|Joseph Henriquez||Elena Chavez|
|Nu Nguyen||Frances Alencastro and Maribel Marquez|
|Rachel Sanchez and Dr.Sephr Eskandari||Carla Noguera|
|Name of Student||Undergraduate / Graduate||Research Mentor / P.I|
|Carla Noguera||Undergraduate||Dr. Jill.P.Adler-Moore|
|Diego Jaime||Undergraduate||Dr. .Robert Talmadge|
|Erika Espinoza||Undergraduate||Dr. .Angel Valdez|
|Frances Alencastro||Graduate||Dr. .Ansel Zhao|
|Giselle Blanco||Undergraduate||Dr. .Ansel Zhao|
|Jessica Jackson||Graduate||Dr. Wei Jen Lin|
|Joseph Henriquezez||Graduate||Dr. Jill.P.Adler-Moore|
|Juan Ruiz||Graduate||Dr. Wei Jen Lin|
|Luladey Ayalew||Undergraduate||Dr. Jun Jun Liu|
|Melissa Arroyomendoza||Graduate||Dr. Nancy Buckley|
|Michael Medrano||Undergraduate||Dr. Ertan Salik|
|Nicole Berry||Graduate||Dr. .Francis Flores|
|Rachel Sanchez||Graduate||Dr. Sephr Eskandari|
|Sarah Saleemi||Undergraduate||Dr. Craig LaMunyon|
|Luladey Ayalew and Dr. Liu||Melissa Arroyomendoza and Dr.Buckley|
|Michael Medrano and Dr. Salik||Dr. Francis Flores and Nicole Berry|
|Erika Espinosa and Dr. Valdes||Frances Alencastro and Dr. Zhao|
|Giselle Blanco and Dr. Zhao||Jessica Jackson and Dr. Lin|
|Dr. Lin and Juan Ruiz|
|Rachel Sanchez and Dr. Eskandari||Dr. LaMunyon and Sarah Saleemi|
Sitting(L-R): Juan Ruiz, Noelle Olson, Natasha Ward, Jessamine Quijano
Standing(L-R): Frances Alencastro and Anna Zelaya
L-R: Frances Alencastro, Natasha Ward, Jessamine Quijano & Anna Zelaya
Jessamine Quijano was awarded the Certificate of Achievement for her Oral Presentation.
Bake Sale: Held by the RISE Club on Jan 6th 2011
L-R: Daisy Cuevas, Carla Noguera, Michael Medrano, Gustavo Ramirez, Frances Alencastro, Jessamine Quijano & Anna Zelaya
Summer Research Experience: For eight weeks during the summer, the students spent every day learning a wide variety of research techniques and worked on independent research projects in the laboratory of their faculty research mentors (see abstracts below from summer research symposium). See table & pictures below for rise students and their P.I/research advisor.
|Name of Student||Undergraduate / Graduate||P.I|
|Anna Zelaya||Undergraduate||Dr.Graciela Brelles-Marino|
|David Zuniga||Undergraduate||Dr.Winny Dong|
|Elysse Gatdula||Graduate||Dr.Angel Valdez|
|Frances Alencastro||Undergraduate||Dr.Ansel Zhao|
|Gustavo Ramirez||Undergraduate||Dr.Graciela Brelles-Marino|
|Jessamine Quijano||Undergraduate||Dr.Steve Alas|
|Juan Ruiz||Undergraduate||Dr. Wei Jen Lin|
|Liliana Nunez||Undergraduate||Dr.Winny Dong|
|Matthew Maestas||Graduate||Dr.Sephr Eskandari|
|Melissa Arroyo-Mendoza||Undergraduate||Dr. Nancy Buckley|
|Michael Peralta||Undergraduate||Dr. Hossein Ahmadzadeh|
|Noelle Olson||Undergraduate||Dr.Christos Stathopolous|
Left: Anna Zelaya and Dr.Graciela Brelles-Marino
Right: Gustavo Ramirez and Dr.Graciela Brelles-Marino
Left: Natasha Ward, Dr.Jill. Adler-Moore, Right: Jessamine Quijano &
Carla Noguera & Elena Chavez Dr. Steve Alas
Left: Standing: Dr.Sephr Eskandari Right: David Zuniga, Liliana Nunez &
Sitting: Matthew Maestas Dr.Winny Dong
Left: Standing: Dr.Nancy.Buckley Right: Dr. Hossein Ahmadzadeh
Sitting: Melissa Arroyo-Mendoza Michael Peralta
Left: Standing: Dr.Ansel Zhao & Right: Noelle Olson &
Frances Alencastro Dr.Christos Stathapolous
Left: Dr.Wei Jen Lin & Juan Ruiz Right: Standing: Elysse Gatdula
Sitting: Dr. Angel Valdez
Skills Workshops: The summer began with a two week, Laboratory Skills Workshops led by Dr Sperry for the Undergraduate-Intensive participants which included experiments in tissue culture, PCR reactions, Southern and Western Blotting, ELISA, DNA sequencing, microarray, purification of green fluorescent protein, SDS-PAGE,and affinity chromatography.
