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Biomedical Sciences Seminar Series

Fall 2008 Poster Presentation
October 31, 2008 at 1 pm
Physical Sciences Lobby

!!Prize for best costume!!!

Poster #1- Abebayehu N Yilma

ACAT-1, a Key Enzyme in Cholesteryl Ester Biosynthesis,
is up-regulated in Human Bronchioepithelial Cells after Challenge with Pseudomonas aeruginosa
Authors: and Edith Porter

Respiratory infectious diseases continue to be among the leading causes of illness and death world wide. A better understanding of innate host defense against microbial invasion may provide alternative strategies to prevent and treat infectious diseases. Previous research in our laboratory showed that host-derived lipids including cholesteryl esters contribute to the inherent antibacterial activity of nasal fluid. Our overall goal is to develop an in vitro system to investigate the regulation of cholesteryl esters production in human bronchioepithelial cells (HBE) and assess the contribution of cholesteryl esters to the bactericidal activity of HBE by blocking the expression of Acyl-coenzyme A:cholesterol acyltransferase one (ACAT-1), a key enzyme of cholesteryl ester synthesis. HBE cells from ATCC were cultured in Keratinocyte-Serum Free Medium (KSFM) supplemented with 50 mg/ml bovine pituitary extract, 5 ng/ml recombinant human epidermal growth factor, and 10 ng/ml cholera toxin. Lipid extracts were prepared from unstimulated cell culture supernatants with chloroform/methanol and subjected to thin layer chromatography and reverse phase HPLC. RNA was extracted from HBE cells 6 hours and 36 hours after infection with Pseudomans aeruginosa, reverse transcribed and subjected to real time PCR using Trizol reagent, transcriptase kit and SYBR GREEN chemistry with probes for GAPDH and ACAT-1, respectively. Our preliminary data demonstrate measurable amounts of free fatty acids and cholesteryl esters in the low ?g/ml range in unstimulated cell culture supernatants, whereby the constitutive secretion of cholesteryl esters appears to diminish with repeated passaging. Furthermore, in real time PCR analysis, mRNA for ACAT-1 was detectable only in HBE cells that had been stimulated with live bacteria for 6 hours but not in control cell. Our HBE model appears to be useful to study the regulation of cholesteryl ester in innate defense and our first results suggest a genuine secretion of these lipids by HBE cells and up-regulation of ACAT-1 mRNA in response to bacterial infection. If successful this research may lead to better treatment of chronic infectious diseases through manipulation of cholesteryl ester production.

Poster #2-Janet Garcia

Predictors of Intimate Partner Violence in a Multi-ethnic College Sample

The purpose of the present study was to examine the relationship between attitudes accepting of IPV, negative traditional gender role beliefs, the ability to recognize IPV when it occurs, and being a perpetrator or victim of IPV in a multicultural college sample, to determine whether such attitudes and beliefs might identify those at risk for IPV. Undergraduate students (N = 183, females = 126) representing different ethnocultural groups (Latino/a, Asian, Caucasian, African American and Mixed race) between the ages of 18 and 53 years (mean age = 21.6, sd = 5.8) completed several self-report measures. The Conflict Resolution Index (CRI) is a 15-item measure of intimate partner violence, based on and similar to the Conflict Tactics Scale (CTS2; Straus et. al, 1996); one version, the CRI-P, assessed whether one had perpetrated IPV. In addition participants observed three video clips depicting different levels of IPV. For each vignette on the video, respondents responded to nine items requiring them to make judgments on the behaviors of the man and woman in the interaction, higher scores were indicative of the ability to accurately judge IPV, and provided another possible indicator of IPV that may be less affected by social desirability. The Intimate Partner Violence Attitude Scale (IPVAS; Smith et al., 2005) measured attitudes accepting of intimate partner violence. The Gender Role Belief Scale (GRBS), developed for this study, is a measure of beliefs about traditional gender roles. Social desirability was also assessed with an eight-item scale. Results showed that the mean score for this sample on the CRI-P was extremely low (X = 6, sd = 1.6), whereas for the SDI the mean score was higher than would be expected (X = 30.4, sd = 4.5). Stepwise regression analysis with gender, ethnicity, GRBS, IPVAS, and SDI scores as predictors and CRI-P and IPV-RI as outcome variables indicated that when CRI-P was used as the outcome measure, social desirability was the only significant predictor (R = .29, β = -.18, p<.05), whereas when IPV-RI was used as the outcome measure, IPVAS was the only significant predictor in the model (R = .36, β = -.29, p<.003). The predictors in this second model accounted for more variance in scores, compared to the first model. The results confirmed that IPV cuts across gender and ethnicity. The results also indicate that social desirability is likely to result in underreporting of IPV, whereas assessing the ability to recognize IPV is less vulnerable to the effects of social desirability. Implications for future research are discussed.


