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Science Poster Day 2009 Dean's Prize Winners!

 

Mr. Sergey Boyarskiy
Ms. Chee Jia Chin
Ms. Inyoung Choi
Ms. Michelle Crespo
Mr. Asghar Haider
Ms. Jennifer Hranilovich
Mr. Sattar Khoshkhoo
Mr. Jun Woo Kim
Ms. Jenny Koo
Ms. Amanda Lea
Mr. Andrew Lechner
Ms. Renata Lerner
Ms. Angela Hui-Chia Liu
Mr. Jason Melehani
Mr. Aaron Meyer
Mr. Rameen Moridzadeh
Ms. Sarah Muradian
Ms. Kristin Owyang
Ms. Michelle Peng
Ms. Tiranun Rungvivatjarus
Ms. Claire Sampankanpanich
Ms. Allison Schwartz
Ms. Christine Thang
Ms. Anna Xu
Ms. Lillian Zhang

Mr. Sergey Boyarskiy
Mentor: Daniel Kamei
Title: The Effects of Hydrophobic Chain Length on Polypeptide Vesicle Formation and Cytotoxicity

Picture Coming Soon!

Many therapeutic candidates that function well in vitro do not reproduce their effects in vivo. This is often caused by, among other reasons, the therapeutics’ insolubility in buffered solutions, inability to penetrate cell membranes, and extracellular enzyme activity which breaks down the molecules. One useful method of circumventing these hurdles is the use of a delivery vehicle which encapsulates the therapeutic in an appropriate solvent separating it from the surroundings, and at the same time presenting a surface that favorably interacts with the target cells. Our group has previously created a material which allows for such encapsulation. We used a peptide block copolymer composed of a hydrophobic leucine block and a hydrophilic, positively charged, lysine or arginine block. These polymers self assemble into bilayer vesicles, and are able to encapsulate therapeutics. Although these vesicles are able to enter cells, some of them have a certain level of cytotoxicity associated with them. We investigated the effects of various physical properties of the vesicles on cell viability. For example, decreasing the hydrophobic chain length from 25 to 10 residues, which is expected to increase the relative concentration of micelles and monomers, was found to decrease cell viability. However, unlike conventional amphiphilic lipid-based molecules, size exclusion studies suggested that it is not the “monomer” form that is primarily responsible for the cytotoxicity, but rather small micellar aggregates.

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Ms. Chee Jia Chin
Mentor: Amander T. Clark
Title: Investigating the Role of PRMT5 in Pluripotency and Human Germ Cell Specification

Inyoung Choi (left) with Chee Jia Chin (right)

Infertility affects approximately 6.1 million women and their partners in the United States. This corresponds to about 10% of the reproductive-age population. Since germ cells are set-aside very early in mammalian fetal life, the molecular mechanisms that govern fetal primordial germ cells (PGCs) specification and development are our primary research focus. Recent evidence suggests that Protein Ariginine Methyltransferase 5 (PRMT5) is a critical component of a novel transcriptional regulatory complex in murine PGCs. The early expression of PRMT5 in human fetal germ cells leads us to hypothesize that PRMT5 is also important in the specification of human PGCs. I am now generating shRNA knockdown virus to further address the role of this protein on both human embryonic stem cells self-renewal and human germ cell development. The efficiency of this lentiviral system was confirmed on BJ cell line (neonatal human foreskin fibroblasts) by observing a greater than 99% reduced transcripts level of PRMT5 via real time-PCR. Overall, results from this research will deepen our insights on the specification of germ cells, the most critical lineage for the continuation and evolution of all species.

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Ms. Inyoung Choi
Mentor: Utpal Banerjee
Title: FGF Signaling in development of glial cells in the eye of Drosophila melanogaster

One of the important processes associated with Drosophila eye development is the migration of the photoreceptor axons from the eye disc into the brain. This action is very tightly regulated, and one of the key aspects to its migration is the ability to reach its correct destination into the brain. Retinal basal glial (RBG) cells that are born in the optic stalk migrate into the eye disc providing the necessary guidance cues to the photoreceptor axons and aiding their migration. Therefore, characterrizing glial cell development and its migration into the eye discs is critical towards addressing questions related to photoreceptor axon guidance. Fibroblast growth factor (FGF) signaling controls many biological processes that include cell proliferation, differentiation, migration, cell motility, wound healing, and malignancy. In this study, I propose a possible involvement of FGF signaling as a regulating element of RBG cell development in the eye. Loss of FGFR, breathless and heartless, and downstream-of-FGF-receptor, Dof, affects the RBG proliferation and migration behavior. Also, over-expression of branchless, breathless and heartless clones resulted in distinct expansion of optic stalk and number of RBG cells present in eye discs. Therefore, these results strongly suggest a role for FGF signaling during RBG development in the eye disc.