Standing (left to right): Noelle, Jenny, Jessamine, Frances, David, Michael
Sitting (left to right): Melissa, Dr. Sperry, Liliana, Carla, Juan
GRE-Preparation Workshops: These workshops were led by Dr.Steve Alas who instructed the students on how to take GRE’s. The students took GRE tests before and after attending this workshop.
PhD. Application Workshops: These workshops were led by Dr.Nancy Buckley where she taught the students how to fill out the PhD application forms and how to write personal assays. She also arranged interviews for the students with the Cal Poly Career Center so that the students could learn how to prepare for interviews and practice for their interviews.
Grant Writing Workshops: These workshops were led by Dr. Jill Adler-Moore. The Grant Writing workshops enabled the students to understand the information about different requirements for various funding agencies, differences between goals and objectives, and some understanding of what information should appear in the Background/Significance section of a grant. This workshop series will continue throughout the year, culminating in their writing a grant on their own independent research project.
Journal Club Workshops: These workshops were led by Dr. Shantanu Sharma who assigned journal articles for the students to read before the workshops and then discussed the journal article with them at the workshop. This resulted in students having a better understanding of the differences between the types of scientific written communication, and the differences between the various sections of a conventional journal article. This activity will continue throughout the year.
Presentation Workshops: These workshops were led by Dr. Jill Adler-Moore. The students were asked to prepare powerpoint presentations on selected topics for each meeting and the presentations were recorded for them to see later to allow them to critique themselves. Evaluations were done for each student by both Dr. Adler-Moore and the students attending the workshops. The students were given the comments after each presentation to help them improve their oral presentation skills.
Graduate Coordinator Visit: Graduate coordinators as well as graduate students from University of California at Irvine, University of California Merced, University of California Riverside, University of California San Diego and City of Hope came to the campus to visit with the RISE students. They gave them information about the graduate programs at their respective campuses, and answered the students’ many questions concerning different aspects of the programs.
Field Trip: The students visited City of Hope and were given a tour of different laboratory facilities on the campus to give them an overview of the type of PhD program that they had and the type of research that was being done at this medical research institution.
Ethics Workshops: The Ethics workshops were led by Dr. Adler-Moore, Dr. Buckley and Dr. Alas and covered topics such as the range of ethical challenges inherent in doing research, why appropriate statistics have to be used in analyzing and presenting research data, and understanding the consequences of falsifying research data.
Seminar Series: The Coordinator of the Seminar Series was Dr.Graciela Brelles-Marino. The speakers were Dr.Marcelo Tolamalsky from California State University Fullerton, former Cal Poly Pomona student Griselda Zuccarino-Catania who is a fourth year PhD candidate at Yale University and Dr. Daniel Martinez from Pomona College. The Seminar Series provided the students with a broader knowledge of the kinds of research that is being done at various institutions and each seminar was followed by informal discussions with the speakers who answered questions about how and why they decided to pursue a scientific PhD career.
The seminar speaker schedule was as follows:
July 17th 2009 (4:00pm-6:00pm) in Bldg 4-2-314
Dr. Marcelo Tolmasky
Department of Biological Science,
California State University Fullerton
Topic: “The problem of antibiotic resistance: new insights in the search for a solution”.
July 24th 2009 (4:00pm-6:00pm) in Bldg 4-1-314
Ms. Griselda Zuccarino-Catania
Ph.D. candidate Yale University
Cal Poly Pomona alumna
Topic: "Studying Memory B cells, and a grad school experience".