Poster #3- Lorillee C. Tallorin

Oxidative Addition of Singlet Oxygen to Pt (II) Thiolato Complex:
A Model System for Oxidation of cis-Platinum Antitumor Drugs

Effective treatment of nephrotoxicity by recognized cis-platinum antitumor drugsrequires a mechanistic understanding of how these drugs initiate toxicity upon binding to cysteine-rich residues of metallothionein (MT), a protein that is predominant in liver and kidneys and responsible for the detoxification of heavy metals.1 The exact mechanism of how these antitumor drugs induce toxicity is unclear. It is believed that oxidation of these metal bound MT cysteine residues may be one of the processes leading to severe side effects of cis-platinum drugs. Therefore, the goal of this study is to determine the mechanism by which metal bound MT cysteine residues are oxidized by cis-platinum drugs. The Selke lab hypothesizes that short-lived peroxidic intermediate(s) in metal-thiolate moieties like Cisplatin oxidize MTs via intermolecular or intramolecular oxygen atom transfer, and may be ultimately responsible for biological damage. Moreover, studies by Gray et al.2 indicate the formation of sulfinite product implying an intramolecular oxygen atom transfer upon photooxidation of a Pt(II) thiolate complex and no intermolecular oxygen atom transfer by the peroxidic intermediate. However, studies currently being conducted by our group indicate that peroxidic intermediate(s) involved in the oxidation of the Pt-S-cys motif are in fact capable of intermolecular oxygen atom transfer and may therefore be involved in oxidative damage. The mechanisms of the peroxidic intermediate(s) produced by these Pt(II) cysteinato complexes may provide clues to understanding the mechanistic intricacies involved in the toxicity of these cis-platinum drugs.


Poster #4-Monica Delgado

Site Directed Mutagenesis to Create Plasmids Encoding an L protein Amino Acid Change in Sendai Virus

The emergence of new viral human pathogens from variants of viruses has highlighted the importance of investigations on the genetic basis of viral infections. Wild-type (wt) Sendai virus causes a localized respiratory tract infection in mice, while a mutant, F1-R, causes a systemic infection. We attributed the difference in type of infection to amino acid changes in the F and M proteins of F1-R. In tissue culture, wild-type virus consistently produces at least 10-fold more viruses than F1-R. However, studies with reverse genetics viruses proved that amino acid substitutions in F1-R M are not responsible for F1-R’s reduced rate of virus production. RGV0, a reverse genetics virus that contains all F1-R F and M amino acid changes produced at least 10-fold more viruses in tissue culture than did F1-R. We suggest that the increased virulence of RGV0 with respect to F1-R is due to the increased rate of virus production in the infected mice. F1-R has a single mutation in each of the P and L genes that encode the viral polymerase complex proteins. Amino acid changes in either or both of those proteins are likely to be the cause of the decreased rate of virus production and decreased virulence of F1-R. This study will test the hypothesis that the mutation in the L gene of F1-R causes the decreased rate of virus production and decreased virulence of F1-R. To address this hypothesis, we will use reverse genetics to construct a virus, RGV22, containing the L gene mutation of F1-R. The RGV22 production rate will be studied in tissue culture via a multiple cycle replication assay and the pathogenicity of RGV22 in mice will be studied using real-time PCR and plaque assays on organ homogenates from infected mice.


Poster #5-Roxana Rangel-Rodriguez

Cation- Interactions: A survey of cationic/aromatic (i,i+4) patterns in ?-helices. Faculty mentor: Dr. Alison McCurdy. Cation- interactions have been shown to play an important role in biological systems. The objective of this research is to gain a better understanding of the stabilizing non-covalent cation- interaction in model helical peptides. Previous studies performed on natural pairs of residues Y/R, Y/K, F/K, F/R, W/K and W/R with four and five residues in between have shown that Y/R interaction had the most stabilizing effect followed by W/R, F/K, Y/K, W/K and F/R. As a means of comparison, a statistical survey that takes into account secondary structure, residue spacing and residue orientation has been performed on aromatic/cationic pairs of residues positioned in a (i,i+4) manner. The survey was made on a non-redundant protein structure database in protein ?-Helices for pair of residues Y/R, Y/K, F/K, F/R, W/K and W/R. Data collected indicates that pairs of residues W/R, Y/R and W/K have a higher tendency to reside in ?-helical chains of protein. Orientation preferences regarding the C-terminal end and N-terminal end of the protein for the (i,i+4) residues will also be presented. In addition, a library of pictures of energetically significant cation- interactions is under construction and an analysis of it will be present it.

