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11th Annual Undergraduate Research Symposium

WOLFE HALL (Room 3246) Saturday, April 1, 2006

 

11th URS Winners
11th Winners
1st Place: Rachael Sullivan and Liz Lawlor
2nd Place: Cecinio Castillo Ronquillo and Patrick Clements


 

Morning Session:
11th AM presenters
Dr. Patricia Komuniecki, Timothy Scott, Ali Hussain, Andrew Stiff,
Rachel Ryland, Patrick Clements and Dr. Emilio Duran.
Afternoon Session
11th PM Presenters
Cecinio Castillo Ronquillo, Josef Froehlich, Elizabeth Lawlor, Rachael Sullivan,
Jonathan Hassel, Jennifer Quinlan, and Ed Briercheck.

 

Abstracts for each presentation follows the timetable

Time

Speaker

Presentation Title

9:25

Welcome, Dr. Patricia Komuniecki, Chair of the Department of Biological Science

9:30

Rachel Ryland

“Investigation into Tissue and Total Body Magnesium Levels in Parkinson’s Patients and Age Match Controls” Advisors: Dr. Lawrence Elmer, Department of Neurology, Medical University of Ohio, and Dr. Patricia Komuniecki, Department of Biological Sciences, The University of Toledo

9:50

Ali Hussain

“Expression of Chitinase in Bacterial and Yeast Expression Systems” Advisors: Dr. Antonio Garafalo, and Dr. Patricia Komuniecki, Department of Biological Sciences, The University of Toledo

10:10

Andrew Stiff

“Enhanced Expression of the Borealin Protein During Mitosis” Advisor: Dr. William Taylor, Department of Biological Sciences, The University of Toledo

10:30

Audrey Kosik

“Investigating Role of Aurora Kinase in Oncogenic Ras Signaling Pathway Using ZM447439” Advisor: Dr. William Taylor, Department of Biological Sciences, The University of Toledo

10:50

Break

11:00

Timothy Scott

“ASegment of the Cauliflower Mosaic Virus Genome is Responsible for Infection in Broccoli” Advisor: Dr. Scott Leisner, Department of Biological Sciences, The University of Toledo

11:20

Patrick Clements

“The Role of HDAC 2 in NF-kB Regulation” Advisor: Dr. Brian Ashburner, Department of Biological Sciences, The University of Toledo

11:40

Elizabeth Lawlor

“Muscarinic Agonists for the Treatment of Schizophrenia” Advisor: Dr. William Messer, Department of Pharmacology, and Dr. Patricia Komuniecki, Department of Biological Sciences, The University of Toledo

12:00

Pizza Lunch

1:00

Josef Froehlich

“Using Non-complementation Screens to Generate Additional mua-2 Alleles in C. elegans” Advisor: Dr. John Plenefisch, Department of Biological Sciences, The University of Toledo

1:20

Jonathan Hassel

“Comparing T-Cell Receptor Usage in Diabetes-Prone NOD Mice to Diabetes-Resistant NOD.IE Transgenic Mice” Advisor: Dr. Anthony Quinn, Department of Biological Sciences, The University of Toledo

1:40

Jen Quinlan

“Identification of cis-acting DNA Elements That Control Tissue Specific mua-1 Expression in C. elegans” Advisor: Dr. John Plenefisch, Department of Biological Sciences, The University of Toledo

2:00

Break/PM Speakers Photo

2:20

Ed Briercheck

“The Use of Aptamers as a Novel Sensor of APC in Developmental Analysis of Drosophila melanogaster” Advisors: Dr. Brooke McCartney, Department of Biological Sciences, Carnegie Mellon University, Dr. Bruce Armitage, Department of Chemistry, Carnegie Mellon University, and Dr. Patricia Komuniecki, Department of Biological Sciences, The University of Toledo

2:40

Rachael Sullivan

“Regulation of NF-?b Activity by Interaction with CBP and p38 Map Kinase” Advisor: Dr. Brian Ashburner, Department of Biological Sciences, The University of Toledo

3:00

Cecinio Castillo Ronquillo, Jr.

