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Wolfe Hall Room 3246 Saturday April 16, 2011
Back: Robert Rominski, Adam Rockacy
URS 2011 Participants
Middle: Robert Fischbach, Eric McCloskey, Allen Maertin, Isaac Perry, Alan Hammer
Front: Adelle Abouarrage, Torrie Klier, Lia Risk, Leigh Anne Autullo, Henrry Malodonado
URS 2011 Winners (L>R): Robert Rominski, Alan Hammer, Robert Fischbach
|8:00-8:25||Coffee, Juice, and Bagels|
|8:25-8:30||Welcome. Dr. William Taylor, Chair, Undergraduate Research Symposium and Dr. John Plenefisch, Associate Chair, Department of Biological Sciences.|
|8:30-8:50||Alexa Elliott, DEK Oncoprotein’s Regulation of NF-κB Activity and DNA Damage-induced Apoptosis. Advisor: Dr. Brian Ashburner, Department of Biological Sciences.|
|8:50-9:10||Kellie Andrews, Menin Regulation of Metabolic Reactions in the Gut. Advisor: Edith Mensah-Osman, Department of Physiology and Pharmacology|
|9:10-9:30||Allen Maertin, Neuronal survival and axonal guidance on the surface of a nanostructure. Advisor: Dr. Guofa Liu, Department of Biological Sciences.|
|9:30-9:50||Eric Andrew McCloskey, Cloning, Expression, Purification, and Crystallization of the Casein Kinase Iε tau Mutant. Advisor: Dr. John Bellizzi, Department of Chemistry.|
Robert Rominski, Studies of Host-Virus Interaction for the Viral Hemorrhagic Septicemia Virus Phosphoprotein and Nonvirion Protein. Advisor: Dr. Douglas Leaman, Department of Biological Sciences.
|10:30-10:50||Adelle Abouarrage, The effects of immunosuppressive drugs on the effector functions of T-cells involved in multiple sclerosis. Advisor: Dr. Anthony Quinn, Department of Biological Sciences.|
|10:50-11:10||Torrie Klier, The Function of DEK in NF-kB Transcriptional Activation. Advisor: Dr. Brian Ashburner, Department of Biological Sciences.|
Henrry Maldonado, P21-Activated Kinase 1 (PAK1) and its Role at Focal Adhesions and Cell Spreading. Advisor: Dr. Maria Diakonova, Department of Biological Sciences.
|11:50-11:50||Alan D. Hammer, The Role of Prolactin and p21-Activated Kinase 1 (Pak1) in Breast Cancer Cell Proliferation, Adhesion, and Spreading. Advisor: Dr. Maria Diakonova, Department of Biological Sciences.|
|11:50-12:10||Leigh Anne Autullo, Essential regulatory regions of the Caenorhabditis elegans tissue attachment gene mua-1. Advisor: Dr. John Plenefisch, Department of Biological Sciences.|
|12:40-1:00||Lia Risk, Investigation of the role of a Divaricata type transcription factor in Zea mays. Advisors: Erich Grotewold, Department of Plant Cellular and Molecular Biology, The Ohio State University and Dr. John Gray, Department of Biological Sciences, University of Toledo|
|1:00-1:20||Robert T. Fischbach, The Interactions Between Pro-Apoptotic Signals and the Cell Cycle. Advisor: Dr. William R. Taylor, Department of Biological Sciences.|
|1:20-1:40||Adam Jason Rockacy, Exploring Rab11-FIP3 in Caenorhabditis elegans. Advisor: Dr. Robert Steven, Department of Biological Sciences.|
|1:40-2:00||Isaac Perry, Does the matrix receptor MUA-3 directly interact with intermediate filaments in Caenorhabditis elegans? Advisor: Dr. John Plenefisch, Department of Biological Sciences.|
|2:00-2:30||Awards Ceremony and Pictures|
DEK Oncoprotein’s Regulation of NF-κB Activity and DNA Damage-induced Apoptosis
Menin Regulation of Metabolic Reactions in the Gut
When ingesting food a variety of signals occur that help with the absorption of nutrients. When looking at diabetes and obesity any problems in the signaling pathways could be related to dysregulated nutrient utilization. GIP is responsible for insulin secretion from the pancreas in response to feeding based on the regulation of glucose. Menin is a protein that is found in humans as well as mice that is encoded by the MEN1 gene. One population of mice that was analyzed contained a knockout of a single allele of menin in the liver. The effects of the knockout in the liver and the differences in regular diet and high fat diet will be looked at by analyzing the expression of GIP, Menin, and FOXO1 in the gut. The other sample population of mice that were looked at were Wild type mice that were fed/fasted for different time periods. The expression of menin, GIP, TGFB, CeaCam1, and FOXO1 were looked at within the fasted mice, the 4 hour fed mice, and the 7 hour fed nice. The use of real time- polymerase chain reaction (RT-PCR) was used to determine expression. It was determined that menin mediates/regulates metabolic reactions in the gut in response to diet.
