Biomed Benchmark - Honors THesis abstracts
DIVISION OF BIOLOGY
AND MEDICINE
G
Reproductive
Trade-Offs Associated with the Innate Immune Response in Drosophila
melanogaster
By: Melissa
Zerofsky
Sponsor: Marc Tatar
Consideration of energetic
trade-offs is important in the study of senescence. An organism that allocates fewer resources to reproduction
in favor of somatic maintenance will age less rapidly. Therefore, upregulation of one system
incurs a cost in another system.
Innate immunity is thought to play a role in senescence, but the nature
of this role is not well understood.
An interesting question is whether induction of the innate immune
response is negatively correlated with other physiological functions.
It has been shown that induction
of the innate immune response in insects is costly. Using transgenic and mutant strains of Drosophila
melanogaster in the imd/Relish
immune pathway, I tested the effects of the humoral branch of the innate immune
response on fecundity in Drosophila females. I demonstrated that induction of
antimicrobial peptides in Drosophila
is negatively correlated with fecundity.
This is the first demonstration of reproductive trade-offs with humoral
immunity in Drosophila.
G
Non-Invasive
polarized light microscopy quantitatively distinguishes the multilaminar
structure of the zona pellucida of living human eggs and embryos.
By: Cory
Pelletier
Sponsor: David Keefe
We quantitively described the
thickness and architecture of the zona pellucida of living human eggs and
cleavage stage embryos, non-invasively with the Polscope; a digital polarizing
light microscope. Patients
involved in this study were undergoing fresh, non-donar infertility treatment
that underwent egg aspiration, fertilization by ICSI or traditional IVF and
cleavage stage embryo (day 3) transfer.
The Polscope imaged the zona of eggs prior to ICSL and in cleavage stage
embryos prior to transfer.
Thickness and
retardance of three zona layers were measured using
Spindle View Image Analysis System, from eight quadrants. Mean and variance in thickness and
retardance were calculated for individual eggs and embryos, between eggs and embryos
of a cohort and across the sample patient population. We found that Cleavage stage (day 3) embryos have thinner
zona (15.2+/-2.9mm) than both mature (20.4 +/- 2mm) and mature (19.5+/-2.2mm) eggs. The zona of embryos is thinner,
primarily due to thinning of the outer layer. The thicker the zona layer the greater itŐs retardance. Considerable variation exists in the
thickness and retardance of zona layers around individual eggs and embryos and
between members of a cohort. The
zona of eggs and embryos from different patients differ in thickness,
retardance and variation. In
conclusion, thickness and organization of zonae pellucidae of human eggs and
embryos varies considerably and can be quantitatively imaged using the
Polscope.
G
CD4-Independent Mechanisms of
HIV-1 infection in Glial Cells and B-Cells
By: Leigh
Baxt
Sponsor: Dr. Walter Atwood and Dr. Peter Shank
The specific aim of our research was to investigate the possible role of syndecans as co-receptors of HIV-1 infection of B-Cells and plial cells. Syndecans are a type of heparan sulfate molecule present on the surface of most cells. They have been shown by Saphire et al. to function as receptors of HIV-1 infection of macrophages. We investigated the expression of syndecans on B-cells and glial cells. We found that of the four syndecans, only syndecans 1 and 4 are present in high amounts on B-cells and glial cells. In addition we have shown that pseudotyped HIV-1 viruses are able to infect a variety of cell types including Hos CD4/CCR5 cells, SVG-CD4/CXCR4 cells, and SVG-A cells. Finally we have shown that incubation of these cells with monoclonal antibodies to syndecan molecules 1 and 4 on the cell surface is able to eliminate infection by the VSV-G envelope-containing pseudotype virus in both SVG cell lines. Incubation with these Abs is also able to abrogate infection by the NL4-3 envelope-containing psuedotype virus in all three cell types.
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Temporal Changes of Vascular
Endothelial Growth Factor Expression Following Traumatic Brain Injury
By: Wendy
Chang
Sponsor:
Joanna Szmydynger-Chodobska
Cerebral edema formation continues to be one
of the major clinical problems in the management of patients with traumatic
brain injury (TBI). However, the
arsenal of effective treatments for cerebral edema is very limited and the
efficacy in reducing intracranial pressure is less than satisfactory. Therefore, it is important to identify
the molecular and cellular mechanisms responsible for progressive swelling of
the brain, and to develop new and effective therapies for the victims of brain
trauma.
Vascular endothelial growth factor (VEGF) has
been identified as one possible player in the development and progression of
edema. VEGF has been postulated to
increase blood-brain barrier (BBB) permeability to low molecular weight markers
and macromolecules by altering tight junction proteins. This investigation analyzed the time
course of VEGF changes at a protein level in rat brains after TBI.
