Student Profiles Archive - Science Poster Day Dean's Prize Winners 2011
Here you will find information about past and present students funded through scholarships administered by the Undergraduate Research Center - Sciences. We are proud of the achievements of our research scholars.
Please click on the program year to get information about the supported students, their mentors and their research projects.
| Faraz Alizadeh
Mentor: Jamie D. Feusner
Title: Self-Reported Anxiety and Brain Response to Fearful Faces in Anorexia Nervosa: an fMRI Study
Prior studies have shown that individuals with anorexia nervosa (AN) often have high degrees of anxiety and/or alexithymia, a condition in which a person has difficulty identifying or expressing emotions. Yet no study thus far has examined the neural correlates of fear processing in AN. To investigate this, we enrolled weight-restored individuals with AN and age and gender-equivalent controls and scanned them with fMRI to compare brain activation while they rated their fear in response to fearful face stimuli. We hypothesized that individuals with AN would have greater amygdala and lower medial prefrontal cortex (mPFC) and anterior cingulate cortex (ACC) activity in response to a fear-provoking stimuli, but that there would be a discrepancy between self-reported anxiety and activity in these regions. Preliminary results for six AN and thirteen healthy control subjects showed higher fear ratings in the AN group (p=0.02). Yet, contrary to our hypothesis, the AN group did not display significantly different amygdala, mPFC, or ACC activity relative to the control group. Despite this, there was a greater correlation in the AN group between reported fear and activation of the amygdala (r=0.88 for AN, r=0.15 for CON; p=.03) and ACC (r=0.99 for AN, r=0.07 for CON; p<0.001). The observation that in these regions mean brain activations were not significantly different between groups despite greater subjective fear in AN (which was highly correlated with activity), may be explained by the possibility of higher baseline activity in AN.
| Rebecca Andersen
Mentor: April D.Pyle
Title: Human Induced Pluripotent Stem Cell Derivatives as a Regenerative Approach to Duchenne Muscular Dystrophy
Duchenne Muscular Dystrophy (DMD) is one of the most common and severe childhood muscular disorders. No effective treatment is available, but one potential therapy involves the use of human pluripotent stem cell (hPSC) derivatives. Once expanded, muscle progenitor cells can be used alone or as a vehicle for correction of dystrophin expression and integration into muscle. We have developed human induced pluripotent stem cells (hiPSCs) from related healthy and DMD-patient fibroblasts for use as a pre-clinical tool and in regenerative applications. We have used these hiPSCs as patient-specific models to test the efficacy of exon-skipping drugs. In order to characterize cells with muscle potential from hPSCs, we determined when muscle gene expression is increased during embryoid body formation and isolated hPSC-derived cells that expressed the myoblast muscle marker NCAM (CD56). These cells were capable of engrafting into the mouse model of muscular dystrophy (mdx) with low efficiency. To isolate progenitor cells with muscle stem cell markers, we have generated a satellite cell (PAX7) reporter vector. As PAX7 is one of the earliest known markers for satellite cells, we expect development of a PAX7 reporter hiPSC line will assist in isolating a more primitive muscle progenitor cell. The identification of signals regulating muscle progenitor cell isolation and maintenance will enhance our ability to generate scalable muscle progenitors from humans, which could be used in regenerative therapies for muscle disorders including DMD.
| Armin Arshi
Mentor: Atsushi Nakano
Title: Controlled Matrix Elasticity Promotes Differentiation of the Cardiac Lineage
Heart disease is the leading cause of death in developed nations. While heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are known to be proliferative, with embryonic stem cells (ESCs) providing an endless reservoir. Though difficult to recapitulate in vitro, cell microenvironments appear to be important in stem cell lineage specification and controlled tissue engineering. Previous studies suggest that mesenchymal stem cells are extremely sensitive to tissue-level elasticity and can be cultured down a specific lineage trajectory with controlled substrate stiffness. Though more recent studies have shown that matrix elasticity can influence cardiac phenotype in neonatal cardiomyocytes, precisely how these factors affect undifferentiated ESCs is poorly understood. Here, we propose an in vitro differentiation system that exploits microenvironments of controlled rigidity to engineer cardiomyocytes with high specificity and efficiency from 2D culture of mouse ESCs. Using an Nkx2.5-neomyocin selection system with synthetic substrates, we demonstrate that stiff microenvironments with feeder cell monolayers facilitate proliferation and maturation of cardiomyocytes. We believe this to be a result of increased mechanotransductive signaling from increased focal adhesion complex formation associated with cell-substrate interactions. Collectively, these findings can help direct the production of functional cardiomyocytes en masse for regenerative medicine purposes.
| Nathanael Bangayan
Mentor: Huiying Li
Title: Characterizing the Propionibacterium acnes Population in the Human Skin Microbiome Using MLST Combined with High-throughput Sequencing
Propionibacterium acnes is a major commensal bacterium found in the human skin microbiota and has been linked to acne pathogenesis. In order to determine whether certain strains of P. acnes are associated with acne pathogenesis, we characterized and compared the P. acnes populations on the facial skin of acne patients and normal individuals. To characterize the P. acnes population structure in a high-throughput fashion, we combined traditional multi-locus sequence typing (MLST) method with 454 high-throughput sequencing. Nine housekeeping genes (lac, oxc, coa, pak, zno, gls, cel, recA, fba) are being analyzed in samples from 12 acne patients, 12 individuals with normal skin, and one mock sample of mixed P. acnes isolates as a control. In addition, phylogenetic trees of various housekeeping genes were built based on 71 P. acnes genomes to validate the use of the chosen MLST genes. The trees indicated that the MLST genes chosen are effective in typing P. acnes strains. This study not only provides the first insights into the microbial population structure of the human skin at the strain level, but also sheds light onto new directions for acne treatment.
| Kay Cadby
Ms. Kay Cadby
Mentor: Mark A. Frye
Title: Sensory Pathology of Preclinical Parkinson's Disease in Drosophila Models
Parkinson's disease (PD) is the second-most common neurodegenerative, age-related disease. The physical symptoms of the disease include resting tremor, rigidity, akinesia, and postural instability, and are due to the accumulation of misfolded proteins and mass cell death of dopaminergic neurons in the substantia nigra. When these progressive symptoms appear in the more advanced stages, the cells in the substantia nigra are beyond recovery. Studies have shown that olfactory and visual dysfunction has been correlated with early idiopathic PD symptoms, and the use of this biomarker, once established, can help develop new methods of early diagnosis. Transgenic fruit flies with overexpression of the wildtype human alpha-synuclein gene have been shown to develop and exhibit Parkinsonian motor dysfunction and alpha-synuclein inclusions with striking similarities to human PD pathology. This study aims to recapitulate these potential early biomarkers for PD and to show that Drosophila models of the disease may prove be even more reliable and efficient of animal models. The Drosophila Odor Trap is an ongoing experiment, and future experimentation is planned with the Olfactory Magnetic Tether system (OMT). Results from Drosophila models may be useful for new drugs that will survive the leap to human patients, a better verifier for new tests to diagnose PD in humans, and inspirational insight for new ideas based on the parallels in PD pathology.
| Ashley Cass
Mentor: Thomas G. Graeber
Title: DNA Copy Number Variations of Glycolytic Pathway Enzymes in Breast Cancer Are Predictive of 18F-flurodeoxyglucose Uptake
In a previous study, we found that the mRNA expression patterns of glycolytic pathways in breast cancer can be used to predict the level of cellular uptake of the glucose analogue 18F-flurodeoxyglucose (FDG). In this study, we extend this approach to DNA copy number patterns. Copy number abnormalities (CNAs) contribute to breast cancer pathophysiologies, differ between tumor subtypes, and are correlated with gene expression and clinical outcome. To better understand the metabolic state of cancer cells, we used several bioinformatic methods to analyze which metabolic pathways regulate breast cancer by studying panels of primary tumors and cell lines. A Weighted Gene Voting (WGV) algorithm was used to predict the FDG uptake of the cell lines. Gene Set Enrichment Analysis (GSEA) was used in order to discover metabolic enrichment in cell lines and tumors. Finally, Rank Rank Hypergeometric Overlap (RRHO) was used in order to find enrichment between two ranked lists of genes. Our results indicate that copy number variation of glycolysis genes is predictive of FDG uptake. This finding supports a model in which the selection pattern of DNA alterations generated by genomic instability are influenced by the linked metabolic function of the genes involved, and offer an additional example of how genomic instability can promote tumor-beneficial metabolism and poor prognosis.
