HURJ Volume 18- Spring 2014

Piecing Together Wellness 1

hurj 2013-2014 hurj editorial board Editor-in-Chief

Meredith Haddix

Brian Tung Focus

Shanaz Hossain

Humanities & Social Sciences

Annie Cho Spotlight

Jessica Terekhov Science and Engineering

Ryan Gallo

Alex Mason-Sharma Layout Editor

Melanie Levine Faculty Advisor

Dr. Michael Yassa

hurj spring issue 2014 contributors Joshua Kassner Rohil Malpani Markakis Davide Pini Rebecca Rimsky

about hurj:

The Hopkins Undergraduate Research Journal provides undergraduates with a valuable resource to access research being done by their peers and interesting current issues. The journal is comprised of four sections - a main focus topic, spotlight, science & engineering, and humanities & social sciences. Students are highly encouraged to submit their original work.

disclaimer:

The views expressed in this publication are those of the authors and do not constitute the opinion of the Hopkins Undergraduate Research Journal.

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Michael Saunders Yunuscan Sevimli Camilla Villasante Helen Zhao

contact us: Hopkins Undergraduate Research Journal Mattin Center, Suite 210 3400 N Charles St Baltimore, MD 21218 hurj@jhu.edu http://hurj.org

a letter from the editors

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The Hopkins Undergraduate Research Journal is the only interdisciplinary undergraduate research journal on campus. Within the pages of this issue you will encounter a brief sampling of some of the amazing research Hopkins students have been doing in the sciences, humanities, social sciences, and engineering.

For each issue the staff of HURJ picks one topic of interest to highlight within the magazine’s Focus section. For the Spring 2014 issue of HURJ, we asked for submissions focusing on the changing face of communication. Within our lifetimes, we’ve seen letter writing become almost obsolete and the rise of emails and text messaging as the primary modes of communication. Communication does not have to be just person-to-person—it can also include the communication of the cells in our body, animal to animal, or one person addressing a group. Understanding various types of communication can be vital in many areas, such as disrupting disease transmission or understanding techniques.

As always, our Spotlight, Science & Engineering, and Humanities & Social Sciences sections highlight research in other fields that are not related to the theme of the issue.

In these sections, you will find diverse articles about robotics, refugees, divinity as a scientific notion, and E. coli.

The articles in this Spring 2014 issue exemplify the diverse research interests of Johns Hopkins University students. We are proud of this collection as a representation of the boundless academic and intellectual talents of our fellow JHU students. We would like to give special thanks to our staff for their continued hard work and to the JHU Student Activities Commission for their support. Take a look inside!

Meredith Haddix

Co-Editor-in-Chief

Brian Tung Co-Editor-in-Chief

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table of contents spring 2014 focus: pg. 8 .......

Bacterial Communication: Quorum Sensing Joshua Kassner

spotlight 6

Towards Robotic Autonomy: Self-Diagnosis, Repair, and Replication Yunuscan Sevimli

humanities & social sciences

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The UEFA as an International Private Regime and the Effects of the Financial Fair Play Regulations on Top Clubs’ Competitiveness Davide Pini

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The Importance of Education and Community-Building to Prevent Mental, Emotional, and Behavioral Problems in Refugee Youth Rebecca Rimsky

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Are Science and Religion Incompatible?: Understanding Divinity as a Scientific Notion Helen Zhao

table of contents science & engineering

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Lift Off of Dehydrated Aqueous Two-Phase Systems from Various Substrates Upon Rehydration Michael Saunders

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Soft-Bodied Robotics: Challenges Ahead, Review of Current Simulation Methods and Materials used in Actuation Rohil Malpani

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Purification and Characterization of Enterotoxigenic Escherichia coli CS1 Pili CooD Protein Camila Villasante (i)

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spotlight

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Towards Robotic Autonomy: Self-Diagnosis, Repair, and Replication Mechanical Engineering Self-sustaining robotic teams working together to prepare the environment of a distant planet for human colonization may sound like the plot of a high-budget Hollywood science fiction, but it is also among the ideas that drive some of the most futuristic robotic research conducted in the basement of Hackerman Hall. In the modestly-sized “lab pod” of the Robot and Protein Kinematics Laboratory (RPKL), researchers are working towards developing robotic teams capable of detecting and repairing malfunctioning members as well as replicating themselves from raw materials.

Today, the robots we have in our houses, offices and factories Self-replicating Robot “Gridlock” during assembly are more capable than ever. We rely on them in many ways, and tend to forget how helpless they actually are without a human looking (such as motors) and electronic components need to be constructed, over their shoulders. They may be adept at basic or specific tasks, and this is another area of open research. The research group has desuch as heating coffee, assembling parts along a production line and veloped various low-complexity, functional motors, but much more vacuuming floors, but they are somewhat useless when it comes to work is required to connect the dots between harvesting raw materihelping themselves or other robots. They rely exclusively on human als and constructing complex electronic components.

The second pillar of robotic autonomy under investigation in intervention for maintenance and repairs. The aim of the research the RPKL is robotic self-replication. The research group’s three genled by Mechanical Engineering faculty Dr. Gregory Chirikjian, with erations of self-repairing robotic teams contributions from Ph.D. candidates (named the Hexagonal Modular DisKendal Ackerman, Joshua Davis, Kris tributed Robotic System, or Hex-DMR) Reynolds; senior undergraduates Jef“The more contact I have with have improved at relying on fellow rofrey Yu, Raymond Tuazon, Ryan Downbots, rather than humans, for repairs. ing, and myself; and freshman Baxter humans, the more I learn.”

Each robot is modular, with important Eldridge, is to develop robots that do subsystems contained in six modules of not need human babysitting. The two four distinct types that can be removed distinct projects the team is working on – The Terminator (Terminator and replaced when a fault occurs. For are the Self-replicating Robot and the 2: Judgment Day, 1991) example, if the robot is not moving corSelf-repairing Robot. rectly, the “drive module,” which conThe Self-replicating Robot Project, tains an omnidirectional wheel and a or “Gridlock” (named for its grid-like DC motor, can be replaced. Each genstructure), was initiated by Matthew eration of the robot bears an end-effector appropriate to that modMoses (Ph.D. ‘12) in 2007. Effectively an ultimate 3-D printer that el’s mode of inter-module connections.

