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Science Poster Day 2008 Dean's Prize Winners!

 

Ms. Sally Ahad
Ms. Elinne Becket
Ms. Tyffany Chen
Mr. Ryan Dosumu-Johnson
Mr. Nathan Jacobs
Ms. Erin Jimenez
Mr. Kristopher Kennedy
Ms. Marie Kim
Mr. Raymond Kung
Mr. Edward Kuoy
Ms. Anne Liu
Ms. Isabel Neacato
Mr. Bac Nguyen
Ms. Susan Realegeno
Ms. Angelica Riestra
Ms. Lauren Sanchez
Mr. Andrew Su
Ms. Anjali Tapadia
Mr. Tuan Tran
Ms. Wendy Tseng
Ms. Marika Watanabe
Ms. Karen Yan
Ms. Sophia Yang
Mr. Siamak Yasmeh
Mr. Michael Yim
Mr. Jesse Zaretsky

 

 

Ms. Sally Ahad
Mentor: Volker Hartenstein
Title: The role of neoblasts, a type of totipotent stem cell, in postembryonic growth and differentiation in Platyhelminths

Platyhelminths (flatworms) possess a unique type of stem cell called a neoblast. Neoblasts are freely moving, totipotent cells that, during the course of the animal’s life, replace cells in organs that are lost through wear and tear. We speculated that the postembryonic growth (increase in cell number) that occurs
in organs largely occurs through the incorporation of neoblasts. To test this experimentally, we gave pulses of bromodeoxyuridine (BrdU) to hatched juveniles of the basal flatworm Macrostomum, followed by chases of different lengths (1-21 days), and then stained the specimens with antibodies against BrdU and a nervous system marker (anti-tyrosinated tubulin) as well as a general nuclear stain (TOTO). If fixed right after the BrdU pulse, neoblasts are restricted to the trunk. After 1 and 2 day pulses, we start seeing migration of neoblasts to the region of the brain; initially, only epidermal and possibly gland cells of the head epidermis show BrdU incorporation. Starting with 3 day chases, we see the first BrdU-positive nuclei in the brain. We are currently analyzing specimens for which longer chases are used. This is the first
analysis addressing the contribution of neoblasts to postembryonic tissue growth. It will contribute significantly to our understanding of the mechanisms that control the behavior of the neoblast, an evolutionarily ancient stem cell system that is most likely the forerunner of somatic stem cells found in
present day animals.

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Ms. Elinne Becket
Mentor: Jeffrey H. Miller
Title: Developing an antibiotic sensitivity profile for Escherichia coli

The increasing number of antibiotic resistant bacteria poses a major health problem. One way to combat this issue is to improve the efficacy of existing antibiotics with combination therapy. Potential targets for such drugs would be bacterial proteins that provide intrinsic antibiotic resistance. We developed a high throughput system using the complete Escherichia coli knockout collection to identify genes whose loss increases sensitivity to subinhibitory concentrations of sixteen antibiotics: ciprofloxacin, rifampicin,sulfamethoxazole, metronidazole, gentamicin, streptomycin, spectinomycin, tetracycline, ampicillin, cephradine, aztreonam, fusidic acid, chloramphenicol, erythromycin, vancomycin, and colistin. Screening using several subinhibitory concentrations of each antibiotic revealed 143 knockout strains (the "sensitivity profile") that were highly sensitive to one or more of the antibiotics. This profile method allows the knockout collection to be compacted into two plates so that the 143 knockout strains can be rapidly screened with many different antibiotics; it also enables us to immediately characterize these antibiotics with respect to their mechanisms of action. The results of these studies may help identify inhibitor
molecules that will target these hypersensitive genes and, consequently, increase bacterial killing efficiencies of antibiotics at levels less toxic to humans.

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Ms. Tyffany Chen
Mentor: Fuyu Tamanoi
Title: Towards identifying a GEF (guanine nucleotide exchange factor) for Rheb: characterization of vps901 and vps902

Rheb is a prominent G protein in the regulation the Target of Rapamycin (TOR) signaling pathway, which is critical in cell growth and cell cycle progression. From our knowledge of G proteins, we speculate that a guanine nucleotide exchange factor (GEF) exists to catalyze Rheb to its active GTP-bound form. We were able to identify a putative GEF complex in Schizosaccharomyces pombe through BLAST analysis and knockout experiments. Our putative GEFs, vps901 and vps902, are speculated to function in a complex and share redundant roles. To study these genes, we made two ectopic thiamine-repressible VPS902 constructs with varying levels of overexpression. Using these constructs in double knockout cells should provide insight into the nature of the synthetic lethality that result in the loss of both vps901 and vps902. While searching for possible GEF phenotypes, we found Cat1 permease mislocalization in the overexpression of vps902 in S. pombe. Cat1 permease has recently been identified to be involved in the TOR pathway. Also, we found that TSC2 mutants have higher resistance to brefeldin A and will soon test for a similar phenotype in vps902 overexpression. Currently, we are checking the thiamine repression in our conditional vps902 constructs. We hope to identify more phenotypes to characterize our putative GEFs and explain their possible role in the critical Rheb/TOR signaling pathway.

