Abstracts Submitted: Oral Presentation (post-baccalaureate students only)
ENGINEERING THE DCRA GENE FROM CHLAMYDIA TRACHOMATIS SEROVAR E INTO A NEW EXPRESSION VECTOR
John Hoskins, Annette Rau, and Jane Raulston, Department of Microbiology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City TN 37614
The sexually-transmitted disease agent, Chlamydia, is an obligate intracellular bacterium that competes with humans for iron. The struggle likely contributes to immune-mediated damage caused by an up-regulation in chlamydial virulence factors. DcrA (Divalent cation-dependent regulator A), is a transcription factor that enables Chlamydia to respond to varying concentrations of iron in the environment by regulating the production of iron-binding proteins and virulence factors. While it appears that DcrA is flexible in which promoter sequences it can bind to, more studies are needed regarding promoter sequence identification. Previous challenges have been encountered in DcrA protein purification, specifically in the removal of a recombinant polyhistidine amino acid tag. Our primary objective was to genetically engineer the Chlamydia trachomatis serovar E dcrA gene into an expression vector that will provide alternative sites for synthetic amino acid tag removal. In addition, the dcrA gene from Chlamydia trachomatis serovar E was sequenced for comparison with other sequenced chlamydial isolates. pET vectors containing an alternative amino acid tag were transformed into E. coli cells for replication. A midi plasmid preparation was performed, followed by digestion with restriction enzymes. Finally, the pET vectors were purified via gel electrophoresis and de-phosphorylated. Chlamydial elementary bodies were lysed, and dcrA was amplified using Polymerase Chain Reaction (PCR). The chlamydial DNA was digested with the same restriction enzymes used to digest the pET vector. The insert and vector were combined and treated with ligase. The reaction mixtures were transformed and the gene identified via PCR. Finally, the pET vector containing dcrA was sequenced. Gel electrophoresis data confirmed that the midi-plasmid prep, restriction enzyme digestions, and PCR products reflected the successful isolation and ligation of dcrA gene into the pET plasmid. The sequence alignment of Chlamydia trachomatis Serovar E has also been confirmed through comparisons with the previously sequenced gene from C. trachomatis Serovar D.
ELECTROCHEMICAL REMEDY AND ANALYSIS FOR THE ENVIRONMENT BASED ON A NEW POLYMER –DNA COMPOSITE MATERIAL
Noor F. Hussain, Larry R. Gordon, and Mian Jiang, Department of Chemistry, East Tennessee State University, Johnson City, TN 37614.
Heavy metals such as Hg, Cd, and Pb have become major problems in the environment. Solid phase micro extraction (SPME) is being used to extract these metals which provide solvent free, easy to recycle means, and has emerged as an innovative environmental technology. In this work a new material, the conducting polymer –DNA composite, has been reported for the first time due its promise in micro extraction, transfer, and desorption of cations under controlled potential conditions. This electrochemical combined SPME provides a platform for controlled recycle, by which the extraction and desorption can be controlled by simply adjusting the potential of the substrate electrode. The composite made by polypyrrole embedded with chromosomal DNA has been chosen as a model SPME material because of its easy preparation. The composite can be formed by oxidation of pyrrole monomers in the presence of chromosomal DNA. In our work we employed electropolymerization to fabricate the composite, because the amount of the resultant coating, i.e. the area of the extraction surface, can be easily controlled by electrolysis period, by applied potential, or current for their optimal ion exchange capacities. Environmental significant Cd, Pb, Hg, Co, Zn, Cu and Bi metal ions can be extracted from the aqueous solution and are able to be transferred to another solution. We can detect the metals in the desorption solution, or the transferred solution by cyclic voltammetry. Further, Cd2+, Pb2+, Hg2+ and Co2+ from a mixture of solutions that can be extracted and detected at the same time. Using Cd2+ as a model, this method has been examined for the optimal adsorption media, the best desorption/detection media and the interference from other cations and anions. Recovery tests have been also carried out with satisfactory results.
This work was supported by Starter Award from SACP (Pittsburgh, PA) and by RDC, Student-Faculty Grants from ETSU (Johnson City, TN).