Using
immunohistochemistry and Western blot analysis we demonstrated that after brain
injury, the expression of VEGF increased steadily in the injured ipsilateral
cortex. The peak of VEGF
expression post-TBI occurred on the fourth day. However, the levels of VEGF remained elevated up to 14 days
after injury. No change in VEGF
levels in the contralateral hemisphere was observed. This time course of increased VEGF expression
corresponded to the late opening of the BBB and to the subsequent development
of edema after TBI.
G
Investigation of the Expression
of C/EP‰ in Antipsychotic-Injected Mice
By: Shawn
Bachan
Sponsor: John Marshall
Abnormalities in the temporal
lobe make it a prime target for schizophrenic research because it houses the
hippocampus, which is the center for learning and memory. The hippocampus communicates with
higher areas of cortex such as the prefrontal cortex (PFC) in the integration
of information, and both cerebral areas are components of the mesolimbic and
mesocortical tracts of the dopaminergic system. Through various studies, it has been shown that typical
antipsychotic drugs such as haloperidol, act as D2 dopaminergic receptors and modulate the expression of
immediate early genes (IEGs) and downstream transcription factors such as cAMP
response element binding protein (CREB).
CCAAT enhancer binding protein beta (C/EBPb) is a transcription factor that
has roles in such processes as learning and memory, hepatocyte differeniation,
and neuronal differentiation. C/EBPb
has been
shown to be involved in the CREB-dependent gene cascade activated in long-term
memory. As a result, like CREB, it
is a possible intracellular target of antipsychotic drug therapy.
[This]
research project aims to determine whether haloperidolŐs action on the D2 receptor modulates the expression of
C/EBPb in the mouse hippocampus and cortex. This was accomplished by
immunohistochemical analyses of brain sections in haloperidol treated and
vehicle treated mice at three different time points (t = 2hrs, 8 hrs, and 24
hrs).
G
The Effects of Parental
Involvement on the Behavior of Students in the Classroom
By: Diana
Comarato
Sponsor: Robert Shaw
Associate
Dean of the College
Much research exists on the amazing impact parental involvement can have on a childŐs achievement in school. Furthermore, there is a great body of research that focuses upon a childŐs emotional, social, and behavioral development as a product of parentŐs influence. Why, then, is there a lack of research focusing on the behavior of children in the classroom, already viewed as a key indicator of a childŐs development, and the impact parental involvement has on that behavior? If parents have such a strong influence on the overall development, wherein lies the body of research promoting parental involvement as a method of improving overall behavioral health of a child in the classroom as opposed to solely improving academic achievement?
Educational progress and achievement should not be weighted to be any less important than it currently is, but rather the void which exists on the impact of parental involvement regarding childrenŐs behavior in the classroom needs to be filled. The research undertaken by this project attempts to take the first step towards linking increased overall child development.
In this child development study, the focus is placed upon the degree of involvement parents/guardians have in their childŐs educational development; with a hypothesis that the less involved parents are in their childŐs school life and environment, the more misbehavior that will appear among these children in a classroom setting.
With past research in mind, the results of this new observational study performed show a general trend for students with lower parental involvement to have higher misbehavior in the classroom. Due to the fact that child development, easily measured through behavior in school, is the result of so many compounding factors, the research presented thoroughly examines issues such as family background, social context variables, ethnicity, maternal employment status, and socioeconomic status.
The
Role of gbb in the Drosophila
ŇStem Cell NicheÓ
By: Melissa
Duan
Sponsor: Kristi
Wharton
Previous
studies have demonstrated defects in reproductive processes in Drosophila
melanogaster
mutant for glass boat bottom (gbb). In this
paper, we characterize the defects associated with these mutants, which include
a disruption in ovary morphology, the number of ovarioles, and stem cell
proliferation. We also
characterize the stem cell and fat body phenotypes in larval gonads. Antibody labeling experiments reveal a
reduction in stem cell proliferation in both larval gonads and adult
ovarioles. While the phenotypes
exhibited by gbb
mutants. Thus the reduction in
fecundity as well as the other patterning defects characterized in the gbb mutants is not accompanied by an
increase in lifespan, but rather serves to weaken the flies.
Ability
of Dendritic Cells to Activate Natural Killer Cells: An In Vitro Study in the MCMV System
By: Tanya
Hamilton
Sponsor: Christine
Biron
Natural
killer cells are essential for host defense during the acute stages of murine
cytomegalovirus (MCMV). Dendritic
cells (DC) produce the cytokines, specifically IFN-a/b and IL-12, necessary for NK cell
activation in vivo
during MCMV infection and mature plasmacytoid dendritic cells (PDC) are able to
rapidly activate NK cells in vitro. In this
study, I further investigate the ability of DC to activated NK cells, and the
role of cytokines and cell-cell contacts in this activation, by using a
coculture system. DC that matured
during MCMV challenge were able to activate NK cells as measured by induction
of IFN-g production and NK cell cytotoxicity, however, na•ve DC
were not. IFN-a/b produced by the DC were critical
for inducing NK cell cytotoxicity in the 129 background, but were unimportant
in the IFN-g response.