 

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Mr. Ryan Dosumu-Johnson
Mentor: Stephanie A. White
Title: AAV-mediated disruption of the zebra finch song circuit using Kir 2.1

Speech plays a pivotal role in the complex social networks fundamental to human civilization. We use songbirds to study the neural mechanisms underlying vocal learning. Progress in studying molecular mechanisms in birds has been limited because current transgenic techniques are not conducive to avian physiology. To overcome this, we are developing a method to genetically interfere with the song control circuit using adeno-associated virus (AAV) to express molecules of interest: GFP and human Kir 2.1. In this ‘proof of principle experiment,’ our goals were two-fold: first to determine the cell type transduced by AAV and, second, to determine the level of transduction sufficient to alter song. Kir 2.1 is an ideal test molecule because its neuronal overexpression is predicted to decrease firing. The song nucleus, HVC, is an ideal locus because it is a master control region for song. Thus far we have shown that our stereotaxic coordinates correctly target HVC, and that the virus is able to infect neurons. Further, the human synapsin I promoter restricts GFP or Kir2.1 expression to neurons. In a small number of birds, bilateral injection of the virus with Kir 2.1 into HVC appears to decrease song structure. If this virus shows cell-type specificity and causes a reliable effect on song, it can be used to further parse out the role of specific neurons in the song circuit.

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Mr. Nathan Jacobs
Mentor: Michael Fanselow
Title: The role of synaptic plasticity in the CA1 region of the hippocampus during incidental spatial learning and memory

Spatial learning and memory is an essential cognitive function for many mammalian behaviors, including navigation and forming memories of specific places. The hippocampus and several of its subregions have specifically been implicated in such spatial learning and memory processes. To understand the specific molecular correlates of spatial learning and memory processes, Tsien et al. (1996) created a transgenic mouse line in which the NR1 subunit of the N-methyl-D-aspartic acid receptor (NMDAR) is knocked out only in CA1 pyramidal neurons. These mice show a complete loss of NMDAR-mediated plasticity in the CA1 region and exhibit spatial learning and memory deficits in both Morris water maze and context fear conditioning tasks. In both of these tasks, spatial learning occurs in the presence of aversive stimuli. The current study aimed to investigate the role of CA1 synaptic plasticity in incidental, or motivationally
neutral spatial learning using a context fear conditioning design called context pre-exposure facilitation of the immediate shock deficit. In this task, mice are pre-exposed to the conditioning chamber, placed back in the chamber the next day and immediately shocked, and then tested for the presence of fear memories on the third day. Knockout mice showed a trend for reduced expression of learned context fear during the post-shock period (KO mice, n=3; control mice n=2; p=0.137, univariate ANOVA), but not during a context test given 24 hours later. A significant reduction in freezing to the context during both the postshock period and the context test in knockout mice would support a role for CA1 plasticity specifically in incidental spatial learning and memory, which reduces the possible confounds of aversive stimuli.

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Ms. Erin Jimenez
Mentor: Carla Koehler
Title: AKT localizes to the intermembrane space in mitochondria isolated from mammalian brain

Recent studies have identified new pathways in the mitochondrial intermembrane space (IMS), including an oxidative folding pathway for the import of proteins and phosphorylation-dependent signaling pathways.
Akt, a major serine/threonine protein kinase capable of inducing pro-survival and proliferative effects, was previously reported to localize to mitochondria in a neuroblastoma tissue culture cell line. We have shown that although Akt is present and phosphorylated in both mouse brain and liver cytosol, Akt only localizes to mitochondria isolated from the brain. Furthermore, the fraction of Akt in the mouse brain mitochondria is active and localizes to the IMS. Use of a phospho-specific antibody that recognizes Akt substrates enabled us to identify two major substrates in mammalian brain mitochondria. One substrate is soluble on the outside of the mitochondria, and the other substrate is inside the mitochondria. We are currently investigating the mechanism of Akt import into the mitochondrion and the identity of the mitochondrial substrates of Akt. This line of research will provide insights into regulation by Akt, as well as a greater
understanding of signaling pathways in the mitochondrial intermembrane space.

