the 4 annual joint molecular biosciences graduate student...

Sponsored By

The 4th Annual

Joint Molecular

Biosciences Graduate

Student Symposium Friday, March 12, 2010

Life Sciences Auditorium, Busch Campus

Student Presentation Guide

Symposium Schedule

10:45am Coffee, Tea, and Juice

10:45am-11:00am Opening remarks

11:00am-12:15pm Student Seminar Session 1

12:15pm Lunch

12:15pm-2:00pm Poster Session/Sponsor Greetings

2:00pm-2:30pm Keynote Address from Dr. Eddy Arnold "Aiming at a rapidly moving target: using structure-based

design to overcome HIV-1 reverse transcriptase drug resistance."

2:30pm-3:45pm Student Seminar Session 2

3:45pm Light Snacks

3:45pm-4:30pm Poster Session

4:30pm Awards

Information about the Joint Molecular Biosciences

Graduate Student Association can be found at http://www.eden.rutgers.edu/~jmbgsa/

Student Posters

Title: “AKT2 is a downstream target of Grm1 " Presenter: Seung-Shick Shin Title: “Cochlear hair cell micro-isolates regulate the soma size of spiral ganglion neurons" Presenter: Felicia Smith Title: “Regulation of Drosophila hematopoiesis by Zfrp8/PDCD2, JAK-STAT and Ken/BCL-6" Presenter: William Tan Title: “The role of PSD-95 and cypin in hippocampal dendrite and spine injury and recovery in response to NMDA-mediated excitotoxicy" Presenter: Chia-Yi Tseng Title: “Metabotropic glutamate receptors (mGlus) and cellular transformation" Presenter: Jessica Teh Title: “Bosea sp. WAO mediates the oxidation of metal sulfides" Presenter: Alexandra Walczak Title: “Efficacy of combination therapies for the treatment of metastatic melanoma" Presenter: Janet Wangari-Talbot Title: “3-D Bioactive scaffolds as stem cell substrates for accelerated wound healing" Presenter: Jing Xu

Student Posters

Title: “Investigating the molecular genetic basis of the mouse mutant vacuolated lens (vl)" Presenter: Bo Li Title: “Isolation of initiating melanoma stem cells from human malignant cell line c8161" Presenter: Elisa Mastrantoni Title: “Bacterial toxin-antitoxin systems as new agents for cancer gene therapy" Presenter: Christopher Mozdzierz Title: “Composition of microbial biofilm communities from diffuse flow deep-sea hydrothermal vents on the East Pacific Rise 9o N" Presenter: Chuck O’Brien Title: “The role of actin regulating WAVE/SCAR complex in maintenance of epithelial organization" Presenter: Falshruti Patel Title: “Preliminary sequence analysis on the effects of distance and selective enrichment on alkane monooxygenase gene diversity in Passaic River sediment" Presenter: Elyse Rodgers-Vieira Title: “Altered binding of eukaryotic elongation factor 3 (eEF3) to the ribosomes: A key to understanding its functional requirement in yeast" Presenter: Arjun N. Sasikumar

Student Presentations

Title: “Modulation of CD154 expression by polypyrimidine tract binding protein (PTB)" Presenter: Rodrigo Matus-Nicodemos Abstract: CD40 ligand (CD40L) is a protein expressed on activated CD4+ T cells, which is crucial for antibody-dependent immunity by B cells. The expression of CD40L is tightly regulated at multiple levels throughout a time course of T cell activation. At the post-transcriptional level the CD40L message is rapidly degraded at early time points of activation followed by a significant increase in message stability at later times of activation (24-48 hr). Previous work from our lab revealed that a cytoplasmic polypyrimidine tract binding protein (PTB)-containing-complex binds to the CD40L 3’UTR at later times of T cell activation. We used a retrovirus RNA interference system to down regulate PTB in D1.1 Jurkat T cell line to address the functional role of PTB in regulating the CD40L mRNA stability and subsequently the surface expression of CD40L. Real-time qPCR measurement of the CD40L mRNA decay revealed that the downregulation of PTB caused a decrease in the half-life of CD40L mRNA. The downregulation of PTB did not significantly change the percentage if CD40L+ cells, but caused an approximate 2-fold decrease in the mean fluorescence (MFI) of CD40L. Cellular fractionation of CD40L mRNA from shCTRL- and shPTB-infected cells revealed a novel role for nuclear PTB in retaining CD40L mRNA in the nucleus. In addition, cytoplasmic PTB appears critical for optimal association of CD40L message with translating polysomes. This work firmly establishes a role for PTB in regulating the turnover of CD40L message and surface protein in activated T cells through processes linked to nuclear and cytoplasmic transport.


