulation by Tbx2

      Chen, Jung-Ren; Department of Pathology (2001-03)
      Tbx2 is a member of an evolutionarily conserved transcriptional regulatory gene family. Little is known about the molecular mechanisms underlying the function of Tbx2. Because connexin43 (Cx43) and Tbx2 are both expressed in neural crest derivatives in pharyngeal arches and because the promoter of Cx43 contains direct repeats of T (Brachyury) half sites, it is hypothesized that Tbx2 regulates Cx43 and other genes important for neural crest cell functions. TBX2 DNA binding affinity was analyzed by eletrophoretic mobility shift assays. Transcriptional regulation of the Cx43 promoter by Tbx2 was analyzed using reporter constructs. These results suggest that Cx43 is a bona fide target gene o(Tbx2 and that Tbx2 negatively regulates Cx43 gene expression by binding to TCACAC sites. Moreover, dye-coupling assays showed that Tbx2 upregulation led to decreased junctional coupling. To identify other genes that may be regulated by Tbx2, a differential gene expression profile was determined using GEM1 microarrays. CellSpace knowledgebase was used to perform functional assignments to 72>x2-regulated genes. This analysis indicated that Tbx2 might be involved in different fundamental cell functions. Tbx2 upregulates proliferation genes, downregulates tenascinC, and upregulates nidogen. In conclusion, together with Cx43 repression, Tbx2 may signal neural crest cells to stop migration and start proliferation. Gene cluster analysis also suggested a potential role for Tbx2 in osteogenesis. In accord, Tbx2 expression was detected in chondrocytes and osteocytes both in long bone and membranous bones.
    • Transformation from Informal Community Group to Community-Based Health Care Organization: A Case Study of Change

      Hanson, Glenda F; Department of Physiological and Technological Nursing (1996-03)
      The purpose o f this study was to examine the transformations of the organization, AID Atlanta in it’s first ten years to determine how and why decisions were made which lead from an informal community group to the creation of the successful, viable, community-based health care organization. Case study methodology was used to conduct the investigation. Sources o f data included primary and secondary documents, direct observations, and systematic interviewing. The theoretical framework for this study was the theory of dissipative structures, as developed by Prigogine (1976) and others within the fields of biology and chemistry. A number o f social scientists have applied this theory to the study of organizational change and transformation. The theory conceptualizes organizations as open systems that exchange energy with the environment, are self determining, and self organizing. Change is conceived as a normal response to an uncertain and complex environment. The study found that AID Atlanta underwent a series o f changes and transformations which enabled it to grow, survive and remain viable. Forces influencing the organization came from both the internal and external environment, with the most powerful force being the AIDS epidemic. Decisions were made by numerous individuals which served to shape the success o f the organization. The clear and constant mission of the organization was a positive sustaining force, and the development of linkages to the community was a key factor in securing necessary resources. Implications o f the study are that decision makers in community-based health care organizations must expect and prepare for change. Knowledge of the experiences of similar successful organizations may lead the administrator to develop strategies which may serve to promote their own success. Strategies shown to promote viability in this study included an open exchange with the internal and external environments, a willingness to change, the use of resources from external connections, articulation o f a vision based on the mission, knowledgeable and experienced leaders, and a strong foundation and heritage.
    • A Transforming Growth Factor From MCG-T14 Mouse Mammary Carcinoma Cells