| Janice Chan
Mentor: Thomas B.Smith
Title: Adaptive Variation As a Biodiversity Surrogate In Efficient Systematic Conservation Planning
Biodiversity is responsible for a wide variety of critical ecosystem services, and thus, possesses economic significance necessitating conservation efforts. As one strategy of conserving biodiversity, systematic conservation planning efficiently applies heuristic algorithms prioritizing biodiversity surrogates as proxies for endangered species distributions in reserve design. In this study, species occurrence as a biodiversity surrogate was used as a control against that of adaptive variation in the form of 1600 genetic and morphological traits. Reserve results generated through ResNet were subsequently evaluated in ArcGIS for the extent to which they actually conferred protection onto 29 endangered species of Ecuador. We found that parks designed to include genetic and morphological variation within seven common species also included habitat for the 29 endangered species, making adaptive variation of common species a good surrogate for endangered species' distributions in Ecuador. Furthermore, it is beneficial to protect adaptive genetic variation within species because it may help species evolve to respond to climate change. Future work should expand these conservation plans for Ecuador by incorporating socioeconomic factors, as successful conservation efforts must account for the economic needs of local stakeholders.
| Anthony Decino
Mr. Anthony Decino
Mentor: David A.Meyer
Title: Learning About Collective Memory From Query Bursts
Query logs provide detailed and useful insight about individual users, but they also provide an unprecedented level of detail about the collective behavior of user communities. Quantitative analysis of such collective behavior can inform search engine design, web content provision, and social scientific theory development. This project is specifically interested in collective memory, namely, memory that is common to the members of a community and often encoded in social artifacts and public discourse. Event-driven query bursts can be identified even in the highly aggregated, and approximate, publicly available Google Trends data. This project compares several possible models for the decay of these bursts by using the Bayesian model selection and the results found overwhelming support for asymptotic decay that follows the power law model rather than the exponential model. As some memory of an event is necessary to frame a relevant query, this research interprets the results as the "forgetting functions" of collective memories.
| Ramzi Dudum
Mentor: Juliana Oh Barba
Title: RBM5 Tumor Suppressor Gene Located At the Lung Cancer Hot Spot 3p21.3, When Over-Expressed, Inhibits Cell Adhesion and Motility
The RBM5 gene is located at chromosomal 3p21.3, a region that is the most frequent and earliest genetic alteration in lung cancer. Therefore, studying this region, and RBM5 in particular, provides a great deal of promise towards developing novel diagnostics/therapeutics to combat lung cancer. The accumulated evidence has corroborated firm characteristics of this gene as a tumor suppressor in both in vitro and in vivo studies. Previously, I have shown that RBM5 knockdown consistently increased gene expression for groups of genes that function in cell adhesion, migration, and motility. These groups are known to be essential in well known cancer metastatic pathways. Among these genes, Rac1 and beta-catenin, two important players downstream of the Wnt signaling pathway leading to increased cell migration, have been the focus of our subsequent functional studies. Having identified this regulation, our focus shifted on investigating the migration and adhesion properties of RBM5. Recent experimental evidence suggests that RBM5 potentially suppresses lung cancer by inhibiting metastasis. Therefore, the present study involves the effects of RBM5 over-expression on cell migration and invasion through wound healing and chamber migration assays. RBM5 up-regulation in two stably transfected lung cancer cell lines displayed decreased rates of proliferation and reduced migration. Further study is needed to validate RBM5's true function in tumor metastasis. Our long-term goal is to develop novel lung cancer therapeutics/diagnostics.
| Peyman Farhangi Oskuei
Peyman Farhangi Oskuei
Mentor: Nasser Ali Farahbakhsh
Title: Volume Regulation in Frog Auditory Hair Cells: Additional Evidence for Water Permeable Channels
When amphibian papillar hair cells (APHCs) of the leopard frog, Rana pipiens pipiens, are osmotically challenged, they exhibit a characteristically asymmetric (rectifying) response: Small decreases in the extracellular solution's osmolarity do not significantly affect the cells' volume; larger decreases produce a relatively slow volume increase in APHCs, while exposure to a hyperosmotic medium leads to rapid shrinking of these cells. Furthermore, the rate of volume change appears to be a function of the rate of extracellular osmotic change. In addition to the slow and perfusion-rate-dependent volume increase, APHCs also exhibit a delayed regulatory volume decrease when exposed to hypo-osmotic solutions: for a 50% reduction in osmolarity, the time-to-peak-volume is 383 ± 51 s and 1285 ± 153 s for the flow rates of 1.6 and 0.23 mls/min. During the remainder of a 30-min-long exposure, APHC volume falls to 78.6 ± 2.7% and 93.1 ± 2.1% of the peak, for the two flow rates, respectively. Upon return to the iso-osmotic solution, the APHC volume undershoots to 74.6 ± 2.3% and 88.6 ± 3.1% of the initial volume, respectively. The regulatory volume increase, however brings the cell volume to 87.1 ± 3.9% and 97.7 ± 3.1% of its initial volume in 30 min. At much lower rates of osmolarity change, APHCs might be able to maintain their shape and thus preserve their level of vitally-important mechanical, electrical and chemical activities. The significance of transmembrane solute transport in amphibian auditory hair cells will be discussed.
| Connie Fung
Mentor: Peter J.Bradley
Title: Trafficking and Function of ISP4: A Novel Toxoplasma gondii Inner Membrane Complex Protein
Toxoplasma gondii is an obligate intracellular pathogen that causes severe neurological disorders in immunocompromised individuals and birth defects in congenitally-infected neonates. T. gondii contains a unique peripheral membrane system called the inner membrane complex (IMC) that facilitates host cell invasion and daughter cell formation, processes crucial to pathogenesis. Recently, our lab has identified a novel family of IMC Sub-compartment Proteins (ISP1/2/3) in T. gondii and showed that they play functional roles in parasite replication. ISP1/2/3 localize to distinct IMC sub-compartments using a targeting mechanism that is contingent upon coordinated myristoylation and palmitoylation of conserved residues in the extreme N-terminus of the proteins. Although a fourth family member (denoted ISP4) was identified, it has not been characterized. We demonstrate that ISP4 localizes to the central IMC sub-compartment, similar to ISP2. Surprisingly, ISP4 lacks the conserved myristoylation and palmitoylation signals present in ISP1/2/3, suggesting that it employs a trafficking mechanism distinct from the other ISPs. Mutagenesis studies of the protein reveal that ISP4 targeting is not dependent upon acylations of the extreme N-terminus, but on palmitoylations of internal cysteine residues instead. We have also disrupted the ISP4 gene, and its relevance to cell division will be discussed. Collectively, these experiments provide new insight into the organization of the IMC, a structure critical to Toxoplasma pathogenesis.
| Agnieszka Grzechnik
Mentor: Guillaume Chanfreau
Title: Mechanisms of Nucleolar Protein Regulation During Zinc Deficiency in Yeast
Zinc plays an important role in many cellular functions and, therefore, maintaining its homeostasis is vital. In the yeast organism Saccharomyces cerevisiae, zinc deficiency causes a down-regulation of RNA Polymerase I subunits. This observed down-regulation is dependent on the degradation of RNA Polymerase I subunits by two vacuolar proteases, Pep4p and Prb1p. However, it is unknown if this down-regulation is general to all nucleolar proteins, or specific to RNA Polymerase I. To determine this, the expression of several nucleolar and nuclear proteins was investigated under zinc-deficient conditions by Western blot analysis. Several other proteins -- Cbf5p, Fpr3p, Reb1p, Rrp5p, and Shq1p - were found to be down-regulated in these conditions and it was further shown that this down-regulation is dependent on Pep4p and Prb1p. These results suggest that the down-regulation of these proteins during zinc-deficiency is mechanistically similar to RNA Polymerase I down-regulation. Future studies will test the levels of other nucleolar proteins to determine whether this down-regulation is specific to nucleolar proteins that associate with RNA polymerase I.
