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Biomedical Sciences Seminar Series

Winter 2011 Poster Presentation
January 14, 2011 at 1 pm
Physical Sciences Lobby

Poster #1- Adrian Esqueda

The Discovery of Novel Inhibitory Compounds Against Human Apurinic/Apyrimidinic Endonuclease 1 (APE1)

Human apurinic/apyrimidinic endonuclease 1 (APE1) belongs to the endonuclease family involved in the base excision pathway (BER). APE1 hydrolytically cleaves the phosphodiester backbone 5 to the AP site, which is produced by DNA lesion-specific glycosylases after the recognition and excision of damaged DNA bases. In combination with alkylating and oxidizing agents, it has been shown that the inhibition of APE1 in certain cancer cells leads to a high degree of DNA damage eventually leading the cell to apoptosis. APE1 has been shown to be over-expressed in specific cancers including colon and breast cancer, making APE-1 a good candidate target for the development of new anti-cancer drugs. Various sets of compounds designed to inhibit APE1 activity were tested using an enzymatic assay and gel electrophoresis.


Poster #2- Rose Bustos

Electron Transfer Rates Determine Product Distribution in P450 Catalyzed Dioxygen Reduction

Developing electrode-driven biocatalytic systems utilizing the P450 cytochromes for selective oxidations depends not only on achieving electron transfer (ET) but doing so at rates that favor native-like turnover. Herein we report studies that correlate rates of heme reduction with ET pathways and resulting product distributions. We utilized single-surface cysteine mutants of the heme domain of P450 from Bacillus megaterium and modified the thiols with N-(1-pyrene)-iodoacetamide, affording proteins that could bond to basal-plane graphite. Of the proteins examined, Cys mutants at position 62, 383, and 387 were able to form electroactive monlayers with similar E1/2 values (-335 to -340 mV vs AgCl/Ag). Respective ET rates (kso) and heme-cysteine distances for 62, 383, and 387 are 50 s-1 and 16 ?, 0.8 s-1 and 25 ?, and 650 s-1 and 19 ?. Experiments utilizing rotated-disk electrodes were conducted to determine the products of P450-catalyzed dioxygen reduction. We found good agreement between ET rates and product distributions for the various mutants, with larger kso values correlating with more electrons transferred per dioxygen during catalysis.


Poster #3- Cynthia Reyes

Optimizing Parameters for Genome-wide MyoD binding +/- AKT1/2 activity

MyoD is a basic helix loop helix (bHLH) transcription factor capable of inducing skeletal muscle differentiation in a variety of cell types. Together with the other myogenic regulatory factors (MRFs), MRF4, Myf5, and myogenin, MyoD directs myogenesis. All four MRFs bind to the E-box consensus sequence (CANNTG) to initiate transcription of muscle specific genes. By acting as effectors of the IGF1/PI3K/AKT pathway, AKTs 1/2 have been shown to promote the association of MyoD at these sites. Chromatin immunoprecipitation/sequencing (ChIP-Seq) assays have identified the sites of MyoD binding throughout the mouse genome, and have revealed a number of unexpected binding sites, including sites with no E-box, or near genes that are not-muscle specific or are not up-regulated, suggesting that the mechanisms that apply to binding at muscle-specific genes may not apply to all binding sites. Our objective is to determine whether or not AKT 1/2 is mechanistically important at only a subset of these sites by inhibiting AKT 1/2 with LY, a PI3-kinase inhibitor. We hypothesize that MyoD will bind at some sites even when AKT1/2 is inhibited. If the hypothesis is correct, one or more additional binding mechanisms must be functional at those sites. A preliminary goal is to define the optimal conditions at which differences in MyoD binding can be observed in the presence or absence of LY. Currently we are working on increasing the efficiency of LY. LY has been administered in 24-hour increments at a concentration of 20L. We tested the effects of re-treating with the same concentration of LY every 12 hours. Through the use of Immunocytochemistry and RT-qPCR we monitored the difference in samples re-treated every 12 hours +/- 20L LY and those re-treated every 24 hours +/-20L LY, in protein and mRNA accumulation, respectively. The data implied that re-treating every 12 hours had similar effects as those treated every 24, with only subtle differences.


Poster #4- Deisy Contreras

Yeast Casein Kinase 2(YCK2) governs the antimicrobial peptide susceptibility of pathogenic fungi, Candida albicans by regulating SSD1 transcription.