For example, in the third can print itself, the robot consists of interchangeable end-effectors generation system, the modules are attached to a central hub with that can move with four degrees-of-freedom. By picking up, moving screw-like connectors.

In this case, an end-effector with a rotational and attaching the given parts in the correct order, the Self-replicating (screwdriver-like) degree of freedom is required.

Robot could assemble a replica of itself, known as its “child.” The To correct a fault that may occur in the team, the robots’ first step child is a “fertile-offspring” – in fact, an almost exact copy of its parwould be to determine that a problem exists and define its specifics. ent, therefore capable of assembling more robots.

In the Hex-DMR, the robotic team members monitor each other to Self-replication, however, goes beyond self-assembly. By changensure that every robot accomplishes its pre-assigned task in the exing its end-effectors, Gridlock is also capable of making silicone pected amount of time.

If an irregularity is sensed, the “sick” robot molds and casting its own structural components using a durable, is visually analyzed by the other robots, who determine whether the two-part plastic resin. In addition to the structure, moving parts

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photos courtesy of Yunuscan Sevimli

Yunuscan Sevimli, Class of 2014

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spotlight

Modularity In most cases, today’s robots are complex electromechanical systems that are intended to be engineered and repaired by humans.

When a complex robotic system fails, a human operator has to follow a series of often complex steps to diagnose the problem, identify the malfunctioning piece of hardware or software, and discover a way to repair or replace the target component. Designing robots with easily replaceable modules can simplify the process of diagnosis and repair.

Gridlock is composed of over one hundred pieces; however, each piece is not necessarily distinct, which lowers the overall complexity of the system.

Additionally, each piece can be manipulated in a similar manner, using a small set of tools. These design choices allow the self-replicating robot to more easily assemble or disassemble parts Scalability of itself or its “child,” unlike robots or To be applied in the field, the conmachines that might require a different cepts developed in the RPKL must be strategy for their every piece. adaptable to robots with different sizFor the Hex-DMR, modularity ales, materials and environmental conlows faults to be addressed at a higher straints. Scalability, therefore, is one of level. If a specific component malfuncthe over-arching design requirements tions, the “doctor” robot can replace for the robots. the module that contains the broken Both the Gridlock and Hex-DMR part with a single, universal tool. This projects require that many components could be considerably easier than reof the final product be scalable. For expairing, for example, an individual wire ample, a method to connect two modor screw. ules is much more useful if applicable In the last few decades, robots have when replicated at twice its size, for become increasingly integrated into Self- repairing Robot “Hex-DMR” attaching a module to a team member use in a robot twice as large. “I have a our lives. As they are used for sweeplove-hate relationship with magnets,” says ing floors, collecting samples from Mars Kendal Ackerman. “Their connecting and and anything in between, our expectations alignment abilities are almost magical, but you just can’t scale them continue to increase, and we require that more complex tasks be pereasily.” In other words, an electro-magnet may be sufficient to keep formed for longer periods of time and with less oversight. The trend two small modules together, but the power required to energize the is poised to continue, and self-sustaining robots will likely represent magnet of a larger robot may not be feasibly stored or supplied on the human intelligence throughout the universe. Whenever should that larger scale. To solve the issues of scalability, numerous mechanical happen, the researchers in RPKL are proud to have contributed. interlocking mechanisms – involving screws, latches and pins – have been developed by the team for different versions of the self-replicating and repairing robots. problem is a broken wheel, a malfunctioning end-effector, a dying battery or something else. Once the problem is identified, the “doctor” robot replaces the module containing the broken component and verifies that the problem is fixed.

Research is being continued to improve the mechanics of the part replacement process and wireless communication between the team members.

Both the self-replicating and selfrepairing robot projects serve as proofsof-concepts whose end goal is not necessarily a directly consumer-marketable product. Despite the striking difference between robots driving on a lab floor and navigating the surface of distant planet, the team believes that the same principles of robotic autonomy can apply to a multitude of diverse situations by maintaining two primary design requirements, scalability and modularity.

Hex-DMR 2nd Generation

Gridlock performing mold making and plastic casting

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Bacterial Communication: Quorum Sensing Joshua Kassner, Class of 2014 Molecular and Cellular Biology Introduction Quorum sensing was first conceptualized when an unusual trend was noticed in a project investigating Vibrio fischeri, a type of bioluminescent bacteria found within the Hawaiian bobtail squid.

These unique bacteria would only shine a bright blue light after the bacteria population reached a specific concentration1. The light would illuminate immediately, as if triggered by a switch. After further investigation, it was discovered that the luminescence resulted from the accumulation of a molecule secreted by the bacteria1. This discovery suggested that bacteria not only communicate with each other but also coordinate population-wide responses to the outside environment.

Quorum sensing involves intercommunication between bacteria through specific chemical signals. These signals, called autoinducers, can be used to coordinate behaviors, such as cell division and biofilm formation, to increase chances of survival. Autoinducer receptors only activate when the signal is present in high concentrations, which result when many quorum-sensing bacteria are in close proximity. Receptor activation ultimately leads to changes in gene expression, a phenomenon that is well documented in biofilm-generating bacteria2. In Staphylococcus aureus (S. aureus), quorum sensing specifically triggers a unique DNA activator called the accessory gene regulator, and its transcripts help create the biofilm formation3. As a result, through autoinducer signaling, bacteria are able to coordinate gene expression with surrounding bacteria to promote survival.

This type interaction offers a novel avenue to counteract the growth of antibiotic-resistant bacteria.

After reading about quo-

sciencelife.uchospitals.edu

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rum sensing, I started to believe that the human immune system must be able to take advantage of this type of bacterial communication. The largest component of the innate immune system, the body’s first line of defense against incoming pathogens, is neutrophils.

These cells use chemical signals to guide their movement and approach pathogens for phagocytosis. Autoinducers have been shown to affect the rate of neutrophil chemotaxis4, thus suggesting that neutrophils are capable of detecting quorum sensing communication.

This study investigated whether autoinducers are involved in neutrophil phagocytosis by testing the growth difference between quorum sensing cultures and cultures with inhibited quorum sensing.