Poster #6-Angelina Hernandez


Chronic stress is known to have detrimental effects on the structure and function of the hippocampus. This brain region plays an important role in spatial learning tasks. Furthermore, glucocorticoids, or corticosterone in rats, are hormones secreted during times of stress that impair spatial learning (Roozendaal et al., 2003). In addition, there have been studies showing that chronic stress is linked to depression or depressive-like symptoms. It has been shown that exercise can prevent the damaging effect of stress on the hippocampus. This may occur through one mechanism involving an upregulation of brain derived neurotrophic factor (BDNF) during exercise (Russo-Neustadt, 2003). In this study, it is hypothesized that stress impairs spatial learning and that this impairment can not only be prevented by exercise, but it can also be repaired by the use of exercise. In addition, it is postulated that chronic stress results in the depressive-like symptom of anhedonia, which is the lack of pleasure seeking. The model for stress in this experiment is the chronic unpredictable stress model that consists of seven stressors: 24 hr illumination, 4 ºC forced swim, 24 hr food deprivation, 5 min tail suspension, 24 hr water deprivation, 20 min footshock, and 2 hr restraint. Rats in the stress group (n=10) and in the stress+exercise group (n=10) received a different stressor each day for three weeks. The rats in the control group (n=10) did not undergo any stress. To test for anhedonia, the one-bottle sucrose intake test was performed on a weekly basis on all three groups. Subsequently, voluntary wheel running was implemented on the stress+exercise group after the three weeks of chronic stress to determine any reparative effects by exercise. The Barnes circular platform maze will be used after voluntary wheel running to verify whether exercise improved spatial learning ability. This maze is a circular maze with 18 evenly spaced holes around the edges. Under one hole, there is a dark escape chamber with new bedding. Rats will use spatial cues, including bright posters and boxes, in a brightly lit room to locate the dark escape chamber hole. Latency will be recorded for each trial (4 per day for 3 days) to indicate the amount of time to reach the escape hole. In support of our hypothesis, chronic stress should result in anhedonia and impaired spatial learning; while exercise should repair this effect. These preliminary results can advance the knowledge of mechanisms involved in preventing neurodegenerative effects due to chronic stress.

Poster #7-Jere A. Wilson

Cloning of Essential Genes of Acinetobacter baumanii in Escherichia

Following the completion of the first microbial genome in 1995 (Heomophilus influenzae), microbial genomics has advanced into a matured discipline with hundreds of bacterial genomes now completed. The availability of the complete nucleotide sequences of bacterial species has stimulated global approaches to identify and understand previously undiscovered gene functions. While it is clear that bacteria possess redundant, or backup functions, there are individual genes that are absolutely required for growth or viability. These are defined as essential genes. The advancement of microbial genomics has facilitated studies on bacterial essential genes whose encoded products could be potential new drug targets. Currently available antibiotics target approximately 20 of the estimated 200 essential gene products in bacteria. The recognition of the utilities of potential antibiotic targets has fostered various innovative approaches for bacterial essential gene identification. Novel antibiotics are needed in order to combat increasing spread of multi-drug resistant strains of bacteria, as well as the threat of bioterrism. In this study we have cloned essential genes from Acinetobacter baumannii to study functions of essential proteins encoded by these genes. Methods: Five essential genes of A. baumannii ATCC 17978 were PCR amplified, and cloned into pLEX5BA expression vector. The identities of the inserts were determined by restriction digest, gel electrophoresis, and DNA sequencing. Results: In this study a total of five essential genes (fabI, ileS, murA, murG, trpS) of A. baumannii were cloned into pLEX5BA, and were confirmed by restriction digest followed by gel electrophoresis and DNA sequencing. Conclusions: We have successfully cloned essential genes of A. baumannii in E. coli. Our results indicated that the pLEX5BA, an E. coli expression vector can provide a viable tool to study the function of A. baumannii essential genes. Cloning of these essential genes and subsequent over-expression and purification of encoded proteins will facilitate discovery of novel antibiotics.