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Mr. Kristopher Kennedy
Mentor:Nicholas C. Brecha
Title: The development of mouse Thy-1.2 YFP expressing retinal cone bipolar cells in vitro

Development of retinal neurons in vitro is poorly understood but will provide a critical step in understanding their differentiation in vitro. The goal of this study is to characterize the development of Thy-1.2 yellow fluorescent protein (YFP)- expressing mouse cone bipolar neurons using a sandwich culture preparation. These animals express YFP in a subset of retinal neurons, which includes cone bipolar neurons. This provides a fluorescent reporter for easy cell identification. Neonatal YFP mouse retinas were dissociated, cultured on coverslips, and inverted into defined media. At various time points between 0 and 35 days, cells were fixed in 4% paraformaldehyde and stained via an indirect immunofluorescence technique. Cone bipolar neurons were labeled with antibodies against calcium-binding protein 1 (CaBP1) and calcium-binding protein 5 (CaBP5). Antibodies against protein kinase C alpha (PKC-alpha) labeled rod bipolar cells. Glutamatergic terminals were labeled with antibodies against vesicular glutamate transporter 1 (VGLUT1). Immunofluorescence was analyzed using confocal microscopy. Colocalization of YFP, cone bipolar cell marker, and VGLUT1 was indicative of a cone bipolar neuron. We observed cone bipolar neurons that developed singular dendrites and glutamatergic terminals in vitro similar to those of cone bipolar neurons in vivo. These results suggest that the sandwich culture preparation provides a long-term in vitro environment where cone bipolar neurons develop functional morphology. This may be useful for future electrophysiological studies of cultured cone bipolar neurons.

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Ms. Marie Kim
Mentor: Robert L. Modlin
Title: E-selectin and ICAM-1 induction by IL-17 on endothelial cells

IL-17 is a proinflammatory cytokine produced by T helper (Th) 17 cells, a novel subset of CD4+ T cells that play a protective role against extracellular microbes and promote neutrophil recruitment. Neutrophils are the most abundant type of white blood cell and migrate out of the vasculature during acute inflammation. IL-17 is expressed in psoriatic lesions, but the role of IL-17 in the development of psoriasis is unclear. To investigate the role of IL-17 in psoriasis, we examined skin lesions from psoriasis patients and determined the levels of IL-17 receptor (IL-17R). We labeled IL-17R-expressing cells using immunohistochemistry and immunofluorescence. The pattern of IL-17R expression was similar to that of
Von Willebrand factor, a marker for endothelial cells. We therefore ypothesized that endothelial cells may be a target of IL-17 activity and promote neutrophil recruitment and inflammation observed in psoriasis. Human umbilical vein endothelial cells (HUVEC) were stimulated in vitro with IL-17 in the presence or
absence of TNFα, a cytokine known to be important in both autoimmunity and inflammation. Preliminary results show that IL-17 works synergistically with TNFα to induce the cell adhesion molecule E-selectin. In addition, while IL-17 alone was unable to induce IL-8 and IL-6, it moderately enhanced the secretion of both cytokines when costimulated with TNFα. This data suggests that IL-17 may activate endothelial cells to promote inflammation in psoriatic lesions

 

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Mr. Raymond Kung
Mentor: Linda Baum
Title: Small molecule high throughput screens for Duchenne Muscular Dystroph

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked recessive musculodegenerative disease caused by a genetic mutation in dystrophin, a cell surface glycoprotein necessary for proper linkage between muscle cell membrane and extracellular matrix. Overexpression of a specific glycosyltransferase, N-acetyl glucosaminyl transferase (CT-GalNAcT), has been shown to inhibit muscular dystrophy by improving membrane-basal lamina interaction in the mdx mouse model, which lacks dystrophin. Through small
molecule high throughput screens on C2C12 mouse muscle cells, we have detected several drugs among a library of FDA-approved drugs that may treat DMD by modulating cell surface glycoproteins. The screens done by ELISA detected increases in the treated cells WFA binding ability, an indirect measure of CTGalNAcT expression. We found a drug that increased C2C12 muscle cell expression of CTGalNAcT, dystrophin-associated proteins, laminin binding proteins, and GalNAc modified proteins, which are essential for proper membrane-basal lamina interaction. This drug also increased WFA binding and
CTGalNAcT expression in mdx cells. We will determine if the drug has similar effects on expression of basal lamina-associated proteins in mdx cells as it had in that of C2C12 cells. Preliminary data showed that this drug does not negatively affect cell viability, fusion, or membrane stability. Subsequent experiments include determining if CTGalNAcT mediates laminin and WFA binding, conducting further screenings of different targets of DMD, and carrying out in vivo experiments on mdx mice.

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Mr. Edward Kuoy
Mentor: Kathrin Plath
Title: A novel approach to transcription factor-induced reprogramming

Induced pluripotent stem (iPS) cells are cells that have been reprogrammed to a pluripotent state similar to embryonic stem cells (ESCs). iPS cells share the same developmental potential as ESCs: they are capable of germline transmission, forming adult chimeric mice, and generating embryoid bodies and teratomas comprised of cells from all three germ layers. The generation of patient-specific iPS cell lines has great therapeutic promise as these cells could be used to study and potentially ameliorate human diseases, and
aren’t limited by technical, ethical or immunological considerations. Recent studies show that murine and human fibroblast cells can be reprogrammed into iPS cells by retrovirally overexpressing four transcription factors: Oct4, Sox2, c-Myc and Klf4. The first demonstrations of their therapeutic potential were recently achieved in humanized sickle cell anemia and Parkinson's disease mouse models. However, in all reprogramming studies, the factors were introduced using viruses that randomly integrate into the genome, which could generate undesirable mutations that ultimately lead to tumors, thus posing a major hurdle to human therapeutic applications. The goal of this project is to achieve transcription factor-induced reprogramming avoiding genomic integration. To this end, we are testing the use of adenoviral vectors that
do not integrate into the genome as a platform for introducing the eprogramming factors. If successful, the iPS technology will bring us a step closer to human therapeutic applications.

