The 16th Annual
NC State University
Undergraduate Research Symposium
Biological Sciences:
Molecular,
Biochemical, Genetics, Cell Biology
Abstracts
Abstracts are listed in alphabetical order by the last name of the
corresponding author.
- Biological Sciences abstracts
Applied Sciences (Crop, Poultry, Animal, and Horticultural Sciences)
Ecology, Environmental, Conservation, Botanical
Molecular, Biochemical, Genetics, Cell Biology
Zoology, Physiology, Behavior, Neurobiology
- Engineering & Technology abstracts
- Humanities, Social Sciences, Psychology abstracts
- Physical and Mathematical Sciences abstracts
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Andrason, Casey E. |
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Department(s):
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Animal Science |
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Research |
Barbara
Sherry/Molecular Biomedical Sciences |
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Title of
Presentation: |
The
Development of a Screening Tool for a Reovirus T1L Mutant |
Interferons are cytokines that provide innate
protection for essentially all cells in response to viral infections. Viruses
can induce interferon-Beta (IFN-Beta), which is then secreted and binds to
other cells to induce antiviral gene expression and protection in those
neighboring cells. Induction of IFN-Beta is particularly important in
protection of cardiac myocytes against reovirus-induced myocarditis
(inflammation of the heart) in mice, which provides a good model for viral
myocarditis in humans. In general, nonmyocarditic reoviruses, such as T3D,
induce IFN-Beta well while myocarditic reoviruses, such as T1L, induce IFN-Beta
poorly. T3D is highly sensitive to the antiviral effects of IFN-Beta, in
contrast to myocarditic reovirus strains which are less sensitive to the
effects of IFN-Beta. We hypothesized that a novel selection procedure can be
developed to select T1L mutants that induce IFN. These T1L mutants could be
used in the future to identify phenotypes that vary concomitantly with
increased induction of IFN. In order to select T1L mutants that induce
interferon, we employed vesicular stomatitis virus (VSV), which is highly sensitive
to the antiviral effects of IFN-Beta. The overall strategy is to use T3D, a
virus known to induce IFN, in varying dilutions to see whether limited reovirus
infection is sufficient to protect against a subsequent challenge with VSV.
After finding a cell culture well that was not killed by VSV, and thus
protected by reovirus, 0.1% NP-40 would be used to lyse the cells and
inactivate the enveloped VSV while leaving the nonenveloped reovirus intact for
isolation and further amplification. After optimizing the assay using reovirus
T3D, the assay would be used to select T1L mutants for further study of
parameters of viral infection that induce IFN.
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Baker, Crystal |
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Department(s):
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Animal Science |
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Research |
Melissa
Ashwell/Animal Science |
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Title of
Presentation: |
Comparative
Mapping of Pig Chromosome 16 and Human Chromosome 5 |
Due to the vast economic impact of the swine industry
on
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Belton, Jon-Matthew |
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Department(s):
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Biochemistry |
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Research |
Trino
Ascencio-Ibanez/Biochemistry Linda Hanley-Bowdoin/Biochemisry |
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Title of
Presentation: |
Immunolocalization
of AL1, geminivirus replication factor, in Arabidopsis Plants Infected with
Cabbage Leaf Curl Virus |
Cabbage Leaf Curl Virus (CaLCuV) is a member of the
Geminiviridae family of plant viruses. Geminiviruses cause agricultural
epidemics world wide, devastating a variety of crops including cassava, tomato,
cotton and maize. Geminiviruses are characterized by their small, circular,
single-stranded DNA genomes with either one or two chromosomes and by their
double icosohedral viral particles. We used immunolocalization assays to study
the viral replication protein, AL1, in wild type and mutant lines of
Arabidopsis thaliana Col-0. Plants were inoculated with Agrobacterium
tumefaciens carrying T-DNA constructs with the A and B genomes of CaLCuV.
Microscopic analysis of vibratome-generated sections of infected plant tissues
revealed the relative concentrations of virus in specific tissues. This
information adds to our understanding of the infection process in the
Arabidopsis model system.
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Brewerk, Kyle D. |
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Department(s):
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Biochemistry |
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Research |
Mitch
Eddy/Gamete Biology Section, Laboratory of Reproductive and Developmental
Toxicology, National Institute of Environmental Health Sciences, NIH, DHHS,
Research Triangle Park, NC |
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Title of
Presentation: |
Novel
Testis-specific Genes on the Mouse X Chromosome |
In male mice, the X and Y sex chromosomes are
inactivated during meiosis and reactivated after meiosis, a feature that
contrasts to autosomes, which remain transcriptionally active. Even during the
post-meiotic period, 87% of genes on the X chromosome remain suppressed. Many
of the genes that are expressed during the post-meiotic period remain unique to
male germ cells and encode proteins required for the development and function
of spermatozoa. Testis-specific genes have been shown to be concentrated on the
X chromosome, implicating its specialized role in the sperm function and
suggesting the X chromosome to be a useful target for gene discovery. Also, as
a large majority of X-linked genes are suppressed in the post-meiotic period,
the genes that are expressed are likely pertinent in fertility. Bioinformatics
approaches were used to identify previously uncharacterized X-linked genes that
are testis-specific and expressed during the post-meiotic phase of
spermatogenesis. Four highly testis-specific genes were discovered and further
characterized by genetic analysis. Three of the genes express starting in the
post-meiotic period. These novel genes currently are identified by their
UniGene numbers, Mm.300779, Mm.350593, and Mm.296422. The gene Mm.300779
encodes a protein containing a Wiskott-Aldrich syndrome protein (WASp) Homology
2 (WH2) domain, involved in actin cytoskeleton regulation in other proteins.
Mm.350593 is similar to Xmr (Xlr-related, meiosis regulated), a gene with an
unknown function that may have a role in DNA recombination or chromatin
condensation. The gene Mm.296422 has high identity to Tes (testis derived
transcript), possibly resulting from being reverse transcribed and integrated
into the X chromosome as an intronless, testis-specific retrogene.
Immunohistochemical staining for rat Tes (testin) demonstrates it is present
only in Sertoli cells in the testis, implying Mm.296422 may replace Tes
function in germ cells. Known interactions of Tes suggest a possible
involvement of gene Mm.296422 in focal adhesion.
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Callahan, Jason M. |
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Department(s):
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Animal Science |
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Research |
Jason
Vittitow/Opthalmology |
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Title of
Presentation: |
Effects of
Latrunculin B on Morphology and the Actin Cytoskeleton of Cultured 3T3 Cells |
Latrunculin B (Lat B), a macrolide isolated from the
marine sponge Latrunculia magnifica,
is a specific and potent actin-disrupting agent that sequesters monomeric
G-actin, leading to the disassembly of actin filaments, cell-cell and
cell-extracellular matrix adherens junctions in several types of cultured
cells. Our objective here is to characterize the dose- and incubation
time-dependent effects of Lat B on actomyosin contractility and cell
morphology. 3T3 cells were plated in 6-well plates coated with Poly-L-Lysine.