In contrast, at high DC concentrations, IL-12 seems to play a role in
the IFN-g production in C57BI/6. However, at low DC concentrations, IL-12 seems to play a
role in the IFN-g response suggesting the possibility of different
activation pathways functioning at different concentrations of DC. When contact between DC and NK cells
was prevented with cells from 129 mice the DC were still able to activate the
NK cells. In contrast, in cells
from C57BI/6 mice, prevention of DC and NK cell contact abrogated the
activation of NK cells. These
results indicated the presence of two pathways, one cytokine-mediated and one
cell-cell contact-mediated, through which DC can activate NK cells with the
cytokine-mediated pathway dominating the 129 mice and the cell-cell
contact-mediated pathway dominating in the C57BI/6 mice in this system.
G
IL-12 production by Dendritic Cells at
the site of infection with Toxoplasma
gandii.
By: Sandy
Wong
Sponsor: George Yap
Protective immunity towards Toxoplasma
gondii infection
require both innate and adaptive immune responses. At the site of infection, antigen-presenting cells (APCs)
such as dendritic cells (DCs) phagocytose the parasite, process the parasitic
antigens, and present them on their cell surface coupled to MHC molecules. These MHC plus antigen complexes prime
na•ve T cells to proliferate into effector cells that
could exert microbicidal activities.
In order to initiate adaptive immunity through T cell activation, DCs
must travel from inflammatory sites to secondary lymphoid organs where the
na•ve T cells reside. DC migration
is mediated not only by the upregulation of adhesion molecules that together
facilitate tissue-specific homing.
Using
FACs analyses and cell surface staining, we have begun to characterize the
immune response profiles of dendritic cells in the spleen, the blood and the
peritoneum on day 4.5 or 5 after intraperitoneal toxoplasma infection. On day 4.5 a population of CDIIc+MHC
IIlow immature DCs (imDCs) appear prominently in the peritoneum but not in the
blood yet later on day 5, a small population of imDCs could be identified in
the blood. Further analysis
revealed that a majority of the imDCs in the peritoneum seem to be of myeloid
(CD11b+B220^CD8a^CD4^) not plasmacytoid nor lymphoid pehnotype. Of these myeloid imDCs, subpopulations
with distinct lymphoid organ homing potential exist in the peritoneum as well
as the blood.
Since
dendritic cells are able to produce IL-12, an important cytokine in TH1
activation, we investigated IL-12 production by intracellular staining. Our experiments showed that only a
minority of the CD11c+ dendritic cells from infected mice spontaneously
produced IL-12 production and that most IL-12 producers in the peritoneum are
MHC IIhigh. From these results, we
propose a hypothetical schema for monocyte to myeloid DC transition,
differentiation and trafficking.
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RNA Interference of HIV-1
Replication
By: Wayne
Chung
Sponsor: Bharat Ramratnam, MD
RNA
interference (RNAi) is the sequence specific degradation of messenger RNA
induced by homologous short, interfering (si) RNA. The purpose of this study is to investigate the
effectiveness of siRNA mediated suppression of HIV-1 replication. We chose the first exon of the HIV-1
transactivator protein tat as the target of our siRNA. We first determined the potency of RNAi inhibition by
transient transfection of 293T cells with synthetic siRNA. Next, we designed a molecular construct
with a short hairpin (sh) RNA expression cassette to allow intracellular
synthesis of siRNA. Finally, we
generated permanent cell lines that stably expressed the shRNA. Inhibition by transient transfection
was 91% compared to controls. The
recombinant adeno-associated virus (AAV) construct, AAV-tat, reduced viral replication in
293T cells by 85%. Stable siRNA
expression was achieved in H9 cells, subsequently reducing HIV-1 replication by
96%. These results show that the
introduction of an engineered expression cassette and the stable expression of
siRNA can lead to long term inhibition of HIV-1 replication, and forms the
basis for further research in RNA interference as a viable therapy against
HIV-1.
Respiratory
physiology of ParkinsonŐs disease during exercise using a magnetometer.
By: Duncan
A. Friedman
Sponsor: Carol
Garber
Study
Objectives: Respiratory dysfunction is a
known cause of death in ParkinsonŐs disease. This study sought to determine if there are any differences
in respiration between patients with mild to moderate PD and age and gender
matched control subjects.
Subjects: Twelve patients with clinically diagnosed PD (Hoehn-Yahr
Stage I-III) with a mean age of 64.11 years were compared to eleven healthy
participants with a mean age of 67.11 years.
Methods: Participants engaged in three levels of submaximal
exercise (0W, 20W, and 30W) for 5 minutes. Data were recorded using an Epri 4-Lead Magnetometer
attached at specific positions on the thorax. These data reflected measures at resting, 0W, 20W, 30W and
recovery levels of exercise. Data
were processed and analyzed statistically.