Staphylococcus Aureus and Hamamelitannin S. aureus bacteria, the same pathogen responsible for methicillin-resistant Staphylococcus aureas (MRSA), bind together to form biofilms inside the human body. Research indicates that the molecules associated with biofilm formation also act as autoinducers and neutrophil chemotactic agents. Initial trials of treatments inhibiting these molecules resulted with negative results. Patients suffering from P. aeruginosa demonstrated no improvements when they were given quorum-sensing inhibitors5. Therefore, the effect of these inhibitors on the human immune system should be further investigated before clinical trials can continue.

Hamamelitannin is a naturally occurring substance found in the bark of Hamamelis virginiana, a type of witch-hazel found in North America. Hamamelitannin has been shown to inhibit RNA polymerase III6, thereby preventing autoinducer production and quorum sensing in S. aureus.

Methods S. aureus sample was streaked onto a 3% TBS plate and incubated at 37°C overnight. The following morning, a colony of cells was collected, washed with PBS, and incubated in growth media until log phase growth was observed. The concentration of the bacterial population was determined using serial dilutions and cell counting techniques. 2 µL aliquots of the growth media and bacteria solution were diluted to 1 × 10-2, 1 × 10-3, 1 × 10-4, 1 × 10-5, and 1 × 10-6 concentrations. The dilutions were plated with 3% TBS and incubated overnight. The resulting colonies were counted the following day.

Neutrophils were isolated from freshly drawn blood by first using Dextran 500 (Pharmacosmos), a dense polysaccharide that causes platelets to separate from the rest of the blood7. The blood drawing protocol was approved by the Homewood Internal Review Board under protocol number HIRB00001033. After removing the platelets, the remaining solution was layered on top of Ficoll Paque Plus (GE Healthcare), a separation media that causes cell stratification after centrifugation at 500 RCF for 35 minutes7. The neutrophil layer was removed, treated with Red Blood Cell Lysis Buffer (Life Technologies), and activated using HBSS buffer solution7. The neutrophil concentration was determined by counting active cells with a hemocytometer.

This experiment consisted of two control trials and one variable trial. The two control trials tested S. aureus growth with and

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without neutrophils, designated (+)neutrophil/(-)hamamelitannin and (-)neutrophil/(-)hamamelitannin respectively, to determine whether the neutrophils had been properly isolated. Neutrophil cells and hamamelitannin were added to the (+)neutrophil/(+)hamamelitannin S. aureus culture in the experimental trial to determine the effect of hamamelitannin on neutrophil phagocytosis. A separate trial comparing the growth of S. aureus with and without hamamelitannin was tested to ensure that the addition of the chemical itself had no effect. Each condition was tested four times using the serial plating method mentioned previously and averaged to determine the rate of bacterial elimination with statistical significance.

Results S. aureus population growth varied under the different conditions, (-)neutrophil/(-)hamamelitannin, (+)neutrophil/(-)hamamelitannin, and (+)neutrophil/(+)hamamelitannin, as shown in Figure 1. Proper neutrophil isolation was confirmed through decreased bacterial growth in the (+)neutrophil/(-)hamamelitannin culture.

This growth decrease was not observed in the (-)neutrophil/(-)hamamelitannin sample and indicates active neutrophil phagocytosis.

Hamamelitannin presence had no overall affect on bacterial growth (Figure 2). The culture with (+)neutrophil/(+)hamamelitannin in Fig. 1 displays a greater decrease in S. aureus growth between

90 and 250 minutes than observed in the (+)neutrophil/(-)hamamelitannin culture. Since hamamelitannin has no overall effect on S. aureus growth, the larger population decrease can be attributed to increased neutrophil phagocytosis caused due to the presence of hamamelitannin.

Conclusions The cultures with lower bacterial concentrations between 90 and 250 minutes indicate the presence of neutrophil activity. The population recovery at 300 minutes can be attributed to neutrophil death since neutrophils only have a six-hour half-life outside of the human body. Cultures with neutrophils and hamamelitannin, (+) neutrophil/(+)hamamelitannin, displayed less S. aureus growth than cultures with only neutrophils, (+)neutrophil/(-)hamamelitannin, as demonstrated in the greater dip between 100 and 250 minutes in graph A. Since hamamelitannin does not affect bacterial growth by itself (Fig. 2), we can conclude that autoinducer inhibition by hamamelitannin led to an increase in neutrophil phagocytosis. This conclusion should be appreciated simply as an observed trend, however, until further trials are completed with blood from multiple subjects and varying concentrations of hamamelitannin. If the later studies find the trend statistically significant, then these results would indicate that quorum sensing inhibition increases the efficiency of neutrophil phagocytosis and enhances the innate immune system.

Acknowledgements This work would not be possible without the help of Dr. Haiquin Zhao, whose lab was used to perform the experiments, Dr. Mary Boyd, who drew the blood used in the trials, and Dr. Jocelyne DiRuggiero, whose advice and guidance as a faculty advisor was instrumental in the design and analysis of this experiment. I would also like to thank Shanaz Hossain, Jennifer Donnelly, and Ziga Vodusek for helping to edit this paper. Finally I would like to thank the Hodson Trust and the PURA committee, for approving and providing funding for my proposal.

Figure 1. S. aureus concentration in various growth conditions

S. aureus (cfu/mL) vs. Time (min)

S. aureus (cfu/mL)

1.00E+08

1.00E+07

Staphylococcal Standard 1.00E+06

S. aureus w/ Neutrophil 1.00E+05

S. aureus w/ Neutrophil/ Hamamelitannin

1.00E+04

-­‐50

0

50

100

150 Time (min)

Figure 2. Growth of S. aureus with and without hammelitannin

200

250

300

350

References 1. Nealson K. “Auto-Induction of Bacterial Luciferase—Occurrence, Mechanism and Significance”. Archives of Microbiology.

1977; 112:73-79.

2. Khmel A. “Quorum-Sensing Regulation of Gene Expression: Fundamental and Applied Aspects and the Role in Bacterial Communication”. Springer Link. Journal of Microbiology, July-Aug. 2006.

3. Yarwood J, Bartels D, Volper E, and Greenberg E. “Quorum Sensing in Staphylococcus Aureus Biofilms.” American Society for MicrobiologyJournal of Bacteriology, Mar. 2004.