The seeding density used was 80,000 cells per well that were cultured to 70%
confluence. Cells were treated with Lat B at 0.625uM, 0.125uM, 0.1875uM,
0.25uM, 0.5uM, 1uM for time periods of 15, 30, 45, 60, 90, and 120 minutes.
After treatment, these cells were fixed with 4% paraformaldehyde for 10 minutes
and stained with phalloidin-TRITC, which stains the actin microfilaments and fluoresces red, and DAPI, stains the nucleus of the cell,
for 1 hour to be able to analyze the effects of the compound. A
phalloidin-TRITC/DAPI solution was made by adding phalloidin to PBS in a 1:800
dilution ratio and DAPI was added with a 1:10,000 dilution ratio. The
morphology of the cell and distribution and organization of the actin
cytoskeleton were visualized using immunofluorescence and digital microscopy.
Lat B induced a pronounced effect on the morphology, intracellular separation,
and disruption of actin filaments in 3T3 cells. The ideal time point and
concentration of Lat B that had a robust effect on the cells was 30 minutes at
0.25 uM. Following compound removal from the media the cells, there was a
period of recovery seen around 60 minutes suggesting that this process is
reversible.
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Carr, Benjamin D. |
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Department(s):
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Microbiology |
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Research |
Jonathan
Olson/Microbiology |
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Title of
Presentation: |
A Campylobacter jejuni Quorum Sensing
Mutant Exhibits Reduced Biofilm Formation |
Campylobacter jejuni is an enteric pathogen transmitted through poultry,
and is estimated to cause more food poisoning yearly than E. coli and Salmonella combined. As a microaerophile that subsists
on metabolic products of many other bacteria, it thrives in the highly
populated environment of the intestine, where oxygen levels are low and
temperature is high. However, the mechanisms of
C. jejuni survival are not well understood in more
hostile environments, outside the gastrointestinal tracts of its human or avian
hosts. Biofilm formation has been suggested as one such mechanism. Biofilm in
this project refers to a community of cells that aggregate and adhere to a
physical interface, such as the liquid-solid interface in a test tube, and
surround themselves with an extracellular polymeric substance, or “slime.” Various
events in biofilm formation are controlled by quorum sensing, a process wherein
microbes use concentrations of small intercellular signaling molecules to
detect a population threshold (the quorum) at which biofilm-associated genes
are up- and down-regulated. It has been suggested that the cell-cell
communication molecule Auto-Inducer II (AI-2) plays a role in C.
jejuni biofilm formation via the
LuxS signaling pathway. This project investigated the role of the luxS gene in
C. jejuni biofilm formation by constructing a luxS mutant strain. A method for quantifying
biofilm formation was developed and used to compare biofilm formation of
wild-type strains to that of luxS mutants in a series of 4-day assays. Results
indicate that biofilm formation is significantly depressed in luxS mutants.
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Drohan, Shannon M. |
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Department(s):
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Gentris
Corporation, Clinical Genetics |
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Research |
Scott
Clark/Gentris Corporation, Clinical Genetics |
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Title of Presentation: |
Linkage
Between CYP2C8 and CYP2C9 Polymorphisms in a Hispanic Population |
Cytochrome P450 (CYP) enzymes are involved in the
metabolism of many clinical drugs and endogenous compounds such as arachidonic
acid and epoxyeicosatrienoic acids. The metabolism of these substrates can be
increased or decreased by isoenzyme genetic polymorphisms. A linkage between
CYP2C8 and CYP2C9 polymorphisms has been previously described in Caucasian
populations (Yasar, 2002). Sixty-nine subjects from four different ethnic
groups were tested for polymorphisms of CYP2C8*2, CYP2C8*3, CYP2C8*4, CYP2C9*2,
and CYP2C9*3 using a novel allelic discrimination assay (Sequence Detection
System 7900HT). A linkage between CYP2C8 and CYP2C9 was confirmed in Caucasians
and was found to be present in Hispanic populations. All subjects carrying a
CYP2C8*3 polymorphism carried a CYP2C9*2 polymorphism with a 100% correlation.
Polymorphisms at these two loci were not found in an Asian population, and
found in only one subject of an African American population. The strong
correlation between CYP2C8 and CYP2C9 single nucleotide polymorphisms may be
clinically relevant when prescribing multiple medications in specific ethnic
groups.
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Edathil, Roshen T. |
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Department(s):
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Structural and
Molecular Biochemistry |
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Research |
William L.
Miller/Biochemistry |
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Title of
Presentation: |
Amplifying
Plasmids for Identifying Transcription Factors Responsible for Activin Induction
of Follicle Stimulating Hormone |
Follicle Stimulating Hormone (FSH) is required for
egg and sperm production in mammals. FSH is an α/ β heterodimer with FSHβ controlling overall
expression. Because of its importance, FSHβ is controlled by more than 6
hormones, one of which is activin-A, a transforming growth factor beta
(TGFβ) family member. Activin typically causes kinases to phosporylate and
activate transcription factors such as Samd2 and Smad3 which partner with Smad4
and other transcription factors to form a nuclear transcription complex on the
FSHβ promoter that induces FSHβ transcription. Our laboratory’s
overall strategy for identifying all the transcription factors that aggregate
to induce FSHβ transcription is to allow these factors to bind DNA
sequences (~ 25 bp in length) from the FSHβ promoter that are known to be
critical for activin action. Once bound, they can be isolated using techniques
that selectively sequester the DNA along with its bound nuclear transcription
factors. Subsequent identification will be performed using HPLC techniques
linked with Mass Spectral analyses. To increase the likelihood that the correct
Smads will bind and form complexes with unknown transcription partners, it is
necessary to amplify plasmids that can be used to increase the production of
Smads 2, 3, and 4 in LβT2 cells. High levels of these Smads will increase
the likelihood that partner proteins will bind the target DNA. Shown on this
poster are figures that describe the nature of the plasmids I amplified this
semester. Furthermore, evidence from EMSAs (electrophoretic mobility shift
assays) show that Smad 4 can bind to the critical FSHβ promoter sequence
currently being studied in our laboratory and that transfection with the
plasmids I prepared increases this binding. Future studies will determine
binding for Smads 2, 3, and other partners.
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Fulp, |
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Department(s):
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Molecular and
Structural Biochemistry |
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Research |
Jeffrey Yoder/CVM-Molecular
and Biomedical Sciences |
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Title of
Presentation: |
Novel
Immune-Type Receptors in the Rainbow Trout |
The purpose of this project is to identify and
characterize the Novel Immune-Type Receptor (NITR) genes in Rainbow Trout Oncorhyncus mykiss. NITR genes encode
cell surface proteins that are hypothesized to play a role in innate immunity.