Poster #8- Ricardo Sanchez

Determination of the Sequence Location for Oxidation Susceptible Protein Thiols for the Validation of the Cysteine Oxidation Prediction Algorithm (COPA)

Under oxidizing conditions a sulfhydryl group can be oxidized into sulfenic acid (S-OH), sulfinic acid (SO2H), sulfonic acid (SO3H) and, most commonly, disulfide bond (S-S). Oxidizers can increase or decrease the function of target proteins by oxidizing protein sulfhydryl groups. It is believed that certain sulfhydryl groups, also known as thiols, on the surfaces of proteins are susceptible to oxidation because of two major factors: the solvent accessibility of the sulfur atom and the presence of atoms that can stabilize the thiolate ion created by deprotonation of the thiol. An algorithm, Cysteine Oxidation Prediction Algorithm (COPA), which predicts the oxidation susceptibility of protein thiols with 80% accuracy, was recently published by our research group. Our goal in this research project is to validate COPA by determining the level of oxidation in proteins with unknown oxidation status. We intend to use COPA to predict the oxidation susceptibility of protein thiols and map reversibly oxidized protein thiols. Mapping will be divided in two procedures. In the first procedure the number of oxidation sensitive thiols in the protein is detected by a SDS-polyacrylamide gel electrophoresis (PAGE) analysis. The susceptible thiols are reacted with methoxypolyethylene glycol-maleimide MW 5,000 kD (MAL-PEG), a well known thiol modification reagent. MAL-PEG binds covalently to the oxidation susceptible thiols increasing the apparent molecular weight of the protein and thus showing as a molecular weight shift on the SDS PAGE gel. The number of such shifts indicates the number of oxidation sensitive thiols present in the protein. In the second procedure, the oxidation susceptible sulfhydryl groups are modified with Oregon green 488 iodoacetamide (OGI), a thiol reactive agent with fluorescent emitting tag. The oxidation insensitive thiols are alkylated with iodoacetamide (IAM). The protein is then fragmented with trypsin so that no protein fragment contains more than one thiol. The fragments containing alkylated thiols are separated by high pressure liquid chromatography (HPLC) and identified by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). COPA predicts that maize phosphoenolpyruvate carboxylase (PEP-C) has two oxidation susceptible thiols. In this research project we intend to determine the accuracy of this prediction.


Poster #9- Caridad Wilson

Response of Breast Cancer Cell Proteome to Hydrogen Peroxide

Hydrogen peroxide is a natural signaling molecule generated by oxidases and mitochondria during respiration and has been shown to mediate several cellular responses. This project is aimed towards investigating the molecular response of human breast cancer cells to hydrogen peroxide. One way to analyze the response is to determine which proteins are up- or down-regulated upon hydrogen peroxide treatment. Previous investigations used two-dimensional (2-D) gel electrophoresis to separate and analyze the proteomic response to hydrogen peroxide treatment in the MCF-7 human breast cancer cell line. One protein exhibited a 3.2 fold increase in the presence of H2O2. The identity of the protein, determined by mass spectrometry, is thought to be THO complex 3 (THOC3), a member of the TREX (transcription/elongation) complex which is involved in mRNA export from the nucleus to the cytoplasm. THO complex 3 has also been suggested to be involved in protein-protein interactions and molecular evidence suggests that it is required for transcription elongation. A Western Blot analysis confirms that the protein is in fact THO complex 3. In future studies, small interfering RNA’s (siRNA’s) will be used to knockdown the THOC3 gene and to determine if the cells can survive hydrogen-peroxide mediated apoptosis. This current study will hopefully lend greater insight into how cells cope with oxidative stress mediated by hydrogen peroxide.

Poster #10-Suzi Sanchez


Using Gene Knockout to determine the essentiality of the ß-Ketoacyl-acyl carrier protein synthase I genes in Acinetobacter baumannii