Bhavya Voleti and Alok Agrawal, Department of Pharmacology, East Tennessee State University, TN 37614
C-reactive protein (CRP) is made in liver and its serum concentration increases in chronic and acute inflammation. Measurement of serum CRP is recommended for use as an indicator of inflammation and predictor of heart complications. Cholesterol-lowering drugs statins also lower CRP. To evaluate statin-mediated CRP reduction and to reassess clinical usefulness of CRP, we investigated regulation of CRP gene expression. Here, we show that pravastatin and nitric oxide (NO) prevent induction of CRP expression in human hepatoma Hep3B cells exposed to inflammatory molecules IL-6 and IL-1β. These findings suggest that the decreased CRP levels in vivo after statin-treatment do not necessarily reflect absence of inflammation, and raise the possibility of utilizing NO-releasing drugs to reduce serum CRP. In addition, we located on the CRP promoter, a regulatory element with overlapping binding sites for transcription factors Oct-1 and NF- қ B (p50-p50 and p50-p65). Basal CRP expression increased dramatically when binding of both NF-қB and Oct-1 was abolished. Results indicate that the binding of Oct-1 to the promoter, facilitated by p50-p50 in a novel way, represses while replacement of Oct-1 by p50-p65 induces CRP transcription. Combined data provide a mechanism for the regulation of CRP expression under acute inflammatory conditions and suggest that the baseline/chronic CRP levels may fluctuate. We propose that the measurement of CRP levels, in normal healthy population and in individuals on statin/NO-therapy, is not as useful as was imagined.
DECTIN-1 EXPRESSION IS INCREASED IN THE BLOOD, SPLEEN, AND KIDNEY OF MICE CHRONICALLY INFECTED WITH CANDIDA ALBICANS
Tammy Ozment-Skelton*, Donald Ferguson#, Johanna Preiszner+, John Schweitzer+, John Kalbfleish**, Gordon Brown##, Siamon Gordon++, and David Williams*
Departments of Surgery*, Microbiology#, Pathology+, and Biometry and Medical Computing**, Quillen College of Medicine, East Tennessee State University, Johnson City, TN ##Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa ++Sir William Dunn School of Pathology, Oxford University, Oxford, UK
Candida albicans is a commensal organism that causes life-threatening systemic infection in the immuno-compromised host. The cell mediated immune response is primarily responsible for host defense against Candida infection; however, the means by which the immune system recognizes and responds to Candida is not fully understood. The Candida cell wall contains multiple pathogen associated molecular patterns (PAMP) that are recognized by pattern recognition receptors (PRR) on immune cells. One such PAMP is 1-3-b-d-glucans. The primary PRR that is known to bind and respond to glucan and is found on cells of the immune system is Dectin-1. The importance of this receptor in the response to Candida infection has yet to be determined. The goal of the present study was to determine the effect of chronic Candidiasis on blood and spleen Dectin-1 expression. ICR/HSD mice were give 1x105 cfu of a laboratory control strain of Candida albicans IV, and blood, spleens, and kidneys were collected at 1, 3, 5, 7, and 14 days post-infection. Mice that received diluent alone were used as controls. These cells were analyzed for Dectin-1 expression by flow cytometry in the blood and spleen and by immunohistochemistry in the kidney. With infection we found that Dectin-1 expression in the blood was significantly increased over control expression by 44.0%, 23.5%, and 58.9% at days 3, 5, and 14 of infection, respectively (p<0.05). Additionally, we found that splenic Dectin-1 expression was increased by 86.5% on day 7 of infection when compared to control levels (p<0.05). Immunohistochemistry revealed an obvious increase in Dectin-1 expression in the kidneys of infected mice at days 3 and 5 of infection. Cellular phenotyping by flow cytometry of blood and splenic leukocytes, and H&E staining of the kidneys revealed an increase in the numbers of Dectin expressing cells, namely neutrophils in the blood and spleen and neutrophils and macrophages in the kidneys. From this we conclude that chronic systemic infection with ....