| Wendy Hsiao
Mentor: Elaine M.Tobin
Title: Interactions of GOLDEN2-LIKE1 and GOLDEN2-LIKE2 Proteins with Circadian Clock Components in Arabidopsis thaliana
In Arabidopsis thaliana, the circadian clock is regulated by core transcriptional components, one of which is encoded by the gene CIRCADIAN CLOCK ASSOCIATED1 (CCA1). CCA1 is expressed in a circadian manner through a 92 bp promoter element. Our analysis has revealed a protein, GOLDEN2-LIKE1 (GLK1), that binds to this element and may play a role in regulating clock responses. Our approach to studying this interaction focuses on 3 key aspects: regulation of circadian clock core components and outputs by GLK1/2, interactions of the GLK1/2 proteins, and clock-associated physiological responses in glk1/2 mutants and GLK1/2 overexpressors. The effects of GLK1 and GLK2 on the expression of three main oscillator components, CCA1, LATE ELONGATED HYPOCOTYL (LHY), and TIMING OF CAB1 (TOC1), indicate GLK1/2 function as negative regulators. Data from qRT-PCR of these genes is presented to support this conclusion. To study the protein-protein interactions of GLK1/2, we used translational fusion proteins of each respective GLK to luciferase (LUC) - ProGLK1/2-GLK1/2-LUC. We will monitor whether the GLK1/2 proteins oscillate with the clock through transient agroinfiltration of leaves of entrained N. benthamiana. Data from yeast interactions will also be shown. Lastly, the physiological effects of loss-of-function glk1, glk2, double mutant glk1 glk2, as well as the gain-of-function Pro35S-GLK1 or GLK2 is considered by comparing hypocotyl length of each line compared to wild-type and 35S-CCA1 controls known to have long hypocotyls.
| Tien-Phat Huynh
Mentor: David B.Teplow
Title: Stabilization of Amyloid Beta-Protein42 Oligomers through PICUP and Tyr "Scanning" Mutagenesis
Alzheimer's Disease (AD) is a neurodegenerative disorder, causing irreversible damage to the brain that culminates in memory loss, cognitive dysfunctions, and eventually death. Amyloid beta-protein (Abeta) forms deposits of fibrillar assemblies, which were long believed to be the key etiologic factors of AD. However, recent studies suggest that pre-fibrillar protein oligomers are the primary toxic effectors causing the disease. Ab exists as either a 40 or 42-residue peptide where the latter isoform appears to be more neurotoxic and clinically relevant. To study the folding dynamics of Abeta42, whose oligomers are metastable, a method for quantitative oligomer frequency distribution was utilized termed PICUP (Photo-induced cross-linking of unmodified proteins). Due to its critical role in PICUP, tyrosine was substituted at different positions along the peptide to produce four variants: F10, Y1; F10, Y20; F10, Y30; and F10, Y42. The project aims to identify variants of Abeta42 that produce stable oligomers amenable for further studies on biophysical and biological analysis. The distribution pattern of PICUP-stabilized Abeta42 based on SDS-PAGE suggests that the F10, Y42 substitution produces the most stable oligomers whereas the other substitutions exhibit significantly less oligomer stability. Further understanding of the oligomers will shed light on key regions within Abeta42 that control the oligomerization process and thereby allow the design of therapeutic agents targeting these structures.
| Norianne Ingram
Mentor: Patricia E.Phelps
Title: Characteristics of Olfactory Ensheathing Cells From Transgenic EGFP-Expressing Rats are Consistent With OECs From Nontransgenic Rats
The evaluation of olfactory ensheathing glial cell (OEC) transplantation into the spinal cord after injury is hindered by the lack of unique OEC markers. Enhanced green fluorescent protein (EGFP)-expressing, transgenic rats contain labeled OECs that can be identified in sections months after transplantation. Immunohistochemistry results show specific EGFP expression in olfactory bulb layers. In sections, the olfactory nerve layer, which contains OECs, showed concentrated EGFP expression. The random insertion of EGFP transgenes, however, may change the regenerative efficacy of the OEC therapy. We used OEC and cerebral cortical neuron (P8) cocultures to evaluate the extent of neurite outgrowth facilitated by OECs derived from either EFGP or nontransgenic rats. Preliminary analyses found that EFGP-expressing OECs stimulate similar neurite outgrowth as nontransgenic OECs. In addition, both types of OECs cause a comparable decrease in the amount of primary neurites grown per neuron, in contrast to a substrate-only control. Potentially, EGFP-derived OECs will allow positive identification of transplanted cells, and the transgene insertion does not appear to alter OEC-induced neuronal growth patterns in culture.
| Saro Kasparian
Mentor: Scott Kitchen
Title: The Role of TIM3 in Regulating the Immune Response in the Context of Cytotoxic T Lymphocytes and Macrophages
During most viral infections, the cytotoxic T lymphocyte (CTL) response is sufficient enough to clear virally infected cells. However, during an HIV infection the normal CTL response is defective in clearing the virus. It has been shown that during HIV infection, there is an up regulation of T cell immunoglobulin and mucin domain protein 3 (TIM3), a protein that inhibits CD8 T cell activation. Not much is known about the role of TIM3 on CD8 cells and we believe that if TIM3 were knocked down on antigen specific CD8 cells, CD8 response to pathogens should improve. We designed a short hairpin RNA molecule to attempt to knock down TIM3 expression. To establish a system to examine the ability of these shRNAs to knock down TIM3, we first needed to identify cell that express it. We screened human peripheral blood mononuclear cells (PBMC) and found that CD8 and CD4 T cells express TIM3 to some degree and activation of PBMCs using traditional methods did not induce TIM3 expression. We also found that macrophages endogenously express TIM3 and found that activation of monocytic progenitors did not induce expression of TIM3. We believe that monocytes only express TIM3 when they are terminally differentiated. HIV is a clever virus able to manipulate the immune system. By discovering how it does this and the role of inhibitory molecules like TIM3, we can create viable treatments to counteract HIV and give the body a chance to clear it.
| Pwint Khine
Mentor: Richard B.Kaner
Title: Thin Film Fabrication of Carbon Allotropes Using Substrate Directed Interfacial Deposition
Carbon nanotubes are mechanically strong, transparent and useful materials for application in sensors, electrical storage, and energy conversion devices. Here we demonstrate a process to grow thin films of carbon nanostructures on any type of substrate. A monolayer of nanostructures, present at a liquid-liquid interface, can be sheared between water and oil. In order to engineer a coating on a solid surface, a spontaneous interfacial surface tension gradient is induced to produce the directional fluid flow. Transparent coatings of nanostructured carbon are dried under ambient conditions and are characterized with a continuously conductive network across the entire coated surface. Scanning electron microscopy shows aligned ropes of single-walled carbon nanotubes and assemblies of single sheets of chemically converted graphene and graphite oxide. Process scale-up, layer-by-layer deposition, and a novel technology for fabricating freestanding films on hydrophobic surfaces are described.
| Jaehyun Kim
Mentor: Dino Di Carlo
Title: Automation of Cellular Sample Preparation Using a Centrifuge-On-A-Chip
The standard centrifuge is a laboratory instrument widely used by biologists and medical technicians for preparing cell samples. Efforts to automate the operations of concentration, cell separation, and solution exchange that a centrifuge performs in a simpler and smaller platform have had limited success. Here, we present a microfluidic chip that replicates the functions of a centrifuge without moving parts or external forces. The device operates using a purely fluid dynamic phenomenon in which cells selectively enter and are maintained in microscale vortices. We identify the hydrodynamic forces responsible for cell entry and maintenance within vortices and the critical particle size for trapping associated with specific geometries. We utilize the system for separating cancer cells from spiked blood samples in a continuous fashion processing liquid volumes at the mL/min scale. We demonstrate fluorescent labeling of intra- and extra-cellular antigens on cells without the need for manual pipetting and washing steps.
| Jin Ki Kim
Jin Ki Kim
Mentor: Judith A.Berliner
Title: Role of Small G-proteins in Regulating Actin-filament Formation by Ox-PAPC in Human Aortic Endothelial Cells
In atherosclerosis, which causes heart attack and stroke, the endothelium allows monocytes to infiltrate the blood vessel, initiating a proinflammatory response. Minimally modified low-density lipoprotein (mm-LDL) accumulates in the vessel wall and causes inflammation in the vessel resulting in atherosclerosis. The major bioactive component of mm-LDL is oxidized phospholipids. We used Ox-PAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-glycerol-3-phosphocholine) as a representative molecule that mimics the effect of mm-LDL. Ox-PAPC activates Vascular Endothelial Growth Factor Receptor Type 2 (VEGFR2) to regulate actin-filament formation and the endothelial cell (EC) monolayer permeability. We hypothesize that Ox-PAPC activation of VEGFR2 regulates the activation of small G-proteins including rhoA. This activation controls actin-filament formation leading to an increase of EC monolayer permeability. In this study, we examined the role of specific small G-proteins by employing siRNA-mediated gene silencing and chemical inhibitors. We used the western blotting technique to measure the relative activation (phosphorylation) of signaling molecules involved in actin-filament formation including myosin light chain (MLC).
