Editors’ Note

December, 2010

Dear UVa Community,

It is with great pleasure that we present you this semester’s issue of The Oculus: Virginia
Journal of Undergraduate Research. As the culmination of eight weeks of peer reviews, this
Fall issue stands as an assortment of some of the most well-written and original pieces of
undergraduate research that can be found at our University. Representing several fields of
study, from history of religion to environmental policy to biomedical engineering, this edi-
tion is truly representative of the breadth and depth of research conducted at the University
of Virginia by students and their faculty mentors.

As this is our last semester as Co-Editors-in-Chief, we wanted to thank everyone involved

in the completion of this journal, from the students who submitted their phenomenal pieces
of research at the beginning of the semester to our insanely dedicated layout editor. Please
note that this publication of undergraduate research could not have been made possible
without the efforts of our motivated and enthusiastic staff of reviewers, who met each week
to discuss, and sometimes debate, the merits of every individual manuscript submitted for
this semester’s issue. This is what we like to call the undergraduate peer-review process,
which is unique from most journals in that there are no faculty advisers reviewing the
manuscripts. Moreover, The Oculus is grateful to the staff at the Center for Undergraduate
Excellence (CUE) for their continued support and collaboration over the years. We give
special thanks to the faculty members who mentored these students in their research en-
deavors, and to you, the readers, for being sufficiently curious to pick up a copy of The
Oculus. Please enjoy!

Sincerely,

Tudor Cisu Mitchell Leibowitz

Co-Editor-in-Chief Co-Editor-in-Chief
 Letter from the President

Dear Readers:

Research conducted by undergraduate students working in close collaboration with dis-

tinguished faculty members has become a hallmark of excellence in America’s top univer-
sities.  Here at U.Va., undergraduate research has added depth and breadth to our core
academic disciplines, enhanced our progress toward interdisciplinary inquiry, and shaped
the interests and aspirations of our students. 

The Oculus presents high-quality research conducted by undergraduate students working

in a broad range of disciplines. This is an undergraduate enterprise entirely. Each semester,
undergraduate student-editors select, edit, and publish the very finest undergraduate re-
search submitted from across the University’s academic departments. Any undergraduate
who has written a paper or conducted a significant research project has the opportunity to
submit work for consideration by the editors. The Oculus is a training ground for under-
graduates who will go on to careers in research and journal publishing.

Thomas Jefferson believed that learning and discovery lead naturally to human betterment.
He wrote, “I look to the diffusion of light and education as the resource to be relied on for
ameliorating the condition, promoting the virtue, and advancing the happiness of man.” 
Our undergraduate students who are dedicated to research, discovery, and scholarship
continue the important work that began when Mr. Jefferson founded this University nearly
200 years ago.

Very truly yours,

Teresa A. Sullivan
President, University of Virginia
 The Oculus
The Virginia Journal of Undergraduate Research

Volume 9 Issue 2

Contents

Around the University: Mentors and Their Students 1

Professor Cassandra Fraser and Ruffin Evans
Identifying Cardiovascular Diseases Through Electrical 3D Modeling of the Heart 3
Joyce Ng, Debbie Padilla, Nivedha Panneer, Wyatt Shields, Hsuan Su
Establishing Legitimacy: Early Christians’ Incorporation of Pagan and Jewish 13
Iconography in the Via Latina Catacombs
Jessica Arden Ettinger
Application of Fingolimod (FTY-720) for TherapeuticArteriogenesis and 21
Microvascular Remodeling
Nikhil Panda
Managing Water Pollution: The United States and Mexico, a Comparative Study 28
Kelsey Kerle-O’Brien
The Influence of Stomatal Morphology on Gas-exchange Processes of Native and 34
Invasive mid-Atlantic Tree Species
Erin Dale

Coeditors-in-Chief
Tudor Cisu and Mitchell Leibowitz

Editoral Staff
Gunjan Amarnani, Upasana Bhattacharya, Ja Hyun Cho,
Michelle Choi, Li “Nelly” Fan, Marina Freckmann,
Jessleen Kanwal, Caroline Plowden, Sharon Rogart,
Catherine Schretter, Ko Eun “Janet” Shin, David Wu, Jason Ya

Layout Editor
David Wu

Photographer
Jessleen Kanwal

The Oculus is published by the Undergraduate Research Network (URN) in

conjunction with the Center for Undergraduate Excellence (CUE). All copyrights are re-
tained by the authors; URN holds the rights to non-exclusive use in print and electronic
formats for all pieces submitted for publication in The Oculus.
 Around the University: Mentors and Their Students
Professor Cassandra Fraser and Ruffin Evans
P rofessor Cassandra Fraser is a Professor of Chem-
istry and Biomedical Engineering at the Universi-
ty of Virginia. She was a Radcliffe Fellow at Harvard
University, serves on the editorial advisory board of
ACS Applied Materials and Interfaces, was a recipient
of the Cavaliers’ Distinguished Teaching Professor
award, and was named a National Associate of the
National Academies for service to the nation. Profes-
sor Fraser is one of U.Va.’s exemplary researchers,
especially in her interaction with undergraduates.
She is proactive in selecting the students who join her
research group in reaching out to those have demon-
strated superior academic performance in introduc-
tory level Chemistry courses. Ruffin Evans is one of
the undergraduates who work in her lab; he started
in his first year at U.Va. Professor Fraser believes that
there is no specific time when one can or should be-
gin researching. She says, “My philosophy is that you
give people opportunities and see what they are ca-
pable of contributing … You set the bar high and they
can jump as high as they want.” Ruffin is currently a
fourth-year double major in Chemistry and Physics,
Fall 2010: Volume 9, Issue 2
and has been awarded numerous research awards
and fellowships as well as the Barry M. Goldwater
Scholarship in 2010.
The approach in the Fraser lab is to always seek
out opportunities, a motto which Ruffin has fol-
lowed. On one occasion, Ruffin, Professor Fraser and
the lab’s creative thinking culminated in a two and a
half week trip to China in the summer of 2009. The
idea to visit China was initiated as a proposal for
the Double-Hoo Grant, which is awarded to pairs of
undergraduate and graduate students intending to
pursue joint research projects. Professor Fraser, Ruf-
fin, and Guoqing Zhang, an outstanding graduate
student in the lab, felt that the experience of going
to China to exchange ideas and share their research
at a number of major universities would be a valu-
able one. Eventually, summer research awards and
support from the Office of Vice President of Research
helped cover costs for the trip. Professor Fraser says,
“In planning the trip, I told our hosts that I would
like for my students to give talks too, to gain expe-
rience.” Both Ruffin and Professor Fraser recall the
1

trip to China with Guoqing as generous guide and
host, as an incredible experience, especially because
it allowed Ruffin, then a second year undergradu-
ate, along with Guoqing, to give scientific talks at the
prestigious University of Science and Technology of
China, to an audience of more than one hundred dis-
tinguished scientists and students.
Professor Fraser believes that “really good stu-
dents should have the chance to take initiative, and
take the lead on something and that is a big part of
science.” Therefore, when she and her team began
exploring computational areas, she asked Ruffin to
lead the initiative. Ruffin now teaches graduate stu-
dents in the Fraser lab how to perform computational
analyses. This leadership experience was invaluable
to Ruffin. The opportunity arose given Ruffin’s inter-
est and study of physics; he has since decided that he
will likely pursue graduate coursework in physics. To
this Professor Fraser says, “My job is to support the
students to become really strong at what they want
to be” and, therefore, she encouraged Ruffin to ex-
plore other interests, while he maintains his relation-
ship with the Fraser lab through his computational
research. Ruffin adds, “I’ve learned skills here that I
would probably never have learned in a physics lab,
but that I may well use. Even the simplest chemistry
techniques have many applications.”
When asked about the advice they would give
to undergraduates, Ruffin replied, “Get involved as
early as you can and apply for everything. It is hard
to be too ambitious. Once you have the opportunity
to do research, really give it your all because no mat-
2 The Oculus: The Virginia Journal of Undergraduate Research
ter what you do in the future, that is probably what
people are going to be looking for. People want good
researchers.” Professor Fraser echoed that sentiment
and advises students to look for mentors who are
supportive and give students unique opportunities.
In terms of research at the University, Professor
Fraser stressed that the key is to take initiative and
be intellectually curious. She adds, “Research makes
what you are learning in the classroom more alive,
because you might think it is a fixed body of knowl-
edge and you are just learning that body of knowl-
edge, but there were people who asked questions and
did experiments to discover and find that and in fact,
it is much more dynamic than that. When you realize
that, and have an idea what it takes to make discover-
ies and generate knowledge and share insights your-
self, you have a much better appreciation of things.”
Additionally, Professor Fraser discussed the im-
portance of broadening the concept of research and
thinking independently. Professor Fraser believes
that “research—even scientific research—can take
many forms. It could be in a lab but you might have
a question about some science question related to
society. You can do a lot of research by just reading
and learning, and talking to people too, and I think it
is important and valuable for everyone to formulate
their own questions. What are you curious about –
not just what your advisor is curious about - and how
would you go about addressing those things? And
that could be coupled to a lab but it doesn’t have to
be.” Ruffin added that students should benefit from
both teaching and research aspects of the University.
He says, “It is very important for undergraduates to
be a part of that and combine the teaching function
with its research function. That is how, ultimately,
human knowledge can move forward.”
The University of Virginia, in the words of its
founder Thomas Jefferson, is based on the “the il-
limitable freedom of the human mind.” Getting in-
volved in research is a small step towards pursuing
that freedom and engaging the mind. Therefore, let
us enquire relentlessly, explore persistently and make
the most of the resources that we have and the oppor-
tunities that we can create from them.
Upasana Bhattacharya
 Identifying Cardiovascular Diseases Through
Electrical 3D Modeling of the Heart
Joyce Ng, Debbie Padilla, Nivedha Panneer, Wyatt Shields, Hsuan Su
Software capable of creating a movie of three-dimensional (3D) spatial vectorcardiography
(VCG) can be used as a clinical tool for screening and diagnosing heart diseases. Cardiolo-
gists support the development of this software because it is a viable substitute for expensive
imaging procedures while decreasing the amount of time used to analyze electrocardiograph
(ECG) signals from separate leads. Two ECG amplifier systems were constructed with five
stages: simple voltage follower, differential operational amplifier, high-pass filter, and two
low-pass filters. Gains and corner frequencies were calculated from theoretical transfer func-
tions and compared to experimental data for the different stages of each circuit. Bode plots
exhibited corner frequencies of 149.4 and 0.97rad-s-1 as well as 147.4 and 1.24rad-s-1 for cir-
cuit-1 and circuit-2, respectively. Non-polarizable electrodes simultaneously recorded digi-
tized ECG analog signals from Leads I and II in the frontal plane, while V4 and V6 were
recorded simultaneously in the horizontal plane. ECG signals in the QRS complex were con-
verted to instantaneous cardiac vectors (ICV) through inverse VCG in each plane and were
subsequently summed together to form a 3D movie. The resulting movie, comprised of 3D
ICVs, traced the contour of biopotentials across the heart. The largest ICV of a healthy heart
showed a general downward direction and pointed into the chest. In comparing the 3D con-
tours of healthy to diseased hearts, the differences in the visual representation indicated
flawed magnitude and movement of biopotentials in diseased hearts. This demonstrates the
efficacy of the software in modeling biopotentials in innovative and unprecedented ways.
Introduction The most commonly used diagnostic tool in the

C ardiovascular disease is the single leading cause

of death in America, accounting for over half a
million deaths annually.1 With recent technologi-
cal and scientific advancements, diagnostic testing
has proven to be a powerful tool in the fight against
cardiovascular disease by allowing for earlier detec-
tion of heart abnormalities. Clinicians are currently
able to conduct a variety of tests and procedures to
reveal different features of the heart, including ECG
to measure bioelectric currents in the heart, and cor-
onary catheterization to assess heart valve patency.
However, many of these procedures and devices are
costly, produce unspecific results, and fail to provide
comprehensive information about the electrical be-
havior of the heart. With a global market value of
$11.9 billion in 2007 and expected growth to $21.4
billion by 2012, cardiovascular diagnostics represents
one of the largest and most rapidly growing fields
within the diagnostic sector.2 The growing market
and the number of annual deaths from faulty diagno-
ses provided students the motivation to improve cur-
rent diagnostic imaging modalities by developing a
3D representation of biopotential propagation across
the heart. Since the aim is to produce 3D represen-
tations from ECG data, only software is required to
implement the program without the use of a new de-
vice. The most important feature of this technology is
its compatibility with existing devices and methods,
thus leading to the tremendous cost-efficiency and
wide profit margin of this pioneering software.
Fall 2010: Volume 9, Issue 2
clinic today is the ECG. By attaching electrodes to
specific locations on the arms, legs and chest, phy-
sicians are able to measure the bioelectric potentials
associated with the contractions of the heart. ECG is
a safe, noninvasive technique that measures the de-
polarization, propagation, and subsequent repolar-
ization of biopotentials throughout the heart. Frontal
plane ECGs consist of bipolar limb leads I, II, and III,
as well as augmented unipolar limb leads aVR, aVL,
and aVF. Horizontal plane ECGs consist of precor-
dial chest leads V1, V2, V3, V4, V5, and V6.3 Together,
these recordings of the frontal and horizontal planes
provide a more complete representation of the bio-
electric events in the heart. One limitation of the ECG
is that an abnormal recording is unspecific in its di-
agnosis since it can be indicative of a number of heart
diseases or irregularities. Hence, ECG is currently
used as a preliminary test and screening tool, from
which more specific diagnostic tests can be imple-
mented. Furthermore, since the ECG records electri-
cal events only for a brief period, a normal ECG re-
cording is not conclusive of a healthy heart. Another
similar technique is the stress test, in which the ECG
signals are recorded while the patient exercises at dif-
ferent levels of physical activity. This allows for de-
tection of arterial blockage, which would result in re-
stricted blood flow and lead to observable changes in
the measured ECG signals. Although this additional
test does enhance diagnosis of diseases, limitations of
stress tests include false negatives.
3
 Another technique that studies the electrical phe-
nomena of the heart is the electrophysiology test in
which catheters are used to detect abnormal heart
rhythm. This technique provides more informa-
tion on heart rhythm that cannot be detected from
ECG signals. By attaching electrodes on the end of
the catheter, which is fed into the heart through an
artery in the arm or leg, this test directly listens to
the electrical activity within different section of the
heart. The information gathered can be used to deter-
mine proper placement of pacemakers. Additionally,
cardiac cells that were producing irregular electrical
signals, resulting in arrhythmia, can be destroyed
through radio frequency waves4. Drawbacks and
risks of this invasive technique include infection or
injury to blood vessel, arrhythmia or damage to the
heart.
The majority of devices and techniques focused
on measuring the electrical features of the heart in-
clude mostly analog, 1-dimensional (1D) represen-
tations of biopotential events of the heart. Yet, there
have been developments that incorporate the 3D na-
Debbie Padilla (left) is a fourth-year majoring in Biomedi-
cal Engineering and her interests include computational
modeling and public health. She has worked the past two
years in Dr. Holmes’ cardiac biomechanics lab with col-
lagen gels to understand how scar forms after a myocar-
dial infarction and how healing may be guided to achieve
desired scar structure. Her current research and capstone
project in Dr. Kelly’s targeted nanomedicine lab involves
the development of a novel rapid bacteria detection device
using bacteriophage, along with a model for disease trans-
mission. After graduation in May 2011, she plans obtain
a Master’s in Public Health or a Master’s in Biomedical
engineering with a concentration in Global Health.
4 The Oculus: The Virginia Journal of Undergraduate Research
ture of electrical propagation within the heart. VCG
is a diagnostic technique where the magnitude and
direction of the electrical currents across the heart
are graphically presented in the form of an ICV. The
spatial VCG is the recording of the time variations of
the ICVs and transforms the ECG signal into a con-
tour. Physicians can then diagnose and determine the
locations of cardiac abnormalities by comparing the
shapes of these contours.5 Spatial VCG was discov-
ered and practiced in the 1950s with the Frank Lead
System, which makes use of V1 to V6 of the horizon-
tal plane and Lead I and Lead II of the frontal plane.6
Other modified lead systems, such as the McFee
and Parungao System, implement different physical
principles to better fit clinical purposes.7 Currently,
by using ECG signals of eight or more electrodes,
the 3D ICVs of P-QRS-T complexes can be mapped
computationally. These 3D ICVs visually represent
the shape of the heart and therefore aid physicians in
detecting diseases more efficiently.8 Since VCG mea-
surements are time-consuming and require multiple
simultaneous lead recordings to develop accurate 3D
Joyce Ng (second from left) is a fourth-year Biomedical En-
gineering major. Apart from BME, she finds computer sci-
ence particularly fascinating and she loves to learn about
people in different cultures, along with her desire to serve
overseas in medicine. She is recently working under the
guidance of both Dr. Owens and Dr. Saucerman on a se-
nior thesis research project on mathematical modeling of
Wnt signaling pathways and epigenetic mechanisms in-
volved in cardiovascular cells differentiation, which will
hopefully be utilized in optimizing protocols for cardiovas-
cular stem cell therapies. After graduation in May 2011,
she plans to attend medical school in Hong Kong, home to
her heart, and continue to pursue her passion in medicine.
Table 1. Transfer Functions for Individual Stages and the Cascaded Circuit This table provides the transfer function used to
calculate the gains for the five stages in the two circuits that were built. It also has the final equation used to calculate the
total gain of the circuits under the column labeled total. The first stage was a voltage follower with a gain of one and the
second stage had a constant gain. Only Stages 3, 4, and 5 depend on the frequency and therefore consistently change.
vectors, it is inconvenient and rarely used in early
examinations. This limits the extent of the diagnostic
use of VCG.
In view of this, the goal of this project was to
develop a simple, user-friendly computational pro-
gram that utilized two sets of two simultaneous ECG
signals to generate an animated 3D QRS contour. By
comparing the resultant 3D contours of healthy and
diseased hearts, this program allowed for instant di-
agnosis at an earlier clinical examination. Currently,
ECGs are used as a screening tool. However, after
successful implementation of this innovative and
cost-efficient software, VCG has the potential to be-
come a new standard in diagnosing heart abnormali-
ties.
Predictions
It was predicted that the 3D contour from the
healthy experimental heart would have a similar ori-
entation to that of the healthy theoretical 3D contour.
Additionally, four different diseased 3D contours
were compared to the healthy experimental 3D con-
tour. It was predicted that the blockages in a bundle
branch block (BBB) would induce irregular propaga-
tion of biopotentials, whereas the macro-action po-
tential, which corresponds to the largest ICV, would
point in a direction different from that of the healthy
contour. In the case of myocardial infarction (MI), the
contour was predicted to be smaller than that of the
Nivedha Panneer (middle) is a fourth-year pursuing a
Bachelor’s of Science in Biomedical Engineering. Her in-
terests include infectious diseases and drug delivery. Her
current capstone research involves the design of an assay
and diagnostic device for the detection of Bordetella per-
tussis, commonly known as whooping cough, as well as
the development of a computational model that studies the
spread and transmission of B. pertussis in a population.
Upon graduation in May 2011, she plans to obtain a Mas-
ter’s in Public Health with a focus on global health and
health education in underdeveloped countries.
Wyatt Shields (right), a Virginia native, is a fourth-year
Biomedical Engineering major and a Materials Science
and Engineering minor. His interests include biomateri-
als, tissue engineering, and ultrasound imaging. His cur-
rent research and senior thesis project in Dr. Saucerman’s
Fall 2010: Volume 9, Issue 2
healthy contour. For myocarditis, the inflamed myo-
cardium was expected to result in an abnormal QRS
complex and a slower propagation time. In the case
of valvular heart disease (VHD), the diseased valves
were hypothesized to cause a non-directional con-
tour orientation.
Materials and Methods
Materials
Materials needed for this experiment include
two ECG preamplifier circuits, two 3-patient cables,
an oscilloscope, a multimeter, a DC power source,
OpenChoice, multiple resistors, capacitors, non-po-
larizable electrodes, alcohol wipes, and wires with
crocodile clips.
Stages of the Circuit and Net Transfer Function
Two ECG preamplifier systems, consisting of five
stages each, were built separately and cascaded as
shown in the Appendix. Stage 1 was a voltage fol-
lower, which served to produce high input imped-
ance. Stage 2 was a differential amplifier, which mul-
tiplied the difference between the two inputs by the
differential gain without implementing positive feed-
back to maintain stability. By using a variable resistor,
the impedances in parallel with the inverting input
properly balanced impedances in series with the non-
inverting input, ensuring that only the cardiac biopo-
tential was amplified. Stage 3 was a high-pass filter,
cardiac modeling laboratory involves modeling the intra-
cellular pathways of cardiac myocytes through beta adren-
ergic receptors. After graduation in May 2011, he plans to
matriculate in a biomedical engineering doctoral program,
specializing in biomaterials with intentions to serve as a
faculty member at an engineering university.
Hsuan Su (second from right) is a fourth year majoring
in Biomedical Engineering with a minor in Engineering
Business. She has past experience working in cancer, bio-
materials, and chemical synthesis labs. For her current
capstone project, she aims to design a novel insole for run-
ning shoes that mimics the properties of barefoot running
and will conduct biomechanics testing in a gait and mo-
tion laboratory. After graduation in May 2011, she hopes
to pursue a lifelong career in the healthcare field anywhere
in the world.
5
 Figure 2. Filtered and Non-filtered ECG signals 2A) Non-filtered Lead II ECG
Figure 1. Bode Plots for Both Circuits 1A)
signals. 2B) Filtered Lead II ECG signals. 2C) Non-filtered V6 ECG signals.
Bode Plot of total gain for circuit-1. 1B)
2D) Filtered V6 ECG signals. Lead II and V6 were used as examples of how
Bode Plot of total gain for circuit-2. The
the software used a Butterworth filter to smooth the ECG signals.
red and blue lines represent the theoreti-
cal and measured gains, respectively.