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Ms. Michelle Crespo
Mentor: Carlos Portera-Cailliau
Title: Metabotropic Glutamate Receptor Activation Induces Elongation of Early Dendritic Protrusions

Michelle Crespo with Dr. Charles Buchanan (Michelle is the first recipient of the Charles Buchanan Dean's Prize Award)

Fragile X syndrome (FXS) is the most common inherited form of mental retardation and autism. Neurons in the neocortex of individuals with FXS as well as Fmr1 knockout (KO) mice exhibit abnormally long dendritic spines and abnormal metabotropic glutamate receptor (mGluR) signaling. To determine whether mGluRs are important in synaptogenesis, we studied whether glutamate-induced elongation of early dendritic protrusions is mediated through mGluRs. We used two-photon time-lapse imaging of layer 2/3 pyramidal neurons transfected with green fluorescent protein in wildtype mice during the first two postnatal weeks. In acute slices of somatosensory cortex, 200 microMolar glutamate was puffed locally over a single dendritic segment. We found that glutamate application causes an elongation of dendritic protrusions within minutes, with about 30% of protrusions exhibiting a significant increase in length. Furthermore, application of an mGluR antagonist during glutamate puffing blocks the effects of glutamate. Our data suggest that a fraction of early dendritic protrusions can respond to glutamate through mGluRs to produce changes in length. This may represent an important mechanism whereby axons recruit dendritic protrusions to make early synapses. To determine whether glutamate-induced growth of protrusions is abnormal in FXS, we intend to repeat these experiments in Fmr1 KO mice. Further investigation of mGluR signaling defects in Fmr1 KO mice will reveal more about the relation between structural and functional abnormalities in FXS during development.

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Mr. Asghar Haider
Mentor: Felix Schweizer
Title: The Antioxidant Ketones Acetoacetate and beta-Hydroxybutyrate Prevent Abeta42-Induced Oxidative Stress

The 42 amino acid long amyloid beta peptide (Abeta42) appears to contribute to neuronal dysfunction and death in Alzheimer’s disease but the underlying pathogenic mechanisms are poorly understood. Oxidative stress, a common finding in Alzheimer brains, is a well-documented cause of neuronal dysfunction and death. Consistent with the oxidative stress hypothesis, our preliminary data show that certain aspects of Abeta42 toxicity can be alleviated by the antioxidant ketones acetoacetate and beta-hydroxybutyrate. We therefore used an ELISA assay to measure levels of oxidized protein in hippocampal tissue. Exposure to Abeta42 (1mcM) for 2 hours was associated with a 23±6% increase in protein oxidation. Pre-treatment with the ketones (1mM) for 1 hour prevented the toxic effects of Abeta42 (4±6% decrease). To understand how Abeta42 might induce oxidative stress, we tried to determine whether Abeta42 entry into neurons was a component of the pathogenic process. Using immunocytochemistry in hippocampal neurons incubated with Abeta42, we did not detect any increase in intracellular Abeta42. This negative result could not be attributed to deteriorating neuronal health as staining for the cytoskeletal protein MAP2 revealed intact morphology but could be due to a limited sensitivity of the technique. Alternatively, we propose that Abeta42 interacts with cell surface receptors to induce oxidative stress. Future experiments will further explore the mechanisms underlying Abeta42 toxicity and the neuroprotective mechanisms activated by ketones.

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Ms. Jennifer Hranilovich
Mentor: Elizabeth Sowell
Title: Testosterone and Sexually Dimorphic Cortical Maturation in Pubertal Boys and Girls

Pronounced cortical thinning occurs during adolescence, a time of great increase in gonadal steroid levels. Thinning has been observed to be sexually dimorphic in trajectory and to show regionally distinct trajectories. The purpose of the present study is to investigate the effect of testosterone on adolescent cortical thinning. Plasma testosterone levels were assayed. Structural MRI was used to investigate 82 adolescents (48 girls). All preprocessing and analysis was performed using FreeSurfer’s automated segmentation software, including a multiple regression analysis of each gender to predict cortical thickness using testosterone level and age. In the girls, testosterone was shown to be widely predictive of cortical thinning (-0.05 < p < -0.0001), independent of age-related variance. In boys, testosterone was shown to be predictive of only scattered cortical thinning (-0.05 < p < -0.0001), independent of age-related variance. Additionally, the less widespread cortical thinning in the boys was counterbalanced by cortical thickening correlated with circulating testosterone levels, (0.05 > p > 0.0001), independent of age-related variance, and largely localized to primary sensory areas. Testosterone appears to differentially predict changes in cortical thickness in pubertal boys and girls. It may be that this is a human correlate of animal studies that have shown puberty to be a time when neural circuits are organized to enable male reproductive behavior.

