The Oculus: Fall 2010 Edition by UVa Oculus

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 edition 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 endeavors, 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 distinguished faculty members has become a hallmark of excellence in America’s top universities. 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 research 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 undergraduates 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 Professor Cassandra Fraser and Ruffin Evans

Identifying Cardiovascular Diseases Through Electrical 3D Modeling of the Heart Joyce Ng, Debbie Padilla, Nivedha Panneer, Wyatt Shields, Hsuan Su

Establishing Legitimacy: Early Christians’ Incorporation of Pagan and Jewish Iconography in the Via Latina Catacombs Jessica Arden Ettinger

Application of Fingolimod (FTY-720) for TherapeuticArteriogenesis and Microvascular Remodeling Nikhil Panda

Managing Water Pollution: The United States and Mexico, a Comparative Study Kelsey Kerle-O’Brien

The Influence of Stomatal Morphology on Gas-exchange Processes of Native and 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 retained 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

rofessor Cassandra Fraser is a Professor of Chemistry and Biomedical Engineering at the University 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. Professor 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 demonstrated superior academic performance in introductory 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 begin researching. She says, “My philosophy is that you give people opportunities and see what they are capable 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,

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 followed. 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, Ruffin, 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 valuable 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 experience.” Both Ruffin and Professor Fraser recall the

Fall 2010: Volume 9, Issue 2

trip to China with Guoqing as generous guide and host, as an incredible experience, especially because it allowed Ruffin, then a second year undergraduate, 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 distinguished scientists and students. Professor Fraser believes that “really good students 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 students in the Fraser lab how to perform computational analyses. This leadership experience was invaluable to Ruffin. The opportunity arose given Ruffin’s interest 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 explore other interests, while he maintains his relationship 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-

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 knowledge and you are just learning that body of knowledge, 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 discoveries and generate knowledge and share insights yourself, you have a much better appreciation of things.” Additionally, Professor Fraser discussed the importance 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 illimitable freedom of the human mind.” Getting involved 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 opportunities that we can create from them.

Upasana Bhattacharya

The Oculus: The Virginia Journal of Undergraduate Research

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. Cardiologists 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 functions 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 circuit-1 and circuit-2, respectively. Non-polarizable electrodes simultaneously recorded digitized 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 converted 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 contours 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

ardiovascular disease is the single leading cause of death in America, accounting for over half a million deaths annually.1 With recent technological and scientific advancements, diagnostic testing has proven to be a powerful tool in the fight against cardiovascular disease by allowing for earlier detection 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 coronary 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 behavior 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 diagnoses provided students the motivation to improve current diagnostic imaging modalities by developing a 3D representation of biopotential propagation across the heart. Since the aim is to produce 3D representations from ECG data, only software is required to implement the program without the use of a new device. 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.

The most commonly used diagnostic tool in the clinic today is the ECG. By attaching electrodes to specific locations on the arms, legs and chest, physicians are able to measure the bioelectric potentials associated with the contractions of the heart. ECG is a safe, noninvasive technique that measures the depolarization, propagation, and subsequent repolarization 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 precordial 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 bioelectric events in the heart. One limitation of the ECG is that an abnormal recording is unspecific in its diagnosis 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 implemented. Furthermore, since the ECG records electrical events only for a brief period, a normal ECG recording 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 different levels of physical activity. This allows for detection of arterial blockage, which would result in restricted 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.

Fall 2010: Volume 9, Issue 2

Another technique that studies the electrical phenomena of the heart is the electrophysiology test in which catheters are used to detect abnormal heart rhythm. This technique provides more information 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 determine 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 include mostly analog, 1-dimensional (1D) representations of biopotential events of the heart. Yet, there have been developments that incorporate the 3D na-

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 contour. Physicians can then diagnose and determine the locations of cardiac abnormalities by comparing the shapes of these contours.5 Spatial VCG was discovered and practiced in the 1950s with the Frank Lead System, which makes use of V1 to V6 of the horizontal 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 measurements are time-consuming and require multiple simultaneous lead recordings to develop accurate 3D

Debbie Padilla (left) is a fourth-year majoring in Biomedical Engineering and her interests include computational modeling and public health. She has worked the past two years in Dr. Holmes’ cardiac biomechanics lab with collagen gels to understand how scar forms after a myocardial 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 transmission. 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.

Joyce Ng (second from left) is a fourth-year Biomedical Engineering major. Apart from BME, she finds computer science 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 senior thesis research project on mathematical modeling of Wnt signaling pathways and epigenetic mechanisms involved in cardiovascular cells differentiation, which will hopefully be utilized in optimizing protocols for cardiovascular 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.

The Oculus: The Virginia Journal of Undergraduate Research

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 program 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 diagnosis 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 become a new standard in diagnosing heart abnormalities.

healthy contour. For myocarditis, the inflamed myocardium 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 contour 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-polarizable electrodes, alcohol wipes, and wires with crocodile clips.

Predictions

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 follower, which served to produce high input impedance. Stage 2 was a differential amplifier, which multiplied the difference between the two inputs by the differential gain without implementing positive feedback to maintain stability. By using a variable resistor, the impedances in parallel with the inverting input properly balanced impedances in series with the noninverting input, ensuring that only the cardiac biopotential was amplified. Stage 3 was a high-pass filter,

Nivedha Panneer (middle) is a fourth-year pursuing a Bachelor’s of Science in Biomedical Engineering. Her interests include infectious diseases and drug delivery. Her current capstone research involves the design of an assay and diagnostic device for the detection of Bordetella pertussis, 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 Master’s in Public Health with a focus on global health and health education in underdeveloped countries.

cardiac modeling laboratory involves modeling the intracellular pathways of cardiac myocytes through beta adrenergic 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.

It was predicted that the 3D contour from the healthy experimental heart would have a similar orientation to that of the healthy theoretical 3D contour. Additionally, four different diseased 3D contours were compared to the healthy experimental 3D contour. It was predicted that the blockages in a bundle branch block (BBB) would induce irregular propagation of biopotentials, whereas the macro-action potential, 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

Wyatt Shields (right), a Virginia native, is a fourth-year Biomedical Engineering major and a Materials Science and Engineering minor. His interests include biomaterials, tissue engineering, and ultrasound imaging. His current research and senior thesis project in Dr. Saucerman’s

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, biomaterials, and chemical synthesis labs. For her current capstone project, she aims to design a novel insole for running shoes that mimics the properties of barefoot running and will conduct biomechanics testing in a gait and motion laboratory. After graduation in May 2011, she hopes to pursue a lifelong career in the healthcare field anywhere in the world.