which blocked low frequency noise whereas Stage 4 ECG and Vectorcardiography
and 5, low-pass filters, blocked high frequency noise. The circuits were used to acquire simultaneous
In order to reduce the baseline 60Hz noise in the ECG ECG measurements to represent the QRS complex
signals and to obtain a sharper pass band, a 1MΩ re- of a specific heartbeat. Frontal plane ECGs were re-
sistor was used in parallel with the capacitor in both corded using Lead I, where electrodes were on the
low-pass filters. Before the electrodes were attached, right and left arms, and Lead II, where electrodes
the patient’s skin was cleaned to remove oil and dead were on the right arm and left leg. The two circuits
skin cells that could disrupt the measured signal. recorded leads I and II simultaneously to produce
The finished ECG preamplifier system was then con- frontal plane ICVs of a specific heartbeat. Horizontal
nected with a 15V DC power supply to measure the plane ECGs were measured using precordial chest
analog ECG signals across the different leads of the
patient. Using Kirchhoff’s Laws, transfer functions
for each stage as well as the overall transfer function
for both circuits were generated and are shown in
Table 1.
From the theoretical transfer functions, the gains
for Stages 3, 4, and 5 were calculated by determin-
ing the respective magnitudes at a range of frequen-
cies, where s was equal to jω. The theoretical gains for
all five stages were multiplied together to obtain the
total gain of the circuit. The total gain was plotted
against time on a log-log scale to construct a Bode
plot. This process was repeated for both circuits. The
experimental gains of the two circuits were obtained
by applying a 100mV sinusoidal input and recording
the peak-to-peak output with an oscilloscope. These
experimental gains were calculated by the relation-
ship:
Figure 3. Frontal Plane ICV This figure illustrates how an
ICV, which is indicated by the red arrow, was constructed
in the frontal plane. The dashed green lines indicate the
perpendicular lines drawn from the lead axes.

6 The Oculus: The Virginia Journal of Undergraduate Research

Figure 4. The ICVs in Three Different Views 4A) 3D QRS ICV loop. 4B) The front plane ICV loop, shown with respect to the x
and z axes. 4C) The horizontal plane ICV loop, shown with respect to the x and y axes.
leads V4 and V6. Using right arm, left arm, and right
leg as negative inputs, V4 and V6 were recorded si-
multaneously to produce horizontal plane ICVs of a
specific heartbeat.
Inverse VCG was performed by transforming the
analog signals from the frontal and horizontal planes
into ICVs, while assuming the heart was stationary.
Since analog ECG signals are 1D, whereas ICVs are
2D, inverse VCG requires two analog signals to gen-
erate one ICV. Frontal plane and horizontal plane
ICVs were computed using the amplitudes within
the QRS complex for leads I and II and leads V4
and V6, respectively. The planes were converted to
Cartesian coordinates and the ICVs were plotted in
MATLAB; refer to the results for further discussion.
Since only two circuits were constructed, the frontal
leads were not recorded at the same time as the hori-
zontal leads. To account for this limitation, boundar-
ies were established to mark the beginning and end
of the QRS complex for ECG recordings in the two
planes. In order to create the 3D ICVs, the number of
sampling points within this interval was required to
be the same in both planes.
Abnormal ECG Data
Abnormal ECG data was obtained from the
Physikalisch-Technische Bundesanstalt (PTB) Diag-
nostic ECG Database. The different types of heart
conditions that were implemented into the software
Fall 2010: Volume 9, Issue 2
included BBB, anterior MI, myocarditis, and VHD.
Frontal leads I and II and horizontal leads V4 and V6
were used from these data files to obtain 2D ICVs of
frontal and horizontal planes, respectively. From this,
3D ICVs were generated. Comparisons were then
made between the contours of the hearts.
Results
Stages of the Circuit and Bode Plots
The overall theoretical and measured Bode plots
for both circuits are shown in Figure 1. The theoreti-
cal corner frequencies for circuit-1 were 149.4 and
0.97 rad-s-1 and for circuit-2 were 147.4 and 1.24 rad-
s-1. The errors between the theoretical and measured
gains for circuits-1 and 2 were 2.64 and 6.30%, respec-
tively. A 100mV peak-to-peak sinusoidal input was
applied to obtain gains since higher input voltages,
such as 1V, distorted the sinusoidal output.
ECG and Vectorcardiography
In order to create a visual representation of the
electrical propagation within the heart, the following
steps were implemented in the software. First, the
data was filtered using a Butterworth filter to smooth
the ECG data, as can seen in Figure 2. To obtain one
frontal plane ICV from two ECG signals, two perpen-
dicular lines were constructed from the amplitudes of
leads I and II, as seen in Figure 3. A resultant ICV was
then constructed from the origin to the intersection
7
 Figure 5. Contour Plots 5A) 3D QRS contour plot. 5B) The contour for the frontal plane,
shown with respect to the x and z axes. 5C) The contour for the horizontal plane, shown with
respect to the x and y axes.
point of the two perpendicular lines. To accomplish
this, the Cartesian coordinates of this intersection
point were determined. Since the Lead I axis cor-
responded to the x-axis of the Cartesian coordinate
system, the x-coordinate of this intersection point
was simply the amplitude of Lead I. Next, to deter-
mine the z-coordinate of the intersection point, the
perpendicular line from Lead II was first extended to
the z-axis. The z-intercept of this line was obtained by
dividing the Lead II amplitude by the cosine of 30 de-
grees. This is analogous to the y-intercept of the lin-
ear slope-intercept equation. The slope of this equa-
tion was the negative reciprocal of the slope of Lead
II. Finally, the z-coordinate could be obtained by in-
serting the previously determined x-coordinate into
the aforementioned linear equation. The z-intercepts
were multiplied by negative one because a positive
Lead II amplitude corresponds to a perpendicular
line with a negative z-intercept and a negative Lead
II amplitude corresponds to a perpendicular line
with a positive z-intercept, as seen in Figure 3. This
entire process was repeated for all data points within
the QRS complex to form a series of frontal ICVs; see
Figure 4B. Using V4 and V6 signals, the developed
software contained code using a similar process to
form a series of horizontal ICVs; see Figure 4C. Last,
the ICVs from both planes were summed in 3D space
to produce a series of 3D ICVs. A contour was formed
by connecting the ends of each subsequent ICV for
the frontal and horizontal planes, as well as in 3D
space; see Figure 5. A movie was generated at an
increased time span for a better visualization of the
contour formation.
The shape and orientation of the experimental
3D contour was used to validate the accuracy of the
software. This was done by comparing the direction
of the largest ICV, corresponding to the peak of the
QRS complex, to the expected direction of the mac-
8 The Oculus: The Virginia Journal of Undergraduate Research
ro-action potential and by comparing the shape of
the experimental to the theoretical contour, as seen
in Figure 6. The expected macro-action potential is
directed down the heart and towards the patient’s
back. This orientation is towards the thicker left ven-
tricle wall that contains more cardiac myocytes. The
experimental macro-action potential exhibited this
orientation, with slight deviations in the frontal and
horizontal contours. In the frontal plane, the only mi-
nor deviation was at the end of the contour. Instead
of completing the loop, the end deviated from the ex-
pected trajectory by curling in the opposite direction.
These deviations could be due to oversampling the
data in which parts of the P or T waves were sampled
instead of solely the QRS complex. In the horizontal
plane, the experimental contour had a smaller loop
within the larger loop, as seen in Figure 5C. Also,
the contour in the horizontal plane of the experi-
mental data exhibited a more circular shape instead
of a pear-shape in the theoretical horizontal plane
contour. These deviations were likely due to minor
differences in the shape and orientation between the
analyzed hearts.
Abnormal ECG Data
The ECG voltage values collected from the PTB
database were about two orders of magnitude great-
er than the ECG voltage values collected experimen-
tally, as seen in Figure 7. The most likely explanation
for this discrepancy was that the database values
were collected at a higher gain, therefore amplifying
the signal more than the experimental values. Along
with the diseased states, a control from the database
was also analyzed using the software; see Figure 7B.
The direction of the largest ICV for the control fol-
lows the same trend as the experimental ICV, which
indicates that both healthy hearts follow the expected
tendency for macro-action potential propagation.
Figure 7. 3D Contours for Healthy and Diseased Hearts 7A) The experimental 3D contour. 7B) The control 3D contour. 7C) The
3D contour for BBB. 7D) The contour for an anterior MI. 7E) The contour for myocarditis. 7F) The contour for VHD. The blue
and pink contours are of healthy and diseased hearts, respectively.

BBB is typically marked by a wider QRS complex
because the travelling time of an impulse from cell to
cell in a BBB heart is longer than through normal car-
diac myocytes. This explains the wide pear-shaped
QRS contour of the patient; see Figure 7C. Although
not specifically mentioned in the patient information,
it was further hypothesized that the patient was diag-
nosed with left BBB because the QRS contour appears
to begin in the right ventricle and travels towards the
left atrium, which is the typical depolarization path
for left BBB. This supported the hypothesis that the
direction of the macro-action potential was affected
by BBB. The frontal plane of the left BBB also has a
counter-clockwise rotation, which matches the pa-
tient’s contour formation.9
Fall 2010: Volume 9, Issue 2
MI is an interruption of blood flow, usually from
an occlusion of fatty thrombotic plaque in a coronary
artery, resulting in ischemia and necrosis of cardiac
myocytes. Once the affected myocardium dies, scar
tissue forms and it can no longer conduct electric-
ity or contract properly.10 As seen in Figure 7D, the
macro-action potential did not fully propagate down
the length of the MI heart, which was illustrated by
the z-to-y ratios of 0.3, 0.24, and 0.53 for the experi-
mental, control, and MI hearts, respectively. Here,
the macro-action potential in the MI heart traveled
43.4% and 54.7% less down the y-axis as compared
to experimental and control, respectively. This sup-
ports the expected propagation of the macro-action
potential for a heart with an anterior MI.
9
 Myocarditis is an inflammation of cardiac muscle
due to infectious diseases. After the original onset of
myocarditis, the response from the immune system
prolongs the disease. It may cause heart failure, heart
rhythm irregularities, or injuries to cardiac myocytes.
There is increased blood flow to the inflamed region
and an elongated QT interval in ECG readings that
represents abnormal depolarization of the heart11.
There may also be electrical conduction blockages
at the AV junction in myocarditis12. As a result, the
biopotentials take longer to propagate through the
QRS complex. As seen in Figure 7E, the predominant
propagation occurs in the horizontal plane with little
change in the z-direction. This widened contour in
the horizontal plane is likely due to longer propaga-
tion time through the QRS complex, which was also
seen in BBB. Since this patient suffers from both myo-
carditis and BBB, it is difficult to discern which dis-
ease could attribute to the wider contour.
In VHD, the valve is either stenosed and restricts
blood flow or is leaky and allows backflow. In both
cases, the heart has to work harder to pump more
blood to maintain normal output. When comparing
the contour of the patient with VHD to the experi-
mental and control contours, the VHD contour ex-
hibits a loop with a wider base. Since electrical ac-
tivity precedes mechanical contraction in the heart,
irregularity in heart contractions reflects abnormality
of electrical stimulation and propagation. As seen in
Figure 7F, the contour does not have a downward ori-
entation as that of the experimental or control, thus
indicating that the largest ICV was not in solely one
direction. This irregularity could lead to arrhythmia.
Discussion
Several assumptions were made throughout the
investigation to compensate for limitations in the
ECG data. These included that the frontal and hori-
zontal plane data sets were precisely simultaneous,
the singular analyzed heartbeat was representative of
all heartbeats for each patient, and that the spatial ori-
entation for each heart was the same. From these gen-
eralizations, improvements could be made to model
the heart more precisely. For instance, electrogram
methods that digitally synthesize simultaneous ECG
signals from incomplete lead data are currently be-
ing investigated.13, 14 This could account for the fron-
tal and horizontal planes being recorded at different
times. Research to account for the disparities in diag-
nosis caused by beat-to-beat variation may also help
to reduce error.15 Limitations of the software include
the inability to account for the variability in the size,
shape, and orientation among individual hearts. This
reduced the effectiveness of comparing the contours
of the diseased conditions from the database to the
healthy experimental contours. Therefore, a screen-
ing early in life, where the heart would most likely be
in a healthy state, would be beneficial to identify later
potential diseased states using the software.
10 The Oculus: The Virginia Journal of Undergraduate Research
Motion artifacts were observed throughout the
experiment, where very small movements led to
increased vibrational effects in the ECG signal. One
main source of motion artifacts was noise introduced
by electrode shifting and skin deformation during
ECG recording. This caused distortion in the signal
obtained from the ECG. To collect frontal and hori-
zontal plane data simultaneously, two additional
ECG circuits would need to be constructed; the four
resulting ECG circuits would then be compiled into
a master circuit. In theory, this master circuit would
be attached to ECG monitors to output the 3D ICV
contour along with the real time ECG data. For this
result, the software must undergo minor alterations
to allow ECG data to be entered and plotted in real
time. This will reduce the error from analyzing a
single heartbeat. A final alteration would be to have
preset various diseased-states such as BBB, MI, myo-
carditis, and VHD into the software. This would al-
low doctors to quickly locate and diagnose heart ab-
normalities in patients without the use of expensive
equipment. Last, additional input from cardiologists,
physicians, and patients would help improve the de-
velopment and final software used to image the 3D
electrical configuration of the heart.
The primary goal of producing software that
plots a 3D contour of the heart from four ECG leads,
using a series of calculated ICVs, was achieved. The
software effectively isolated the QRS complex of the
heartbeat and performed the necessary calculations
for graphic visualization of the biopotential propa-
gation. The final result would allow cardiologists to
analyze the heart in an unprecedented way, through
electrical configuration, to diagnose a range of car-
diac diseases. With further development, this inno-
vative tool has the potential to impact the medical
industry worldwide.
References
1. “Deaths and Mortality” Center for Disease Con-
trol and Prevention. http://www.cdc.gov/nchs/
fastats/deaths.htm. Accessed 27 April 2010.
2. Cardiovascular Medicine: Diagnostics, Drugs,
and Devices (HLC041B). BCC Research. As re-
ported by http://www.redorbit.com/news/
health/1145566/bcc_research_ market_data_in-
dicates_cardiovascular_diagnostics_drug_and_
devices/index.html. Accessed 27 April 2010
3. Yanowitz, F.G. The Standard 12 Lead ECG. Univ.
of Utah School of Medicine. library.med.utah.
edu/kw/ecg/ecg_outline/Lesson1/index.html
4. McDaniel, N. Electrophysiology Testing. Univ.
of Virginia Health System. http://www.health-
system.virginia.edu/internet/childrens-heart/
pted/pcrd003.cfm Accessed 24 April 2010.
5. Dahlin, L.G. et al. Vectorcardiography is superior
to conventional ECG for detection of myocardial
injury after coronary surgery. Scandinavian Car-
diovascular Journal 2001. 35: 2:125-128.
6. Frank, E. An Accurate, Clinically Practical System
For Spatial Vectorcardiography. Circulation 1956.
13:737.
7. Liebman, J. What’s old, what’s new in nonar-
rhythmia clinical electrocardiography. Journal of
Electrocardiology. 37: 1: 152-165. October 2004.
8. Allenstein, B. J. Direct spatial vectorcardiography-
clinical application. Dis Chest 1956;30:359-372.
9. Riera, A. R. P. et al. “The History of Left Septal
Fascicular Block: Chronological Considerations
of a Reality Yet to be Universally Accepted” In-
dian pacing and electrophysiology journal. 8: 2:
114-128. 2008
10. Pfeffer, M. A., and Braunwald, E. “Ventricular
remodeling after myocardial infarction. Experi-
mental observations and clinical implications.”
National Institutes of Heath, 81.4 (1990): 1161-72.
11. Diaz-Peromingo, J.A., A.I. Marino-Callejo, C.
Gonzalez-Gonzalez, J.F. Garcia-Rodriguez, M.E.
Ameneiros-Lago, and P. Sesma-Sanchez. Tuber-
culous myocarditis presenting as long QT syn-
drome. Eur J Int Med. 11:340-342, 2000.
12. Yilmaz, A., K. Klingel, R. Kandolf, and U. Sech-
tem. Imaging in inflammatory heart disease: from
the past to current clinical practice. Hellenic J Car-
diol. 50:449-460, 2009.
13. Strommer, P. J., Lekkala, J. O., Malmivuo, J. A.
“Accurate Digital Synthesiser for Simulating Vec-
torcardiogram.” Medical and Biological Engineering
& Computing (1981): 250-4.
14. Kachenoura, A. et al. “Using Intracardiac Vector-
cardiographic Loop for Surface ECG Synthesis.”
EURASIP Journal on Advances in Signal Processing
(2008): 1-8.
15. Aidu, E. A. I., Trunov, V. G., and Titomir, L. I.
“Biophysical Model for Beat-to-Beat Variations of
Vectocardiogram.” Measurement Science Review 9.3
(2009): 64-6.

Fall 2010: Volume 9, Issue 2 11

Appendix

Figure 8. Circuit Schematic Diagram of the original circuit with the five stages. Circuit-1 and circuit-2 were built using this
schematic. The values of resistors and capacitors were measured by a multimeter and are listed in Table 2 and Table 3 for
circuit-1 and circuit-2, respectively. RA, LL, and RL refer to the right arm, left leg, and right leg, respectively.

Table 2. Circuit-1 Resistor and Capacitors Values Table 3. Circuit-2 Resistor and Capacitors Values
The above table lists the resistor values, in kilo- The above table lists the resistor values, in kilo-
ohms (k-ohms), and capacitor values, in nano- ohms (k-ohms), and capacitor values, in nano-
Farads (nF) for circuit-1. Farads (nF) for circuit-2.

12 The Oculus: The Virginia Journal of Undergraduate Research

 Establishing Legitimacy:
Early Christians’ Incorporation of Pagan and
Jewish Iconography in the Via Latina Catacombs
Jessica Arden Ettinger
Through the inclusion of non-Christian motifs in early Christian artwork, it is clear that the Chris-
tians used art as a means of establishing the legitimacy and superiority of their religious beliefs
over those of others during an age of intense religious rivalry. The incorporation of pagan motifs
was a logical choice for the Christians, not a blasphemous one, as might initially be assumed. In
order to gain followers, they needed to communicate through the widely recognized language of
pagan iconography. The early Christians’ uses of Jewish art were more complex, as they used it to
both legitimize and differentiate themselves from Judaism. Claiming Jewish ancestry provided
the early Christians with a more respected position in the religious world. In order to prove them-
selves superior to their predecessors, they illustrated typological connections between the Old
Testament and New Testament stories, confirming Christianity’s role as the fulfillment of Judaism.