NITRs are encoded by a complex multi-gene family in multiple fish species and
are proposed to play a role in the detection of virally infected and
transformed (cancer) cells. Although 36 NITR genes have been identified in
zebrafish, only 4 NITR genes have been characterized in rainbow trout
suggesting additional NITR genes remain to be identified in this agriculturally
important species. We have utilized genomics and PCR strategies to identify
partial sequences of additional NITR genes and transcripts in trout. The
definition of these genes in trout will contribute to a better understanding of
innate immunity in this species and may advance aquatic medicine by developing
biomarkers for infection or cancer.
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Grimes, Shavon |
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Department(s):
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Research |
Cynthia N.
Cudaback/Marine, Earth and Atmospheric Sciences |
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Title of
Presentation: |
The Other Side of Fertilizer |
I have reseached the harmful effects of fertilizers
in aquatic systems. I found that fertilizers act as plant nutrients to aquatic
plants causing algae to rapidly grow. This rapid growth is caused by the two
main chemical (phosphate and nitrates) used in fertilizer and in the natural
plant process. As the algae begins to grow so does the amount of bacteria
anaerobic. These anaerobic bacteria uses oxygen to break down algae. When algae
starts to decompose it uses up all oxygen needed to make aquatic life
sustainable. In attempt to prove that fertilizers are harmful to aquatic
systems by depleting it of its oxygen, I have conducted two trails of ten day
periods where each day I add specfic amount of the two main chemicals found in
fertilizer (Phosphates and nitrates). In my results I found that water
contaminated with phosphates had 4ppm of oxygen, some water with nitrates had
4ppm, and water with both phosphates and nitrates had 8ppm of oxygen.
Phosphates and Nitrates are harmful when separate, but when they act as a team
they cause greater damage.
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Hancock, Brynne L. |
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Department(s):
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Microbiology |
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Research |
Eric S.
Miller/Microbiology |
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Title of
Presentation: |
Conjugation and Genetic Recombineering Systems for Vibrio parahaemolyticus and its phage
KVP40 |
Conjugation is a method of gene transfer that can be
used to mobilize plasmids from cell to cell. Genetic recombineering is a
technique used to introduce genes from one organism into another organism, or
from one DNA to another DNA, by homologous recombination. The aim of this
project was to develop conjugation and recombineering systems for use with Escherichia coil and Vibrio parahaemolyticus. Once these
procedures are optimized, genetic systems for V. parahaemolyticus and its lytic phage KVP40 will be available.
Mobilization of pRK2013 from E. coli
to E. coli worked with a transfer
efficiency of 1.0. Mobilization of pRK2013 from E. coli into V.
parahaemolyticus was shown to be relatively inefficient, with the exception
of a single experiment resulting in a transfer efficiency of 0.04. Tri-
parental matings from E. coli to E. coli ranged in frequency from 0.01 to
0.04 and tri-parental matings into V.
parahaemolyticus were not successful. Genetic recombineering is now being
used to introduce nadV from KVP40
into the related E. coli phage T4
genome. By introducing the KVP40 nadV
gene into T4 it can be studied in the context of T4 – E. coli infection processes. The nonessential rapid lysis genes
(rI, rII, and rIII) from the T4 genome have been selected as alternative sites
where nadV can be inserted into the
T4 genome by genetic recombineering. The use of homologous recombination to
insert nadV into the T4 genome is in
progress.
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Heron, Linda L. |
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Department(s):
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Microbiology |
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Research |
Tim
Petty/Microbiology |
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Title of
Presentation: |
Development of
a Vaccine Virus that can be Produced in a New Cell Line |
I am currently developing a smallpox vaccine using
the modified vaccinia Ankara (MVA) virus that can be produced commercially in a
new cell line. MVA was made by repeatedly infecting chicken fibroblasts with
vaccinia virus. As a result, the viral DNA lost genes needed for growth in
other cell types, and MVA only grows well in baby hamster kidney cells and
chicken fibroblasts. The vaccinia virus gene, SPI-1 (serine proteinase
inhibitor 1) is missing from MVA. The SPI-1 gene is required for vaccinia virus
to grow in human lung carcinoma A549 cells, which cannot be infected by MVA. In
preliminary tests, I infected BHK and A549 cells with MVA, vaccinia virus, or
cowpox virus to ensure that the stock of MVA did not grow in A549 cells. The
vaccinia and cowpox viruses were positive controls. My hypothesis is that the
absence of the SPI-1 gene prevents MVA from infecting A549 cells. To test this
hypothesis, I used homologous recombination to insert the SPI-1 gene into MVA,
together with an enhanced green fluorescent protein (egfp) marker gene. The
egfp gene will cause infected BHK cells to fluoresce green and will be used so
the recombinant MVA/SPI-1 can be separated from the parent virus and purified.
Once recombinant virus has been purified, whether MVA/SPI-1 will grow in A549
cells will be determined in comparison with MVA as the negative control, and
vaccinia virus as the positive control.
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Huang, Cheryl T. Eaves, Brittney K. |
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Department(s):
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Molecular and
Structural Biochemistry |
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Research |
Edward C.
Sisler/Molecular and Structural Biochemistry |
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Title of
Presentation: |
Cyclopropene
Compounds Counteracting Ethylene at the Receptor Level in Bananas |
The use of cyclopropene compounds is known to
counteract ethylene responses by preventing ethylene binding at the receptor.
At various concentrations, compounds protect bananas for 12-36 days at 23C,
depending on the cyclopropene, by a 24 hour exposure. Bananas then respond to
ethylene and appear to give the normal ethylene response. Some volatile
compounds that have an X group are reacted with aqueous acid and X to form a
salt. X renders the compound non-volatile. As a salt, the compounds can be
applied outdoors and remain on the plant tissue to inactivate the receptor,
allowing an extended life. Readily soluble in diethyl ether, the compound is
mixed with the aqueous phase and diethyl ether allowed to evaporate. Bananas
were treated in the gas phase by pipetting a diethyl ether solution of the
compound on to filter paper and sealed in a jar with the banana. Bananas were
alternately treated by swabbing the aqueous compound on half of the banana as a
comparison of treated versus untreated. After a 24 hour exposure, the jar is
vented and exposed to 333 µl/L of ethylene gas for 15 hours. Disappearance of
chlorophyll was determined and firmness recorded. Bananas remained insensitive to
ethylene for 32-34 days. Minimum concentrations of cyclopropene compounds
required for protection by a 24 hour exposure ranged from 5.3 nl/L to 248 nl/L
depending on the size of the compound’s side chain and for a protection period
of 32-34 days. Both gaseous and aqueous salt compounds were shown to be
effective inhibitors of ethylene responses.