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Ms. Anne Liu
Mentor: Jeffrey H. Miller
Title: Developing an antibiotic sensitivity profile for Escherichia coli

The increasing number of antibiotic resistant bacteria poses a major health problem. One way to combat this issue is to improve the efficacy of existing antibiotics with combination therapy. Potential targets for such drugs would be bacterial proteins that provide intrinsic antibiotic resistance. We developed a high throughput system using the complete Escherichia coli knockout collection to identify genes whose loss increases sensitivity to subinhibitory concentrations of sixteen antibiotics: ciprofloxacin, rifampicin,sulfamethoxazole, metronidazole, gentamicin, streptomycin, spectinomycin, tetracycline, ampicillin, cephradine, aztreonam, fusidic acid, chloramphenicol, erythromycin, vancomycin, and colistin. Screening using several subinhibitory concentrations of each antibiotic revealed 143 knockout strains (the "sensitivity profile") that were highly sensitive to one or more of the antibiotics. This profile method allows the knockout collection to be compacted into two plates so that the 143 knockout strains can be rapidly screened with many different antibiotics; it also enables us to immediately characterize these antibiotics with respect to their mechanisms of action. The results of these studies may help identify inhibitor
molecules that will target these hypersensitive genes and, consequently, increase bacterial killing efficiencies of antibiotics at levels less toxic to humans.

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Ms. Isabel Neacato
Mentor: Manuel L. Penichet
Title: The analysis of an anti-hTfR IgG3-avidin fusion protein alone and in combination with other potential drugs for cancer immunotherapy

A great obstacle in cancer treatment is the development of resistance to chemotherapeutic drugs. One way of overcoming this problem is through combination treatment strategies. We previously developed a mouse/human chimeric antibody fusion protein composed of avidin genetically fused to a human IgG3 specific for the human transferrin receptor (anti-hTfR IgG3-Av). Anti-hTfR IgG3-Av alone has intrinsic cytotoxic activity and can deliver biotinylated agents into malignant non-biotinylated cell lines. In the present study, four potential chemotherapeutic non-biotinylated drugs; DHMEQ (an NF-kB inhibitor), NPI- 0052 (a proteasome inhibitor), VX-680 (an aurora kinase inhibitor), and HXR9 (an inhibitor of the HOX/PBX dimer formation), were tested alone and in combination with anti-hTfR IgG3-Av in the malignant B-cell lines IM-9 and U266. The goal was to evaluate whether the therapeutic effect of this
fusion protein could be enhanced when combined with these drugs. The effective drug concentration of each therapeutic was determined by a dose-response analysis using a [3H]-thymidine incorporation assay. Sub-toxic doses were chosen and tested in combination with various concentrations of anti-hTfR IgG3-Av. Preliminary data show that each component alone has cytotoxic effects in both cell lines. The combination treatment of anti-hTfR IgG3-Av and HXR9 in IM-9 cells demonstrates enhanced cytoxicity compared to either agent alone. These studies are important for the potential discovery of a novel therapeutic regime for the treatment of B-cell malignancies.

 

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Mr. Bac Nguyen
Mentor: David E. Krantz
Title: A novel neurotransmitter transporter required for sexual behavior in Drosophila

Neurotransmitter transporters play an integral role in the function of the nervous system. They store neurotransmitters in synaptic vesicles and also remove them from the synaptic cleft. Changes in the activity of both vesicular and plasma membrane transporters have been shown to alter the concentration of
neurotransmitter in the synaptic cleft. Thus, regulated changes in the function of both vesicular and plasma membrane transporters has the potential to profoundly impact both synaptic transmission and behavior. We have identified a novel vesicular transporter gene, DVX, that is expressed in the mushroom bodies (MBs) of Drosophila melanogaster, a structure required for learning and some sexual behaviors. Through transposon-mediated mutagenesis, we have generated a DVX mutant fly line. To determine the function of DVX, the mutants have been subjected to a series of behavioral assays. Preliminary results have shown that DVX mutants have a reduced ability to learn and a striking increase in activity during copulation. We have quantified these behaviors and found that males have a reduced ability to mount and maintain position and females show increased rejection of the male. Identifying a phenotype for DVX will help determine how changes in transport activity can contribute to complex behaviors potentially relevant to neuropsychiatric illness.