The recent emergence of multi-drug resistant strains of bacterial pathogens has revived the imminent need for the development of novel antibiotics. Most currently used classes of antibiotics target the products of essential genes. Consequently, identification and validation of essential genes within bacterial species is important to discovery of novel antibiotics. Acinetobacter baumannii epitomizes the current trend, being documented in multiple sources as the causative agent in an array of nosocomial infections. It is believed that A. baumannii dedicates a large portion of its genome to pathogenecity and that it has acquired a considerable amount of foreign DNA through horizontal gene transfer. We propose to use a recombination based gene knockout method to identify essential genes. The A. baumannii 17978 chromosome contains homologous copies of the fabB gene known to be essential in related organisms. This gene codes for the ?-ketoacyl-acyl carrier protein synthase I, an essential enzyme in bacterial type II fatty acid synthesis. In order to determine whether one or both copies of the fabB genes are essential, a suicide vector has been validated and used to knockout both copies of the gene individually. Methods: The linear cloning vector pET-30 ek/LIC was circularized using restriction enzymes to remove the LIC sequence from each end followed by ligation using T4 DNA ligase. The circularized vector was transformed into NovaBlue Escherichia coli competent cells via electroporation. It was then recovered using a miniprep DNA purification kit. The recovered plasmid was subsequently transformed into A. baumannii 17978 via electroporation. The cell supernatant was plated on media containing kanamycin to positively select for cells that had gained resistance via expression of a kanamycin gene from the pET-30 ek/LIC plasmid. Since the pET-30 ek/LIC plasmid was unable to proliferate within this strain it was used to construct the suicide vector. Primers were designed to specifically amplify the middle regions of the genes of interest while incorporating ligation independent cloning (LIC) sequences flanking the inserts. The PCR products were purified and ligated into the vector through complementary base pairing of the LIC overhangs. Using an electroporation technique the suicide vector was transformed into the strain of interest. Colonies that were able to grow on selective media were screened for the presence of the suicide vector following extraction of all plasmid DNA. Colony PCR will be used to confirm the presence of the pET-30 ek/LIC suicide vector within the chromosomal DNA. Results: Using electroporation we were able to successfully transform into the A. baumannii strain using the pWH1266 vector as a control to test transformation efficiency and cell competency. Cells that had taken up the plasmid were selected for on LB containing tetracycline 10 ?g/ml. The plasmid was subsequently re-isolated from these colonies and its identity was confirmed via restriction digest and gel analysis. We were able to successfully recover the modified circularized form of pET-30 ek/LIC after transformation into E.coli competent cells. The pET-30 ek/LIC was subsequently shown to be incapable of replication within A. baumannii 17978, making it an appropriate suicide vector. Furthermore, sequencing results indicate that fabB genes can be successfully cloned into pET-30 ek/LIC. Clones containing portions of the non-essential AIS gene, fabB1 gene, and fabB2 gene were all successfully constructed and recovered for use in knockout experiments. Independent transformations into A. baumannnii with each of these clones produced cells that had gained the ability to grow on selective media containing 30 µg/ml kanamycin. Based on restriction gel analysis of recovered plasmid DNA samples, these recombinant plasmids were not detectable in the A. baumannii transformants screened. Our results indicate that fabB1 or fabB2 genes are not essential when knocked out individually, presumably due to the cellular function that the remaining copy serves within the cell.


Poster #11- Alex Valle

Reactive and Dissociative Mechanisms of Propyl Radicals in the Troposphere

Ozone in the polluted urban troposphere—associated with high smog episodes—is a primary contributor to the health issues seen in those susceptible to respiratory distress. Alkyl peroxy radicals play a significant role as intermediates in the production of ozone in the troposphere. Our research focuses on alkyl peroxy radical formation and understanding its role in the chemical mechanisms governing ozone formation. Specifically, we have studied the reaction dynamics of the alkyl radical precursor using pyrolysis techniques at low to moderate pressures (1 – 50 Torr). The mid-infrared spectra following pyrolysis of 1- and 2-iodopropane were collected using a long path length absorption/FTIR apparatus. Pyrolysis was performed as a function of temperature and added oxygen. Spectral results indicate that 1-iodopropane forms propene, ethene, and methane. Pyrolysis of 2-iodopropane forms only propene. Even in the presence of added oxygen, the reactions products were identical, and we observed no evidence for formation of propyl peroxy radical. We used ab initio techniques to determine stationary points on the potential energy surface including transition states for each the reaction. Molecular dynamic trajectory calculations were performed at various temperatures to determine the probability of dissociation of the propyl radical to products before addition of oxygen to form propyl peroxy radical. At 300K, more than half of trajectories were dissociatve. At 900K, all trajectories lead to dissociation. If correct, our results indicate that the mechanism for ozone production from C¬3 and larger alkanes an alkene intermediate, and not formation of an alkyl peroxy radical intermediate.

Poster #12- Sergio Rivas

The study of expander graphs that are Ramanujan

The theory of expander graphs is a hot topic in mathematics and computer science, as these graphs have a wide range of applications. One of these applications is to communications networks. Think of a communications network as a graph where each vertex represents a computer in a network and two vertices are connected by an edge if they can communicate directly with one another. When designing a communications network one wishes that any message originating from anywhere within the graph will propagate quickly. There are various measurements, which describe how well a graph spreads messages. One such measurement is called the spectral gap of a graph. A graph is k-regular if every vertex of the graph has k edges incident to it. The spectral gap of a connected, k-regular graph X is given by k– ?1(X), where ?1(X) is the second largest eigenvalue of the graph X. One wishes to construct infinite families of regular graphs where the spectral gap of each graph is bounded away from zero by a fixed amount. That is, one wants to make ?1(X) as small as possible. But at the same time one wishes to minimize the number of edges in each graph of the family. Some of the best graphs that have the property of being highly connected are families of Ramanujan graphs. One way to construct these families of graphs is via group theory and representation theory.

Caridad Wilson
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