Neuronal survival and axonal guidance on the surface of a nanostructure
A lesion within the central nervous system (CNS) can lead to aberrant neuronal connectivity and possible paralysis. The lesion site inhibits migration of neurons and guidance of axons across it. Thus we proposed a mechanism for neurons and axons to cross the lesion site and possibly restore proper connectivity. We combined the complex fields of nanotechnology and neuroscience to help develop this mechanism. The matrix of the nanostructure is a gold coated, polycarbonate porous matrix. We show that the nanostructure is able to hold the axon guidance molecule, netrin-1. E15 mouse cortical cells also are able to adhere directly to the matrix and survive. The matrix when holding netrin-1 has a greater effect on axonal outgrowth. Netrin-1 conditioned neurons have significantly longer axons than the HEK conditioned neurons. The matrix with netrin-1 also plays a role in anti-apoptosis mediating cell survival. Neurons conditioned in netrin-1 media had less apoptotic cells than neurons conditioned in HEK media.
Cloning, Expression, Purification, and Crystallization of the Casein Kinase Iε tau Mutant
Studies of Host-Virus Interaction for the Viral Hemorrhagic Septicemia Virus Phosphoprotein
and Nonvirion Protein
Viral hemorrhagic septicemia virus (VHSv) is a highly contagious fish rhabdovirus responsible for large-scale fish kill offs worldwide. A new strain of VHSv, denoted IVb, has been identified recently in the Great Lakes region, potentially threatening the billion-dollar sport and farm fishing industries of the region. Fish infected with VHSv exhibit a variety of symptoms, including loss of swim bladder control and, sometimes, internal and external hemorrhagic lesions. We have proposed that, like other pathogenic viruses, VHSv has developed mechanisms to evade the host innate immune system and counteract the production of Type I interferons (IFN). To better understand the mechanisms used by the virus we have cloned its individual genes for use in cell-based studies of innate immune signaling. The studies described herein focus on the Phosphoprotein (P) and Nonvirion (NV) proteins that have been ectopically expressed in EPC and BF2 fish cell lines. Our data demonstrate that ectopic expression of P protein decreased IFN-Stimulated Gene (ISG) induction by IFN, implicating a role in regulating responses to this important cytokine. Our data also suggest that expression of P decreases IFN production in cells expressing activated components of the cellular virus response pathway. These data implicate P as a potential regulator of VHSv innate immune evasion. Previous publications on the NV protein suggested that it might be essential for viral pathogenicity. In our hands, ectopic expression of NV in EPC cells increased ISG promoter activation by IFN and suggested that expressing NV in EPC cells could stimulate the production of IFN. This suggests that the innate immune system in fish may recognize NV mRNA or protein as a pathogen-associated molecular pattern. Future studies will be aimed at further determining whether NV and P dictate viral evasion or mediate modulation of the fish innate immune system as a means of enhancing VHSv replication and dissemination.
The effects of immunosuppressive drugs on the effector functions of T-cells involved
in multiple sclerosis
Multiple Sclerosis (MS) is a neurodegenerative disease of the central nervous system (CNS) with different forms of relapsing and remitting symptoms leading to ascending paralysis. The pathology of MS includes autoreactive T-cells that recognize components of the myelin sheath in the CNS and release proinflammatory cytokines that induce demyelination and axonal degeneration, which leads to neurological symptoms and eventual loss of functional neuronal tissue. There are no cures for MS, but it can be treated with immunosuppressive and/or immunomodulatory drugs. Mitoxantrone (MX), an anticancer drug, has immunosuppressive characteristics that are therapeutic in progressive MS. MX has significant impact on the relapse rate and progression of the disease by decreasing levels of proinflammatory cytokines produced by T-cells, including IL-2 and IFN-gamma. Although MX is well tolerated, the drug has a long-term risk for malignancies and cardiotoxicity. Analogues of MX, Pixantrone (PX) and BBR3378, were developed and shown to have antitumor activity similar to MX, but lack cardiotoxicity. Work in our lab and others, has shown that PX and BBR3378 are effective in suppressing ascending paralysis in mouse models of MS. In this study, we compared the immunomodulatory activity of MX, PX, and BBR3378 on T-cell function. All three drugs were able to inhibit anti-CD3 induced T-cell proliferation and cytokine production, including IL-13, IL-17, and IFN-gamma. However, BBR3378 was much more efficient at blocking the production of Il-17 and IFN-gamma at levels below the cytotoxic range. These findings suggest that BBR3378 should be considered as an alternate for treatment of MS.
