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We hypothesize that the loss of miR-137 in colon cancer results in an overexpression of oncogenes, such as Msi1, contributing to colon cancer initiation and progression. Using miRNA-prediction programs PicTar and TargetScan, we identified predicted targets of miR-137. Colon cancer cell line HCT-116 was transfected with miR-137 mimic for 24 to 48 hours and total RNA was isolated. RNA was then converted to cDNA using reverse-transcription PCR and cDNA expression of our genes of interest was measured using quantitative real-time PCR (qRT-PCR). By identifying additional miR-137 targets, we hope to gain a better understanding of colon cancer etiology. Additionally, understanding the role of miRNAs such as miR-137 is important to developing miRNA-based molecular therapy, deliverable to tumors via Dr. Xu’s patented nanoparticle.
THE ROLE OF MTORC1 SIGNALING IN SPERMATOGONIAL STEM CELLSMaria Montoya, Brian Hermann.
University of Texas at San Antonio, San Antonio, TX.
Mammalian target of rapamycin complex 1 (mTORC1) is a multisubunit protein kinase complex that senses the cellular environment in order to determine whether conditions are optimal for protein synthesis and cell proliferation.
There is evidence that suggests mTORC1 may also play a critical role in the self-renewal versus differentiation-fate decision in spermatogonial stem cells (SSCs). SSCs are the unipotent stem cells responsible for sperm production in the testis and male fertility. This project will investigate the effects of inhibiting mTORC1 in cultured SSCs. We hypothesize that the SSC population will be depleted due to uncontrolled stem cell differentiation when mTORC1 signaling is inhibited. To test this hypothesis, we will inhibit mTORC1 in SSCs using RNA interference (knockdown) of RAPTOR, the substrate recognition component of mTORC1. For this purpose, we will optimize mouse RAPTOR knockdown using short-hairpin RNAs. Currently, we are cloning an expression plasmid that will contain the entire mouse Raptor cDNA, which we will use to express RAPTOR protein by transient transfection into human cells.
Cotransfection of this expression construct with knockdown plasmids followed by RAPTOR western blot analysis will optimize RAPTOR knockdown. We anticipate that this approach will confirm which knockdown target efficiency depletes RAPTOR and allow us to proceed to test the consequences of mTORC1 loss in SSCs. Ultimately, this experiment will determine whether mTORC1 signaling is required for normal SSC fate determination and, consequently, confirm its role in spermatogenesis and male fertility.
PEDF GENE THERAPY USING BONE MARROW-DERIVED MACROPHAGES: A NEW MEAN FOR TREATING
PROSTATE CANCERDalia Martinez-Marin, Thomas Nelius, Megan Howard, Stephanie Filleur.
Texas Tech University Health Sciences Center, Lubbock, TX.
Macrophages have been described as one of the main inflammatory components involved in prostate cancer (PCa) initiation, progression, and metastases. PEDF (pigment epithelium-derived factor) is an antiangiogenic factor with differentiation activities and was recently suggested as an immune-modulating factor. PEDF expression has been shown to be downregulated in PCa compared to normal tissues. In previous studies we demonstrated that PEDF reexpression in PCa cells curbs tumor growth in vivo and prolongs the survival of tumor-bearing mice. More recently, we have showed that PEDF induces the migration of macrophages and stimulates their differentiation towards a tumor-cytotoxic phenotype leading to phagocytosis of tumor cells. While these results emphasize on PEDF anti-tumor properties, the delivery of PEDF still remains challenging. The objective of the present study is to investigate PEDF gene therapy using bone-marrow derived macrophages (BMDMs) as a novel therapeutic modality for advanced PCa. Our hypothesis is that PEDF expression will induce the migration and differentiation of BMDMs into a tumorcytotoxic phenotype, block tumor growth and metastases formation, and prolong survival. To validate this hypothesis, we are isolating BMDMs from C57BL/6 mice. After differentiation, macrophages are treated with or without PEDF, or transduced with PEDF or control lentiviruses, then analyzed for tumor-promoting or tumor-cytotoxic differentiation.
In the present proposal, we will use the transgenic adenocarcinoma of the mouse prostate and the LNCaP-derived CL1 orthotopic xenografts to investigate the effect of PEDF expression by BMDMs on tumor growth and survival. This project may lead to development of improved therapeutic approaches for treatment of prostate cancer.