T houghts concerning cultural exchange during

the medieval period often conjure images of
Christian and Muslim artistic interchange during
time period into their already shared iconographic
programs. The famed Via Latina catacombs, located
just outside Rome, are no exception to these artistic
the Crusades. Buildings like the Church of Hagia So- instances of shared symbolism and mixed meanings.
phia display the amalgamation of architectural and
artistic styles as regions were conquered by various
new leaders. However, one must recognize that the
precedent for borrowing and readapting artistic mo-
tifs was established underground as early as the mid-
fourth century CE, when Jews, early Christians and
pagans were commonly buried together in the same
spaces.1
In order to determine the belief-systems of tomb
occupants, historians today examine the iconog-
raphy of the tomb’s extant frescoes to extrapolate
greater meanings. However, if present, inscriptions
and motifs are often quite difficult to discern, which
leaves many tombs with religiously-unidentified oc-
cupants. The arbitrariness and difficulty of such an
identification process is compounded by the fact that
Jewish and Christian artistic programs of the mid-
fourth century shared many stories and artistic styles
of depiction. Moreover, early Christians commonly
reappropriated previously distinctive Jewish sym-
bols, such as the emblematic Menorah.2 In addition,
both religions were known to incorporate the well-
circulated and recognizable pagan imagery of the

1  Leonard Victor Rutgers, “Archaeological Evidence for

the Interaction of Jews and Non-Jews in Late Antiquity,”
American Journal of Archaeology 96.1 (1992): 110.
2  Rutgers 111.
Jessica Arden Ettinger is a fourth-year student from Po-
tomac, Maryland, double-majoring in Art History and
English. She produced this paper for Professor Lisa Reil-
ly’s seminar, The Medieval Mediterranean, during the
Spring of 2009. Currently, she is working on a seminar
paper for Professor Ramirez-Weaver, involving an inves-
tigation of Jewish life in medieval Prague through objects
linked to Jewish life-cycle events. In addition, Ms. Ettinger
is in the process of completing an undergraduate thesis on
twentieth-century American artist Edward Hopper. Out-
side of school, she trains year-round as an equestrian, and
competes both for the Virginia Riding team and indepen-
dently. After graduating in May, Ms. Ettinger plans to
attend law school and pursue a career in Art Law.

Fall 2010: Volume 9, Issue 2 13

While multiple attempts have been made to identify
the occupants’ religious affiliations through tomb
iconography, more information about the general
religious groups of the time period can be gleaned
from studying the overlaps in their artistic programs,
including a sharing of stories and figural depictions.
The inclusion of non-Christian motifs in early
Christian art stems from the need to legitimize their
religious beliefs, convert non-believers, and differ-
entiate themselves from their other religious con-
temporaries. By using pagan art forms, the Christian
minorities were able to communicate with the mas-
sive pagan population, a population which naturally
recognized their own pagan artistic program very
quickly. Early Christian’ reasoning for using Jewish
art was two-fold: in an effort to uphold the unofficial
doctrines of the Church – namely that Christianity
replaced and fulfilled Judaism3 – early Christians af-
filiated themselves with Judaism to legitimize their
fledgling beliefs, and then used Old Testament sto-
ries to produce typological connections to the coming
of Christ. The artistic program presented in the Via
Latina catacombs embraces all these elements of early
Christian iconography. The analysis and comparison
of the frescoes in several cubicula of the catacombs
illuminate the intricate relationships between these
three religious groups during the fourth century CE.
The Via Latina catacombs provide a case-study
example of the relationships amongst pagans, Jews,
and early Christians because it are one of the earli-
est Christian tomb sites and serve to document the
infant steps of artistic evolution in the creation of the
new religion. In addition, the size, wealth, and gran-
deur of these catacombs provide a broad selection of
artistic images from which to choose and compare,
both amongst themselves and with other images of
the same or nearby time periods. Moreover, the im-
ages are well preserved, which facilitates their analy-
sis. Two important aspects of the catacombs have
been their fame and accessibility, which have allowed
many professionals to photographically document
the frescoed walls and allowed researchers world-
wide access to the catacombs through the Internet.
The early Christians supported three tenets,
which continue to this day to be unofficially en-
dorsed. These cornerstone ideas dramatically influ-
enced the sharing and reappropriation of both pagan
and Jewish motifs in Christian art: (1) Christianity
functioned as the replacement for Judaism; (2) it was
necessary for Judaism to come before Christianity;
(3) the Old Testament foretells of the birth, life, death
and resurrection of Jesus Christ.4 Such ideas created
a need for the early Christians to explain why Chris-
tianity had fulfilled and surpassed Judaism, and how
the Hebrew Bible foretold of such subordination.
3  Henry Claman, Jewish Images in the Christian Church: Art
as Mirror of the Jewish-Christian Conflict, 200-1250 C.E. (Ma-
con: Mercer UP, 2000) 11.
4  Claman 11.
14 The Oculus: The Virginia Journal of Undergraduate Research
In an age without mass communication, art be-
came the easiest way to spread new ideas to a large
number of people with the intention of attracting
adherents and establishing a new community.5 This
method was popular with all religious groups of the
time periods alike, as equally with pagans as with
Jews and early Christians. However, the latter two
groups were minorities in a prolifically pagan world,
so they relied upon established pagan iconographic
programs in order to convey their ideas to the largest
audience possible.6 The use of art to circulate new be-
liefs, however, was a competitive venture. The fourth
century was an age of religious rivalry, in which
“Jews and Christians had to show that their proto-
types of salvation were no less attractive and no less
powerful”7 than those of their pagan predecessors.
The Roman emperors Constantine I and Licinius
passed the Edict of Milan in February 313 CE, which
effectively granted religious toleration throughout
the Roman Empire. This edict was particularly im-
portant because it guaranteed early Christians their
legal rights, including the opportunity to organize
churches and publically proselytize.8 Without the
fear of incessant religious persecution after 313 CE,
early Christians became more aggressive in their ef-
fects to convert the still-pagan Roman population.
Rather than remain a minority group, like the Jewish
people, the early Christians aggressively wished to
gain supporters and burgeon into a mainstream reli-
gion to rival paganism.
Jews and early Christians were familiar with
the pagan artistic program because the vast major-
ity of the Roman population was pagan. Knowing
this, both the Jews and the early Christians strategi-
cally used pagan art in order to convey their ideas
to the largest audience possible. The Jews targeted
an audience of potential pagan-Roman converts,
while the early Christians targeted both potential
pagan-Romans and Jewish converts. The Jews set
the precedent for the early Christians to follow. In
the synagogue at Dura-Europos, dated to approxi-
mately 245 CE, an image of Orpheus, the divine pa-
gan lute-player, doubles as an image of King David,
the Jewish monarch. Art historians explain that an
image such as this is permissible because it fits the
biblical description of David, and because artists re-
lied on pre-established representations:9 the intended
concept was much more important than the physical
5  Jaś Elsner, “Archaeologies and Agendas: Reflections on
Late Ancient Jewish Art and Early Christian Art,” The Jour-
nal of Roman Studies 93 (2003): 125.
6  Claman 28.
7  Elias J. Bickerman, “Symbolism in the Dura Synagogue:
A Review Article,” The Harvard Theological Review 58.1
(1965): 145.
8  “Edict of Milan,” Encyclopedia Britannica, 2009, 28
Apr. 2009 <http://www.britannica.com/EBchecked/top-
ic/382119/Edict-of-Milan>.
9  Jacob Neusner, “Judaism at Dura – Europos,” History of
Religions 4.1 (1964): 85.
similarity to a pagan prototype.10 Similarly, an image
in Dura-Europos shows Moses holding the club com-
monly seen in the hands of Hercules.11 The club itself
retains the power of its heroic pagan predecessor, but
is now imbued with the biblical association of Mo-
ses’ staff. Moses touches the club/staff to the parted
Red Sea, magically making the waters come together
again and swallow the pursuant Egyptian forces. The
act seems not unlike one of the twelve labors of Her-
cules, in which the club helped the mythological hero
defeat multiple enemies. The Jews promoted Hercu-
les’ club as a symbol of physical strength and power,
but embraced it through an association with Moses,
the emblematic leader of the Hebrew nation. In this
case, as that of the Orpheus/David, the Jews main-
tained artistic convention while re-evaluating the
value of the pagan motif, and borrowed the pagan
symbol’s physical construction while shedding its ex-
planation.12 This trend proved particularly attractive
to the early Christians.
Early Christians were familiar with the pagan
artistic program and followed the Jews’ example of
using pagan motifs to communicate their religious
ideas to the generally pagan population of Rome.
With respect to the Via Latina catacombs, the early
Christian patrons limited the majority of pagan deco-
rations to cubiculum N and cubiculum O. The former
is decorated solely with images of Hercules and his
labors, while the latter contains scenes with the Ro-
man goddesses Ceres and Proserpina.13 The juxtapo-
sition of these pagan images with the nearby Judeo-
Christian art perhaps indicates split religious beliefs
amongst family members who wished to be buried
in the same tomb; some may have honored Roman
gods while others were recent converts to Christi-
anity.14 The similarity with which all the images are
painted, whether pagan or monotheistic, signals that
the entire tomb was probably decorated by the same
workshop, and potentially by the same artist.15 This is
interesting because it means that artists were not nec-
essarily of the same religious beliefs as their patrons
but were certainly proficient in the stories and shared
artistic programs of the three competing religions.
The proficiency of the workshop or artist also proves
the success of art as a means of communicating the
different doctrines to a large number of people.
The decorations of cubiculum N and cubicu-
lum O were carefully selected because the patrons
believed them to symbolically convey Christian
meaning. One of the cubiculum focuses specifically
on Hercules, a mythological figure early Christians
10  Neusner 86.
11  Neusner 89.
12  Neusner 91.
13  Beverley Berg, “Alcestis and Hercules in the Catacomb
of Via Latina,” Vigiliae Christianae 48.3 (1994): 219.
14  Robin Margaret Jensen, Understanding Early Christian
Art (London; New York: Routledge, 2000) 90.
15  Berg 229.
Fall 2010: Volume 9, Issue 2
would have recognized through a plethora of marble
copies of Greek sculptures and friezes throughout
Rome. Hercules, while a pagan hero, is of specific
interest to the early Christians because he is semi-
divine, like Christ.16 Most convincing, however, is
Hercules’ infamous, willing self-sacrifice upon the
pyre at Mount Oite, marking his transformation into
a divine figure.17 Early Christians unreservedly drew
a parallel between this story and that of Christ’s cru-
cifixion. The labors of Hercules offered similar op-
portunity. The inclusion of the serpent in the image
of Hercules in the Garden of the Hesperides led early
Christians to recall the Garden of Eden, and Christ’s
role as the absolver of the sins unleashed upon the
world by its first two occupants.18 Christ functioned
as a New Adam, because his sacrifice reversed the
troubles and tribulations the first Adam put upon the
world through his original sin. The early Christians
did not hesitate to see a parallel between Hercules’
physical slaying of the serpent in the Garden of the
Hesperides and Christ’s symbolic overcoming of
the serpent from the Garden of Eden. Cubiculum N
also includes an image of Hercules rescuing Alcestis
from the Underworld. This scene can be interpreted
as a story corresponding to Christ’s apparent death
and resurrection – both Christ and Hercules have
the power to come back from the dead. This scene
appears between an image of Jonah, in the adjacent
cubiculum M, and the Raising of Lazarus, adjacent
on the opposite side, in cubiculum O.19 While many
scholars assert that there is no planned orientation to
convey connections between the tales, this juxtaposi-
tion seems more than coincidental. All three stories
embrace the theme of resurrection and take place
over three-day periods – Jonah is regurgitated from
the whale after three days; Lazarus is raised from the
dead three days after his death; and Christ is resur-
rected three days after his crucifixion.
The ceiling of Cubiculum O contains images of
Ceres and Proserpina, Roman goddesses who sym-
bolically represented items of importance to early
Christians and were associated with death and re-
newal.20 Ceres, the goddess of fertility and agricul-
ture, embodies the Earth’s annual life-cycle of death
in the winter and rebirth in the spring.21 Similarly,
Proserpina, the Roman version of the Greek goddess
Persephone, is a life-death-rebirth deity because Ha-
des, King of the Underworld, holds her captive for
one-third of the year,22 and the rest she spends with
her mother on Earth. Early Christians likely chose to
16  Berg 229.
17  Manfred Lurker, Dictionary of Gods and Goddesses,
Devils and Demons (London; New York: Routledge and
Paul, 1987) 149.
18  Berg 229.
19  Berg 219.
20  Berg 230.
21  Lurker 77.
22  Lurker 280.
15
include these two pagan goddesses in the catacombs
because of the nature of the setting; the idea of sal-
vation and resurrection in the after-life is incredibly
important in a tomb. The early Christians believed it
was more important to represent the concept of spiri-
tual rebirth than to focus on specifically monotheistic
figures. Essentially, “the magic emblems of eternal
life”23 were the same in the eyes of the pagans, Jews,
and early Christians.
While the reappropriation of pagan iconography
allowed early Christians to artistically communicate
with a wider variety of people, borrowing from the
Jewish artistic program legitimized their newfound
beliefs. While early Christians assumed that the Old
Testament was an accurately recorded history of the
past, they felt that their interpretation of the events
was the most valid.24 Early Christians sought to ex-
pose the true meaning behind the sacred original
text, which they believed involved the message of
the coming of Christ. However, prior to circulating
a different interpretation of such a sacred text, early
Christians first had to affirm their right to do so by
acknowledging their Jewish ancestry. As Henry Cla-
man astutely notes, “As a newly developing religious
group, Christianity had no claim to legitimacy other
than its Jewish heritage.”25 The Jewish people were a
centuries-old religious group by this time, and while
anti-Jewish attitudes were prevalent, early Christians
associated themselves with their Jewish ancestry as
an essential resume-builder. In artistic contexts, ear-
ly Christians associated images of their savior with
those of powerful Jewish leaders.
Frederick Bargebuhr’s comparison of the image
of Christ Raising Lazarus and Moses leading the Isra-
elites out of Egypt and into the Promised Land26 best
explains the early Christians’ desire to associate Jesus
Christ with pre-established Jewish leaders.27 Christ
23  Bickerman 145.
24  Jack P. Lewis, “Noah and the Flood: In Jewish, Chris-
tian and Muslim Tradition,” The Biblical Archaeologist 47.4
(1984): 228.
25  Claman 13.
26  There have been several debates surrounding the na-
ture of the figure in the latter image. Many art historians
believe it to be Moses, due to the background images of a
figure climbing a mountain the background, and a column
of fire – likely indicative of Moses receiving the Ten Com-
mandments, and the column of fire which helped Moses
guide the Israelites out of Egypt. Bargebuhr is of the opinion
that the figure in the image from the Via Latina catacombs
is in fact Moses, but the reader should be aware of the dis-
crepancy of opinions before agreeing or disagreeing with
his comparison. I believe that SCALA, Florence/ART RE-
SOURCE, N.Y., who supplied the image to ARTstor, from
which it was retrieved to be included in this paper, mistak-
enly titled the image as the “Raising of Lazarus”. The error
has been reported, although it still appears as such in the
ARTstor database.
27  Frederick Perez Bargebuhr, The Paintings of the “New”
Catacomb of the Via Latina and the Struggle of Christianity
Against Paganism, (Heidelberg: Winter, 1991) 63.
16 The Oculus: The Virginia Journal of Undergraduate Research
Raising Lazarus is one of the few images containing
a story of Christ in the Via Latina catacombs, perhaps
because the early Christians felt that Christ’s miracles
needed to gain theological legitimacy through an in-
tended identification with profound Hebrew leaders
such as Moses. Side by side examination of the two im-
ages reveals extreme similarities in the main leaders’
poses, the crowds, the settings, and the background
images. Both Moses and Christ raise their right arms
and orchestrate with uniquely similar wands. Their
figural positioning is perfectly parallel: each steps
forward with his left foot while balancing with the
toe of his right foot still on the ground. A figure be-
hind either Christ or Moses, respectively, touches
the enlightened leader’s shoulder, as if guiding his
actions. The settings are simultaneously barren, save
for a house atop a set of stairs towards which both
figures and crowds approach. In both backgrounds, a
column of fire hovers above each leader.
The likenesses between the two leaders stresses
their equivalent power and spiritual enlightenment.
The replicated figural positions show a physical em-
bodiment of authority, while the column of fire in
the background asserts that G-d speaks to both lead-
ers with equal form, power, and respect. However,
the differences between the two figures show a con-
scious step towards a typological connection of the
two tales. In the image with Moses, the background
includes the figure of a man rushing up the side of
a mountain, a likely allusion to the story of Moses
receiving the Ten Commandments atop Mount Sinai.
The inclusion of this motif confirms Moses’ identity.
The column of fire, which was recorded in the Old
Testament to help Moses guide the Israelites’ out of
slavery in Egypt, affirms the setting. The reappear-
ance of the column in the Raising of Lazarus is then
a continuation of the Jewish tradition in the stories of
early Christians, and confirms Judaism as the prede-
cessor of Christianity. In addition, the house in the
image with Moses is empty, perhaps representative
of the Jews wandering towards the Promised Land
but not yet enjoying it, while the house in the scene
with Christ contains a swathed figure. The figure in
the latter scene confirms the setting as the story of
Lazarus, but is perhaps also symbolic of Christianity
fulfilling and improving upon the Jewish tradition.
Moreover, Christ’s miracles surpass those of Moses,
who is not recorded to have resurrected anyone from
the dead. Christ therefore gains legitimacy through
an in-depth association with Moses and simultane-
ously proves superior through a visual representa-
tion of just one of his miracles.
At the same time that early Christians used their
Jewish ancestry to establish themselves as people of
a legitimate faith, they also felt the need to differen-
tiate themselves from their religious competitors. In
an age of intense religious rivalry, the early Chris-
tians needed to express why they should gain more
followers than the other contemporaneous religious
groups. However, at this point in time no established
Christian iconographic program existed.28 Images
that art historians currently associate with Christian
art, such as the Madonna and Child Enthroned, or
the Deposition, were either minimally circulated or
simply did not yet exist. Instead, early Christian art
relied on Jewish Old Testament stories to convey ty-
pological connections for the coming of Christ and
his miracles. Typological connections refer to the
Christian belief that the Old Testament predicted the
coming of Christ and his miracles. Early Christians
felt that communicating this assertion through art
was a way of differentiating themselves from their
Jewish ancestors.29 The age-old consistency in repre-
sentation of the most famous Old Testament stories
facilitated the comparisons early Christians wished
to make. While many of the images in the Via Latina
catacombs aimed to make such comparisons through
traditional representation, the images of Noah and
the Flood, and the Sacrifice of Isaac are two of the
most prominent and worthwhile to study.
The story of Noah is a typologically rich tale.
Noah, a pious carpenter, is confronted by G-d and
told to build an ark, in which to gather his family and
two of every kind of animal, because G-d is going to
send the flood waters to wipe out sinful humanity.
As Jack Lewis points out, both the gospel of Luke
and that of Matthew associate the Flood and Noah
with the coming of Christ. Such an association sug-
gests that Christ, like Noah, will aid in the erasure
of humanity’s sins.30 Noah is an intrinsic symbol of
salvation,31 and a figure associated with those who
are pushed by G-d’s gracious right hand into the
realm of Heaven. Noah’s Ark later evolved into an
allegory of the Church, “by which all mankind is
saved.”32 Moreover, the notion of an all-encompass-
ing Flood that cleansed the Earth was seen by early
Christians as a parallel to baptism;33 the world is
purged of humanity’s sin in the same way that a baby
is purged of original sin through symbolic and physi-
cal submersion in water. These connections between
the Old Testament and New Testament were facili-
tated by a consistent iconographic means of depict-
ing Noah springing forth from the ark in the pose of
an orant. An orant, as Leslie Ross explains, is a fron-
tally depicted figure with arms extended, raised in
prayer.34 Such figural gestures were typically painted
for funerary art as symbols of piety and salvation.
This depiction of Noah is seen at the Via Latina cata-
combs as well as at the Catacomb of SS Pietro and
28  Bickerman 147.
29  Claman 26.
30  Lewis 225.
31  Leslie Ross, Medieval Art: A Topical Dictionary, (West-
port: Greenwood, 1996) 187.
32  Jennifer Speake, The Dent Dictionary of Symbols in Chris-
tian Art, (London: Dent, 1994) 12.
33  Bargebuhr 59.
34  Ross 190.
Fall 2010: Volume 9, Issue 2
Marcellino and the Catacomb of Priscilla. The consis-
tent gesture and setting facilitate the identification of
Noah, the symbolism that his story conveys, and the
consequential parallelism to the story of Christ.
Early Christians viewed the story of the Sacrifice
of Isaac as another rich with typological meaning.
In this story, G-d tests Abraham’s piety by request-
ing that he sacrifice his only son, Isaac. Isaac will-
ingly lays himself on the altar, and just as Abraham is
about to slay him, an angel rushes down and explains
G-d’s orders were merely a test. As Alison Smith so
aptly explains, “[The sacrifice of Isaac] presented,
to the early churchman’s symbol-seeking mind, an
almost exact parallel to the passion of Christ.”35 So
perfect is Jesus’ faith in G-d, like that of his forefather
Abraham, that he does not challenge his own sacri-
fice but trusts that it is all within G-d’s plans.36 Differ-
ent, however, is the fact that Isaac survives his poten-
tial sacrifice, while Christ does not – Christ physically
fulfills the offering to G-d that Isaac escaped,37 which
parallels Christianity’s fulfillment of Judaism. On a
macro-level, Christians saw themselves, as followers
of Christ, to be both the fulfillment and improvement
upon the Jews. Similar to how the story of Noah was
represented consistently in early Christian art, the
sacrifice of Isaac is artistically rendered by the same
formula for hundreds of years. The earliest depictions
appear on ancient Pentateuch illustrations and were
continued in catacomb decoration.38 The earliest-
surviving representations of Isaac’s sacrifice follow a
formula in which Isaac kneels on the ground beside
the altar, and Abraham wields a dangerous looking
knife.39 This formula set the precedent for representa-
tions of the scene in the same and later centuries. The
traditional representation is so strong that the model
transmitted across thousands of miles and appears
on sarcophagi as far away as Spain.40 By the sixth cen-
tury, the formula had even reached North Africa.41
What is, perhaps, most important to recognize
about the repetition of artistic motifs in the same, for-
mulaic style for hundreds of years is that the continu-
ation represents the early Christians’ desire to pro-
duce an iconographic program that was widespread
and recognized amongst multiple groups of different
religious affiliation. Essentially, they aimed to pro-
duce an artistic program that rivaled and ultimately
surpassed the pervasive and prevalent iconographic
program of the pagans in Rome during the fourth
century and also to consequently establish Christ as
the ultimate Savior.
35  Alison Moore Smith, “The Iconography of the Sacrifice
of Isaac in Early Christian Art,” American Journal of Archaeol-
ogy 26.2 (1922): 159.
36  Speake 76.
37  Bargebuhr 61-62.
38  Joseph Gutmann, “The Sacrifice of Isaac in Medieval
Jewish Art,” Atribus et Historiae 8.16 (1987): 68-69.
39  Smith 161.
40  Smith 4.
41  Gutmann 68.
17
 The artistic evolution of a hand-held object of
power interestingly summarizes the cycle of shared
iconography amongst different but contemporaneous
religious groups in order to legitimize, convert, and
differentiate. Thomas Matthews explains that, “The
wand is not incidental, but a standard and necessary
feature in early Christian art.”42 The artistic trend be-
gins with Hercules, whose club helps him perform
the twelve labors dictated to him by King Eurystheus,
in addition to providing him with protection and the
power to rescue Alcestis from the underworld. Both
these scenes of Hercules appear in the Via Latina cat-
acombs. The Jews, a minority group exposed to the
vast displays of pagan iconography around them in
Rome, were familiar with the club’s association with
power and appropriated it into their own iconogra-
phy. As previously mentioned, Moses is seen carry-
ing a club at the synagogue at Dura-Europos.43 The
Jewish people, recognizing the power and divine as-
sociations of Hercules, found an equivalent figure, in
Moses, in their own scripture but utilized Hercules’
attributes to help convey the parallelism. A compari-
son between Hercules and the Hydra, from the Via
Latina catacombs, and Moses Crossing the Red Sea,
from the synagogue at Dura-Europos, affirms this
assertion. The evolution of the hand-held object of
power is further propagated by early Christians seiz-
ing upon the motif and reappropriating it for their
own uses. In early Christian art, the club gradually
grows into a more delicate and slender wand, yet is
still intended to “convey magic power by touch.”44 In
the Via Latina catacombs, artists depict both Moses
and Christ as carrying the same wand in scenes of the
Raising of Lazarus and the Crossing of the Red Sea.
The early Christians assert Christ as their equivalent
to an enlightened Jewish leader such as Moses and
once again provide the attributes to cement the asso-
ciation. The Via Latina catacombs contain the entire
cycle of this evolution. The concept is represented in
the labors of Hercules as a powerful weapon symbol-
ic of combating evil and sin; Hercules is then replaced
with Moses, an enlightened ancestral leader who also
carries a wand – modified slightly because he has dif-
ferent religious beliefs and consequently different
powers; and finally, Jesus carries the same wand as
Moses but can perform an even bigger miracle than
the parting of the sea – he can resurrect the dead and
provide visible proof of salvation. The evolution of
the wand parallels the Christian understanding of
the evolution of their faith: upon recognizing Jesus
as the ultimate Messiah, or Savior, they evolved from
their Jewish beliefs and embraced the most powerful
of all faiths. Essentially, both the Jewish and Christian
religions built on symbolic representations of power
42  Thomas F, Matthews, The Clash of the Gods: A Rein-
terpretation of Early Christian Art, (Princeton: Princeton UP,
1993) 57.
43  Neusner 89.
44  Matthews 57.
18 The Oculus: The Virginia Journal of Undergraduate Research
from their predecessors by borrowing and modifying
iconography in order to convey the same message in
their own specific context.
Through the inclusion of non-Christian motifs in
their artwork, the early Christians clearly used art as
a means of establishing the legitimacy and superior-
ity of their religious beliefs over those of others dur-
ing an age of intense religious rivalry. The incorpora-
tion of pagan motifs was a logical, not blasphemous,
choice for the early Christians. In order to gain fol-
lowers, they needed to communicate legibly, which
was accomplished through the widely recognized
language of pagan iconography. The early Chris-
tian’s use of Jewish art was more complex than their
use of the pagan iconography, since they used it to
both legitimize and differentiate themselves from Ju-
daism. Claiming Jewish ancestry provided the early
Christians with a more respected position in the reli-
gious world. In order to prove themselves superior to
their predecessors, they illustrated typological con-
nections between the Old Testament and New Tes-
tament stories, confirming Christianity’s role as the
fulfillment of Judaism.
Annotated Bibliography
1. Bargebuhr, Frederick Perez. The Paintings of the
“New” Catacomb of the Via Latina and the Struggle of
Christianity Against Paganism. Heidelberg: Winter,
1991.
Bargebuhr’s book begins with a comprehensive
description of the Via Latina catacombs that is im-
measurably beneficial to the understanding of the
importance of the iconography within the under-
ground structure. This style of detailed analysis
carries over to the chapters that follow, in which
he identifies the pagan iconography, as well as
the Old and New Testament scenes painted in
the catacombs. His conjectures about the frescoes’
symbolism are logical but indefinite, which he ex-
plains is a result of how “the paintings are indeed
very different in many traits … from those of
other catacombs,” making comparisons and con-
fidence very difficult. Nevertheless, his book is
an intelligent look into the catacombs, providing
a strong geographic understanding of the layout
and the iconography.
2. Berg, Beverly. “Alcestis and Hercules in the Cat-
acomb of Via Latina.” Vigiliae Christianae 48.3
(1994): 219-34.
Berg focuses on the controversial iconography of
Cubiculum N in the Via Latina catacombs. Rather
than use the iconography of the tomb to draw
suppositional conclusions about its occupant,
Berg analyzes the images of Hercules and his la-
bors within themselves, and the surrounding ico-
nography. She perceptively analyzes the pagan
icons in terms of potential Christian symbolism,
and compares Hercules to other figures within the
tomb to corroborate her argument.
3. Bickerman, Elias J. “Symbolism in the Dura Syna-
gogue: A Review Article.” The Harvard Theological
Review 58.1 (1965): 127-51.
Bickerman’s work in decoding the symbolism of
the iconography in the synagogue at Dura-Euro-
pos helps to explain the competitive relationship
between the pagans, Jews and Christians of the
late second and third centuries CE. She ingenious-
ly explains the confusing sharing of iconographic
motifs amongst these religions as the simple use
of a widespread repertoire of artistic representa-
tion. This practice continues into the fourth and
fifth centuries, as the “age of religious rivalry”
continued, and the early religious groups had not
yet formed an iconographic program distinctly
their own.
4. Claman, Henry N. Jewish Images in the Christian
Church: Art as Mirror of the Jewish-Christian Con-
flict, 200-1250 C.E. Macon: Mercer UP, 2000.
Claman’s discussions of the relationship between
and interconnectedness of Jewish and Christian
artwork stems from his assertions that the Church
unofficial recognizes three tenets: that Christian-
ity replaced Judaism; Judaism had to come before
Christianity; and that the Old Testament portends
the arrival of Christ on Earth as the Messiah.
These three doctrines guide and define Claman’s
astute discussion of how Christians used Jewish
iconography both to legitimize their fledgling
religion and simultaneously differentiate them-
selves from their Jewish predecessors. With this
foundation, he later goes on to discuss the history
of Jewish and Christian interactions from the late
fourth through mid-thirteenth centuries.
5. “Edict of Milan.” Encyclopedia Britannica. 2009.
Encyclopædia Britannica Online. 28 Apr. 2009
<http://www.britannica.com/EBchecked/top-
ic/382119/Edict-of-Milan>.
This succinct article provides a general summary
of the creation and importance of the Edict of Mi-
lan. Although it does not go into great depth con-
cerning the specific legal changes that took place,
the article does explain that the edict established
religious freedom throughout the Roman Empire.
6. Elsner, Jaś. “Archaeologies and Agendas: Reflec-
tions on Late Ancient Jewish Art and Early Chris-
tian Art.” The Journal of Roman Studies 93 (2003):
114-28.
Elsner’s article stresses the interconnectivity of
the early Christians’ artistic programs and the
programs of those preceding them. He chastises
those art historians and readers that assume a
particular symbol can conclusively state that a
piece of artwork is definitely of a specific ethnic
or religious group. He stresses the “complex mix-
ture” that produces such iconographic cycles as
those at Dura-Europos or the Roman catacombs.
With or without sufficient evidence to concretely
determine the origins of certain artistic programs,
Fall 2010: Volume 9, Issue 2
it is imperative that the art historian keep in mind
the multiple influencing factors leading to its pro-
duction.
7. Ferguson, George Wells. Signs & Symbols in Chris-
tian Art. with Illus. from Paintings of the Renaissance.
New York: Oxford UP, 1959.
Ferguson’s dictionary is perfectly organized with
direct, comprehensive abstracts, accompanied by
helpful illustrations alongside each entry. He pro-
vides both explanations of biblical stories and a
wide array of symbols prevalent in religious of art
from the first through twenty-first centuries.
8. Gutmann, Joseph. “The Sacrifice of Isaac in Me-
dieval Jewish Art.” Artibus et Historiae 8.16 (1987):
67-89.
Gutman’s article analyzes and discusses the simi-
larities in style of the portrayal of the sacrifice
of Isaac over multiple centuries. The image of
Abraham with knife raise, and Isaac tethered but
submissive, reappears in artwork over and over
again. She asserts that this form begins in Penta-
teuch illustrations and catacomb images, and is
maintained so as to become readily recognizable.
In addition, the consistency of its portrayal allows
it to be used typologically so as to insinuate the
sacrifice even when in other contexts.
9. Jensen, Robin Margaret. Understanding Early
Christian Art. London; New York: Routledge,
2000.
Jensen’s book explores the most prevalent of ear-
ly Christian images, paying specific attention to
those extant in the Roman catacombs. She begins
with static, non-narrative images, such as the fish,
and The Good Shepherd. Following these, she dis-
cusses the early frescoes of biblical stories, such as
Jonah and the whale and the sacrifice of Isaac. She
also discussed pagan iconography in relation to
its juxtaposition with Christian iconography.
10. Lewis, Jack P. “Noah and the Flood: In Jewish,
Christian, and Muslim Tradition.” The Biblical Ar-
chaeologist 47.4 (1984): 224-39.
In this article, Jack Lewis performs an interesting
comparison of the history of the story of Noah and
the Flood and in the Jewish, Christian and Mus-
lim traditions. Similar to the iconographic depic-
tions of the sacrifice of Isaac, those of Noah and
the ark are repeated in the same form throughout
history and across religious barriers. Such an ac-
ceptance conveys a shared acknowledgement of
legitimacy of the Jewish bible between Christians
and Muslims.
11. Lurker, Manfred. Dictionary of Gods and Goddesses,
Devils and Demons. London; New York: Routledge
and Paul, 1987.
Lurker’s dictionary provides a thorough explana-
tion of the mythological stories and histories be-
hind the majority of the Roman and Greek deities
and demons prevalent in famous literature and
artwork.
19
12. Mathews, Thomas F. The Clash of Gods : A Rein- typological interpretation. Through the multiple
terpretation of Early Christian Art. Princeton: Princ- examples Smith provides, the reader realizes that
eton UP, 1993. the story knows no boundaries; it is found every-
Matthew’s book is one of the penultimate re- where from in Byzantine iconography to the sar-
sources for anyone studying the interactions cophagi of Rome, Gaul and Spain, and beyond.
and relationships between the pagan, Jews and 17. Speake, Jennifer. The Dent Dictionary of Symbols in
Christians of the early medieval time period. Of Christian Art. London: Dent, 1994.
particular interest is Matthews’ discussion on the Speake’s compilation provides concise and easily
use of “the wand” and its development through- navigated entries of the most important symbols
out art, its power bestowed with Hercules, con- and stories in Christian artwork. Unfortunately, it
tinued with Moses, and ultimately legitimized lacks any information regarding the pagan sym-
through Christ’s use. He also pays great atten- bols that are quite prevalent in early Christian
tion to the symbols of the chariot and the donkey, artwork. However, its entries for biblical stories
and Christ’s changing appearance over time (the are sufficient and it provides interesting insight
“Christ Chameleon”). It is written with an eye to- regarding the plethora of documented symbols.
wards accessibility and the attention of a wide au-
dience, which makes it a uniquely enjoyable and
reliable source.
13. Neusner, Jacob. “Judaism at Dura - Europos.” His-
tory of Religions 4.1 (1964): 81-102.
In his article, Neusner analyzes the appearance
of pagan iconography in the synagogue at Dura-
Europos. He takes into account the suggestion
of artistic evolution that takes place from Jewish
observance and reappropriation of late pagan art.
He cites the club of Hercules being transferred to
the staff of Moses as an important example of this
evolution.
14. Ross, Leslie. Medieval Art : A Topical Dictionary.
Westport: Greenwood, 1996.
Ross’s dictionary identifies and documents the
most prevalent symbols in medieval artwork. It
restricts itself to non-pagan symbols, which is
frustrating, given the occurrence of many pagan
icons in early medieval works. Otherwise, it is a
generally complete source for Christian and non-
religious symbols of the era.
15. Rutgers, Leonard Victor. “Archaeological Evi-
dence for the Interaction of Jews and Non-Jews
in Late Antiquity.” American Journal of Archaeology
96.1 (1992): 101-18.
Rutgers emphasizes the occasionally overlooked
fact that Christians were generally hot-headedly
anti-Jewish during the same period that there ex-
isted an unprecedented amount of artistic motif-
sharing and exchange. She sites these attitudes as
the source of the desire to immediately reappro-
priate Jewish symbols while still depending on
them for religious legitimization and subsequent
power.
16. Smith, Alison Moore. “The Iconography of the
Sacrifice of Isaac in Early Christian Art.” American
Journal of Archaeology 26.2 (1922): 159-73.
Smith’s article hinges on the categorization and
cross-comparison of multiple ‘types’ of Isaac sac-
rifices. She asserts that Christians promoted the
same style of representation because they saw
in it “an almost exact parallel to the Passion of
Christ,” and wished to promote and circulate this