4. Zimmermann S, Wagner C, Müller W, Brenner-Weiss G, Hug F, Prior B, Obst U, and Hänsch G. “Induction of Neutrophil Chemotaxis by the Quorum-Sensing Molecule N-(3-Oxododecanoyl)-lHomoserine Lactone.” National Center for Biotechnology Information. U.S. National Library of Medicine, 27 Mar. 2006.

5. Köhler T, Perron G, Buckling A, van Delden C. “Quorum Sensing Inhibition Selects for Virulence and Cooperation in Pseudomonas aeruginosa”. 2010.

6. Kiran M, Adikesavan N, Cirioni O, Giacometti A, Silvestri C, Scalise G, Ghiselli R, Saba V, Orlando F, Shoham M, and Balaban N. “Discovery of a Quorum-Sensing Inhibitor of Drug-Resistant Staphylococcal Infections by Structure-Based Virtual Screening” Molecular Pharmacology. May 2008; 73:1578-1586.

7. Oh H, Siano B, and Diamond S. “Neutrophil Isolation Protocol”.

Journal of Visualized Experiments. 2008.

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The UEFA as an International Private Regime and the Effects of the Financial Fair Play Regulations on Top Clubs’ Competitiveness Davide Pini, Class of 2015 Economics

the owners to overspend for certain top players, the UEFA intervened in order to relieve them of this obligation. The long-term goals these policies are trying to achieve include: • Improving the investment in the development of young players within the team.

• Ensuring an appropriate level of management of the clubs’ accounts.

• Creating modern, safe and comfortable infrastructures (stadiums) for the supporters.

• Allowing more fair competition within different clubs not based on their wealth and liquidity.

The regulations require the teams to reduce their losses to no more than €45 million over three years; by 2018, the annual expenditures can exceed returns by no more than €10.2 million. The penalties the UEFA can award to the violators include points’ deduction, restriction on players’ registration to any UEFA competition, fines, disqualification from tournaments and the withholding of monetary awards earned in UEFA competitions. In order to enforce these rules, the newly instituted UEFA Club Financial Control Body has the power to assign the proper penalties to the delinquent clubs. Due to the UEFA’s cross-borders and non-public nature and the difficulties in enforcing its own rules, the author considers it an example of a private international regime. This paper will attempt to evaluate the efficiency of the UEFA in enforcing its own regulation.

It will consider the expenditures and returns in the players’ trading market of three of the major clubs in the continent in the period 1995-2013, in order to highlight eventual changes in their managing patterns: F.C. Inter (Italy), Barcelona (Spain) and Manchester United (England). This choice is due to the prestige these three teams enjoy and the massive costs their owners have undertaken in recent years.

The enforcement of the financial regulations will not start until the 2013-14 season, but it will take into account the profits/losses over the previous two years. Therefore, rather than evaluating the effective power of the UEFA to assign penalties, the data will show how the establishment of these rules is affecting the major clubs’ accounting strategies. The results I expect to see is a drastic reduction on expenditures on top players, which will confirm their consideration of the UEFA’s ability to enforce its regulations.

In September 2009, UEFA President Michel Platini announced the introduction of the UEFA Financial Fair Play Regulations, a revolutionary milestone in the history of world soccer. For the first time, major football clubs would be required to constrain their expenditures to their returns. The disproportionate debts the European major leagues have incurred over the past 20 years have made this reform necessary. The clubs of the Premier League had accumulated a total debt of 3.1 billion pounds in 2009. The Spanish Liga and the Italian Serie A had also amassed substantial debts. This paper will analyze to what extent the UEFA has been able to enforce these new financial restrictions and the impacts these policies have on the competitiveness of these clubs. The analysis will highlight the power of the UEFA as an international private regime to enforce its own rules, building on the ongoing scholar debate regarding this type of regime.

This will be approached within a principle-agent framework focusing on the existing relations between the clubs (agents) and their multiple principals’ respective interests (the owners and the fans), which are often in contrast with the rules the regime is trying to enforce. Measurements of the effects of these rules on competitiveness will be based on data from 2007-2012; the analysis will compare the results of teams that have maintained sustainable expenditures even before the introduction of these regulations and of teams that have drastically cut their expenditures since 2009 in order to respect the new UEFA’s parameters. The findings of the paper will be important because the introduction of regulations over the major clubs’ accounts constitutes a new chapter of the never-ending debate between Keynesian and Hayekian political economists over the freedom of the market and the role of government.

The Union of European Football Association (UEFA) is the governing and administrative body for European soccer, one of the six continental confederations of the International Federation of Association Football (FIFA). Due to the prestige of the major European clubs and the amount of money involved, the UEFA is by far the most important of the six. Founded in 1954 in Basel, Switzerland, it now includes fifty three national associations from Europe and Asia (Kazakhstan, Russia). The current President, Michel Platini, is a former French football star of the 1980s and was elected in 2007. During his campaign he promised to include in the future Champions League, an annual competition with the elite clubs of the continent, more teams from smaller countries and, most importantly, to enforce clubs to undertake sustainable expenditures. The first agreement over limiting expenditures was made in September 2009. Its main goal was to ensure clubs live within their means; since teams’ supporters often pressured Table 1. Clubs considered in this analysis with respective debt/surplus

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The second section of the paper will focus on how these rules have affected the competitiveness of the different teams. The analysis will involve both clubs with sustainable accounts already respecting the Fair Play rules and less rigorous ones still far from meeting the UEFA’s guidelines. Although the regulators have enforced these policies in order to reduce the gap between top and medium-low teams, critics have raised concerns regarding the efficiency of this strategy. Top clubs earn higher returns on merchandise, television broadcast and sponsorships; as a consequence, they will still be able to over-spend the smaller teams even under the Financial Fair Play regime. Arab investors with virtually unlimited monetary resources have recently acquired clubs like Manchester City, Paris Saint-Germain and Malaga. In the past couple years they have been buying some of the top players in the market for incredibly high sums (i.e.

Sergio Agüero to Manchester city for €45 million, Thiago Silva to PSG for €42 million). The general belief is that these clubs are trying to cheat the restrictions, recruiting the best footballers before the Financial Fair Play’s rules fully apply. In order to measure the change in competitiveness due to t h e enforcement of these rules, the paper will consider the results achieved by various teams from the main three European leagues (England, Italy and Spain) in the period 20072012. For each nation, a team

Courtesy of Christopher Johnson via Flickr

humanities & socialhumaniti scienceses

with annual budgetary surpluses and a team that used to run deficits and has tried to contain its expenditures since the introduction of the Fair Play restriction will be picked for comparison. This will give a concrete measure of the general trend in competitiveness and the effects the new rules has had on it. The expected result is to observe a reduction of the gap between the indebted top teams and the well managed smaller ones in terms of points and win percentage per season after 2009. This analysis will consider the following teams (see Table 1).