| Patrick Kim
Mentor: Dong Sung An
Title: Efficiency in CCR5 HIV-1 Co-Receptor Knockdown using aiRNA and shRNA Construct
The chemokine receptor CCR5 on macrophages and T cells has been known to be a vital co-receptor for R5 tropic HIV-1 viral attachment and infection. Dispensable for human physiology but crucial in HIV-1 infection, CCR5 has become a popular subject in therapeutic approaches. Down-regulation of CCR5 through RNA interference using siRNA constructs can treat HIV-1 infection and viral prolongation. A previously identified unique shRNA, is very potent in down-regulating the CCR5 gene, but the more efficient siRNA would have improved potency while eliminating any degree of cytotoxity from off-target silencing, prevalent with highly active promoters. Based on this unique shRNA sequence and the theory behind aiRNAs, two unique siRNAs were designed to compare their efficiencies in CCR5 knockdown with the shRNA constructs they were derived from. An aiRNA and a modified shRNA construct with a single-nucleotide mutation were both cloned to be expressed using the highly active U6 pol III promoter in a lentiviral vector and were analyzed to determine their efficiency in CCR5 gene down-regulation in human T lymphocyte lines. Both constructs, designed to reduce off-target silencing due to asymmetry while still maintaining a strong gene silencing potency, were efficient in down-regulating CCR5; however, they were not as potent as the shRNA constructs they were modeled from. Nonetheless, the original shRNA with a single-nucleotide mutation still managed to produce efficient knockdown of the CCR5 co-receptor.
| Grace Lee
Mentor: April D.Pyle
Title: PTEN Regulates the Pluripotency Network and Lineage Fate Choice in Human Embryonic Stem Cells
Every division, human Embryonic Stem Cells (hESCs) are faced with the decision to increase the pool of self-renewing, pluripotent cells or to differentiate into cells with more restricted capabilities. Understanding the regulation of cell fate decisions in hESCs is necessary to improve our ability to culture as well as tailor lineage differentiation. One pathway known to regulate proliferation and differentiation in multiple stem cell systems is PI3K/AKT. To study PI3K/AKT in hESCs, we targeted PTEN, a tumor suppressor that negatively regulates PI3K/AKT, using short hairpin RNA. In this study, we compare in vitro and in vivo differentiation of knockdown (KD) and control (NT) H9 hESCs. We demonstrate that, although KD hESCs have inefficient terminal differentiation across all three germ layers in vitro, KD hESCs exhibit greater neural differentiation compared to NT hESCs in vivo. Under in vitro directed differentiation conditions, KD hESCs maintain high expression of the pluripotency markers OCT4 and Nanog. In vivo, KD hESCs form teratomas with greater size and more proliferation than NT teratomas. Increased AKT activation in hESCs does not lead to nuclear localization of beta-catenin, but activation of S6 increased expression of pluripotency markers. Improving our understanding of the extrinsic pathways regulating pluripotency signals will improve our ability to regulate the pluripotent state and our ability to tailor lineage differentiation for use in regenerative medicine.
| Sung-Ling Dean Lee
Sung-Ling Dean Lee
Mentor: Alvaro Sagasti
Title: Identification of Cis-Regulatory Elements that Drive Somatosensory Neuron-Specific Expression in Zebrafish
In vertebrates, thermal, chemical, and mechanical stimuli are detected by a diverse repertoire of somatosensory neurons (SN). Such diversity is generated by the transcriptional regulation of key developmental and functional genes. Thus far, no transcription factors (TF) have been identified that direct the SN-specific expression of these genes. To begin describing the transcriptional regulatory circuit that defines SN gene expression, we are analyzing the genomic regions upstream of SN-specific genes, such as tropomyosin receptor kinases (Trk) and transient receptor potential (TRP) channels. We hypothesized that TF binding sites are shared by the promoter regions of these genes. To isolate these shared sites, we subcloned regions of the genome upstream of the TrkA and TrpA1 genes into green fluorescent protein reporter transgenes. We subjected reporters that drove expression in zebrafish SNs to deletion analyses and identified two minimal cis-regulatory elements upstream of TrkA and another upstream of TrpA1a. Using bioinformatic analyses, we identified several short consensus sequences (5-10 base pairs) within these elements, implicating them as candidate regulatory sequences that drive SN-specific expression. To test the function of these sequences, we disrupted each with point mutations and are currently characterizing their effects on reporter expression. These candidate SN-specific TF binding sites will help elucidate the mechanisms of transcriptional regulation that contribute to vertebrate somatosensory specification.
| Alice Ma Li
Alice Ma Li
Mentor: Joan S.Valentine
Title: Subcellular Localization and Potency of Superoxide Dismutase-Mimicking Compounds in Saccharomyces Cerevisiae
Several laboratories and biotechnology companies manufacture superoxide dismutase (SOD)-mimicking compounds, which reportedly possess SOD activity in vivo. Some of these compounds, particularly manganese porphyrin ligands, have been shown to improve the growth of mutant yeast strains lacking copper/zinc-superoxide dismutase (Sod1p), or sod1-delete yeast. However, the mechanisms by which such mimics exert their effects upon the mutant yeast remain unknown. To investigate how Mn porphryin mimics, particularly MnTE-2-PyP, MnTnHex-2-PyP, MnTnBu2-PyP and MnTM-2-PyP, operate inside yeast, we first assessed their potencies by examining how well each mimic could rescue common phenotypes of the sod1-delete mutant, namely its auxotrophies for lysine and methionine. Based on the distinct manner of rescue exerted by the mimics on the auxotrophic phenotypes, our data suggested MnTE-2-PyP, MnTnBu-2-PyP and MnTnHex-2-PyP accumulate inside the mitochondria while MnTM-2-PyP more evenly distributes between the mitochondria and cytosol. Experiments directly measuring each compound's concentrations inside yeast cells' mitochondria and cytosol supported this hypothesis. Further analysis of our results indicated that, with respect to all four mimics, the degree of mitochondrial localization correlates with lipophilicity. By illuminating these Mn porphyrin mimics' biological mechanisms of action, our data explores methods to both increase drug biological availability in mammalian cells and reduce some harmful effects of oxidative stress in tissue.
| Xian Liu
Mentor: Amander T.Clark
Title: Investigating the Role of Apobec1 in DNA Demethylation of Embryonic Stem Cell Derived Germ Cell
Primordial germ cells are early progenitor cells that give rise to haploid gametes. In mammals, the epigenetic reprogramming of primordial germ cells (PGCs) involves demethylation of DNA, modification of histone tails and higher order chromatin remodeling. Newly formed murine PGCs begin with high levels of DNA methylation and during the next five days of PGC development, chromatin reorganization occurs and the genome starts to demethylate. Failure of epigenetic reprogramming and DNA demethylation can result in infertility or birth defects. In this study, we specifically focus on the loss of DNA methylation, which is a covalent modification of the DNA nucleotide cytosine. To facilitate studying of this molecular event, our lab established a differentiation model of mouse embryonic stem cells (mESCs) to generate PGCs and examine DNA demethylation in vitro. Epigenetic changes, such as global DNA demethylation and enrichment of the epigenetic mark, histone 3 lysine 27 trimethylation have been found in the in vitro derived PGCs. We are currently investigating the role of a known DNA demethylase, Apobec1 in the formation of early PGCs through retrovial short hairpin RNA (shRNA) mediated knockdown. Our goal is to examine the effect of disrupting Apobec1 mediated DNA demethylation on germ cell formation and identify the targeting sites of DNA demethylation.