Fall 2010: Volume 9, Issue 2

Figure 1. Bode Plots for Both Circuits 1A) Bode Plot of total gain for circuit-1. 1B) Bode Plot of total gain for circuit-2. The red and blue lines represent the theoretical and measured gains, respectively.

Figure 2. Filtered and Non-filtered ECG signals 2A) Non-filtered Lead II ECG signals. 2B) Filtered Lead II ECG signals. 2C) Non-filtered V6 ECG signals. 2D) Filtered V6 ECG signals. Lead II and V6 were used as examples of how the software used a Butterworth filter to smooth the ECG signals.

which blocked low frequency noise whereas Stage 4 and 5, low-pass filters, blocked high frequency noise. In order to reduce the baseline 60Hz noise in the ECG signals and to obtain a sharper pass band, a 1MΩ resistor was used in parallel with the capacitor in both low-pass filters. Before the electrodes were attached, the patient’s skin was cleaned to remove oil and dead skin cells that could disrupt the measured signal. The finished ECG preamplifier system was then connected with a 15V DC power supply to measure the 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 determining the respective magnitudes at a range of frequencies, 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 relationship:

ECG and Vectorcardiography The circuits were used to acquire simultaneous ECG measurements to represent the QRS complex of a specific heartbeat. Frontal plane ECGs were recorded using Lead I, where electrodes were on the right and left arms, and Lead II, where electrodes were on the right arm and left leg. The two circuits recorded leads I and II simultaneously to produce frontal plane ICVs of a specific heartbeat. Horizontal plane ECGs were measured using precordial chest

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.

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 simultaneously 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 generate 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 horizontal leads. To account for this limitation, boundaries 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) Diagnostic ECG Database. The different types of heart conditions that were implemented into the software

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 theoretical corner frequencies for circuit-1 were 149.4 and 0.97 rad-s-1 and for circuit-2 were 147.4 and 1.24 rads-1. The errors between the theoretical and measured gains for circuits-1 and 2 were 2.64 and 6.30%, respectively. 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 perpendicular 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

Fall 2010: Volume 9, Issue 2

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 corresponded 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 determine the z-coordinate of the intersection point,Â 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 degrees. This is analogous to the y-intercept of the linear slope-intercept equation. The slope of this equation was the negative reciprocal of the slope of Lead II. Finally, the z-coordinate could be obtained by inserting the previously determined x-coordinate into the aforementioned linear equation.Â 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-

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 patientâ&#x20AC;&#x2122;s back. This orientation is towards the thicker left ventricle 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 minor deviation was at the end of the contour. Instead of completing the loop, the end deviated from the expected 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 experimental 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 greater than the ECG voltage values collected experimentally, 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 follows the same trend as the experimental ICV, which indicates that both healthy hearts follow the expected tendency for macro-action potential propagation.

The Oculus: The Virginia Journal of Undergraduate Research

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 cardiac 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 diagnosed 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 patientâ&#x20AC;&#x2122;s contour formation.9

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 electricity 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 experimental, 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 supports the expected propagation of the macro-action potential for a heart with an anterior MI.

Fall 2010: Volume 9, Issue 2

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 propagation time through the QRS complex, which was also seen in BBB. Since this patient suffers from both myocarditis and BBB, it is difficult to discern which disease 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 experimental and control contours, the VHD contour exhibits a loop with a wider base. Since electrical activity 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 orientation 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 horizontal plane data sets were precisely simultaneous, the singular analyzed heartbeat was representative of all heartbeats for each patient, and that the spatial orientation for each heart was the same. From these generalizations, 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 being investigated.13, 14 This could account for the frontal and horizontal planes being recorded at different times. Research to account for the disparities in diagnosis 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 screening 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.

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 horizontal 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, myocarditis, and VHD into the software. This would allow doctors to quickly locate and diagnose heart abnormalities in patients without the use of expensive equipment. Last, additional input from cardiologists, physicians, and patients would help improve the development 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 propagation. The final result would allow cardiologists to analyze the heart in an unprecedented way, through electrical configuration, to diagnose a range of cardiac diseases. With further development, this innovative tool has the potential to impact the medical industry worldwide.

References

1. “Deaths and Mortality” Center for Disease Control 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 reported by http://www.redorbit.com/news/ health/1145566/bcc_research_ market_data_indicates_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.healthsystem.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 Cardiovascular Journal 2001. 35: 2:125-128.

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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 nonarrhythmia clinical electrocardiography. Journal of Electrocardiology. 37: 1: 152-165. October 2004. 8. Allenstein, B. J. Direct spatial vectorcardiographyclinical 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” Indian pacing and electrophysiology journal. 8: 2: 114-128. 2008 10. Pfeffer, M. A., and Braunwald, E. “Ventricular remodeling after myocardial infarction. Experimental 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. Tuberculous myocarditis presenting as long QT syndrome. Eur J Int Med. 11:340-342, 2000. 12. Yilmaz, A., K. Klingel, R. Kandolf, and U. Sechtem. Imaging in inflammatory heart disease: from the past to current clinical practice. Hellenic J Cardiol. 50:449-460, 2009. 13. Strommer, P. J., Lekkala, J. O., Malmivuo, J. A. “Accurate Digital Synthesiser for Simulating Vectorcardiogram.” Medical and Biological Engineering & Computing (1981): 250-4. 14. Kachenoura, A. et al. “Using Intracardiac Vectorcardiographic 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.

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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 The above table lists the resistor values, in kiloohms (k-ohms), and capacitor values, in nanoFarads (nF) for circuit-1.

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

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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 Christians 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 themselves 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.

houghts concerning cultural exchange during the medieval period often conjure images of Christian and Muslim artistic interchange during the Crusades. Buildings like the Church of Hagia Sophia 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 motifs was established underground as early as the midfourth 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 iconography 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 occupants. The arbitrariness and difficulty of such an identification process is compounded by the fact that Jewish and Christian artistic programs of the midfourth century shared many stories and artistic styles of depiction. Moreover, early Christians commonly reappropriated previously distinctive Jewish symbols, such as the emblematic Menorah.2 In addition, both religions were known to incorporate the wellcirculated and recognizable pagan imagery of the

time period into their already shared iconographic programs. The famed Via Latina catacombs, located just outside Rome, are no exception to these artistic instances of shared symbolism and mixed meanings.