20 The Oculus: The Virginia Journal of Undergraduate Research

 Application of Fingolimod (FTY-720) for Therapeutic
Arteriogenesis and Microvascular Remodeling
Nikhil Panda
Background: The application of small-particle conjugated drug delivery is a promising
method of stimulating collateral arterial growth and neovascularization. It is hypoth-
esized that the localized delivery of FTY-720 encapsulated in PLAGA nanoparticles to isch-
emic skeletal muscle will restore blood flow to the tissue and induce vascular remodeling.
Methods and Materials: In this experiment, mice were injected with FTY-720, a small syn-
thetic stimulant of arteriogenesis and angiogenesis, directly and intramuscularly into isch-
emic gracilis muscles caused by hindlimb arterial ligation. Perfusion recovery was moni-
tored for 18 days post-ligation. The gracilis muscle was harvested on day 18 and changes in
collateral main feeder vessel diameter were determined by quantifying the diameter of
smooth-muscle-alpha actin positive (SMA+) vessels. To further characterize vascular remod-
eling, the number of BS1-lectin+ and SMA+ vessels per muscle fiber (MF) was quantified.
Results: The average perfusion recovery of the FTY-720 treated hindlimbs was similar to contra-
lateral controls, with significant increases in perfusion at days 14 and 18; no significant increases
in the rate or amount of perfusion at other time points were observed. The 22% average increase
in collateral main feeder vessels was not found to be significant; however, there was a significant
increase in diameter within the FTY-720 treatment group relative to the non-ischemic control. The
increase in SMA+ and lectin+ vessels per MF resulting from the FTY-720 treatment was determined
to be 17% and 19%, respectively; however, these results were not statistically significant. The ob-
served increase in SMA+ and lectin+ vessel profiles per MF due to ligation was also insignificant.
Conclusions: It was concluded that the intramuscular injection of FTY-720 to localized vascular
occlusions was not effective in restoring blood flow to ischemic tissue. The observed trend in-
creases between treatment groups suggests this method is promising for similar future research.

Introduction

O ver eight million Americans, or one in every

sixteen people, suffer from peripheral arterial
disease (PAD)1,2. PAD is diagnosed as limited blood
flow in the upper and lower leg as a result of vascular
occlusions, which in turn induces pain and can lead
to loss of tissue in or even amputation of the leg. As
current attempts at minimizing PAD, such as angio-
plasties, are invasive procedures that do not generate
new vasculature, there exists a need for a treatment
for the disease.
The stimulation of collateral arterial growth is a
method that could be applied for treatment of PAD.
In animal and small-scale clinical trials, the deliv-
ery of arteriogenic and angiogenic stimulants and
growth factors, such as vascular endothelial growth
factor (VEGF), has shown promising results. One
other such synthetic stimulant, fingolimod (FTY-720),
is structurally similar to sphingosine-1-pohsophate

Nikhil Panda is currently a fourth-year pre-medical stu-

dent from Chattanooga, TN studying Biomedical Engi-
neering and Engineering Business. While his research is
within BME, Nikhil has spent time working within both
obstetrics clinical settings and medical device develop-
ment. Nikhil plans to matriculate in medical school upon
graduation in May.