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Ms. Susan Realegeno
Mentor: Otto Yang
Title: Identifying HIV-1 target cell entry versus productivity with a dual reporter virus

The Human Immunodeficiency Virus (HIV-1) infects cells of the immune system; however, our understanding of how HIV-1 crosses mucosal barriers to reach primary target cells is limited. In order to identify target cells, we designed an HIV-1 reporter virus that enables us to distinguish between cells
initially infected and cells productively infected. To generate this virus, we co-transfected two expression plasmids into 293T cells. One plasmid contains the entire HIV-1 genome with the exception of the vpr gene, which was replaced by the reporter gene HSA. The second plasmid encodes an enhanced green
fluorescent protein (EGFP)-Vpr fusion protein, which gets expressed and packaged in trans into the viral particles produced in the 293T cells. The recombinant viral particles, therefore, contain the gene that codes for HSA (but no HSA protein) and the EGFP-Vpr fusion protein (but not the sequence that encodes it). Confocal microscopy has shown productive infection of HIV-1 permissive cells with the recombinant virus via HSA expression and cells only initially entered by the virus via EGFP-Vpr protein expression. We plan to use this dual reporter virus to study cells that are initially infected versus cells productively infected in a colon mucosal culture model for mucosal HIV-1 infection. Identification of HIV-1 primary target cell types can direct further studies of HIV-1 pathogenesis and may suggest vaccine targets or drug therapies designed to prevent viral propagation.

 

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Ms. Angelica Riestra
Mentor: Sherie L. Morrison
Title: Production of murine-human recombinant IgG3 antibodies against the glucoronoxylomannan (GXM) polysaccharide component of Cryptococcus neoformans capsule

Cryptococcus neoformans is an opportunistic fungus that causes life-threatening meningitis in immunocompromised persons. Available treatments are not effective since 10-20% of treated individuals still die from the disease and treatment in AIDS patients does not eradicate the infection. Passive antibody (Ab) therapy is an alternative treatment that has shown promise, with mouse IgG1 extending the life span of C. neoformans-infected mice. However, preliminary studies in our laboratory showed that mouse-human chimeric IgG3 also extended the survival of BALB/c mice infected with C. neoformans and C57BL/6J mice infected with Cryptococcus gatii when the antibodies (Abs) are administered as immune complexes and at lower doses than those used in previous studies. In order to determine the unique characteristics of IgG3 that may contribute to this previously unreported protection, two mutant mouse-human IgG3 chimeric Abs against the glucoronoxylomannan (GXM) polysaccharide component of C. neoformans capsule were constructed. One Ab contains an N297Q mutation in the CH2 region which will prevent its glycosylation. The other Ab had the long hinge region characteristic of IgG3 replaced with a shorter IgG1 hinge. Both Abs have been successfully expressed in NSO/1 cells and are currently being purified using protein A
affinity chromatography. Once both Abs have been characterized, their effects will be tested in vitro and in vivo. These results will provide insight about the mechanisms involved in IgG3-mediated protection against Cryptococcus infection.

 

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Ms. Lauren Sanchez
Mentor: M. Luisa Iruela-Arispe
Title: The effects of progesterone receptor deletion in the vascular endothelium

The hormone progesterone has an important physiological role in the female reproductive system. The progesterone receptor (PR) has been implicated in diverse roles in both sexes, including neuroprotection and vascular homeostasis. Studies have also suggested an anti-inflammatory role for PR, which may account for the increased incidence of autoimmune and cardiovascular diseases in women, especially postmenopause. To study the PR-mediated inflammatory response, we are evaluating the role of PR in the
vascular endothelium, as the vascular endothelium has a critical role in mediating the recruitment of inflammatory cells to sites of infection. We have generated a knockout mouse model, the VENPR, in which PR is conditionally deleted in the vascular endothelium. To evaluate the role of endothelial cell-specific PR expression in modulating the inflammatory response to infection, we utilized a mouse model of localized infection in the uterus. The uteri of VENPR female mice were injected with Chlamydia muridarum and evaluated post-infection by histological and FACS analysis. Histological analysis demonstrates increased uterine inflammation compared with control mice, while FACS analysis of hematopoietic cells isolated from infected uteri demonstrates that there is an increased neutrophil response in VENPR mice at earlier
stages of infection and diminished T-cell response at later stages of infection compared to controls. These results support PR as an anti-inflammatory agent and suggest that PR supplements the ability of the immune system to clear an existing infection.