Results: No significant difference existed between groups in the 5
breathing measures examined (VT, VC, Frq, VT/VC, and VE/MVV). General trends were observed in each of
these measures. PD patients had
consistently higher VT means and consistently lower Frq means. VE plots were very similar between
groups. VE/MVV means were higher
at each level of exercise for PD patients.
Conclusion: PD patients in the early stages of the disease maintain a
similar minute ventilation as control counterparts. This achieved through a relative increase in breathing
frequency in an effort to compensate for decreased tidal volume. Patients might exhibit these
differences due to lack of subcortical control over respiratory control areas
in the brain stem. Although no
measure achieved significance, further research might confirm the general
trends seen by this study and demonstrate a significant difference in between
PD patients and matched controls.
G
Molecular Mechanisms of
Parathyroid Hormone (PTH) Binding
By: Kimberly
J. Hyde
Sponsor: Dale Mierke
The parathyroid hormone (PTH) plays an essential role in the regulation of calcium ion homeostasis in the blood. The importance of this ion in diseases such as osteoporosis and hyperparathyroidism has brought about the need to have a complete understanding of how this hormone binds to its preceptor (PTH1). Once the precise intermolecular interactions that are responsible for the activation and suppression of this hormone system are know, it will be possible to design pharmaceutical agents that can mimic or antagonize the action of PTH, leading to a number of therapeutic solutions for the diseases that are affected by the parathyroid hormone and its receptors. The existing model of the receptor-ligand complex for PTH is by no means complete. New photoaffinity cross-links have been developed that play an important role in the understanding how the ligand binds to the receptor. Here, we have incorporated this new information and performed molecular dynamics (MD) simulations on the ligand-receoptor complex to determine the molecular mechanism of PTH binding. The starting structure for these studies originated from experimental data, including a series of photoaffinity labeling experiments, structure predictions based on homology analysis with proteins of known structures, and spectroscopic structural studies of PTH (1-34) and extracellular domains of PTH1. The N-terminus of PTH is located on the extracellular side in the center of the seven transmembrane helix core of the receptor. Ser1, Ile5, Met8, and His9 have side chains that project towards the core of the receptor while Ser3, Leu7, and Leu11 have side chains that project towards the extracellular matrix (ECM). The C-terminus of PTH (1-34) interacts with the N-terminal and EC1 domains of PTH. The hydrophobic face of the C-terminal alpha helix of PTH (1-34) (residues Vsl21, Leu24, Leu28, Val31) interacts with N-terminal domain of PTH1. Electrostatic interactions also occur between Arg20/E177 and Lys26/E254. Further studies are needed to supplement this model. This model provides further insight into the molecular mechanism of ligand binding and receptor activation, which may eventually allow for rational design of drug candidates to regulate this important hormone system.
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Respiration and Locomotion of
the Painted Turtle
By: Rebecca
Kessler
Sponsor: Donald Jackson
Reptile
anatomoy is such that their hypaxial musculature is shared between their
locomotor and respiratory systems.
This may limit the capacity of the animals to breathe and move simultaneously. Chelonians, however, possess unique
anatomical characteristics that may uncouple the antagonistic functions of the
hypaxial musculature such that they can maintain proper ventilation while
swimming. This hyposthesis was
tested by measuring tidal volume (VT), oxygen consumption (V02) and carbon dioxide production
(V02 ) in the semi-aquatic painted
turtle, Chysemys picta, both at rest and while swimming at 13.5, 20, and 26 cm/s in aquatic
flume. Both VT and V02 increased significantly from
resting values of 17.3 + 2.6 ml kg-1 and 0.69 + 0.07 ml 02 min-1
kg-1, respectively, compared to those
at 20 cm/s (20.1 + 2.1 ml kg-1
and 1.86 + 0.26 ml 02 min-1 kg-1,
respectively). V02 was also significantly elevated from
resting at 26 cm/s (1.92 + 0.67 ml 02
min-1 kg-1). Instantaneous frequency (f1) remained constant, while the
respiratory quotient (RQ) showed a statistically insignificant decreasing
trend. Alveolar ventilation (VA) showed an insignificant increase, while
air convection requirement (VE/VCO2) remained relatively unchanged. These data suggested that painted
turtles can effectively ventilate their lungs during increased levels of
exercise.
Encapsulated
Cell Therapy for Removal of Uremic Toxins: Uric Acid
By: Jan
Markus Bruder
Sponsor: Michael
Lysaght
This thesis will be limited to reporting the findings on uric acid; the results however were obtained in conjunction with similar studies conducted for the degradation of urea and creatinine and have to be viewed as part of the framework of this larger project.
