Biotechnology by 761BJA

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The Biotechnology Laboratory provides a unique technology experience for a large portion of the
Jefferson student body. The program provides both a biotechnology training and research program
designed to transform traditional secondary molecular biology studies into an applications program that
will help students to experience the power of newly discovered research tools associated with
recombinant DNA technologies. The laboratory’s primary mission is to provide a laboratory research-
based program that allows its students to experience topics from bacterial transformation to DNA
mapping. In addition, the program provides students with the experience of new, leading-edge
technologies, such as bioinformatics, western blotting, rtPCR, and DNA sequencing.

Student:       Nathan Allen                            Description: This project is designed to
                                                       determine the microorganisms that inhabit the
Firm:          George Mason University                 area. Using extractions done by Kate Smith,
                                                       DNA from dive four samples was amplified to
Mentor:        Dr. Patrick Gillevet                    determine the presence and abundance of life in
                                                       the pushcore. This was done by means of the
Title:         Determination of Unique                 Polymerase Chain Reaction, using the primers
               Ecosystems in Gulf of Mexico            27F and 1492R. To ensure accuracy, additional
               Oil Seeps                               extractions were completed on the Bush Hill
                                                       samples using Bio 101, and similar results were
Background: Below the floor of the Gulf of             achieved. Serial dilutions, ranging from 1:10 to
Mexico lie vast oil fields. At certain locations,      1:10,000, were made with the samples, and
where the earth between the oil and the ocean is       PCRs done with each, to find the ones that
thin, oil slowly seeps up through the ground and       produced optimal results. The best PCR
is discharged into the water. The oil is at the        products, which contained the fluorescent
same temperature as the surrounding water,             version of the primer 27F, were then
which is very cold. This is one of the primary         fingerprinted. Due to the presence of distinct
differences between the oil seeps and the              peaks present in the fingerprint analysis, it was
thermal vents that exist elsewhere on the planet.      apparent that many organisms were present in
Another difference is that the bacteria that form      some abundance. Another PCR was then
the base of the food chain are methane digesting       performed using non-fluorescent primers, and
bacteria, as opposed to those at the                   these products were ligated, and then
hydrothermal vents, which are dependent upon           transformed, and placed on plates. Each distinct
sulfide compounds. Certain organisms thrive in         colony produced on this agar plate was then
this environment, such as giant tube and ice           used in a PCR reaction. The PCR, using the
worms, and many microorganisms. Because of             primers M10F and M10R, was successful for
the large amount of available oil, oil companies       many of the colonies. These products will be
are anxious to drill in the area. However,             used for sequencing. Once the sequencing is
environmental groups are concerned that if the         done, it will be possible to compare the
oil platforms disrupt or destroy the area around       sequences found with the NIH database to
the oil seeps, entire ecosystems may be lost.          determine what each of the organisms is, and if
                                                       any might be previously unknown life-forms.

Mentorship Projects - 2001                                         Biotechnology           Exhibit   1
Student:       Lisa An                               to initiate transcription. These general
                                                     transcription factors are required to assemble at
Firm:          Lombardi Cancer Center,               the promoter with RNA polymerase II, the
               Georgetown University                 enzyme that transcribes most eukaryotic genes.
               Medical Center                        This complex binds at the TATA box, which is
                                                     a short sequence of DNA that mostly contains
Mentor:        Dr. Luyuan Li, Assistant              thymine and adenine nucleotides.
                                                     Description: This study is aimed at identifying
Title:         Promoter of Vascular Endothelial      and locating the promoter of the VEGI gene.
               Growth Inhibitor                      The sequence of about 10 kilobases upstream
                                                     from the start codon of the open reading frame
Background: Vascular endothelial growth              is known. Five possible promoter regions have
inhibitor (VEGI) is a member of the tumor            been identified through a computer program on
necrosis family (TNF). It has various functions,     the Berkeley Genome Project website. The
including cell cytotoxicity, immune responses,       sequence was inputted into the program that
and regulation of cell proliferation. VEGI,          compared it to other known promoters to
member of this family, has about 20-30%              generate a list of possible promoter regions.
sequence homology to the TNF superfamily and         These five possible promoter regions were
is expressed mainly in endothelial cells.            amplified through polymerase chain reaction
Endothelial cells are involved in many different     (PCR). Then these DNA fragments were
biological activities including angiogenesis.        inserted into a vector called pGL3-basic with a
During this process, endothelial cells proliferate   Firefly luciferase reporter gene and transfected
and migrate toward the stimulus of                   into mammalian cells. Also, the cells were
angiogenesis, such as a cancer cell. They            transfected with an internal control vector and
eventually form capillaries; however, this type      normalizing vector called pRL-TK, in addition
of pathological angiogenesis seen in tumors          to the pGL3-basic construct. The pRL-TK
continues with no check even though new blood        vector contains the Renilla luciferase reporter
vessels have already formed. Previous research       gene. This vector will serve as a normalizing
has shown that VEGI is an endothelial cell           vector to take into account the variability of the
specific negative regulator of angiogenesis.         transfection from well to well. During the
        The gene for VEGI encodes a protein          luciferase assay, separate readings for the
consisting of 174 amino acids. There are four        Renilla and Firefly luciferase reactions were
isoforms of the protein, including a secreted        made.
form and a form that has characteristics of a                This study found that the positive
transmembrane protein. It has been shown that        regulator region of the VEGI promoter is found
murine colon cancer cells that express the           about 300 base pairs upstream from the start
soluble form of VEGI have significant reduction      codon and that the promoter is not endothelial
of vascularization. The VEGI protein can be          cell specific. This research has great
highly valuable in the future of angiogenesis-       significance because once the promoter is
based cancer therapies.                              known, further studies may be conducted on
        The promoter of a gene is the site           how the gene is regulated.
upstream from the start of the open reading
frame where general transcription factors bind

Mentorship Projects - 2001                                      Biotechnology            Exhibit    2
Student:       Bic Cung                             such a plasmid confers on Shigella the ability to
                                                    invade and move inter- and intracellularly. A
Firm:          Walter Reed Army Institute of        32 kb region on the invasion plasmid encodes
               Research                             the major proteins required for invasion. This
                                                    invasion-associated region has two operons: a)
Mentor:        Dr. Malabi Venkatesan                the Ipa proteins, which are secreted invasion
                                                    plasmid antigens or virulence factors; and b) a
Title:         Construction and Analysis of         type III secretion system with the mxi/spa
               ipaB Mutants by Making               genes, which encode approximately 20 proteins
               Deletions in Two Separate and        whose function is to present the effector
               Independent Regions of the           molecules (Ipa proteins) on the bacterial
               Protein                              surface.