 

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Mr. Andrew Su
Mentor:Osvaldo Rey
Title: Structure-function characterization of the G-protein calcium-sensing receptor agonist-binding domains

A new dimension in Ca2+ signaling has recently emerged with the identification and cloning of the G protein-coupled extracellular Ca2+-sensing receptor (CaR). The CaR is an allosteric protein that recognizes and responds to two different agonists, Ca2+ and aromatic amino acids, with the production of different
intracellular Ca2+ ([Ca2+]i) oscillations. An increasing amount of experimental evidence indicates that the CaR is a signal transducer of great biological importance implicated in the control of Ca2+ homeostasis, gastric acid secretion, intestinal transport, homing of embryonic cells and in the progression and spread of colorectal, breast, ovary and parathyroid cancers. In order to define the mechanism mediating the activation of the CaR by Ca2+ or aromatic amino acids, several point mutations were introduced in the extracellular agonist-binding domain of the receptor. HEK-293 cells transiently transfected with cDNAs encoding either wildtype or mutant CaR were loaded with the Ca2+ fluorescence indicator fura-2 and the [Ca2+]i oscillations were examined using a digital imaging system. Our results indicate that mutations within the CaR Venus Flytrap motif affect both Ca2+ and aromatic amino acid-mediated [Ca2+]i oscillations, suggesting that the binding sites for both agonists partially overlap. Thus, these studies provide critical information to help further understand the mechanism mediating the activation of this receptor by two
different agonists.

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Ms. Anjali Tapadia
Mentor: Gaston Pfluegl
Title: Fine mapping of Lrp11 Trans-eQTL on mouse chromosome 5 using SNP markers

Here we report the preliminary results of the effort to fine-map a previously identified 3.5Mbp region on mouse chromosome 5 (Chr5) associated with variation in Lrp11 expression levels in 110 MF1 outbred mice. The UCLA Undergraduate Genomics Research Initiative is using genotype data to locate the specific causal variation within the 3.5Mbp region for Lrp11. In mice, Lrp11 expression is associated with elevated LDL levels, which in humans has been implicated in atherosclerosis and coronary heart disease. Based on the C57BL/J6 standard inbred mouse strain, we designed polymerase chain reaction (PCR) primer pairs and dye-labeled custom sequencing primers for nine equidistant single nucleotide polymorphism (SNP) sites in the 3.5Mbp region selected to be polymorphic across eight inbred strains. Thus far we have tested primers for two SNPs by PCR amplification and sequencing on the eight standard inbred strains, of which one successfully identified the polymorphism in all eight strains. We plan to further test our designed primers on the remaining seven SNPs to confirm polymorphisms across all eight strains. This will enable us subsequently to genotype the 110 MF1 mice using the designed primers to fine map and identify the candidate gene(s) within the Lrp11-associated region on Chr5. These results should prove informative for understanding interactions among the human LRP11-associated region, LRP11, LDL levels, and aid in atherosclerosis and coronary heart disease prevention, diagnosis, and treatment.

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Mr. Tuan Tran
Mentor: April Pyle
Title: Regulation of pluripotency in human embryonic stem cells

Human embryonic stem cells (hESCs) are unique in possessing pluripotency, the ability to differentiate into each germ layer (mesoderm, endoderm, and ectoderm). Disrupting pluripotency could improve our understanding of how differentiation is regulated. Our first approach is to examine the cell fate regulator Cripto-1 (CR-1). CR-1, also known as teratatocarcinoma-derived growth factor-1 (TDGF-1), is a coreceptor for Nodal, a protein in the transforming growth factor-beta (TGF-β) pathway. CR-1 has been shown to enhance cardiomyocyte and inhibit neuronal differentiation of mouse embryonic stem cells (mESCs). Our goal is to determine whether CR-1 functions similarly in hESCs by utilizing RNA interference (RNAi). In a second approach to studying regulation of pluripotency, we generated a directed protocol to differentiate hESCs into skeletal muscle cells. This approach could improve the development of treatments for diseases including Muscular Dystrophy (MD). Advancing our knowledge of how pluripotency is regulated in hESCs will improve our ability to ultimately establish safe protocols for cellbased therapy.

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Ms. Wendy Tseng
Mentor: Yin Tintut
Title: Oxidized phospholipids induce interleukin-6 expression via the protein kinase A pathway in osteoblasts

Picture coming soon!

Cardiovascular disease is associated with the development of osteoporosis, a disease where bones become fragile due to an increased rate of bone resorption compared to bone formation. We previously found that
differentiation of bone-forming osteoblasts is inhibited by oxidized lipoproteins, which trigger the pathogenesis of atherosclerosis. Since differentiation of bone-resorbing osteoclasts requires cytokines produced by osteoblasts, we hypothesize that oxidized phospholipids also regulate bone resorption by
altering osteoblastic expression of cytokine including interleukin-6 (IL-6). In this study, we investigated the regulatory mechanism of IL-6 expression by ox-PAPC, an active component of oxidized lipoproteins. Treatment of murine calvarial preosteoblasts with ox-PAPC increased IL-6 mRNA expression and
activated IL-6 promoter-reporter constructs. The ox-PAPC-induced IL-6 promoter activity and mRNA expression were inhibited by the protein kinase A (PKA) inhibitor H89, but mutations in the NF-kB or C/EBP binding sites did not attenuate ox-PAPC-induced IL-6 promoter activity, suggesting that PKA but
not NF-κB or C/EBP mediates the ox-PAPC effect. Since H89 also blocks Rho-associated protein kinase II (ROCK-II), we next used a specific inhibitor of ROCK-II. Cotreatment of cells with ox-PAPC and the ROCK-II inhibitor failed to attenuate ox-PAPC activation of IL-6 promoter, confirming that PKA but not
ROCK-II mediates ox-PAPC induction of IL-6. In summary, these data suggest that oxidized lipoproteins may contribute to osteoporosis by promoting bone resorption in addition to inhibiting bone formation.