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 Potomac, Maryland, double-majoring in Art History and English. She produced this paper for Professor Lisa Reilly’s seminar, The Medieval Mediterranean, during the Spring of 2009. Currently, she is working on a seminar paper for Professor Ramirez-Weaver, involving an investigation 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. Outside of school, she trains year-round as an equestrian, and competes both for the Virginia Riding team and independently. After graduating in May, Ms. Ettinger plans to attend law school and pursue a career in Art Law.

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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 differentiate themselves from their other religious contemporaries. By using pagan art forms, the Christian minorities were able to communicate with the massive 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 affiliated themselves with Judaism to legitimize their fledgling beliefs, and then used Old Testament stories 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 earliest 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 grandeur 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 images are well preserved, which facilitates their analysis. 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 worldwide access to the catacombs through the Internet. The early Christians supported three tenets, which continue to this day to be unofficially endorsed. These cornerstone ideas dramatically influenced 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 Christianity 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. (Macon: Mercer UP, 2000) 11. 4 Claman 11.

In an age without mass communication, art became 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 beliefs, however, was a competitive venture. The fourth century was an age of religious rivalry, in which “Jews and Christians had to show that their prototypes 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 important 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 effects 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 religion to rival paganism. Jews and early Christians were familiar with the pagan artistic program because the vast majority of the Roman population was pagan. Knowing this, both the Jews and the early Christians strategically 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 approximately 245 CE, an image of Orpheus, the divine pagan 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 relied 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 Journal 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/topic/382119/Edict-of-Milan>. 9 Jacob Neusner, “Judaism at Dura – Europos,” History of Religions 4.1 (1964): 85.

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similarity to a pagan prototype.10 Similarly, an image in Dura-Europos shows Moses holding the club commonly 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 Moses’ 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 Hercules, in which the club helped the mythological hero defeat multiple enemies. The Jews promoted Hercules’ 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 maintained artistic convention while re-evaluating the value of the pagan motif, and borrowed the pagan symbol’s physical construction while shedding its explanation.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 decorations 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 Roman goddesses Ceres and Proserpina.13 The juxtaposition of these pagan images with the nearby JudeoChristian 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 Christianity.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 necessarily 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 cubiculum 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.

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 semidivine, 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 crucifixion. The labors of Hercules offered similar opportunity. 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 juxtaposition 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 resurrected three days after his crucifixion. The ceiling of Cubiculum O contains images of Ceres and Proserpina, Roman goddesses who symbolically represented items of importance to early Christians and were associated with death and renewal.20 Ceres, the goddess of fertility and agriculture, 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 Hades, 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.

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include these two pagan goddesses in the catacombs because of the nature of the setting; the idea of salvation 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 spiritual 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 expose 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 Claman 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, early 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 Israelites 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, Christian and Muslim Tradition,” The Biblical Archaeologist 47.4 (1984): 228. 25 Claman 13. 26 There have been several debates surrounding the nature 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 Commandments, 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 discrepancy of opinions before agreeing or disagreeing with his comparison. I believe that SCALA, Florence/ART RESOURCE, N.Y., who supplied the image to ARTstor, from which it was retrieved to be included in this paper, mistakenly 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.

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 intended identification with profound Hebrew leaders such as Moses. Side by side examination of the two images 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 behind 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 embodiment of authority, while the column of fire in the background asserts that G-d speaks to both leaders with equal form, power, and respect. However, the differences between the two figures show a conscious 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 reappearance 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 predecessor 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 simultaneously proves superior through a visual representation 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 differentiate themselves from their religious competitors. In an age of intense religious rivalry, the early Christians needed to express why they should gain more followers than the other contemporaneous religious

The Oculus: The Virginia Journal of Undergraduate Research

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 typological 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 representation 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 suggests 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-encompassing 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 physical submersion in water. These connections between the Old Testament and New Testament were facilitated by a consistent iconographic means of depicting Noah springing forth from the ark in the pose of an orant. An orant, as Leslie Ross explains, is a frontally 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 catacombs as well as at the Catacomb of SS Pietro and

Marcellino and the Catacomb of Priscilla. The consistent 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 requesting that he sacrifice his only son, Isaac. Isaac willingly 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 sacrifice but trusts that it is all within G-d’s plans.36 Different, however, is the fact that Isaac survives his potential 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 earliestsurviving 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 representations 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 century, the formula had even reached North Africa.41 What is, perhaps, most important to recognize about the repetition of artistic motifs in the same, formulaic style for hundreds of years is that the continuation represents the early Christians’ desire to produce an iconographic program that was widespread and recognized amongst multiple groups of different religious affiliation. Essentially, they aimed to produce 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.

28 Bickerman 147. 29 Claman 26. 30 Lewis 225. 31 Leslie Ross, Medieval Art: A Topical Dictionary, (Westport: Greenwood, 1996) 187. 32 Jennifer Speake, The Dent Dictionary of Symbols in Christian Art, (London: Dent, 1994) 12. 33 Bargebuhr 59. 34 Ross 190.

35 Alison Moore Smith, “The Iconography of the Sacrifice of Isaac in Early Christian Art,” American Journal of Archaeology 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.

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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 begins 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 catacombs. 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 iconography. As previously mentioned, Moses is seen carrying a club at the synagogue at Dura-Europos.43 The Jewish people, recognizing the power and divine associations of Hercules, found an equivalent figure, in Moses, in their own scripture but utilized Hercules’ attributes to help convey the parallelism. A comparison 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 seizing 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 association. The Via Latina catacombs contain the entire cycle of this evolution. The concept is represented in the labors of Hercules as a powerful weapon symbolic 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 different 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 Reinterpretation of Early Christian Art, (Princeton: Princeton UP, 1993) 57. 43 Neusner 89. 44 Matthews 57.

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 superiority of their religious beliefs over those of others during an age of intense religious rivalry. The incorporation of pagan motifs was a logical, not blasphemous, choice for the early Christians. In order to gain followers, they needed to communicate legibly, which was accomplished through the widely recognized language of pagan iconography. The early Christian’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 Judaism. Claiming Jewish ancestry provided the early Christians with a more respected position in the religious world. In order to prove themselves 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.

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 immeasurably beneficial to the understanding of the importance of the iconography within the underground 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 explains is a result of how “the paintings are indeed very different in many traits … from those of other catacombs,” making comparisons and confidence 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 Catacomb 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 labors within themselves, and the surrounding iconography. 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.