A part of the human normal flora, Candida albicans is an opportunistic pathogen that causes serious superficial and disseminated fungal diseases in immunocompromised populations. Since C. albicans interacts with various host cells during the initiation of superficial or disseminated infection, this organism most likely has acquired to express niche specific genes to adhere, penetrate, and progress the infections. Previous genome-wide transcriptional analyses from our lab have shown that C. albicans up-regulates different sets of genes when it interacts with human oral epithelial cells compared to when it is grown on tissue culture plates. Following functional analysis on one of the up-regulated genes, Yeast casein kinase I homologue (YCK2) has shown that C. albicans mutant strain lacking YCK2 functional activity has significantly reduced capacity to damage oral epithelial cells in vitro and is hypersusceptible to antimicrobial peptides (Park et al, Eukaryotic cell, 2009). This study suggested that YCK2 is most likely one of the key genes governing C. albicans virulence. These findings led us to identify the mechanisms by which YCK2 governs the resistance of C. albicans to antimicrobial peptides. Antimicrobial peptides are small peptides secreted by the innate immune system and have antimicrobial activity. C. albicans is known to develop resistance by inducing the mRNA level of SSD1, whose function is completely unknown in this organism. First, we tested two casein kinase homologues (YCK3 and CKA2) whose mutants are available for antimicrobial peptide susceptibility to determine whether or not there exists any functional overlap. Both YCK3 and CKA2 mutants have shown similar antimicrobial peptide susceptibility compared to that of the wild type strain. These results suggest that YCK2 is the main casein kinase homologue that governs antimicrobial peptide susceptibility of C. albicans. We have also assessed whether or not YCK2 also governs the mRNA expression of SSD1, which is the key gene responsible for C. albicans resistance to antimicrobial peptides by quantitative RT PCR analysis. SSD1 mRNA expression was significantly decreased (2-4 fold, from three biological replicates) in yck2 mutant strains, which imply that SSD1 transcription is dependent on the presence of functional YCK2. The findings from this study suggest that YCK2 governs C. albicans resistance to antimicrobial peptides by positively regulating the transcription of SSD1. More detailed mechanisms on how the signals from different environments are transferred via YCK2 to the downstream transcriptional activators that enhance the gene expression of SSD1 will be further delineated in our future studies.

Poster #5- Jennifer Uyere

Functional Analysis and Characterization of the Yeast Casein Kinase 3 Gene in Comparison to the Yeast Casein Kinase 2 Gene in C. albicans

Candida albicans is a part of the human normal flora on the mucosal surface, but often causes serious life-threatening disease in immunocompromised patients. In individuals with compromised immune systems, Candida is able to cause a variety of infections. Most notably, C. albicans is the most abundant cause of oropharyngeal candidiasis and hematogenously disseminated disease. The pathogenic mechanisms by which C. albicans causes candidiasis are not well understood, therefore, treatments are limited and thus not very effective. Several studies have revealed that the initial step to causing infection is that C. albicans invades the epithelium; hence, the ability of C. albicans to damage epithelial cell lining is a major key virulence factor involved in the initiation of the disease. Previous studies in our laboratory have shown through transcriptional analysis that the C. albicans yeast casein kinase 2 homologue, YCK2, is remarkably up-regulated and plays a key functional role in the interaction between C. albicans and oral epithelial cells. In addition to finding YCK2 in C. ablicans and its function, another casein kinase homologue, YCK3, was discovered. At the genomic level, YCK3 shares over 80% homology to YCK2. Moreover, the function of casein kinase activity in C. albicans is not completely understood. It has been previously shown that in Saccharomyces cerevisiae, a non-pathogenic fungus that shares a great deal of homology to C. albicans, YCK2 is required for morphogenesis. Consequently, it has been hypothesized that YCK2 also plays an important role in morphogenesis in C. albicans, and thus influences virulence. The purpose of my research is to elucidate the functional relationship between YCK2 and YCK3 to aid in further understanding the fungal pathogenesis pathways, as well as to explore the specific phenotype brought about by the YCK3 gene. This will be investigated through an array of studies including gene deletion and overexpression analysis, generation of GFP-fusion constructs, restriction mapping, antimicrobial peptide sensitivity assays, and various other in vitro tests.