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Ms. Marika Watanabe
Mentor: Harumi Kasamatsu
Title: Disulfide and non-disulfide interactions between bacterial proteins and SV40 Vp1

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Simian virus 40 (SV40) is a DNA tumor virus that infects monkey cells. Vp1 is a major capsid protein of SV40 which forms pentamers through transient disulfide-linked intermediates and then into the icosahedral
capsid. However, the mechanism of how Vp1 forms the capsid remains unclear. It was shown that the mammalian heat-shock proteins Hsp70 and Hsp40 facilitate Vp1 folding, and cysteines 49 and 87 are crucial for proper Vp1 folding. We found that bacterial proteins co-purify with Vp1 and predict that they
may chaperone the folding of Vp1. To identify such bacterial proteins, the amino-terminal 103 residues of Vp1 (tVp1), which include the Cys49 and Cys87, along with those carrying respective mutations, were expressed in Escherichia coli, and were purified either under native (N) or denaturing (D) conditions. Systematic analysis using non-reducing and/or reducing polyacrylamide gel electrophoresis followed by Western blot analysis using αVp1 antibody show that: 1) 15kDa bacterial protein covalently, but not
through disulfide linkage, cross-links with tVp1; 2) 12kDa, 20kDa, and 25kDa bacterial proteins also bind to tVp1 via disulfide linkages; 3) tVp1 (N) is purified as multimers of tVp1 and further truncated tVp1s. Their cleavage is enhanced in the absence of Cys87. Identification of these bacterial proteins that interact
with tVp1 should elucidate how they mediate the folding of SV40 Vp1 and may lead to discovery of mammalian homologs of these proteins.

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Ms. Karen Yan
Mentor: Ren Sun
Title: Use of artificial neural networks to study optimal drug combinations on multiple myeloma-derived cell lines

Multiple myeloma (MM) is a plasma cell neoplasm that cannot be cured despite the use of conventional chemotherapeutic agents. Standard treatment for patients with MM consists of a combination of vincristine, doxorubicin (adriamycin), and dexamethasone (VAD). Unfortunately, the well-described toxicities and high drug resistance reduce the efficacy of the current drug therapy in terms of high patient relapse and low survival rates. We are currently investigating the potential synergy between a combination of VAD and two
promising new drugs that have been described in previous studies: rapamycin and bortezomib (valcade). Using primary T cells as a control, we measured the drug response profiles for all five drugs using dexamethasone-sensitive (MM.1S) and dexamethasone-resistant (MM.1R) MM-derived cell lines. We hope
to use Artificial Neural Network (ANN), an information processing paradigm inspired by the highly interconnected neurons in the biological nervous system, to generate a model that describes the effect of various drug combinations on cell survival and ultimately predict the optimal combination of drugs to use
for multiple myeloma. Previously, we successfully used ANN to predict the optimal drug combination to treat non-small cell lung cancer cells in vitro. Ultimately, this research could potentially help us gain a better understanding of the differences in signaling pathways between other subpopulations of drugresistant MM-derived cell lines using a luciferase reporter on doxorubicin resistant MM-derived cell lines.

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Ms. Sophia Yang
Mentor: Yung-Ya Lin
Title: In vivo studies on early lesion detection using feedback-enhanced MRI

Contrast enhancement in MRI is an important goal for early cancer detection since tumor and healthy tissues are chemically and magnetically similar. Through the use of feedback magnetic fields, we have been able to create new pulse sequences which can achieve greater differentiation over conventional methods. This past year, we proposed a new method in our framework of “Feedback-Enhanced Magnetic Resonance,” termed “Self Nutation by Feedback Fields.” Preliminary mathematical simulations using the
generalized Bloch equation and in vivo imaging on mice with early-stage human colon cancer have suggested the novelty and applicability of this conceptually innovative approach. To test this method for early detection, the cancer cell line Colo 205 was grown in the thigh of a laboratory mouse. After 24 hours,
biomarkers consisting of superparamagnetic iron oxide (SPIO) nanoparticles (50nm) coated with protein-A and linked to mouse anti-carcinoembryonic antigen (CEA) were injected intravenously to bind to the cancer, and MR images were acquired. The magnetic field of the nanoparticles interacted with the imaging magnet, radiofrequency, and feedback pulses to remain robust against small field inhomogeneities, or fluctuations in tissue composition. This is significant since the downfall of the conventional method, T2* imaging is the inability to differentiate between magnetically similar tissue. By comparison, the Self Nutation method demonstrates greater constrast, suggesting that this method can be used for further studies on early lesion detection.