If the expected results hold, they will confirm that the Financial Fair Play Regulations have a negative impact on the competitiveness of top clubs and that the goal of leveling the ground between wealthy and poor teams is achievable through the enforcement of these rules.

The Enforcement and Establishment of Rules by Private International Regimes: Traditional Approaches An international private regime is defined as “a complex, highlyinstitutionalized form of interfirm cooperation that is usually wellcoordinated with the relevant state or states.” The main duties of an international private regime (i.e. UEFA) toward its constituent members (the football clubs) include providing services (the Champions League and its monetary returns), setting standards (the Financial Fair Play Regulations) and enforcing rules (the institution of the UEFA Club Financial Control Body). A lot of concerns have been raised over the effective capabilities of international private regimes to enforce their own rules. Philipp Pattberg has focused his analysis on rules and regulations, asserting that private governance has to enforce rules in order to gain legitimacy; it cannot center on “spontaneous, un-coordinated, behavior such as market interactions.. This is a key concept for the analysis, which confirms the UEFA’s status as a private international regime.

There is a general consensus that private regimes are able to enforce rules based on their legitimacy, accountability and capacity. The paper will build on Taylor St. John’s observation that international private regimes are not a mere cooperation between different private actors; they require a long-term recognition of their legitimacy. This is why, entering this paper, I believe the UEFA will be successful in enforcing the Financial Fair Play Regulations. Due to the great amount of money and prestige its competitions (Champions League, Europa League) provide to the major European clubs, the penalties they will incur for breaking the rules will be sufficient incentives to prevent the teams from doing so. What can undermine the UEFA capability is in fact a drop in legitimacy. The UEFA President Michel Platini has so far taken the side of the poorer and less renowned leagues in the continent, upsetting many European elite clubs. If these teams suddenly create a cartel and refuse the authority of the UEFA, maybe creating their own elite league as they have proposed in the past, the ability of enforcing rules by the international regime will obviously end.

A major threat to the UEFA legitimacy is the unilateral decisions it takes in order to have its decisions enforced, often without consulting either national governments or private firms (mainly sponsors) affected by the consequences of its policies. Piotr Szwedo focused on the requirements the UEFA imposed on the Polish government after

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Graph 1. Inter FC Purchases and Sales of New Footballers

Graph 2. FC Barcelona Purchases and Sales of New Footballers

the country was picked to host Euro 2012, the continental competition between national teams held every 4 years. He argues that “states that are highly affected by [the UEFA’s] decisions do not have at their disposal many effective tools of control.” The lack of transparency involved in these decisions, which are then imposed to the national federations, can create a desire for independence from the UEFA also at national levels. Elite clubs not willing to constrain their expenditures could use their influence to fuel this feeling at the respective national federations. They could then eventually separate from the UEFA and create their own league, undermining the regime’s legitimacy.

Another debate has risen regarding the effects of globalization on the enforcement power of international private regimes. Gunther Teubner argues that globalization creates problems for the law of the regime, which contradict the rules followed by each member in its own country. This leads to the creation of separated, autonomous sectors, which act independently from economy and politics. In the UEFA’s case, this might be an explanation as to why clubs that borrowed heavily to repay their debts find themselves having trouble

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hurjspring spring2012: 2014:issue issue14 18 hurj adapting to the new limits imposed by the Financial Fair Play. Wyn Grant is of another opinion; he believes that globalization due to the UEFA’s legislative expansion has led to an ideological contrast between a “profit maximizing understanding of football [defended by the elite, indebted clubs] and a more community oriented, democratic vision [supported

by Platini].”

What make the UEFA’s enforcement extremely difficult are the conflicting interests of the different principals of the clubs (agents). In fact, while the governing body of European soccer is trying to limit Graph 1 their losses and help them achieve financial stability, the clubs must also respond to their owners and to their supporters. The owners sometimes share the UEFA’s same objective of reducing losses, but, more often, they look at their teams as a mean to increase their personal popularity and visibility. To do so, club owners are willing to lose money to build great squads and please the supporters.

The fans, on the other hand, always have an interest which is diametrically opposite to the UEFA’s, hoping for the purchases of the best footballers on the planet without worrying for the clubs’ finances. This is why for this particular international private regime, enforcement capability is extremely hard: the agents have to Graph 2 cope with these conflicting interests of their different principals.

Kristoffer W. Eriksen and his colleagues have showed, through the use of tournament theory, how in a given regime the presence of a prize (the participation to UEFA’s competitions) leads to an effort of the different agents to accomplish the principal’s goal. Lewis Evans and Neil Quigley, focusing on the financial sector, argues that the most efficient type of regime is based on high liability left to the shareholders, as long as the creditors are willing to compensate their risks. This position suggests that as long as the clubs’ owners are willing to take on the risks of excessive indebtedness in exchange for the image returns top players will yield, the UEFA’s regimes will be efficient. Any distorted intervention to change the agents’ behaviors will not be beneficial. Gary J. Miller instead highlights the importance of the incentives used by the principals to make sure the agents’ interests adhere to his own one. This paper will show how the access to the prestigious Champions Leagues is a powerful incentive used by the UEFA to transform the clubs’ other principals in agents themselves. The owners receive monetary returns and popularity for participating in this tournament, while the supporters are delighted by the team’s fame and potential success of competing at the highest international level.