July 31st 2009 (4:00pm-6:00pm) in Bldg 4-1-314
Dr. Daniel Martinez
Department of Biology
Topic: "WMD: Do hydra use giant viruses to keep algae hostage?”
Summer Research Symposium: A research symposium was organized by RISE which included students from various student summer research programs: i.e. RISE, CCRAA,McNair and HHMI. The symposium was a one day event, with two concurrent sessions held throughout the day. The students each had 20 minutes to give an oral presentation on their summer research. The scientific abstracts for the RISE students who participated in this symposium were as follows:
Comparing The Memory Immune Response to Systemic Candida albicans in Wild Type, Heterozygote, and Knockout Mice.
Student: Melissa Arroyo-Mendoza
Research Advisor:Dr. Nancy Buckley
Cannabinoids, such as delta-9-tetrahydrocannabinol (THC), the psychoactive component of marijuana, is known to modulate resistance to bacterial, viral and protozoan infections. However, very little is known about the effects of cannabinoids on yeast infections. Recently, in our laboratory, we have found that THC (4-64 mg/kg) decreases the memory immune response to a systemic Candida albicans (C. albicans) infection in female c57BL/6 mice. In this paradigm the mice were chronically injected with THC (i.p.) on days 1-4, 8-11 and 15-18. On day 2, the mice received 0.75x106 C. albicans cells/ml (100l/mouse, i.v.). On day 19, the mice received 5x106 C. albicans cells/ml (100l/mouse, i.v.). THC is known to bind to two known cannabinoid receptors, the central cannabinoid receptor (CB1R) and the peripheral cannabinoid receptor (CB2R). CB1R is predominantly expressed in the central nervous system and thus is considered to mediate the psychoactive effects of THC. CB2R is mainly expressed in cells of the immune system, and thus thought to mediate the immune effects of THC. Thus, the goal of the present study is to determine the role of CB2R in the THC effect on the memory immune response to systemic C. albicans infection. To carry out this study, we will use CB2R wild type, CB2R heterozygote and CB2R knockout mice, all on a c57BL/6 background. In our study we will also include male mice. We first studied the memory paradigm in these mice as follows. Male and female CB2R wild type, CB2R heterozygote and CB2R knockout mice received 0.75x106 C. albicans cells/ml (100l/mouse, i.v.) on day 1. On day 18, the mice received 5x106 C. albicans cells/ml (100l/mouse, i.v.). The mice were monitored daily for weight and morbidity and survival will be determined at the end of the study (day 33). Thus far, day 17, mice are not displaying differences in weight or morbidity. At the completion of this study, we will chronically treat the male and female CB2R wild type, CB2R heterozygote and CB2R knockout mice with THC and the memory response paradigm as described previously. Our findings will help us elucidate whether the THC effects seen on the memory paradigm are mediated by CB2R or not.
A two-gene phylogeny of Chelidonura and the validity of some Caribbean species
Research Advisor: Dr.Angel Valdez
Several species of Chelidonura are known from tropical and subtropical regions. In the Caribbean a number of new species have been described during the last few years, mainly based on external morphology and coloration. Some authors have suggested that at least some of this new diversity constitute color forms of other species. The present project aims to 1) determine genetic divergence between the newly described species in order to verify how many of these are valid and 2) provide a preliminary phylogeny for Chelidonura based on 16S and H3 gene data, including some sequences from GenBank. The molecular phylogenies obtained support the need to synonymize most of the newly described Caribbean species, except for Chelidonura cubana, which is distinct. The phylogenies contain some geographic structure that appears to indicate a diversification in Chelidonura before the closure of the east-west main communication, as some clades contain a mixture of Atlantic and Indo-Pacific species. The pan-tropical Chelidonura hirundinina constitutes at least two distinct clades, likely separated by the formation of the Panama Isthmus.
Effect of Bisphenol A on Human Mesenchymal Stem Cell Differentiation
Research Advisor: Yuanxiang (Ansel) Zhao
Adult stem cells are undifferentiated cells found throughout the body among differentiated cells in tissues. The primary role of adult stem cells is to maintain and repair the tissues in which they are found. Scientists and researchers are interested in stem cells because of their ability to self-renew for long periods of time and to differentiate into specialized cells. Human mesenchymal stem cells (hMSCs) differentiate into several cell types including bone cells (osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), muscle cells, and neurons. hMSCs are an excellent model to study the toxicity of pharmaceutical drugs and the potential effects of environmental chemicals on the body.