Poster #6- Luiz Galdino

Study of Freezing Point and Melting Point Changes of Type I Antifreeze Protein Aqueous Solutions

Antifreeze proteins (AFPs) inhibit the growth of ice crystals resulting in the survival of many organisms in subzero environments. Understanding the mechanism of ice growth inhibition will lead to successful non-destructive cryogenic applications such as conservation of organs for transplantation and food in frozen state. Other applications such as agriculture in subzero regions and even oil pipeline clogging prevention are also potential beneficiaries of such studies. Our current studies using cold-temperature microscopy demonstrate the growth inhibition of ice crystals in AFP and NaCl joint aqueous solutions under various NaCl and AFP concentrations. It is shown that type I AFP, a sub-class of antifreeze proteins, in sodium chloride aqueous solutions is able to cooperatively reduce the freezing point. We hypothesize that in the presence of electrolytes, thermal hysteresis induced by AFP is independent on the presence of NaCl and/but with a lower melting point. This suggests the study of bulk property is as critically important as micro-crystal analysis in understanding the ice growth inhibition mechanism of antifreeze proteins since lower melting point of bulk solution can significantly lower freezing of water. We propose a mechanism that can explain how this inhibition in saline solution occurs and how KCl and AFP solutions correlate to our proposal. Additionally, ice growth inhibitions in non-electrolyte solutions and bulk melting properties observed using micro-imaging will be presented as well.


Poster #7- Marcela Leyva


Understanding the Mechanism of Decrease Production of F1-R Sendai Virus

Sendai virus is a negative sense RNA virus which causes a respiratory infection in mice similar to the infection in humans caused by the Human Parainfluenza virus. A variant of Sendai virus call F1-R causes a systemic infection which makes it more virulent than wild type (Wt) virus. To identify which mutations are responsible for the F1-R systemic phenotype, reverse genetics was use to create variants of Sendai virus with various combinations of the F1-R gene mutations. One such variant, RGV0, has all the same mutations as F1-R except for mutations in the P and L gene. Animal studies showed that RGV0 causes a systemic infection in mice, but it appeared to be more virulent than either wt or F1-R. Tissue culture studies showed that RGV0 is replicates more like wt, consistently producing at least 10-fold more viruses then F1-R. This lead us to suggest that RGV0 is more virulent than wt because it causes a systemic infection, and that it is more virulent than F1-R because produces more viruses than F1-R . Given that the only difference between F1-R and RGV0 is that RGV0 lacks the P and L gene mutations it is believed that the mutations in the P and/ or L gene are responsible for the decrease production of F1-R. To gain a better understanding of the mechanism of decreased virus production in F1-R infections and determine if the decrease is due to an overall decrease in viruses being produced or a decrease in the production of infectious versus non-infectious virus, we have assayed the viruses via hemagglutinin, egg infectious dose assays, tissue culture infectious does assays, plaque assays and multiple round of replication assays. The results thus far may indicate that the decrease is in infectious virus production. We are currently developing both one-step and two step QRT-PCR protocols that can be used to accurately quantify the number of overall viruses and the ratio of infectious to non-infectious viruses produced during infections of the different variants of Sendai virus. An internal RNA control that utilizes the same primers and generates a product of the same size, but with a different internal sequence, has been made and it will be spiked into each experimental sample to correct for variations in amplification efficiency and for the presence of inhibitory substances. The two products will be distinguished based on the use of differentially labeled fluorescent probes. The results of the QRT-PCR assays will be compared to the results of a plaque assays, an egg infectious dose assays, a HA assay as well as a two step RT-PCR to determine if the difference in virulence is due to the amount of infectious virus being produced.


Poster #8- Melo Encinas

Modeling the dynamics of layered mussel beds

Cellular automata (CA) models have been used in studying predator-prey dynamics of marine mussel beds, Mytilus californianus. In two-dimensions (2D), these models span gradients of tidal height and wave exposure, with each grid cell having a state representing mussel size. State transitions represent mussel recruitment, growth, and mortality, and vary with gradient location and neighborhood effects. These models have been effective in describing mussel zonation; however, the current 2D approach cannot be used to investigate mussel bed formation due to hydrodynamic stress from storms. This project aims to study size and frequency of gap formation, as well as the distribution of gaps over the range of tidal heights and wave energies. C++ will be used to add a third dimension to the existing 2D model with the following main layers: basal layer (mussels attached to rock), mesolayer (mussels attached to each other), and top layer (mussels exposed to predators). The model will incorporate known mechanisms affecting layered mussel beds, such as detachment of basal layers due to sedimentation and increased hydrodynamic stress due to hummock formation. The model will be used to predict the distribution and size of mussel bed tear-outs due to storms for comparison to field observations. The ability to model realistic complex layered populations, such as mussel beds, will serve as a paradigm for other layered biological systems, such as microbial films, tissues, and forests. (Supported by NIH NIGMS MBRS-RISE grant GM61331 and NSF EF-0827595.)