| Ramin Morshed
Mentor: Dean Buonomano
Title: Spontaneous and Learned Network Activity in Primary Sensory and Motor Cortex
Examining neural dynamics, or how activity flows through neuronal networks, is important for understanding how the brain processes information. Functional and anatomical differences between cortical areas suggest that neural dynamics in such regions may be variable. To examine this, we compared spontaneous and evoked network activity in rat organotypic slices that contained either primary sensory or motor cortex. Intracellular recordings in layer 2/3 pyramidal neurons were used to measure spontaneous activity. Sensory networks displayed a higher frequency of Up-states (periods of prolonged network activity) while motor networks demonstrated a greater rate of neuronal spiking which was not attributable to differences in intrinsic excitability. To investigate whether network dynamics can be shaped by experience in vitro, we delivered a stimulation pattern encoded with temporal information to slices for 2 hours via implanted electrodes. Evoked responses from layer 2/3 pyramidal neurons were acquired before and after stimulation training. Both cortical areas revealed changes in the distribution of polysynaptic activity after training, illustrating experience-dependent plasticity in neural dynamics. Our results demonstrate that spontaneous network activity can significantly differ across the brain, and that neural dynamics in cortical circuits can be altered by experience. We are currently investigating whether the timing interval used during stimulation is reflected in the distribution of polysynaptic activity in these networks.
| Partia Naghibi Mahmoudabadi
Partia Naghibi Mahmoudabadi
Mentor: Benjamin S.Williams
Title: Fabrication of Terahertz Quantum Cascade Laser with Integrated Waveguide Probe
The terahertz region of electromagnetic wave spectrum spanning from 300 GHz to 10 THz (the transition from electronics to photonics) has recently attracted considerable attention based on its numerous applications in different fields such as astronomy, biology, medical imaging, homeland security and others. Quantum cascade lasers (QCLs) have been developed in the past ten years as important sources of terahertz radiation. Despite having impressive output power, a divergent output radiation beam pattern has been one of the important challenges for QCLs. As a solution, we have proposed a QCL with slightly different design. In this research, we've designed and fabricated a metal-metal QCL with addition of an integrated coupling radial probe on one facet using micro-fabrication techniques. The laser is designed to be mounted in a micro-scale full-height rectangular waveguide. The radial probe would help transitioning the laser's output into a waveguide TE10 mode. The waveguide will terminate in a diagonal horn antenna which will improve the laser's highly divergent output radiation beam. Achievement of a quality radiation beam pattern would make QCLs able to be employed in various terahertz instruments.
| Don Nguyen
Mentor: Kent L. Hill
Title: Role of Receptor-type Adenylate Cyclases in Trypanosoma brucei Cell-cell Communication and Social Behavior
Trypanosoma brucei is a parasitic protozoan responsible for African sleeping sickness, a fatal disease that causes significant human mortality and limits economic development in sub-Saharan Africa. Recent studies have shown that T. brucei responds to surface contact by exhibiting a social behavior termed social motility (SoMo). In SoMo, individual trypanosomes actively recruit one another to form macroscopic communities. These communities then migrate en masse across a surface and away from the site of inoculation to form characteristic radial projections. These radial projections can change their trajectory to avoid contact with other projections, indicating the coordinated sensing and response to extracellular signals. Within other eukaryotic organisms, cAMP signaling is responsible for regulating many different physiological processes, including social behavior. Here, we report that RNAi knockdown of adenylyl cyclases 1 and 2 (AC1+ 2), or adenylyl cyclase 6 (AC6) results in a "hyper SoMo" phenotype characterized by an increased number of projections formed as compared to control cell lines. These results demonstrate a clear role for adenylyl cyclases in the regulation of SoMo. Currently, we are working to understand the specific mechanisms these proteins employ to regulate SoMo. Investigating the role of adenylyl cyclases in social behavior will provide further insight regarding how T. brucei senses external signals within its hosts, and how other pathogens communicate and exhibit social behavior.
| Haidang Nguyen
Mentor: Ronald M. Harper
Title: Heart Failure Patients Show Global and Regional Volume Loss in a Motor and Movement Regulatory Brain Region
Heart failure (HF) patients demonstrate reduced motor and cognitive skills that contribute to inadequate coping with the disease. The putamen contains dopaminergic neurons that serve many of the affected functions but the localized tissue integrity of this structure in HF is still unclear. We used a 3.0-Tesla MRI scanner to collect two high-resolution T1-weighted brain scans from 16 HF and 32 control subjects. Both T1-weighted image volumes were realigned, averaged, and reoriented into a common space, followed by manual outlining of the putamen structures. The putamen tracings were normalized for head-size, global volumes determined, and surface models created for three-dimensional surface morphometry. We compared the demographic data between groups using independent samples t-tests and Chi-square, evaluated global putamen volumes using independent samples t-tests, and examined regional volume differences using surface models with two-sample t-tests, based on surface morphometry. No significant differences in age or gender emerged between groups. HF patients showed significantly reduced right and left putamen volumes over controls, containing significant localized volume losses in medial-caudal, bilateral rostral, and mid-dorsal regions. Putamen structures exhibit localized and global volume reductions in HF over control subjects. Regional volume losses in HF subjects provide a basis for deficits in motor functions. The processes underlying the localized volume reduction may result from hypoxic and ischemic processes.
| Kaylin Nguyen
Mentor: Stephen Smale
Title: Evolutionary Divergence within the Nuclear Factor-kappaB Family of Transcription Factors
Studying the evolution of the immune system has lead to important advances in immunology. Understanding how protein families such as NF-kappaB evolved could provide insights into the development and selective pressures surrounding the evolution of conventional adaptive immunity. The activity of the NF-kappaB family is crucial in mounting normal immune responses and in lymphocyte survival and activation. The phenotypes of gene-knockout models of NF-kappaB family members suggest the proteins play distinct roles in the regulation of cell survival, lymphocyte function, and immune regulation. Of particular interest are the differences in phenotypes between c-Rel- and RelA-deficient mice. RelA-deficient mice are embryonic-lethal while c-Rel-deficient mice appear to develop normally. It is unknown how c-Rel and RelA regulate diverse genes in the same cell types despite similar biochemical properties. The binding kinetics of c-Rel and RelA proteins from model organisms representing different points in evolution were analyzed using EMSA off-rate experiments. We hypothesized that the divergence of c-Rel and RelA coincided with the evolution of adaptive immunity and that relatively subtle differences in DNA-binding properties were responsible for the functional diversification of the proteins. Preliminary results suggest that c-Rel developed after the evolution of conventional adaptive immunity with further studies needed to understand the significance of the divergence of c-Rel and RelA in vertebrate evolution.
| Amber-Chantelle Ocampo
Mentor: Rachelle H. Crosbie
Title: Sarcospan Improves Muscle Physiology in mdx Mice
Duchenne muscular dystrophy is a degenerative muscle disorder characterized by mutations in the dystrophin gene, which results in the disbanding of the dystrophin glycoprotein complex (DGC). The DGC provides structural stability to the sarcolemma during muscle contraction. One of the core components of the DGC is the transmembrane protein sarcospan (SSPN). We have previously shown that SSPN over-expression in dystrophin deficient (mdx) mice ameliorates the dystrophic pathology, indicative of functional restoration of the extracellular-intracellular linkage across the sarcolemma. In this study we have investigated the effect of SSPN over-expression on muscle strength and recovery potential in mdx mice by electrophysiological stimulation of isolated extensor digitorum longus (EDL) muscles. Our results show that SSPN over-expression in mdx mice improves strength and increases recovery rate. To move these findings closer to an actual therapy, we have engineered recombinant adeno-associated virus to express SSPN (rAAV6/CMV-SSPN). We show here that rAAV6/CMV-SSPN can direct expression of SSPN and increase levels of utrophin and its associated protein components at the sarcolemma in mdx mice. Taken together, these results support the therapeutic value of SSPN in dystrophic muscle and bring us closer to developing an applicable therapy for muscular dystrophy patients.