The purpose of this research was to investigate the effect of Bisphenol A (BPA) on adipogenesis, the process in which undifferentiated cells differentiate into fat cells, in hMSCs. BPA is a chemical compound used primarily in the production of high performance polycarbonate plastics. Many consumer products including toys, drinking containers, eyeglass lenses, sports safety equipment, medical devices, and consumer electronics are made using polycarbonate plastics. Previous work using 3T3-L1 cells, a mouse cell tissue culture line, has shown that BPA in combination with the hormone insulin accelerated the conversion of 3T3-L1 fibroblasts into adipocytes . These results suggest that exposure to environmental chemicals, such as BPA, may be a significant factor to the world-wide obesity epidemic. In this study, hMSCs were treated with varying concentrations of BPA and cell pellets were collected at 24, 48, 72, and 168 hours. Future applications in this study involve performing RT-PCR from isolated RNA using primers specific for genes which are molecular markers for cell differentiation into adipocytes.
Bactericidal applications of different magnesium oxide materials
Research Advisor: Dr.Winny Dong
The purpose of the presentation is to present necessary information to accurately describe the sol-gel method and the background behind the aerogel. First a description of the aerogel and some of its properties will be given. The differences between commercial MgO and the MgO Aerogel will be examined next. A general outline into the entire aerogel process examined. The mixing steps will be presented next. A discussion of the supercritical drying procedure and the background will be given. Finally, a general description of the analysis procedures will be discussed. Specifically, the BET and FTIR procedure will be discussed
Biofilm Formation on Experimental Alloys for Human Prosthetics
Student: Jessamine Quijano
Research Advisor: Steve Alas
Human prosthetics are being utilized more frequently as the populations that require their use expand. These cohorts include an increasing elderly population, American soldiers returning with war injuries, and members of the general public who suffer serious injury from accidents. The development of modern human prosthetics has resulted in more biocompatible implants, but they lack longevity due to prosthetic loosening that is caused by metal corrosion or chronic infection. One major area of concern is the formation of antibiotic resistant biofilm on the prosthetic surface during infections. Using Pseudomonas aeruginosa, a bacteria that commonly creates biofilm formation in patient implants, I investigated the biocompatibility of novel titanium alloys with regard to their susceptibility to biofilm propagation. I examined stainless steel (SS), commercially pure titanium (Ti), Ti-6Al-4V (Ti64) and 3 novel titanium alloys that contained either 0.05% Boron, 0.4% Boron, or 1.0% Boron. To determine the amount of biofilm growth, I performed crystal violet staining. Ideal growth conditions were obtained using flasks with 50mL of TSB media for 24 hours at 37˚C or using a biofilm reactor with 350mL TSB media, and allowing incubation for 48 hours at 37˚C. My preliminary results show that the Ti64 alloy permits less biofilm formation than SS and Ti in flask conditions by P. aeruginosa, using crystal violet staining. Using the biofilm reactor, I determined that P. aeruginosa resulted in less biofilm formation on Ti64 than SS, Ti, and the other alloys tested. In order to characterize the viability of the cells comprising the biofilm, I used the BacLight staining procedure and fluorescent microscopy. I determined that the biofilm created contained a large percentage of dead cells. However, more analysis must be done to quantify biofilm viability grown on the various novel alloys, as this is an indicator of prosthetic longevity. At this stage, my data indicates that the Ti-6Al-4V alloy may allow less biofilm formation than traditional metals and, thus, may be a better alternative to stainless steel and pure titanium as a prosthetic biometal.