Background: Shigellosis, a severe and highly        Description: A short sequence (approximately
infectious disease that kills more than one         15-amino acid in length) in ipaB has been
million people yearly, is caused by the             shown to react to antibody from subjects
enteroinvasive bacteria Shigella. Infection is      challenged with Shigella. To test its function in
transmitted via the oral-fecal route, whereas the   IpaB protein using molecular techniques. The
bacteria travel down the gastrointestinal tract     altered IpaB protein, so obtained, is compared
(GIT) and penetrate the epithelial lining of the    with a normal wild type IpaB protein for
large intestine, causing bacillary dysentery.       function by using it to complement a Shigella
Clinical manifestations of shigellosis may          strain SF620, which contains an IpaB mutation.
include but are not limited to watery diarrhea,     SF620 is noninvasive in epithelial cells in tissue
frequent passage of bloody stools with mucus        culture. When a normal IpaB protein is
and acute abdominal pain in combination with        introduced into SF620, using a plasmid clone of
rectal tenemus, fever, tenderness of left colon     the ipaB gene, invasion is restored in epithelial
upon palpation and presence of faecal               cells. Function (or lack thereof) of the deleted
leucocytes.                                         sequence is determined by an invasion assay
         The ability of Shigella to invade          done with SF620 complemented with plasmid
epithelial cells is encoded by a 220 kb plasmid     clones encoding normal or altered IpaB.
termed the “invasion plasmid.” The presence of

Mentorship Projects - 2001                                     Biotechnology             Exhibit   3
Student:       Bic Cung                              Description: The ability of Shigella to invade
                                                     epithelial cells is encoded by a 220 kb plasmid
Firm:          Walter Reed Army Institute of         termed the “invasion plasmid”. The presence of
               Research (WRAIR)                      such a plasmid confers on Shigella the ability to
                                                     invade and move inter- and intracellularly. A
Mentor:        Dr. Malabi Venkatesan                 32 kb region on the invasion plasmid encodes
                                                     the major proteins required for invasion. This
Title:         Analyzing Functions of ipaB           invasion-associated region has two operons: a)
               Epitopes in Shigella                  the Ipa proteins, which are the secreted invasion
                                                     plasmid antigens or virulence factors and b) a
Background: Shigellosis, a severe and highly         type III secretion system with the mxi/spa
infectious disease that kills more than one          genes, which encode approximately 20 proteins
million people yearly, is caused by the              whose function is to present the effector
enteroinvasive bacteria Shigella. Infection is       molecules (Ipa proteins) on the bacterial
transmitted via the oral-fecal route. The            surface.
bacteria travel down the gastrointestinal tract           A short region on ipaB has been shown to
(GIT) and penetrate the epithelial cells lining      react to antibody from subjects challenged with
the distal colon and rectum, causing bacillary       Shigella. To correlate the function of those
dysentery. Shigella secrets some 15 proteins         regions with structure in ipaB, the mutants of
that are crucial for invasion (effector molecules)   ipaB are created and then transformed into an
through the mxi-spa system. The four most            ipaB mutant of Shigella (SF620) and tested for
abundant proteins are Ipa (Invasion plasmid          restoration of biological activity. Various
antigen) A, B, C and D. All four Ipa proteins        mutants of ipaB are constructed using
are crucial to Shigella invasion, and most are       Strategene QuickChange Mutagenesis Kit. PCR
multifunctional. For example, IpaB also plays a      is the method by which the mutants are
role in forming contact with host cells using        constructed. Primers are designed with the
receptors. IpaB has also been implicated as the      desired mutations (ranging from single-base
factor causing apoptosis in macrophages              change to deletion of several amino acids). The
because bacteria without IpaB (or mutated IpaB)      mutants are tested for accuracy by DNA
have been observed to be unable to escape            sequencing. Mutants of ipaB are then
phagocytic vesicle.                                  transformed into SF620 and the function of the
                                                     mutants are assessed by Western Blot and
                                                     invasion Assay.

Mentorship Projects - 2001                                      Biotechnology            Exhibit   4
Student:       Melanie Dispenza                     and possibly help generate them from ES cells.
                                                    Different conditions were investigated in an
Firm:          National Institutes of Health        attempt to reach optimal generation of DA
                                                    neurons, which could apply to finding a better
Mentor:        Dr. Jonathan Auerbach                therapy or cure for Parkinson’s Disease.

Title:         Embryonic Stem Cell-Derived          Description: Stage V ES cells were plated with
               Dopaminergic Neurons Co-             fresh embryonic LGE in culture-specific media.
               cultured with Striatal Explants      A chicken plasma/thrombin clot was used to
                                                    hold the tissue in place, and the cultures were
Background: Parkinson’s Disease is a                incubated at 37º C in a rotating drum. Cultures
neurodegenerative disease caused by the death       were removed from incubation at different time
of dopamine-producing (dopaminergic) neurons        intervals, placed in a fixative, and stained with
located in the substantia nigra of the brain,       fluorescence-conjugated antibodies for
resulting in the debilitation of movement and       observance of tyrosine hydroxylase-positive
motor skills. As common treatments are not          (TH+) neurons with a fluorescent microscope.
efficient enough and only slow the progression      The number of TH+ neurons produced in each
of the disease, new therapies are being             culture was used as a measure of survival of DA
investigated. One possible treatment would be       neurons. Data was also correlated with
replacing dying neurons with cells grown in a       electrophysiological analysis of the synaptic
lab from embryonic stem (ES) cells. ES cells        properties of the DA neurons. If mature DA
can differentiate to form any type of cell found    neurons can be grown, they must also form
in the human body, and new research has been        functional synapses with the striatum in order to
applied to the manipulation of stem cell            be effective therapy for Parkinson’s patients.
differentiation specifically into dopaminergic      (Electrophysiological data will be correlated
(DA) neurons. The most efficient way to do this     with this project in a separate assay).
remains to be elucidated, as different procedures           Results from this project were
and conditions are being proposed and               cumulative, with one set of data determining the
researched.                                         next set of experimental cultures. Several
         This project attempts to grow DA           different growth conditions were tested with
neurons from ES cells together with striatal        these co-cultures, using different media and
brain tissue, specifically the lateral ganglionic   growth factors such as cyclic adenosine
eminence (LGE). In the midbrain, the striatum       monophosphate (cAMP), brain-derived
is the target area of DA neurons in the             neurotrophic factor (BDNF), and neurotrophin 3
substantia nigra. The hypothesis is that the        (NT3) in order to reach optimal generation of
striatal cells from the LGE tissue will help        DA neurons.
support the survival and growth of DA neurons