Fall 2010: Volume 9, Issue 2 21

(S1P), a known agonist of sphingolipid receptors,
and a relatively strong immunosuppressant3. S1P-1
receptors are key components in the regulation of
cell-signaling pathways, specifically in endothelial
cell differentiation, proliferation, and migration4.
Although S1P has shown potential as a therapeutic
treatment in occlusive vascular diseases, its associ-
ated costs and short half-life make it a poor option
for effective and efficient treatment5. Therefore, FTY-
720, a more practical pharmaceutical with low costs
and longer half-life, is a more viable option for cur-
rent research.
Difficulties in the successful in vivo application
of small synthetic materials, such as FTY-720, still
remain. While in vitro testing reveals therapeutic ad-
vantages, direct introduction in vivo did not prove ef-
fective8. As such, stimulating agents are introduced
within polymeric biomaterials, such as poly-lactic-
co-glycolic-acid (PLAGA)6,7. These biodegradable
and biocompatible materials help maintain the ben-
eficial properties of stimulating agents, such as rela-
tively long half-life, and growth factors while also
creating a controlled-release drug delivery profile.
The prevalence of small-particle conjugated drug de-
livery continues to increase in current studies.
The aim of this experiment is to evaluate the ther-
apeutic efficacy of FTY-720 treatments to stimulate
collateral arterial growth in ischemic mice hindlimbs.
Specifically, the hypotheses that the direct introduc-
tion of FTY-720 encapsulated in PLAGA nanopar-
ticles results in increased blood flow recovery in the
lower hindlimb, apparent arteriogenesis of major
arteriolar vessels, and significant angiogenesis and
neovascularization will each be investigated.
Methods and Materials
In this study, the arteriogenic and angiogenic
effects of FTY-720 (conjugated with PLAGA micro-
capsules) were examined upon direct intramuscu-
lar injection into the gracilis muscle of the ischemic
hindlimb of BALB/cJ mice. To quantify the resulting
arteriogenesis, the ischemic:contralateral perfusion
ratio of equivalent areas within each hindlimb was
determined from Laser Doppler Perfusion Imaging
(LDPI) analysis, and the diameters of major collateral
vessels within the gracilis muscle were measured.
Further arteriogenic and angiogenic capabilities of
FTY-720 were determined by calculating the number
of smooth muscle coated vessels and capillaries per
muscle fiber within the gracilis muscle.
Mouse Hindlimb Ischemia Model8
All methods herein complied with University of
Virginia Animal Care and Use Committee (ACUC)
guidelines. Unilateral hindlimb ischemia was in-
duced in BALB/cJ mice by ligating the femoral ar-
tery. Prior to the surgical procedure, the station coun-
tertop was covered with sterile drapes, the surgery
plate was sterilized with ethanol spray, and surgical
22 The Oculus: The Virginia Journal of Undergraduate Research
instruments were autoclaved. Mice were each anes-
thetized by an intraperitoneal (IP) injection of 1.56
mL/kg body weight ketamine HCl, 0.52 mL/kg xy-
lazine, 0.173 mL/kg atropine, and 3.12 mL/kg sterile
saline. The hair covering the dorsal and ventral sides
of the left hindlimbs were removed by applying and
removing Nair. The bare skin was then cleared with
alcohol and betadine topical anesthetic was applied.
A 1.5cm incision no more than 5mm from the sagit-
tal plane was made along the subdermal artery/vein
pair on the left hindlimb. After isolating the artery/
vein and nerve bundle through the disconnection
of subdermal connective tissue, these three struc-
tures were separated from each other. Two sutures
were then tied on the femoral artery approximately
2.0 millimeters downstream of the epigastric artery
to completely occlude blood flow to the hindlimb
and foot. The right leg of each mouse was left in its
normal physiological state to serve as a contralateral
control for ligated limbs.
FTY-720 Nanoparticle Fabrication5
PLAGA nanoparticles loaded with FTY-720 were
created using an oil-in-water emulsion solvent evap-
oration technique. Specifically, 40g of polyvinyl al-
cohol (PVA) were dissolved in 1000mL cold distilled
water and stirred overnight. Following filtration,
180mg of uncapped 50:50 PLAGA were dissolved
in 6mL methylene chloride (MC). The FTY-720 was
dissolved in MC and then added to the PLAGA/
MC solution and vortexed for 1min. After placing
the solution on ice for 5min, it was sonicated at 60W
for 150sec. This MC/PLAGA/payload solution was
added to 24mL of 4% PVA, in two portions with in-
termediate vortexing. After placing the solution on
ice for 5min, it was sonicated at 60W for 150sec. The
solution was then stirred for 12hours on a stir plate in
a hood to allow evaporation of MC and nanoparticle
stabilization. To isolate the nanoparticles and remove
residual PVA, the suspension was repeatedly centri-
fuged for 60 min at 20,000 rpm at 4˚C. The nanoparti-
cle solution was resuspended again in distilled water
(10mL) and sonicated for 30s. To remove large aggre-
gates, the nanoparticle solution was centrifuged at
1000rpm for 10min at 4˚C. A sample of the nanoparti-
cle suspension was analyzed by a submicron-particle
sizer (Multisizer IIe, Beckman Coulter, Inc., Palo Alto,
CA) and by scanning electron microscopy (SEM). The
pellet was then isolated, lyophilized, and frozen at
-80˚ C.
Nanoparticle Delivery to the Hindlimb8
Mice will each be anesthetized by the (IP) injec-
tion described previously. A solution of 1.5mg FTY-
720 loaded PLAGA nanoparticles resuspended in
125µL phosphate buffered saline (PBS) was delivered
by intramuscular injection to 5 equally spaced locations
along the gracilis muscle in the ischemic hindlimbs
three days post-ligation (day 4). The non-ischemic
 Figure 1. Characterization of FTY-720 nanoparticles. A: SEM images of fabricated nanoparticles.
Analysis in ImageJ resulted in an average particle diameter of 115nm B: Number-weight distribu-
tion of nanoparticle diameter, resulting in an average particle diameter of 207nm.
hindlimb received no injections. Control samples, in
which control PLAGA were applied, were adminis-
trated as described above.
Laser Doppler Perfusion Measurements5
Animals anesthetized by IP injection animals were
placed on a surgical heating pad inside a small cham-
ber that eliminates ambient light. Feet were aligned
under the scan head of the Lisca PIM laser Doppler
imager in a single plane. The regions of interest were
scanned at a resolution of 256×256 pixels to produce
a color perfusion image. The LDPI software was then
used to quantify the mean voltage per region, which
in turn was used to calculate relative perfusion.
Muscle Preparation and Harvest5
Gracilis muscles for excision and cross-sectioning
were superfused with Ringer’s solution containing
10-4 M adenosine for 30min. The abdominal aorta
was retrograde cannulated following euthanization.
Following blood removal with a PBS (pH=7.4) per-
fusion, muscles were perfusion-fixed with 4% para-
formaldehyde in PBS (4˚C) for 30min at 100 mmHg.
Specimens for cryosectioning were excised.
Whole Muscle Processing and Analysis5
Whole-mount gracilis muscles were treated with
type I collagenase (3 mg/ml, Sigma, St. Louis, MO)
in PBS for 30 minutes to digest the type I collagen
in the connective tissue between muscle fibers. After
being washed in PBS for 10 minutes, specimens were
placed in a 1:200 Cy3-conjugated monoclonal anti-
smooth muscle alpha-actin (anti-SMA) (clone 1A4,
Sigma, St. Louis, MO) solution of 0.1% saponin and
2% bovine serum albumin (BSA) in PBS at 4°C for
3 nights. The tissues were washed in 0.1% saponin
in PBS once for 60 minutes and in PBS twice for 30
minutes on a shaker plate. Samples were stored in a
50/50 mixture of PBS and glycerol until mounted on
microscopic slides for confocal microscopy. Whole
mounted muscles were observed with a ×4 objective
on a Nikon TE-300 inverted microscope (Nikon, Mel-
ville, NY). Using ImageJ software (NIH, Bethesda,
Fall 2010: Volume 9, Issue 2
MD), SMA+ vasculature was analyzed for diameter
measurements of large collateral vessels. For inves-
tigation of the angiogenic effects of the FTY-720,
specimens will be bisected about their midlines per-
pendicular to muscle fiber (MF) direction. BS-1 lec-
tin+ vessel profiles per MF, SMA+ vessel profiles per
MF, metrics that indicate the nature of the vascular
remodeling response, were determined.
Quantification Metrics and Statistical Analysis8
To summarize the purposes of the protocols de-
scribed previously, there were three main metrics
used to quantify the arteriogenic and angiogenic ef-
fects of FTY-720. The ischemic:contralateral perfusion
ratio was used to determine blood perfusion recov-
ery, the diameter of the major collateral vessel im-
aged from the harvested and excised gracilis muscle
was used to examine vascular remodeling, and the
SMA+ and lectin+ vessel profiles per muscle fiber
were used to investigate arteriogenesis and angio-
genesis. The LDPI was analyzed using a two-way
variance analysis, while changes in arteriolar collat-
eral diameter and SMA+ and lectin+ vessel profiles
per muscle fiber were analyzed by two-way repeated
measures ANOVA with Holm-Sidak multiple pair-
wise comparison. Statistical testing was performed in
SigmaStat 2.0 using p<0.05 to evaluate significance.
Results
Characterization of FTY-720 Nanoparticles
To characterize the size and structure of the PL-
AGA nanoparticles loaded with FTY-720, scanning
electron microscopy was performed (FIG. 1). Analy-
sis shows that the nanoparticles applied in this exper-
iment were spherical in shape and structure. Using a
submicron particle analyzer, the number-weight dis-
tribution of average nanoparticle diameter was found
to be 207nm. Separate analysis in ImageJ software
where individual nanoparticles were measured, the
average diameter was 115nm. While variance exists
between these values, they nonetheless characterize
the spherical structure and order of magnitude of
size of the FTY-720 nanoparticles.
23
Figure 2. Blood perfusion recovery of mice with induced hindlimb ischemia. Laser Doppler Perfusion Imaging software
was applied to calculate the ischemic:controlateral average perfusion ratio. No increase in the rate of recovery was ob-
served. Significant increases (p<0.05) in perfusion was seen at days 14 and 18.
Hindlimb Blood Perfusion Recovery Analysis
The regeneration of blood flow to the lower
hindlimb was quantified by measuring the perfusion
recovery in the ischemic and contralateral hindlimbs.
Measurements using the LDPI software were made
prior to (labeled day -1) and immediately post-li-
gation (labeled day 0), as well as days 2, 4, 7, 9, 14,
and 18. At each time point, the ischemic:contralateral
perfusion ratio was determined to evaluate the over-
all recovery of blood flow in the ischemic hindlimb
(FIG. 2). While the overall trend suggested a thera-
peutic effect, treatment with PLAGA loaded FTY-720
nanoparticles did not show a significant increase in
the rate of blood flow regeneration. Greater perfu-
sion was seen at days 14 and 18; no significant in-
creases in perfusion were seen at other time points.
Vasculature Remodeling Analysis: Arteriogenesis
Harvested gracilis muscles excised 18 days post-
ligation were imaged using confocal microscopy to
evaluate the arteriogenic effects of the FTY-720 treat-
ment. Using ImageJ software, the diameter of the
collateral main feeder vessels was measured (FIG.
3a). Results indicated that the average diameter of
the collateral main feeder vessels in the ischemic
hindlimbs was 102.54µm and 124.97µm for the PL-
AGA and FTY-720 treatments, respectively (FIG. 3b).
Statistical testing revealed that this 22% increase was
not significant. The change in diameter relative to the
non-ischemic hindlimb, however, was found to be
significant in the FTY-720 treatment, with a 72% in-
crease. The PLAGA treatment did not yield a signifi-
cant increase in diameter of the collateral main feeder
relative to the non-ischemic hindlimb.
24 The Oculus: The Virginia Journal of Undergraduate Research
Vasculature Remodeling Analysis: Angiogenesis
Bisected gracilis muscle specimens were imaged
to examine the BS-1 lectin+ and SMA+ vessel profiles
per MF. Using ImageJ software, the number of lec-
tin+ and SMA+ vessels per MF for the PLAGA and
FTY-720 treatments and for the ischemic and contra-
lateral control hindlimbs was determined (FIG. 4).
While a trend increase was observed, the increase in
SMA+ and lectin+ vessels per MF resulting from the
FTY-720 treatment – 17% and 19%, respectively – did
not prove to be statistically significant. Similarly, the
observed increase in SMA+ and lectin+ vessel pro-
files due to ligation was also insignificant.
Discussion
Conclusions
At the onset of this experiment, it was expected
that the direct intramuscular injection of PLAGA
nanoparticles loaded with FTY-720, a drug character-
ized by arteriogenic and angiogenic capabilities5,9, in
the ischemic hindlimbs of mice would yield statisti-
cally significant therapeutic results. Specifically, an
increased rate and amount of blood flow recovery
and a significant increase in the average diameter
of the collateral main feeder vessel in the ischemic
hindlimb were expected upon LDPI and confocal
imaging analysis. Additionally, FTY-720 treatments
were expected to induce a significant generation of
arterioles and venules as well as microvesseles in the
ischemic hindlimb9. Previously performed experi-
ments have shown that these results are plausible5,8.
The results and statistical analysis of the data
indicate that the treatment of FTY-720 yielded an in-
creased average perfusion ratio at the final two time
points, representative of increased blood flow recov-
ery to the ischemic hindlimb. The overall recovery
profile is not entirely consistent with the hypotheses
 Figure 3. Arteriogenic effects of FTY-720 treatment. A: Representative image of collateral main feeder ves-
sel in ischemic hindlimb gracilis muscle and ImageJ measurement technique. B: Change in average diameter
of collateral main feeder vessels. The FTY-720 treatment did not yield a significant increase (p=0.505) in the
average diameter. The change in diameter between the non-ischemic and ischemic hindlimbs in the FTY-720
treatment was significant (p=0.007).

made at the onset of the study and the significant
increase in perfusion at days 14 and 18 was not ex-
pected. A potential explanation for this sudden in-
crease could relate to the release of FTY-720 from the
PLAGA nanoparticles in vivo. It is known that the re-
lease of a material, such as a synthetic material like
FTY-720, is a function of its degradation and erosion
profiles as well as the hydrophilicity of the mate-
rial11. Furthermore, as PLAGA is characterized as a
bulk-eroding copolymer, the release of a hydrophilic
material like FTY-720 would not be marked by a con-
stant release profile5,12. As such, the observed increase
in perfusion at days 14 and 18 might be a result of a
bulk-release profile of FTY-720 from the PLAGA co-
polymer13. Further repetitions of this experiment are
required to verify at what time point this influx of
FTY-720 occurs.
The observed average diameter of the collateral
main feeder vessels in ischemic hindlimbs increased
significantly relative to the non-ischemic controls
with the FTY-720 treatment, a result consistent with
the aforementioned conclusions from LDPI analy-
sis. As perfusion measurements revolve about the
ligated:contralateral average perfusion ratio, it is log-
ical that the observed increase in blood flow recovery
might result from an increase in the diameter of this
vessel14.
Characterization of the arteriogenesis and angio-
genesis induced by FTY-720 resulted in increased av-
erage diameter of the collateral main feeder vessels
as well as SMA+ and lectin+ vessels per MF. While
these trend increases exist, it is difficult to validate
initial hypotheses regarding the efficacy or efficiency
of FTY-720 as a therapeutic agent for vascular remod-
eling as statistical analysis of the data showed there
was no significant change as a result of treatment.
These results imply that the vessel development in-
duced by FTY-720 is not significantly greater than
the natural remodeling response. In ischemic tissues,
there is an increase in the pressure gradient within the
vasculature as a result of an occlusion15. The resulting
change in the blood flow profile and increased shear
experienced at the fluid-vessel interface activates the
growth-factor receptors in the ischemic tissue. The
subsequent increased interactions between growth
factors and their upregulated receptors activate sig-
naling transduction pathways associated with cell
growth, proliferation, and differentiation16. Each of
these cell-fate responses is characteristic of new ves-
sel development and growth.
In summary, while the data obtained in this ex-
periment did not successfully confirm the hypoth-
eses regarding the therapeutic ability of FTY-720 via
vasculature remodeling, the desired trend increases
in the collected data were observed.
Limitations
The difficulty in acquiring significant data can be
attributed to limitations in the design of the experi-
ment and analysis techniques. Regarding the former,
the sample sizes of treatment groups was initially
n=8. Four mice in the PLAGA and FTY-720 treatment
groups were euthanized at various times during the
study as a result of poor recovery from the initial
hindlimb ligation surgery. The resulting relatively
small sample size (n=4) introduced a significant
amount of variance and deviation during statistical
testing. Furthermore, the timeline for this experiment
was such that only one trial was completed to evalu-
ate the therapy of the FTY-720 treatment.
In regard to error in analysis techniques, it was
difficult to measure significant increases in perfusion
recovery due to significant noise in collected LDPI
data. This also led to large values of variance and
standard deviation in statistical analysis testing. Ad-
ditionally, it was difficult to compare the results from

Fall 2010: Volume 9, Issue 2 25

Figure 4. Analysis of FTY-720 induced arteriogenesis and angiogenesis. A: Confocal images of the bissected and cross-sec-
tioned gracilis muscle 18 days post-ligations show SMA+ vessels, arterioles and venules (left), lectin+ microvessels (middle),
and the combined SMA+/lectin+ vessels per muscle fiber of the ischemic and non-ligated hindlimbs for PLAGA and FTY-
720 treatments; no significant increase was observed as a result of ligation (p=0.915) or treatment (p=0.412). C: Bar graph of
lectin+ vessels per muscle fiber; no significant increase was observed as a result of ligation (p=0.085) or treatment (p=0.233).

the LDPI perfusion analysis and the characterizations
of vasculature remodeling. LDPI data is such that
blood flow throughout the entire hindlimb is quan-
tified17. Measurements of average collateral main
feeder vessel diameter as well as SMA+ and lectin+
vessel profiles per MF, however, are metrics localized
to the targeted area of the gracilis muscle. As such, if
further trials of this experiment yielded statistically
significant results, it would become challenging to
draw consistent conclusions from these tests.
Future Research & Refinements
In addition to further experimental trials, there
are several components of this experiment that merit
further investigation. One of the most compelling of
these is characterizing the release of FTY-720 from
the PLAGA copolymer. By quantifying the kinetics
of the drug delivery to the ischemic hindlimb, FTY-
720 concentration, nanoparticle fabrication and load-
ing, and methods of drug delivery become additional
variables for optimization in further experiments.
Future plans include developing a mathematical and
computational time-dependent model of the release
profile of FTY-720 to surrounding tissue.

26 The Oculus: The Virginia Journal of Undergraduate Research

 A second goal for future trials is to successfully
differentiate between the therapeutic arteriogenesis
and angiogenesis induced by FTY-720 treatment and
the innate immune and remodeling responses. The
original protocol for this experiment involved quan-
tification of the number of F4/80+ cells per MF bun-
dle area to evaluate the degree to which FTY-720 is an
immunosuppressant18. Confocal imaging of F4/80+
stained slides did not result in the collection of mean-
ingful data. As such, it is unknown if the observed
trend increase in the observed arteriogenesis and an-
giogenesis was truly a result of FTY-720 treatment.
Acknowledgments
The support for this research came from grants
received by Dr. Price from the National Institute of
Health. The author would like to thank Dr. Price and
the Department of Biomedical Engineering for this
opportunity, and especially Caitlin Burke for her
help and guidance throughout the study.
References
1. Allison MA, Ho E, Denenberg JO, Langer RD,
Newman AB, Fabsitz PR, Criqui MH. (2007).
Ethic-specific prevalence of peripheral arterial
disease in the United States. Am J Prev Med, 32,
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2. Rajagopalan S, Mohler ER, Lederman RJ, Gold-
man, CK, et. al. (2003). Regional angiogenesis
with vascular endothelial growth factor in pe-
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3. Keul P, Tolle M, Lucke S, Heusch G, et. al. (2007).
The sphingosine-1-phosphate analogue FTY720
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cient mice. Arteriosclerosis, Thrombosis, and Vascu-
lar Biology, 27, 607-13.
4. Westhoff T, Schmidt S, Glander P, Liefeld L, et. al.
(2007). The impact of FTY720 (fingolimod) on va-
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5. Burke CW. (2009). A platform for therapeutic in-
sonation: targeted drug delivery and non-thermal
albation. Dissertation Proposal. The University of
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6. Chappell JC, Song J, Klibanov AL, Price RJ. (2008).
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blast growth factor-2 by ultrasonic microbubble
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7. Panyam J, Labhasetwar W. (2002). Biodegradable
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8. Lawrence RT, Archer BP, Burke CW, Meisner JK,
RJ Price. (2009). Investigation of phthalimide neo-
vascular factor-1 for therapeutic arteriogenesis in
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The immunomodulator FTY720 and its phos-
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27
 Managing Water Pollution:
The United States and Mexico, a Comparative Study
Kelsey Kerle-O’Brien
In 1990, the United States developed a system of permit trading to control air pollution from coal
fired power plants. In 1992, Mexico adapted this system to manage water pollution, by establish-
ing a system of tradable permits for water use. Why does Mexico rely on an incentive based sys-
tem of taxes and tradable permits for water pollution management while the U.S. continues to
implement direct regulation? I argue that cultural values and path dependency on previous leg-
islation combine to prevent the U.S. from moving to Mexico’s market based approach. The ex-
tent to which each nation initially centralized water pollution policy molded future legislation.
When President Carlos Salinas de Gortari signed the 1992 Law of National Waters, he left the
Federal Bureau of Environmental Protection and the National Water Commission with consider-
able power. It was only after a strong federal oversight was in place that Mexico began to devolve
the power to manage water to local user groups. By contrast, the U.S. has shown a pattern of de-
centralizing policy control to state and local governments from the onset of legislation. The EPA
has gradually attempted to gain more control in water management, but overlaps in jurisdiction
among state governments, and federal agencies have become entrenched in American policy.
Introduction ing water pollution differ in Mexico and the United
G eorge H.W. Bush’s 1990 Clean Air Act Amend-
ments established a cap and trade program for
SO2 from coal fired power plants. The United States
States. Next, I discuss possible alternative explana-
tions for the differences in the two nations. Then, I
compare how the history of how water pollution
successfully achieved a 50% reduction in overall
emissions through two strategies of this program:
(1) declining yearly caps on emissions, culminating
in the establishment of a cap of 8.10 million tons by
2010 and (2) a system of trading SO2 allowances. The
success of this innovative, market based system for
environmental protection in reducing emissions and
driving technological innovation drove the Euro-
pean Union member nations and other governments
around the world to adopt similar policies for air pol-
lution control. Mexico, however, is one of only two
countries in the world (Chile is the second) with a
formal permit trading program to protect and pre-
serve scarce water resources (Thobanl, 1997, 161-79).
Why does Mexico rely on an incentive based system
of taxes and tradable permits for water pollution
management while the U.S. continues to implement
direct regulation through government standards?
In this paper, I argue that cultural values and path
dependency on previous legislation combine to pre-
vent the U.S. from moving to Mexico’s market based
approach. These factors also explain how Mexico was
able to initially pass legislation based on economic
incentives to control pollution. To begin with; I pres-
ent a brief overview of how the policies for manag-
Kelsey Kerle-O’Brien is a fourth-year History major with work with Alternative Spring Break, through which she
a concentration in Latin American Studies and a minor in has traveled to San Diego, CA and Ecuador. In the fall,
Technology and the Environment. She has served as the she plans to pursue her Master’s in Public Policy. For fun,
Secretary of Habitat for Humanity since December 2008, Kelsey enjoys studying art history, working out, watching
as Vice President of Advocacy in 2009, and co-President Disney Channel, doing arts and crafts, recycling and par-
in 2010. Her passion for volunteer work is demonstrated ticipating in Outdoors Club trips to ski and hike.
not only in her commitment to Habitat but also in her