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Mr. Sattar Khoshkhoo
Mentor: Carlos Portera-Cailliau
Title: Investigating the Role of BDNF in Developmental Decorrelation of Cortical Firing Patterns

Activity in the neocortex in mice is highly synchronized during early postnatal ages; however, gradually this synchronized activity is replaced with sparse and uncorrelated firing of individual neurons throughout development. This developmental decorrelation of network dynamics is likely to be important for efficient sensory encoding in the neocortex, and perturbations to this transformation of network dynamics may play a role in neuropsychiatric disorders such as epilepsy, autism, and mental retardation. Previous findings from our lab showed no correlation between peripheral sensory input and the developmental decorrelation of activity, which suggested a potential role for internally generated molecular causes for this transformation. Brain derived neurotrophic factor (BDNF) has been shown to play a critical role in synaptic and functional circuit maturation and plasticity; therefore, it is a good molecular candidate for further investigations. Using in-utero electroporation to overexpress BDNF in layer 2/3 neocortical neurons in mice followed by in-vivo two-photon calcium imaging, we showed that overexpression of BDNF may hasten the developmental decorrelation of activity. However, the extent to which BDNF contributes to this process remains largely elusive and it illustrates the need for future studies of this nature.

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Mr. Jun Woo Kim
Mentor: Ben Wu
Title: Incorporation of Multicellular Spheroids in Three-Dimensional Tumor Models Using Polymeric Scaffolds for Screening Cancer Drugs

Jun Woo Kim with Vice Provost Judi Smith

Previous studies have demonstrated that culturing cells in 3D biocompatible polymeric scaffolds produces more in vivo-like characteristics with respect to secretion of angiogenic factors, composition of extracellular matrix proteins, cellular growth behavior as well as drug resistance. Other groups have shown that spherical aggregates of cancer cells, or spheroids, better mimic the three-dimensional cell-to-cell interactions by capturing the morphology of real-life tumors. In this study, we have developed a novel in vitro model in which U251 (glioma cell line) spheroids were incorporated into 3D poly(lactic-co-glycolic) acid (PLGA) scaffolds. We hypothesized that this model would possess improved representation of the in vivo environment by combining the features of previous models and thus enable more effective drug screening. To identify the drug resistance of the new system, IC50 values of scaffolds seeded with spheroids (“Sphscaff”) were compared with those of scaffolds seeded with dispersed cells (“Monoscaff”) and cells seeded onto 2D tissue-culture plates (“Plate”). For doxorubicin, a widely used anticancer drug, Sphscaff exhibited approximately three-fold and twenty-fold higher drug resistance than Monoscaff and Plate, respectively. When the PLGA scaffolds were functionalized with arginine-glycine-aspartic acid (RGD) peptide conjugation instead of collagen coating, Sphscaff had fewer spheroids intact and showed significantly lower drug resistance. These results together suggest that incorporation of spheroids into 3D polymeric scaffolds serves as an improved in vitro model for screening cancer therapeutics.

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Ms. Jenny Koo
Mentor: Owen Witte
Title: Optimizing Gene Expression of Lentiviral Vectors in Chronic Myelogenous Leukemia Mouse Models after Bone Marrow Transplantation

The Philadelphia Chromosome has been found in 95% of chronic myelogenous leukemia and results in the expression of oncoprotein Bcr-Abl p210. We are developing a mouse model to demonstrate Bcr-Abl gene knockdown using RNAi, by infecting cells with miRNA-containing lentivirus. There is, however, some question regarding the activity of the cytomegalovirus promoter currently used to drive the expression of our lentiviral vectors. Studies have shown that CMV promoter activity is restricted to specific stages of mouse-embryonic-stem cell differentiation and also decreases over time in bone marrow. Since the Philadelphia Chromosome is present in multiple bone marrow cell lineages of CML patients and originates in the HSC, it is important for the miRNA to be consistently expressed in all bone marrow cells. There is concern that gene expression will likewise be silenced in differentiated bone marrow cells. Alternatively, the elongation-factor-1-alpha promoter has demonstrated strong expression at every stage of mES cell differentiation. To compare the expression profiles of the promoters in bone marrow transplants, we substituted the CMV promoter on the vector with an EF1-alpha promoter. To this end, both promoters show similar expressivity in lentivirally-infected cell cultures, and in vivo investigation is currently underway. Optimizing expression of lentiviral vectors in bone marrow will enable consistent knockdown of the Bcr-Abl using RNAi, and thus increase the potential of using miRNA-mimics for cancer therapy.