Mentorship Projects - 2001                                     Biotechnology            Exhibit   5
Student:       Megh Duwadi                          powerful technique of differential-display
                                                    polymerase chain reaction (DD-PCR), these
Firm:          Walter Reed Army Institute of        modifications in expression are being detected.
               Research                             The identification of gene expression patterns
                                                    unique to ischemic SC injury thus allows
Mentor:        Drs. Joseph B. Long and              neuroprotective therapeutic strategies to be
               Debra L. Yourick                     targeted and performed, reducing the effects of
                                                    glutamate mediated- and other types of neuronal
Title:         Alterations in Gene Expression       loss, a primary result of SC trauma.
               in Response to Ischemic Spinal       Methodology for the completion of these studies
               Cord Injury in Rats                  was investigated, modified, and applied to SC
                                                    tissue extracted from SC-injured rats.
Background: Traumatic injury to the central                  Ischemic SC injury was induced in rats
nervous system (CNS) is a leading cause of          by injection of the endogenous opioid peptide
death in civilian and military populations alike.   dynorphin A. Upon insult, dynorphin A
The amino acid glutamate (Glu) is the major         lowered lumbosacral blood flow, elevated
excitatory neurotransmitter in the CNS. Upon        cerebrospinal fluid lactic acid concentrations,
insult of ischemic spinal cord (SC) injury, a       and caused flaccid hindlimb paralysis and
model of CNS trauma offering ready                  striking neuropathological changes. Anaerobic
recognition of the degree of neuronal injury by     metabolism was increased in association with
the obvious measure of hind limb performance,       ischemia.
an overactivation of Glu receptors leads to                  DD-PCR is a relatively new technique,
substantial increases in extracellular Glu and      which, unlike conventional methods of
intracellular Ca2+. These changes have been         measuring modifications in gene regulation,
shown to largely mediate neuronal degeneration.     allows for the comparison of similar tissue types
Attenuation of Glu release minimizes further SC     and the identification and isolation differentially
damage; however, it is imperative that this         expressed genes. RNA isolated from dynorphin
procedure be performed near the onset of injury     A- and saline-injected spinal cord tissue (injured
to be successful. Identifying significant changes   and noninjured) was reverse transcribed from an
in neuronal gene expression may allow the           anchor primer containing a poly (dT) region,
targeting of specific downstream events caused      which targets the 3’ ends of mRNA. Low
by alterations in gene regulation, and              stringency, competitive PCR followed to induce
consequently, a greater window for SC injury        mismatching – it is this process that
treatment. The present study, which examines        differentiates DD-PCR from related procedures.
the possibility of therapeutic intervention in      By utilizing DD-PCR to analyze genes found in
downstream changes in gene expression               injured SC tissue, changes in gene expression
following CNS trauma in the form of ischemic        levels upon incidence can be readily detected.
SC injury, will perhaps lead to novel therapy in    Differentially expressed genes appear as bands
CNS injury.                                         present in the track from only one cell type.
                                                    Fragments can be excised from the DD gel,
Description: Previous studies involving             identified using GenBank and BLAST
ischemic SC injury have shown that dramatic         databases, and used to prepare gene tags for the
changes in neuronal gene regulation occur both      study of gene expression level alterations.
during and after incidence. By utilizing the

Mentorship Projects - 2001                                      Biotechnology            Exhibit   6
Student:      Erwin P. Gianchandani               was analyzed by “GLIMMER,” a computer
                                                  program written to recognize coding regions of
                                                  microbial DNA. The possible genes that were
Firm:         The Institute for Genomic
                                                  found were subsequently fed into computer
              Research (TIGR)
                                                  algorithms – Blast-Extend-Repraze (BER)
                                                  pairwise alignments and Hidden Markov
Mentor:       Dr. William C. Nelson
                                                  Models (HMMs) – that matched the ORFs with
                                                  genes of known functions. The resulting data
Title:        Re-Annotation of Helicobacter
                                                  were then combined by “autoBYOB,” which
              pylori and Analysis of
                                                  made preliminary gene assignments by
              Bioinformatics Techniques
                                                  classifying each gene into one of 113 different
                                                  role-specific categories.
Background: Helicobacter pylori is a
                                                           In addition to curating the “autoBYOB”
flagellated, motile, curved microaerophilic
                                                  assignments – gene name, type, and role, etc. –
Gram-negative rod that accounts for more than
                                                  the start sites selected by “GLIMMER” were
90 percent of all duodenal ulcers and up to 80
                                                  checked. It was also important to identify
percent of all gastric ulcers. The bacterium is
                                                  frameshifts or other disruptions within the
also attributed to mucosal-associated-lymphoid-
                                                  reading frames.
type (MALT) lymphoma as well as Sudden
                                                           Finally, upon conclusion of the re-
Infant Death Syndrome (SIDS) and morning
                                                  annotation process, a number of computer-
sickness. In 1997, TIGR successfully
                                                  generated outputs became critical in further
completed the sequencing and annotation of H.
                                                  understanding H. pylori, including the
pylori strain 26695. The published genome was
                                                  following: a “GC”-skew allowed for the
composed of a 1,667,867-basepair circular
                                                  identification of the origin of replication within
chromosome that was predicted to have 1,590
                                                  the organism; a trinucleotide skew helped
coding regions. However, nearly one-third of
                                                  distinguish those genes that may have been
these open reading frames (ORFs) had no
                                                  transferred into the genome from other bacteria;
matches to global databases of known genes.
                                                  a software program developed since 1997
Although many questions about H. pylori’s
                                                  analyzed rho-independent terminators and assist
pathology were answered with the release of the
                                                  in the understanding of operon structures; and
26695 genome, discovering the “unknowns”
                                                  repeat finders located those sequences that occur
was still essential to the development of cures
                                                  more than once within a genome and that may
for diseases of the likes of SIDS and MALT.
                                                  be ordered into paralogous families.
This project served to re-annotate the entire
                                                           Data analysis was conducted thereafter
sequence of strain 26695 using the enhanced
                                                  to make a distinction between the 1997
resources available today in the field of
                                                  annotation and the 2000 re-annotation. In
Bioinformatics. Specifically, the objectives
                                                  addition to investigating new or different gene
were as follows: identifying some of the 499
                                                  classifications, a step-by-step comparison
unknown genes from 1997 by matching them
                                                  between the two genomes during the annotation
with genes of known type and function;
                                                  process – number of genes identified by
verifying previous gene assignments, including
                                                  “GLIMMER” versus number of genes identified
updating names and references; and learning the
                                                  by 1997 technology, original number of
improvements in bioinformatics techniques by
                                                  database “hits” of the two annotations, and the
comparing the new and old data.
                                                  first classifications by “autoBYOB,” etc. –
                                                  validated the hypothesis.
Description: The sequence data from 1997

Mentorship Projects - 2001                                   Biotechnology            Exhibit   7
Student:       Brenda Goguen                          piscicida. Other causes of the fish lesions in
                                                      Virginia waters have been proposed to be the
Firm:          George Mason University, Prince        result of endosymbiotic bacteria associated with
               William Campus                         the putative amoeboid stages of P. piscicida.