28 The Oculus: The Virginia Journal of Undergraduate Research

policy developed for both countries. I will discuss
the strengths and weaknesses to both approaches to
water pollution management. Finally, I look ahead to
see how the opening of the Mexican permit trading
system to Western states in the United States may
have water policy implications in the future.
The 1972 Clean Water Act and the 1992
Law of National Waters
The 1972 Federal Water Pollution Control Act is
the legislative basis for what is now known as the
Clean Water Act (CWA) in the United States. The
Clean Water Act serves as the primary regulatory
measure for control of water quality in the U.S. This
policy, implemented by Richard Nixon, established
a system of technology-based effluence standards
to place quantitative limits on pollution discharges.
Because the goals set by the CWA— such as the com-
plete reduction of all pollution entering fishable and
swimmable waters across the nation by 1983 and wa-
terways by 1985.—were too idealistic, amendments
were passed in 1977 and 1987. These amendments
extended the deadline for the attainment of good wa-
ter quality and they implemented new measures to
restrict toxic pollution and pollution from non-point
sources (Kraft, 2003, 107-111).
By contrast, the 1992 Law of National Waters in
Mexico decentralized management of irrigation wa-
ter to newly created “user groups” and established a
market based system for water management. A ma-
jority of Mexico’s water pollution is focused in agri-
culture. More than 5.5 million hectares are irrigated
for used in farming. The user groups, which were
primarily composed of farmers, were responsible for
implementing federal water policies at the local level.
Within four years, 372 user associations had formed
to manage the distribution and quality of water by
collecting fees for permits and by delineating the
area covered by each permit (Garrido, 2010, 36-38).
The impetus for this major policy change was the be-
lief that local stakeholders would be better equipped
to prevent “contamination by pesticides and nitrates
contained in irrigation runoff… and dumping of un-
treated sewage,” than federal officials removed from
the problem (Sanchez, 1997, 267).
The trading of permits is based on the economic
principles of scarcity and surplus: “a farmer sells a
specified volume of his surplus groundwater or sur-
face water for a season to a neighboring farmer or sev-
eral farmers collectively sell to a nearby town.” The
user associations grant water rights to “existing hold-
ers” without charge, and are required by law to level
uniform charges to all buyers. The Federal Bureau of
Environmental Protection (PROFEPA) in Mexico and
the National Water Commission (CNA), an interme-
diary administrative body, monitor the exchange of
permits to ensure that the environmental quality of
water bodies remains at a high level. Permit holders
Fall 2010: Volume 9, Issue 2
can lose their water rights if the CNA finds that they
have severely damaged an ecosystem’s health by pol-
luting (Thobanl, 1997, 161-79).
A particular strength of U.S. policy for water
pollution management is the strong central role the
federal government plays in funding the construc-
tion of local waste management plants. According to
OECD data, in 1995 only 21.8% of Mexico’s popula-
tion was connected to public waste treatment plants,
compared to 71.4% of the U.S. population1 (Statistics
Portal). Ninety percent of toxic waste in Mexico has
an unknown destination, with 80% assumed to en-
ter Mexican waterways. The U.S. figure, by contrast,
is only 3%. Although this discrepancy is primarily
due to the strength of U.S. waste treatment facilities,
it is important to note that the U.S. does export sig-
nificant amounts of toxic waste to Mexico. The su-
periority of U.S. technology for waste management
is demonstrated by the fact that the U.S. chemical
industry only generates ten times more toxic waste
than the Mexican chemical industry, although it
is about twenty times larger (Castro-Acuña, 1997,
1). The Clean Water Act requires the EPA to over-
see installation of the “best possible technology” for
managing waste in industries known to discharge
pollution. The vague language of the CWA, which
only requires waste management technology to be
implemented when it comes “at a reasonable cost,”
however, does give officials discretion in interpreting
policy (Hunter, 1996).
Historic Institutionalism and Cultural
Values
Several theoretical explanations could also argu-
ably account for policy differences in the two nations.
Possible alternative approaches include: (1) studying
the role played by political institutions in both na-
tions, and (2) looking at the success of interest groups
in lobbying to have their demands met. First, politi-
cal institutions alone cannot account for the discrep-
ancies between Mexican and American policy. The
United States of America and the United Mexican
states have very similar government structures- Mex-
ico has a Presidential system, three branches of gov-
ernment (federal, judicial, executive), and federalism,
with 32 state governments sharing political jurisdic-
tion (OECD, 2003). Despite this similarity, Jacob S.
Hacker’s model of “historical institutionalism” may
provide some of the explanation for why the U.S. has
not shifted to a market based system for regulating
water pollution. He cites W. Brian Arthur’s descrip-
tion of path dependency, which states that “policies
frequently provide incentives that encourage indi-
viduals to lock into a particular path of policy devel-
opment, creating social commitments that may be
quite difficult to reverse” (77).
1  By 2006, this percentage had grown to 36.1 in Mexico,
but comparative data was lacking for the U.S.
29
 The Clean Water Act is, in many ways, not
“locked in place,” but rather, extremely flexible. EPA
and state officials have considerable autonomy in
choosing and implementing the goals of the legisla-
tion. In 1999, for example, the EPA shifted the focus
of its regulation “away from compliance with indi-
vidual discharge permits” to broader efforts at mak-
ing national waters swimmable and fishable (Free-
man, 2000, 181). But why does this autonomy exist?
To answer this question, I analyzed the extent to
which each nation initially centralized water pollu-
tion policy. When President Carlos Salinas de Gortari
signed the 1992 Law of National Waters, he left the
Federal Bureau of Environmental Protection and the
National Water Commission with considerable pow-
er. It was only after a strong federal system of control
was in place that Mexico began to devolve the power
to manage water to local user groups. By contrast,
the U.S. has shown a pattern of decentralizing policy
control to state and local governments from the onset
of legislation to control water pollution.
Second, in my research, I found no arguments or
statistical evidence suggesting that interest groups
are responsible for the development of water pollu-
tion policy in Mexico. Interest groups in the United
States have always played a strong role in lobbying
for environmental protection, but they have tradi-
tionally demanded more aggressive implementation
of the laws in place, for example, engaging in civil
suits to force the Environmental Protection Agency
(EPA) to uphold the laws of the Clean Water Act or
lobbying for stricter protection of a threatened river.
The passage of legislation in both the U.S. and Mexi-
co was brought about by the emergence of a popular
consensus demanding change. In Mexico, the people
wanted cleaner waters as part of a larger overall
strategy to become competitive in the new global
economy. American motivations for passing water
pollution legislation have shifted over time from pro-
moting human health, to encouraging recreation, to
preserving the aesthetic beauty of nature, to today’s
objective of protecting ecosystems.
Thus the values explanation developed by Sey-
mour Lipset in American Exceptionalism: A Double
Edged Sword, can also help explain why the United
States has not adopted Mexico’s market based ap-
proach to improving water quality. Lipset argues that
the strength of American religious beliefs and an em-
phasis on equality of opportunity have created an at-
mosphere of moralism in politics. As I will further ex-
trapolate below, Americans, who receive high quality
water for free through municipal distribution, would
be opposed to a market based system which does not
allocate evenly across geographic and income lines,
as found in Mexico. In “Mobilizing International
Norms,” Amy Gurowitz describes how the burdens
of globalization pushed Japan to place a high value
on becoming competitive in the international market.
The pressures placed on Mexico by globalization ac-
30 The Oculus: The Virginia Journal of Undergraduate Research
count for the willingness of the country to adopt an
economic approach to water pollution management
at the expense of social equality.
If the United States enacted a market based ap-
proach to air pollution management, then can a
values explanation truly account for the lack of this
same system in the water policy arena? Initially, I
struggled with this question. After further research,
however, I concluded that the sheer complexity of
attempting to manage water pollution prevents the
market based solution of the 1990 Clean Air Act and
creates a unique moral situation. Point source pollu-
tion for both air and water is relatively steady and
is traceable to a single discharge source so it can be
more easily regulated. The Clean Air Act Amend-
ment gave the EPA the authority to distribute trad-
able permits granting the right to emit point source
pollution into the air. The Mexican CNA works with
user groups to grant permits for the right to use the
water itself. There is no single point source contribut-
ing to water pollution to an extent comparable to the
coal fired power plants in air pollution or car exhaust
tailpipes (which are regulated by national standards,
like water pollution, not a national trading system).
By contrast, the episodic nature of non-point source
pollution in water, coming in sheets from rainfall and
snowmelts makes it difficult to control with efflu-
ence standards. Under the 1987 CWA amendments,
the responsibility for developing management plans
for non-point pollutions was given to the states. In
Mexico, permit holders are expected to manage their
irrigation in sustainable manner which will prevent
water pollution from fertilizer runoff. If the U.S. can-
not rely on a market based system for allocation, oth-
er economic incentives to prevent pollution are much
less feasible.
History
Heavy industrialization, in addition to inadequate
methods for waste disposal, made pollution a criti-
cal problem for U.S. waterways in the 19th century.
The1899 Refuse Act was passed out of the necessity
of protecting navigation, which was being obstructed
by the heavy volume of solid waste in commercial
waterways. The first U.S. Water Pollution Control Act
was passed in 1948 after the Public Health Service be-
gan voicing concerns over dangerous contaminants
found in national waters. This legislation granted
federal funding to state and local governments for
research and development of water pollution control
in interstate waters. This trend of decentralization
to the state and local level continued with the 1965
Water Quality Act, which required each state to draw
up minimum water quality standards and pollution
management plans (Freeman, 2000, 169-176). Con-
siderable state discretion for what constituted good
water quality, however, often led to continued pol-
lution. The Cuyahoga River, for example, was clas-
sified for “waste disposal” (Kraft, 2003, 107-111). The
fire on the Cuyahoga River in 1969 dramatized the
deplorable conditions in national rivers and created
a public consciousness of water pollution. Pictures of
Earth distributed by the successful 1968 Space Pro-
gram helped cultivate an aesthetic desire to preserve
the beauty of the natural world. In the wake of this
cultural movement for environmental change, the
Clean Water Act went into effect in 1972.
Similarly, Mexican water pollution policy also de-
veloped in the wake of massive amounts of pollution
caused by industrialization. The creation of Mexican
policy, however, was motivated by the pressure of
globalization, not by the cultivation of a value for the
ecological world. In 1946, the Mexican government
bought and subsidized mines in order to promote
rapid industrialization at the expense of environmen-
tal quality. Prior to the emergence of a national wa-
ter policy in 1992, the unchecked dumping of waste
by industries had made more than 75% of all river
basins polluted enough to present a risk to human
health (Sanchez, 1997, 260-76). After his election in
1988, President Carlos Salinas de Gortari made the
deregulation of the Mexican state his top priority.
He privatized enterprises and shifted the economy
to exports in order to make Mexico more competi-
tive with the rise of globalization. His 1992 Law of
National Waters combined efforts to improve water
quality with an “irrigation management and transfer
program” to draw national and international inves-
tors into the water management system (Garrido,
2010, 36-38).
Policy Outcomes
The initial decision made by Congress to decen-
tralize authority to the states in the 1948 Water Pol-
lution Control Act continues to influence American
water policy today. Water quality standards remain
under state control, even after the 1987 Clean Wa-
ter Act Amendment, the most current reform in the
United States. To prevent disasters like the Cuyahoga
River fire, the EPA does now hold states accountable
to the minimum quality standard that waters be fish-
able and swimmable. Under the CWA, states are
required to clean up rivers within 10 years. Priority
for clean-up is given to water bodies heavily relied
on for drinking water, or to those known to contain
endangered species. Clean-up plans are reviewed by
the EPA every three years (Freeman, 2000, 169-209).
One of the primary strategies state governments
have for controlling water pollution is to calculate To-
tal Maximum Daily Loads, or the maximum amount
of pollution a body of water can receive in a day and
still meet quality standards. States grant National
Pollutant Discharge Elimination System (NPDES)
permits based on these calculations of allowable
emissions (United States Environmental Protection
Agency, 2000). The considerable discretion granted
to states in choosing quality levels for water bodies
results in standards which are, on average, lower
Fall 2010: Volume 9, Issue 2
than a national standard would have been if it had
been implemented by the federal government. State
governments are closer to their constituents, and
thus more likely to be influenced by the pressures of
interest groups. Some states have begun to charge a
fee for the obtainment of NPDES permits, in order
to fund the hiring of more enforcement personal and
improve pollution monitoring.
The EPA has gradually gained more control
over water management, but overlap in jurisdiction
among state governments and federal agencies has
become entrenched in American policy, complicating
efforts to prevent water pollution. The report, “The
EPA Needs a Better Strategy to Identify Violations of
Section 404 of the Clean Water Act,” reveals many
of the flaws of U.S. decentralization in water pollu-
tion policy. Section 404 gives the EPA the authority
to override permits to “dredge or fill” bodies of water
granted by the Army Corps of Engineers when the
permits can be shown to threaten vulnerable ecosys-
tems, such as wetlands. Because the EPA only has a
small number of agents to send to the field for moni-
toring, it primarily relies on “complaints, tips, and
referrals” to learn about CWA violations. The lack of
a system for communication with state officials and
Army Corps of Engineer officers also constrains ef-
forts at monitoring. A national record of waterways,
as found in Mexico, could serve as a model for im-
proving the EPA’s methodology for enforcing regu-
lations. “A system to track repeat and flagrant vio-
lators” could help integrate efforts across state and
agency lines (Brass, 2009, 6). Prior to the passage of
the 1972 Federal Water Pollution Control Act, only
one potential violator of water pollution laws had
been taken to court. In 1991; however, the EPA “pros-
ecuted 3,109 cases, resulting in $28 million in penal-
ties,” revealing increased federal efforts at enforce-
ment (Freeman, 2000, 179-181).
In order to become more competitive in the new
global economy, the Mexican government encour-
ages foreign investment, and prioritizes the gain of
capital along with the expense of other values such as
social equality and environmental protection. When
water is managed by the market instead of the gov-
ernment, it is unlikely to be allocated equally across
geographic boundaries and income groups. Rural
families without access to water are more likely to pay
a large fee for water use and have an increased likeli-
hood of catching diseases transmitted by water. Prox-
imity to water raises the average income of families
and improves the standard of living (Sanchez, 1997,
260-276). Market allocation also gives advantages to
“larger domestic and foreign producers” over small-
er, more locally based farmers. In addition, the 2000
EU Water Framework Directive encourages member
states not to treat their water bodies as a “commer-
cial good like any other,” but rather as a “heritage
which must be protected (Salman, 2006, 50). Critics
of economic incentives for pollution frequently cite
31
the moral argument that permit trading systems and
taxation carry the implication that it is acceptable to
pollute, once the right to do so has been purchased.
Strict fines are more likely to imply immorality.
Effluent taxes and tradable pollution permits are
means of making expensive pollution control more
cost effective in Mexico, where many local govern-
ments cannot afford to build waste treatment plants.
People would not value the benefits of high quality
waterways if the costs of preventing pollution were
too high (Freeman, 2000, 198-201). Taxation is espe-
cially effective under circumstances in which little is
known about the size of reduction needed or the cost
of reduction. Under the “polluter pays” principle, the
price of pollution needs to be high enough to provide
an incentive for industries to practice sustainable
water use. Mexico taxes for pollution on the basis
of “receiving body, location, volume and pollution
content,” but offers discounts for industries showing
improvements in the treatment of waste. The U.S. has
no such charge (Goldberg, 1998).
Conclusion
In the period from 1961-1973, an irrigation district
in the United States began dumping saline ground-
water into the Colorado River, and degrading the
quality of freshwater available further downstream
for Mexico. The U.S. and Mexico have been em-
broiled in negotiations over the quantity and qual-
ity of groundwater withdrawn for each nation along
their shared border since 1973 (Beach, 2000, 119). As
demonstrated by this conflict, the trans-boundary na-
ture of water pollution will ensure that domestic wa-
ter pollution policies in both nations will forever be
intertwined. As Western states like California begin
to enter Mexico’s permit trading market, an opening
will be created for both the U.S. and Mexico to learn
from one another and reform their policy approaches
for controlling water pollution (Sanchez, 1997, 260-
76). By investing in the Mexican infrastructure for
water management, the U.S. can funnel wealth and
sophisticated pollution control technology into the
country. In return, solutions to many of the problems
of the Clean Water Act may be found by studying
Mexican policies.
An integrated approach, consisting of the
strengths of both policy approaches towards pollu-
tion management, would first incorporate other poli-
cy arenas, such as energy, transportation, agriculture
and tourism to protect the environment from a vari-
ety of angles into future legislation. For example, the
1977 Amendments to the Clean Water Act authorized
the Department of Agriculture to subsidize up to 50%
of the costs for farmers to manage irrigation effec-
tively enough to reduce non-point source pollution
(Freeman, 2000, 179). This integrated approach could
also emphasize public participation in any changes
made to existing water pollution policy. Seventy-five
percent of family owned industries in Mexico City
32 The Oculus: The Virginia Journal of Undergraduate Research
do not follow environmental regulations because
their owners lack the education to understand their
responsibilities (Castro-Acuña, 1997, 1). To alleviate
this problem, the National Water Commission is giv-
en the responsibility to foster a “culture of water” to
teach the Mexican people about the value of preserv-
ing water, especially considering the scarcity of water
across many areas of the nation (Salman, 2006, 80-81).
The ability to participate in pollution management
gives people a greater stake in protecting natural re-
sources. Additionally, if the public is given copies of
and information about water pollution management
plans, they can also provide commentary informa-
tion to help policy makers improve.
References
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Hewitt, Anja Kurki, Joe A. Oppenheimer, Aaron
T. Wolf, and Edy Kaufman. Trans-boundary Fresh-
water Dispute Resolution: Theory, Practice and Anno-
tated References. Tokyo: UN UP, 2000. Print.
2. Brass, Ira, Dan Engleberg, Byron Shumate, Dani-
elle Tesch, Andre Von Hoyer II, and Michael
Wagg. EPA Needs a Better Strategy to Identify Viola-
tions of Section 404 of the Clean Water Act. Rep. no.
10-P-0009. U.S. Environmental Protection Agen-
cy, 26 Oct. 2009. Web. 28 June 2010.
3. Castro-Acuña, Carlos Mauricio. “A Discussion of
Water Pollution in the United States and Mexico.”
Journal of Chemical Education 74.12 (1997): 1413-
421. Print.
4. “EPA’s Clean Water Act Section 404(c): Veto Au-
thority.” U.S. Environmental Protection Agency.
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gpo.gov/websites/epagov/www.epa.gov/
OWOW/wetlands/facts/fact14.html>.
5. Freeman III, A. Myrick. “Water Pollution Policy.”
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209. Print.
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and Mexico.” (2010): 36-38. Source OECD. Web. 4
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7. Goldberg, Eduard. Water Management: Perfor-
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gov/OWOW/wetlands/facts/fact13.html>.
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Paris: OECD, Organisation for Economic Co-op-
eration and Development, 2003. Print.
13. Rieu-Clarke, Alistair. International Law and Sus-
tainable Development: Lessons from the Law of Inter-
national Watercourses. London: IWA, 2005. Print.
14. Salman, Salman M.A., and Daniel D. Bradlow.
Regulatory Frameworks for Water Resources Man-
agement: a Comparative Study. Washington, D.C.:
World Bank, 2006. Print. Law, Justice, and Devel-
opment Ser.
15. Sanchez, Roberto A. “Water Conflicts Between
Mexico and the United States: Towards a Trans-
boundary Regional Water Market?” Ed. Esther J.
De Haan, Jan Rinzema, and Andre Nollkaemper.
The Scarcity of Water: Emerging Legal and Policy Re-
sponses. Ed. Edward H. P. Brans. London: Kluwer
Law Internat., 1997. 260-76. Print.
16. “Section 404 of Clean Water Act: Program Ques-
tions and Overview.” U.S. Environmental Protec-
tion Agency. Web. 28 June 2010. <http://perma-
nent.access.gpo.gov/websites/epagov/www.
epa.gov/OWOW/wetlands/facts/fact12.html>.
17. “Statistics Portal.” Organisation for Economic Co-
operation and Development. Web. 05 July 2010.
<http://www.oecd.org/statsportal/0,3352,
en_2825_293564_1_1_1_1_1,00.html>.
18. Thobanl, Mateen. “Formal Water Markets: Why,
When, and How to Introduce Tradable Water
Rights.” The World Bank Research Observer 12.2
(1997): 161-79. Print.
19. United States. Environmental Protection Agency.
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Goals of the Clean Water Act. EPA, July 2000. Web. 4
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20. Vaughn Switzer, Jacqueline. “Water Quality:
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Fall 2010: Volume 9, Issue 2 33