Existing treatment options for end-stage-renal-disease (ESRD) remain unsatisfactory; dialysis is attended by a 20% annual mortality rate and a low quality of life while donor organs are available for less than 2% of the patient population. Accordingly, we have been investigating a novel treatment protocol utilizing encapsulated cell therapy to clear three major uremic toxins: urea, creatinine, and uric acid. Genetically modified bacteria that contain plasmids carrying the genes for the enzymes urease, uricase and creatininase may provide patients with an alternative treatment to supplement dialysis. In one therapy format, capsules containing these modified strains could be given orally and reduce the level of uremic toxins during their passage through the GI system.
Previously, we have reported that a genetically modified and encapsulated strain of E. coli DH5 (containing the urease plasmid pkAU17 from Klebsiella aerogens Đ generously provided by Scott Mulrooney) effectively degrades urea in-vitro with kinetics and mass removal rates which appear realistic for therapeutic application. As will be shown in this paper, in our hands at least, genetically modified E. coli DH5 cells had no effect on uric acid levels, a finding that contradicts previous reports in the literature.
More recently, we have evaluated the capacity of free and encapsulated bacteria as well as enzymes (including uricase) to degrade uric acid in vitro as well as in vivo.
G
Conservation Under the Citizen
Suit Provision of the Endangered Species Act, 1998-2003.
By: Carter
Romansky
Sponsor: Sharon Swartz
Section 11 of the Endangered Species Act gives private citizens the power to bring suit against the government when they feel that regulatory agencies are not performing their duties to the extent mandated by the Act. Because of administrative disfavor for the Act and limited funding from Congress, the implementation of the ESA has largely fallen out of the hands of the Fish and Wildlife Service and increasingly into the hands of the courts. This mode of enforcement has important implications for the way the Act operates in this country and for the potential survival of endangered species in North America. In this study I have examined the practical record of the entire federal court system over the last five years with respect to enforcement of the Endangered Species Act. I found that fish are the most often litigated type of species, that river systems are the most often litigated type of habitat, and that agriculture is the most often litigated type of human activity. ESA citizen suits occur almost exclusively in the western half of the United States, especially on federal lands in the Sonoran Desert, throughout California, and in the Columbia and Snake River Valleys of the Pacific Northwest. The appearance of the ESA in courts, then, appears to be just another front in the perennial struggle for the control of water rights in the West and indicates a fundamental shift in the way that this law is administered in this country. Further, all the date taken together indicate that the ESA becomes less effective as habitats become more degraded (the exact opposite of what should happen if we hope to have an effective means for protecting species and habitat in this country), that court mandated activities do not provide an adequate platform from which to launch a nationally coherent policy to protect threatened and endangered species, and that the protection of overall biodiversity through the Act is secondary to the protection of the basic functional integrity of ecosystems.
The
Effect of Monoclonal Antibody b E11 Treatment on the Function of CD98 Heterodimer: Growth
Inhibition via Inhibition of Amino Acid Transport
By: Andy
Su
Sponsor: Nancy
Thompson
Despite
recent advances in the study of cancer, medical treatments of cancer remain
imperfect due to an incomplete understanding of the processes that permit,
facilitate and otherwise contribute to the abnormal growth and proliferation of
cells that lead to cancer.
Traditionally, most researchers have focused their studies on how DNA
damage and mutation disrupt the machineries regulating cell cycle. While many tumor phenotypes display the
malfunctioning of cell cycle control, altered expression of genes that are not
involved in the regulation of cell cycle also contribute to cancer phenotypes. We are interested in expanding the knowledge
base in such type of gene product and in investigating the role of amino acid
transporters in cancer progression.
We
study the role of CD98 activation molecule and associated amino acid
transporter LAT1 (L-amino acid transporter 1) in cancer growth. LAT1, which is a 40kDa light chain,
forms the CD98 heterodimer when associated with the CD98hc, an 80kDa
glycoprotein (Mannion et al., 1998). We
have previously observed that LAT1 increases system L amino acid transport in
mouse hepatocytes, and these cells showed a growth of advantage in limiting
conditions (Campbell et al., 2001). Due
to these and other observations, we hypothesize that the alteration of LAT1
expression confers a growth or survival advantage by increasing amino acid
transport activity when amino acid availability is limited.
The
goal of my Honors Thesis Project is to assess the relationship between amino
acid transport and CD98 function in cell growth. The study examines the relationship between amino acid
transport mediated by CD98 and tumor growth and proliferation over an
observation period while cancer cell lines were treated with blocking
antibodies to CD98hc provided by Dr. Ira Herman (Papetti and Herman,
2001). The b E11
antibody, a monoclonal antibody specific to the CD98hc, inhibited the growth of
several cancer cell lines in a dose dependent manner in vitro (Papetti and Herman, 2001). However, their study did not address
whether the b E11 antibody had any affect on amino acid transport activities and if
any causal link could be established between growth inhibition and a change in
amino acid transport activities.