| Beatriz Osuna
Mentor: Alison R. Frand
Title: Role of the MAM-domain Proteins MLT-9 and ZC13.3 in the Molting Cycle of Caenorhabditis elegans
Caenorhabditis elegans periodically sheds and remakes its exoskeleton (the cuticle) in order to grow. During this molting process, epidermal cells fully renovate local extracellular matrices (ECM) and related cell-ECM and cell-cell attachments. Because the C. elegans exoskeleton is a collagenous ECM similar to human skin and connective tissue, findings about the regeneration of the nematode exoskeleton will likely apply to ECM homeostasis in vertebrates, which is a critical process for normal development and wound repair. This study investigates the mlt-9 and zc13.3 genes, which were previously identified in an RNAi-based screen for larvae unable to shed cuticles. Both genes encode annotated secreted proteins with MAM domains, which are characteristic of adhesive molecules. We hypothesize that interactions between the MAM domains of MLT-9 and ZC13.3 contribute to dynamic changes in adhesion among epidermal cells and the ECM critical for molting. Genetic and cell biological analysis will be used to test this hypothesis. Specifically, mlt-9 and zc13.3 null mutants are being examined for defects in the cyclic detachment and reattachment of epidermal cells to one another and to the cuticle. Thus far, several distinct mutations in mlt 9 and zc13.3 have been obtained, confirmed by PCR-based genotyping, and out-crossed to wild-type C. elegans. Specific phenotypes observed to date are consistent with the hypothesis that MLT-9 and ZC13.3 regulate cell-ECM adhesion during the molting cycle.
| Olga Prikhodko
Mentor: Carmen Bertoni
Title: Restoration of Dystrophin Expression by Inducing Readthrough of Premature Stop Codons
Nonsense mutations cause a number of genetic diseases, such as Ataxia Telangiectasia and Duchenne muscular dystrophy (DMD). Induction of readthrough of premature stop sites generates full-length gene transcripts, leading to functional gene products. Compounds that interfere with premature transcript termination can rescue gene expression and alleviate the phenotype. Two such compounds, RTC13 and RTC14, identified at UCLA using a high throughput screen, were shown to have potential therapeutic effect for DMD. DMD is characterized by the absence of dystrophin protein in skeletal muscle resulting from mutations, 13% of which are nonsense mutations. We have tested the ability of RTC13 and RTC14 to restore dystrophin expression in muscles of the mdx mouse model for DMD. Intramuscular injection of RTC13 restored dystrophin expression as evidenced by immunostaining analysis. Injections of compound RTC14 did not result in significantly higher dystrophin levels compared to injections of vehicle. Our data demonstrate that RTC13 can restore dystrophin expression in different muscle groups after intraperitoneal injection. Acute and chronic toxicity studies have shown no significant difference between mice injected with RTC13, vehicle, or uninjected mice. Future studies are focused on optimizing the drug structure for more efficacious restoration of dystrophin expression. These studies will enable us to advance this newly identified compound into clinical application for this disease.
| Allison Quan
Mentor: Robert Wayne
Title: Origins of Invasive Red Swamp Crayfish (Procambarus clarkii) in the Santa Monica Mountains
Non-native species are widely recognized as serious ecological and economic threats. Although native to the southeastern United States, the red swamp crayfish (Procambarus clarkii) has expanded across the world by both accidental and intentional means. In the Santa Monica Mountains, the presence of the omnivorous crayfish is associated with the absence or reduced abundance of native amphibians, such as the Pacific Treefrog (Pseudacris regilla). The origins of P. clarkii in southern California are unknown; however genetic analysis can be used to determine sources of invasion. From 2007 to 2011, 93 crayfish were sampled from streams in the Santa Monica Mountains, surrounding areas and possible source populations such as baitshops and petshops. Sequence data were generated from the 16S rRNA subunit and cytochrome oxidase I (COI) mitochondrial genes and compared to sequence data from native regions. In addition, DNA samples were collected from the stomachs of 17 individuals in an effort to verify the predation behavior of P. clarkii. Twenty-three haplotypes were identified among 115 samples and showed possible source populations from Florida, Texas and New Mexico. A high number of haplotypes within crayfish from the Santa Monica Mountains suggested multiple introductions. No evidence of tadpole DNA was evident from stomach content analysis. By identifying the original source and specific methods of introduction, further expansion of P. clarkii in the ecosystems of Southern California can be slowed and prevented in the future.
| Erik Reinertsen
Mentor: Nabil J.Tawil
Title: 3D Fibrin Composition Affects Human Fibroblast Proliferation and Fibrinolysis
Fibrin is widely investigated as a tissue engineering scaffold because it is biocompatible, biodegradable, and formed at the site of injury in normal wound healing. One potential application of fibrin is to enhance wound healing by delivering cells and biomolecules to sites of injury. It is important to characterize how fibrin composition affects cell behavior, in order to enhance fibrin delivery vehicles. To test the hypothesis that fibrin composition affects cell behavior relevant to wound healing, human foreskin fibroblasts (HFF) were cultured in fibrin scaffolds of varying concentrations (5-20 mg/mL). Proliferation, cell morphology, gel degradation, and urokinase plasminogen activator activity were assessed. Controls included 2D cell culture and acellular fibrin. Gels were incubated at 37°C with media. Time points were taken days one, three, and five. HFF proliferation was evaluated by labeling cells with Calcein AM (Invitrogen) and reading average fluorescence intensity. Mechanical stiffness of the fibrin gel was determined using indentation testing. HFFs proliferated most in 10 mg/mL gels. HFFs degraded fibrin gels after 5 days. Fibrinolysis did not occur in 10 mg/mL gels, correlating with higher cell density. Soluble uPA activity was not altered in fibrin cultures, but was lower in 2D culture. Future work includes studying other proteases, and mechanisms of cell-fibrin interactions, starting from the alpha-v beta-3 integrin. Results from such studies could enhance the design of fibrin-based therapies for wound healing.
| Logan Roberts
Mentor: David E. Krantz
Title: Using the Drosophila Vesicular Monoamine Transporter to Identify Novel Drugs for Treating Parkinson's Disease
Parkinson's disease (PD) is a neurodegenerative condition characterized in part by the death of dopaminergic neurons in the substantia nigra resulting in severe motor deficits. Recent studies show that increasing the activity of the vesicular monoamine transporter (VMAT) may ameliorate motor dysfunctions and protect against cell death. However, mechanisms underlying PD and an approach for pharmaceutically regulating VMAT activity are still uncertain. VMAT is vesicular protein essential for regulating the packaging of all cytosolic monoamines including dopamine, serotonin, and octopamine. Research show that octopamine is critical for promoting the central pattern generator which controls repetitive behaviors like locomotion. This study investigated whether reduced locomotion could be rescued by potentiating the octopaminergic circuit via VMAT. The hypothesis is that molecules capable of increasing VMAT activity may rescue locomotor deficits and serve as neuroprotectants. Pharmacological screens were conducted to identify drugs capable of increasing Drosophila VMAT (dVMAT) function in dVMAT mutant larvae as measured by locomotion. DVMAT mutants show severely retarded locomotion and drugs capable of rescuing this were selected for further screening. This screen revealed 41 agents that significantly improve locomotion; some of which may act directly through dVMAT. These agents have the potential to serve as novel therapeutic agents for PD as well as further our understanding of the mechanisms underlying PD.
| Joseph Rodrigues
Mentor: Stephen Smale
Title: Characterization of the Evolutionary Divergence of the NF-kappaB Family of Transcription Factors by Binding Affinity Analysis
The NF-kappaB transcription factor family plays a key role in regulating innate and adaptive immunity. Homodimers of two of the five family members--c-Rel and RelA--utilize similar residues to contact DNA consensus binding sites yet interact with both common and distinct sets of genes. Insight into the evolution of the NF-kappaB family can elucidate the mechanisms behind the differential function of each member. Phylogenetic analysis indicated that c-Rel and RelA duplicated from an ancestral Rel between the divergence of chordate species Sea Lamprey and Elephant Shark. The divergence is concurrent with a whole genome duplication key to the subsequent development of adaptive immunity similar to that found in mammals. Previous analysis demonstrated that differential binding affinity caused by 46 unique residues within the highly conserved Rel Homology Region determined gene activation specificity for Interleukin-12b. We hypothesized that the duplication of an ancestral Rel into c-Rel and RelA led to a divergence in binding affinity necessary for the development of the adaptive immune system. Binding affinity of c-Rels and RelAs from seven chordate organisms was analyzed by EMSA off-rate analysis. However, results indicate that the divergence of DNA binding affinity occurred between Zebrafish and Frog. Thus it is unlikely the divergence in binding affinity led to the initial formation of the adaptive immune system. This suggests something else remains to be learned about the mechanistic differences between c-Rel and RelA.