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Joe, Daniel |
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Department(s):
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Plant
Pathology and Chemistry |
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Research |
Steve
Lommel/Plant Pathology Stephan
Franzen/Chemistry Dan
Feldheim/Chemistry Dick
Guenther/Plant Pathology |
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Title of
Presentation: |
Conformational Dynamics and Structural Integrity of
Red Clover Necrotic Mosaic Virus as an Anticancer Drug Delivery Vector |
Advances in targeted therapies is a promising
approach to cancer treatment that minimizes side effects by selectively
blocking carcinoma cells, rather than indiscriminately killing both diseased
and healthy cells. Viruses were chosen as a vector for targeted drug delivery
because of its nature to selectively invade cells and, in some cases, maintain
structural integrity under physiological conditions. A soil-born plant virus,
Red clover necrotic mosaic virus (RCNMV), has been shown to exploit these viral
properties to function as a nano-vessel for chemotherapeutic agents like
doxorubicin and a delivery vector to cervical carcinoma (HeLa) cells. Its
structural integrity in vitro and after infusion has not been investigated. A
spectral method frequently used to investigate protein dynamics is electron
paramagnetic resonance (EPR). Experiments were performed to determine if
nitroxide spin labels could be attached to lysine residues located on the outer
surface of the viral protein coat. After determining that labels could be
attached to the wild-type virus, experiments were performed to determine if
doxorubicin-infused virus could also be labeled. Analysis of EPR signal
confirmed that the infused virus could be labeled while maintaining stability.
Current experiments are using this method to determine the shelf life of
infused virus. Another question related to infusion is its affect on the
infectivity of the virus. The preferred method to determine infectivity is to
serially dilute a test sample and inoculate the samples on a local lesion host
plant, Chenopodium quinoa. A comparison of the lesions from the wild-type virus
to that of the infused virus found an almost 100 fold reduction in infectivity.
The characterization of the effect of infusion on RCNMV is an important step in
the process of determining if this use of viruses may lead to the development
of improved anticancer drug delivery approach with high selectivity and low
systematic toxicity.
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Joshi, Prashant J. |
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Department(s):
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Microbiology |
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Research |
Amy
Grunden/Microbiology Casey
Theriot/Microbiology |
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Title of
Presentation: |
Characterization of Suspected Prolidase from Pyrococcus furiosus homolog II and Pyrococcus horikoshii homolog II |
Pyrococcus furiosus and Pyrococcus horikoshii are
hyperthermophilic archaea that proliferate in deep-sea thermal vents. Prolidase is a protein that has been isolated
and characterized from P. furiosus and has been shown to be effective in
neutralizing toxic organophosphate (OP) nerve agents, such as the biowarfare
agents sarin and soman. Prolidases not
only have the ability to hydrolyze the (C-N) peptide bond in dipeptides that
have a prolyl residue at the C-terminus (NH2-X-/-Pro-COOH), which is their cellular
function, they are also capable of cleaving the O-P, C-F, and P-F bonds found
in toxic OP nerve agents and pesticides.
Pyrococcus prolidase is highly thermostable but shows little
activity below 50oC. Genome analyses of P. furiosus and P.
horikoshii led to the identification of two sets of homologs of the
previously characterized P. furiosus prolidase, termed Pf prolidase
homologs 1 and 2. The P. furiosus and P. horikoshii prolidase
homolog 2 genes have been amplified, cloned into expression vectors, and
recombinantly expressed in Escherichia coli. These homologs are of
interest because they have 2 out of the 5 metal binding sites that are
conserved in typical prolidases. P. furiosus homolog 2 is 43% similar to
the characterized P. furiosus prolidase, while P. horikoshii homolog 2 has 42% similarity. To determine if these homologs functions as
prolidases, metal and substrate preferences were determined for each homolog by
using aerobic and anaerobic conditions for prolidase activity assays. The results
of the aerobic and the anaerobic assays show little to no activity with both
the preferred substrate, leucine-proline, and metal, Co2+, of the
characterized P. furiosus prolidase.
Using bioinformatic tools such as BLAST and Clustal alignment we have shown
that the homologs show some homology to another protease, methionine
aminopeptidase. Future studies, including a methionine aminopeptidase assay,
will be done to further aid in determining the function of these proteins and
the role they play in hyperthermophilic organisms.
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Killela, Patrick J. |
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Department(s):
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Genetics |
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Research |
Fang
Bao/Genetics and Robert G. Franks/Genetics |
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Title of
Presentation: |
Reverse Genetic Analysis of the SEUSS-LIKE Family
of Transcriptional Regulators |
SEUSS and three SEUSS-LIKE
genes (SLK1, SLK2 and SLK3)
comprise a family of transcriptional co-regulators in Arabidopsis thaliana that share sequence similarity. The seuss mutants have been shown to exhibit
defects in floral morphology, however loss-of-function mutant phenotypes for
the other members of the SLK gene
family are not yet characterized. We want to determine what degree of
functional similarity these genes share. We are screening available T-DNA
insertion lines to identify loss-of-function alleles in SLK1, SLK2 and SLK3. We are verifying the insertion sites for these
T-DNA mutants by sequencing amplified flanking genomic DNA sequences.
Additionally, we are using quantitative real-time PCR to determine if RNA
transcript levels from these genes are affected by the T-DNA insertions. We
will also characterize any floral morphology defects in these T-DNA alleles.
Future work includes generating double and triple mutant combinations to
examine possible functional redundancy between members of this gene family.
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Lee, Winnie Y. Culver, Carolyn A. Eads, Dawn |
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Department(s):
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Microbiology |
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Research |
Scott M.
Laster/Microbiology |
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Title of
Presentation: |
Suppression of Prostaglandin Production by
Adenovirus Correlates with Coordinated Translocation of cPLA2 and
Vimentin-Containing Intermediate Filaments |
Adenovirus is a double-stranded DNA virus that can persist
in humans for seven to ten years and cause respiratory diseases,
gastroenteritis and conjunctivitis. Our laboratory is interested in
understanding how adenovirus is able to escape the immune system for this
length of time. We have found that adenovirus can suppress the activity of
cPLA2, an enzyme key to the production of inflammatory lipids. These
experiments were performed to understand the molecular basis for this effect.
Since cPLA2 can bind vimentin, and adenovirus can trigger intermediate filament
collapse, we hypothesized that cPLA2 might be caught in this collapse and
transported to an inappropriate position in the cell. To test this hypothesis,
we used immunofluorescence with antibodies specific for vimentin, cPLA2, and
the adenovirus E1A protein. Our experiments revealed condensation of vimentin
around the nucleus four hours after infection with adenovirus was initiated,
while intranuclear vimentin appeared within eight hours. We found that cPLA2
switched its intracellular location in a similar manner, first appearing in the
perinuclear region and later within the nucleus. Since it is unlikely that
cPLA2 can be active from these intracellular locations, we believe these data
support the hypothesis that adenovirus inhibits the activity of cPLA2 by its
mislocation within the cell.