Title: “MicroRNA modulators of C. elegans aging and healthspan" Presenter: Mehul Vora Abstract: Identification and characterization of genes involved in promoting healthy aging is a key focus of aging biology and offers a dual advantage of improving the period of healthy aging as well as extending understanding the cellular processes involved in delaying age-onset diseases. MicroRNAs (miRNAs) are a newly appreciated class of conserved [1] regulatory non-coding RNAs that are implicated in fundamental and diverse biological processes, including diseases of aging like cancer and neurodegeneration. miRNAs have been proposed to buffer against variation and stochastic event in order to maintain biological robustness. Age-related loss of miRNA-mediated regulation may lead to large and detrimental deviations from robustness that are characteristic of aging. Indeed, in a genome wide survey of how miRNA expression changes during adult life, we find that about half of C. elegans miRNAs are significantly regulated during aging (most of them are down-regulated) [2] In an effort to determine whether miRNAs may modulate aging in general, we are currently conducting the first comprehensive screen to identify miRNAs that modulate the aging process. Assays that measure the accumulation of age-pigments (fluorescent lipofuscin and advanced glycation end products) in whole animals have shown that 17 mutant miRNA strains exhibit abnormal levels compared to same age wildtype animals during early adulthood (Day 4). Since age pigments are thought to reflect the physiological age rather than the chronological age [3], these miRNAs may be responsible for maintaining physiological homeostasis through life. 5 tested strains also exhibited significantly altered age pigment levels at mid-late (Day 9) or late (Day11) adulthood. 4/5 age-regulated miRNAs (mir-1, mir-34, mir-71, mir-238) also showed a significant decline in swimming prowess compared to wild type animals at late age (Day 10), although their swimming in early adulthood is similar to wild type. Significantly, these mutants also accumulate higher age pigments early in life, perhaps indicating that a "poor" start leads to decline in integrity later in life. We have also identified a few miRNA mutants that have lower age pigment levels late in life. Some of these have multiple indicators of strong healthspan.

In conclusion, some miRNAs may play an important role in maintaining homeostasis and general integrity of the aging animal. Deciphering the gene networks that they regulate may suggest new strategies for positive healthspan intervention.

Student Posters

Title: “Cyanobacterial metabolomics: Liquid-chromatography mass spectrometry (LC-MS) based analysis of fermentative metabolism and biofuel production in the model cyanobacterium Synechoccous sp. 7002" Presenter: Nicholas Bennette Title: “Autism-associated haplotype of ENGRAILED2 is a functional unit recruiting transcription factors" Presenter: Jiyeon Choi Title: “Interacting partners of ESC1, a protein involved in silent chromatin positioning” Presenter: Chia-Ching Chou Title: “An in vitro assay for acupuncture" Presenter: Ka Po Chu Title: “Functional characterization of NOS1AP schizophrenia risk allele" Presenter: Anna Dulencin Title: “Role of the eukaryotic elongation factor 1Bγ in vesicular trafficking of carboxypeptidase Y" Presenter: Anthony Esposito Title: “Transglutaminase 2 and the toxicity of misfolded α-synuclein" Presenter: Hilary Grosso