“The Taxonomy of Conus through Molecular Phylogenetics” Advisors: Dr. Baldemero Olivera, Department of Biology, The University of Utah, and Dr. Patricia Komuniecki, Department of Biological Sciences, The University of Toledo

3:20

Break/Judges’ meeting

3:30

Awards ceremony


Abstract 11th URS


Ed Briercheck
“The Use of Aptamers as a Novel Sensor of APC in Developmental Analysis of Drosophila melanogaster”
Advisors: Dr. Brooke McCartney, Department of Biological Sciences, Carnegie Mellon University, Dr. Bruce Armitage, Department of Chemistry, Carnegie Mellon University, and Dr. Patricia Komuniecki, Department of Biological Sciences, The University of Toledo

Proteins are the key to unlocking the cellular mechanisms of organism development. Thus, the need for proper identification of proteins and their interactions in vivo has arisen. Using Drosophila melanogaster as a developmental model, our lab has determined that the Adenomatous polypolis coli (APC) protein family plays a significant role in development. To determine mechanisms of APC function it is crucial to be able to localize APC and other protein interactions within subcellular structures without disrupting the functionality of the endogenous protein. Satisfaction of these requirements may be found in oligonucleotide aptamers. Aptamers are short single-stranded DNA molecules which specifically bind to the target protein. In order to act as a sensor, the aptamers are tagged by base pairing interactions with fluorescently labeled peptide nucleic acids (PNA). To select for aptamers that specifically bind to APC2 we purified the APC2 protein, which was mixed with a synthetically produced randomized DNA pool. We then subjected this mixture of APC2 protein with the DNA pool to nonequlibrium capillary electrophoresis of equilibrium mixtures (NECEEM), allowing us to definitively separate free DNA, APC2 bound DNA, and free APC2. We are currently amplifying selected aptamers via PCR and will clone the optimum sequences. One technical hurdle of aptamer biosensors is the control of fluorescence. It is imperative aptamers show differential fluorescence when bound and unbound. A PNA-linked Dapoxyl fluorophore, showed significantly more fluorescence when bound to the protein than when free, making this a viable technique for future work on the aptamers for APC2.

Cecinio Castillo Ronquillo, Jr.
“The Taxonomy of Conus through Molecular Phylogenetics”
Advisors: Dr. Baldemero Olivera, Department of Biology, The University of Utah, and Dr. Patricia R. Komuniecki, Department of Biological Sciences, The University of Toledo

There are 500-700 different species of venomous cone snails (Conus). Past studies have established that Conus venoms are a remarkable source of pharmacological agents for studying receptors and ion channels in the nervous system. Some of these toxins from Conus have been used for diagnostic and therapeutic purposes. One, Prialt (ziconotide) was recently approved by the FDA as a drug for intractable pain. Conus species can be classified into related groups that are generally referred to as clades that are based on shell morphology, hunting patterns, environment and, recently, molecular sequences using mitochondrial markers. Several dissected Conus species were used to isolate DNA. Sequences of 12S and 16S gene segments in mitochondrial DNA, intron 9 from the gamma-carboxylase gene of Conus and cytochrome oxidase sub 1 gene were obtained by molecular cloning from each cone snail species. A consensus sequence was obtained for each species and was used to obtain information to clarify the taxonomy. Phylogenetic trees based on the sequences provided by 12S and 16S mitochondrial markers were made using parsimony and Bayesian analysis. Combining all the consensus sequences of the fish-hunting cone snails and the mollusk-hunting cone snails yielded an accurate phylogenetic tree based on the separation of the species’ hunting patterns as well as the grouping of species based on the proposed clades. This approach provides reliable lineage relationships between the species and will be a meaningful way to discriminate between closely related, but pharmacologically distinct, peptides contained in the Conus venom based on the primary sequence of the species alone.