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Ms. Amanda Lea
Mentor: Daniel Blumstein
Title: Heritable Vigilance is Genetically Correlated with Locomotor Performance

Animals must allocate some proportion of their time to detecting predators. In birds and mammals, such anti-predator vigilance may be influenced by a variety of intrinsic (e.g. species, stress hormone levels, body size, and body condition) and extrinsic (habitat type, visibility, group size, and time of year) factors. Despite hundreds of prior studies focusing on vigilance, we report the first evidence that vigilance behavior is heritable. Using a restricted maximum likelihood procedure, we found that, in yellow-bellied marmots (Marmota flaviventris), the time allocated to vigilance while foraging is genetically correlated with locomotor ability (slower animals are less vigilant while foraging). Interestingly, the heritability of vigilance (h2 = 0.093) is much less than the heritability of locomotor ability (h2 = 0.248), suggesting high environmental variation or a history of directional selection eliminating genetic variation in vigilance. The genetic correlation is substantial (r = 0.528), and demonstrates the ability of variable but equally successful genotypes to maintain unexpected genetic variation. An analysis of breeding values demonstrates no current selection on either trait; a finding consistent with the hypothesis that there are two ways to solve the problem of foraging in exposed areas: be very vigilant and rely on early detection coupled with speed to escape, or reduce vigilance to minimize time spent in an exposed location. This “locomotor ability-wariness” syndrome allows slow animals to compensate behaviorally for their impaired locomotor ability.

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Mr. Andrew Lechner
Mentor: Hanna Mikkola
Title: PDGF-B Signaling as a Regulator of Balanced Hematopoiesis During Fetal Development

The unique regenerative ability of hematopoietic stem cells (HSCs) highlights their potential in treating hematological disorders. However, in order to generate and expand HSCs in vitro, we must first understand how hematopoiesis is regulated during development. Studies in our lab have shown that the mouse placenta is a unique hematopoietic organ supporting de novo HSC generation in the chorionic plate and possible HSC expansion in the placental labyrinth, while preventing differentiation. The structural integrity of the labyrinth is compromised in a mouse model lacking Platelet Derived Growth Factor-B (PDGF-B) or its receptor, PDGF-R-Beta. PDGF-B and Beta deficient embryos are anemic, die perinatally, and display dilated fetal vasculature and a reduction of trophoblasts in the placental labyrinth. Recently, we have found that the absence of the PDGF-B results in an imbalance in HSCs/progenitors in the placental microenvironment. The PDGF-B-/- phenotype includes the accumulation of actively proliferating progenitors in the dilated vasculature as determined by expression of c-kit, CD41, and Ki67. These blast-like cells also appear to prematurely differentiate into ter119+ erythroid cells. The presence of macrophages with the blast-like cells suggests this ectopic erythropoiesis is a macrophage assisted process. Future experiments will determine if this ectopic erythropoiesis results from the inability of the PDGF-B-/- placenta to protect HSCs from premature differentiation or if the anemic conditions recruit the placenta to act as an erythropoietic organ.

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Ms. Renata Lerner
Mentor: Marie-Francoise Chesselet
Title: Quantitative Analysis of DARPP-32 Immunoreactivity and Striatal, Cortical and Corpus Callosal Volume Measurements in CAG 140 Knock-In Huntington’s Disease Mouse Models

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder, characterized by progressive cognitive, emotional and motor deficits. Animal models are indispensable for developing therapeutics for the disease and a key step in such studies involve the characterization of the animal model to ensure pathology relevance to the human disease. This study aimed to extend previous CAG 140 Knock in (KI) mice model characterization, by replicating striatal stereological volume measurements, and further measuring corpus callosal and cortical volumes to establish neurodegeneration patterns in 20-26 month old CAG 140 KI mice. Levels of 32kD -Dopamine and cAMP- Regulated Phospho-Protein (DARPP-32), an important regulator of dopamine signaling in striatal neurons, were also measured via fluorescence immunostaining in 20-26 month old CAG 140 CAG KI mice to determine striatal cellular dysfunction. Results have confirmed striatal degeneration, and indicated additional atrophy in the cortex and the corpus callosum. A reduction in striatal-specific DARPP-32 also suggested cellular dysfunction in the mice. In the future, these characterizations will be extended to 4 and 12 month old mice to generate a time-course of HD pathology of this mouse model, in an effort to improve drug screening studies.

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Ms. Angela Hui-Chia Liu
Mentor: Paul Mischel
Title: Therapeutic Implications of Dep-1 in Co-Activation of Receptor Tyrosine Kinases