| Varun Shahi
Mentor: David E. Krantz
Title: A Pharmacological Screen in Drosophila may Identify Novel Drugs to Treat Mood and Attention Deficit Disorders
Drugs that target aminergic signaling are the mainstay for the treatment of depression, anxiety and attention deficit disorder, but methods to identify new targets are limited. We are using Drosophila to identify novel drugs that potentiate monoamine signaling. In flies, as well as mammals, VMATs are required to package into synaptic vesicles all monoamine neurotransmitters, including dopamine and serotonin. dVMAT mutants show several behavioral deficits including reduced larval locomotion. The locomotion deficit of the dVMAT mutant provides a sensitized background to screen for novel drugs that potentiate amine release or post-synaptic signaling. We screened a panel of 1040 drugs and identified 42 compounds that increase larval locomotion. In a secondary screen comparing the effects of dVMAT null and hypomorphic alleles, we showed that 11 of the 42 are likely to act via increasing aminergic signaling, and at least one may employ a novel mechanism to potentiate amine release. A second set of drugs is likely to act post-synaptically to directly activate aminergic signaling pathway. We will now validate our results in rodents. Our screen represents an important new way to identify potentially new drugs and targets for the treatments for depression, anxiety and attention deficit disorder.
| Justin Sharim
Mentor: Peyman Golshani
Title: Mechanisms of Pathological High Frequency Oscillations in the Pilocarpine Model of Epilepsy
Temporal lobe epilepsy is a neurological disorder characterized by recurrent unprovoked seizures. Transient bursts of high frequency oscillations (HFOs) in the 140 -- 200 Hz frequency band, called ripples, have been observed in the non-epileptic brain. These oscillations are normal, and may play a role in the consolidation of memory and in synaptic plasticity. In epileptic patients and in animal models of temporal lobe epilepsy however, much higher frequency oscillations of 200 -- 600 Hz, called fast ripples, have been observed. The presence of these pathological HFOs correlates with encoding and retrieval deficits, and is thought to be a marker for where seizures originate. However, the underlying cause of the pathological frequencies is still unclear. To uncover the underlying cause of pathological HFOs, we have recorded in vivo local field potentials simultaneously with whole-cell membrane potentials from CA1 pyramidal neurons in the hippocampus of control and epileptic mice (1-3 months after status epilepticus) under urethane anesthesia. Spectral analysis demonstrated that the anesthesia blocks the pathological HFOs. A proportion of epileptic mice however did show the presence of high amplitude spikes within ripples. We are currently conducting these recordings in non-anesthetized mobile mice running on a spherical treadmill. Understanding the synaptic mechanisms underlying these rapid coherent rhythms will give insight into the pathogenesis of the epileptic state, and may give us insights into new forms of treatment.
| Lindsey Sharpe
Mentor: Andrea M. Kasko
Title: Saccharides as a Viable Alternative to Peptides in Mediating Cell Adhesion
The role of the extracellular matrix (ECM) in cell growth and adhesion is a primary concern in tissue engineering. A variety of materials can promote cell adhesion. For example, both ECM proteins and peptide fragments bind to integrin receptors on cell surfaces, and therefore are often incorporated into cell scaffolds to promote cell adhesion. As an alternative, we propose the incorporation of saccharide residues may promote cell adhesion in synthetic ECMs, either directly or indirectly. Sugar residues may bind directly to cell surface receptors such as selectins, CD44 or CD168. Indirectly, sugar residues may be able to bind to ECM proteins that interact directly with cell surface receptors. In addition to promoting cell adhesion, the incorporation of monosaccharides into hydrogel scaffolds may increase water content and mechanical properties. Although oligosaccharide synthesis can be challenging, the incorporation of monosaccharide-containing macromers into hydrogels is relatively easy. In this investigation, 6-O-acryloxy galactopyranose was synthesized and incorporated into polyethylene glycol hydrogels in various concentrations. The effect of composition on the volumetric swelling ratio and complex modulus was quantified. Fibroblasts were seeded onto these scaffolds. The cells adopt an extended morphology, and cell adhesion qualitatively increases with increasing galactose content. These results suggest that the incorporation of sugars may be a viable alternative to peptides to promote cell adhesion to artificial ECMs.
| Shayne Siegman
Mentor: Tatiana Segura
Title: Design and Characterization of mu-Porous Hyaluronic Acid Hydrogels for in vitro Gene Transfer to Mouse Mesenchymal Stem Cells
The ability to locally deliver DNA sequences to cells in both in vitro and in vivo environments is a promising technique that can be used in gene therapy and regenerative medicine. Delivery of naked DNA has been successfully accomplished but is limited by low levels of transfection efficiency, therefore the development of DNA/polymer nanoparticles is one proposed solution to such a problem. One approach to delivering DNA nanoparticles is through their encapsulation within hydrogel scaffolds. Traditionally, hydrogels contain nano-sized pores that impede cell migration and require degradation by the cell in order for migration to occur. We proposed the use of hyaluronic acid hydrogels containing mu-sized interconnected pores that allow for cellular migration and easier access to encapsulated DNA nanoparticles for higher and more sustained transfection. In this study, we examine the effects of pore size and DNA concentration on gene transfer and expression kinetics. We optimized a previously developed sphere-templating method to produce 30, 60, and 100 micrometer pore size hydrogels and found that pore size affected gene expression while having no significant effect on cell viability. Gaussia luciferase reporter plasmid was used to confirm transfection up to 10 days and a live/dead cell viability kit was used to assess toxicity. We believe that the knowledge gained through this in vitro model can be utilized to design and better predict scaffold-mediated gene delivery for local gene therapy in an in vivo model.
| Shanna Swartwood
Mentor: Bennett G. Novitch
Title: Functional Characterization of Hypaxial Motor Neurons and the Formation of Respiratory Motor Circuits in Mammalian and Avian Species
Motor neurons (MNs) serve as the final effectors of body movement. In vertebrate embryogenesis, spinal MNs arise from a pool of ventral progenitor cells and then differentiate into a variety of specialized subtypes, each with unique molecular properties, cell morphologies, and muscle targets. The development and organization of several MN classes have been well characterized, such as the MNs innervating the limbs and back muscles. However, MNs involved with respiratory function remain poorly defined. Using immunohistochemistry and retrograde tracing techniques in both mouse and chick, we identify a near-contiguous population of MNs spanning the cervical, thoracic, and lumbar segments of the spinal cord which innervate muscle groups directly affecting inflation and deflation of the lungs. Interestingly, despite the highly divergent respiratory systems found in mammalian and avian species, we find that all respiratory MNs share a common set of molecular, topographic, and dendritic arborization patterns reminiscent to the Hypaxial Motor Column (HMC). These observations suggest an evolutionarily conserved mechanism may be driving respiratory motor circuit formation across vertebrate species, and opens up the possibility of using different animal models, such as chick, for studying respiratory motor function with relevance to human health and disease.
| William Temple
Mentor: Benhur Lee
Title: Human Tolerogenic Dendritic Cells Induced by Galectin-1 Promote FoxP3+ Regulatory T Cell Differentiation and Suppress T Cell Proliferation
Dendritic cells (DCs) are sentinels of the immune system that regulate immune activation and immune tolerance. Galectin-1, a known immunoregulator and modulator of dendritic cell activity, is present in peripheral tissues at sites of monocyte to dendritic cell differentiation. Thus, we asked if galectin-1 exposure induces DCs to have tolerogenic function. Here, we show that DCs differentiated in the presence of galectin-1 produced tolerogenic DCs. Similar to classical tolerogenic DCs, galectin-1 conditioned tolerogenic DCs suppressed immune responses by downregulating costimulatory molecule CD86, secreting anti-inflammatory cytokine IL-10, and inducing FoxP3+regulatory T cells. Furthermore, these galectin-1 differentiated DCs retained two of the central functions of DCs, exhibiting high phagocytic ability and enhanced migratory capability. Interestingly, flow cytometric analysis revealed that galectin-1 conditioned DCs could suppress T cell proliferation in a tolerogenic DC-T cell contact autonomous manner. Further analysis of galectin-1 induced tolerogenic DCs holds therapeutic potential for autoimmune diseases and transplant rejection pathologies that involve an overactive adaptive immune response.