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EVALUATION OF THE TOXICITY OF VIRAL VACCINES ON THE CENTRAL NERVOUS SYSTEM OF RAINBOWTROUT Erin Larragoite, Luca Tacchi, Irene Salinas.
University of New Mexico, Albuquerque, NM.
Vaccines must be innocuous or cause minimal side effects to humans and animals. Many viruses have a tropism for the central nervous system (CNS). Viral vaccine formulations may consist of a live, attenuated viral strain that can potentially reach the CNS. Since the 1970’s, fish vaccines have been widely used to control aquatic diseases.
The infectious hematopoietic necrosis virus (IHNV) is known to cause fatal infections in salmonids worldwide and to have neurotropic manifestations. The aim of this study is to evaluate the safety of a live, attenuated IHNV vaccine in rainbow trout delivered by intramuscular injection (i.m.) or intranasally (I.N.). Rainbow trout were vaccinated once (trial
1) I.N. or i.m. with the IHNV vaccine or phosphate buffer saline (PBS). Trout received a booster vaccination 28 days after primary immunization in trial 2. Five fish from each group of both trials were sampled 1, 4, 7, 14, 21, and 28 days post immunization (dpi). Brain tissue samples were collected and the presence of IHNV was detected by RT-qPCR.
Three i.m. trout 4 dpi and 1 i.m. trout 21 dpi tested positive for IHNV in the first trial while 1 i.m. and 1 I.N. specimen tested positive for IHNV 4 dpi and 28 dpi, respectively. Presence of IHNV correlated with expression of IL-1B in the brain. Nasal vaccination appears to be a safer vaccination route than injection in our IHNV trout model. Future studies will develop nonlethal toxicity tests using magnetic resonance imaging (MRI) to track vaccine fate in the central nervous system.
DIRECT INTERACTION BETWEEN DISHEVELLED AND DISCS LARGE INDUCES MICROTUBULE
POLARIZATION IN DROSOPHILAJoshua Garcia, Christopher Johnston.
University of New Mexico, Albuquerque, NM.
Asymmetric cell division, which generates cellular diversity, requires properly regulated cell polarity. Cell polarity is essential for proper development of adult tissues, and human diseases such as cancer have been linked to defects in cell polarity. The mechanisms involved in cell polarity remain incompletely described. Two proteins that influence cell polarity in a range of cellular systems include Dishevelled (Dsh) and Discs large (Dlg), which are thought to induce cell polarity partly by regulating microtubule stability. Adenomatous polyposis coli (APC), a tumor suppressor protein, interacts with Dsh and Dlg, enhancing microtubule stability. This suggests a possible interaction between these proteins even though a direct functional interaction between Dsh and Dlg has not yet been identified. We have identified an interaction between the PDZ domain of Dsh (DshPDZ) and an internal Hook domain of Dlg (DlgHook).
We hypothesize that a functional protein complex forms between Dsh-Dlg accompanied by APC that is crucial for microtubule polarization. The objective of this project is to define the molecular basis for the Dsh-Dlg interaction and analyze its role in microtubule dynamics. We will first examine binding of DshPDZ to a series of DlgHook truncations to determine the minimal binding sequence. Additionally, we will determine the X-ray crystal structure of the Dsh-Dlg complex to define the molecular basis of this interaction. Lastly, we will examine the function of the Dsh-Dlg complex in regulating microtubule polarization in cultured Drosophila cells. Overall, we look to identity the binding site and the function associated with this complex.
DEFINING MOLECULAR MECHANISMS THAT LINK OBESITY WITH BREAST CANCER AGGRESSIVENESSElizabeth Fisher, Ignacio Camarillo.
Purdue University, West Lafayette, IN.
Breast cancer in women is the second leading cause of cancer death in the United States. Obesity (body mass index of 30) is associated with increased breast cancer risk and morbidity. The molecular mechanisms leading to tumor formation and progression are not yet fully understood. Our experiments focus on defining protein markers that play a role in epithelial to mesenchymal transition (EMT), a process by which epithelial cells become invasive and can acquire stem-like properties. Toward this goal, expression of adiponectin receptors (AR1 and AR2) and EMT markers in mammary tumor tissue of lean and obese female Spraque-Dawley rats, both on high fat diets, were evaluated through western blots. Obese rats predominately developed invasive tumors, verified by histology. The EMT markers quantified include E-cadherin, a protein responsible for cell-cell contact, and N-cadherin and vimentin, markers that indicate that cells have entered a mesenchymal state. In brief, our results reveal an increase in expression of
STRUCTURAL AND SEQUENCE ANALYSIS OF A BACTERIOPHYTOCHROME FROM THE MYXOBACTERIUM
STIGMATELLA AURANTIACAKevin D. Gallagher, Hardik K. Patel, Nicole C. Woitowich, Svetlana E. Kovaleva, Rachael St. Peter, Wesley B.
