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«Strengthening the Nation through Diversity, Innovation & Leadership in STEM San Antonio,Texas · October 3-6, 2013 Get Connected! Connect with the ...»

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MeHg exposure elevates the levels of intracellular Ca2+ concentration ([Ca2+]i) in two phases. Phase 1 consists of Ca2+ released from intracellular storage organelles, such as the endoplasmic reticulum and mitochondria. Phase two (P2) results from an influx of Ca2+ through ion channels. Investigators have expressed that cell death is preceded by elevated levels of [Ca2+]i explaining how MeHg ultimately can cause cell death. However, the application of Ca2+ channel antagonists slows the onset of P2 and cell death following MeHg exposure, suggesting expression of Ca2+permeable membrane proteins makes cells susceptible to MeHg-induced cytotoxicity. In this study, we will investigate how in vitro MeHg exposure of F11 cells affects the RNA expression of high-voltage Ca2+ channels, low-voltage Ca2+ channels, and transient receptor potential ankyrin 1 through RT-PCR assay. The reason to use this cell line is to immortalize the gene products of single neurons that are sensitive to MeHg. Under those circumstances, changes in RNA expression may suggest which Ca2+-permeable ion channels make DRG-like neurons more susceptible to the neurotoxicity of MeHg.

PHARMACOLOGY

SAT-627

RAC1 INHIBITION AMELIORATES OBSERVED COGNITIVE DEFICITS IN FMRP-DEFICIENT FEMALE MICE

Stacy Nguy, Maria V. Tejada-Simon.

University of Houston, Houston, TX.

Identifying the causes and pathogenic mechanisms of neurodevelopmental diseases such as fragile X syndrome (FXS) is complex. It has been reported that, in FXS, there is an unusually high density of long, thin, immature dendritic spines accompanied by aberrant long-term plasticity. Our lab has recently demonstrated that, in the brain of FXS mice, there is an over-activation of Rac1, a protein of the Rho GTPase subfamily implicated in neuronal and dendritic spine development. This mouse model also exhibits alteration of synaptic plasticity and deficits in cognition. Since this particular disease is linked to the X chromosome, most of the existing studies, including ours, make use of male knock-out mice. Thus, this study was undertaken to provide analogous studies in FXS female mice. Expression of Rac1 levels was determined in Fmr1-KO female mice. Furthermore, Fmr1-KO female mice were treated with a Rac1 inhibitor and subjected to behavioral and morphological studies to determine whether 1) similar alterations to the ones observed in males exist, and 2) modification of the levels of Rac1 could rescue these deficiencies. We observed that pharmacological inhibition of Rac1 indeed ameliorates cognitive deficits observed in untreated Fmr1-KO females, as measured in a fear-conditioning paradigm. This suggests that Rac1 activity may contribute to FXS etiology in both male and female and that Rac1 could be involved in the mechanism responsible for the deficient neuronal morphology, perturbed synaptic plasticity, and cognition impairment associated with this disorder.

145 UNDERGRADUATE POSTER ABSTRACTS

SAT-610

CHARACTERIZING A NEW PATHWAY IN NUCLEAR QUALITY CONTROL

Joshua Sanchez, Richard Gardner.

University of Washington, Seattle, WA.

Proteins have a three dimensional structure that allows for their specific activity and function. While a protein’s structure is usually stable, it can be damaged by physical and/or chemical stresses. Such damage results in misfolding and is often accompanied by exposure of hydrophobic residues. Exposed hydrophobicity can lead to the aggregation of misfolded proteins, which is associated with many prominent neurodegenerative diseases. To avoid these deleterious consequences, cells have evolved protein quality control (PQC) systems that degrade misfolded proteins. In eukaryotes, PQC utilizes ubiquitin ligases to mark proteins with ubiquitin for proteasome degradation. By studying the yeast PQC ubiquitin ligase San1, which is found in the nucleus, we discovered that San1 recognizes misfolded proteins with exposed hydrophobicity minimally equal to 5 contiguous hydrophobic residues. Interestingly, we found that there is another PQC degradation system that recognizes exposed hydrophobicity at a lower threshold than San1. In this study we are trying to understand how this new pathway functions. We took multiple constructs with varying degrees of exposed hydrophobicity to define how broad the substrate specificity for the pathway is.

Intriguingly, we found 2 separate pathways, 1 that was ubiquitin dependent and 1 that was ubiquitin independent.

Importantly, we found that the ubiquitin dependence changed within two similar constructs that differed by a 24 amino acid deletion. We are exploring this sequence to identify which region defines ubiquitin dependence and independence. Through this study, we hope to gain a better understanding of PQC degradation systems and their methods to preventing severe pathologies.