Title: “Immobilized glycan mimics for peripheral nerve regeneration" Presenter: Shirley Masand Abstract: Despite the innate regenerative potential of the peripheral nervous system following injury, functional recovery is limited due to a non-supportive microenvironment and improper axon targeting. Two carbohydrates– HNK-1, an epitope from the human natural killer cell, and 2,8 polysialic acid (PSA) – are favorably involved in preferential motor reinnervation, neuron survival, and acceleration of regrowth. Peptide mimics of these molecules, which are simpler in structure and easier to produce, have been covalently grafted onto monomeric type I collagen, which immobilizes the peptide for well-controlled presentation following self-assembly of the collagen into a fibrillar gel. Peptide-grafted collagen gels significantly enhanced neurite outgrowth from chick dorsal root ganglia and rat embryonic spinal cord neurons in vitro. In vivo, the materials were evaluated using a mouse femoral nerve injury model, where a polyethylene cuff is used to bridge a 5-mm transection. The lumen of the cuff was filled with saline, native collagen, or peptide-grafted collagen. Animals were videotaped before injury, and then weekly for 15 weeks. Single-frame motion analysis was used to quantitatively evaluate gait. Significant improvement in functional recovery was observed in animals with nerve grafts comprising peptide-modified hydrogels compared to collagen and saline conditions. Additionally, evaluation of motor axon targeting efficiency with retrograde labels and nerve pathology with immunohistochemistry has demonstrated the efficacy of using this biomaterial as a scaffold for a bioartificial nerve guidance conduit.


Title: “Regulation of junctional stability by Arp2/3-dependent actin nucleation: Teaching old dogs new tricks" Presenter: Yelena Bernadskaya Abstract: The cell movements of morphogenesis require rearrangement of the actin cytoskeleton and turnover of the epithelial cell junctions. Our lab studies this process in C. elegans with a focus on the GEX pathway, which promotes actin nucleation through the Arp2/3 complex. All of the components of the GEX pathway are highly conserved from nematodes to humans. In the C. elegans embryo, we found that Arp2/3-dependent actin nucleation is required to increase the protrusion activity of the leading edge of specific cells during epidermal morphogenesis. Our studies also indicate a role for Arp2/3-dependent actin nucleation in epithelial junctional stability and turnover. The adherens junction is composed of cadherin, β-catenin, and α-catenin, with α-catenin being the component that can bind to filamentous (F) actin. It was previously thought that this interaction was very static and directly linked the junction to the actin cytoskeleton. Recent work suggests that the junction is a highly dynamic structure. It was recently shown that α-catenin is not able to bind both beta-catenin and F-actin simultaneously, and cycles between a monomeric and dimeric form. The current model suggests that Arp2/3-dependent actin nucleation is required for the establishment of the adherens junction. Then, as local concentration of α-catenin increases, α-catenin is able to dimerize and dissociate from β-catenin. This dimeric form of α-catenin then binds to F-actin, promoting filamentous actin formation and inhibiting branched actin nucleation by Arp2/3. However, my data suggests that Arp2/3-dependent branched actin nucleation is required not only for the establishment of the junction, but is also continuously required for the local enrichment of α-catenin at the junction and may be indirectly

affecting the interaction between epithelial cell junctions and actin. This leads us to propose a new model in which Arp2/3 dependent actin nucleation plays a positive role during junctional establishment and maintenance.


Title: “Mesenchymal stem cells induce T regulatory cell differentiation via indoleamine 2,3 dioxygenase metabolites" Presenter: Andrew L'Huillier Abstract: It has been previously reported that Mesenchymal Stem Cells (MSCs) have immunosuppressive effects both in vitro and in vivo. Conflicting results exist concerning the mechanisms of this immunosuppression, and it is clear that no one of these mechanisms is solely responsible. It has also been reported that these mechanisms differ between species. Immunosuppression by human MSCs can be mostly reversed using the tryptophan analog 1-MT. This suggests that indoleamine 2,3-dioxygenase (IDO), a protein which human MSCs can be induced to express high levels of, is a major mechanism of this suppression. IDO is a tryptophan catabolizing enzyme. Traditionally its suppressive effect has been attributed to tryptophan starvation, though other models such as the bioactivity of tryptophan metabolites have been proposed. IDO is thought to play a role both in natural immune regulation as well as in immune escape by tumors. Methods: Human CD4+ T cells isolated from human PBMCs were co-cultured with IDO metabolites or human MSCs producing IDO, and were supplemented with known immunosuppressive factors. To determine the effects of the interactions of stem cells and T cells via IDO, apoptosis and T regulatory cell induction were measured. Apoptosis was measured by DNA content using PI staining, while T regulatory cell induction was measured by three color flow cytometric analysis and q-PCR. To determine the immunosuppressive abilities of MSC derived Tregs, treated T cells were co-cultured 1:1 with irradiated allogeneic PBMCs. Proliferation was determined by measuring incorporation of tritiated thymidine. Results: We have found that metabolites produced by IDO from MSCs synergize with TGF-b and Retinoic Acid to induce CD4+ CD25+ Foxp3+ T regulatory cell

differentiation from naïve CD4+ T cells. Furthermore, we show these Treg cells to have immunosuppressive functions independent of the MSCs that produce them.