Mentor:        Dr. Patrick M. Gillevet                Description: The purpose of this research was
                                                      to molecularly characterize microorganisms
Title:         Molecular Characterization of          associated with HABs, including potential fish
               Potential Fish Pathogens in            pathogens in Chesapeake Bay waters where
               Waters Where Reported                  reported Pfiesteria outbreaks and fish kills have
               Pfiesteria piscicida Outbreaks         occurred, to clearly show what the casual agent
               Have Occurred                          is. Bottom soil sediments from five river
                                                      systems (Potomac, Patuxent, Chester, Choptank,
Background: Over the past ten years, harmful          and Pocomoke) that discharge into the Bay were
algal blooms (HABs) have occurred in Atlantic         taken prior to this study. In order to molecularly
slope waters, including in the Chesapeake Bay         determine the variety of protists and bacteria in
region, killing millions of fish and causing          the rivers, the DNA extracted from the soil
human debilitation. It has been hypothesized          sediments was amplified through polymerase
that these events can be attributed to two            chain reaction (PCR) with fluorescently labeled
endotoxins released by the dinoflagellate             primers specific to protist and bacterial small-
Pfiesteria piscicida. In the protist kingdom, P.      subunit (SSU) regions of the DNA. There is an
piscicida represents a new family, genus, and         abundance of natural length variation in the
species of dinoflagellate that was first identified   SSU rRNA genes in microorganisms which is
and named the causal agent of fish deaths in          directly related to phylogeny. Through the use
Pamlico Sound, North Carolina in 1988. It has         of an amplicon length heterogeneity fingerprint
been proposed that P. piscicida has about 24          after PCR with fluorescent primers, it was
varying growth stages, including stellate             possible to differentiate between genera and to
amoeboid and flagellated zoospore stages.             determine the microheterogeneity of protists and
Additionally, it is hypothesized that in its          bacteria in the samples from the five rivers.
flagellated form, P. piscicida releases               Analysis of the fingerprints found that P.
endotoxins which may lead to ulcerated lesions        piscicida was present in small amounts in only
in fish and to large scale fish kills.                the Pocomoke, Patuxent, and Choptank Rivers,
         The presence of these endotoxins poses a     preliminarily indicating that P. piscicida may
concern for ecological and human health and for       not have had a role in the fish kills.
commercial and recreational fisheries.                Additionally, other DNA taken from
However, growing evidence exists that the fish        experimental fish tanks has been amplified by
kills in the Chesapeake Bay have not been the         PCR and fingerprinted for both the protist and
result of the proposed toxins excreted by P.          bacterial regions of the DNA. Analysis of this
piscicida or Pfiesteria-like organisms. Studies       data remains to be completed. The fingerprints
have reported that the proposed amoeboid forms        showing the bacteria from all samples will be
of P. piscicida, in fact, do not exist for the        used to help determine if the fish death is the
organism. Thus, stellate amoebae may be               result of endosymbiotic bacteria associated with
mistaken for the reported amoeboid forms of P.        the putative amoeboid stages of P. piscicida.

Mentorship Projects - 2001                                       Biotechnology            Exhibit   8
Student:       Tina Gupta                           highly unstable state, with a half-life of 20-30
                                                    minutes. The phosphorylation of p53, which
Firm:          Walter Reed Army Institute of        stabilizes the transcription factor, may be
               Research                             involved in the expression of the mustard
                                                    associated protease. Curcumin, a powerful anti-
Mentor:        Prabhati Ray, Ph.D.                  cancer agent, prevents the phosphorylation of
                                                    p53, and other biological molecules.
Title:         Protease Response to Alkylation
               Damage in Cultured Human Skin        Description: Protease expression was
               Cells                                identified with Western blotting analysis using a
                                                    polyclonal rabbit antibody raised against the HD
Background: Nitrogen mustard (HN-2, bis-(2-         induced protease from NHEK purified
chloroehtylthioethyl)-ether) and sulfur mustard     previously in this laboratory. The intracellular
(HD, bis-(2-chloroethyl) sulfide) are alkylating    calcium chelator, BAPTA-AM reduced HD
agents. HD has been used as a chemical warfare      induced protease expression. Topical
agent with severe consequences in the past, and     application of HD on hairless guinea pigs
was used more recently in the Iran-Iraq conflict.   verified in vivo protease induction. A light
Mustard-induced blister formation is an             microscopy study demonstrated that mustard
epidermal event accompanied by a separation of      exposed NHEK had remarkable morphological
the dermis from the epidermis due to the            changes. Treatment of cells with the protease
disruption of the connective tissues, possibly by   antisense oligonucleotides blocked
the action of some protease(s) at the epidermal-    morphological alterations. Western blot
dermal junction. Recent studies have                analysis of antisense treated cells is
demonstrated induction of a tumor-suppressant       forthcoming. NHEK incubated in curcumin,
nuclear phosphoprotein, p53, in mustard             may inhibit protease expression, however results
exposed NHEK. Typically, p53 exists in a            remain inconclusive.

Mentorship Projects - 2001                                     Biotechnology           Exhibit   9
Student:       Edward Kim                           FABP’s may have some role in prostate and
                                                    breast cancer.
Firm:          Walter Reed Army Institute of
               Research                             Description: Not much is known on how the
                                                    levels of FABP’s are regulated in cancer cells.
Mentor:        Dr. Rina Das                         A comparison was done between normal
                                                    prostate cells and cancer cells as a model
Title:         Regulation of Fatty Acid Binding     system. This study examined the levels of
               Proteins in Breast and Prostate      FABP’s in several prostate normal and cancer
               Cancer Cells                         cell lines in order to establish a correlation
                                                    between FABP levels and how several growth
Background: Epidemiological studies on
                                                    regulators affect them. The levels of expression
cancer of the prostate gland have shown a
positive relationship between the consumption       of mRNA for selected FAPB’s were analyzed
of dietary fats and development of prostate         using primers for RT-PCR. These levels could
cancer. The objective of the research was to        then be further affected towards or away from
determine the effects of cell growth regulators     the normal cell levels by the addition of growth
such as anti-sense, fatty acids, and cancer         regulators such as cancer fighting drugs, fatty
fighting drugs, on the expression of Fatty Acid     acids, and anti-sense specific for each FABP.
Binding Proteins (FAPB’s) and how these             Cancer cells would be grown in different growth
FABP levels in turn regulate the proliferation or   regulators, such as different fatty acids,
the apoptosis, systematic death, of prostate and    harvested, then analyzed for FABP expression.
breast cancer cells. FABP’s serve as                These levels would then be compared to a
transporters of bioactive lipids and whose up or    standardized control culture of cancer cells to
down regulation may play a role in cancer           determine the amount of change.
growth in prostate and breast cancer cells.                  The addition of the cancer fighting drugs
Several studies suggest that FABP’s increase the    Curcumine, MK 886, and NDGA to the cancer
solubility of fatty acids in the cell cytoplasm     cells brought the levels of both groups of
causing a new diffusion of fatty acids from the     FABP’s towards control levels due to the fact
plasma membrane to the intracellular membrane       that they are inhibitors of the arachidonic acid
compartments, making them more easily               metabolic pathway. Also, by adding anti-sense
available to cause cancer proliferation. The
                                                    of Liver and Breast, which work by blocking the
levels of these FAPB’s can be found by
measuring the mRNA expression levels in             expression of L-FABP and B-FABP
various strains of cancer cells, which are          respectively, the levels of other FABP’s varied
systematically grown in different mediums and       in response to the change in concentration of
growing conditions. In preliminary research it      one FABP. The addition of fatty acids such as
was found that Liver-FAPB and Intestine-FABP        linoleic and arachidonic acids did increase the
were up-regulated, in increased levels, in cancer   growth of cancer cells, but did not return the
cells, while Adipose-FABP, Epidermal-FAPB,          expected results. This shows that the levels of
and Heart-FABP were down-regulated, in              FABP’s were directly affected by the
decreased levels, compared to levels in normal      proliferation of cancer and normal cells. The
cells. FABP’s could act as potential markers for    detection of FABP levels in cancer cells and in
detection of prostate or breast cancer. By          the medium in which they grow can provide a
adding various cell growth regulators, FABP         means of identifying the aggressiveness of a
levels fluctuated accordingly, which show that      patient’s prostate or breast cancer.