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Little, Perry E. Petullo,
Brian T. |
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Department(s):
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Biology Biomedical
Engineering |
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Research |
Peter L.
Mente/Biomedical Program-Engineering |
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Title of
Presentation: |
Proteoglycan
Quantification of Experimentally Induced Post-Traumatic Osteoarthritis in Pig
Patellae |
The objective of this research was
to compare proteoglycan levels in pig patellae following an injury as an
indication of breakdown in the extra-cellular matrix and to model
osteoarthritic degeneration. Pig patellae were impacted in vitro with either
2000N (high), 1000N (medium), or received no impaction (control) and were then
put in culture at varying times of 0, 3, 7, or 14 days. The cartilage tissue
was removed from the bone and stained with Safranin-O. These tissues were then
analyzed in terms of staining intensity to determine proteoglycan levels as a
function of impaction level, time following impaction and location within the
cartilage relative to the impaction. Results of the testing indicated a trend
of higher to lower proteoglycan levels when moving medially and superficially
towards the center of impaction which suggests more degeneration in the
extra-cellular matrix closer to the site of impaction. There was also a general
decrease in proteoglycan levels with more time in culture which is indicative
of matrix breakdown but no significant effect was observed. There were no
significant differences in staining between control and medium impactions but
there was a significant decrease in staining following high impactions.
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Lomax, Aaron W. |
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Department(s):
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Plant Biology |
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Research |
Imara
Perera/Plant Biology |
|
Title of
Presentation: |
Characterization
of Gravity Sensing Mechanism |
Plant gravitropism is a complex process involving
different signaling pathways. We have shown previously that inositol
1,4,5-trisphosphate (InsP3), a second messenger generated in the
phosphoinositide (PI) pathway, increases in response to gravity in the pulvinus
of inflorescence stems of cereal grass stems and in inflorescence stems of
Arabidopsis. Furthermore, transgenic Arabidopsis plants expressing the human
type I inositol polyphosphate 5-phosphatase (InsP 5- ptase), an enzyme which
hydrolyzes InsP3, showed a delayed gravitropic response compared to wild type
plants (Perera et al., 2006 Plant Physiol. 140:746). This work is focused on
characterizing the role of InsP3 and its connection to other events in the
gravity signaling cascade such as amyloplast sedimentation and auxin
redistribution. To study the interaction between amyloplast signaling and the
PI pathway we used the starchless (pgm1) mutants. The pgm mutant has reduced
root , hypocotyl and inflorescence gravitropism (Caspar and Pickard 1989, Plant
177:185; Weise and Kiss 1999). To compare pgm-1 to wild type and InsP 5-ptase
plants we monitored inflorescence bending using time lapse photography. Wt
plants were shown to be the most responsive to gravitropism, re-orienting
between 90-120 minutes, while pgm-1 showed the most reduced response to
gravitropism finally re-orienting between . 5-ptase showed an intermediate
response re-orienting between 120-180 minutes. Pgm-1 inflorescences and seedlings
showed normal basal levels of InsP3 compared to wild type seedlings and
inflorescence.
|
Lopez, Harry |
|
|
Department(s):
|
Plant Biology |
|
Research |
Jeffrey W.
Gillikin/Plant Biology Rebecca S. Boston/Plant
Biology |
|
Title of
Presentation: |
Characterization of Maize Ubiquitin Fusion
Degradation Protein (Ufd1) during Seed Development and ER Stress |
Quality control in the endoplasmic reticulum (ER)
relies in part on moving misfolded proteins from the ER to the cytosol for
degradation. This process, known as ER-associated degradation (ERAD), requires
retrotranslocation of the degradation substrate and subsequent delivery to the
proteasome. As part of a larger goal to characterize the multiprotein machinery
responsible for early ERAD steps, we used bioinformatic tools to identify a
putative maize homolog of ubiquitin fusion degradation protein (Ufd1), a
cytosolic protein implicated in other systems in the ERAD process. Immunoblot
analysis using antibodies directed against a truncated version of the maize
Ufd1 protein revealed specificity toward both the recombinant Ufd1 protein from
maize and a band of the expected size from plant extracts. Ufd1 accumulation
was characterized by comparing predictions from mRNA expression data with
signals from immunoblots of different plant organs. To assay for changes in
Ufd1 accumulation during ERAD, we used maize lines that synthesize mutant
storage proteins and show induction of both molecular chaperones and components
of the ERAD machinery. Endosperm tissue from seeds with the normal complement
of storage proteins was compared with that from mutants for Ufd1, molecular
chaperones and mitochondrial protein controls over a time course of seed
development. Levels of Ufd1p changed during endosperm development and showed a
different accumulation pattern than the ER-resident molecular chaperone BiP.
Supported by the NSF-AGEP program, the RISE program at UPR-Cayey and the U.S.
Dept. of Energy.
|
Lowder, Casey D. |
|
|
Department(s):
|
Biological
Sciences |
|
Research |
Yang Ju
Im/Plant Biology Wendy F.
Boss/Plant Biology Amy M.
Grunden/Microbiology |
|
Title of
Presentation: |
Overexpressing
Pyrococcus furiosus Superoxide Reductase
in Arabidopsis Enhances Drought and Herbicide Tolerance |
Reactive oxygen species (ROS) are produced during
plant metabolism and in response to environmental stimuli. ROS, such as
superoxide, hydrogen peroxide, and oxygen radicals, can be used as signaling
molecules in plants and are usually kept in check by antioxidants. However, if
ROS are allowed to build up over time, cell damage may result. Pyrococcus furiosus superoxide reductase
(SOR) is a heat-stable archaeal enzyme that reduces superoxide without the
production of oxygen unlike plant superoxide dismutase (SOD). Our lab has
previously expressed functional P.
furiosus SOR in Arabidopsis, and SOR expressing Arabidopsis showed
increases in heat tolerance. Our hypothesis was that the presence of SOR would
also increase plant performance when exposed to other stresses such as drought
and herbicides. These stresses generate excess ROS, which can be reduced more
efficiently by the SOR reducing system. When Arabidopsis plants were exposed to
drought conditions we found that our 2 independent transgenic lines, SOR3 and
SOR9, appeared to be more resistant than wild type. The rate of water loss of
detached leaves from SOR3 and SOR9 was 22.4 ± .7% and 26 ± .2%, respectively,
lower than wild type and GFP vector control. Furthermore, when treated with
paraquot, a superoxide generating herbicide, transgenic SOR plants showed an
increased resistance compared to the wild type. This study leads us to believe
that the SOR reducing system in plants plays an active role in reducing ROS in
response to drought and herbicidal stress and thereby increases survival.
