Department of Biological Sciences

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.

Henrry Maldonado, P21-Activated Kinase 1 (PAK1) and its Role at Focal Adhesions and Cell Spreading. Advisor: Dr. Maria Diakonova, Department of Biological Sciences.

DEK Oncoprotein’s Regulation of NF-κB Activity and DNA Damage-induced Apoptosis
Alexa Elliott
Department of Biological Sciences
Faculty Advisor: Dr. Brian Ashburner

Abstract:
                NF-κB is a transcription factor that plays a role in many important cellular processes by regulating expression of a variety of genes involved in immune and inflammatory responses. Abnormal regulation of NF-κB affects the cell cycle and cell survival, thus it can play a part in the development of cancer and other diseases. NF-κB is able to activate transcription through interactions with transcriptional co-activator and co-repressor proteins. More specifically, the co-repressor protein, DEK interacts with the p65 subunit of NF-κB to help regulate NF-κB activity. As a co-repressor, DEK’s interaction with NF-κB inhibits NF-κB-mediated gene transcription. As DEK is overexpressed in various human tumors and implicated in many autoimmune diseases, this research is aimed to better understand the function of DEK in regulating NF-κB and apoptotic pathways. Cultures of p53 HCT116 (colon cancer cell line) were maintained and showed DEK knockdown. Short hair-pin RNA (shRNA) was used to knockdown the expression of the targeted DEK protein and western blot analysis to measure the level of knockdown of this protein. Through RNA isolation, Polymerase Chain Reaction (PCR), and gel electrophoresis, the effects of DEK on the expression of various genes were tested. Different time treatments (Untreated-30 hours) of Doxorubicin and western blotting with the same cell cultures were completed to analyze NF-κB and apoptotic activity in the presence and absence of DEK. This research is aimed to further determine the contribution of the suppression of DEK on apoptosis and the negative regulation on NF-κB in response to DNA damage.

Menin Regulation of Metabolic Reactions in the Gut
Kellie Andrews
Department of Physiology and Pharmacology
Faculty Advisor: Edith Mensah-Osman

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
Allen Maertin
Department of Biological Sciences
Faculty Advisor: Guofa Liu 

ABSTRACT

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
Eric Andrew McCloskey
Dr. John Bellizzi
University of Toledo Department of Chemistry

Abstract
                To understand the biochemistry of sleep one must first grasp the concept of circadian rhythms. In the course of evolution, organisms have become entrained to run in synchrony with a twenty-four hour internal clock. The basis of regulation for this clock is the sensing of light and dark by the suprachiasmatic nuclei (SCN) in the hypothalamus of the brain. In response to signals from the SCN, circadian oscillator proteins including casein kinase I (CKI) regulate the cycle of sleep and wake. The epsilon (CKIε) and delta (CKIδ) isoforms of CKI each have different effects on the period of circadian rhythms. The CKIε tau mutant was identified in a Syrian hamster that demonstrated a circadian rhythm of twenty hours. The preliminary goals of this project encompass cloning, expression, purification, and crystallization of the CKIε tau mutant. A wild type CKIε expression construct has been created and manipulated using site-directed mutagenesis in order to make the mutant kinase. Optimal procedures for expression and purification of mutant CKIε have been determined empirically. Further analysis of the three-dimensional structure of CKIε together with enzyme kinetics assays will allow for an unprecedented look into the mechanisms by which the tau mutant of CKIε attenuates circadian rhythms, and assist in the development of novel drug therapies for people suffering from the effects of sleep-based disorders.

Studies of Host-Virus Interaction for the Viral Hemorrhagic Septicemia Virus Phosphoprotein and Nonvirion Protein
Robert Rominski
Advisor: Dr. Douglas Leaman

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
Adelle Abouarrage, Anthony Quinn
Department of Biological Sciences

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
Torrie Klier
Dr. Brian Ashburner
Biology Department

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 [1]. 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 [1]. 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 [1]. 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 [5]. DEK is commonly overexpressed in autoimmune diseases and several types of invasive cancers including retinoblastoma, colon cancer, and bladder cancer [4]. Further research has shown DEK to interact with p65 and repress NF-kB activity in a concentration dependent manner [5]. 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 [2]. 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 [4]. 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
Henrry Maldonado
Research Supervisor: Dr. Maria Diakonova
University of Toledo Department of Biological Sciences