 The Influence of Stomatal Morphology on
Gas-exchange Processes of Native and Invasive
mid-Atlantic Tree Species

Erin Dale
This study measured the effect of stomatal structure on gas-exchange processes in three differ-
ent ozone (O3) concentrations (20ppb, 80ppb, 160ppb) and compared the stomata morphologies
across five native and eight invasive tree species from the mid-Atlantic region. Two leaves from
103 trees were measured under 400x magnification after preparation and staining with safra-
nin O dye. Significant variations in stomatal complex size, guard cell width, number of contact
cells, and stomatal density were measured across species. The stomatal complex size, guard cell
width, guard cell length, and number of contact cells were all positively correlated to one anoth-
er. Novel correlations between stomatal type and guard cell width, number of contact cells, and
stomatal complex size were discovered. No significant morphological differences were found
between native and invasive species groups. Stomatal characteristics were found to correlate to
ozone uptake, cumulative ozone uptake, and net photosynthesis, but not stomatal conductance.
Cumulative ozone uptake at the medium ozone treatment departed from other trends and sug-
gests protective mechanisms in trees to avoid ozone uptake. At the high ozone treatment, the
traits that contributed to net photosynthesis changed from stomatal density to guard cell width
and stomatal complex size. This switch could be means for a new competitive advantage in
environments where ozone levels are growing. Individual tree species also exhibited variation
in physical stomatal characteristics and their strength of influence on gas-exchange processes.
Introduction

P lants that come into contact with the air possess

specific pore structures called stomata located on
the epidermis of a leaf, which regulate gas-exchange
between the plant and its environment (Zeiger 1987). 
When a stoma opens, CO2 enters the leaf and can be
used in photosynthesis. However, leaf cells have large
vacuoles filled with water at higher concentrations
than the atmosphere that allow water to diffuse out in
a process called transpiration. Therefore, stoma must
modulate their opening and closing to optimize the
ratio of the amount of carbon gained vital to metabo-
lism to the amount of water lost necessary to prevent
desiccation. The aperture of a stoma is surrounded
by two guard cells that control the opening and clos-
ing of the pore. First studied by Hugo von Mohl in
1856, the morphology of guard cells is now classified
as either kidney or dumbbell shaped (Meidner 1987;
Sack 1987; Hetherington and Woodward 2003).  This
divergent trait may have influenced the broad diver-
sification of plants 30 million years ago after a period
of aridification, as dumbbell stoma can respond to
increased light and resist drought better than kidney
shaped cells (Hetherington and Woodward 2003).
Erin Dale is a fourth-year student double majoring in En- would like to thank the NSF for funding the project, and
gineering Science and Biology.  With these interests she the staff and students at Blandy for their support, guid-
hopes to pursue a career in environmental conservation ance, and friendship.  She is also extremely grateful for
upon graduation in May 2011.  This research was com- her mentor Eric Elton, a PhD student in the Department
pleted at Blandy Experimental Farm in Boyce, Virginia in of Environmental Sciences at the University of Virginia,
the Research Experience for Undergraduates program. She who was essential to the completion of this project. 

34 The Oculus: The Virginia Journal of Undergraduate Research

 Stomata have been shown to be an evolutionary
adaptation to a terrestrial environment (Baranova
1983; Ziegler 1987). Aquatic plant species lack sto-
mata, and plants with only specific areas interacting
with the atmosphere will have stomata only on those
aerial surfaces. And while the cuticle of a leaf pre-
vents desiccation and lowers the transpiration rate,
it also creates a barrier disabling the diffusion of CO2
leading to the critical importance of stomata (Ziegler
1987). These structures also have been thought to
provide a basis for taxonomic classification of plant
species (Baranova 1983; Hetherington and Wood-
ward 2003). Originally, four types of morphologi-
cal differences were identified in stomata based on
the characteristics of the stomatal complex (Ziegler
1987). The stoma itself includes the pore aperture
and the two guard cells surrounding the pore and
can range from 10-80 micrometers in length (Heth-
erington and Woodward 2003). Three apertures in
an hourglass shape define the structure of the pore.  and Fricker 1996). This is done to conserve water and
The central aperture in the middle of the epidermis
is the smallest opening, and the wider pores come in
contact with the air and the mesophyll (Sack 1987).
The stomatal complex consists of the stoma and the
subsidiary cells that touch the guard cells. Subsidiary
cells are specialized, having a different structure than
the rest of the cells on the leaf (Sack 1987). Baranova
(1983) discovered a new classification of stomatal
complex called laterocytic, and in 2005, the four com-
mon categories were classified with even more depth
(Carpenter 2005). The various classifications of sto-
mata developed, along with pertinent terminology,
are illustrated in Figure 1.
The rate of carbon assimilation in a leaf vacillates
as the stomata vary their apertures diurnally (Ten-
hunen et al. 1987). Many factors influence the open-
ing and closing of stomata and the manner in which
this occurs is influenced by stomatal morphology.
The turgor of the plant, ionic salt fluxes, as well as
the intracellular CO2 concentration, all act as inter-
nal stimuli that stomata react to. The turgor pressure
of the plant is the amount of pressure exerted on a
plant cell wall due to the expansion and depression
of the cell’s vacuole. A plant with high turgor has low
osmotic pressure, creating a more rigid plant. The
stomata open less frequently if there is a low turgor
pressure because the openings of stomata result in
continued loss of water; this loss could be detrimen-
tal to a plant lacking sufficient moisture in the soil.
Such action can be explained by the biomechanics
and morphological differences of stomatal complex-
es (Sharpe et al. 1987). Plants in a consistently water-
stressed environment often possess smaller stomata
that are more capable of maintaining the openness
of pores at lower turgor pressures than epidermal
cells or larger guard cells; this lessens the mechanical
disadvantage large guard cells normally experience
with neighboring cells (Sharpe et al. 1987; Willmer
and Fricker, 1996). In addition, mature stomata do
Fall 2010: Volume 9, Issue 2
not have plasmodesmata, which aid in cell-to-cell
transfers, essentially isolating the stomata from sur-
rounding cells. This sequestering allows the cells
to more easily differentiate between varying turgor
pressures (Sack 1987). Stomata are also influenced
by ion fluxes in and out of their pores, specifically
potassium salts. To deal with the energy demands of
actively transporting potassium and the mechanical
opening of the stomata, guard cells contain over ten
times the amount of mitochondria compared to epi-
dermal cells, but have much smaller and many fewer
chloroplasts (Meidner 1987; Sack 1987). Also unique
to guard cells is the presence of starch found in the
stomata because cells have very little ability to buffer
the influx of potassium and change in pH. This starch
can be converted into malate, which is found in abun-
dance to help maintain pH (Outlaw 1987). When in-
tracellular CO2 levels are high, plants have a tenden-
cy to open the stomata less (Morison 1987; Willmer
decrease transpiration rates, because water is lost at
1.6 times the amount that carbon is gained (Hether-
ington and Woodward 2003).
External light, humidity, and CO2 levels influence
the direction of stomatal pores. Stomata gas-exchang-
es are measured by stomatal conductance, which is
the speed at which water evaporates from the pores
and is directly related to the relative size of the stoma-
tal aperture. In general, stomatal conductances tend
to correlate closely to available light and air humidity
levels. The stomatal response to light can vary with
wavelength, as blue light causes pores to open more
than red light, but pores are open the longest during
the brightest times of day (Zeiger et al. 1987). As for
humidity, low humidity results in lower CO2 assimi-
lation (Schluze et al. 1987). This has led to the stoma-
tal physiological adaptation in plants that are xero-
phytes, where guard cells lay below the epidermis for
protection and significantly change the morphology
of stomata (Sack 1987). Extremely low humidity \,
or quick changes from low to high light or high to
low light, can result in patchy stomatal conductance,
where small areas of stomata respond to the envi-
ronmental cues differently. This variability amongst
stomata in close proximity to each other echoes the
heterogeneity of stomata across species (Mott and
Buckley 1998). For CO2 levels, on average, the higher
the CO2 concentration, the lower the stomata density
(Woodward et al. 2002). It should be noted however,
that stomata are driven by an aggregation of these
factors at any one time, and there is great difficulty
in determining the contribution of any one lone fac-
tor. For example, an increase in light will lead to the
opening of stomata and a subsequent increase of
photosynthesis metabolism. As photosynthesis oc-
curs, intracellular CO2 decreases and also leads to
the opening of stomata for CO2 uptake (Sharkey and
Ogawa 1987). Numerous internal and external cues
stimulate the opening and closing of stomata.
35
Figure 1. Four main subcategories of stomatal types (Carpenter 2005)

Another component of the atmosphere that af-
fects plants due to diffusion through the stomata
is ground-level O3 gas. While a beneficial layer of
stratospheric O3 protects the Earth from ultraviolet
rays from the Sun, the O3 in the troposphere acts a
pollutant and greenhouse gas. Ozone is derived from
nitrogen oxides and volatile hydrocarbons, both of
which humans contribute to through automotive
emissions (Krupa et al. 2000). In the Eastern part of
the United States, the ambient levels of O3 range from
30ppb to 50ppb, but in some areas reach over 100ppb
(Patterson et al. 2000). This air polluting gas hinders
plant maturation, decreases tree growth by ten per-
cent (Patterson et al. 2000), and lowers water-use ef-
ficiency (Krupa et al. 2000). Through model predic-
tions, O3 will also decrease net primary production by
16% at current ambient levels (Patterson et al. 2000).
However, most models ignore non-stomatal conduc-
tances and recent data depicts non-stomatal conduc-
tances should be incorporated into calculations be-
cause it can account for up to 63% of total O3 flux of a
given area (Hogg et al. 2007). Nevertheless, stomatal

36 The Oculus: The Virginia Journal of Undergraduate Research

conductance has been thoroughly studied because it
is the dominant method in which O3 affects individ-
ual plants. It has been found that low soil moisture
reduces stomatal conductance and thus decreases
O3 uptake (Patterson et al. 2000). Also, short bursts
of intense O3 levels or constant low levels can injure
plants, though the response varies across species.
Certain tree types such as the Fraxinus americana are
particularly sensitive, while others appear to be able
to resist or localize the negative effects of O3 uptake
(Krupa et al. 2000). Despite the acknowledgement of
the array of reactions to O3, there have been no stud-
ies focusing on how the morphology of stomata af-
fects the uptake of this harmful agent.
With an assortment of stomatal features that vary
between plant species, little research on the differ-
ences and similarities of stomata between native and
invasive species has occurred. Invasive species are
species that have been introduced into a region that
is not within their natural home range. In the new
environment, they survive, reproduce, and persist.
Often times, invasive species can outcompete the
native species of the region and transform the com-
position and diversity of the area. With the damage
O3 is currently causing to trees, those with a higher
susceptibility may be driven out by those that can
better compete in high ambient O3 concentrations,
again changing the landscape of the mid-Atlantic.
Such disruptions can affect not only ecosystem inter-
actions, but also economically impact humans (Mack
et al. 2000).
The goals of this study addressed the questions
surrounding the relationship of stomatal morphol-
ogy to gas-exchange processes, including:
1. Observing if stomatal morphology varies
among tree species.
2. Determining the effects of stomatal morpho-
logical differences on gas-exchange.
3. Determining if the influence of stomatal mor-
phology on gas-exchange varies by species.
4. Distinguishing whether invasive and native
species exhibit different stomatal morpholo-
gies.
Materials and Methods
Thirteen tree species were used in this experiment
from a common garden planted during the spring of
2008 at Blandy Experimental Farm located in Boyce,
Virginia (Clarke County, 39º 09’N, 78º 06’W). Eight
native species planted include: Acer rubrum (AR),
Carya glabra (CG), Celtis occidentalis (CO), Liriodendron
tulipifera (LT), Prunus serotina (PS), Quercus alba (QL),
Quercus rubra (QR), and Robinia pseudoacacia (RP). The
five invasive species included: Acer platanoides (AP),
Ailanthus altissima (AA), Paulownia tomentosa (PT),
Pyrus calleryana (PC), and Quercus acutissima (QA).
There were a total of 103 trees to be analyzed, origi-
nally planted in a randomized complete block design
with reduced numbers due to seasonal mortality.
Fall 2010: Volume 9, Issue 2
Leaf sampling
A single fully expanded sun-leaf from each tree
was selected for measurements, starting with Block 1
and concluding with Block 3 trees. This was repeated
for each tree for a total of two leaves from each tree
in all three blocks.
Leaf clearing and scoring
((The leaves from each tree was cleared, dyed,
and mounted onto slides. After gathering one
healthy leaf from a tree, a protocol from Carpenter
(2005) was used to prepare the leaf for use under a
compound light microscope. First, an approximately
one centimerter (cm) square was cut from the leaf, in-
cluding part of the mid-vein for identification of leaf
surface after clearing. The leaf was placed into a 5%
potassium hydroxide solution until it was complete-
ly submerged for 24 hours. Then the leaf was rinsed
in deionized water and placed into a fresh solution
of 5% potassium hydroxide for another 24 hours. Af-
ter rinsing the leaf in deionized water again, it was
placed it in 5% acetic acid for 5 minutes. The leaf was
net transferred to a beaker of Clorox bleach and sat,
completely submerged, until the leaf was clear. This
process took about 5 minutes on average depending
on the species. To dehydrate the leaf, it was placed it
in an ethanol gradient of 50% ethanol, 75% ethanol,
and 100% ethanol. Then it was placed in a 1% safra-
nin O dye solution for three days. After three days,
the leaf sat in 100% ethanol for 10 minutes and was
then adhered, abaxial side up, to a microscope slide
using glycerin jelly and heat. The edges of the cover-
slip were coated with clear fingernail polish to ensure
a semi-permanent mount.
After mounting the leaves, they were examined
under a compound light microscope (Fisher Scien-
tific MicroMaster II, 12-561-4D) at 400x magnifica-
tion. Digital photographs were taken of stomata of
each species with a small digital camera. Once the
leaves were on the slides, they were measured for a
variety of stomata morphological characteristics con-
sisting of: stomata type, total number of contact cells,
presence of lateral subsidiary cells, presence of polar
subsidiary cells, presence of specialized contact cells,
radial or bilateral orientation of contact cells walls,
stomata complex size, guard cells width, guard cell
length, and stomatal density. Stomata length was
measured as a horizontal length reaching from the
end of each subsidiary cell that flanked the midpoint
of the stomata. Two stomatal densities were taken
from each slide and consisted of the number of sto-
mata present in one 400x magnification frame. A to-
tal of 206 leaf samples were collected, two from each
tree in the common garden. On each of the collected
leaves, 25 stomata were measured and two density
measurements were recorded.
To relate these morphological traits to O3 uptake
totals, Eric Elton gathered and analyzed data from
these trees using leaf-chamber experiments during
37
 Figure 2. Digital photographs of stomata from each tree species. Ailanthus altissima (AA), Acer platanoides (AP),
Acer rubrum (AR), Carya glabra (CG), Celtis occidentalis (CO), Liriodendron tulipifera (LT), Pyrus calleryana (PC), Prunus
serotina (PS), Paulownia tomentosa (PT), Quercus acutissima (QA), Quercus alba (QL), Quercus rubra (QR), Robinia pseu-
doacacia (RP)

the summers of 2008 and 2009. The leaves were ex-
posed to three O3 treatments (20ppb, 80ppb, 160ppb)
for eight hours in cuvettes. Measurements on gas-
exchange processes included O3 uptake per second,
cumulative O3 uptake over the course of one day
(COU), net photosynthesis (An), and stomatal con-
ductance (gs).
Statistical Analysis
All data collected were analyzed using SAS 9.1
(version 9.1.3; SAS Institute, Cary, NC, USA). Repeat-
ed measure mixed model ANOVAs were run for the
dependent variables: the number of contact cells, size
of the stomatal complex, guard cell length, guard cell
width, and stomatal density. For all of these analyses,

38 The Oculus: The Virginia Journal of Undergraduate Research

 Table 1. List of all species, dominant stomata type, and native or invasive status
the fixed independent variables were status (native
or invasive) and stomatal type (laterocytic, stepha-
nocytic, paracytic). Random variables included block
and tree species nested within the status. The unit of
replication was a leaf from each tree subject, nested
within species. Pearson correlation coefficients were
run for the number of contact cells, guard cell width,
guard cell length, and stomatal complex size. Step-
wise multiple regression models with entrance crite-
ria of p=0.15 were used for gas-exchange processes. A
separate regression was run for ozone uptake, COU,
An, and gs. The independent variables included:
number of contact cells, the percentage of radial cells,
the percentage of polar cells, guard cell width, guard
cell length, the presence of specialized cells, stomatal
density, and stomatal complex size.
Fall 2010: Volume 9, Issue 2
Results
Stomatal morphology among tree species
Stomata type
All tree species exhibited a dominant stomata
type. For example, A. altissima was prominently ano-
mocytic while L. tulipifera was predominantly latero-
cytic, and Q. alba was primarily stephanocytic (Fig.
2, Table 1). The three main types of stoma (i.e., para-
cytic, laterocytic, stephanocytic) exhibited significant
correlation to other measured traits including num-
ber of contact cells (F2, 250= 511.62, p= <0.0001), stoma-
tal complex size (F2, 252=21.3, p=<0.0001), and guard
cell width (F2, 252=3.49, p=0.032). Laterocytic types
had a mean of 4.656 contact cells, 14.057 µm stomatal
complex size, and 2.861 µm guard cell width. Para-
cytic types had a mean of 2.0939 contact cells, 12.770
µm stomatal complex size, and 2.679 µm guard cell
width. Stephanocytic types had a mean of 5.504 con-
39
Table 6. Relationships between stomatal type and other stomatal traits L=laterocytic S=stephanocytic P= paracytic
tact cells, 15.255 µm stomatal complex size, and 2.971
µm guard cell width. All types had no significant dif-
ferences between guard cell length (Figs. 3, 4, 5, Table
6).
The native species consisted of five laterocytic
types and three stephanocytic types. The invasive
species included one anomocytic, one laterocytic,
and three stephanocytic stomatal types.
Stomata density
The stomatal density varied across species rang-
ing from 36.38 stomata in one reference frame
(.1662mm2) of P. calleryana to 103.33 stomata for Q.
alba (X2=2.31, p=0.0103) (Fig. 6, Table 5). In native and
invasive species however, there was no significant
density difference, as invasive species had an aver-
age of 61.9 and native species had an average of 61.4
(F1, 12=0.32, p=0.5829) (Fig. 11).
Stomatal complex size, length, and width
Stomatal complex size also exhibited significant
differences across species. Ailanthus altissima had an
average stomatal complex size of 20.9 µm and C. occi-
dentalis of 11.8 µm (Fig. 7, Table 7) (X2=2.13, p=0.0088).
Variation existed across tree species in regards to
guard cell length with a range between A. altissima of
11.3 µm and A. platanoides average of 6.4 µm, but was
not significant (X2=2.26, p=0.1020) (Fig. 9, Table 7).
Guard cell width displayed distinctions in stomatal
40 The Oculus: The Virginia Journal of Undergraduate Research
morphologies, as A. altissima had a mean of 3.95 µm
and C. occidentalis had a mean of 1.97 µm (X2=2.22,
p=0.0066) (Fig. 8, Table 5).
When comparing native and invasive species,
however, there was no significant variation between
the two groups in regards to stomatal complex size
(F1, 11= 0.23, p= 0.6394), guard cell length (F1, 11=0.87,
p=0.3699), and guard cell width (F1, 11=1.38, p=0.2656)
(Fig. 11).
Contact cells
For the number of contact cells surrounding the
guard cells, there were significant differences across
species between a range of 5.372 µm in C. glabra and
3.964 µm in L. tulipifera (X2=1.79, p=0.0366) (Fig. 10,
Table 5). There were no significant differences be-
tween invasive and native species.
Correlations between traits
Stomatal complex size, length, width, and num-
ber of contact cells were all significantly positively
correlated to each other (Table 2). Guard cell length
and width were most strongly correlated (r=0.76031,
p=<0.0001) while guard cell length and contact cell
number were the weakest positively correlated traits
(r=0.16162, p=0.006). Stomatal density exhibited nega-
tive correlations to stomatal complex size (r=-0.29541,
p=0.0427), guard cell width (r=-0.38874, p=<0.0001),
and guard cell length (r=-0.38652, p=<0.0001). All the
Figure 3. Relationship between stomatal type and number of contact cells (F2,250= 511.62, p= <0.0001) L=laterocytic
S=stephanocytic P= paracytic