Poster #9- Orlando Perez

Wheel-running exercise enhances the activation of N-methyl-D-aspartic acid receptors in the rat hippocampus

It has been well documented that both antidepressant medications and physical exercise lead to increased expression of hippocampal brain-derived neurotrophic factor (BDNF). Neurotrophins are macromolecules that play an important role in survival, growth, and plasticity-related signaling. Previous studies have suggested that BDNF expression is part of an activation/phosphorylation mechanism for amino acid derivative N-methyl-D-aspartic acid (NMDA). NMDA acts as specific agonist for the NMDA receptor, which in turn imitates glutamate neurotransmitter at the receptor. The phosphorylation of NMDA receptors is known to activate phosphoinositide 3-kinase (PI3-K), a family of enzymes used for intracellular signaling for cell growth, survival, and trafficking, and is also known to activate BDNF expression. Sprague-Dawley rats (n = 10) were allowed access to running wheels in their home cages for 3 weeks; control animals were sedentary throughout the experiment. All animals were singly housed and received food and water ad libitum. Rats were sacrificed at the end of the study, hippocampi extracted and frozen in dry ice. Tissues were processed for Western analysis, antibodies were purchased from Cell Signaling against Phospho-NMDA and total-NMDA. Our results revealed that the expression of hippocampal NMDA was increased in exercised rats. Our study has suggested that exercise increases hippocampal NMDA expression, compared to sedentary control animals. Knowledge about the cellular mechanisms of survival-enhancing interventions such as exercise will inform the development of more efficacious pharmaceutical treatments.


Poster #10- Sierra Claytor


The fox squirrel (Sciurus niger) is native to the central and eastern United States and the southern prairie provinces of Canada. It was introduced by humans into many cities within California in the late 1800s and early 1900s. It is likely that each of the introductions was composed of a small number of fox squirrels, however, it remains unknown whether the introduced animals came from one or several regions within the natural geographic range of the species. Information about the genetic diversity, population structure, and potential sources of northern and southern California fox squirrels is investigated by sequencing the D-loop region of the mitochondrial DNA because of its small genome size and conserved arrangement of genes. As the introductions of fox squirrels to California are very recent, it is expected that the mtDNA haplotypes present in California represent the effective minimum number of founders from each introduction. The source of the introductions of S. niger into California has been speculated for some time, yet conclusive evidence is lacking regarding the historical source of these introductions. Some researchers are concerned that range expansion by the non-native fox squirrel may displace the native western gray squirrel (Sciurus griseus). This study was conducted to better understand the population genetic structure of the introduced fox squirrel. ;


Poster #11- Evelyn Barrios


Poster #12- Eduardo Cabrera

Optimization of Analytical Techniques for the Detection of Oxygenated Polycyclic Aromatic Hydrocarbons on Environmental Surfaces

Polycyclic aromatic hydrocarbons (PAH) are formed in incomplete combustion processes including the burning of fossil fuels, and tobacco. Heavy PAH (those that contain 4 or more fused benzene rings) tend to reside on environmental surfaces such as particulate matter, soils and sediments. Photodegradation of PAHs may induce carcinogenic activity by forming oxygenated PAH with ketone, hydroxyl, or epoxide functional groups. Benzo[a]pyrene (BaP) is a PAH that has been studied extensively due to its carcinogenic potential. In the presence of sunlight and oxygen, oxygenated BaP derivatives, including isomeric benzo[a]oyrene-diones, are formed. Consequently, it is imperative to develop sensitive and selective analytical techniques to identify and quantify oxy-PAH on environmental surfaces. This paper will discuss preliminary results designed to optimize the extraction an oxy-PAH (BaP-dione) from silica gels that serve as surrogates for environmental dust, soil and sediment. The silica will be spiked with a known concentration of BaP-dione and dried. To extract the BaP-dione from the silica gel, a range of methanol : methylene chloride solutions will be used to reconstitute the BaP-dione in an ultrasonic bath. An Accela high performance-liquid chromatograph (HPLC) equipped with a photodiode array (PDA) detector will be used to measure the concentrations of BaP-dione in solution after extraction. The results of these experiments will be used to determine the optimal ratio of methanol to methylene chloride for the extraction of oxy-PAH from environmental surfaces. The results of this study will further help to understand the risk of oxy-PAH present in actual environmental samples. ;