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3. Bickerman, Elias J. “Symbolism in the Dura Synagogue: 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-Europos helps to explain the competitive relationship between the pagans, Jews and Christians of the late second and third centuries CE. She ingeniously explains the confusing sharing of iconographic motifs amongst these religions as the simple use of a widespread repertoire of artistic representation. 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 Conflict, 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 Christianity 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 themselves 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/topic/382119/Edict-of-Milan>. This succinct article provides a general summary of the creation and importance of the Edict of Milan. Although it does not go into great depth concerning 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: Reflections on Late Ancient Jewish Art and Early Christian 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 mixture” 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,

it is imperative that the art historian keep in mind the multiple influencing factors leading to its production. 7. Ferguson, George Wells. Signs & Symbols in Christian 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 provides 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 Medieval Jewish Art.” Artibus et Historiae 8.16 (1987): 67-89. Gutman’s article analyzes and discusses the similarities 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 Pentateuch 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 early 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 discusses 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 Archaeologist 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 Muslim traditions. Similar to the iconographic depictions 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 acceptance 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 explanation of the mythological stories and histories behind the majority of the Roman and Greek deities and demons prevalent in famous literature and artwork.

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12. Mathews, Thomas F. The Clash of Gods : A Reinterpretation of Early Christian Art. Princeton: Princeton UP, 1993. Matthew’s book is one of the penultimate resources for anyone studying the interactions and relationships between the pagan, Jews and Christians of the early medieval time period. Of particular interest is Matthews’ discussion on the use of “the wand” and its development throughout art, its power bestowed with Hercules, continued with Moses, and ultimately legitimized through Christ’s use. He also pays great attention to the symbols of the chariot and the donkey, and Christ’s changing appearance over time (the “Christ Chameleon”). It is written with an eye towards accessibility and the attention of a wide audience, which makes it a uniquely enjoyable and reliable source. 13. Neusner, Jacob. “Judaism at Dura - Europos.” History of Religions 4.1 (1964): 81-102. In his article, Neusner analyzes the appearance of pagan iconography in the synagogue at DuraEuropos. 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 nonreligious symbols of the era. 15. Rutgers, Leonard Victor. “Archaeological Evidence 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 existed an unprecedented amount of artistic motifsharing and exchange. She sites these attitudes as the source of the desire to immediately reappropriate 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 sacrifices. 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

typological interpretation. Through the multiple examples Smith provides, the reader realizes that the story knows no boundaries; it is found everywhere from in Byzantine iconography to the sarcophagi of Rome, Gaul and Spain, and beyond. 17. Speake, Jennifer. The Dent Dictionary of Symbols in Christian Art. London: Dent, 1994. Speake’s compilation provides concise and easily navigated entries of the most important symbols and stories in Christian artwork. Unfortunately, it lacks any information regarding the pagan symbols that are quite prevalent in early Christian artwork. However, its entries for biblical stories are sufficient and it provides interesting insight regarding the plethora of documented symbols.

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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 hypothesized that the localized delivery of FTY-720 encapsulated in PLAGA nanoparticles to ischemic 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 synthetic stimulant of arteriogenesis and angiogenesis, directly and intramuscularly into ischemic gracilis muscles caused by hindlimb arterial ligation. Perfusion recovery was monitored 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 remodeling, 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 contralateral 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 observed 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 increases between treatment groups suggests this method is promising for similar future research.

Introduction

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 angioplasties, 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 delivery 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 student from Chattanooga, TN studying Biomedical Engineering and Engineering Business. While his research is within BME, Nikhil has spent time working within both obstetrics clinical settings and medical device development. Nikhil plans to matriculate in medical school upon graduation in May.

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(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 associated costs and short half-life make it a poor option for effective and efficient treatment5. Therefore, FTY720, a more practical pharmaceutical with low costs and longer half-life, is a more viable option for current research. Difficulties in the successful in vivo application of small synthetic materials, such as FTY-720, still remain. While in vitro testing reveals therapeutic advantages, direct introduction in vivo did not prove effective8. As such, stimulating agents are introduced within polymeric biomaterials, such as poly-lacticco-glycolic-acid (PLAGA)6,7. These biodegradable and biocompatible materials help maintain the beneficial properties of stimulating agents, such as relatively long half-life, and growth factors while also creating a controlled-release drug delivery profile. The prevalence of small-particle conjugated drug delivery continues to increase in current studies. The aim of this experiment is to evaluate the therapeutic efficacy of FTY-720 treatments to stimulate collateral arterial growth in ischemic mice hindlimbs. Specifically, the hypotheses that the direct introduction of FTY-720 encapsulated in PLAGA nanoparticles 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.

instruments were autoclaved. Mice were each anesthetized by an intraperitoneal (IP) injection of 1.56 mL/kg body weight ketamine HCl, 0.52 mL/kg xylazine, 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 sagittal 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 structures 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.

Methods and Materials

In this study, the arteriogenic and angiogenic effects of FTY-720 (conjugated with PLAGA microcapsules) were examined upon direct intramuscular 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.

FTY-720 Nanoparticle Fabrication5 PLAGA nanoparticles loaded with FTY-720 were created using an oil-in-water emulsion solvent evaporation technique. Specifically, 40g of polyvinyl alcohol (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 intermediate 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 centrifuged for 60 min at 20,000 rpm at 4˚C. The nanoparticle solution was resuspended again in distilled water (10mL) and sonicated for 30s. To remove large aggregates, the nanoparticle solution was centrifuged at 1000rpm for 10min at 4˚C. A sample of the nanoparticle 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.

Mouse Hindlimb Ischemia Model8 All methods herein complied with University of Virginia Animal Care and Use Committee (ACUC) guidelines. Unilateral hindlimb ischemia was induced in BALB/cJ mice by ligating the femoral artery. Prior to the surgical procedure, the station countertop was covered with sterile drapes, the surgery plate was sterilized with ethanol spray, and surgical

Nanoparticle Delivery to the Hindlimb8 Mice will each be anesthetized by the (IP) injection described previously. A solution of 1.5mg FTY720 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

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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 distribution of nanoparticle diameter, resulting in an average particle diameter of 207nm.

hindlimb received no injections. Control samples, in which control PLAGA were applied, were administrated as described above. Laser Doppler Perfusion Measurements5 Animals anesthetized by IP injection animals were placed on a surgical heating pad inside a small chamber 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) perfusion, muscles were perfusion-fixed with 4% paraformaldehyde 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 antismooth 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, Melville, NY). Using ImageJ software (NIH, Bethesda,

MD), SMA+ vasculature was analyzed for diameter measurements of large collateral vessels. For investigation of the angiogenic effects of the FTY-720, specimens will be bisected about their midlines perpendicular to muscle fiber (MF) direction. BS-1 lectin+ 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 described previously, there were three main metrics used to quantify the arteriogenic and angiogenic effects of FTY-720. The ischemic:contralateral perfusion ratio was used to determine blood perfusion recovery, the diameter of the major collateral vessel imaged 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 angiogenesis. The LDPI was analyzed using a two-way variance analysis, while changes in arteriolar collateral diameter and SMA+ and lectin+ vessel profiles per muscle fiber were analyzed by two-way repeated measures ANOVA with Holm-Sidak multiple pairwise 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 PLAGA nanoparticles loaded with FTY-720, scanning electron microscopy was performed (FIG. 1). Analysis shows that the nanoparticles applied in this experiment were spherical in shape and structure. Using a submicron particle analyzer, the number-weight distribution 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.