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.              
Student Researcher: Alan D. Hammer
Research Advisor: Dr. Maria Diakonova
Department: Biological Sciences

                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
Leigh Anne Autullo
Laboratory of Dr. Plenefisch
Department of Biological Sciences

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.  
Lia Risk⁽¹⁾, Erich Grotewold⁽²⁾ and John Gray⁽¹⁾, 
(1) Dept. of Biological Sciences, Univ. of Toledo, OH 43606,  
(2) Dept. of Plant Cellular and Molecular Biology, Plant Biotechnology Center, The Ohio State Univ., Columbus, OH 43210 email contact: jgray5@utnet.utoledo.edu  

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
Robert T. Fischbach
Faculty Advisor: Dr. William R. Taylor

Abstract
The epothilones are a new class of antineoplastic drugs that disrupt the mitotic spindle and induce cell death. Based upon the known mechanisms of other antineoplastic drugs, there are three spindle disruption mechanisms known. Drugs such as Vinblastine® inhibit the mitotic spindle from forming, while drugs such as Monastrol arrest cells in mitosis due to the formation of a single spindle pole. The epothilones appear to act on the mitotic spindle by stabilizing tubulin, as does Taxol®. Evidence suggests that Aurora B Kinase (AURKB) monitors incorrect kinetochore-microtubule interactions; thus, if the epothilones do not stabilize tubulin then AURKB will have no effect on the pathway, and a cell will remain in mitotic block until death. The drug ZM447439 inhibits AURKB. By manipulating the inhibition of AURKB, a cell that previously remained in mitotic block would exit mitosis. Utilizing the ZM447439 variable, Epothilone B does activate the spindle assembly checkpoint via the stabilization of tubulin. Cells treated with 2.5uM ZM447439 and 20nM Epothilone B did not have a prolonged mitotic block, and the addition of ZM447439 to arrested cells immediately caused the cells to exit mitosis. In cells treated with 20nM Epothilone B, the mean length of mitotic block until the onset of apoptosis was 20.75 (+/- (6.97)) hours. The addition of sub-lethal TRAIL (10ng/mL) drastically decreased time between mitotic arrest and onset of apoptosis, or 5.24 (+/- (1.52)) hours. This seems to suggest a synergistic combination of extrinsic and intrinsic death pathways.

Title of Project: Exploring Rab11-FIP3 in Caenorhabditis elegans
Student Researcher: Adam Jason Rockacy
Research Supervisor: Dr. Robert Steven
Department: Biological Sciences

Abstract:
Signals or messages within the neural network of the nervous system are passed from one neuron to another mainly via chemical communication; different classes of neurotransmitters including neuropeptides play critical roles. The nematode Caenorhabditis elegans has been chosen as a model organism for study due to its unsegmented, transparent, and bilaterally symmetrical body that complements the ease of which cellular analyses may be performed. Every somatic cells differential fate in C. elegans has been mapped and shown to be invariant; an aspect that allows for congruent genetic and molecular analyses. Prior research revealed C. elegans UNC-73 proteins are required for nervous system development and function through their roles as activators of the Rho GTPase family; one of eight main family members of the Ras superfamily of small GTPases. There are two RhoGEF activating domains encoded by unc-73 and mutations affecting the RhoGEF-2 domain result in animals with a lethargic movement phenotype likely the result of a neurotransmission defect. Utilization of a yeast-two hybridization screen revealed Rab11-FIP3 as a protein that potentially interacts with UNC-73. Rab11-FIP3 has been shown to bind to the chaperone complex NSF-γSNAP which supports the claim that Rab11-FIP3 may be an intermediate in regulating neurotransmitter receptors and further suggesting a role for UNC-73 at the synaptic vesicle. Cell-specific RNAi, fusion PCR rescuing experiments, and locomotion assays were utilized in an effort to characterize the Rab11-FIP3 phenotype. If similarities between mutant FIP3 and UNC-73 phenotypes present themselves, we will further examine if UNC-73 directly interacts with FIP3.

Does the matrix receptor MUA-3 directly interact with intermediate filaments in Caenorhabditis elegans?
Isaac Perry
Laboratory of Dr. John Plenefisch
Department of Biological Sciences

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.