Bactericidal applications of different magnesium oxide materials
Research Advisors: Dr.Winny. Dong, Dr.Tanya. Faltens
The goal of this research is to investigate the bactericidal properties of magnesium oxide (MgO) xerogels and aerogels. MgO has been shown to have bactericidal properties against Escherichia coli and Staphylococcus aureus cultures. Experiments suggest that these bactericidal properties result from surface defects, such as unsaturated surface oxygen ions. It is thought that contact with the MgO surface inhibits bacterial growth by interrupting vital bacterial cell functions. Surface defects in MgO also serve as active sites for a variety of chemical reactions, such as methane reformation, H2S removal and ethylene polymerization. In this work, the sol-gel method is used to synthesize amorphous MgO xerogels and aerogels that have a highly porous structure, giving them a greater surface area and larger concentration of structural defects than crystalline MgO. Of the three types of materials studied, crystalline MgO has the lowest surface area, ambiently dried xerogels have a higher surface area, and supercritically dried aerogels have the highest surface area of the three. If our hypothesis is correct that MgO surface defects are where the biocidal activity takes place, we should observe a correlation between bactericidal activity and the surface structures obtained by the different preparation methods. Additionally, sol-gel synthesis allows metal ions to be easily incorporated into the amorphous MgO structure. Addition of metal ions that are known to have bactericidal properties of their own may farther enhance the bactericidal properties of MgO. Future applications of amorphous magnesium oxide include its potential for use as a bactericide on spacecraft on extraterrestrial missions and as a non-toxic additive to food packaging for food safety. Methods of creating and testing materials that incorporate MgO for these applications will be presented.
Functional Role of Glycine 93 in the Sodium/Iodide Symporter
Sulfhydryl Modification of the g-Aminobutyric Acid Transporter 1 (GAT1)
Student: Matthew Maestas
Research Advisor: Dr. Sephr Eskandari
The sodium/iodide symporter (NIS) is responsible for the transport of iodide across the basolateral membrane of thyroid follicular cells. This process is the rate-limiting step in the biosynthesis of the thyroid hormones thyroxine and tetraiodothyronine. NIS is driven by the movement of sodium ion down its electrochemical gradient, which in turn drives iodide against its electrochemical gradient thereby increasing the intracellular concentration of iodide. This process has a stoichiometry of 2 Na+ : 1 I- making the process electrogenic in nature. In contrast, Na+/perchlorate or Na+/perrhenate are transported with a 1:1 stoichiometry making the transport of those ions electroneutral. Interestingly, when glycine 93 is replaced with other amino acids, the transport process can become electroneutral or even render the transporter non-functional. By characterizing the functional properties of various NIS G93 mutants, we have gained a better understanding of the role position 93 plays in the transport cycle.
g-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter within the central nervous system (CNS). The principle role of the GABA transporters (GATs) is to maintain a low resting level of GABA in the brain extracellular fluid, and to transport synaptically-released GABA back into the presynaptic neuron, as well as into surrounding glial cells. The latter function serves to terminate GABAergic neurotransmission. Functional, biochemical, and structural data suggest that the GABA transporters contain 12 transmembrane domains (TMs) with cytoplasmic amino and carboxy terminii. In GAT1, three cysteine residues are exposed to the extracellular milieu. Two of the cysteine residues (C164 and C173) are in close proximity to each other in the extracellular loop between TM3 and TM4 and are thought to form a disulfide bridge. The third cysteine, cysteine 74, is believed to be partially buried within TM1 although still accessible to modification by sulfhydryl reagents introduced into the extracellular fluid. We have examined the accessability of C74 from the extracellular fluid by using the membrane-impremeant sylfhydryl reagent, methanethiosulfonate ethyltrimethylammonium (MTSET). Transporter labeling with MTSET leads to inhibition of transport activity. These preliminary studies will lay the foundation for future stoichiometric labeling of GAT1 with the aim of quantifying transporter density at the cell surface. Such an approach will then allow us to reliably estimate the physiological turnover rate of the transporter.
Comparison of Influenza Infection in Male vs. Female Outbred Swiss Webster Mice
Research Advisor: Dr.Adler
Other Researchers: Hana Kim, Chris Petro, Juan Romero, and Jon Olson
The influenza virus (INFV), which infects three to five million people every year worldwide (WHO, 2009), is an enveloped, single stranded, RNA virus, composed of 10 proteins, including three envelope surface glycoproteins that are the components of many commercial INFV vaccines. The INFV surface glycoproteins include, Neuraminidase (NA), Hemagglutinin (HA), and M2, with the HA and NA being the predominant proteins. NA and HA proteins are constantly mutating, giving rise to new viral strains each year to which the general population has no immunity. This constant mutating of HA and NA necessitates needing to prepare new influenza vaccines each year. The HA proteins adhere to the α-2,3 sialic acid receptors on epithelial cells in the human airway causing initial localization of the virus at this site and then spread to the lungs (lower respiratory tract). To improve the design of INFV vaccines, several groups have begun developing vaccines which target the M2 protein because this protein is conserved amongst the different INFV strains, providing a vaccine that could potentially protect against various strains of INFV. Our laboratory has been investigating the efficacy of a liposomal M2 vaccine in female inbred BALB/c mice and we are now ready to test this vaccine in outbred Swiss Webster male and female mice. The present experiment was done to initiate these studies by first determining: (1) localization of the INFV in the airway and/or lungs of male and female murine outbred Swiss Webster mice; and (2) comparing the viral burden in infected organs of male and female outbred murine Swiss Webster mice.