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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 observed. 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-ligation (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 overall recovery of blood flow in the ischemic hindlimb (FIG. 2). While the overall trend suggested a therapeutic effect, treatment with PLAGA loaded FTY-720 nanoparticles did not show a significant increase in the rate of blood flow regeneration. Greater perfusion was seen at days 14 and 18; no significant increases in perfusion were seen at other time points. Vasculature Remodeling Analysis: Arteriogenesis Harvested gracilis muscles excised 18 days postligation were imaged using confocal microscopy to evaluate the arteriogenic effects of the FTY-720 treatment. 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 PLAGA 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% increase. The PLAGA treatment did not yield a significant increase in diameter of the collateral main feeder relative to the non-ischemic hindlimb.

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 lectin+ and SMA+ vessels per MF for the PLAGA and FTY-720 treatments and for the ischemic and contralateral 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 profiles 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 characterized by arteriogenic and angiogenic capabilities5,9, in the ischemic hindlimbs of mice would yield statistically 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 experiments 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 increased average perfusion ratio at the final two time points, representative of increased blood flow recovery to the ischemic hindlimb. The overall recovery profile is not entirely consistent with the hypotheses

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Figure 3. Arteriogenic effects of FTY-720 treatment. A: Representative image of collateral main feeder vessel 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 expected. A potential explanation for this sudden increase could relate to the release of FTY-720 from the PLAGA nanoparticles in vivo. It is known that the release 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 material11. 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 constant 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 copolymer13. 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 analysis. As perfusion measurements revolve about the ligated:contralateral average perfusion ratio, it is logical that the observed increase in blood flow recovery might result from an increase in the diameter of this vessel14. Characterization of the arteriogenesis and angiogenesis induced by FTY-720 resulted in increased average 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 remodeling as statistical analysis of the data showed there was no significant change as a result of treatment. These results imply that the vessel development induced 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 signaling transduction pathways associated with cell growth, proliferation, and differentiation16. Each of these cell-fate responses is characteristic of new vessel development and growth. In summary, while the data obtained in this experiment did not successfully confirm the hypotheses 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 experiment 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 evaluate 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. Additionally, it was difficult to compare the results from

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Figure 4. Analysis of FTY-720 induced arteriogenesis and angiogenesis. A: Confocal images of the bissected and cross-sectioned 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 FTY720 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 quantified17. 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, FTY720 concentration, nanoparticle fabrication and loading, 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.

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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 quantification of the number of F4/80+ cells per MF bundle 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 meaningful data. As such, it is unknown if the observed trend increase in the observed arteriogenesis and angiogenesis 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, 328-33. 2. Rajagopalan S, Mohler ER, Lederman RJ, Goldman, CK, et. al. (2003). Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease. Circulation, 108, 1933-38. 3. Keul P, Tolle M, Lucke S, Heusch G, et. al. (2007). The sphingosine-1-phosphate analogue FTY720 reduces atherosclerosis in apolipoprotein E-deficient mice. Arteriosclerosis, Thrombosis, and Vascular Biology, 27, 607-13. 4. Westhoff T, Schmidt S, Glander P, Liefeld L, et. al. (2007). The impact of FTY720 (fingolimod) on vasodilatory function and arterial elasticity in renal transplant patients. Nephrology Dialysis Transplantation, 22, 2354-58. 5. Burke CW. (2009). A platform for therapeutic insonation: targeted drug delivery and non-thermal albation. Dissertation Proposal. The University of Virginia: Department of Biomedical Engineering. 6. Chappell JC, Song J, Klibanov AL, Price RJ. (2008). Targeted delivery of nanoparticles bearing fibroblast growth factor-2 by ultrasonic microbubble destruction for therapeutic arteriogenesis. The FASEB Journal, 22, 1147.1. 7. Panyam J, Labhasetwar W. (2002). Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Advanced Drug Delivery Reviews, 55, 329-347.

8. Lawrence RT, Archer BP, Burke CW, Meisner JK, RJ Price. (2009). Investigation of phthalimide neovascular factor-1 for therapeutic arteriogenesis in occlusive vascular disease. Capstone Thesis. The University of Virginia: Department of Biomedical Engineering. 9. Xin C, Ren S, Eberhardt W, Pfeilschifter J. (2006). The immunomodulator FTY720 and its phosphorylated derivative activate the smad signaling cascade and upregulate connective tissue growth factor and collagen type IV expression in renal mesangial cells. British Journal of Pharmacology, 147, 164-74. 10. Banai S, Jaklitsch MT, Shou M, Lazarous DF, et. al. (1994). Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs. Circulation, 89, 2183-89. 11. Gopferich A. (1996). Mechanisms of polymer degradation and erosion. Biomaterials, 17, 103-114. 12. Adachi K, Kohara T, Nakao N, Arita, M. (1995). Design, synthesis, and structure-activity relationships of 2-substituded-2-amino-1,3-propanediols: discovery of a novel immunosuppressant, FTY720. Bioorganic & Medicinal Chemistry Letters, 8, 853-56. 13. Burkersroda F, Schedl L, Gopferich A. (2002). Why degradable polymers undergo surface erosion or bulk erosion. Biomaterials, 23, 4221-31. 14. Celermajer DS, Sorensen KE, Gooch VM, Sullivan ID, et. al. (2003). Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. The Lancet, 340, 1111-15. 15. Whitesides T, Hanel T, Morimoto K, Harada H, et. al. (1975). Tissue pressure measurements as a determinant for the need of fasciotomy. Clinical Orthopedics and Related Research, 113, 43-51. 16. Miller-Jensen K, Janes KA, Brugge JS, Lauffenburger DA. (2007). Common effector processing mediates specific responses to stimuli. Nature, 448, 604-8. 17. Wardell K, Jakobsson A, Nilsson, GE. (1993). Laser doppler perfusion imaging by dynamic light scattering. IEEE Transactions on Biomedical Engineering, 40, 309-16. 18. Sanchez T, Estrada-Hernandez T, Paik J, Wu MT, et. al. (2003). Phosphorylation and action of the immunomodulator FTY720 inhibits vascular endothelial cell growth factor-induced vascular permeability. Journal of Biological Chemistry, 278, 47281-90.