To investigate whether the growth inhibition is a consequence of
decreased amino acid transport mediated by b E11 blockade of CD98, the
activities of amino acid transport and cell proliferation of cancer cell lines
treated with b E11 or non-immune IgG1 K over several days were compared. Current data suggests that there is a
statistically significant decrease in amino acid transport level due to b E11 treatment, but it remains
uncertain if the growth inhibition can be attributed to the decrease in
transport activity.
Novel
Mechanisms for the Reduction of Hepatitis B Virus e Antigen Expression
By: Genie
Bang
Sponsor: Shuping
Tong
Background: Hepatitis B virus (HBV) expresses a secreted protein
called HBeAg that is not essential for viral replication but may not promote
persistent viral infection. On the
other hand, the host immune response against HBeAg helps clear HBV infected
cells. Thus, rise of anti-HBe
antibody in HBV patients is usually associated with emergence of viral variants
that express low HBeAg or no HBeAg at all. So far only two types of HBeAg variants have been well
characterized: core promoter mutants (with reduced transcription of HBeAg mRNA)
and precore mutants (with nonsense mutation in the HBeAg coding sequence). The generation of mature HBeAg requires
removal of the N-terminal signal paptide, C-terminal basic sequence, and
formation of disulfide bond between two cysteine residues (cys-7 and
cys61). Naturally occurring
mutations have been found in anti-HBe patients at precore codon 17, encoding
the Đ3 residue of the signal preptide cleavage site. This mutation was proposed to impair cleavage and hence
reduce HBeAg formation. Mutation
of either of the two cysteine residues was found to greatly impair HBeAg
secretion and/or antigenicity, but whether such mutations will impair viral
replication has not been studied.
Aims: We wished to determine 1. whether the naturally occurring
codon 17 mutation indeed impaired HBeAg expression, and whether it affected
viral replication capacity; 2. whether mutations affecting the two cysteines
affect viral replication.
Methods: Site-directed mutants were generated, converted to tandem
dimer, and transfected to human hepatoma cells. HBeAg expression was measured and normalized against HBsAg,
which should not be affected by the mutations. Viral DNA replication was detected by Southern blots.
Results: The codon 17 mutation did not impair HBeAg expression, but
greatly reduced viral DNA replication.
A precore codon 29 mutation, which often accompanies codon 17 mutation
in patients, was found to restore the replication capacity of the codon 17
mutant. It did not affect the
HBeAg expression level. Mutation
of the cys-7 impaired HBeAg expression as anticipated, but also abolished viral
DNA replication. This is
consistent with the importance of the position occupying the loop of
encapsidation signal, a structure essential for viral replication. Mutation of Cys61 to ser or arg, while
abolished HBeAg expression, was tolerated for viral replication. However, a cys to arg change at this
position abolished viral particle secretion.
Conclusion: 1. Contrary
to prediction, precore codon 17 mutation did not impair HBeAg expression. 2. The co-variation at codon 29 is required to maintain high
replication capacity of codon 17 mutant.
3. Đ7 cys mutant is
replication defective and hence unlikely viral survival mechanism under
anti-HBe. 4. Cys61 mutation will abolish HBeAg
expression, retain viral replication, and maintain or disrupt viral secretion
depending on the exact nature of the substitution.
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Bat Flight Kinenatics and
Ecological Correlations.
By: Daniel
VanMeter
Sponsor: Sharon Swartz
The
dynamic flight patterns of aerial animals far exceed the capabilities of many
modern aircraft. Understanding bat
flight and the role of dynamically varying camber will aid in achieving the greatest
amount of lift with the least cost in modern airfoils. The goals of this experiment were first
to demonstrate the relationship between camber and lift, and second, to
investigate camber variation over the wing beat cycle at different wind
speeds. Camber increases drag at
higher speeds, and so it is expected that camber will decrease with increasing
speed. In this experiment, flight
videos of the bat Miniopterus australis in a wind tunnel were entered into the PEAK Motus
system and a series of points critical to estimating wing camber and lift were
digitized. A series of segmental
distances and a vector angle were used to estimate wing camber and lift. Lift, as estimated by vertical
acceleration, and camber were positively related over time. This relationship was not perfect,
however, with a change in camber sometimes offset from the change in lift. Vertical acceleration, and camber were
positively related over time. This
relationship was not perfect, however, with a change in camber sometimes offset
from the change in lift. Vertical
acceleration always decreased during the downstroke from a positive value to a
negative value. Vertical
acceleration during the upstroke returned to a positive value. Adjusted camber, however, did not show
such a clear trend. The lack of a
perfect correlation between camber and lift could be the result of other
mechanisms of lift generation such as wing flapping. At higher speeds camber remained relatively constant, but at
the slowest speed, camber increased significantly during the downstroke. Further data will illustrate whether
trends in this study can be applied to other bat species or even other
individuals. Computer flow models
could directly investigate how camber affects flow, and could be better put
into context through integration with studies of other flight characteristics,
such as angle of attack.
