The 5th Annual

NC State University

Undergraduate Summer Research Symposium

 

HHMI Student Research Internship (SRI) Program

 


Abstracts are listed in alphabetical order by the last name of the corresponding author.

 

 

 

 

 

Student Author(s): 

Batchelor, Trevis H

Home Institution:

North Carolina Wesleyan College

Program:

HHMI SRI Program

Department(s):

Clinical Sciences

Research Mentor(s): 

Steven E. Suter/Clinical Sciences

Title of Presentation:

Detection of Epstein-Barr Virus (EBV)-like Sequences in Normal Dogs and Dogs with Lymphoma

 

 

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is implicated in the development of numerous human malignancies, including Burkitt’s lymphoma. EBV, found naturally in humans and some primates, will infect canine cells in vitro. Using PCR, EBV-like genomic sequences were detected in peripheral blood leukocytes isolated from normal dogs in Taiwan. Additionally, using immunocytochemistry, two extremely abundant small non-polyadenylated RNAs found in latently infected cells called EBERs, were also identified. In Burkitt’s lymphoma, EBV causes up regulation of the oncogene c-myc via a reciprocal translocation of the c-myc locus to sites controlling immunoglobin gene  expression. Some dogs with naturally occurring lymphoma, whose incidence has been steadily increasing over the past 20 years, also have c-myc perturbations leading to c-myc protein over-expression. The aim of this study was to determine if EBV-like sequences could be detected in normal dogs or dogs with lymphoma in the United States. Specifically, genomic PCR was used to detect a published human EBV Bam HI genomic fragment. Also, RT-PCR was used to detect EBV-encoded EBER-1 and EBER-2. We did not detect EBV genomic sequences in dogs with and without lymphoma. Our initial EBER RT-PCR experiments suggest that some of the dogs used in our study may be infected with an EBV-like virus. Although preliminary, these initial results are the first to document the existence of an EBV-like latent viral infection in dogs from the United States which lends credence to the notion that the increase in the incidence of lymphoma in dogs may be due, in part, to an infectious agent. 

 

 

 

 

Student Author(s): 

Desmarais, Jamie A.

Home Institution:

North Carolina State University

Program:

HHMI SRI Program

Department(s):

Zoology

Research Mentor(s): 

Jane  Lubischer/Zoology

Title of Presentation:

Neonatal Animals Show  Markers of Schwann Cell Reactivity after Denervation of  Soleus Muscle

 

 

The understanding of neural plasticity (the ability of  the body to repair itself after injury to neurons) is  important to the progress of investigations in aging,  development, and neurodegenerative diseases such as  Amyotrophic Lateral Sclerosis (Lou  Gehrig's Disease).  It has been shown that plasticity  at the neuromuscular junction in neonatal rats is limited in comparison to adult animals, and that this  response is linked to the fact that neonatal terminal Schwann cells die after denervation of muscle.  Terminal Schwann cell reactivity is key for  reinnervation of denervated muscle in adult rats, and involves a change in gene expression (including the upregulation of a protein known as 4E2) and the extension of processes.  Schwann cell reactivity after denervation of the soleus muscle was examined in  neonatal animals at ages where reinnervation rarely occurs.  After denervation and before Schwann cell death (1-2 days after surgery), the soleus  muscles were removed.  Immunohistochemical procedures (antibody labeling) were used to visualize Schwann cells (using anti-S100) and to test for the expression of 4E2 by terminal Schwann cells.  Preliminary results showed evidence of the expression of 4E2 by terminal Schwann cells after denervation in rats aged 14 days,  suggesting that neonatal animals show potential for neural plasticity.  Further questions remain about what other factors are preventing reinnervation, as well as the identification of a developmental time course of Schwann cell reactivity.  Further  studies may examine if reinnervation will occur given the right trophic factors present to prevent Schwann cell death after denervation in neonatal animals.

 

 

 

 

 

Student Author(s): 

Gebreselassie, Nebiat G.

Home Institution:

Salem College

Program:

HHMI SRI Program

Department(s):

Microbiology

Research Mentor(s): 

Eric S. Miller/Microbiology

Title of Presentation:

Escherichia coli Detection Using Green Fluorescent Protein Tagged T4 Phages

 

 

The emergence of antibiotic-resistant pathogenic bacteria and bioterrorism have elevated the need for rapid methods of bacterial detection. One way is to engineer reporter bacteriophages that infect their specific and viable host cells. Our purpose is to construct a GFP recombinant derivative of  bacteriophage T4 which would enable us to detect their host cells Escherichia coli.