Fall 2010: Volume 9, Issue 2

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 establishing a system of tradable permits for water use. 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? I argue that cultural values and path dependency on previous legislation combine to prevent the U.S. from moving to Mexico’s market based approach. The extent 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 considerable 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 decentralizing 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 States. Next, I discuss possible alternative explanations for the differences in the two nations. Then, I compare how the history of how water pollution

Kelsey Kerle-O’Brien is a fourth-year History major with a concentration in Latin American Studies and a minor in Technology and the Environment. She has served as the Secretary of Habitat for Humanity since December 2008, as Vice President of Advocacy in 2009, and co-President in 2010. Her passion for volunteer work is demonstrated not only in her commitment to Habitat but also in her

work with Alternative Spring Break, through which she has traveled to San Diego, CA and Ecuador. In the fall, she plans to pursue her Master’s in Public Policy. For fun, Kelsey enjoys studying art history, working out, watching Disney Channel, doing arts and crafts, recycling and participating in Outdoors Club trips to ski and hike.

eorge H.W. Bush’s 1990 Clean Air Act Amendments established a cap and trade program for SO2 from coal fired power plants. The United States 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 European Union member nations and other governments around the world to adopt similar policies for air pollution 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 preserve 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 prevent 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 present a brief overview of how the policies for manag-

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 Law of National Waters

and the

1992

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 complete reduction of all pollution entering fishable and swimmable waters across the nation by 1983 and waterways by 1985.—were too idealistic, amendments were passed in 1977 and 1987. These amendments extended the deadline for the attainment of good water 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 water to newly created “user groups” and established a market based system for water management. A majority of Mexico’s water pollution is focused in agriculture. 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 belief that local stakeholders would be better equipped to prevent “contamination by pesticides and nitrates contained in irrigation runoff… and dumping of untreated 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 surface water for a season to a neighboring farmer or several farmers collectively sell to a nearby town.” The user associations grant water rights to “existing holders” 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 intermediary administrative body, monitor the exchange of permits to ensure that the environmental quality of water bodies remains at a high level. Permit holders

can lose their water rights if the CNA finds that they have severely damaged an ecosystem’s health by polluting (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 construction of local waste management plants. According to OECD data, in 1995 only 21.8% of Mexico’s population 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 enter 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 significant amounts of toxic waste to Mexico. The superiority 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 oversee 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 Values

and

Cultural

Several theoretical explanations could also arguably account for policy differences in the two nations. Possible alternative approaches include: (1) studying the role played by political institutions in both nations, and (2) looking at the success of interest groups in lobbying to have their demands met. First, political institutions alone cannot account for the discrepancies between Mexican and American policy. The United States of America and the United Mexican states have very similar government structures- Mexico has a Presidential system, three branches of government (federal, judicial, executive), and federalism, with 32 state governments sharing political jurisdiction (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 description of path dependency, which states that “policies frequently provide incentives that encourage individuals to lock into a particular path of policy development, 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.

Fall 2010: Volume 9, Issue 2

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 legislation. In 1999, for example, the EPA shifted the focus of its regulation “away from compliance with individual discharge permits” to broader efforts at making national waters swimmable and fishable (Freeman, 2000, 181). But why does this autonomy exist? To answer this question, I analyzed the extent to which each nation initially centralized water pollution 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 power. 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 pollution policy in Mexico. Interest groups in the United States have always played a strong role in lobbying for environmental protection, but they have traditionally 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 Mexico 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 promoting 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 Seymour Lipset in American Exceptionalism: A Double Edged Sword, can also help explain why the United States has not adopted Mexico’s market based approach to improving water quality. Lipset argues that the strength of American religious beliefs and an emphasis on equality of opportunity have created an atmosphere of moralism in politics. As I will further extrapolate 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-

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 approach 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 pollution 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 Amendment gave the EPA the authority to distribute tradable 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 contributing 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 effluence 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. cannot rely on a market based system for allocation, other economic incentives to prevent pollution are much less feasible.

History

Heavy industrialization, in addition to inadequate methods for waste disposal, made pollution a critical 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 began 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). Considerable state discretion for what constituted good water quality, however, often led to continued pollution. The Cuyahoga River, for example, was classified for “waste disposal” (Kraft, 2003, 107-111). The

The Oculus: The Virginia Journal of Undergraduate Research

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 Program 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 developed 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 environmental quality. Prior to the emergence of a national water 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 competitive 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 investors into the water management system (Garrido, 2010, 36-38).

Policy Outcomes

The initial decision made by Congress to decentralize authority to the states in the 1948 Water Pollution Control Act continues to influence American water policy today. Water quality standards remain under state control, even after the 1987 Clean Water 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 fishable 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 Total 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

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 pollution 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 ecosystems, such as wetlands. Because the EPA only has a small number of agents to send to the field for monitoring, 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 efforts at monitoring. A national record of waterways, as found in Mexico, could serve as a model for improving the EPA’s methodology for enforcing regulations. “A system to track repeat and flagrant violators” 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 “prosecuted 3,109 cases, resulting in $28 million in penalties,” revealing increased federal efforts at enforcement (Freeman, 2000, 179-181). In order to become more competitive in the new global economy, the Mexican government encourages 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 government, 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 likelihood of catching diseases transmitted by water. Proximity 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 smaller, more locally based farmers. In addition, the 2000 EU Water Framework Directive encourages member states not to treat their water bodies as a “commercial good like any other,” but rather as a “heritage which must be protected (Salman, 2006, 50). Critics of economic incentives for pollution frequently cite

Fall 2010: Volume 9, Issue 2

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 governments 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 especially 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 groundwater into the Colorado River, and degrading the quality of freshwater available further downstream for Mexico. The U.S. and Mexico have been embroiled in negotiations over the quantity and quality of groundwater withdrawn for each nation along their shared border since 1973 (Beach, 2000, 119). As demonstrated by this conflict, the trans-boundary nature of water pollution will ensure that domestic water 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, 26076). 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 pollution management, would first incorporate other policy arenas, such as energy, transportation, agriculture and tourism to protect the environment from a variety 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 effectively 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

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 given the responsibility to foster a “culture of water” to teach the Mexican people about the value of preserving 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 resources. Additionally, if the public is given copies of and information about water pollution management plans, they can also provide commentary information to help policy makers improve.