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Mr. Siamak Yasmeh
Mentor: Rick B. Delamarter
Title: Assessing the effective dose of BMP-2 and BMP-7 in demineralized bone matrix products for spinal fusion

Demineralized bone matrix (DBM) based product, a grafting material derived from cadaver bone, is commonly used to assist the union of vertebrae in spinal fusion procedures. Bone morphogenetic proteins (BMPs), the osteoinductive component of DBM, promote fusion by inducing differentiation of boneforming
cells (osteoblasts). BMP-2 and BMP-7 levels in DBM have been shown to vary across production lots and to correlate positively with fusion success; however, the BMP quantities necessary for fusion are unclear. The purpose of this study was to determine the average effective dose (ED80) of BMP-2, BMP-7,
and BMP-2 + BMP-7 average required for 80% fusion success. ELISA was used to assay BMP-2 and BMP-7 levels in 10 production lots of a single DBM product. Posterolateral fusion was then performed on 40 athymic rats using each of the 10 lots (n=4 rats per lot). An aliquot of DBM was implanted between the L4-L5 transverse processes of the spine. Fusion success was determined using radiographic analysis and physical examination of excised spines. Dosing curves based on BMP levels and fusion outcomes were constructed using probit analysis. BMP-2 and BMP-7 levels correlated well with fusion success (p=0.01). The ED80s for BMP-2, BMP-7, and BMP-2 + BMP-7 average were 100, 141, and 107 pg/mg DBM, respectively. To improve fusion outcomes in clinical applications, DBM lots should be screened for effective BMP quantities (ED80s) prior to commercial distribution.

 

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Mr. Michael Yim
Mentor: Michael S. Levine
Title: The effect of CAG repeat length on GABAergic inhibition in the R6/2 mouse model of Huntington’s disease

Picture coming soon!

Huntington’s disease (HD) is a neurodegenerative disorder caused by a trinucleotide (CAG) repeat expansion in the gene encoding the huntingtin protein. The length of this repeat is inversely correlated with
the age at onset and severity of the disease. In humans, repeat lengths greater than ~80 lead to a juvenile onset with more severe symptomology, including seizures. We performed experiments using the GABAA receptor antagonist picrotoxin in R6/2 transgenic mice at 40 days of age, when these mice begin to show an overt behavioral phenotype. In order to assess the role of the CAG repeat length, R6/2 mice and wildtype littermates carrying 110, 150, 210 and 310 CAG repeats were examined. An increased latency to seizure in the mutant mice as CAG repeat length increased was observed. The CAG110 and CAG150 groups were affected similarly, while the CAG210 mice were affected less and the CAG310 mice were indistinguishable from wildtypes. In addition, corticostriatal spontaneous excitatory postsynaptic currents showed a reduction in frequency dependent upon CAG repeat length. The CAG150 mice were most affected, the CAG110 and CAG210 mice were affected less, and the CAG310 mice were indistinguishable from wildtype mice. Together, these findings substantiate the importance of CAG repeat length in clinical studies of HD. Also, there may be a neuroprotective effect in the transgenic mice at higher repeat lengths.

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Mr. Jesse Zaretsky
Mentor: Utpal Banerjee
Title: A search for new hematopoietic markers: Characterization of gene expression patterns in an uninvestigated Drosophila lymph gland structure using enhancer-trap reporters

The Drosophila hematopoietic system serves as a model of vertebrate hematopoietic development and innate immunity. The Drosophila lymph gland is a larval structure that acts as a regulated site of proliferation and differentiation of blood cell progenitors. Blood cell differentiation happens in a spatially
restricted manner in the primary lymph gland lobes, forming three distinct zones of maturation. The goal of this study was to identify genes that play a developmental role in the secondary lymph gland lobes, which are relatively uncharacterized with regard to hematopoietic function and genetic program. We used GFPand GAL4-insertion based enhancer-trap systems as reporters of gene expression, and visualized expression patterns in the lymph gland using confocal fluorescence microscopy. Eight out of forty-eight fly insertion
lines exhibited reporter gene expression that was spatially and temporally restricted within the primary and/or secondary lymph gland lobes. Two of these showed expression in the cortical zone of the primary lobes that predates the earliest immunohistochemical differentiation markers. Four other reporters seemed to express in the posterior half of the secondary lobes, which may point to a new unique population of cells. In the future, we hope to use these newly characterized enhancer-traps as markers to aid our investigation of the development, function, and regulation of hematopoiesis in the primary and secondary lymph gland lobes.

 

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