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The Effects of the Financial Fair Play Regulations on Clubs’ Competitiveness: Traditional Approaches The debate over the effects on clubs’ competitiveness due to the UEFA intervention to reduce the huge debts of the national leagues exemplifies the never ending debate between Hayekian and Keynesian thinkers over the proper role of the government (i.e. the UEFA) in the economy. Clinton R. Long, of neo-liberal positions, believes that although the aim Graph 3 of the UEFA is to increase the

teams’ efficiency, doing so with Graph 3. Manchester United F.C. Purchases and Sales of New Footballers heavy intervention will undermine their long-term ability to stay competitive. Johan Lindholm is also openly against the intro- F.C. Inter - Italy The Football Club Internazionale Milano S.P.A. is one of the two duction of these regulations; he argues that the salary cap imposed by the break even requirement of the Financial Fair Play Regulations professional soccer teams based in Milan. Founded in 1908, it is the goes against the competition law of the European Union. Peter Ken- only Italian team to have never been relegated in Second Division nedy on the other hand is supportive of the UEFA intervention, as- (Serie B) and has seen many international successes during its hisserting that the way in which football clubs are managed has noth- tory (3 Intercontinental Cups, 3 UEFA Cups, 3 Champions League). ing to do with capitalism. Their huge debts have reduced football Inter has won its most recent won the Champions League in 2010 clubs to mere parasites, constantly refinanced by the external wealth under the leadership of the famed Portuguese coach José Mourinho. of their owners. Based on the evidence provided so far, this paper The team owner and President Massimo Moratti has been the head cannot take a final position on this aspect yet. The analysis of the of the team since 1995 and, although often criticized by its supportaforementioned teams’ results over the past five years in terms of ers, has given tremendous financial support to the team. During his points achieved and winning game percentage will provide a useful 18-year tenure, the club has registered losses for € 1.3 billion, with quantitative measure which will show how the Financial Fair Play Moratti personally spending more than € 750 million. Inter player trading market during Moratti’s era has often been characterized by Regulations have affected football clubs’ competitiveness. huge expenditures on the purchasing side, with little returns from sales. The situation has drastically changed since 2009, as the Graph Influence of Financial Regulation on Clubs’ Expenditures Since the UEFA Financial Regulations were established in 2009, 1 illustrates.

As observed in the data, during this 20 year span, the return the owners of the major European clubs have been dealing with the threat of upcoming enforcement of penalties for highly indebted from sales exceeded the expenditures in purchases only 4 seasons: teams. Such penalties will be awarded starting in the 2013-14 season, 2004/05, 2009/10, 2010/11 and 2011/12. Three out of four occurred so as of right now it is not possible to directly evaluate the enforce- after the establishment of the financial regulations. ment power of the European football’s governing body. Nonetheless, a change in the managing behaviors of the prestigious clubs indi- F.C. Barcelona - Spain The Futbol Club Barcelona, familiarly known as Barcelona, rectly proves the UEFA’s ability to have its rules respected. A drastic drop in expenditures exceeding returns would prove that the major takes the name from the capital of the Spanish autonomous region of teams consider the UEFA’s jurisdiction in penalizing teams accord- Catalonia. It is one of the most successful teams in the world, which ing to the Fair Play Regulations. We will look at the returns and costs has earned many national and international trophies during its hisF.C. Inter (Italy), F.C. Barcelona (Spain) and Manchester United F.C. tory. Founded in 1899, it has an historical rivalry with the other top (England) have incurred during their trading player sessions in each Spanish team, Real Madrid. Unlike other major clubs on the contiof the last 18 seasons, covering the period from 1995 to 2013. Al- nent, Barcelona is owned by the supporters, who acquire memberthough these costs and returns do not represent the overall finan- ships (not shares like normal limited companies) and operate the cial situation of the clubs, for one must also consider broadcasting, team through the election of an assembly of delegates (the governing commercial and ticketing incomes, they are good indicators of the body) and of a President. This doesn’t negatively affect its revenues effectiveness of the establishment of the UEFA regulations. If these though. According to Deloitte, in the 2011/12 season, Barcelona’s three case studies highlight a substantial change in the clubs’ spend- revenue amounted to an astonishing € 483 million, the second highing behaviors, this will prove the consideration that the members of est in the world after Real Madrid (the other top club managed in the UEFA have for the regime’s rules and, indirectly, its enforcement an analogous way). Nonetheless, the team’s accumulated net debt in 2012 was at €364 million, with a loss over the season of €21 million capability. (which still represents a great improvement from the €83 million lost

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humanities & social sciences the previous year). In fact, as graph 2 illustrates, Barcelona, regardless of the many successes enjoyed by its stellar squad, has been dealing with expenditures constantly exceeding revenues.

In the last 18 seasons, Barcelona revenues from sales on the players’ transfer market exceeded the expenditures only 4 times: 1995-96, 1999-00, 2002-03 and 2005-06. Since the introduction of the UEFA Financial Regulations in 2009, the club has spent €245,080,000 on new players, while the revenue from sales amounted to €109,296,000, with a net difference of €135,784,000.

Manchester United F.C. - England Manchester United Football Club is a professional soccer team based in Old Trafford, a district of Manchester. Founded in 1878, it is currently the team who has won more Premier Leagues (19) on top of many international victories including the 2007-08 UEFA Champions League. Forbes considers Manchester United to be the most valuable sports team in the world, with an estimated enterprise value (equity plus debt) of €3.3 billion dollars. Manchester United is also considered the most popular team in the world, with more than 200 officially recognized fan clubs located in 24 countries. The total number of supporters is estimated at 333 million. The current manager of the team is Sir Alex Ferguson, who has been in charge since 1986. Under his tenure, the club has enjoyed its most successful era, with 24 major trophies achieved. In 2005, the Glazer family acquired 98% of the team’s share for £800 million (approximately $1.5 billion). The majority of the cash to complete the acquisition came from loans, mostly covered with Manchester United’s assets, de facto creating huge debts in the club’s book accounts. The UEFA’s Financial Regulations are now requiring the team to re-enter its means, and this has had a concrete impact on the club’s recent player transfers policies (graph 3).

Manchester United’s expenditures on new players have exceeded the returns from sales 15 times out of the last 18 seasons. The exceptions are 1995/96, 2003/04 and 2009/10, the season after the approval of the UEFA Financial Regulations. This latter season saw an incredibly high return of £91.299.200, mainly from the £82.720.000 sale of Cristiano Ronaldo to Real Madrid, the highest price ever paid for a single player in the history of football. The following seasons however Manchester constantly spent more on new players than what it

Table 2. Arsenal and Stoke City respective Net Debt from 2007-2012

Table 3. Arsenal and Stoke City Winning Percentage and Points Achieved 2008-2012

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hurj hurjspring spring2012: 2014:issue issue14 18 gained from sales. Considering the period after the establishment of the Financial Fair Play, the expenditures exceeded the purchases by £32.445.600.