               The strategy taken used a plasmid containing T4 homology: 422 base pairs (bp) of rllA and 451 bp of the flanking gene rllB. A GFP-Kan reporter cassette was inserted between the rll homology regions using  the Polymerase Chain Reaction (PCR), Gene Splicing by Overlap Extension (gene SOEing), and recombineering. An alternative  strategy used the PCR  and TOPO -TA cloning to insert just the GFP reporter gene, with only 45 bp of flanking rllA and rllB homology, in to the plasmid pCR2.1-TOPO.

               A T4 phage infection of the of the cells containing the "rllA-GFP-rllB" plasmid constructs should yield recombinant phages that fluoresce to detect their viable E. coli  hosts.

 

 

 

 

Student Author(s): 

Kumsa, Doe

Ferree, Sandra

Home Institution:

Salem College

Program:

HHMI SRI Program

Department(s):

Chemical Engineering

Research Mentor(s): 

Henry Lamb/ Chemical Engineering

Title of Presentation:

Growth of Recombinant E. Coli BL21(DE3)(pAD1) for GFP Production

 

 

The growth kinetics of the recombinant E. Coli were examined using conventional and high-throughput screening techniques. The E. Coli strain BL21 (DE3)(pAD1) has been altered through a plasmid containing the gene for GFP and conferring kanamycin-resistance.  Experiments were conducted using conventional shake flasks and using a micro-titer plate reader with 24- and 96- well plates. The base growth media was Luria-Bertani broth to which glucose was added with initial concentrations in the  1-10g/L range. Cells grew on the base media to a final optical density of 1.2 in the 24- and 96- well plates. Growth on LB broth was rapid initially but slowed markedly after approximately 3h due to the depletion of readily metabolized carbohydrates. Addition of glucose at 1g/L extended the log phase to ~ 4 h, and the culture achieved the same final OD. Higher initial glucose concentrations, however, had a negative impact on final OD. This effect was traced to the production of acetic acid by the cells which lowered the pH of the growth medium. Similar trends in final OD and pH with initial glucose concentration were observed in growth experiments conducted in 150-ml shake flasks, although the final OD values were in the 4-6 range. Future experiments will investigate potential oxygen mass transfer limitations in the 24-well plate experiments and the influence of kanamycin on specific growth rate.  

 

 

 

 

Student Author(s): 

Lough, Lea C.

Home Institution:

Fayetteville State University

Program:

HHMI SRI Program

Department(s):

Food and Science

Research Mentor(s): 

Sophia Kathariou/Food Science

Carol D'lima/Food Science

Title of Presentation:

Identification and Characterization of Plasmids from Multiantibiotic Resistant Strains of Campylobacter coli from Turkeys in North Carolina

 

 

 

Campylobacter is the leading bacterial cause of human gastroenteritis in the United States and is commonly found in poultry at retail. Most illnesses are due to Campylobacter jejuni and Campylobacter coli. Findings show that Campylobacter coli has gained resistance against a number of antibiotics while in the gut of birds. Turkeys in North Carolina are frequently colonized with Campylobacter coli strains that are resistant to multiple antibiotics. Farmers treat the birds with different antibiotics and this may result in Campylobacter coli that has acquired resistance to these antibiotics. If such resistant strains were to infect humans, they could cause problems since these drugs (ciprofloxacin and erythromycin) are also used for treatment of human infection. Resistance can be due to a point mutation in the bacterium’s chromosome or it can be conferred by a plasmid, which carries the resistance gene. However, the plasmid content of multi-antibiotic resistant Campylobacter from turkeys has not been characterized. Plasmids from different  strains of two multilocus sequence typing (MLST) groups (ST1126 and ST1101) were isolated to determine if they contributed to the resistance. All strains were resistant to streptomycin, kanamycin, nalidixic acid, ciprofloxacin, erythromycin, and tetracycline. Plasmids were extracted from the isolates and visualized on a gel. Plasmids that appeared similar were restricted with BglII, Hind III, and Sau3A I to try to determine if these plasmids were indeed identical. Isolates from ST1126 had three sets of strains with the same plasmid(s) and group ST1101 had six sets of strains with the same plasmids. Plasmid curing, transformation, and conjugation were pursued in order to determine the stability of the plasmid(s) in certain strains, and their potential to become transferred to other Campylobacter strains. The findings suggest extensive presence of plasmids in these organisms, with potential to contribute to resistance to multiple antibiotics.