Patrick Clements
“The Role of HDAC 2 in NF-kB Regulation”
Advisor: Dr. Brian Ashburner, Department of Biological Sciences, The University of Toledo

NF-kB is an important and conserved protein involved in the mammalian immune response, cell growth, and survival pathways. NF-kB is a transcription factor that binds to DNA, allowing for expression of specific genes involved in these processes. The understanding of NF-kB has significant implications for biomedical research, as increased NF-kB activity has been shown to aggravate or even cause such conditions as cancer and inflammatory disease. The ability of NF-kB to act is based on the accessibility of its DNA binding sites. DNA is wrapped around nucleosome proteins made of histone octamers. Histone deacetylase (HDAC) proteins regulate which regions of DNA are unwound and accessible to transcription factors like NF-kB. Our lab group focuses on the role that HDAC proteins play in NF-kB regulation, with my project centering specifically on HDAC 2. A tetracycline-inducible lentiviral system was established to create short hairpin RNA (shRNA) to specifically knockdown HDAC 2 expression. My tasks this year have focused on isolating and testing clones of these cells to find optimal HDAC 2 knockdown. Once a cell line with strong HDAC 2 knockdown potential has been established, our group will look at the activity of NF-kB in relation to this knockdown. NF-kB activity will be analyzed using Western blotting, luciferase reporter gene assays, and chromatin immunoprecipitation (ChIP) assays. Identifying the role of HDAC 2 in NF-kB activity will provide a better understanding of NF-kB signaling pathways, as well as provide foundations for further research and therapeutic treatment involving NF-kB regulation.

Josef Froehlich
“Using non-complementation screens to generate additional mua-2 alleles in C. elegans
Advisor: Dr. John Plenefisch, Department of Biological Sciences, The University of Toledo

In C. elegans, mutations in the mua genes can cause a number of problems in the attachment of the muscles to the cuticle. It is believed that mua-2 works in the hypodermis, encoding proteins that interact with hemidesmosomes and/or intermediate filaments. In order to determine the mua-2 gene products, a positional cloning strategy is underway. For this to succeed, the genes to be cloned have to be genetically mapped to a defined chromosomal interval. For mua-2, the chromosomal interval has to be reduced in size. As of this point, only two weak alleles of mua-2 exist. By generating new alleles, we hope to better characterize mua-2. A null mutation could affect more tissues or have additional effects than currently observed. Non-complementation screening is being used to generate new alleles. Males heterozygous for mua-2 are crossed with dpy-18 hermaphrodites mutagenized either by exposure to EMS, or exposure to gamma irradiation source. Non-dpy mua-2 F1 hermaphrodites are screened for, these should be of genotype + rh174/dpy-18 M. These are then mated with wild type males to recover the dpy-18 M chromosome, the M being the possible new allele. The chromosome will then be backcrossed to remove dpy-18, and the new mua allele will be balanced with eDp6. The non-complementation screens are an efficient and effective way of generating new alleles.

Jonathan Hassel
“Comparing T-Cell Receptor Usage in Diabetes-Prone NOD Mice to Diabetes-Resistant NOD.IE Transgenic Mice”
Advisor: Dr. Anthony Quinn, Department of Biological Sciences, The University of Toledo

The earliest T-cells arising in the islets of Langerhans in the pancreas of NOD (Non-Obese Diabetic) mice, which develop spontaneous Type I Diabetes (TID), are directed against GAD65 p530-543. It was determined that the majority of the p530-specific T cells in the NOD mice utilize Vß4 for the TCR (T-Cell Receptor) gene family. The prevention of responses by the T-cells may be sufficient to prevent TID in NOD mice. There are two ways to accomplish this: 1) Regulatory T-cells can be used to inhibit the response of the islet-invasive p530-43 specific T cells. 2) Alternatively, deletion of pathogenic clones would also be expected to provide protection. The addition of a 2nd MHC Class II molecule added via transgenesis protects NOD mice from disease, as in the NOD.IE mice. Our data suggests the NOD.IE mice down-regulate or delete the pathogenic clones specific for the p530-43 fragment, which utilize Vß4-J ß2.7 genes. NOD.IE mice do mount a response to p530-543; however, they utilize a different repertoire. Therefore, the Vß4-J ß2.7 TCR can be linked to TID as a pathogenic receptor and the deletion of this receptor can provide immunity in the NOD.IE mice.