Swiss Webster female mice (n=14/group) were infected intranasally (i.n.) with a 1:4 dilution of H1N1 (A/PR/8/34) and monitored for disease signs, including weight loss, activity level, and grooming. When the disease signs were severe, 7 mice/group were sacrificed and blood as well as organs (lungs, trachea, brain, liver, spleen, kidneys) were collected from these infected mice and used to determine the inhibition agglutinating antibody titer (serum) and the viral burden in the organs (foci assay). The remaining 7 mice/group were monitored for disease signs and morbidity for 6 days. Swiss Webster male mice (n=5/group) were infected i.n. with a 1:16 dilution of H1N1 (A/PR/8/34) and sacrificed on day 4 or 5 when they had severe disease signs and blood and organs were collected for subsequent analysis.
The results showed that the INFV infected female mice all died by day 6 but had produced antibodies to the INFV with a hemagglutination inhibition antibody titer of 5.3×103 HAIU/mL. In addition to the female mice, the male mice also produced antibodies to the INFV with a hemagglutination inhibition antibody titer of 1.6×103 HAIU/mL which was significantly lower than the female titer. When the lungs were examined, the viral burden in the lungs of the females was lower than that seen in the males (4.7×104 foci/g vs 8.0×104 foci/g) suggesting a possible difference in the immune response of the sexes. This difference was noted in the inoculum dose that was administered to infect the males vs the females, since the females needed to be challenged with a greater amount of the virus to elicit the same degree of infection as the males. Future studies will be done to evaluate the viral burden in the remaining collected tissues. We will then determine the efficacy of the liposomal M2e vaccine for protecting immunocompetent and immunosuppressed female and male Swiss Webster mice against H1N1 (A/PR/8/34) challenge.
Retrocyclin Interactions with Heptad Repeat Regions of HIV-1 gp41
Student: Michael Peralta
Research Advisor: Dr. Shantanu Sharma
Other Researchers: Jonathan Reyles, Yoanna Wei
Retrocyclin (RC) is an antimicrobial peptide that potently inhibits HIV-1 infection. It works against many HIV-1 subtypes by binding to the gp41 heptad repeat (HR) region. This bound complex prevents six-helix bundle formation, which is a precursor critical to cell entry and infection. The goal of our research is to determine residue-specific binding interactions between the RC-HR complex that will help us in designing more effective antimicrobial peptides.
We will achieve this goal using a methodology developed in collaboration with researchers at the UCLA School of Medicine. We will obtain dynamical and structural information from computational and experimental data that complement each other (PNAS 2009). The computational data will give us a molecular view of the complex that can be used to determine interactions that stabilize the docked protein complex, while experimental data will yield binding affinities and allow us to test the fidelity of our computational predictions. Currently, we have used a Monte Carlo Method (MCM) to find the most energetically favorable RC-HR conformations and refined our complexes using molecular dynamics (MD) simulations.
The aforementioned hybrid approach will aid us in developing mutant RCs that can bind with greater than wild-type affinity. Our last step will be to synthesize the native and mutant RC peptides and measure their respective binding affinities to HR using Surface Plasmon Resonance spectroscopy, and obtain additional experimental data via analytical ultracentrifugation and isotope-enhanced FTIR.
Expression and function of Clostridial autolysin in E.coli
Research Advisor: Dr.Wei Jen Lin
Clostridium botulinum is an anaerobic, Gram-postive bacteria that is responsible for the disease botulism, a deadly neurological and paralyzing disease. Botulism is usually derived by eating infected food or through wounds. The ability to form spores gives them the advantage to survive in extreme conditions of drought, heat, or lack of nutrients and regrow when conditions are suitable. The severe consequences that the botulinum neurotoxin (BoNT) poses to human and animal health makes it a major concern to public health officials and national defense regulation.