Glioblastoma Multiforme (GBM) is the most prevalent and aggressive cancer of the adult CNS. Molecular targeted therapy for GBM aims to suppress receptor tyrosine kinase (RTK)-driven proliferation signals, in particular through the PI3K pathway. However, GBM is characterized by striking molecular heterogeneity and shows poor response to treatment of kinase inhibitors. Previous research (Strommel et al.) proposes the co-activation of multiple RTKs as a novel paradigm for cancer resistance to RTK inhibitors. Multiple concomitantly activated RTKs provide redundant inputs to drive and maintain downstream PI3K signaling even in the presence of single kinase inhibitor. Dep-1, density enhanced phosphatase 1, is a receptor-like protein tyrosine phosphatase that acts on several RTK substrates, including EGFR, Met, PDGFR, and VEGFR; genetic aberrations of Dep-1 are also observed in breast, colon, and lung cancers. We therefore hypothesize Dep-1 as an alternative therapeutic target for co-activation of RTKs in GBM. Immunoprecipitation analysis on GBM cell lines T98 and Ln229 shows physical association between Dep-1 and EGFR, PDGFR, and Met. Immunoblot analysis and RTK array of transient siRNA Dep-1 knockdown models also show negative correlation between Dep-1 expression and phosphorylation levels, or activation, of multiple RTKs. In addition, preliminary functional assay shows increased proliferation of Ln229 Dep-1 knockdown cells compared with control-siRNA-treated cells. This data suggests Dep-1 may play a tumor suppressor role in GBM by its action on multiple RTKs.

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Mr. Jason Melehani
Mentor: Kent Hill
Title: MudPIT Proteomic Analysis of the T. brucei Flagellar Skeleton Reveals Many Novel Proteins

Jason Melehani with Vice Provost Judi Smith

The flagellum drives motility of Trypanosoma brucei, the causative agent of African sleeping sickness. RNAi induced depletion of individual proteins within the flagellum disrupts flagellum substructures, generation of a productive motor force and cell division. In the bloodstream life cycle state, this often results in lethality. Attachment of the trypanosome flagellum to Tsetse fly host tissues as well as social motility studies in vitro suggest that the flagellum also plays a role in sensing. However, the exact mechanism by which the flagellum influences these processes is unclear. We have taken a proteomic approach to identify proteins within the flagellar skeleton. Previous inventories of flagellar proteins in T. brucei were incomplete since they employed flagellar skeletons extracted with high salt concentrations that disrupted flagellum ultrasturcture. Here we report that improvements on flagellar skeleton preparation preserve flagellum substructures, yielding a preparation that closely resembles the flagellum in intact cells. MudPIT proteomic analysis of the flagellar skeleton resulted in the identification of 867 proteins, including 467 proteins not identified in previous datasets. This dataset is referred to as the T. brucei flagellar skeleton proteome (“TbFSP”). Comparison to earlier flagellar proteomic datasets from T. brucei shows that significant variability exists in the number and identity of proteins and that our proteomic analysis encompasses this variability. Evaluation of flagellar localization and investigation of function are underway for many interesting targets.

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Mr. Aaron Meyer
Mentor: Daniel Kamei
Title: Concentrating DNA Using Two-Phase Aqueous Micellar Systems

The concentration of cancer biomarkers, such as DNA, prior to a subsequent detection step may facilitate the early detection of cancer, which could significantly increase chances for survival. In this study, the partitioning behavior of mammalian genomic DNA fragments, as well as oligonucleotides, were experimentally and theoretically investigated in a two-phase aqueous micellar system. The micellar system was generated using the nonionic surfactant Triton X-114 and phosphate-buffered saline. Partition coefficients were measured under a variety of conditions and compared with our theoretical predictions, demonstrating that the partitioning behavior of DNA fragments in this system is primarily driven by repulsive, steric, excluded-volume interactions that operate between the micelles and the DNA fragments, but is limited by the entrainment of micelle-poor, DNA-rich domains in the macroscopic micelle-rich phase. Furthermore, the volume ratio, that is, the volume of the top, micelle-poor phase divided by that of the bottom, micelle-rich phase, was manipulated to concentrate DNA fragments in the top phase. Specifically, by decreasing the volume ratio from 1 to 1/10, we demonstrated proof-of-principle that the concentration of DNA fragments in the top phase could be increased 2- to 9-fold in a predictive manner.

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Mr. Rameen Moridzadeh
Mentor: Volker Hartenstein
Title: Characterizing the role of JAK-STAT signaling in Drosophila melanogaster Intestinal Stem Cells of the posterior midgut

Although the mammalian intestinal stem cell (ISC) niche has been relatively well characterized, serving as an exemplar of niche function and regulation, the preliminary studies of adult Drosophila midgut ISCs leave several questions of fundamental importance unresolved. While the presence of ISCs in the adult midgut is well-established, neither the origin of the adult ISCs nor the existence of midgut stem cells in earlier developmental stages is determined. Previous studies have shown that expression of the transcription factor escargot (esg) serves as a marker of ISCs in the adult posterior midgut. To this end, we have conducted a differential gene expression report across the development of the midgut to establish unique markers for cluster cells. In late larval stages, numerous clusters consisting of approximately six cells appear in the posterior midgut. In our preliminary studies, we have found that esg is also expressed in all the larval cell clusters, suggesting the reality of a larval ISC. Furthermore, we have determined cluster cells are divided into two putative populations: stem cells, which express a STAT92E-GFP reporter for JAK-STAT activity in all the larval cell clusters, and transit amplifying (TA) cells. By studying the expression of JAK-STAT components, we have discovered larval ISCs intrinsically signal JAK-STAT activity. In future studies, we aim to establish conclusively the role of JAK-STAT signaling in ISC lineage progression.