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An Evaluation of Astrogliosis
and Synaptic Alterations in Temporal Cortex Tissue of Patients Diagnosed with
Mesial Temporal Sclerosis
By: Krister
Jones
Sponsor: Edward Stopa, M.D.
The
most common neuronal cell dysfunction observed in patients with temporal lobe
epilepsy is mesial temporal (AmmonŐs horn) sclerosis. Synchronous hyperactivity in the hippocampus of patients
diagnosed with mesial temporal sclerosis has been shown to cause neuronal cell
loss, decreased synaptic density, and increased mossy fiber sprouting within
the affected hippocampus (Proper et al., 2000).
In the present study, temporal cortex tissue removed through partial
temporal lobectomy of patients suffering from pharmaco-resistant temporal lobe
epilepsy was evaluated using synaptophysin, glial fibrillary acidic protein
(GFAP), and GAP-43 immunoreactivity.
The degree of immunoreactivity was quantitatively measured using
computerized image analysis.
Results indicate that while levels of GFAP in temporal cortex are
similarly elevated to those found in hippocampus with mesial temporal sclerosis
in temporal cortex, there may not be the widespread degeneration of synapses
seen in the hippocampus of patients with this disease. This finding may lead to further
determination of the mechanisms by which the epileptic seizure is either caused
or spread in the hippocampus and subsequently affects larger brain regions.
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Costs of Resistance Associated
with Genetically Engineerd Cold Tolerance in Arabidopsis thaliana.
By: Matthew
W. Jackson
Sponsor: Johanna Schmitt
Traditionally researchers
investigated costs of resistance to disease, herbivores, and herbicides in
plants instead of environmental stresses.
Much of this research focuses on the costs and benefits of constitutive
expression versus inductive expression in resistance pathways. Using Arabidopsis thaliana (Brassicaceae), a primarily
selfing winter annual found in temperate regions throughout the Northern
Hemisphere, I used genetically engineered Arabidopsis thaliana that, in each line,
constitutively overexpress an individual CBF gene to investigate the costs of
resistance to cold tolerance across variable environments. A drop in temperature induces an
endogenous cold-acclimation pathway controlled by a family of transcriptional
activators, the CBF gene family.
Consisting of three genes (CBF1, 2 and 3), this family activates a
cascading reaction of cold-tolerance genes that prepare the pant to survive in
cold environments. I investigated
the effects of cold-acclimation temperatures 4űC, and cold-shock temperatures,
-6 to 8űC, on the fitness of engineered and non-engineered with a two-by-two
factorial design. Plants with
cold-shock exposure produced too few fruits for analysis; however, interesting
effects on lifespan occurred. For
plants without cold-shock exposure, engineered plants showed no difference in
fitness across no cold-acclimation and cold-acclimation environments, unlike
the control plants. CBF2, 3 showed
significant costs of resistance in all environments; however, CBF1 showed no
cost of resistance in a no cold-acclimation environment, and it yielded
significantly greater fitness than the controls in a cold-acclimation
environment. Interestingly,
theCBF1 gene showed no cost of resistance in a normal, no cold-acclimation
environment and a clear fitness benefit in a cold-acclimation environment.
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Slinity Fluctuations and
Competitive Dynamics in Salt Marsh Bacterial Communities
By: Lexi
Weintraub
Sponsor: Jennifer Hughes
Theory predicts that
environmental variability can play a significant role in determining community
structure. I examine the effects
of salinity fluctuations on the competitive dynamics of salt marsh bacterial
communities. I measured the
maximum growth rates of 6 strains of bacteria isolated from a fresh and a
brackish marsh at multiple salinities and incorporated these data into a
model. The model generated
predictions about the effects of salinity fluctuations on dynamics of
competition between strains.
Maximum
growth rate measurements suggested that salinity plays a role in structuring
marsh bacterial communities.
Strains isolated from the fresh marsh had relatively higher growth rates
at low salinities, while strains from the brackish marsh specialized at
salinities approximately equal to the site from which they were isolated. The model was unable to predict the
results of fluctuations. The data
suggest that multiple interactions occurred between strains.
Fluctuations
affected competitive dynamics in all cases, and generated an overall increase
in community evenness in all competitions. These results support the hypothesis that environmental
variability influences community structure and promotes diversity.
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Comparison of 2 Methods of
Fabrication of Poly (fumaric-co-sebacic) Anhydride Microspheres for Oral Delivery
of Insulin
By: Emily
Bubbers
Sponsor: Edith Mathiowitz
Poly (fumaric-co-sebacic)
anhydride (P(FA:SA)) is used to encapsulate insulin for oral delivery to Type diabetics. The
purpose of this study is to reduce the size of P(FA:SA) microspheres to less
than 2 µm in diameter in order to increase the number of microspheres absorbed
by the small intestine. Reducing
the size of the spheres allows the microspheres to traverse into the blood
stream either via the PeyerŐs patches, paracellularly or transcellularly. As the microsphere degrades, carboxyl
groups are formed on the surface, which bind with the glycoproteins in the
intestinal lumen, increasing the residence time in the gastrointestinal tract.