Mentorship Projects - 2001                                     Biotechnology           Exhibit   10
Student:       Jonathan Lasken                      tested as one type of fertilizer and the sludge
                                                    was tested as another. The fertilizer made from
Firm:          ARCTECH, Incorporated                the sludge is termed actosol®-x and the one
                                                    from the supernatant is termed actosol®-s. Both
Mentor:        Mr. Randy Reed                       of these products were mixed to a forty to one
Title:         The Effect of Humic Acid-Based                Actosol®-x, actosol®-s, water, and
               Soil Amendments on the               Professional actosol®, a professionally
               Premature Stages of Plant Radish     manufactured and used fertilizer, were each
               Growth                               applied to five out of twenty pots, all of which
                                                    were filled with potting soil. Each pot was
Background: Recently the Department of              planted with three seeds and their germination
Defense has found it necessary to develop a         rates and shoot growth were recorded. The
more environmentally friendly way of disposing      plants were allowed to grow for thirty-five days
of its surplus propellant. The previous method,     at which point, the plants were uprooted from
termed open detonation, was the destruction of      the ground and both above ground and below
the munitions in a pit. Under the Department of     ground biomass were taken. This was done to
Defense’s “R3 Reduce/Recycle/Reuse” plan            see the effects of the various actosols® on the
ARCTECH has developed the Actodemil™                preliminary plant growth, both in shoot growth
technology. The Actodemil™ technology is            (above ground) and root growth (below ground).
used to convert the propellant from missiles,                Through this experiment, the various
often TNT or another highly explosive               actosols® were proven to be effective on their
substance, into a humic acid based fertilizer.      respective plants. Another purpose of this
This technology has been proven at Hawthorne        experiment was to try to infer what is contained
Army Depot, Nevada, and the University of           in the supernatant. It is known that the sludge
Nevada Las Vegas has done some preliminary          consists primarily of humic acid, but the
testing on the fertilizer, declaring it safe for    contents of the supernatant were unknown. This
commercial use. However, little testing has         experiment shone some light on what is
been done on the effectiveness of this fertilizer   contained in the supernatant. There are three
to this point.                                      extensions of this experiment. In the first
                                                    extension, the same experiment will be
Description: The direct resultant of the            conducted in sandy soil, and in the second
Actodemil™ technology is called actosol®-m          experiment, the actosol®-m will be turned into a
(actosol®-mixture). To separate the supernatant     fertilizer of its own and used on an additional
from the humic acid the pH of the actosol®-m is     set of pots along with the other four fertilizers
lowered to below 2 and the actosol®-m is            used in the experiment outlined above. The
centrifuged. Sludge forms at the bottom of the      final extension would be to mix dilutions of the
centrifuged container and supernatant forms         sludge and supernatant to find the most effective
above. In this experiment, the supernatant was      combination of the two.

Mentorship Projects - 2001                                     Biotechnology          Exhibit   11
Student:        Swan Lee                               resuscitation, it is important to characterize the
                                                       BBB changes resulting from combined
Firm:           Walter Reed Army Institute of          traumatic brain injury and hemorrhagic
                Research                               hypotension, to evaluate where loss of BBB
                                                       integrity might affect the efficacy of the
Mentor:         Dr. Debra Yourick                      resuscitation fluid.

Title:          Changes in the Blood Brain             Description: Evans blue dye (EB) was used to
                Barrier After Inducing Fluid           evaluate the blood brain barrier to the
                Percussion Injury and                  movement of molecules, during or after
                Hemorrhagic Shock                      combined injury. The dye concentration was
                                                       measured and compared in brains from rats that
Background: Traumatic brain injury is one of           were uninjured, fluid-percussion injured only,
the most frequent causes of morbidity and              hemorrhaged only, or injured and hemorrhaged.
mortality on the battlefield, and remains the                   Male Sprague-Dawley rats were injured
leading cause of traumatic death in the United         with a fluid-percussion device, and
States. The pathophysiological features of brain       subsequently, rats were hemorrhaged to a mean
injury seen in humans can be recreated through         arterial blood pressure of 40 mmHg over a
rodent fluid percussion injury (FPI), which is         period of 15 minutes, and maintained at that
considered to be one of the most effective             level for 60 minutes. After injection of the
models of traumatic brain injury. The aspect of        Evans blue dye, rats were resuscitated to 80
particular interest in this study is the destruction   mmHg with a lactated Ringer’s solution or
of the blood brain barrier (BBB). The BBB is           autologous blood for 60 minutes. Brains were
the barrier that exists between the blood and the      removed, dissected, and EB was extracted in a
cerebrospinal fluid which prevents the passage         50% trichloroacetic acid solution.
of various substances from the bloodstream to          Concentration of EB was determined using a
the brain. Traumatic brain injury disrupts the         fluorescence plate reader with appropriate
BBB, which can cause a complete breakdown of           excitation and emission wavelengths for the
the BBB, or cause the brain to be more                 dye. The dye content in plasma and brain was
susceptible to leakage of solutes. Since the state     determined, and a ratio calculated for relative
of the BBB influences the effectiveness of             permeability, specifically, percent extravasation.

Mentorship Projects - 2001                                         Biotechnology           Exhibit    12
Student:      Meredith Lowe                       Description: The purpose of the research was
                                                  to determine the effects of mutations in the
Firm:         Food and Drug Administration        NS4B gene on dengue virus replication, viral
                                                  release, and protein synthesis. It is hypothesized
Mentor:       Dr. Robin Levis                     that the seven nonstructural proteins in the
                                                  dengue genome play a large role in dengue virus
Title:        The Role of the NS4B                replication, but so far little is known about the
              Nonstructural Protein in Dengue     individual proteins. Minimal information about
              Virus Replication                   the NS4B nonstructural protein has been
                                                  published, so that protein was the focus of the
Background: The goal of this project is to        experiment.
determine the role of the NS4B nonstructural              In this experiment, two cell lines were
protein in dengue virus replication. The dengue   transfected with six lethal virus mutants of the
virus is spread among humans by mosquitoes        NS4B nonstructural protein and one wild type.
and is especially prevalent in subtropical and    One cell line was a control; the other had been
tropical regions. However, epidemics have         genetically altered to express NS4B in trans.
occurred throughout history all over the world,   Both cell lines were incubated and samples were
including the United States. There are an         extracted every six days for 24 days. Three
estimated 100-300 million cases of dengue         assays were performed on the samples: Northern
infection per year in recent years. The dengue    blot analysis to test virus replication, reverse-
virus causes 2 different diseases: primary        transcription polymerase chain reaction (RT-
dengue fever (bone-crushing disease), and         PCR) to test viral release, and an
dengue hemorrhagic fever shock syndrome           immunofluorescence assay (IFA) to detect
(DHFSS). There is currently no vaccine or cure    NS4B and other viral protein synthesis. This
for any of these diseases.                        experiment is one step towards understanding
                                                  how the dengue virus replicates, which is crucial
                                                  for development of a vaccination.