 

 

 

 

Student Author(s): 

Nelson, Jessica M.

Home Institution:

Salem College

Program:

HHMI SRI Program

Department(s):

Molecular and Structural Biochemistry

Research Mentor(s): 

Raquel Hernandez/Molecular and Structural Biochemistry

Title of Presentation:

Characterization of Furin Protease Sensitive Sindbis Virus Mutants

 

 

Sindbis virus is an arbovirus member of the Togaviridae family.  The inner and outer capsid shells of the virus particles are composed of three structural proteins (Capsid/E1/E2).  The E1 glycoprotein ectodomain is composed of two disulfide-bridge constrained domains necessary for virus infectivity, and the E2 glycoprotein is responsible for receptor binding.  Ectodomain mutants with furin protease sensitive sites were produced previously in both membrane  glycoproteins.  These mutations should cause the release of the specific protein domain after correct folding in the ER, generating truncated proteins that impede virus assembly and result in the formation of irregular virus structures. The phenotypes associated with the introduction of  these motifs produced in virus from mosquito cells were analyzed. When compared to wild type, mutants consistently showed a lower virus titer with high heat resistance profiles that suggest the virus structure  was similar to wild type, indicating infectious virus structures have limited tolerance for the truncated proteins. 

 
 

 

 

 

Student Author(s): 

Pradhan, Arjana

Home Institution:

Salem College

Program:

HHMI SRI Program

Department(s):

Microbiology

Research Mentor(s): 

Michael Sikes/Microbiology

Title of Presentation:

Identifying a Unique Transcriptional Promoter Upstream of TCR Dß2

 

 

T cells are essential members of our immune system, helping to defend against pathogens that invade our  bodies.  T cells recognize individual pathogens via the T cell receptor (TCR), which is composed of two proteins called the alpha and beta chains. Each T cell’s TCR contains a unique pathogen-binding region called the variable domain. As billions of new T cells develop each day, they must assemble the DNA that encodes this variable domain from a large pool of gene segments called Variable (V), Diversity (D), and Joining (J) by the process of V(D)J recombination.  Evidence has shown that V(D)J recombination at TCRß is controlled by transcriptional promoters associated with each V and D gene segment.  The aim of my project is to locate the position of an additional promoter upstream of the second Dß (Dß2).  This involves creating reporter constructs in which DNA fragments of varying size are isolated from the Dß2 upstream sequence and transferred into a plasmid that carries the firefly luciferase gene.  The plasmids are then transfected into a T cell line, and the cells are then assayed for the ability of the inserted Dß2 DNA to promote luciferase expression.  With the luciferase assay of our constructs, we expect to define the minimal length and location of this new Dß2 promoter. Future experiments will test its role in V(D)J recombination during T cell development. Such findings will enrich our understanding of the immune system.      

 

 

 

 

 

Student Author(s): 

Quintero-Varca, Tatiana

Home Institution:

Meredith College

Program:

HHMI SRI Program

Department(s):

Molecular Biology

Research Mentor(s): 

Amy Grunden/ Molecular Biology 

Title of Presentation:

Expression and Biochemical Characterization of Pyrococcus horikoshii Prolidase Homolog 1 and Homolog 2 for Potential Use in Organophosphorous Nerve Agent Detoxification 

 

 