Title: “Functional genomics screen to identify cellular regulators of HIV-1 latency" Presenter: Leia Miller Abstract: Approximately 33 million people around the world are living with HIV/AIDS today. HIV-infected individuals must adhere to anti-retroviral therapy for the duration of their lives and these drugs not only have harsh side effects but can eventually be rendered obsolete via the emergence of drug-resistant virus within individuals. The majority of infections of CD4+ T cells by HIV result in active viral replication within those cells and subsequent cell death. However, not all CD4+ T cells infected with HIV support active viral replication providing the virus an opportunity to lie dormant within those cells. This phenomenon is referred to as latency and represents the only true barrier to HIV eradication due to the fact that latently infected cells are not susceptible to anti-HIV pharmaceuticals or to the cytolytic effects of the host immune system. Despite the fact that many cellular and viral factors have been implicated in contributing to the establishment and maintenance of HIV latency, there are no successful treatment regimens to date capable of purging the latent reservoir. It is evident that a more fundamental understanding of the mechanisms involved in the regulation and maintenance of latency is necessary in order to develop more specific, effective therapies. Therefore, we propose a functional genomics screen to identify novel cellular regulators of HIV latency. To accomplish this, we are employing a lentiviral vector based shRNA library to screen approximately 38,000 genes in a latency model cell line to determine their role in the regulation of this process. Follow-up experiments will be performed to confirm that the genes identified in the screen truly modulate HIV latency and to delineate the mechanisms involved for the identification of novel drug targets.


Title: “In vivo functional analysis of ENGRAILED2 (EN2) ASD Associated Haplotype" Presenter: Silky Kamdar Abstract: Autism Spectrum Disorders (ASD) is a complex neuropsychiatric disorder with a strong genetic basis. EN2 - a homeobox transcription factor - has been one of the candidate genes associated with ASD. Previous work done in our lab strongly indicates the two intronic SNPs of EN2 as ASD risk alleles. The major alleles A of rs1861972 and C of rs1861973 were observed to be significantly over transmitted to off springs affected with ASD (P-value < 0.000001), while the minor alleles G and T respectively were under transmitted. In vitro functional studies such as Luciferase assays and Electro Mobility Shift Assays demonstrate differential gene expression as well as binding of nuclear proteins with the ASD associated A-C haplotype (A-C) as compared to the other common G-T haplotype (G-T). The above data demonstrates that A-C, an ASD risk allele, is functional in vitro. After observing the above results, the next important step is to test for A-C function in vivo. EN2 has a dynamic expression pattern throughout CNS development, which leads us to ask if the A-C haplotype is functional at more than one developmental time points in more than one cell type, and if the haplotype always increases gene expression during CNS development? To investigate the in vivo functional role of rs1861972- rs1861973 during CNS development, transgenic mouse lines were generated for both A-C and G-T. 6 lines for A-C and 8 lines for G-T with varying copy numbers were successfully established. Histological analyses done on all the lines at postnatal and adult stages suggest a similar spatial expression pattern between A-C and G-T. RT-qPCR analyses done to measure expression level differences between the two haplotypes, revealed that A-C is functional in vivo and it increases gene expression in postnatal (96% increase, P-value<0.0001) and adult stages (200% increase, P-value<0.00001). Similar analyses to be done in the embryonic

stages will help determine when, where and how the ASD-associated A-C haplotype functions during development.