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Ms. Sarah Muradian
Mentor: Harley Kornblum
Title: The Role of PTEN in Regulating Self-Renewal, Radiation Resistance, and Multipotency in Neural Stem and Progenitor Cells

Mutations in the tumor suppressor gene Pten are often found in brain tumors and have also been implicated in autism and macrocephaly. While previous research has shown that PTEN loss in mouse neural stem cells increases cell proliferation and size, questions remain regarding the role of PTEN in regulating other stem cell-like properties including self-renewal, multipotency, and resistance to radiation. We hypothesize that loss of PTEN in mouse neural stem cells and progenitors results in increased self renewal, multipotency, and radiation resistance, thereby making these cells more stem-like and possibly more predisposed to transformation into cancer. To test this hypothesis, we knocked down PTEN in mouse postnatal day zero cortical cells via silencing RNA techniques. At each passage, cells were assayed for sphere formation, radiation response, and differentiation ability. Our data indicate that cells lacking PTEN had increased capacity for self-renewal and resistance to radiation. Our preliminary data also suggests that PTEN loss likely enhances multipotency, specifically neurogenesis. Taken together, our results indicate that PTEN loss may influence stem-like character in neural stem and progenitor cells; further studies are needed to establish a potential role of PTEN loss in oncogenesis.

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Ms. Kristin Owyang
Mentor: Julian Antonio Martinez
Title: The Role of pico, the Drosophila Homologue of Human Grb10, in Larval Growth and Fat Body Function

Signal transduction pathways are central for the regulation of tissue growth. Growth factor receptor binding protein 10 (Grb10) is an adapter protein that connects cell membrane receptors with downstream signaling components to regulate processes such as metabolism and growth. However, the exact mechanism by which Grb10 regulates growth and developmental processes remains unclear. Our goal is to gain a better understanding of the signaling pathway through which Grb10 regulates growth using Drosophila melanogaster as a model organism. We used RNA interference and the GAL4/UAS system to spatiotemporally knock down pico expression. We find that this alters larval and wing growth, and significantly increases apterous (ap) expression in Drosophila fat body. The latter is particularly intriguing because ap is implicated in fat body histolysis, and expression of its mammalian homologue, ISL1, is negatively correlated with abdominal fat. Preliminary results are consistent with the idea that pico and ap interact in the same signaling pathway. We hypothesize that pico may interact with ap to regulate Drosophila fat body function, and believe this regulation may be related to the mechanism by which pico regulates growth. We are performing additional knockdown experiments to identify the tissues in which pico is required for regulation of ap expression and larval growth. These results will provide insight into the role of Grb10 signaling pathways in growth disorders and related diseases.

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Ms. Michelle Peng
Mentor: Deborah Farber
Title: Interaction of a Novel Protein, ZBED4, with Scaffold Attachment Factor B1

Pictures coming soon!

Rod and cone photoreceptors are directly involved in maintaining the integrity of the retina. Mutations in the genes coding for the visual pigments localized in the cones lead to a variety of congenital color vision deficiencies, color blindness, and cone dystrophy. A novel ZBED4/KIAA0637 gene was discovered in our laboratory using representative differential analysis subtraction of adult normal and cone degeneration dog retinal cDNAs and microarray analysis. We characterized the ZBED4 protein product and found that it eluted from a nickel column with an estrogen receptor co-repressor, the scaffold attachment factor B1 (SAFB1). Here we verify whether ZBED4 and SAFB1 are interacting proteins using immunoprecipitation and co-localization techniques. Since SAFB1 overexpression has been shown to inhibit the proliferation of breast cancer cells by acting as an estrogen receptor alpha (ER alpha) co-repressor, we studied the interaction of ZBED4 and ER alpha and discovered that they also co-localize. It is possible that ZBED4 could function together with SAFB1 as co-repressors of ER alpha in the retina. The elucidation of ZBED4 functional characteristics will be important for the future development of therapies involving vision restoration.