The enzyme prolidase is a proline dipeptidase, which cleaves dipeptides having proline as the C-terminal residue. Prolidase has also been reported to hydrolyze the model organophosphorus (OP) nerve agent diisopropylfuorophosphate (DFP). The prolidase from the hyperthermophilic archeaon, Pyrococcus furiosus, has been biochemically and structurally characterized. The potential use of Pyrococcus furiosus prolidase for detoxification of OP nerve agents is particularly attractive due to its extreme thermostability; however, its cobalt-dependence for activity and low activity at temperatures below 50 °C currently limit its utility in OP nerve agent detoxification. Recently it was determined that the hyperthermophilic archeaon Pyrococcus horikoshii has a prolidase gene equivalent to the previously characterized P. furiosus prolidase as well as two other prolidase homolog genes. For future evaluation of the use of P. horikoshii prolidase homolog 1 (55% similar to P. furiosus prolidase) and homolog 2 (42% similar to P. furiosus prolidase) genes for detoxification of OP nerve agents, these genes were cloned into the T7 RNA polymerase-based _expression vector pET 21b. Over-_expression of the P. horikoshii prolidase homolog 1 and homolog 2 proteins in E. coli strain BL21(λDE3) was evaluated in small scale _expression experiments using both LB and autoinduction media. Thermostability and activity studies were conducted using the recombinant P. horikoshii prolidase homologs 1 and 2. It was determined that both proteins were thermostable and that prolidase homolog 1 had significant activity when cobalt is present in the reaction mixture and when the dipeptide Leucine-Proline is used as the substrate.

 

 

 

Student Author(s): 

Smith, Arianna L.

Barnum, Chelsea V.

Buerkle, Shauna C.

Home Institution:

North Carolina State University

Program:

HHMI SRI Program

Department(s):

College of Veterinary Medicine

Department of Clinical Studies

Laboratory of Equine Medicine

Research Mentor(s): 

Betta A. Breuhaus/Clinical Sciences

Douglas Heath LaFevers/Clinical Sciences

Title of Presentation:

Obesity-associated Insulin Resistance in Mature Horses

 
 

 

 

As a result of the reoccurring and serious nature of laminitis, it is imperative to determine causal factors.  Prior studies have shown a link between insulin resistance and laminitis.  The purpose of this study is to determine the prevalence of insulin resistance in specific breeds of horses.  Horses were separated into four groups based on breeds prone to obesity and body condition score.   Obesity-prone breeds include Morgans, Paso Finos, Peruvian Pasos, and Saddlebreds; non-obesity prone breeds include Thoroughbreds and Standardbreds.  Intravenous Glucose Tolerance Tests (IVGTT) were administered to determine insulin resistance and glucose sensitivity.  It is expected that breeds predisposed to obesity will exhibit higher levels of insulin resistance, regardless of body condition score.  Higher levels of insulin resistance in obesity-prone breeds may put them at greater risk of developing laminitis.

 

 

 

 

Student Author(s): 

Welsh, Alice B.

LaMontagne, Lauren N.

Selgrade, Brian P.

Home Institution:

North Carolina State University

Program:

HHMI SRI Program

Department(s):

Biomedical Engineering and Biological Sciences

Research Mentor(s): 

Peter L. Mente/Biomedical Engineering

Title of Presentation:

Cartilage Cracking Resulting from Shear and Normal Loading

 
 

 

 

Osteoarthritis is a joint disease that affects over 20 million Americans. A major risk factor for  osteoarthritis is a previous injury to a joint which causes cracking and eventual destruction of the articular cartilage. Joint injuries generally result

from a very complex loading including both normal loads and shear loads. In this study, we simulated shear and normal impact injuries to articular cartilage of porcine patellae. We wanted to test the hypothesis that shear impactions were more damaging to the cartilage than normal impactions. Impactions were carried out using two servo-hydraulic load frames, one to apply axial loads, and another that created shear forces by displacing the patella perpendicular to the axial force. The damaged  patellae were stained with India ink, photographed, and the cracks in the cartilage were traced using the computer program Metamorph; the number of cracks, net crack length, area of impaction, and crack density were recorded.  One of the difficulties in comparing normal and shear injuries is that the area of damage from a normal impaction is much smaller than the area of damage resulting from a shear impaction.  While  a shear impaction induces a significantly greater total crack length than the normal impaction (p .0001), the crack density of both normal and shear impaction is not significantly different (p = .35). Cartilage cracks are only one potential measure of damage; these numbers do not take into account other consequences such as subsurface cracks and the  effects on the cartilage cells.  Further study of the

affects of different types of impactions on cartilage may lead to a better understanding of how cartilage damage leads to cartilage degeneration and osteoarthritis.

 

 

 

 

 

 

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