Mentorship Projects - 2001                                   Biotechnology           Exhibit   13
Student:       Greg Mattingly                       adult anesthsized rat regarding the role of 7
                                                    nAChR subtype can be confirmed by
Firm:          Department of Pharmacology,          electrophysiological studies of single neurons in
               Georgetown University Medical        the DMV that project to the fundus; (2) to
               Center                               pharmocologically characterize 7 nAChR on
                                                    single DMV neurons; (3) to determine whether
Mentors:       Dr. Niaz Sahibazada,                 nAChR subtypes other than the 7 subtype are
               Dr. Richard Gillis                   present on DMV neurons that project to the
Title:         Modulation of Intragastric                   In order to identify which DMV neurons
               Pressure and Fundic Tone in the      project to the fundus, Sprague Dawley rats
               Dorsal Motor Nucleus of the          (postnatal day 14) were anesthesized with
               Vagus of the Rat                     methoxyflurane, cut open in the stomach area,
                                                    and a retrograde DiI tracer was applied to the
Background: The native 7 nicotinic                 fundus. The rats (19-28 days old) were then
acetylcholine receptor (nAChR) is very              decapitated and brainstem slices 250 um thick
common in both the central and peripheral           were cut using the vibrating microtome. After
nervous systems. They have a high                   being immersed in a physiological solution,
permeability to calcium, can act presynaptically    cells bearing the retrograde DiI tracer were
to modulate neurotransmitter release, and can       identified visually by flourescence optics.
also participate in postsynaptic signalling.        Using the whole-cell patch clamp method, the
Although the 7 nAChR has been studied and          cells were voltage clamped and acetylcholine,
classified as homomeric when expressed on a         an nAChR agonist, was applied via bath
Xenopus oocyte, it has never been characterized     application. When a viable cell was obtained
in its native state. Previous studies have linked   (one that responded well to acetylcholine), 7
the 7 nAChR to an area in the central nervous      antagonists such as -BGTX and
system known as the Dorsal Motor Nucleus of         methyllycaconitine were pressure injected into
the Vagus (DMV) due to the effects of an 7         the cell, and the change in current was recorded.
antagonist, -bungarotoxin (-BGTX), on the         It was found that the currents of the DMV cells
fundus area of the stomach. -BGTX blocks           studied were only partially attenuated by -
fundic tone, indicating that the neurons            BGTX and MLA, indicating that the native 7
projecting to the fundus contain 7 subunits.       receptor is indeed heteromeric. The other
                                                    subunit(s) remains to be determined.
Description: The purpose of my project is to
(1) determine whether results obtained from the

Mentorship Projects - 2001                                     Biotechnology           Exhibit   14
Student:       Vasiliki Michopoulos                Description: The specific aim in this study
                                                   was to identify additional downstream PPAR
Firm:          National Institutes of Health       (peroxisome proliferator-activated receptor
                                                   gamma) target genes by using in silico methods.
Mentor:        Dr. Zlatko Trajanoski               Genes with PPAR responsive elements in their
                                                   promoter regions were identified by screening
Title:         In silico Identification of         annotated sequence databases for PPAR binding
               Peroxisome Proliferator-            motifs.
               Activated Receptor Gamma                     In order to identify PPAR targets,
               Transcriptional Targets             database searches were performed and three
                                                   computational techniques were used: 1)
Background: Obesity is an increasing health        Searching for short sequence patterns within the
problem reaching epidemic proportions in most      database entries, 2) Searching for patterns using
western societies. The prevalence of obesity in    a position weight matrix (PWM) from the
much of Europe is 15-20% of the middle-aged        TRANSFAC database and programs, 3) Patterns
population and is higher in the United States,     were searched using a program and a newly
especially in minorities such as African or        constructed position weight matrix (PWM). Due
Mexican Americans where the prevalence in          to the context-sensitivity and the possibility of
women is about 40%. Obesity is the main            using specialized PWMs, the method of choice
precursor state of diabetes. Other diseases such   for in silico identification of PPAR targets was
as hypertension, dyslipidaemia, and                "TargetFinder", leading to a total of 1206
cardiovascular diseases are also attributable to   putative targets.
obesity. Recently, a protein member of the                  This computational approach enabled the
nuclear hormone receptor super-family              identification of putative target genes for
designated as PPAR (peroxisome proliferator-      PPAR. These target genes can be responsible
activated receptor gamma) was discovered and       for setting the adipogenic program in motion
its central role in fat cell differentiation was   and hence, enable a more rational design of new
identified.                                        classes of drugs to control obesity and
                                                   eventually other diseases.

Mentorship Projects - 2001                                    Biotechnology          Exhibit   15
Student:      Diane Oh                             including A-FABP, E-FABP, and H-FABP were
                                                   severely down-regulated (3-50 fold) in cancer
Firm:         Walter Reed Army Institute of        cells compared to normal cells. This suggests
              Research                             that there may be a distinct balance between
                                                   these two groups of FABP whose up or down
Mentor:       Dr. Rasha Hammamieh                  regulation in cells may play a role in prostate
                                                   and breast cancer.
Title:        Expression and Purification of
              Fatty Acid Binding Proteins in       Description: In order to discover the effects of
              E. coli                              up or down regulating these FABPs in cancer
                                                   cells, the FABPs themselves must first be
Background: Intracellular transport of             isolated for use. The purification of FABPs was
bioactive lipids is a critical component in the    done by using the PinPointTM Xa-1 T-Vector to
process by which molecules continuously            clone and express the cDNA that codes for
stimulate proliferation through interaction with   specific FABPs in E. coli. After transformation
nuclear receptors. Transport and utilization of    of the vector into E. coli, the transformation
lipids are mediated by an important family of      cultures were grown and the DNA was isolated
cytoplasmic proteins, known as fatty acid          from cells from each culture. In those DNA
binding proteins (FABPs). Several studies          samples in which the cDNA insert had been
suggest that FABP increase the solubility of       inserted into the vector successfully, the DNA
fatty acids in the cell cytoplasm, causing a net   was sequenced and checked with existing
diffusion of fatty acids from the plasma           sequences in the GeneBank to ensure that the
membrane to the intracellular membrane             cDNA had been inserted into the vector in the
compartments.                                      correct orientation. IPTG, a protein inducer, was
         The members of this multigene family of   then added to the E. coli cultures from which the
FABPs consist of at least seven types whose        successful plasmid samples were isolated to
amino acid sequences have been obtained from       induce the production of the FABP. The
protein purified from tissues or from cDNA         proteins produced were fusion proteins. The
sequences. The designations for each of the        PinPointTM Xa-1 T-Vector carries a segment
FABPs have been derived from the tissue from       encoding a peptide that becomes biotinylated in
which it was originally isolated and include       E. coli and subsequently functions as a
adipocyte (A-FABP), heart or muscle (H-            purification tag. Using an avidin resin, the
FABP), brain (B-FABP), epidermis or psoriasis-     protein was specifically removed from an
associated (E-FABP), liver (L-FABP), intestine     extract by binding of the covalently linked
(I-FABP), and myelin or P2 (P2-FABP).              biotin on the fusion to the monomeric avidin
         Within this family of FABPs, however,     linked to the resin. Elution from the resin was
are two separate classes. In one class is L-FABP   accomplished by incubation with biotin under
and I-FABP, which were elevated 5-9 fold in        nondenaturing conditions. This produced the
most cancer cells compared to normal primary       purified FABP.
cells. In contrast, the other class of FABPs