Ali Hussain
“Expression of chitinase in bacterial and yeast expression systems”
Advisors: Drs. Antonio Garofalo and Patricia R. Komuniecki, Department of Biological Sciences, The University of Toledo.

Parasitic nematodes cause economic loss by infecting humans, livestock and crops. Ascaris suum is a parasitic nematode that infects swine. As-p50, a family 19 chitinase, was discovered as one of the proteins in the perivitelline fluid of infective A. suum third-stage larvae. C08B6.4, an orthologue of As-p50 in Caenorhabditis elegans, a free-living nematode, has similar temporal and spatial expression in the L1 stage of C. elegans. The similar localization and timing of expression of C08B6.4 and As-p50 suggested that the putative chitinases might have similar functions. This project was designed to determine whether C. elegans family 19 chitinases have anti-fungal activity. The bacterium, Rosetta gami, was used to express the chitinase protein via the use of the T7 RNA polymerase promoter and IPTG was used for induction. In addition, the X-33 strain of the methylotropic yeast, Pichia pastoris, was used to generate the recombinant chitinase protein. The native C. elegans secretory sequence was utilized to induce secretion of the recombinant protein from the P. pastoris into the growth medium. In addition, X-33 cells were also transformed with the pPICZ-alpha recombinant vector which has the yeast secretory sequence but not the C. elegans secretory sequence. Chitinase activity was present using glycol-chitin as substrate. The yeast expression system proved to be more efficient in expression of chitinase than the bacterial system.

Jen Quinlan
“Identification of cis-acting DNA elements that control tissue specific mua-1 expression in C. elegans
Advisor: Dr. John Plenefisch, Department of Biological Sciences, The University of Toledo

Movement of Caenorhabditis elegans is dependant on the way its muscle is attached to the outer cuticle. Many mutants have been identified where, initially, their movements and muscle differentiation are normal, but then the muscles somehow detach from the cuticle and paralysis occurs. These mutants identify a class of genes required for growth and maintenance of functional muscle attachments. These genes are named mua, for muscle attachment defective. I will be taking a closer look at mua-1, particularly, by identifying DNA elements that control its expression in specific tissues during the development of C. elegans. This gene is expressed in three tissues – the pharynx, the epidermis, and the uterus (during the larval to adult molt only). This project is designed to use different potential enhancers of mua-1 in an attempt to determine which enhancers are expressed where in C. elegans. The polymerase chain reaction (PCR) will be used to generate expression constructs. Since the nucleotide sequence of mua-1 is known, fragments can be amplified directly using the PCR technique and ligated into a plasmid vector upstream of GFP (green fluorescent protein) using standard molecular techniques. The resulting recombinant plasmids, all carrying the identical genomic DNA segment, can then be microinjected into C. elegans or propagated in E. coli bacteria. GFP will fluoresce wherever the amplified fragment turns on expression. The ultimate goal of my project is to find out what is controlling mua-1 – how its expression is being controlled, why it is being turned on during such a specific time in the uterus, and what makes sure it is turned on. It is hypothesized that mua-1 is an element important for localization to the nucleus.

Audrey Kosik
“Investigating Role of Aurora Kinase in Oncogenic Ras Signaling Pathway Using ZM447439”
Advisor: Dr. William Taylor, Department of Biological Sciences, The University of Toledo