      Cheng, Charles; Department of Cell and Molecular Biology (1983-04)
      Mitogenic activity was assayed in the harvest fluid concentrates (HFC) from. human· mammary cell.lines ·(T47P, HSO 578T, MDA-157, HBL-100 and BT-20) and a mouse mamamry carcinOinii (MCG-T14) cell line. Data showed that the HFC from four of the human cell lines (T47D,.HSO 578T, MDA-157, and HBL-fOO) and the mouse cell line (MCG-Tl4) were sour.ces of mitogenic activity. The mitogenic activity from the HFC was not due to the action of the serine protease, plasminogen activator. The mitogenic substance was also not a cell degradation product. The HFC from the human mammary carcinoma line BT-20 contained a non-dialyzable inhibitory activity which su~pressed the activities of the mitogerts in fetal bovine serum. Attempts to isolated the mitogenic activity from HFCs proved impractical due to proteolytic breakdown. However, a transforming growth factor (TGF) from acid-ethanol extracts of MCG-T14'mouse mammary carcinoma ' ' cells was isolate~ and·partially characterized. This factor stimulated thymidine uptake in BALB/c 3T3 cells and promoted anchorage-independent growth of NRK-49F cells in soft agar. The mitogenic activity, designated T14-TGF, stimulted thymidine uptake in conflue.nt-quiescent BALB/c 3T3 cells to the same extent as tha,t exhibited by 5% calf serum. T14-TGF was potentiated by EGF in its ability to promote colonial growth of NRK-49F cells. In competition binding assays, T14-TGF and EGF competed for binding to BALB/c 3T3 and A431 cells. However, EGF was a more efficien~ competitor than T14-TGF in all experiments and T14-TGF exhibited only partial inhibition of EGF-binding to A431 cells. This suggests that T14-TGF may have contained subfractions which did not compete for EGF binding sites. Nerve growth factor, fibroblast growth factor, and multiplication stimulating activity did not compete with T14-TGF for binding .. 1 sites .on BALB/c 3T3 cells. In addition, iodinated T14-TGF did not bind to NR6/6 3T3 cells which lack EGF receptors. It was concluded that Tl4-TGF must interact with EGF receptors specifically~ 2 T14-TGF was purified to apparent electrophoretic homogeneity by electroelution from 15% SDS-urea polyacrylamide gelso The factor was basic (pi 8.3), had anapparent moleculaJ; weight of 14,000 and was selectively inactivated by trypsin. In addition, T14-TGF exhibited no proteolytic activity and contained no carbohydrate residues. The biological activity of T14-TGF was resistant to inactivation by acid or heat and· denaturation by guanidine HCl. T14-TGF was completely inactivated by treatment with dithiothreitol which indicated that it required one or more intact disulfide .bridges for activ~ty. A unique characteristic of T14 ... TGF was that this factor bound very strongly to. both DEAE or carboxymethyl f:on excha'Iigers. T14-TGF contained high proportions of basic amino acid residues lys~ne and arginine.
    • Transient Self-Association of Beta2-Adrenergic Receptors

      Lan, Tien-Hung; Department of Pharmacology and Toxicology (2014-03)
      G-protein coupled receptors (GPCRs) constitute the largest family of cell surface receptors. Through binding to ligands such as hormones, peptides, neurotransmitters, or photons, GPCRs are activated and regulate a variety of physiological responses. It has been widely accepted that GPCRs can self-associate as dimers or higher-order oligomers even though protomeric GPCR is capable of activating heterotrimeric G proteins and recruiting arrestins. Although GPCR complexes have been suggested to possess distinct functional properties such as receptor trafficking, receptor phosphorylation, biased downstream signaling, and allosteric communication, the stability and the structural mechanisms for the dimerization of class A GPCRs have not been extensively studied. The β2 adrenergic receptor (β2AR) is a prototype of class A GPCRs and is probably the most extensively studied among the currently available high resolution crystal structures of GPCRs. However, lack of clear dimer interface and the controversy of the stability of β2AR dimeric complexes brings to the question that whether β2AR self-associate as a stable dimer.

      Hellebuyck, Rafael Adriel; Department of Biostatistics and Epidemiology (2019-01-08)
      Within the medical field, the demand to store and analyze small sample, large variable data has become ever-abundant. Several two-sample tests for equality of means, including the revered Hotelling’s T2 test, have already been established when the combined sample size of both populations exceeds the dimension of the variables. However, tests such as Hotelling’s T2 become either unusable or output small power when the number of variables is greater than the combined sample size. We propose a test using both prepivoting and Edgeworth expansion that maintains high power in this higher dimensional scenario, known as the “large p small n ” problem. Our test’s finite sample performance is compared with other recently proposed tests designed to also handle the “large p small n ” situation. We apply our test to a microarray gene expression data set and report competitive rates for both power and Type-I error.