Mentorship Projects - 2001                                    Biotechnology          Exhibit   16
Student:       Rita Portocarrero                     membrane to trigger a reaction, signaling the
                                                     start of mitosis. Many are present in the dentate
Firm:          National Institute for                gyrus, though how they affect neurogenesis is
               Neurological Disorders and            unknown. Most likely, the receptors for these
               Stroke                                growth factors are present on the dividing cells;
                                                     this would suggest that they directly signal cell
Mentor:        Dr. Heather Cameron                   proliferation.
                                                              Five rat brains were labeled with [3H]
Title:         Effect of Growth Factors on           thymidine, a radioactive substance that labels
               Proliferating Neurons in the          cells in the S phase of mitosis, and euthanized
               Adult Dentate Gyrus                   by Dr. Heather Cameron. Sections of these
                                                     brains were put on slides and
Background: After birth, most neurons in the         immunohistochemically stained for epidermal
central nervous system no longer proliferate,        growth factor receptor (EGFR), insulin-like
remaining in the brain until they die. This          growth factor receptor (IGFR), and fibroblast
characteristic makes it extremely difficult to       growth factor receptor 1, 2, and 3 (FGFR1,
reverse damage done to the brain, because the        FGFR2, FGFR3). Then the slides were dipped
cells cannot replace themselves. In the 1960s,       in photographic emulsion, and allowed to sit in
scientists discovered that in the dentate gyrus, a   the dark for three weeks, so the cells that had
region of the hippocampus, which controls            been probed by thymidine would be visibly
learning and memory, cells proliferate               labeled under the microscope. Finally, the
throughout adulthood. The direct signals             slides were analyzed, and the thymidine labeled
controlling proliferation are not known, but         cells counted, to determine whether or not the
several growth factors have been found in the        specific receptors are on the dividing cells. The
dentate gyrus, leading scientists to believe that    results show that all five receptors were present
they may be regulating proliferation.                on some proliferating cell, suggesting that they
                                                     directly trigger the start of mitosis in neurons.
Description: Growth factors are ligands that
attach to specific receptors on the cell

Mentorship Projects - 2001                                      Biotechnology           Exhibit   17
Student:       Andrew Ra                              occurs, it is unknown what role, if any, cells
                                                      such as oligodendrocytes take in the
Firm:          Georgetown University,                 remyelination process.
               Department of Neuroscience
                                                      Description: Studies have identified an
Mentor:        Dr. Jean R. Wrathall                   oligodendrocyte progenitor cell (OPC)
                                                      population in the adult central nervous system.
Title:         Proliferation of Cells After           These cells are as abundant as microglia and
               Spinal Cord Injury                     there is evidence to show that these progenitor
                                                      cells may aid in the remyelination of axons in
Background: Spinal cord injury was once               the spinal cord. However, this work was done in
viewed to be incurable. Victims of spinal cord        vitro and thus it is not known if these cells
injury could expect themselves to live in             actually aid the spinal cord in the healing
wheelchairs for the rest of their life, helpless      process. In the study that we are conducting, we
and without any hope of recovery. However,            used bromodeoxyuridine, or BrdU for short, to
recent discoveries in the field of neuroscience       show the time course during the first week
have given those victims of spinal cord injury        following spinal cord injury and the distribution
hope. Studies indicate that the spinal cord is        of cell division in the rat spinal cord at 2
able to take steps to heal itself after spinal cord   millimeters and 4 millimeters rostral and caudal
injury. After spinal cord injury, oligodendrocyte     to the epicenter. Some of the cells that divide
apoptosis, loss of myelin occur at the injury site    after spinal cord injury are likely to be
up and down the spinal cord and in various            oligodendrocyte precursor cells that will be
animal models it is observed that demyelination       involved in remyelinating axons after injury.
occurs most significantly during the first week       Identifying when and where these cells divide is
after spinal cord injury. Also remyelination          the first step towards studying how they may be
appears to occur at approximately 14 days post        involved in the healing process. BrdU is thus
injury and by one month most axons have               incorporated in the experiment because it has
become remyelinated, however, not to the level        been found to be able to detect the S phase of
of myelination observed before spinal cord            cell division.
injury. Although it is known that remyelination

Mentorship Projects - 2001                                       Biotechnology           Exhibit       18
Student:      Elizabeth Reynolds                   Description: We have investigated muscarinic
                                                   receptor 2 [M2] gene expression differences in
Firm:         National Institute of Mental         dorsolateral prefrontal cortex [DLPFC] of
              Health, Clinical Brain Disorders     patients with schizophrenia or affective
              Branch                               disorders (patients who committed suicide or
                                                   had bipolar disorder) using in situ hybridization
Mentor:       Dr. Jeremy M. Crook                  histochemistry [ISHH]. This includes: mRNA
                                                   density analysis of autoradiograpic images
Title:        Post Mortem Studies of               produced from in situ hybridization and silver
              Muscarinic Receptor 2 Gene           grain analysis of M2 mRNA on tissue on slides.
              Expression in Dorsolateral           To identify the localization of M2 receptor
              Prefrontal Cortex of Patients with   protein we have also performed
              Schizophrenia                        immunohistochemistry on tissue from 5 normal
                                                   subjects included in ISHH. From film based
Background: Schizophrenia is a mental              studies no significant difference was found
disorder characterized by auditory                 between mRNA levels of patients with
hallucinations, delusional thought and paranoia,   schizophrenia or affective disorder and normal
distortion of personality, inappropriate impulse   controls. The lack of a difference in mRNA
and emotional response, and difficulty in          levels between schizophrenics and normals
learning and memory. Schizophrenia effects         along with findings of Crook et al. 1999, and the
1% of the world’s population. Schizophrenia        1999 Intel entry of Hilary Glidden who report
has been found in patients from anywhere in        reduced [3H]AF-DX 384 binding to receptors in
their early teens to well into their seventies.    the DLPFC and caudate-putamen, respectively,
Most cases of Schizophrenia are diagnosed          of subjects with schizophrenia, suggests
between the ages of 15 and 45. Studies have        changes to muscarinic receptor M2 are post
shown that males and females have equal            transcriptional. Preliminary results from the
chances of being affected by schizophrenia.        slide-based study show low levels of muscarinic
The cause of schizophrenia is currently            receptor 2 mRNA associated with pyramidal
unknown, however several factors are suspected     neurons in DLPFC. Immunohistochemistry
to contribute to a persons chances of developing   revealed immunostaining throughout the
schizophrenia. While genetics is most likely an    neuropil with no staining around dendrites or
underlying cause of schizophrenia,                 cell bodies. This suggests muscarinic receptor
environmental factors such as fetal trauma or      M 2 is presynaptic.
infection, season of birth, and viral pandemics
may also be important (Harrison, 1999).