|
Morckel, Allison R. |
|
|
Department(s):
|
Biological Sciences |
|
Research |
Nanette M. Nascone-Yoder/Molecular
Biomedical Sciences |
|
Title of
Presentation: |
The Role of
Retinoic Acid in Xenopus laevis Gut Development |
The synthesis of Retinoic Acid (RA) is important in
the development of many organs. Treatment of frog embryos (Xenopus laevis) with
DEAB, a chemical that inhibits the activity of aldehyde dehydrogenases,
including the retinaldehyde dehydrogenases that act in the synthesis of RA,
causes digestive organ deformities. To further test whether a deficiency in RA
underlies these deformities, the activity of alcohol dehydrogenases, which also
function in the synthesis of RA, was inhibited by treatment with ethanol. In
addition, the function of an RA receptor was also inhibited by treatment with
RO 41-5253. Both ethanol and RO-41-5253 elicted phenotypic changes in the
digestive organs similar to those induced by DEAB, providing strong evidence
that DEAB affects RA synthesis, and that normal RA levels are essential for
normal gut development. By further analyzing the changes in gene expression
patterns that occur in chemically treated embryos by in situ hybridization,
this work also provided insight into the mechanisms by which these chemicals
affect gut development.
|
Myers, Amber D. |
|
|
Department(s):
|
Microbiology |
|
Research |
Hosni M.
Hassan/Microbiology |
|
Title of
Presentation: |
Oxidative Stress and the Antioxidant Defense
Responses in Streptococcus mutans |
Streptococcus
mutans is one of the
most cariogenic of all oral Streptococci and the leading cause of dental
caries, an oral disease that is caused by the microbial production of acid that
breaks down tooth enamel. S. mutans
are facultative anaerobes that get their energy through anaerobic metabolism,
regardless of the presence of oxygen. Because oral Streptococci are constantly
exposed to air, it is important to understand how S. mutans respond to oxidative stress. In order to survive under
exposure to air, oral bacteria must have developed defense systems against
reactive oxygen species (ROS) produced by activated macrophages and
neutrophils. This experiment compares the growth of S. mutans under shaking aerobic, static aerobic, and anaerobic
environments and the activity of enzymes such as superoxide dismutase (SOD) and
catalase when grown under those three conditions. It appears that exposing S. mutans to higher concentrations of
oxygen results in greater expression of SOD and catalase, as defense against
toxic ROS. This research also examines the effect of different anti-microbials
and different concentrations of anti-microbials on S. mutans. S. mutans
appear to be resistant to the chosen toothpastes tested as well as paraquat.
However, they are least resistant to hand-soap. Understanding the physiology of
S. mutans and their responses to
different environmental conditions, ROS, and anti-microbials will help us to
learn to fight off their survival in the oral cavity and thus potentially
decrease the occurrence of dental caries caused by S. mutans.
|
Park, Ji-Seon |
|
|
Department(s):
|
Biochemistry |
|
Research |
Robert B.
Rose/Biochemistry |
|
Title of
Presentation: |
The Use of DCoH/HNF-1 Fusion Protein as a Tool to
Analyze the Activities of a Bifunctional Protein |
DCoH (dimerization cofactor of HNF-1) is a
bifunctional protein which functions as a metabolic enzyme in the cytoplasm and
a transcriptional coactivator in the nucleus. In the nucleus, DCoH interacts
with the transcription factor HNF-1-alpha (hepatocyte nuclear factor-1-alpha)
and stabilizes HNF-1-alpha dimers. Significantly, mutations in HNF-1-alpha are
the most common cause of Maturity-onset diabetes of the young (MODY). We have
generated a DCoH/HNF-1 fusion protein to determine: 1) whether the two functions
of DCoH are independent and 2) how DCoH increases the transcriptional activity
of HNF-1. The DCoH and HNF-1 coding sequences were linked by Polymerase Chain
Reaction (PCR) using a primer overlap strategy and ligated into a mammalian
expression vector. The DCoH/HNF-1 fusion protein will be tested for enzymatic
activity to determine whether formation of the interaction with HNF-1 might
regulate the enzymatic activity of DCoH. One study has concluded that DCoH does
not lose its enzymatic activity when it is bound to HNF-1. However, this study
did not determine whether the DCoH/HNF-1 complex was stable throughout the
study. Structural studies of the DCoH/HNF-1-alpha complex indicate an active
site residue of DCoH is involved in interaction with HNF-1, suggesting the
DCoH/HNF-1 complex should be enzymatically inactive. The fusion protein will
prevent dissociation of the complex during the enzyme assay. The fusion protein
will also allow us to determine how DCoH increases the transcriptional activity
of HNF-1. Our current hypothesis is that DCoH increases the half-life of HNF-1.
|
Pistole, Carla M. |
|
|
Department(s):
|
Microbiology |
|
Research |
Amy M.
Grunden/Microbiology |
|
Title of
Presentation: |
Catalase and Ascorbate Peroxidase Activity in
Transgenic Arabidopsis thaliana
Expressing Archaeal Superoxide Reductase |
As science pushes mankind beyond terrestrial
boundaries, it becomes essential to design sustainable life support systems for
use in manned space missions. Plants play an integral role in maintaining
atmospheric conditions that are suitable for human life, yet they themselves
are also susceptible to the harsh conditions found in space exploration.
Notably, plants produce reactive oxygen species (ROS), such as superoxide, in
response to environmental stress; normally these ROS act in signal pathways,
but under sustained adversity they can accrue in tissues and cause cell death.
To combat this, plants produce superoxide dismutases (SOD), which degrade
superoxide into hydrogen peroxide and oxygen. However, when plants experience
sustained environmental stress, the oxygen produced by superoxide dismutases
can induce further, detrimental generation of ROS. This issue was corrected
through engineering transgenic Arabidopsis
thaliana strains with the enzyme superoxide reductase (SOR) from the
hyperthermophilic archaeon, Pyrococcus
furiosus . SOR, like SOD, converts superoxide to hydrogen peroxide. Unlike
SOD, superoxide reductase does not generate oxygen that can then produce more
ROS. In addition, P. furiosus SOR is
heat-stable, thus enabling the plant to manage high ROS levels under adverse
environmental conditions. Yet increased SOR activity yields a secondary
problem, as it causes amplified production of hydrogen peroxide. Though not as
toxic as superoxide, hydrogen peroxide can also induce tissue damage.
Fortunately, Arabidopsis naturally
produces the enzymes catalase and ascorbate peroxidase, which convert hydrogen
peroxide into harmless byproducts. Our hypothesis was that stressed Arabidopsis would upregulate production
of catalase and ascorbate peroxidase in response to the increased hydrogen
peroxide levels produced by the archaeal SOR enzyme. Through protein activity
assays it was found that the catalase enzyme was indeed upregulated in the
SOR-expressing Arabidopsis plants
compared to wildtype, under both control and drought stress conditions.
Conversely, there was no change in activity levels of ascorbate peroxidase.
|
Quintero-Varca, Tatiana |
|
|
Department(s):
|
Microbiology |
|
Research |
Amy M.