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Ms. Tiranun Rungvivatjarus
Mentor: Ivan Lopez
Title: Superoxide Dismutase-2 Upregulation in the Developing Cerebellum of the Rat after Chronic Mild Carbon Monoxide Exposure (0.0025% in air)

Tiranun Rungvivatjarus with Vice Provost Judi Smith

This study tested the hypothesis that chronic, mild prenatal CO exposure (25 parts per million) subverts the normal development of the rat cerebellar cortex. Pregnant rats were chronically exposed to carbon monoxide (CO) from gestational day E5 to E20. During postnatal period, rat pups were grouped as follows: Group A: prenatal exposure to CO only; group B: prenatal exposure to CO then exposed to CO from postnatal day 5 (P5) to P20; group C: postnatal exposure only, from P5 to P20, and Group D, controls (air without CO). At P20, immunocytochemical analysis of Superoxide dismutase-2 (SOD-2), an important anti-oxidant enzyme found exclusively in the mitochondria, was assessed in the cerebellar cortex of the four groups. Result shows that SOD-2 immunoreactivity increased in cerebellar cortex cells of the 3 CO-exposed groups. This up-regulation of SOD-2 indicates that exogenously supplied CO during prenatal period promotes oxidative stress. There were also differential responses to prenatal vs. postnatal CO exposure. Following prenatal only CO-exposure, there was a persistent increased in SOD-2 immunoreactivity, suggesting oxidative stress in pups at P20. The continuation of this cellular environment up to day 20 after CO exposure indicates that the condition is chronic and that prenatal exposure is more damaging. Postnatal exposure without prenatal exposure shows the least impact, whereas prenatal followed by postnatal exposure exhibits the most pronounced outcome among the groups.

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Ms. Claire Sampankanpanich
Mentor: Ren Sun
Title: Characterizing the Interaction of Murine Gammaherpesvirus 68 Open Reading Frame 34 and poly(C) Binding Protein 1

Epstein-Barr virus and human herpesvirus 8 belong to the Gammaherpesvirinae subfamily and are important human pathogens involved in tumor development in epithelial, hematopoeitic, and endothelial cell cancers. Gammaherpesviruses characteristically establish lifelong latent infection, persistently counteract host immune responses, and undergo sporadic lytic replication in compromised host immune systems. Herpesvirus genes mediating the lytic cycle are temporally classified into immediate-early, early, and late expression kinetics. While the lytic cycle has been thoroughly studied, regulation of late gene expression after DNA replication is not well understood. Previous studies identified Murine gammaherpesvirus 68 (MHV-68) ORF34, a viral trans factor required to stimulate late gene promoters after viral DNA replication during lytic infection. From cellular-viral PPI Y2H screens, ORF34 was found to interact with cellular poly(C) binding protein 1 (PCBP1), which belongs to the hnRNP family of RNA- and DNA-binding proteins. Preliminary findings indicate that PCBP1 overexpression significantly inhibits MHV-68 viral replication, while PCBP1 knockdown enhances viral replication. Studies of PCBP1 activity show an inhibition of late viral promoter but not early promoter, suggesting that PCBP1 regulates viral replication during the late stage of lytic replication. We seek to characterize how the protein-protein interaction between cellular poly(C) binding protein 1 and viral ORF 34 regulates viral late gene expression and affects viral replication.

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Ms. Allison Schwartz
Mentor: Ann Hirsch
Title: A New Strain of Bacillus simplex is a Plant Growth Promoting Rhizobacteria That Promotes Growth and Root Architectural Change in Lotus japonicus by Utilizing Bacterially Derived Phytohormones

Plant growth promoting rhizobacteria (PGPR) are important for plant growth and viability, interacting with plant roots to supply nutrients, phytohormones, and anti-pathogenic compounds in exchange for carbohydrates. We present our findings that a new strain of Bacillus simplex is a PGPR. PCR analysis of the 16S ribosomal DNA of strain 30N-5 was used to identify the bacterium as B. simplex. We hypothesize that B. simplex 30N-5 enhances the growth of the model legume Lotus japonicus by secreting auxin and cytokinin, two important phytohormones. We found using two different assays that B. simplex secretes auxin. Also, we present evidence that it expresses ACC deaminase, an enzyme that degrades the phytohormone ethylene, an antagonist of auxin. We determined that L. japonicus MG-20 biomass is significantly enhanced upon inoculating roots with B. simplex, and furthermore, that root architecture is altered such that more lateral roots and shorter primary roots develop. Inoculating seedlings of the L. japonicus hit1-1 mutant, which is defective in a cytokinin receptor, rescued the phenotype. The production of auxin by B. simplex and the rescue of the hit1-1 mutant strongly suggest that both phytomormones are involved in this positive growth effect. Based on these results, we conclude that Bacillus simplex 30N-5 is a PGPR that enhances growth by altering root architecture. Future studies will elucidate the molecular mechanisms behind the PGPR plant growth effect.