References

1. Beach, Heather L., Jesse Hammer, J. Joseph. Hewitt, Anja Kurki, Joe A. Oppenheimer, Aaron T. Wolf, and Edy Kaufman. Trans-boundary Freshwater Dispute Resolution: Theory, Practice and Annotated References. Tokyo: UN UP, 2000. Print. 2. Brass, Ira, Dan Engleberg, Byron Shumate, Danielle Tesch, Andre Von Hoyer II, and Michael Wagg. EPA Needs a Better Strategy to Identify Violations of Section 404 of the Clean Water Act. Rep. no. 10-P-0009. U.S. Environmental Protection Agency, 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): 1413421. Print. 4. “EPA’s Clean Water Act Section 404(c): Veto Authority.” U.S. Environmental Protection Agency. Web. 28 June 2010. <http://permanent.access. gpo.gov/websites/epagov/www.epa.gov/ OWOW/wetlands/facts/fact14.html>. 5. Freeman III, A. Myrick. “Water Pollution Policy.” Public Policies for Environmental Protection. Ed. Paul R. Portney and R. N. Stavins. 2nd ed. Washington, DC: Resources for the Future, 2000. 169209. Print. 6. Garrido, Alberto. “Agricultural Water Pricing: EU and Mexico.” (2010): 36-38. Source OECD. Web. 4 July 2010. 7. Goldberg, Eduard. Water Management: Performance and Challenges in OECD Countries. Paris: Organisation for Economic Co-operation and Development, 1998. Print. 8. Hildering, Antoinette. International Law, Sustainable Development and Water Management. Delft, The Netherlands: Eburon, 2004. Print. 9. Hunter, Susan, and Richard W. Waterman. Enforcing the Law: the Case of the Clean Water Acts. Armonk, N.Y.: M.E. Sharpe, 1996. Print. 10. “Issue Resolution Procedures: Clean Water Act/ Section 404(q).” U.S. Environmental Protection Agency. Web. 29 June 2010. <http://permanent. access.gpo.gov/websites/epagov/www.epa. gov/OWOW/wetlands/facts/fact13.html>.

The Oculus: The Virginia Journal of Undergraduate Research

11. Kraft, Michael E. Environmental Policy and Politics. 2nd ed. New York: Longman, 2001. Print. 12. OECD Environmental Performance Reviews: Mexico. Paris: OECD, Organisation for Economic Co-operation and Development, 2003. Print. 13. Rieu-Clarke, Alistair. International Law and Sustainable Development: Lessons from the Law of International Watercourses. London: IWA, 2005. Print. 14. Salman, Salman M.A., and Daniel D. Bradlow. Regulatory Frameworks for Water Resources Management: a Comparative Study. Washington, D.C.: World Bank, 2006. Print. Law, Justice, and Development Ser. 15. Sanchez, Roberto A. “Water Conflicts Between Mexico and the United States: Towards a Transboundary Regional Water Market?” Ed. Esther J. De Haan, Jan Rinzema, and Andre Nollkaemper. The Scarcity of Water: Emerging Legal and Policy Responses. Ed. Edward H. P. Brans. London: Kluwer Law Internat., 1997. 260-76. Print. 16. “Section 404 of Clean Water Act: Program Questions and Overview.” U.S. Environmental Protection Agency. Web. 28 June 2010. <http://permanent.access.gpo.gov/websites/epagov/www. epa.gov/OWOW/wetlands/facts/fact12.html>. 17. “Statistics Portal.” Organisation for Economic Cooperation 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. Office of Water. Final TMDL Rule: Fulfilling the Goals of the Clean Water Act. EPA, July 2000. Web. 4 July 2010. <http://www.epa.gov/owow/tmdl/ finalrule/factsheet1.pdf>. 20. Vaughn Switzer, Jacqueline. “Water Quality: From Ground to Tap.” Environmental Politics: Domestic and Global Dimensions. New York: St. Martin’s, 1994. 169-86. Print. 21. Weiss, Edith Brown, and Nathalie Bernasconi-Osterwalder. “International Investment Rules and Water: Learning from the NAFTA Experience.” Fresh Water and International Economic Law. Ed. Laurence Boisson De Chazournes. Oxford: Oxford UP, 2005. 263-88. Print.

Fall 2010: Volume 9, Issue 2

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 different 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 safranin 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 another. 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 suggests 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

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 metabolism 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 closing 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 diversification 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 Engineering Science and Biology. With these interests she hopes to pursue a career in environmental conservation upon graduation in May 2011. This research was completed at Blandy Experimental Farm in Boyce, Virginia in the Research Experience for Undergraduates program. She

would like to thank the NSF for funding the project, and the staff and students at Blandy for their support, guidance, and friendship. She is also extremely grateful for her mentor Eric Elton, a PhD student in the Department of Environmental Sciences at the University of Virginia, who was essential to the completion of this project.

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Stomata have been shown to be an evolutionary adaptation to a terrestrial environment (Baranova 1983; Ziegler 1987). Aquatic plant species lack stomata, 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 prevents 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 Woodward 2003). Originally, four types of morphological 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 (Hetherington and Woodward 2003). Three apertures in an hourglass shape define the structure of the pore.Â 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 common categories were classified with even more depth (Carpenter 2005). The various classifications of stomata 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 (Tenhunen et al. 1987). Many factors influence the opening 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 internal 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 cellâ&#x20AC;&#x2122;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 detrimental to a plant lacking sufficient moisture in the soil. Such action can be explained by the biomechanics and morphological differences of stomatal complexes (Sharpe et al. 1987). Plants in a consistently waterstressed 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

not have plasmodesmata, which aid in cell-to-cell transfers, essentially isolating the stomata from surrounding 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 epidermal 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 abundance to help maintain pH (Outlaw 1987). When intracellular CO2 levels are high, plants have a tendency to open the stomata less (Morison 1987; Willmer and Fricker 1996). This is done to conserve water and decrease transpiration rates, because water is lost at 1.6 times the amount that carbon is gained (Hetherington and Woodward 2003). External light, humidity, and CO2 levels influence the direction of stomatal pores. Stomata gas-exchanges 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 stomatal 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 assimilation (Schluze et al. 1987). This has led to the stomatal physiological adaptation in plants that are xerophytes, 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 environmental 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 factor. For example, an increase in light will lead to the opening of stomata and a subsequent increase of photosynthesis metabolism. As photosynthesis occurs, 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.