Grunden/ Microbiology |
|
Title of
Presentation: |
Expression and Biochemical Characterization of Pyrococcus horikoshii Prolidase Homolog
1 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 50C 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) for detoxification of OP nerve agents, this
gene was cloned into the T7 RNA polymerase-based expression vector pET 21b.
Over-expression of the P. horikoshii
prolidase homolog 1 protein 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 homolog 1. It was determined that the protein was
thermostable and that it had significant activity when cobalt is present in the
reaction mixture and when the dipeptide Methionine-Proline is used as the
substrate.
|
Reed, Rachel A. |
|
|
Department(s):
|
Molecular Biomedical
Sciences |
|
Research |
Nanette
Nascone-Yoder/Molecular Biomedical Sciences |
|
Title of
Presentation: |
The Effect of Rho Kinase in the Gut Morphogenesis
of Xenopus laevis |
Rho GTPase and its effector enzyme
|
Ricks, Jennifer L. Overton, Matthew H. Loo, LiNa Overton, Kenneth W. |
|
|
Department(s):
|
Biochemistry
and Chemistry Biochemistry
and Microbiology Chemistry Chemical and
Biomolecular Engineering |
|
Research |
Richard
Guenther/Chemistry and Plant Pathology Stefan
Franzen/Chemistry |
|
Title of
Presentation: |
Modified RCNMV as a Method for Delivering
Doxorubicin to Cancer Cells |
Most current chemotherapy drugs attack rapidly-dividing
cells, whether healthy or malignant. These indiscriminate treatments can result
in debilitating side effects. However, if therapies were targeted to cancer
cells, then many of these side effects could be avoided. A targeting method
under investigation is using the plant virus Red Clover Necrotic Mosaic Virus (RCNMV) to selectively deliver
Doxorubicin, a chemotherapy drug, to cancer cells. The structure of RCNMV
allows the virus to carry significant numbers of small drug molecules to cells.
RCNMV is targeted to malignant cells by attaching a small peptide to the viral
protein coat that binds to a receptor on the cancer cell surface. The cell
takes in the virus, which releases the drug and inhibits DNA replication within
the cell. Intake of Doxorubicin by RCNMV was measured by fluorescence
spectroscopy. We have found that this treatment works effectively against human
cervical carcinoma (HeLa) cells, and we are now investigating its effect on
human breast cancer cells that express another receptor, HER2. A future area
for research includes using peptide-labeled RNCMV to treat
Doxorubicin-resistant cancer cells.
|
Shelton, Julie E. |
|
|
Department(s):
|
Microbiology |
|
Research |
Amy M.
Grunden/Microbiology Casey M.
Theriot/Microbiology |
|
Title of
Presentation: |
Overexpression of PHD-like Zinc Finger Region of
Aminoacylase in Pyrococcus furiosus
|
Aminoacylase is an aminopeptidase that hydrolyzes
N-acyl-L-amino acids to produce a carboxylate and L-amino acid. Thermostable
aminoacylases are of interest to pharmaceuticals because they produce only
L-form amino acids instead of racemic mixtures that arise from chemical
syntheses, since D-form amino acids can be cytotoxic. Previously characterized
aminoacylase from Pyrococcus furiosus
is a metalloenzyme requiring Zn2+ for maximal activity. The P. furiosus aminoacylase gene shows two potential transcriptional
start sites, one producing a 441 amino protein (full-length aminoacylase) and
the second a 383 amino acid protein (truncated aminoacylase). A native version
of truncated P. furiosus aminoacylase
has been purified and characterized showing measurable aminoacylase activity;
however, the recombinant version had poor activity. Recently, reverse transcriptase
PCR experiments demonstrated that transcripts encoding both full-length and
truncated aminoacylase genes are produced in P. furiosus. The full-length aminoacylase gene contains an extra
174 bases, coding for a 58 amino acid extension on the N-terminus of the
truncated aminoacylase. This extended region contains 2 predicted metal binding
sites (CxxC) and (CxxxC) (amino acid positions 6-9 and 17-21 in the extended
sequence), which could be important to aminoacylase function. Searching the
SMART website, this extended region has only shown homology to a plant
homeodomain (PHD) finger. A PHD finger is a C4HC3 zinc-finger-like motif
involved in chromatin-mediated transcriptional regulation in eukaryotes.
Because of this zinc finger homology, it is hypothesized that the full-length
version may serve as a protein scaffold to help hold the metal in place to
create a functional aminoacylase. To study this PHD-like finger region, we
amplified the extended region of aminoacylase, cloned it into the pET vector,
and recombinantly expressed it in Escherichia
coli. For future studies, the PHD-like finger protein will be purified and
used to produce antibodies for purification and characterization of the native
full-length P. furiosus.
|
Smith, Ashley M. |
|
|
Department(s):
|
Toxicology |
|
Research |
Andrew D.
Wallace/Toxicology |
|
Title of
Presentation: |
Promoter Analysis of the Human Pregnane X Receptor
(PXR) Gene |
PXR is a nuclear transcription factor, highly expressed
in the liver, which is activated by binding to pesticides, environmental
contaminants, and many prescription drugs. Activation of PXR has been shown to
be responsible for the induction of a growing number of key metabolizing
enzymes such as cytochrome P450 3A4 (CYP3A4). Expression of metabolizing
enzymes is dependent on PXR levels in the cell, but little is known about the
mechanisms regulating PXR expression. The hypothesis of this research is that
key regulatory elements in the human PXR promoter control the constitutive and
inducible expression of the human PXR gene. This project aims to identify
promoter regulatory elements important for the constitutive and inducible
expression of the human PXR. To study the PXR promoter regulatory elements, a
2.3 kb portion of the PXR promoter was PCR generated with primers containing
restriction sites and cloned into a TA plasmid. By direction cloning, the 2.3
kb region of the PXR promoter will be ligated into the pGL4.13-Luciferase
reporter plasmid. The HepG2 human liver cell line will be transiently
transfected with the PXR-Luciferase reporter plasmid and promoter activity will
be measured. Activation of PXR has been shown to be responsible for inducing
key metabolizing including phase I enzymes such as CYP3A4, phase II enzymes,
and a number of transport proteins. Understanding was controls PXR expression
is important, because a number of studies have indicated that the magnitude of
the CYP3A4 induction is dependent on PXR expression levels. The regulation of
these key pathways by PXR can lessen the effectiveness of therapies or increase
drug toxicity. Also inappropriate metabolism of endogenous steroids such as
testosterone or estradiol may lead to disruption of normal endocrine pathways.
|
Tull, R. Read |
|
|
Department(s):
|
Biological
Sciences |
|
Research |
Nanette M.