The BoNT toxin is produced during cell growth but the mechanism through which the toxin is releaseed is not known, but can occur through lysis of the cell or a protein-secretion pathway. Our previous studies show that autolysins or cell wall hydrolases may have caused cell leakage during early growth phases and led to early release of the toxin. The early release of toxin and other cellular constituents appears to be unique for Clostridium. In this study, we will investigate the role of these autolysins in relationship to the early release of the toxin. We proposed to clone, express, and purify individual autolysins in the E. coli strain. The function of each autolysin in cell lysis and the release of cellular constituents will be examined in C. botulinum and E. coli where no cell leakage was observed in early growth phases. The first autolysin we studied is the lysozyme which cleaves the backbone of the cell wall peptidoglycan. Gene specific primers was designed and the gene was amplified by the polymerase chain reaction. After verification of the gene sequence, the DNA will be cloned to an expression vector, pET30, for expression and purification from an E. coli strain. The role of the lysozyme in early cell leakage will be further examined. The success of this experiment would give us a better understanding of the toxin-release mechanism and potentially, a method to inhibit its release.
Early stage plant-microbe interactions: The search for an alternative Nod-factor perception mechanism in M. truncatula seeds.
Gustavo A. Ramirez
Research Advisor: Graciela Brelles-Marino,
Other Researchers: Raudhah Rahman
Atmospheric nitrogen is the largest pool of biologically active nitrogen in terrestrial ecosystems but it is only available to relatively few prokaryotes that are able to directly incorporate N2 into their metabolism. Soils are usually poor in nitrogen and require vast amounts of synthetic fertilizer for acceptable crop yields. The rhizobia-legume symbiosis provides the plant with ammonia, a product of the reduction of N2, which is readily incorporated into plant metabolism. This symbiotic interaction implies an ecologically friendly way of fertilizing crops and remediating nitrogen depleted soils. This complex molecular dialogue between Medicago plants and their rhizobial symbionts is mediated by prokaryotic-excreted signal molecules called Nod Factors (NFs). NFs trigger developmental changes in the plant, culminating in the development of a novel structure, the nodule, where N2 is reduced. The mechanism for NF perception is poorly understood. Research has exclusively focused on the perception of these factors by the roots. It has been suggested that treatment of Medicago truncatula seeds prior to symbiont inoculation yield a significant increase in nodule number (Macchiavelli and Brelles-Mariño, 2004). The objective of this work is to study putative NF perception by M. truncatula seeds by using traditional and molecular techniques. Initial experimental analysis of transgenic Medicago truncatula seeds carrying the pMtENOD11-gusA gene fusion has been made. The seeds were exposed to a 10-8 NF suspension. Subsequently, the seeds were processed for Gus staining with an initial vacuum infiltration for 15min and further overnight incubation at 37 ̊C. Following the staining procedure, the seeds were prepared for microtome slicing and microscopy analysis. The expression of early ENOD genes results in blue color. Preliminary results have revealed ENOD11 gene expression in seed tissue. Microscopy analysis has shown ENOD11 gene expression primarily along the embryonic cortical cells. Further investigations are being carried out to confirm these results.
Testing Alternative Methods for Immuno-Fluorescence Microscopy
Noelle E. Olson
Research Advisor: Christos Stathopoulos
Other Researcherss: Athina Rodou
Bacteria cells maintain life by using various interaction mechanisms. Secretion of proteins to extracellular space is one such mechanism used for communication, nutrient acquisition, and spreading disease. One type of secretion mechanism used by gram negative bacteria is the autotransporter pathway. Autotransporters are virulence proteins that play key roles in the establishment of bacterial infections. Studying these proteins will provide necessary insight on how bacteria infect host cells, which could help in development of vaccines and antibodies. Our model for studying secretion is Temperature-sensitive hemagglutinin (Tsh), an autotransporter of pathogenic Escherichia coli cells. In our previous studies, Tsh mutants were analyzed for secretion defects by employing various molecular biology techniques. For those samples which showed a different phenotype than the wild type, we studied their surface localization by carrying out immunofluorescence microscopy using antibodies specific for Tsh proteins. Our lab currently prepares our samples for the indirect immunofluorescence antibody test using a time-intensive protocol, so we are currently testing a more efficient adapted protocol. This experimental protocol has been proven suitable for the subcellular localization of proteins, but our aim is to confirm that it is appropriate for our experiments in which cell surface localization is assessed. A first trial of the experimental protocol demonstrated similar fluorescence signals as our old protocol, with no signs of contamination from the periplasm. The new protocol appears to offer several advantages over the original, in that it provides better visualization of proteins as well as greater ease and decreased length of preparation. Additional trials to confirm our results are ongoing.