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Figure 1. Four main subcategories of stomatal types (Carpenter 2005)

Another component of the atmosphere that affects 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 percent (Patterson et al. 2000), and lowers water-use efficiency (Krupa et al. 2000). Through model predictions, O3 will also decrease net primary production by 16% at current ambient levels (Patterson et al. 2000). However, most models ignore non-stomatal conductances and recent data depicts non-stomatal conductances should be incorporated into calculations because it can account for up to 63% of total O3 flux of a given area (Hogg et al. 2007). Nevertheless, stomatal

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conductance has been thoroughly studied because it is the dominant method in which O3 affects individual 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 studies focusing on how the morphology of stomata affects the uptake of this harmful agent. With an assortment of stomatal features that vary between plant species, little research on the differences 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 composition 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 interactions, but also economically impact humans (Mack et al. 2000). The goals of this study addressed the questions surrounding the relationship of stomatal morphology to gas-exchange processes, including: 1. Observing if stomatal morphology varies among tree species. 2. Determining the effects of stomatal morphological differences on gas-exchange. 3. Determining if the influence of stomatal morphology on gas-exchange varies by species. 4. Distinguishing whether invasive and native species exhibit different stomatal morphologies.

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, originally planted in a randomized complete block design with reduced numbers due to seasonal mortality.

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, including 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 completely 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. After 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% safranin 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 coverslip 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 Scientific MicroMaster II, 12-561-4D) at 400x magnification. 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 consisting 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 stomata present in one 400x magnification frame. A total 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

Fall 2010: Volume 9, Issue 2

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 pseudoacacia (RP)

the summers of 2008 and 2009. The leaves were exposed to three O3 treatments (20ppb, 80ppb, 160ppb) for eight hours in cuvettes. Measurements on gasexchange processes included O3 uptake per second, cumulative O3 uptake over the course of one day (COU), net photosynthesis (An), and stomatal conductance (gs).

Statistical Analysis All data collected were analyzed using SAS 9.1 (version 9.1.3; SAS Institute, Cary, NC, USA). Repeated 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,

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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, stephanocytic, 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. Stepwise multiple regression models with entrance criteria 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.

Results

Stomatal morphology among tree species Stomata type All tree species exhibited a dominant stomata type. For example, A. altissima was prominently anomocytic while L. tulipifera was predominantly laterocytic, and Q. alba was primarily stephanocytic (Fig. 2, Table 1). The three main types of stoma (i.e., paracytic, laterocytic, stephanocytic) exhibited significant correlation to other measured traits including number of contact cells (F2, 250= 511.62, p= <0.0001), stomatal 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. Paracytic 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-

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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 differences 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 ranging 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 average 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. occidentalis 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

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 between invasive and native species. Correlations between traits Stomatal complex size, length, width, and number 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 negative 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

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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

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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)

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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 negative 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 positively correlated with the exception of the number of contact 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 morphological 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 total An (R2=0.2374, F= 27.4, p= <0.0001) (Tables 3, 4). The traits that correlated to An varied with different ozone treatments (Table 3). In the low treatment,

Fall 2010: Volume 9, Issue 2

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 native 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-

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 negative 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 impacted by stomatal attributes including: A. rubrum (decreasing 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, partial 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. pseudoacacia (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.

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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)

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Table 2. Pearson correlation coefficients between stomatal traits of all species

Three species, however exhibited significant stomatal trait correlation (Table 7). Guard cell length positively (partial R2=0.3841), width negatively (partial 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), radial 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 negatively (partial R2=0.5745) influenced Q. rubra significantly (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 inversely 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 treatment 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 positive correlation to the presence of polar cells (partial R2=0.0871) (F=3.08, p=0.0871).

The COU of A. rubrum, C. occidentalis, P. serotina, Q. acutissima, and R. pseudoacacia are affected by stomatal morphology (Table 7). A. rubrum was negatively correlated to guard cell length (partial R2=0.7382, F= 19.74, p=0.003). C. occidentalis was positively correlated 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 correlated 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 (partial 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 correlation between COU and stomatal traits, but there was an inverse relationship between COU and stomatal 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 characteristics of 13 mid-Atlantic trees across species. By gath-

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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

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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

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 competitive 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 laterocytic stomata types, while all three Quercus spp. possessed stephanocytic stomata types (Table 1). Genetic controls account for many of the stomatal attributes 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 classification criteria. The variation in mean number of contact cells, stomatal complex size, and guard cell width expressed 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 relationship 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 Âľm, guard cell width= 3.95 Âľm, guard cell length= 11.34 Âľm). 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 relationship between all of these traits indicates that

the larger the guard cells, the larger the stomatal complex. This suggests that the correlation between guard cells and stomatal complex may be a morphological response to the physical demand of stoma size. This positively proportional trend was apparent 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 Liriodendron tulipifera oscillated often between two and four contact cells. This trend implies that larger guard cell length correspond to two contact cells (paracytic stomatal 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 physical 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 gasexchange 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 stomatal 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 Azadirachta indica (Kundu and Tigerstedt 1998), but very little research has been completed on direct correlations between An and stomatal characteristics of mid-Atlantic trees. This study did find a direct influence 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

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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

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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 influenced An at the low and medium treatments, but not at the high treatment. This may illustrate that at extremely high concentrations of ozone, normal stomatal morphological influences no longer play a role in net photosynthesis. This follows the hypothesis that at ozone levels above 150ppb, stomatal regulation 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 influenced 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 morphological 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. calleryana 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 occidentalis 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.

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 physical 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 conductance. Cumulative ozone uptake and stomatal morphology The presence of specialized cells strongly influences overall COU, but when separated by individual 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 other treatment levels. This departure from the overall trend suggests that certain species possess ozone protective mechanisms and that these mechanisms are influenced by different traits than normally functioning 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).

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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 hypothesis that invasive species should have a higher stomatal density trend than native ones. Since pore size and stomatal density are usually correlated, a relatively smaller aperture would also be predicted in invasive species (Hetherington and Woodward 2003), but this is not supported. It suggests that morphological 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 different 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 correlations 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 morphological traits of individual species that had not been well researched. Though much was known about stomatal conductance and gas-exchange, very little was known about the effects of stomatal morphological differences in these processes. It also presented relationships 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 different ozone concentrations that had not been previously 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 discovered are steps that bring scientists closer to fully understanding and more closely modeling the complex 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 anthropogenic effects society is having on mid-Atlantic tree species.

Acknowledgements

Thank you to NSF, UVA, and Blandy Experimental 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 encouragement and fellowship.

References

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