Influence of Financial Regulations on Club’s Competitiveness The critics of the introduction of the UEFA Financial Regulations have raised concerns regarding the negative impact they may have on the top clubs’ performances. In order to verify to what extent these claims may hold true, this section of the paper will compare the performances of 5 different teams in the period 2007-12 in terms of the percentage of games won and points achieved over the span of five seasons. The choice involves two teams per each of the three main leagues of the continent; one which has undertaken sustainable management strategies even before the introduction of the new rules and one which instead has constrained its expenditures in order to reenter the UEFA parameters. In order to confirm Clinton’s claims, we should expect to see a reduction in the gap between the small, wellmanaged clubs, and the big, highly indebted ones.

English Premier League – Stoke City vs. Arsenal Stoke City Football Club is an English professional team based in Stoke-on-Trent, Staffordshire. Although it is the second oldest professional club in the world, its recent performances have excluded it from the soccer elite. The club was able to join the Premier League only in 2008, after 23 years of absence from the top league of English football. Currently coached by Toni Pulis, it is one of the few Premier League clubs which was already respecting the UEFA’s parameters in 2009 and which has kept since then its accounts in good orders.

Arsenal Football Club is one of the most successful and renowned team in the Premier League. Founded in 1866, it is based in the district of Holloway, London. It has won the Premier League 13 times during its history and it is always considered one of the top contenders for national and international trophies. The current coach Arséne Wenger has been in charge since 1996. Famous for his scouting talent and appreciation for young players, usually acquired for lower prices before they reveal themselves as worldclass footballers, Wenger has still looked upon a huge soar in Arsenal’s debts in recent years

(Table 2). These data highlight Arsenal’s effort to reduce its net debt in order to achieve the financial sustainability required by the UEFA. Arsenal has been able to constrain its liabilities in part through the selling of top players like Cesc Fabregas, Robin Van Persie and Samir Nasri. In order to see if the existing gap between these two teams has been reduced since the introduction of the Financial Rules, we must consider the Premier League’s game won/

game played ratio and points achieved along

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the period 2008-2012. We are going to omit the season 2007-08 because Stoke City was playing in the second division (Table 3).

The data do not highlight a significant reduction in the gap between Arsenal and Stoke City.

Arsenal has achieved an average 71.25 points per season, with a standard deviation of 2.58. Stoke City’s mean is 45.75 points per

season, with a standard deviation Table 4. A.C. Milan and S.S. Lazio Profit Losses 2007-2012 of 0.83. Both teams have kept constant results along these seasons and no significant closing up between top and low-middle clubs could be observed.

Italian Serie A – Lazio vs. Milan In 2011 the Italian Serie A’s clubs total debts amassed to a total of € 2.6 billion. S.S. Lazio and A.C. Milan are valid representatives of two very different management strategies. SS. Lazio is one of the three profes sional football clubs located in Table 5. A.C. Milan and S.S. Lazio Winning Percentage and Points Achieved 2007-2012 Rome. Founded in 1900, during the 1990s it was considered one of the Seven Sisters of the Italian UEFA’s requirements (Table 4). league when it achieved many national and international trophies.

The analysis will compare these two team’s performances over The former President and owner Sergio Cragnotti created one of the the 2007-12 period. As shown by the data, S.S. Lazio has been rungreatest teams in Europe, acquiring some of the best international ning constant surpluses in recent years even before the UEFA’s new players in the market such as Siniša Mihajlović, Marcelo Salas and requirements; A.C. Milan is still struggling to enter the parameters Pavel Nedvěd. Due to a financial scandal which involved Cragnotti’s and has been forced to sell some of its best footballers (Table 5). food company Cirio, the club suddenly ran out of money. Between Rather than a reduction in the gap between Milan and Lazio, the 2002 and 2004 it was administered by a bank pool, until a new en- data highlight a similar pattern of improvement for the two teams. trepreneur Claudio Lotito relieved the club’s majority. Since then, the The difference between the two at the end of the 2007-08 season was club has kept a tight control over its finances, in order to avoid exces- of 18 points. In 2012 the gap remained constant, although they both sive debt accumulation. The result is that in the last 5 years the club registered a significant improvement of 16 points over the span of 5 has been always able to achieve profits, with a total surplus over the years. The great variability in the results is confirmed by S.S. Lazio’s period 2007-12 of €18,463,667. (σ = 8.39 with a 54 points mean) and A.C. Milan’s (σ = 6.57 with a A.C. Milan has been following diametrically opposite manage- 74 points mean) standard deviations. It is interesting to notice how rial policies under the current owner Silvio Berlusconi. Founded in A.C. Milan’s performances improved after introduction of the UEFA 1899, it has been in the media tycoon’s hands since 1986, when the regulations, achieving the best results during the last two seasons. club was under risk of bankruptcy. Berlusconi invested amounts of money never seen before in recruiting the best players on the globe, Spanish Liga – Granada vs. Valencia bringing A.C. Milan to unprecedented heights. During these 27 La Liga is widely considered the most spectacular football years, A.C. Milan has won 8 Italian Serie A, 5 Champions League league in the world. Regardless of the quality of the games and of and many other minor national and international trophies. Berlus- the huge amount of broadcasting and sponsorship money poured on coni revolutionized the global trading market for players, due to the the major teams however, the total debt contracted by the clubs has huge amounts of money he invested in the team. Nonetheless, A.C. reached the astonishing amount of €3.53 billion. The analysis will Milan has been constrained by the UEFA regulations to cut on ex- compare Granada C.F., representative of the smaller, well-managed penditures. Last summer, for example, the team’s arguably two top teams, and Valencia C.F., a bigger, highly indebted team struggling to players, Thiago Silva and Zlatan Ibrahimovic went to Paris St. Ger- meet the UEFA’s requirements. main for £55,440,000. Regardless of these sales, the club still faced Granada Club de Fútbol was founded in 1931; a small club hard times in achieving a profit, in part due to the high salaries of with few ambitions, it lived its golden era during the 1970s, when the players. Regardless of the efforts undertaken, A.C. Milan is still it reached the highest placement in its history in la Liga (6th). The running huge losses every year, which Berlusconi is covering with club found itself on the brink of bankruptcy in 2009; Gino Pozzo, his personal wealth. Nonetheless the team has started a new lower the owner of the Italian club Udinese Calcio, saw an opportunity for salary policy which should eventually lead to the achievement of the profit. A partnership was signed between the two clubs, allowing

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Table 6. Valencia C.F. Winning Percentage and Points Achieved 2007-2012

Udinese’s youngsters to practice and play on loans for the Andalusian team. The club has qualified for the second division’s playoffs and was eventually promoted to la Liga for the first time in 35 years in 2011. This was accomplished through an unusual financial transparency which has made Granada one of the few solvent clubs in the nation, with a 2012 deficit of mere € 200,000.