SAT-611

ANALYSIS OF MITOCHONDRIAL HAPLOTYPES, APOE, AND TOMM40 GENOTYPE WITH ALZHEIMER’S

DISEASE Marcus Florez, Elias Michaelis, Xinkun Wang.

University of Kansas, Lawrence, KS.

Mitochondrial dysfunction and decline is well known to be a prominent event in the development of Alzheimer’s disease (AD). It has also been strongly suggested that both nuclear and mtDNA variations contribute to mitochondrial dysfunction and AD pathology. Inheritance of the nuclear DNA variant of APOE, the APOE e4, has been known to be a leading risk factor in AD. The TOMM40 gene, also located on chromosome 19, has been recently associated with AD.





Cybrid evidence strongly suggests a role for mt genes in the development of AD. However, previous studies that have investigated mtDNA variation and the risk of AD have gotten mixed results. The objective of this study is to examine the TOMM40, APOE, and haplogroup status of AD and non-AD patients in order to gain a better understanding of the genetic factors that influence AD pathology. This seems to be the first study to assess the TOMM40, APOE, and haplogroup status in association with developing AD. Thus far, primer/probe sets were developed to genotype 8 single nucleotide polymorphisms that distinguish nine common European haplotypes. Using real-time PCR, the haplogroup of 218 DNA samples from AD and non-AD patients were determined. The haplotype data will be combined with genotyping data for APOE and TOMM40, and statistical analysis will soon be used to assess the risk of AD, severity of AD, and age of onset of AD with respect to haplogroup status, sex, and APOE and TOMM40 genotype.

FRI-610

A LIVE-CELL REPORTER THAT DIFFERENTIATES BETWEEN QUIESCENT AND CYCLING CELLS

Lindsay Henderson1, Brian Rasnow2, Curtis Lim1, Chris Shumate2, Alexander Zambon1.

University of California, San Diego, La Jolla, CA, 2Etaluma Inc., Carlsbad, CA.

1 One approach to stem cell biology’s current limitation to generate sufficient yields of therapeutic target cells is to direct the proliferative expansion of desired cell types after they commit to a specific lineage. Our lab focuses on targeting signaling pathways that trigger cardiac myocyte (CM) proliferation during heart development. Fetal CMs proliferate until the perinatal period where they become arrested in G0/G1. This study’s objective was to generate a fluorescent-based genetically encoded reporter capable of differentiating between arrested and actively cycling CMs. The Ki67 promoter (Ki67p) previously developed by our lab can distinguish proliferating from quiescent cells.

However, using Ki67p to drive GFP as a reporter gene results in fluorescent daughter cells due to GFP’s long half-life.

We hypothesized using Ki67p to drive expression of the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system would enable detection of quiescent (G0) CMs and CMs in G1/S and S/G2/M. The promoter driving the existing FUCCI reporters is constitutively active and cannot distinguish quiescent from proliferating cells that have entered G1.

–  –  –

PHYSIOLOGY/PATHOLOGY

FRI-615

EFFECTS OF MATERNAL TOBACCO SMOKE EXPOSURE ON ESTROGEN SERUM LEVELS AND SIGNALING

IN RAT ADIPOSE TISSUE

Danielle Holliday, Isis Trevenzoli, Lisa Joss-Moore.

University of Utah, Salt Lake City, UT.

Despite anti-smoking campaigns, the prevalence of pregnant smokers remains high, reaching 18% in the United States and 40% among Native Americans. Maternal tobacco smoking (MTS) results in obesity, specifically among adult male offspring. Obesity is associated with several co-morbidities and adipose tissue dysfunction. Adipose tissue functions are regulated by estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Lipoprotein lipase (LPL) and perilipin are target genes for ERs. We hypothesized that MTS will alter serum estrogen levels, ERα and ERβ abundance, and LPL and perilipin mRNA levels in rat offspring adipose tissue in a sex and adipose depot specific manner. To test this hypothesis, pregnant Sprague Dawley rats were exposed to tobacco smoke or room air from embryonic day 11 until birth (MTS and control groups). The MTS pups were cross-fostered to control dams at birth and both offspring were raised until weaning (postnatal day 21), when they were killed to collect serum and visceral and subcutaneous adipose tissue samples. ELISA, western blotting and real time RT-PCR will be used to determine the serum estrogen, ERα and ERβ abundance, and LPL and perilipin mRNA levels, respectively. Our data thus far show MTS decreased serum estrogen levels in both male offspring (-30%, p 0.05) and female offspring (-29%, p 0.05). We speculate that the decrease of serum estrogen levels may be associated with obesity development in the MTS model. We further speculate that the ER abundance will be decreased only in adipose tissue of male offspring, which is consistent with fat accumulation.