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Ms. Christine Thang
Mentor: Rick Delamarter
Title: Gaussia Luciferase (Gluc) in Monitoring Implanted Adipose-Derived Stem Cells in vivo

Luciferase-mediated bioluminescent imaging is a powerful ex vivo technique assessing in vivo biological processes. Purpose is to evaluate Gaussia luciferase (Gluc) in monitoring implanted exogenous adipose-derived stem cells (Ad-HMSCs, xenograft) viability in vivo and after rhBMP-2 exposure. Used in spinal tissue engineering, Ad-HMSCs can differentiate into osteoblasts, generating bone. We hypothesize higher photon measurement of Gluc expression correlates with increased cell survival. Ad-HMSCs were isolated using standard culture techniques, maintained in Preadipocyte media, and introduced to lentivirus encoding Gluc. Posterolateral spinal fusion was performed in rats. After surgical exposure, cells were implanted into paraspinal muscle across L4-L5 transverse processes. Implants were 5x10^6 Ad-HMSCs on Absorbable Collagen Sponge (ACS, 1.0cmx0.5cmx0.5cm), 5x10^6 Ad-HMSCs/ACS with 0.003mg/ml rhBMP-2 engineered/exposure, 5x10^6 Ad-HMSCs/ACS mixed with 0.003mg/ml rhBMP-2, or 0.003mg/ml rhBMP-2/ACS. Urine was collected by experimenter bladder expression daily over first two weeks then every other day until sacrifice two months post-surgery. Urine Gluc expression was assessed via a luminometer in relative light units per second (RLU/s). Results show average RLU/s highest from rats with 5x10^6 Ad-HMSCs+rhBMP-2/ACS, then 5x10^6 Ad-HMSCs/ACS engineered with rhBMP-2, then 5x10^6 Ad-HMSCs/ACS (no rhBMP-2), and lowest with rhBMP-2/ACS (no stem cells) (p<0.05). Average RLU/s values agree with expected BMP-induced cell survival compared to control conditions. Results demonstrate ex vivo reporter potential of Gluc to assess in vivo viability of implanted adipose-derived stem cells in spinal tissue engineering.

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Ms. Anna Xu
Mentor: Mirella Dapretto
Title: Neural Mechanisms Underlying the Segmentation of Word and Tone Sequences in Adults

Previous behavioral research examining language acquisition has implicated statistical learning in the parsing of continuous streams of both words and tones (i.e., word and tone segmentation). Indeed, these investigations have revealed that 8-month old infants as well as adults can utilize statistical regularities (e.g. transitional probabilities) and prosodic cues (e.g., stress as conveyed by increased pitch, amplitude, and duration ) to identify word boundaries within continuous speech and to segment analogous sequences of non-linguistic stimuli such as tones. However, these studies have neither addressed the neural correlates of statistical learning nor clarified issues of domain-specificity of the input during statistical learning and initial language acquisition. Here, using functional magnetic resonance imaging (fMRI), we adapted the paradigm from the infant behavioral literature to examine the neural substrates of the statistical learning of linguistic and non-linguistic stimuli. We revealed left-lateralized blood oxygenation level-dependent (BOLD) signal increases over time in temporal and inferior parietal cortices for both types of stimuli, indicating that implicit detection of statistical boundaries occurred. Moreover, we observed greater BOLD signal increases over time during exposure to the speech streams than to the tone streams in the left superior temporal gyrus, suggesting that the greater experience of our participants at processing speech significantly affects how the brain segments linguistic versus non-linguistic sequences.

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Ms. Lillian Zhang
Mentor: Dr. Hong Wu
Title: Delta-Catenin Modulation of Gamma Secretase Function in Alzheimer’s Disease

Alzheimer’s disease is a progressive neurodegenerative disorder, characterized by extracellular amyloid beta plaques and intraneuronal neurofibrillary tangles, which results in severe atrophy of the cerebral cortex and cognitive dysfunction. Genetic mutations in the presenilin-1 protein (PSEN1) result in familial Alzheimer’s disease (FAD). PSEN1 is the active site for the gamma-secretase complex which cleaves the amyloid precursor protein (APP), and gives rise to amyloid beta peptides. FAD mutations in PSEN1 increase the ratio of the abnormal amyloid beta-42 peptide (AB-42) to the normally produced AB-40 peptide by affecting gamma-secretase function. AB-42 is more prone to aggregation than AB-40 and is the main component of amyloid plaques. Delta-catenin (d-cat), a neuronal specific protein important for cognitive development of the brain, has been known to interact with PSEN1, and may influence the production of amyloid beta peptides. Our studies suggest that in vivo plaque deposition is considerably amplified in d-cat knock-out mice, which exhibit significant cognitive deficits. Furthermore, in vitro studies reveal that d-cat appears to effectively modulate gamma-secretase activity. These results indicate that d-cat interaction with PSEN1 may positively alter cleavage of APP by gamma-secretase, thereby decreasing the ratio of AB-42 to AB-40. Given that this ratio is important in understanding Alzheimer’s disease pathogenesis, these results may highlight a potential avenue for d-cat mediated disease therapy.

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