Title: “Composition of microbial biofilm communities from diffuse flow deep-sea hydrothermal vents on the East Pacific Rise 9o N" Presenter: Chuck O’Brien Abstract: The goal of this project was to identify the relationship between chemical data, macrofaunal colonization and microbial biofilms at deep-sea hydrothermal vents. We deployed and retrieved colonization substrates made of acrylic mesh (TAMS: Temporal Autonomous Multi-disciplinary Substrates) from two diffuse flow vent sites (Marker 19 and Tamtown - in flow treatments) and from control sites (no flow treatments) on the East Pacific Rise (EPR) at 9oN. We measured the concentration of H2S and temperature from each site, counted the number of tubeworms (vestimentiferans) on each colonization mesh, and assessed the composition of the microbial biofilms. To this end, we extracted total genomic DNA from the colonization meshes for denaturing gradient gel electrophoresis (DGGE) analysis. Then, we selected samples exposed to different chemical regimes (in flow and no flow), presence/absence of tubeworm colonists, and unique DGGE patterns to construct 16S rRNA gene clone libraries. We amplified the 16S ribosomal RNA genes by PCR, gel purified it, and ligated it into a TOPO TA cloning vector for transformation into TOP10 E. coli cells. We profiled each clone by restriction fragment length polymorphism (RFLP) to identify unique taxa for sequence identification. Our data indicate that the in flow substrates were colonized primarily by Epsilonproteobacteria, while the no flow substrates were dominated by Gammaproteobacteria.


Title: “Development and preclinical validation of a preimplantation genetic diagnosis (PGD) method for the prevention of MELAS Syndrome" Presenter: Jessyca Campos Abstract: Mitochondrial Encephalomyopathy, Lactic Acidosis with Stroke-Like Episodes (MELAS) syndrome is caused by mutations in mitochondrial (mt) DNA and has no known treatment for the underlying fatal disease. Preimplantation genetic diagnosis (PGD) involves testing DNA in embryos produced by in vitro fertilization prior to embryo transfer back to the uterus of the intended mother, and may provide the only method available to prevent inheritance. In the present study, we establish a quantitative real-time PCR (qPCR) test that is capable of accurate quantification of the 3243A>G mutation load in single or few cells. Second, we establish that oocytes and embryos from the intended MELAS mutation carrier mother posses a variety of 3243A>G mutation loads. This was critical to establish that embryos with low mutation load existed and could be selected over embryos with high mutation load. Although oocytes displayed similar mutation loads (from 58 to 61%), the cleavage, morulae, and blastocyst stage embryos displayed a variety of mutation loads (from 7 to 86%), indicating the potential to select low MELAS mutation load embryos by PGD. Finally, we establish that cells from within the same embryo provide consistent results. Standard deviations of mutation loads within each embryo were low (from 0.5 to 2.4%), indicating faithful representation of the entire embryo by a single biopsy, and demonstrating that this technology could accurately determine the MELAS mutation load in embryos suitable for clinical use. In conclusion, we have developed and demonstrated the validity and feasibility of the first method that is now available for clinical use to reduce the risk of inheritance of 3243A>G derived MELAS syndrome.


Title: “Regulation of mTORC1 signaling through FKBP38-mediated mTOR targeting to mitochondria" Presenter: Ming-Feng Lin Abstract: As a major growth regulator, mTORC1 pathway is frequently associated with oncogenesis and is widely recognized as a key anticancer drug target. mTOR is known to distribute to several organelles, including ER and mitochondria. However, the physiological significance of mTOR subcellular localization remains poorly understood. Here we show that mTOR localization within the cytoplasm is regulated by serum and nutrients. Starvation causes mTORC1 to relocate to mitochondria from ER. mTOR mitochondrial localization is mediated by three novel mitochondrial localization sequences (MLSs). Mutations in MLS motifs disrupt mTOR mitochondrial localization and the ability of starvation to inhibit mTORC1 signaling. FKBP38, an integral mitochondrial membrane protein and an endogenous mTOR inhibitor, interacts with MLS motifs and targets mTOR to mitochondria. Additionally, inactivation of TSC or activation of Rheb disrupts mTOR localization to mitochondria. Altogether, these observations reveal a novel, FKBP38-dependent spatial control of mTORC1 signaling in response to growth signals.

the 4 annual joint molecular biosciences graduate student...

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