THE AFFECT OF ADOLESCENT-PARENTAL COMMUNICATION ON ADOLESCENT SELF-MONITORING
Kelli Ferguson and Dr. Chris Dula, Deptartment of Psychology, East Tennessee State University, Johnson City, TN 37614 Dr. Chris Qualls, Department of Psychology, Emory & Henry College, Emory, VA 24327
Much of the research regarding self-monitoring has been conducted using college students as participants (as cited in Pledger, 1992). However, adolescents are an important group to study, as developmental changes affect self-monitoring and self-esteem. The role of family communication in relation to an adolescent’s degree of self-monitoring should be examined, as the family has been found as one of the most important influences on adolescent development (White & Matawie, 2004). Self-monitoring is defined as self-observation and self-control which are guided by situational cues to social appropriateness (Snyder, 1974). Individuals may fall into one of two categories, high self-monitors or low self-monitors, based on the extent to which they adjust their behavior according to situational cues. High self-monitors continuously assess cues in a situation and adjust their behavior accordingly while low self-monitors tend to act similarly across different situations (Snyder, 1980). Like adults, adolescents vary in their levels of self-monitoring (Lapsley, Jackson, Rice, & Shadid, 1988). An adolescent’s identity formation and role-taking ability has been found to be affected by family communication (Barnes & Olson, 1985). Cooper et al. suggest that adolescents who experience the support of their families may feel freer to explore issues of identity (as cited in Barnes & Olson, 1985). Further, communication differences in mother-adolescent and father-adolescent relationships may play a role in adolescent self-monitoring as past research has found that these relationships differed substantially (Barnes & Olson, 1985). The present study was designed to examine the influence of family communication on adolescent self-monitoring, as well as to add to the limited amount of research concerning adolescent self-monitoring. It was hypothesized that 1) adolescents who rate communication with their parents as less open, would be more likely to be high self-monitors, and that, 2) overall, adolescents will report more open communication with their mothers. Participants in the study consisted of 30 adolescents (6 males and 22 females), ranging in age from 11 to 14 years (M = 12.64, SD = .83) from a middle school in Southwest Virginia, who completed the Parent-Adolescent Communication Scale: Adolescent Form, and the Junior Self-Monitoring Scale. The hypothesis that adolescents who rate communication with their parents as less open would be more likely to be high self-monitors was partially confirmed. In an examination of overall parent-adolescent communication, level of communication did not appear to affect self-monitoring. However, a closer examination of the means showed that this difference was almost exclusively the result of differences in high and low self-monitoring for communication with the father. In particular, low self-monitors reported better communication with their fathers than did high self-monitors. Mother-adolescent communication did not appear to affect adolescent self-monitoring. Analyses confirmed the second hypothesis that adolescents would report more open communication with their mothers. Results will be discussed in terms of their relation to the existing literature and theory, and with regard to future directions for study.
ATTITUDES TOWARD SUICIDE ATTEMPTERS
Kandi Shearer, East Tennessee State University, Department of Psychology, Johnson City, TN 37614
A Survey Examining The Attitudes In A College Population Toward Suicide Attempters
Suicide has become an increasingly important health concern over the past few decades due the drastic increase in its occurrence. The American Association of Suicidology (2001) estimated that 30,622 people living within the United States commit suicide each year, and it has been shown that for every death by suicide, between 8 and 20 unsuccessful attempts are made. With this many suicidal attempts being made, understanding people’s reaction to and attitude towards this behavior becomes crucial. Past research on suicide indicates that a culture’s attitude toward suicide has a distinct impact on that culture’s rates, with accepting attitudes resulting in higher death rates. Past studies investigating attitudes held towards suicide attempters show that while suicidal ideation may be high, acceptance of this behavior varies greatly, from understanding and support to rejection and criticism. The situations surrounding the attempts have also been shown to have an impact on the reactions of others. This study attempted to isolate the college population to see whether their attitudes were primarily negative or understanding in nature and to see if attitudes varied depending on the circumstances. A survey was administered to assess demographic characteristics, the suicide ideation levels of the participants, and the perceptions formed after reading a short report of a suicidal attempt. Four different suicidal scenarios were included, two rational and two irrational attempts, and they were distributed randomly over the sample. Results from the 360 surveys (111 male, 249 female) indicated that contrary to most research, college men and women did not have significantly different levels of suicidal ideation. Additionally, men and women did not differ in their perceptions formed toward suicide attempters. However, suicide ideators were significantly more likely to view suicide attempters as more intelligent, more justified in their actions, more likable, more trusting, and more likely to be a personal friend. Circumstances surrounding the suicide attempts, be they rational or irrational, did not have a significant impact on the perceptions formed by the participants. Results further indicated that participants who reported knowing a suicide attempter were more likely to be suicide ideators themselves. These findings signify that acceptance of suicidal behavior is positively correlated with one’s own level of suicidal ideation. The understanding of these attitudes is an essential aspect to address when developing prevention programs for suicide attempts and suicide in the future.