| Shivani Thaker
Mentor: Utpal Banerjee
Title: Oncogenic Activation and Mitochondrial Component Inactivation Leads to Increase in Lactate Dehydrogenase Enzyme Activity and Up-regulation of LDH Gene in Drosophila
While cells normally break down glucose depending on oxygen availability and undergo oxidative phosphorylation with a high ATP yield in normoxic conditions, cancer cells are unique in they undergo the typically anaerobic pathway of glucose metabolism, regardless of the presence of oxygen. This phenomenon is known as the Warburg effect, or "aerobic glycolysis," resulting in lactate production and a low ATP yield. A key enzyme participating in the final step of the aerobic glycolysis pathway is lactate dehydrogenase (LDH). We used LDH as an indicator of a cell's metabolic shift to aerobic glycolysis and studied the effect of oncogenic activation and inactivation of mitochondrial genes on LDH activity and expression. Using a LDH activity histochemical stain, we observed that cells expressing various oncogenes show increased LDH enzyme activity and used a GFP enhancer trap to show that this increase was due to differential LDH up-regulation. Cells with various inactivated mitochondrial genes also showed LDH up-regulation. We used clonal analysis to show that LDH up-regulation was hypoxia-independent and most of the oncogenic backgrounds showed cell autonomous LDH up-regulation. Establishing a model for studying cancer cell metabolism in Drosophila with its unique genetic reagents can provide a useful vehicle for further study of mechanisms behind the Warburg effect. Interfering with the unique manner of glucose metabolism in cancer cells may have important implications in possible treatments to specifically target these cells.
| Elizabeth Wang
Mentor: Michael S. Levine
Title: Increased AMPA Receptor-Mediated Synaptic Activity in a Mouse Model of Schizophrenia
A common mutation in the Disc1 (Disrupted-in-Schizophrenia-1) gene occurred in a Scottish family with a high rate of schizophrenia. The mutation results in the expression of a truncated Disc1 protein, which may confer susceptibility to psychiatric disease. Studies in humans and rodent models of schizophrenia have also shown concomitant disruptions in neurotransmitter systems such as glutamate and GABA. In particular, NMDA receptor function is reduced. Here, we examined if mutant human Disc1 (hDisc1) expression alters glutamatergic and GABAergic synaptic transmission in the medial prefrontal cortex in mice. Whole-cell patch clamp recordings of pyramidal neurons from mice expressing the truncated hDisc1 protein were compared to those of wildtype (WT) mice. Cells from mutant hDisc1 males recorded at -70 mV, in the presence of a GABA receptor antagonist, showed a significant increase in the frequency of small-amplitude AMPA-receptor mediated spontaneous excitatory postsynaptic currents (sEPSCs) compared to WT cells. In contrast, cells from mutant and WT males showed similar average frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs). No differences in sEPSCs and sIPSCs were seen in cells from mutant females. Our findings indicate that mutant hDisc1 expression in male mice increases glutamatergic synaptic activity and this effect is gender-specific. We propose that increased AMPA receptor function may compensate for deficits in NMDA receptor function in this model. Future research should explore this avenue.
| Catherine Yao
Mentor: Hanna K. A. Mikkola
Title: Defining the Transplantable Human Hematopoietic Stem Cell with Specific Cell Markers
Hematopoietic stem cells (HSCs) are blood tissue-specific stem cells with unique properties of multipotency, self-renewal, and engraftment ability. They are used successfully in transplant treatments of many blood disorders such as leukemias and immune deficiencies. Because of the limited availability of HLA-matched donors, there is great need for the expansion of engraftable human HSCs in culture to supplement transplant therapies. However, inadequate knowledge of the definitive human HSC phenotype is a significant barrier to understanding HSC self-renewal and recapitulating these functions ex vivo. To address these issues, our goal is to identify cell markers that isolate engraftable human HSCs for further study of the mechanisms that regulate expansion. Through gene expression analysis, we identified a novel surface marker GPI-80 that isolates a subpopulation of HSPCs that can differentiate into various blood cell types in colony-forming assays, as well as self-renew in co-cultures. During in vivo engraftment assays in irradiated mice, only GPI-80+cells display reconstitution ability, indicating that GPI-80 marks a population further enriched for human HSCs than any other cell marker to date. We are performing shRNA knockdown assays of GPI-80 to study the role of this protein in HSC function. These studies give insight into the cellular mechanisms of unique HSC properties and necessary signaling pathways required to expand HSCs in culture for therapeutic purposes.
| Mary Youssef
Mentor: Paul Mischel
Title: Molecular Inhibitor Fatostatin Reduces Glioblastoma Cell Proliferation and Viability
Glioblastoma (GBM) is one of the most deadly cancers, with an average patient survival rate of 12-18 months after diagnosis despite surgery, radiation and chemotherapy, making it increasingly urgent to find new molecular targets and effective drugs. Recently, The Cancer Genome Atlas identified that RTK/PI3K/Akt signaling is activated in about 88% of GBM; this pathway has been recognized as a major driver in promoting malignant cancer cell growth in several cancers. Recently, our group demonstrated that RTK/PI3K/Akt signaling promotes GBM cell growth through sterol regulatory element-binding protein 1 (SREBP-1) mediated fatty acid synthesis, and identified SREBP-1 as a potential molecular target. The purpose of this research is to select effective lipid synthesis inhibitors, and to test their anti-cancer function. Small molecular inhibitor fatostatin was shown to effectively inhibit lipid synthesis. It is promising to test its anti-cancer function. Proliferation assays and cell counts indicate that fatostatin treatment inhibited cell proliferation and increased cell death in a dose-dependant manner. These data suggest that alteration of lipid and cholesterol homeostasis through pharmacological inhibitors can suppress glioblastoma growth, providing a new avenue for treating GBM.
| Carl Yu
Mentor: Julio L.Vergara
Title: Electrophysiological and Optical Comparion of ClC-1 Conductance and Localization in Normal and Myotonic Skeletal Muscle Fibers
The actual density of chloride channels (ClC-1 channels) in the surface and transverse tubular system (TTS) membranes of mammalian skeletal muscle fibers is still unknown. The hypothesized localization of ClC-1 channel among the two membrane compartments has been controversial. However, characterizing the properties of ClC-1 channels and determining the density of ClC-1 channels in each membrane compartment of normal and myotonic mice (HSALR) is important as it can provide insights about the disease. Therefore, a combination of electrophysiological and optical techniques was used to obtain data pertaining to the physiological and pathophysiological properties of ClC-1 channels in normal (C57BL) and HSALR, respectively. In the experiments, fluorescence signals and membrane potentials were obtained from enzymatically-dissociated flexor digitorum brevis (FDB) muscle fibers and interossei (IO) of control and myotonic mice. The fibers were stained with the potentiometric dye di-8-ANEPPS, and voltage-clamped with a two-microelectrode technique to stimulate and record the membrane potential and change in fluorescence. Subsequently, the comparison of electrical and optical data obtained from normal and HSALR fibers showed that HSALR fibers have depressed chloride conductance; and the presence of attenuating optical signal in both animals indicate that there are ClC-1 channels located in the transverse tubules. Consequently, a mathematical model can then utilize the data to determine the pathophysiological properties of myotonia.
| Madeline Yung
Mentor: Jeffrey H. Miller
Title: Identification and Characterization of a Novel DNA Repair Protein in Bacillus Anthracis
Mutations in the genome result in a wide range of phenomena, from natural selection to the development of cancer. In bacteria, DNA mutagenesis is responsible for the growing concern of antibiotic resistance in modern healthcare. However, many of the pathways by which mutations occur in bacteria, even in the anthrax-causing bacterium Bacillus anthracis, remain unknown. Elucidation of these pathways is vital to controlling anthrax's lethality and diminishing its potential as a bioweapon. While searching for new DNA repair genes in B. anthracis, we discovered that disruption of the gene BAS4289 resulted in a much higher mutation rate than in wild-type. Further mutation assays showed that BAS4289 knockout mutants have a mutational footprint similar to that of mutS mutants. MutS codes for a DNA repair gene involved in the DNA mismatch repair pathway, which corrects base substitution and frameshift mutations. Finally, sequence homology shows that this gene codes for a 5' to 3' DNA helicase. Its closest relative, a recD gene in Escherichia coli, aids in homologous recombination. However, the BAS4289 mutational spectrum and its additional uncharacterized N-terminal region suggest that this gene codes for a protein involved in mismatch repair rather than homologous recombination. The different functions of these two homologous proteins suggest a mechanism for the evolution of DNA repair in bacteria. Characterization of DNA repair pathways sets the foundation for the development of techniques to combat antibiotic resistance.