Working with Fungi: Species Identification and Susceptibility to Antifungal Drugs
Student: Carla Noguera
Research Advisor: Dr. Jill.P.Adler-Moore
Other Researchers: Davin Hahka
Fungi are members of a diverse group of Eukaryotic microorganisms such as yeast, molds and mushrooms that have unicellular and/or multicellular structures. Referred to as “saprophytes” or “saprobes” they contain a diversity of macroscopic and microscopic forms. They are found throughout nature and have a significant role decomposing organic matter. Their reproduction can be either via asexual or sexual spores. They are used as a source of food such as mushrooms and for fermentation of several food products. However they may also produce bioactive compounds toxic for animals and humans and can also infect vulnerable individuals such as those with suppressed immune systems, causing them to be more susceptible to fungal infections. Since there are many different kinds of fungi, one has to use biochemical, microscopic and macroscopic procedures to accurately diagnose the specific fungus causing the infection. Once the fungus is identified, antifungal drugs and sometimes immunostimulating agents are used to control the infection. In the present study, we tested the in vitro antifungal activity of AmBisomeâ, a liposomal formulation of amphotericin B, as well as the same type of liposome without any amphotericin B (i.e. empty liposomes). The empty liposome formulation has been reported to have some immunomodulatory activity in vivo. We used the in vitro microtiter both minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) assay to evaluate the antifungal activity of these agents alone and in combination. The target fungus (0.9-1.2 X 10^4 spores/well) was Aspergillus flavus and Asperigillus fumigatus. The results showed that for A. flavus, the AmBisome MIC was 1.5625 X 10^4 spores/well. Although, when one half AmBisome was combined with one half empty liposomes, the MIC decreased to 3.123 X 10^3 spores/well. For A. fumigatus, the AmBisome MIC was .78125 X 10^4 spores/well. When one part AmBisome was combined with 3 parts empty liposomes, the AmBisome MIC was the same as the AmBisome alone MIC as it was also .78125 X 10^4 spores/well. The empty liposomes alone had no antifungal activity. It is hypothesized that in this in vitro system, the empty liposomes in high concentration competed with the AmBisome for binding sites on the fungal cell wall interfering with the ability of AmBisome to bind to the fungal cell wall and kill the fungus. Future studies will focus on investigating the antifungal effects of the combination of AmBisome and empty liposomes in mouse fungal infection models and on fungal infected macrophages. These future studies are based on the premise that the AmBisome has direct antifungal activity, and the empty liposomes can stimulate the fungal infected macrophage immune cells to become more fungicidal.
RISE Activities Fall Quarter 2009(September-December 2009)
Fall 2009: RISE Seminar Series
October 30, 2009
“Role of Biomedical Physics in Neurosciences and Medicine”
Dr. Amir Huda
Director, Biomedical Physics Program
Department of Physics
California State University, Fresno
"What's exciting about smooth muscle cells?"
Mr. Albert Gonzales (Cal Poly Alumnus),Ph.D. Candidate
Department of Biomedical Science
Colorado State University at Ft.Collins
December 4, 2009
“Uncovering the Molecular Basis for the Antioxidant-like
Activity Exerted by the Trace Mineral Manganes”
Dr. Chandra Srinivasan
Department of Chemistry and Biochemistry
California State University Fullerton
Grant Writing Workshops and Journal Clubs: These activities continued every third Friday during the Fall Quarter. In the Grant Writing Workshops led by Dr. Adler-Moore, the students were given examples of successful grants. They worked in teams to analyze each of the Objectives and Methodology in a given grant and then asked to reformat the grant as a flow sheet to demonstrate that they understood how each Objective in the grant would be achieved. The students were given additional journal articles to read and to discuss at the Journal Club Workshops.
Poster Workshops: Dr. Alas led these workshops on Tuesday evenings. The students were taught how to format an effective poster, and these workshops culminated in their preparing a poster on their research for presentation at a scientific meeting.