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