Activating Ras mutations are present with alarming frequencies in malignancies of the pancreas (80-90%), colon (25-60%), and lung (25-60%). The Aurora-B kinase has recently been implicated in transformation by oncogenic Ras. The mechanism was not determined however Aurora-B may enhance Ras signaling and/or perturb chromosome segregation. We tested the hypothesis by inhibiting Aurora kinase activity in NIH3T3 (WT, Ras, or Raf activated) cells using ZM447439 and probing for phospho MEK, MAPK, and MLK3 only to observe a reduction at the highest concentration of drug. This may be due to direct inhibition of MEK, therefore, inhibiting Aurora-B does not reduce signaling of Ras to the downstream targets we have tested. Using NARF2 cells containing IPTG inducible p14ARF we were also able to test the effects of ZM on p53 and p21. Aurora-B may be involved in disrupting the p53 response. In preliminary experiments we have found no change in p53, but a reduction in p21. This does not explain the transformation results previously reported. Another study suggests that tetraploidy can promote transformation. In vivo tetraploidy gave rise to malignant tumors in mice. We hypothesized that tetraploidy induced by overexpressing Aurora kinase was the cause of augmenting Ras transformation. We transfected 10T1/2 cells with Ras and induced tetraploidy which resulted in the formation of fewer foci from tetraploid than diploid cells. Therefore, Aurora-B augments Ras transformation by an unknown mechanism. Further investigation must take place in order to determine where Aurora kinase has a role in Ras induced neoplastic transformation.

Elizabeth Lawlor
“Muscarinic Agonists for the Treatment of Schizophrenia”
Advisor: Dr. William Messer, Department of Pharmacology, and Dr. Patricia Komuniecki, Department of Biological Sciences, The University of Toledo

Schizophrenia afflicts approximately 1% of the population in the USA and is associated with hallucinations, delusions, blunted emotion, and cognitive deficits. Current research is directed toward the development of new therapies that address the many aspects of the disease. Two paradigms useful for evaluating new therapies for schizophrenia include prepulse inhibition (PPI) of the startle response and delayed spatial alternation. PPI is an attenuation of the response to a strong stimulus (pulse) when it is preceded by a weaker non-startling stimulus (prepulse). The PPI paradigm measures sensorimotor gating deficits. Preliminary testing indicates that apomorphine, a dopamine agonist, induces the PPI deficit, providing a useful system for evaluating antipsychotics. The antipsychotics haloperidol and clozopine are effective in reversing the effects of apomorphine in the PPI paradigm. The novel muscarinic agonist CDD-0304 was evaluated for its efficacy in reversing the PPI deficits induced by apomorphine. CDD-0304 (at 1.0 and 3.0 mg/kg, s.c.) significantly reversed the apomorphine-induced PPI deficits. Delayed spatial alternation tasks test animals’ ability to retain information and carry out a task. Delayed spatial alternation is utilized to evaluate working memory, which may be related to activity of the prefrontal cortex. In order to establish a paradigm for assessing cognitive enhancement, the effects of N-methyl-d-aspartate (NMDA) receptor antagonists, such as PCP and MK-801, were tested in a delayed spatial alternation task. MK-801 impaired performance of the delayed spatial alternation task in a dose-dependent manner. The paradigm should be useful in evaluating the ability of muscarinic agonists to enhance cognitive function.

Rachel Ryland
“Investigation into Tissue and Total Body Magnesium Levels in Parkinson’s Patients and Age Match Controls”
Advisors: Dr. Lawrence Elmer, Department of Neurology, Medical University of Ohio, and Dr. Patricia Komuniecki, Department of Biological Sciences, The University of Toledo

Parkinson’s disease (PD) is a neurodegenerative movement disorder of the brain. Specifically, dopamine-producing nerve cells of the substantia nigra lose their function either by death or impairment. These play a role in controlling coordinated movement. The cause of Parkinson’s disease is currently unknown but it is now thought that a combination of both environmental and genetic factors influence its development. Magnesium is the second most plentiful free divalent cation in the body, and plays a role in regulating the transmission of action potentials in conjunction with NMDA glutamate receptors. A previous study in this lab demonstrated a two-fold increase in tissue magnesium levels compared to age match controls. Fifty patients, 25 PD patients and 25 age match controls, will be asked to donate tissue samples via buccal swabs and two 24-hour urine samples in order to determine tissue magnesium concentrations as well as total body magnesium concentrations. A magnesium injection will be administered between the two 24-hour urine collections in order to determine any deficiencies in the body. Atomic absorption spectrophotometry and protein analysis will be used to determine the concentration of magnesium per milligram of protein for both the tissues samples and the urine samples. Previous work indicated a correlation between decreased magnesium concentrations in the tissue samples of PD patients, and the present study is designed to explore that correlation further.