The Function of DEK in NF-kB Transcriptional Activation
Nuclear Factor Kappa B (NF-kB) is an evolutionarily conserved transcription factor that regulates the transcription of genes whose products are involved in the immune response, cellular differentiation, and cell survival . In the canonical pathway, NF-kB exists primarily in a p50-p65 heterodimer. When bound to its inhibitor, IkBa, the nuclear localization sequence of p65 is masked and the complex is contained in the cytoplasm . IkBa degradation is stimulated through the activation of IKK. With IkBa degraded, NF-kB undergoes nuclear translocation, where it is able to bind to kB sequences on DNA and induce or repress gene expression . The results of NF-kB binding to DNA depend on its association with cofactor proteins. The nuclear protein, DEK, functions as a transcriptional co-repressor to NF-kB . DEK is commonly overexpressed in autoimmune diseases and several types of invasive cancers including retinoblastoma, colon cancer, and bladder cancer . Further research has shown DEK to interact with p65 and repress NF-kB activity in a concentration dependent manner . NF-kB and DEK have both been shown to affect the pathway of the tumor suppressor p53. p53 functions against oncogenic activation and genotoxic effects, generally resulting in apoptosis . While the activation of NF-kB functions to oppose the apoptotic effects of p53, cells with decreased levels of DEK were suspect to increased p53 activation and apoptosis . The purpose of my thesis project was to examine the effects of DEK expression on the regulation of genes controlled by NF-kB in the presence or absence of p53.
P21-Activated Kinase 1 (PAK1) and its Role at Focal Adhesions and Cell Spreading
P21-Activated Kinase (PAK) plays a role in cell migration and cell adhesion through many pathways, including phosphorylation of Ser273 on paxillin, a focal adhesion associated protein involved in adhesion dynamics. Here we explore PAK1 dependant phosphorylation of paxillin downstream of the hormone Prolactin (PRL) through JAK2 signaling. We utilized T47D cells, a breast cancer cell line stably overexpressing GFP, GFP-tagged PAK1 WT or PAK1 Y3F which is deficient for tyrosyl phosphorylation by JAK2. We showed localization of Ser273 phosphorylated paxillin at small focal complexes as well as in ruffles in all T47D clones in response to PRL. We demonstrated that PRL treatment (500ng/ml, 48hrs) increases the percent of cells with well developed focal adhesions for T47D PAK1 WT cells, but decreases them in Y3F PAK1. We assessed changes in cell morphology by using shape factor. With it we demonstrated that T47D PAK1 Y3F cells are less spread than WT PAK cells treated with PRL (500ng/ml, 48hrs). With this data combined we conclude that Ser273 phosphorylated paxillin localizes to ruffles and small focal complexes in response to PRL, and tyrosyl phosphorylation of PAK1 by JAK2 promotes T47D cell adhesion and spreading.
Title: The Role of Prolactin and p21-Activated Kinase 1 (Pak1) in Breast Cancer Cell Proliferation,
Adhesion, and Spreading.
Breast cancer cells can produce the hormone prolactin, suggesting that prolactin may play a role in cancer progression. Prolactin activates the kinase Jak2, which then activates another kinase called Pak1. Pak1 plays a role in multiple processes including cell proliferation, motility, and adhesion. We hypothesized that prolactin activates Pak1 via Jak2 and manipulates the processes of cell adhesion, spreading, and proliferation. We used T47D breast cancer cells stably expressing GFP, GFP-tagged Pak WT, or tyrosine deficient Pak mutant Y3F. We assessed the proliferation of these cells in response to prolactin by MTT assays. T47D-Pak WT cells had increased proliferation rates while T47D-Pak Y3F clones had decreased rate compared to control cells. Adhesion was tested in the presence or absence of prolactin by allowing cells to adhere to collagen IV coated plates for 1 hour. Cells treated with prolactin had decreased ability to adhere to collagen IV than cells without prolactin treatment although T47D-Pak1 WT cells were more adhesive than control and T47D-Pak1 Y3F cells. Live imaging of this same procedure confirmed that T47D-Pak WT cells spread and adhered more efficiently than T47D-Pak Y3F and control cells. Prolactin makes cells more motile and less spread. To assess changes in cell morphology, we determined shape factor from immunofluorescent images stained for F-actin. In 1 hour of prolactin treatment, T47D-Pak WT cells were less round and more elongated compared to Y3F and control cells. We concluded that Pak1 WT promotes cell proliferation and cell adhesion and decreases cell spreading in response to prolactin.