CHLAMYDIA TRACHOMATIS ENTERS A VIABLE BUT NON-CULTIVABLE STATE WITHIN HERPES SIMPLEX VIRUS TYPE 2 (HSV-2) CO-INFECTED HOST CELLS.
Jennifer Vanover, Srilekha Deka, Jennifer Kintner, Judy Whittimore, and Robert V. Schoborg, Department of Microbiology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614
Co-infection with various sexually transmitted disease pathogens, including C. trachomatis and HSV-2, is a common event in the human population. Because the consequences are not well understood, we have established a tissue culture model of chlamydia/HSV-2 co-infection in our laboratory. Transmission electron microscopic (TEM) analyses indicated that, by 10 hours post-HSV infection, reticulate bodies (RBs) in co-infected HeLa cells had become swollen, aberrantly shaped and electron-lucent. Also, HSV-2 co-infection abrogated production of infectious chlamydial progeny in passage experiments. Both of these observations are consistent with the hypothesis that HSV-2 super-infection of the host cell drives C. trachomatis into a persistent state similar to that observed when chlamydiae are challenged by nutrient depletion, interferon gamma or penicillin. However, the reduction in chlamydial infectivity observed during co-infection could also be explained by a developmental cycle delay. To distinguish between these two possibilities, we examined a number of “molecular markers” of chlamydial persistence in co-infected cells. Western blot analyses indicated that accumulation of chlamydial major outer membrane protein (MOMP) was significantly decreased while accumulation of chlamydial heat-shock protein 60-1 (HSP60-1 or GroEL) was either steady or increased in co-infected compared to singly infected controls. PCR experiments indicate that, at 20 hours post-HSV infection, chlamydial genome copy number was similar in co-infected and singly infected cells. These data demonstrate that the abrogation of chlamydial infectivity by HSV is not due simply to a developmental cycle delay. Semi-quantitative RT-PCR experiments demonstrate that chlamydial groEL, ftsK, ftsW, dnaA, and unprocessed 16S rRNA transcript levels were not altered by HSV-2 super-infection. Although the ftsK/ftsW transcript expression profile do not match that observed in some other in vitro models of chlamydial persistence (most notably gamma interfe....
CHOOSING TO BE FREE: THE JAMAICAN REVOLT CONSPIRACY, 1776
Tabetha Garman, Department of History, East Tennessee State University Johnson City, TN 37614
In July of 1776, the white planters of Hanover Parish, Jamaica uncovered a massive insurrection plot hatched within the Creole slave elite. For the first time in Jamaican history, the conspiracy involved members of the slave class previously considered trustworthy by white society: the drivers, artisans, and domestics believed to be most loyal to their white masters.
Modern historians have taken the view that the Creoles had merely taken advantage of the distraction of British troops on the mainland colonies to mount their attack. However, this view underestimates the intellect of this unique class of slave, and fails to take into account several aspects of the slave conspiracy. By examining letters written by members of Jamaican white society, historian's accounts of the Creole slave elite, accounts of the trial of those convicted of conspiracy, and expectations of freedom that both the North American and island slave populations believed would result were the Americans to win the Revolutionary War, a clearer understanding of why the Creoles choose July of 1776 for their revolt is revealed. This paper will endeavor to show that this unique class of slave had every reason to believe the rhetoric espousing freedom from tyranny that circulated in abundance throughout the British Colonies would apply to them were they to succeed in overthrowing their white masters.