Timothy Scott
“A Segment of the Cauliflower Mosaic Virus Genome is Responsible for Infection in Broccoli”
Advisor: Dr. Scott Leisner, Department of Biological Sciences, The University of Toledo

Every year plant viral infections result in millions of dollars due to crop disease in the United States alone. An understanding of plant resistance to some of the more harmful viruses would greatly enhance protection of crops and prove extremely beneficial to the agricultural industry. Because virus-host interactions are complex, it is an advantage to use a model pathogen, such as Cauliflower mosaic virus (CaMV). CaMV is a reverse-transcribing virus that infects plants. CaMV particles contain a double-stranded circular DNA genome that encodes seven genes (labeled I-VII). CaMV chimeras along with their parental viruses can be propagated in turnips. After propagating the virus in turnips, tissue extracts can be prepared and used to infect other hosts by mechanical inoculation. Our lab has previously determined that gene VI is the most crucial portion of the CaMV genome controlling infection of a specific Arabidopsis ecotype (Tsu-0). However, it was unclear if this same region was responsible for controlling infection of other Brassica species. To test this, several chimeras, in addition to their parental viruses, were inoculated to Broccoli Romanesco. Interestingly, our preliminary data indicate that while gene VI was important, a larger region of the CaMV genome is required for systemic infection of Broccoli than for Tsu-0. These data indicate that CaMV-host interactions are unique to the pathosystem examined.

Andrew C. Stiff
“Enhanced Expression of the Borealin Protein During Mitosis”
Advisor: Dr. William Taylor, Department of Biological Sciences, The University of Toledo

Cancer is the second leading cause of death in the United States, killing approximately 500,000 people every year. One of the characteristics of cancer is uncontrolled cell proliferation and a way to understand this is by examining the ceil cycle. The process by which cell divide their nuclear and cytoplasmic contents is called mitosis, composed of the steps interphase, prophase, metaphase, anaphase, telophase, and cytokinesis. Borealin is a recently identified protein required for chromosome segregation and cytokinesis. We identified two spliced variants transcribed for the borealin gene. We tested whether these variants encoded a protein of similar size by transient transfection of epitope tagged versions. We observed that both variants led to the production of a protein with the same molecular weight. Interestingly, treatment of cells with nocodazole caused higher levels of the Borealin protein to accumulate. Since nocodazole arrests cells in mitosis, this suggests that the Borealin protein is stabilized during mitosis. Other experiments using LLnL, a drug that inhibits the proteasome, have shown Borealin may be degraded by the proteasome, however this is a preliminary finding. Future experiments are aimed at measuring the stability of the Borealin protein during mitosis and determining the mechanism of regulation.

Rachael Sullivan
“Regulation of NF-?kb Activity by Interaction with CBP and p38 Map Kinase”
Advisor: Dr. Brian Ashburner, Department of Biological Sciences, The University of Toledo

NF-kB is a transcription factor that controls expression of genes that play a role in inflammatory and immune responses, cell growth and differentiation, and protecting cells from programmed cell death. NF-kB activity is dysregulated in different types of cancer and chronic inflammatory disorders. The main form of NF-kB is a dimer of p50 and p65 proteins. NF-kB is found in its inactive form in the cytoplasm, bound to the IkB inhibitory protein of the IKK complex. Stimuli such as viruses, UV light, inflammatory cytokines, and bacterial toxins can induce NF-kB activity by initiating degradation of the IKK complex. After degradation of the complex, NF-kB is freed and can translocate into the nucleus to activate transcription. Although NF-kB activity is largely controlled through its nuclear translocation, it is also controlled through its interaction with other coactivator and corepressor proteins in the nucleus. My project focuses on the role of the p38 MAP kinase in mediating the interaction between the p65 subunit of NF-kB and the CBP coactivator. Chromatin immunoprecipitation assays are being used to assess the recruitment of NF-kB, CBP, and p38 to the NF-kB-regulatory promoter in the presence or absence of p38 inhibitors. Through these experiments, the lab hopes to gain insight into how p38 regulates NF-kB activity.

 

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Last Updated: 6/26/15