Mentorship Projects - 2001                                    Biotechnology          Exhibit   19
Student:       Won Suh                                requires living organisms (lab animals or tissue
                                                      culture) to grow.
Firm:          Uniformed Services University
                                                      Description: This project focused on the
Mentor:        Allen L. Richards, PhD                 detection and quantitation of O. tsutsugamushi
                                                      in laboratory and animal acquired samples
Title:         Rapid Molecular Diagnosis of           utilizing a TaqMan assay. The assay takes the
               Scrub Typhus Utilizing TaqMan          polymerase chain reaction (PCR) technique one
               Assay                                  step further. It incorporates a specific
                                                      nucleotide probe with a fluorescent dye in order
Background: Scrub typhus is a bacterial               to provide for an extremely sensitive assay. The
disease endemic to the Asia-Pacific region,           dye and a quencher molecule are bound to each
which has, if left untreated, up to a 50%             end of the probe, which attaches specifically to
mortality rate. Its etiologic agent is Orientia       the targeted region of the O. tsutsugamushi 47
tsutsugamushi (previously known as Rickettsia         kDa antigen gene being amplified by the Taq
tsutsugamushi), a bacteria transmitted by the         polymerase. In addition to the Taq enzyme’s
bite of a chigger, a trombiculid mite in its larval   ability to make new DNA it also “chews up” the
phase. Some symptoms of scrub typhus include          probe in front of it releasing the fluorescent dye
an eschar at the site of the bite, fevers,            from close proximity to the quencher molecule,
headaches, swollen lymph glands, a rash that          allowing the fluorescence emitted to be detected
spreads to the arms and legs, and at times            by the AB1 Prism 7700 SDS machine. The
pneumonitis. Although this disease has been           assay has been successful in the detection of
around for a long time, it was not much               laboratory grown O. tsutsugamushi strains. In
publicized until U.S. soldiers began to report        addition, it was found to be effective in
infections during World War II and the Vietnam        detecting O. tsutsugamushi in mouse blood. In
War. Antibiotic treatments are available              order to quantify the results of the experiments,
including one of the tetracyclines or                 a standard curve was made with dilutions of the
chloramphenicol. However, there have been             plasmid VR 1012 containing the O.
recent reports of drug resistant strains of the       tsutsugamushi Kato strain 47 kDa antigen gene
bacteria. Working with O. tsutsugamushi is            produced in the laboratory. Blood from humans
rather difficult and expensive, because unlike        infected with O. tsutsugamushi have not been
other types of bacteria, O. tsutsugamushi cannot      tested yet, but this is the next step planned in
be cultured on plates (artificial media), but         evaluating and optimizing this new rapid
                                                      molecular diagnostic assay for scrub typhus.

Mentorship Projects - 2001                                       Biotechnology           Exhibit   20
Student:       Grace Wan                             for drug-induced arrhythmias. One such
                                                     mutation is found on chromosome 7 in HERG, a
Firm:          Georgetown University Medical         potassium channel gene that encodes the rapid
               Center                                component of two major repolarizing potassium
                                                     currents, the delayed rectifier (IKr) and the
Mentor:        Dr. Steven N. Ebert                   inward rectifier (IK) current densities.
                                                     Preliminary studies have shown that women are
Title:         The Genetic and Molecular             more likely to suffer from mutations on the
               Mechanisms Underlying Female          HERG gene which alters the IKr density and in
               Susceptibility to Drug-Induced        turn results in LQTS. This would explain why
               Cardiac Arrhythmia                    women are more likely to suffer from TdP.

Background: It has been shown that women             Description: It is logical to assume that male
are at a far greater risk of developing torsades     sex hormones may play a role in the length of
de pointes (TdP) in response to drugs such as        the QT interval and that this effect is regulated
antihistamines, antibiotics, antimalarials, and      by an effect on HERG. In this study, the effects
antiarrhythmics, which act as potassium channel      that the androgen, dihydrotestosterone (DHT),
blockers. TdP is a type of cardiac arrhythmia        has on HERG mRNA and protein were
occurring in the setting of a lengthened QT          examined. We determined whether there is an
interval, which reflects prolonged cardiac           increase in the concentration of the mRNA and
repolarization and may result in ventricular         the HERG protein in castrated male rabbit hearts
fibrillation and cardiac arrest. It is yet unknown   that have been treated with either placebo slow
why women are more susceptible to TdP,               release pellets or DHT slow release pellets. If
although it is suspected that the naturally longer   concentrations of HERG protein increase in
baseline electrocardiographic rate corrected QT      DHT treated hearts relative to placebo heart, this
(QTc) interval may be a factor. The QTc              will support the hypothesis that androgens
interval in males begins to shorten at the onset     chronically modulate delayed rectifier
of puberty when the level of androgens in males      potassium current activity through an effect on
begins to rise and returns to equal that of          HERG gene expression. If concentrations of
women’s at about age 50. This suggests that          HERG mRNA is also increased due to DHT
one or more of the male hormones may be              treatment, then the molecular mechanisms of
responsible for the QTc shortening, and              androgen action may also affect HERG gene
relatively lower risk of drug-induced TdP in         expression. Experiments were designed to
men. Molecular mechanism studies have shown          distinguish between the actions of DHT on
at least six chromosomal loci that have been         HERG mRNA and on HERG protein.
linked to congenital long QT syndrome (LQTS)

Mentorship Projects - 2001                                      Biotechnology           Exhibit   21
Student:       Leslie White                         primer. Allele sizes from the three subspecies
                                                    were compared with the use of an ABI
Firm:          National Zoo, Molecular              automated sequencer and data were analyzed
               Genetics Laboratory                  using the computer program Arlequin.
                                                    Comparisons of allele sizes and frequencies help
Mentor:        Dr. Miyoko Chu                       to determine the degree of genetic divergence
                                                    between the Coastal Plains Swamp Sparrow and
Title:         PCR Optimization and Genetic         the other subspecies and also to estimate levels
               Comparison of Three Melospiza        of gene flow.
               georgiana Subspecies Using
               Microsatellite DNA

Background: Swamp Sparrows (Melospiza
georgiana) are migratory birds that breed in
North America. The Coastal Plain Swamp
Sparrows (M.g. nigrescens) are morphologically
distinct from the other two subspecies, and they
breed in saltwater marshes, rather than
freshwater marshes. They are also restricted to a
distinct range in the marshlands of Maryland,
Delaware, Pennsylvania, and New Jersey, and
their populations are declining. If the Coastal
Plain Swamp Sparrows are genetically distinct
from other subspecies of Swamp Sparrows,
actions should be taken to protect them from
extinction. Previous projects suggest that any
divergence of the three subspecies must have
occurred recently, as they could not be
distinguished based on mitochondrial DNA.
Studies of microsatellite DNA, or segments of
non-coding repeated sequences, could reveal a
more recent divergence.

Description: In order to compare
microsatellite DNA, the Swamp Sparrow DNA
samples must be amplified through PCR. There
are no primers that have been designed to work
specifically on Swamp Sparrow DNA, so a
major component of the project was optimizing
PCR conditions so that Song Sparrow primers
would amplify Swamp Sparrow DNA. The
factors that were altered most were the amount
of MgCl2 added to the PCR solution and the
annealing temperature.
        When optimal PCR conditions were
determined for a primer, Swamp Sparrow DNA
from the three different subspecies was
amplified using fluorescent versions of the
Mentorship Projects - 2001                                     Biotechnology          Exhibit   22
Mentorship Projects - 2001   Chemical Analysis   Exhibit   23
Mentorship Projects - 2001   Chemical Analysis   Exhibit   24

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