Essential regulatory regions of the Caenorhabditis elegans tissue attachment gene mua-1
The mua-1 gene encodes a predicted transcription factor that is necessary for attachment of the epidermis to the cuticle and the attachment of the uterus to the body wall of Caenorhabditis elegans. It is expressed in the pharynx, epidermis, and uterus. This latter expression is present only at the larval-adult switch and correlates with formation of the uterine "seam" that attaches the uterus to the body wall and the timing of vulva formation. Little is known about how this uterine expression is developmentally controlled. Previous experiments have shown an area of 500base pairs in the mua-1 promoter is crucial for uterine expression. Sequence analysis has identified four specific regions in the 500base pairs as potential transcription factor binding sites. To verify the functional significance of these four sites, mutations consisting of deletions ranging from eight to eleven base pairs have been generated in each site. If the site is essential for uterine expression, the mutation will cause mua-1 expression to be abolished as assayed by a GFP transcriptional reporter construct. Promoter mutations were generated in the pOT27 mua-1p::gfp reporter plasmid by PCR, and the constructs re-introduced back into worms by microinjection. These transgenic worms are viewed under a fluorescent microscope to observe the GFP expression pattern, which reflects the activity of the mutated promoters. Confirmation of specific sites will allow us ultimately to identify proteins that bind these sites and regulate uterine expression of mua-1 and identify the signaling pathway that activates its expression.
Investigation of the role of a Divaricata type transcription factor in Zea mays.
The first member of the DIVARICATA (DIV) sub-family of MYB transcription factors (TFs) was discovered in Antirrhinum majus and found to play a role in dorsoventral symmetry. The DIV TF sub-family is small but well conserved in plants and study of a DIV TF in tomato revealed that it appears to affect cell division and expansion. Little or no study of DIV TFs has been performed in monocots. This family of TFs exhibits two MYB DNA-binding domains of which the second contains a characteristic SHAQKY motif. We surveyed the repertoire DIV TFs in maize and related grasses and found that there are at least seven complete DIV genes, none of which has studied to date. We report here a phylogenetic comparison of this subfamily in monocots. In addition we availed of the Ac/Ds tagging project at Cornell and identified a Ds insertion in exon2 of one of these genes (ZmDIV6). The insertion is immediately adjacent to the SHAQKY motif and thus likely to knock out gene function. We have generated constructs of ZmMYB3,4 and 6 that will be of use in the study of their DNA binding affinities, structures, and in generating antiserum for use in chromatin-immunoprecipitation (ChIP) type experiments aimed at identifying their target genes. In addition we will report on the expression pattern of these genes based on analysis of published RNA-Seq data.
The Interactions Between Pro-Apoptotic Signals and the Cell Cycle
Title of Project: Exploring Rab11-FIP3 in Caenorhabditis elegans
Does the matrix receptor MUA-3 directly interact with intermediate filaments in Caenorhabditis elegans?
Intermediate filaments (IFs), one of three types of cytoskeletal elements, are essential for mechanical stress resistance and locomotion in C. elegans. IFs attach to and connect hemidesmosomes (HD) at the basal and apical surfaces in the epidermis and are an essential component of force transfer from the muscles that lie below the epidermis to the cuticle that lies above, ultimately resulting in movement. Mutations in epidermal IFs result in loss of epidermal tissue integrity and paralysis of the worm. This phenotype is mimicked by mutations in the mua-3 gene that result in partial paralysis. Previous studies have shown that MUA-3 protein localizes to the epidermal HDs, that the primary mutant defect is a separation of the epidermis from the cuticle, and that MUA-3 is a trans-membrane protein with characteristics of a matrix receptor. To determine if MUA-3 directly interacts with IFs in vivo, I have GFP tagged the cytoplasmic domain of MUA-3 and am expressing it in animals that have ectopic epidermal accumulations of RFP tagged IF, examining the animals for co-localization of the two proteins in the ectopic IF accumulations. This work complements on-going in vitro approaches. The cytoplasmic domain of MUA-3 was ligated in frame with the GFP of the L4058 expression vector, and the 2kb dpy-18 promoter was ligated into promoter drop-in site, to drive epidermis specific expression of the MUA-3::GFP reporter. This construct was then co-introduced with the IFA-2::RFP over-expression vector into C. elegans by microinjection. Results will be presented.