SAT-614

HISTONE MODIFICATIONS ON WNT-RESPONSIVE AND STEM CELL SELF-RENEWAL GENES

Julio Silva1, Susan Krum2, Gustavo Miranda-Carboni3.

David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA, 2Orthopaedic Hospital 1 Research Center, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, 3Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA.

The Wnt10b/β-catenin/Hmga2 axis has been shown to be active in several subtypes of triple-negative breast cancers (TNBC), such as mesenchymal stem-like (MSL), basal-like (BL), and is capable of predicting significant survival outcome. HMGA2 can predict metastasis. However, the role of Wnt10b/β-catenin/Hmga2 and the underlying mechanism(s) for the aggressiveness of TNBC remain unknown. We found that WNT10B/β-catenin signaling is deregulated in human TNBC and leads to upregulation of HMGA2. The Wnt inhibitor ICG-001 disrupts CBP interaction with β-catenin, leading to loss of HMGA2-mediated proliferation of TNBC cells by disrupting the protein complex in the promoter regions of Wnt responsive elements (WRE). To investigate the mechanistic role of the β-catenin/ CBP complex in mediating potential changes to histone modifications on WREs, chromatin immunoprecipitation (ChIP) assays were performed in cells exposed to ICG-001. Using antibodies against histone H3, Histone 3 lysine 4 trimethylation (H3K4me3), Histone 3 lysine 27 trimethylation (H3K27me3), Histone 3 lysine 18 acetylation (H3K18Ac), and Histone 3 lysine 9 acetylation (H3K9Ac) in MDA-MB-231 cells exposed to ICG-001, revealed rules of a chromatin consistent with transcriptionally “closed” confirmation. Decreased changes of transcriptionally active chromatin markers near HMGA2, BMI-1, AXIN2, and c-MYC are consistent with observed loss of proliferation. The results suggest that ICG-001 interference of the β-catenin/CBP complex at WREs alters the histone modification, thus making the chromatin compact and transcriptionally inactive. Our findings may alter therapeutics for the highly metastatic TNBC tumors by specific inhibitors to chromatin modifications.

147 UNDERGRADUATE POSTER ABSTRACTS

FRI-619

THE THERAPEUTIC USE OF ATROPINE IN DIABETIC NEUROPATHY

Andre Mota, Nigel Calcutt.

University of California, San Diego, La Jolla, CA.

Diabetic neuropathy, a neurological complication of diabetes, is associated with significant rates of morbidity and mortality and affects both the somatic and autonomic divisions of the peripheral nervous system. Manifestations of diabetic neuropathy include sensory loss, pain, and autonomic dysfunction. Limited therapeutic approaches currently exist for diabetic degenerative neuropathy as pain management procedures do not address the roots of this diabetes-generated affliction. Thus, we aim to investigate the efficacy of approaches to treating diabetic neuropathy. Specifically, we will focus on the use of atropine, an alkaloid and commonly-used drug that affects the autonomic nervous system. To investigate the therapeutic potential of atropine in treating diabetic neuropathy, insulindeficient (type 1) diabetes was induced in mice via injection of streptozotocin (STZ), producing insulin deficiency and hyperglycaemia within days. By applying atropine to the eyes and feet of mice and later assessing cutaneous innervation in skin biopsies, we will be able to detect any differences in epidermal innervation by measuring intraepidermal and sub-epidermal nerve fibers (IENF and SNF). Corneal nerve fibers will also be measured throughout the study in live animals by corneal confocal microscopy (CCM). These assays will provide accurate, quantifiable information about degenerative neuropathy in the small sensory fibers innervating the skin and cornea. We predict that atropine will prevent onset of degenerative neuropathy in diabetic mice, supporting use of this drug as a possible therapeutic approach to the treatment of diabetic neuropathy.

SAT-616

PLATELET DYSFUNCTION IN A MURINE MODEL OF DIET INDUCED OBESITY

Kali Dale1, Trevor Fidler2, Dale Abel2.

University of Minnesota Morris, Morris, MN, 2University of Utah, Salt Lake City, UT.

1 Patients with type 2 diabetes mellitus (T2DM) and metabolic syndrome (MS) have increased thrombosis. This increase in thrombosis is thought to contribute to microvascular and macrovascular complications in diabetic patients.



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