Insulin loaded microspheres were fabricated
using the phase inversion nanoencapsulation (PIN) method, either with or
without the PIN apparatus. A
surfactant was also added to those microspheres made without the PIN
apparatus. The spheres were
characterized using scanning electron microscopy (SEM) and Coulter Small Volume
Particle sizing. Insulin release
profiles were obtained via in vitro release studies. In vivo studies were
performed on diabetic rats to test the ability of the microspheres to maintain
blood glucose levels in the presence of food. Microspheres fabricated
with surfactant demonstrate the smallest mean diameter by volume, deliver more
insulin to the body and maintain blood glucose levels for prolonged periods of
time. These microspheres can be an effective method of controlled
delivery of insulin. An oral
delivery system for insulin will negate the need for daily injections of
insulin and provide a more constant level of insulin in the blood stream,
rather then bursts from subcutaneous injections.
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Design of a Novel Immunoisolated
Islet Delivery Device Utilizing Microfabrication, Polymer and Cellular
Technology
By: Peter
A. Rachlin
Sponsor: Jeffrey Morgan
Transplantation of islets of
Langerhans is a potential treatment of Type I diabetes that aims to restore
normal glucose homeostasis. Microencapsulation of islets could enable
transplantation in the absence of immunosuppression, which would be beneficial
as the side effects associated with immunosuppression outweigh the potential
benefits of islet transplantation.
However, cell-encapsulating technology is not without challenges,
including problems concerning the geometrical characteristics of the implant
device, the tissue density and spatial arrangement of the encapsulated cells,
and the islet cellsŐ oxygen availability.
Housing and maintaining approximately 700,000 islets in a practical
implant remains as one of the major hurdles to overcome in making implantable
immunoisolation devices a clinical reality. The aim of this study is to pursue these engineering
challenges and design a novel immunoisolated islet delivery device as an
alternative to current therapy using microfabrication techniques and polymer
and cellular technology. Porous
and topographically complex polyurethane membranes and alginate hydrogels were
manufactured utilizing a soft lithography approach. SU-8 polymer-coated silicon wafers were employed as master
chips, providing a surface array of hexagons 50 µm to 200 µm deep, with radii
from 25 to 300 µm on centers from 75 to 900 µm. A battery of negative replicates of these wafers was
produced using polydimethylsiloxane silicone elastomer (PDMS). Solutions of polyurethane and alginate were
polymerized on the PDMS templates to yield membranes and hydrogels,
respectively. Both the
polyurethane membranes and the alginate hydrogels successfully replicated the
complex surface topography of the various PDMS stamps. These two products were subsequently
envisioned to function as three-dimensional scaffolds for tissue-engineered
materials, specifically an islet delivery device. Polyurethane scaffolds were loaded with fluorescent beads
approximating the size of islets and immunoisolated with a coating of alginate
hydrogel. With the concept proven
to be viable, isolated rat islets were successfully loaded into a polyurethane
scaffoldŐs hexagonal wells with precise and predetermined dimensions and then
the scaffold was immunoisolated with a coating of alginate. The islets remained functional and
entirely contained in the polyurethane-alginate housing. Microfabrication technology appears to
be an integral component in the effort to make a reality the possibility of
replacing damaged or diseased organs with artificial tissues engineered from a
combination of living cells and biocompatible scaffolds. The development of photolithography to
pattern biological molecules onto surfaces, novel tools to move silicon-based
fabrication to polymer-based constructs and the move from two-dimensional to
three-dimensional fabrication each provide essential tools in the development
of in vitro
systems to study the control of cells through their environment, the production
of scaffolds to support tissue architecture, and the fabrication of living
hybrids of cells and scaffolds that demonstrate the incremental steps toward
reconstruction of tissue function.
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Metabolic Cross-Feeding in
Two-Species Bacterial Communities
By: Ian
T. Carroll
Sponsor: Jennifer Hughes
Metabolic cross-feeding between
bacteria occurs in natural assemblages and artificial communities, including
cultures of closely related Escherichia coli strains. Laboratory communities with
cross-feeding relationships support coexistence in cultures supplied with a
single limiting nutrient for which response competition generates testable
predictions about community structure.
The rate of supply of the limiting resource is predicted to determine
whether competitive exclusion or coexistance occurs. For increasing rates of nutrient supply, the population
sizes are predicted to equilibrate at a common ratio. Results from experimental manipulations of two independently
derived pairs of cross-feeding E. coli grown in chemostat culture qualitatively agree
with model predictions.
Exploring
Cytokines and Innate Immune Kinetics in the Epidermis; Development of a
Non-Invasive Luminescence Based Assay to Quantify Bacterial Load
By: Roxanne
J. Wadia
Sponsor: Jeffrey Morgan