Ozarowski, Cynthia N. Hernández, Aaron E. Schirmer, Emina A. Stojkovic.
Northeastern Illinois University, Chicago, IL.
Bacteriophytochromes (BphPs) are red-light photoreceptors found in various photosynthetic and nonphotosynthetic bacteria. They are unique in their ability to undergo reversible photoconversion between spectrally distinct red (Pr) and far-red (Pfr) light absorbing forms. They are composed of a photosensory module covalently linked to an effector domain, which is usually a histidine kinase. BphPs require biliverdin (BV) as an organic cofactor for photoactivity.
We recently solved the crystal structure of a BV-bound BphP from a nonphotosynthetic myxobacterium, Stigmatella aurantiaca (SaBphP1). Unlike classical BphPs, SaBphP1 undergoes limited Pr/Pfr photoconversion. The complete photoconversion of SaBphP1 can be restored by mutating threonine (Thr289) to a histidine (His) in the photosensory module. In classical BphPs, the conserved His stabilizes the D-ring of BV in the Pr state. Based on structural analysis of SaBphP1 in the wild-type and mutant (Thr289His) forms, we propose that Thr is too far away to form a hydrogen bond with the D-ring of BV resulting in the partial Pr-Pfr photoconversion of the wild-type protein. When Thr is converted to His in SaBphP1 (Thr289His) mutant, increased photoactivity is observed due to the ability of His to hydrogen bond with the D-ring. In related BphP’s, RpBphP2 and RpBphP3 from R. palustris, mutating conserved His to Thr abolishes photoconversion. Mutations were also introduced to highly conserved residues (Asp208His and Tyr262Phe) in SaBphP1 that stabilize BV by forming hydrogen bonds. Our structural and sequence analyses of SaBphP1 and related BphPs from R. palustris, RpBphP2 and RpBphP3, emphasize the importance of His289 interactions with BV for complete Pr/Pfr photoconversion.
PH SENSITIVE REGULATION OF YEAST TORC1: THE ROLE OF V-ATPASEDustin Martinez, Karlett Parra.
University of New Mexico, Albuquerque, NM, 2University of New Mexico School of Medicine, Albuquerque, NM.
1 Vacuolar proton ATPase (V-ATPase) is a membrane protein complex that uses energy from ATP hydrolysis to pump protons into organelles against a concentration gradient. V-ATPase can affect cell function directly through its proton pumping activity or indirectly by regulating the activity of other proteins. It is known that V-ATPase activity is necessary for activation of the target of rapamycin complex 1 (TORC1), which is a major regulator of cell growth. However, it is unknown exactly how V-ATPase activates TORC1. We propose using budding yeast as a model system to study the V-ATPase-TORC1 axis. We showed that the yeast TORC1 activity is compromised in V-ATPase mutants grown at pH 6 or above, whereas it is normal when cells are grown under more acidic conditions. This result suggests that the extracellular pH interplays with V-ATPase to regulate TORC1. We hypothesize that cellular pH homeostasis controls TORC1. It is our aim to determine whether cytosolic or vacuolar pH regulates TORC1. We are using fluorometric assays employing BCECF, a pH-sensitive fluorescent dye, and pHluorin, a pH-sensitive green fluorescent protein, to measure yeast vacuolar and cytosolic pH respectively. The TORC1 activity will be evaluated by measuring phosphorylation of Sch9p, a direct TORC1 substrate. The correlation between the TORC1 activity and the vacuolar or cytosolic pH will be determined in wild-type and V-ATPase mutant cells that are grown in medium buffered to various pHs, ranging from pH 4.0 to pH 6.0.
IDENTIFICATION OF LEGIONELLA PNEUMOPHILA EFFECTOR PROTEINS THAT BIND ON HUMAN RAB5PROTEIN Nikki Jamie Cruz, Mary-Patricia Stein.
California State University Northridge, Northridge, CA.
Legionella pneumophila is a Gram-negative bacterium that normally lives in a fresh-water amoeba. Inhalation of aerosolized water containing Legionella pneumophila can cause severe pneumonia in immunocompromised
59 UNDERGRADUATE POSTER ABSTRACTS