Poster #13- Christine De La Fuente

Identifying the Phenotype of C. albicans Mutant Strains Against Protamine Sulfate Candida albicans is an eukaryotic microorganism that is part of our normal flora. Often times C. albicans causes either superficial or disseminated candidiasis in immunocompromised patients. Thus, its ability to become an opportunistic pathogen is one of the important topics in medical mycology to study. This virulence arises during uncontrolled colonization in which overgrowth of C. albicans leads to infection and disease known as candidiasis. Innate immunity has an important role in controlling the overgrowth of C. albicans by mediating first line host defenses, one of which is the secretion of antimicrobial peptides. Antimicrobial peptides, found on our skin and mucosal surfaces, are present to control invasive C. albicans. Previous studies found that C. albicans strains that are resistant to antimicrobial peptides have an increased expression of the SSD1 gene (Gank 2008). Our previous studies have also suggested that Yck2p, a yeast casein kinase homologue known to be involved in the glucose sensing pathway, also plays an important role in C. albicans resistance to antimicrobial peptides. From the previous finding, we hypothesized that Yck2p regulates the expression of SSD1 by mediating the signal transduction pathway. One of the known mechanisms how Yck2p transfers the environmental signal is through the glucose sensing pathway. When glucose is present in the environment Rgt4p, an upstream glucose sensor, gets activated and as a result will phosphorylate and activate Yck2p. Rgt11p, a downstream transcriptional regulator, is affected by Yck2p. The objective of this study is to test whether or not the glucose sensing pathway mediated by Yck2p also regulates SSD1 expression and further governs antimicrobial peptide resistance in C. albicans. In this experiment the antimicrobial susceptibility test was performed using a plate dilution assay for the mutant strains of the glucose sensing pathway components (RGT4, RGT11, and YCK2) and assess their susceptibility to antimicrobial peptide. If the mutant strains show either similar or greater susceptibility to the antimicrobial peptide, it will provide evidence that the regulation of SSD1 expression and antimicrobial peptide resistance is likely regulated by the glucose sensing pathway in C. albicans.


Poster #14- Abdullah Madany


Poster #15- Phil Soto

Do the F1-R M Gene Mutations contribute to the Enhance Cleavability of F and Plaque Formation of F1-R

Sendai virus (Sev) causes a respiratory tract infection in rodents. The Wild-type (wt) is restricted to the lung epithelium while a variant, F1-R, is able to cause a systemic infection. By making a reverse genetics virus, RGV0, that contained all F1-R F and M gene mutations, our lab previously proved that mutations in the fusion (F) and matrix (M) genes of F1-R were solely responsible for the pantropic phenotype of the virus. RGV0 was able to cause a systemic infection, thus proving that some combination of F and M gene mutations was responsible for the systemic infection. In previous studies it has been shown that F1-R is able to undergo multiple rounds of replication and has the ability to form plaques in LLC-MK2 cells without the addition of trypsin. Since the mutations in the F gene result in enhance cleavability of F we hypothesize that M protein should not contribute to the ability of F1-R to undergo multiple cycles of replication or form plaques in the absence of trypsin. To test the hypothesis that the F1-R M gene mutations do not contribute to the ability of F1-R to undergo multiple rounds of replication and form plaques in LLC-MK2 in the absence of trypsin, a reverse genetics virus, RGV19, will be created by performing mutagenesis on a previously mutagenized plasmid, inserting all of the F1-R F gene mutations as well as reverting an M gene mutation. 4 of 6 mutations have been done at present and once completed this virus will be subject to tissue culture plaque and multistep replication assays in the presence and absence of trypsin to determine if the M gene mutations are needed for the virus to behave like F1-R in these assays. By understanding factors affecting pathogenicity of Sendai virus, we can gain insight into the pathogenicity of RNA viruses that infect humans.

Poster #16- Amber Hannah

Expanding our Knowledge of Syringe Exchange Program Participants

Syringe exchange programs (SEPs) are a key component to harm reduction strategies which target intravenous drug users (IDUs). Majority of the research on SEPs focuses on program structure and the reduction of HIV transmission. However, there is a paucity of literature regarding the individuals these programs serve. The present study will examine client level variables such as housing status, exchange pattern, and referral source at a SEP in downtown Los Angeles-Homeless Heath Care Los Angeles, Harm Reduction Center (HRC). Upon each visit, clients enrolled at HRC will complete the Homeless Health Care Encounter Form. Data will be analyzed so as to link individual variables to client retention among other outcomes. The researchers anticipate that findings from this study will inform our knowledge about clients of syringe exchange programs locally and beyond.

Caridad Wilson
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