Figure 4. Relationship between stomatal type and stomatal complex size (F2,252=21.3, p=<0.0001) L=laterocytic
S=stephanocytic P= paracytic

Fall 2010: Volume 9, Issue 2 41

 Figure 5. Relationship between stomatal type and guard cell width (F2,252=3.49, p=0.032) L=laterocytic
S=stephanocytic P= paracytic

Figure 6. Comparison across species of stomatal density (X2=2.21, p=0.0135)

42 The Oculus: The Virginia Journal of Undergraduate Research

 Table 5. ANOVA tables for number of contact cells, stomatal complex size, guard cell width, guard
cell length, and stomatal density

individual tree species also followed these trends,
except for L. tulipifera which demonstrated a nega-
tive correlation between contact cells and guard cell
length (r =-0.46649, p=0.0216).
Traits in invasive species were also all positively
correlated. Native species were significantly positive-
ly correlated with the exception of the number of con-
tact cells and guard cell length (r=0.0732, p=0.3118).
Stomatal morphology and gas-exchange
Net photosynthesis and stomatal morphology
Of the traits measured, multiple morphologi-
cal characteristics were correlated to gas-exchange
processes including net photosynthesis (An). None
of the characteristics were positively correlated to
An. Stomatal density negatively influenced the to-
tal An (R2=0.2374, F= 27.4, p= <0.0001) (Tables 3, 4).
The traits that correlated to An varied with differ-
ent ozone treatments (Table 3). In the low treatment,

Fall 2010: Volume 9, Issue 2 43

 Figure 11. Comparison of stomatal traits (number of contact cells F1,11= 0.16, p=0.6927; guard cell width, F1,11=0.23
p=0.6394; stomatal complex size, F1,11= 1.38, p=0.2656; guard cell length, F1,11= 0.96, p=0.386) in invasive and na-
tive species
both the increasing percentage of radial cells (partial
R2=0.1849) and density (partial R2=0.1058) resulted in
a decreased An (p= 0.0115, F= 5.33). At the medium
treatment, guard cell width strongly and positively
(partial R2= 0.6059) and stomatal density slightly
and negatively (partial R2= 0.0532) contributed to An
(F= 27.06, p=<0.0001). At the high treatment, An was
influenced positively by guard cell width (partial
R2=0.1213) and negatively by stomatal complex size
(partial R2=0.1778) (F=5.76, p=0.0082).
In one of the species, C. occidentalis, there was a
significant negative correlation between stomatal
complex size (partial R2=0.3097), density (partial
R2=0.6385), and the presence of polar cells (partial
R2=0.0514) (F=1015.55, p= 0.023) (Table 7). Stomatal
morphology of native and invasive species affected
An through different traits (Table 8). Invasive species
were impacted positively by the increasing length
of guard cells (partial R2=0.2414, F=9.23, p=0.005)
and native species by radial cells (partial R2=0.3123)
and stomatal density (partial R2=0.0627) (p=<0.0001,
F=16.8).
Ozone uptake and stomatal morphology
Ozone uptake was positively correlated to guard
cell width (partial R2=0.0235), but negatively related
to the presence of specialized cells (partial R2=0.0271)
and guard cell length (partial R2=0.226) (F=2.77,
p=0.0465) (Tables 3, 4). However, when the ozone
data was separated into the high, low, and medium
treatments that were assigned to the trees, other ef-
44 The Oculus: The Virginia Journal of Undergraduate Research
fects emerged (Table 3). The high and the medium
treatments of ozone were not significantly correlated
to stomatal traits. At the low ozone treatment, which
most resembled a normal functioning leaf, the nega-
tive correlation to specialized cells was still present
and slightly stronger than the total ozone uptake per
second (partial R2=0.1059, F= 6.43, p= 0.0174).
Ozone uptake of five species was directly impact-
ed by stomatal attributes including: A. rubrum (de-
creasing stomatal complex size, partial R2=0.06988,
F= 16.24, p= 0.005), C. occidentalis (increasing guard
cell length, partial R2=0.982; decreasing width, partial
R2=0.0162; decreasing radial cells, partial R2=0.0018;
F= 20482, p=0.005), P. serotina (decreasing stomatal
complex size, partial R2=0.5818; increasing guard cell
width, partial R2=0.0374; decreasing radial cells, par-
tial R2=0.1442; decreasing density, partial R2=0.2176;
F= 51.29 p =0.001), Q. acutissima (decreasing radial
cells, partial R2=0.4875; decreasing stomatal complex
size, partial R2=0.1656; F= 5.65, p= 0.042), and R. pseu-
doacacia (decreasing radial cells, partial R2=0.1838;
decreasing number of contact cells, partial R2=0.5726;
F= 9.32, p= 0.015) (Table 7). There was no significant
effect in either the native or invasive species groups
(Table 8).
Stomatal conductance and stomatal morphology
Overall, total stomatal conductance (gs) values
could not be explained by any stomatal attributes.
Also, there were no significant contributions at the
high, low, and medium ozone treatments.
 Figure 7. Comparison across species of stomatal complex size (X2=2.13, p=.0088)

Figure 8. Comparison across species of guard cell width (X2=2.22, p=0.0066)

Fall 2010: Volume 9, Issue 2 45

 Table 2. Pearson correlation coefficients between stomatal traits of all species
Three species, however exhibited significant sto-
matal trait correlation (Table 7). Guard cell length
positively (partial R2=0.3841), width negatively (par-
tial R2=0.1098), and radial cells positively (partial
R2=0.343) influenced A. rubrum (F= 8.55, p=0.021).
Stomatal complex size negatively (partial R2=0.0721),
guard cell length negatively (partial R2=0.3797), ra-
dial cells positively (partial R2=0.4305), contact cells
positively (partial R2=0.0937), and specialized cells
negatively (partial R2=0.0158) influenced P. serotina
(F= 73.26, p=0.003). The number of contact cells nega-
tively (partial R2=0.5745) influenced Q. rubra signifi-
cantly (F= 6.75, p=0.048). There were no significant
effects on native and invasive species (Table 8).
Cumulative ozone uptake and stomatal
morphology
The presence of specialized cells changes in-
versely to the COU across all three ozone treatments
collectively (F=4.46, p=0.0143), as well as in the low
and high treatments (Table 3). The medium treat-
ment departs from this trend (Table 4). The medium
treatment regression has a negative slope suggesting
that at the medium ozone level, the stomata are not
uptaking an increasing amount of ozone as they do
at both the low and high treatments. This difference
could be driven by the negative correlation of the
number of contact cells (partial R2=0.0933) and posi-
tive correlation to the presence of polar cells (partial
R2=0.0871) (F=3.08, p=0.0871).
46 The Oculus: The Virginia Journal of Undergraduate Research
The COU of A. rubrum, C. occidentalis, P. serotina,
Q. acutissima, and R. pseudoacacia are affected by sto-
matal morphology (Table 7). A. rubrum was negative-
ly correlated to guard cell length (partial R2=0.7382,
F= 19.74, p=0.003). C. occidentalis was positively cor-
related to guard cell length (partial R2=0.9694) and
negatively to stomatal complex size (partial R2=0.025)
(F= 177.03, p=0.006). P. serotina was negatively cor-
related to specialized cells (partial R2=0.1179), radial
cells (partial R2=0.2483), density (partial R2=0.0239),
and stomatal complex size (partial R2=0.5378), and
positively correlated to guard cell length (partial
R2=0.056) (F= 36.39, p=0.0070). Q. acutissima was
negatively correlated to stomatal complex size (par-
tial R2=0.5117, F= 7.33, p=0.0300). R. pseudoacacia
was negatively correlated to contact cells (partial
R2=0.5622) and radial cells (partial R2=0.1910) (F=
9.15, p=0.0150).
In native species, there was no significant correla-
tion between COU and stomatal traits, but there was
an inverse relationship between COU and stoma-
tal complex size (partial R2=0.1218) and specialized
cells (partial R2=0.1471) in invasive species (F= 5.15,
p=0.013) (Table 8).
Discussion
Stomatal morphology among tree species
The results of this study identified and compared
the variation of morphological stomatal characteris-
tics of 13 mid-Atlantic trees across species. By gath-
Figure 9. Comparison across species of guard cell length (X2=2.26, p=0.1020)

Figure 10. Comparison across species of number of contact cells (X2=1.79, p=0.0366)

Fall 2010: Volume 9, Issue 2 47

48 The Oculus: The Virginia Journal of Undergraduate Research
Fall 2010: Volume 9, Issue 2
Table 7. Stepwise multiple regression data: the influence of stomatal traits on individual tree species
COU= cumulative ozone uptake An= net photosynthesis gs= stomatal conductance

49
Table 8. Stepwise multiple regression data: the influence of stomatal traits on native and invasive trees
COU= cumulative ozone uptake An= net photosynthesis gs= stomatal conductance
ering data on a large number of species, this study
can categorize potential similarities and differences
within a community to predict possible areas of com-
petitive advantage.
Stomatal type
As a common taxonomic differentiation (Ziegler
1987, Baranova 1983), the morphological stomatal
type was classified and retained within genus. For
example, A. rubrum and A. platanoides both had lat-
erocytic stomata types, while all three Quercus spp.
possessed stephanocytic stomata types (Table 1).
Genetic controls account for many of the stomatal at-
tributes measured in this study. Therefore, including
closely related native and invasive species led to no
significant difference between these species groups.
Stomatal type is defined by the arrangement of
subsidiary cells around the guard cells. But, each
type also showed a significant correlation to other
morphological traits, not included within classifica-
tion criteria. The variation in mean number of contact
cells, stomatal complex size, and guard cell width ex-
pressed significant relationships in these species. No
previous study has linked any stomatal trait other
than the number and orientation of subsidiary cells
to stomatal type (Carpenter 2005, Baranova 1983).
Stomatal density
Native and invasive species did not differ in
stomatal density counts, but did display significant
variation across species. Stomatal density was found
to negatively correlate to guard cell length. This re-
lationship has also been published in past studies
(Ohsumi et al. 2007, Kundu and Tigerstedt 1998), but
the negative relationships between stomatal complex
size and guard cell width have not been previously
studied. Larger stoma on a leaf correlate to lower
stomatal density, which was evident across species
such as A. altissima (stomatal density= 42.3, stomatal
complex size= 20.94 µm, guard cell width= 3.95 µm,
guard cell length= 11.34 µm).
Stomatal complex size, length, and width
Other morphological traits including stomatal
complex size, guard cell length, and guard cell width
exhibited significant correlations. The positive re-
lationship between all of these traits indicates that
50 The Oculus: The Virginia Journal of Undergraduate Research
the larger the guard cells, the larger the stomatal
complex. This suggests that the correlation between
guard cells and stomatal complex may be a morpho-
logical response to the physical demand of stoma
size. This positively proportional trend was appar-
ent in all species, except L. tulipifera, between guard
cell length and number of contact cells (r= -.46649,
p=0.0216). The number of subsidiary cells in Lirioden-
dron tulipifera oscillated often between two and four
contact cells. This trend implies that larger guard cell
length correspond to two contact cells (paracytic sto-
matal type), while smaller stoma lengths correspond
to four subsidiary cells (laterocytic stomatal type).
Stomatal morphology and gas-exchange
This study also focused on the influence of physi-
cal stomatal traits on gas-exchange across species in
order to help predict variation in gas-exchange. As
gas-exchange can differ across species (Novak et.al.
2005), in turn, the method in which species respond
to pollutants such as ozone also may vary. While the
effects of ozone (Dizengremel et al. 2008, Booker et
al. 2009, Pfleeger et al. 2010), as well as the effects of
stomatal traits on gas-exchange (Russo 2010), have
been studied, this is the first study to link the role
of morphological traits to the effect of ozone on gas-
exchange processes across multiple tree species.
Net photosynthesis and stomatal morphology
A measure of An and its possible correlation to
stomatal traits were analyzed to help explain stoma-
tal modulation of gas-exchange. The leaves that the
stomatal measurements were measured from were
different from the leaves in which the gas-exchange
measurements were collected, although they were
from the same tree. A positive correlation between
An and stomatal density was observed in Azadi-
rachta indica (Kundu and Tigerstedt 1998), but very
little research has been completed on direct corre-
lations between An and stomatal characteristics of
mid-Atlantic trees. This study did find a direct influ-
ence of stomatal density on An, but contradicts other
studies on trees from other areas of the world. This
study found an inverse relationship between An and
stomatal density at overall, low, and medium ozone
treatments (Table 4). This result indicates that the
stomata on leaves with a lower stomatal density are
Table 3. Stepwise multiple regression data for stomatal trait influences on overall and distinct ozone
treatments H=high treatment L= low treatment M=medium treatment COU= cumulative ozone uptake An=
net photosynthesis

Fall 2010: Volume 9, Issue 2 51

 Table 4. Stepwise multiple regression equations for gas-exchange processes H=high treatment L= low treatment
M=medium treatment COU= cumulative ozone uptake An= net photosynthesis
more efficiently assimilating CO2 than stomata on
leaves with higher stomatal densities. Density in-
fluenced An at the low and medium treatments, but
not at the high treatment. This may illustrate that at
extremely high concentrations of ozone, normal sto-
matal morphological influences no longer play a role
in net photosynthesis. This follows the hypothesis
that at ozone levels above 150ppb, stomatal regula-
tion is no longer under the control of the plant due to
injury (Elton). The switch from An being influenced
by stomatal density and the percentage of radial cells
at low treatments to stomatal density and guard cell
width at medium treatments to stomatal complex
size and guard cell width could provide an avenue
for a change in competitive advantage. The An of trees
growing in higher ozone environments will be influ-
enced by different stomatal characteristics than those
in low ozone environments. With different stomatal
contributors, different trees may succeed in these two
atmospheric environments on account of stomatal
morphology.
Ozone uptake and stomatal morphology
It was also hypothesized that certain morphologi-
cal traits would be more highly correlated with O3
uptake and in combination these traits would explain
more of the variance in O3 uptake than they would
alone (Patterson et al. 2000). This variation in number
and type of traits was apparent across species (Table
7). It was predicted that the stomata with the longest
guard cell length and shortest width would have the
most O3 uptake. This was thought to be because the
larger the aperture, the more stomatal conductance,
leading to an increase in O3 uptake (Patterson et al.
2000; Sack 1987). The data presented in this study did
not corroborate this idea. Both A. altissima and P. cal-
leryana possessed the highest mean guard cell lengths
(Fig. 9, Table 5), but ozone uptake was not influenced
by stomatal traits in either species (Table 7). Celtis oc-
cidentalis possessed stomata with the shortest guard
cell,but, they were most prominently influenced by
length in regards to ozone uptake (partial R2= 0.982).
Therefore, this species contradicts the expected trend.
52 The Oculus: The Virginia Journal of Undergraduate Research
Also, guard cell length and width were the strongest
positively correlated traits (Table 2). A smaller width
was hypothesized to most resemble a dumbbell
shaped guard cell, which is known to more efficiently
and quickly respond to light and increased opening
(Hetherington and Woodward 2003). However, the
dumbbell shaped stomata seen in the Quercus spp.
were closer to the average guard cell width across
species. The departure of C. occidentalis and Quercus
spp. may be due to the fact that these traits have not
been well studied in past literature. Also, this study
does not address all possible influences on stomatal
regulation.
Stomatal conductance and stomatal morphology
Stomatal condutance, though a measure of water
flux, estimates the passage of gas through a leaf. Little
variation or influence of stomatal attributes appeared
across species. These findings support other studies
that have discovered little correlation to g and physi-
cal stomatal traits (Ohsumi 1998, Russo et.al. 2010).
The lack of correlation may be explained because
other environmental factors such as light availability
and air humidity strongly influence stomatal conduc-
tance.
Cumulative ozone uptake and stomatal morphology
The presence of specialized cells strongly influ-
ences overall COU, but when separated by individu-
al tree speices, only Q. acutissima and P. serotina were
influenced by specialized cells. A slightly significant
negative relationship appeared at the medium ozone
treatment. At the medium ozone treatment, the
leaves responded to ozone differently than at the oth-
er treatment levels. This departure from the overall
trend suggests that certain species possess ozone pro-
tective mechanisms and that these mechanisms are
influenced by different traits than normally function-
ing stomata at the high and low ozone treatments.
The stomata have been proposed to resist ozone by
closing faster at these specific levels (Unsworth and
Black 1981; Krupa et.al. 2000).
Native and invasive species
This study consistently found that there was no
statistical difference between native and invasive
traits (Fig. 11). This lack of significant differences
could be because some native and invasive species
are closely related. These results rejected the hypoth-
esis that invasive species should have a higher sto-
matal density trend than native ones. Since pore size
and stomatal density are usually correlated, a rela-
tively smaller aperture would also be predicted in in-
vasive species (Hetherington and Woodward 2003),
but this is not supported. It suggests that morpho-
logical stomatal traits do not provide an advantage
to invasive species as they invade and settle in a new
environment and pollutant ozone does not influence
competition in the mid-Atlantic region. Interestingly,
three native species, A. rubrum, C. occidentalis, and
P. serotina all departed from the total gas-exchange
trait influences significantly for three of the four dif-
ferent gas-exchange measurements (ozone uptake,
COU, gs, An). For these species, the R2 values were
well above 0.5 implying that a substantial portion of
effects could be attributed to stomatal characteristics
in these species.
Conclusion
In conclusion, this study provided significant cor-
relations between stomatal characteristics (stomatal
complex size, guard cell width, guard cell length,
stomatal density, number of contact cells) and their
variation across species and identified morphologi-
cal traits of individual species that had not been well
researched. Though much was known about stoma-
tal conductance and gas-exchange, very little was
known about the effects of stomatal morphological
differences in these processes. It also presented re-
lationships between stomatal attributes (stomatal
complex size, guard cell length, guard cell width,
stomatal density, number of contact cells, percentage
of radial cells, percentage of polar cells, presence of
specialized cells) to O3 uptake, COU, gs, and An at dif-
ferent ozone concentrations that had not been previ-
ously researched in these species. A shortcoming of
this study was that the gas-exchange measurements
and stomatal measurements were taken from the
same tree, but not the same leaf. The correlations dis-
covered are steps that bring scientists closer to fully
understanding and more closely modeling the com-
plex physical mechanisms of stomatal gas-exchange
modulation. By clarifying how different plants with
differently structured stomata respond to different
levels of O3, we can begin to quantify some of the an-
thropogenic effects society is having on mid-Atlantic
tree species.
Fall 2010: Volume 9, Issue 2
Acknowledgements
Thank you to NSF, UVA, and Blandy Experimen-
tal Farm for funding this project. I would like to give
a special thanks to my mentor, Eric Elton, for his
patience and willingness to teach. I also extend my
appreciation to all the staff, faculty, and students at
Blandy Experimental Farm this summer for their en-
couragement and fellowship.
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54 The Oculus: The Virginia Journal of Undergraduate Research

The Oculus
Volume 9, Issue 2