Nascone-Yoder/Molecular Biomedical Sciences |
|
Title of
Presentation: |
Gene Expression in Left-right Asymmetric
Morphogenesis of the Xenopus Digestive
System |
A concentration gradient of retinoic acid is known to
influence important left-right asymmetries within the developing embryo, such
as the looping and rotation of the gut tube. In specific areas of the embryo, CYP26A1,
a retinoic acid hydroxylase, degrades retinoic acid to generate these
gradients, affecting cell proliferation, apoptosis, or other cellular events.
The transcription factor Pitx2 is also known to influence embryonic left-right
asymmetries and is expressed in a left-side only, asymmetric pattern during
embryonic development. We have shown that the genes for CYP26A1 and PitX2 are
expressed in complementary patterns on the left side of the developing Xenopus
laevis embryo, and that ectopic expression of either the CYP26A1 or PitX2 gene,
by microinjecting mRNA encoding these genes into early stage embryos, causes
abnormal left-right development of the gut tube. Although these data suggest
that these two genes interact within the same signaling pathway, little is
understood about how they might actually affect one another's expression.
Interestingly, in situ hybridization studies showed that injection of ectopic
CYP26A1 mRNA suppresses the expression of PitX2, whereas injection of ectopic
PitX2 mRNA induces the expression of CYP26A1. Histological and
immunohistochemical staining help demonstrate the intercellular effects that
PitX2 and CYP26A1 may have on the developing gut. These results provide
valuable information concerning the development of left-right asymmetry during
vertebrate digestive system morphogenesis.
|
Wolf, Rebecca L. |
|
|
Department(s):
|
Animal Science |
|
Research |
Jill A.
Barnes/Molecular Biomedical Sciences |
|
Title of
Presentation: |
The Role of Hsc70 as a Co-Activator for the
Estrogen Receptor in MCF-7 Human Breast Cancer Cells |
Breast cancer is the second leading cause of death
among women, so finding new targets for treatment remains a top priority.
Estrogen Receptors (ER) play a role in the growth of breast cancer tumors and
their transcriptional activity is enhanced by the presence of co-regulatory
proteins. The presence of the inducible form of heat shock protein 70 (Hsp70),
a protein chaperone, in breast cancer tumors has been highly correlated with
estrogen induced cell proliferation and ER+ status. Cells also have a
constitutive form of Hsp70, Hsc70. Because of their similarities, these
experiments were designed to determine if Hsc70 also acted as a co-activator of
the estrogen receptor. To test this hypothesis, two stably transfected MCF-7
human breast cancer cell lines (DD6 and AA10) were made in which the levels of
Hsc70 (basal versus overexpression) can be regulated by doxycycline. These
Hsc70 cell lines were compared to another cell line (C14) which can overexpress
Hsp70. Growth curves were used to assess the effects of estrogen on cell
proliferation with or without the overexpression of Hsc70 and Hsp70. Western
blot analysis was used to verify the level of expression of Hsc70 in each cell
line. Real-Time PCR was used to determine the effect of Hsc70 on the estrogen
responsive pS2 gene. Results showed estrogen enhanced cell growth when Hsc70
was overexpressed, which was similar to what was seen in the Hsp70 overexpressing
cell line. Real-Time PCR showed a promising co-activator relationship in the
DD6 clone, but this was not observed in the AA10 clone.
|
Wunsch, Christina H. Medearis, Sarah |
|
|
Department(s):
|
Poultry
Science |
|
Research |
Matthew D.
Koci/Poultry Science |
|
Title of
Presentation: |
Expression of
Mx in response to Avian Influenza |
The threat of avian influenza (AI) affects not only
the health of our chicken industry, but can impact the health of our society as
well. Recent outbreaks of AI in
|
Zachary, Christopher L. |
|
|
Department(s):
|
Plant Biology |
|
Research |
Deyu Xie/Plant
Biology Lili
Zhou/Plant Biology |
|
Title of
Presentation: |
Molecular Characterization of Antioxidant
Proanthocyanidins in Genetically Engineered Tobacco Plants |
Proanthocyanidins are potent antioxidants with
multiple medicinal and nutritive benefits to human health, such as anti-cancer
and anti-arthrosclerosis. The goal of our research is to identify and
characterize the presence of proanthocyanidins in transgenic tobacco. We
genetically transformed tobacco plants by introducing the PAP1 and ANR genes,
which led to the formation of proanthocyanidins. To our knowledge, this is the
first time that proanthocyanidins have been identified in this type of
transgenic tobacco plant. We grew the plants in the campus greenhouse to a
sufficient size and harvested the leaf tissues at different times for our
studies. Protocols for extraction, purification, HPLC profiling, and
histological assay were developed for the identification of proanthocyanidins
in the transgenic plants. We ground the fresh leaf tissues in liquid nitrogen
and then extracted proanthocyanidins using seventy percent acetone, which was
later evaporated under reduced pressure. The leftover water phase containing
proanthocyanidins was extracted with a chloroform reagent to remove any
liposoluble substances, followed by an extraction with ethyl acetate. We
analyzed proanthocyanidins contained in the ethyl acetate phase by using thin
layer chromatography and visualized the separation using a DMACA reagent. This
helped us to distinguish monomeric, dimeric, and different oligomeric
proanthocyanidin molecules from the transgenic leaf tissues. HPLC-MS profiling
identified the presence of monomers epicatechin and epiafzelechin, dimers
procyanidin B2 and epicatechin-epigallocatechin and a trimer of
epicatechin-epicatechin-epicatechin. The identification of other unknown
oligomeric and polymeric proanthocyanidin molecules is under way. Genetic
characterization of the coupled PAP1 and ANR transgenic plants is currently
being carried out by PCR analysis. The application of proanthocyanidins from
transgenic tobacco plants will be discussed in the poster presentation.
|
Zelin, Samantha L. |
|
|
Department(s):
|
Microbiology |
|
Research |
Robert M. Kelly/Chemical
and Biomolecular Engineering |
|
Title of
Presentation: |
Expression and Isolation of Thermophilic
Sugar-Degrading Enzymes from Thermotoga
maritima and Caldicellulosiruptor
saccharolyticus |
Enzymes from extreme thermophiles enable the
expansion of biocatalysts by expanding the range of temperatures that can be
utilized. Particularly, the polysaccharide-degrading enzymes glycosyl
hydrolases from these microorganisms can be exploited in the various
applications in detergent production, sugar chemistry, lipid and oil chemistry,
and food processing. Recombinant forms of glycoside hydrolases from the
thermophiles Thermotoga maritima and Caldicellulosiruptor saccharolyticus
have been expressed and purified from Escherichia
coli to better comprehend their activity on different substrates.
Mannanases from T. maritima were
produced and studied on different glucomannans and locus bean gum, while
annotated β-xylosidases from C.
saccharolyticus are currently being produced in E. coli. Initial efforts have shown inclusion bodies being formed
during enzyme production, which have slowed efforts to study the enzymes.
[ 2007 Undergraduate Research Symposium
Main Page ]
Last modified Februrary 2007 by Sharon E. Hunt, WordHunting