Valencia Club de Futból is an historical Spanish powerhouse.

Founded in 1919, it is recognized as the third great Spanish team after Barcelona and Real Madrid. Valencia has triumphed in la Liga six times and has seen a particularly prominent dominance in the early 2000s, when the team reached the Champions League final for two years in a row (2000-01). Due to mismanagement and overspending the club’s debts reached the level of €547 million in 2009. After the UEFA’s regulations were introduced, Valencia found itself constrained to sell its best players in order to cover part of the amount owned to creditors. In the summer of 2010, David Villa (to Barcelona) and David Silva (to Manchester City) were sold for a total £60,500,000.

The following year, the captain Juan Mata followed the same path, moving to Chelsea for £23.5 million. A comparison between these two teams is not possible. The lack of available data doesn’t allow us to look into the debts contracted over the years by these clubs and the absence of Granada from the Liga until 2011 makes the competiveness confrontation unfeasible. Nonetheless, in order to analyze the effect of the UEFA’s requirements on competitiveness, the only statistics we really need are the ones regarding Valencia’s performances in the period 2007-2012. The forced sales of the team’s top players are a consequence of the UEFA’s decisions. Observing Valencia’s trend during these crucial years will show if its competitiveness has been affected by the financial regulations (Table 6).

Looking at the data we can conclude once again that the introduction of the UEFA Financial Regulations has not, in the short run, significantly affected the performances of this highly indebted team.

Valencia has achieved on average 62.3 points per season, with a standard deviation of 7.43. The club achieved its best results during the 2009-10 and 2010-11 seasons, right after the new rules’ introduction.

Although the data highlight a drop in 2011-12, insufficient evidences are provided to conclude that the regime’s decisions have undermined Valencia’s competitiveness in a significant way.

Conclusion and Evaluation The main purpose of this paper was to evaluate to what ex-

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hurj 2012: issueissue 15 18 hurjfall spring 2014: tent the UEFA is able to enforce its own rules and to highlight the impact the Fair Play Financial Regulation have had so far on the clubs’ competitiveness.

Since these newly introduced regulations will not come into full effect until the 2013-14 season, a direct analysis of the UEFA’s ability to assign the prescribed punishment to the delinquent clubs was not possible. Instead, this research adopted an indirect ap proach, focusing on variations in the clubs’ expenditures in the player trading market since the establishment of these regulations in 2009. Inter F.C.’s behavior and Manchester United’s £82.720.000 sale of Cristiano Ronaldo testify how clubs are taking UEFA’s regulations seriously, which suggests that they recognize it as a private international regime. Inter has run surpluses 3 times in the last 4 seasons, while in the previous 14 years, sales exceeded purchases only one other time. Manchester United sold one of the best footballers in order to reduce its debts and reenter the UEFA’s parameters. The only club who seems to have so far ignored the upcoming requirements is F.C. Barcelona. F.C. Barcelona’s purchases in the last 4 seasons have always exceeded the returns from sales, with an overall loss of €135,784,000. Based on these data, claiming that the clubs fully recognize the UEFA as a legitimate private regime could not be made. Out of the 12 post-regulation seasons (4 per team) considered by this analysis, sales exceeded purchases only 4 times. This 33% is higher compared to the previous 42 seasons (14 per team), when sales exceeded expenditures only 8 times (19%).

Regardless of this positive trend, however, not enough evidence has emerged from the data to conclude in an absolute way that the UEFA’s enforcement power is fully recognized by the clubs. There are, however, some important observations to be made.

Clubs’ expenditures on the player trading market are not a comprehensive measure of the financial situation of the club. One must also consider salaries, merchandising and sponsorship returns, sales of broadcasting rights and many other factors that influence the teams’ balance accounts. The author of this paper has chosen to consider the player trading market because it is the most relevant financial components to the actual game of football and because of the greater availability of data than any other components of the clubs’ finances. Acquiring the best players obviously increases a team’s competitiveness, pleases the supporters and ensures high returns on merchandise. For these reasons, many clubs’ owners have lost sight of their means in the past and have spent incredibly high sums in this market, nearly bankrupting their own teams. The UEFA’s aim is to reduce the occurrence of such events. The reason why clubs like

Table 7. Barcelona F.C., Manchester United F.C. and F.C. Inter Main Sources of Revenues

hurjspring spring2012: 2014:issue issue14 18 hurj Graph 4

Inter F.C. changed their behavior since the Financial Regulations have been established, while F.C. Barcelona has kept its yearly expenditures on purchases constantly higher than sales’ revenues, lies in the other factors contributing to the overall balance sheets. Deloitte claims that in the 2011-12 season Barcelona accumulated revenues of €483 million, against Manchester United’s €395.5 and Inter’s “mere” €185.9 million. These big discrepancies are mainly due to the greater shares of merchandise and ticketing returns that Barcelona and Manchester United enjoy (Table 7).

Barcelona and, to a lesser extent, Manchester United can afford to have expenditures on new players’ purchases exceeding sales because their higher revenues allow them to cover the net losses. Furthermore, F.C. Inter, unlike the other two clubs, receives its greatest share of revenues from selling broadcasting rights; this factor is less volatile than Match Day or Commercial Revenues, because it is mostly based on the numbers of supporters following the respective teams. Inter has always received the core of its revenues from broadcasting and can therefore afford to reduce its spending on new players. Barcelona and Manchester United instead fuel their balance sheet with Match Day (tickets) and Commercial Revenues (merchandise), which are highly dependent on the quality of the team and on the popularity of the players acquired. Relying on higher revenues and being dependent on yearly sensational acquisitions of new footballers to keep those revenues high explain F.C. Barcelona’s behavior, apparently indifferent to the UEFA’s regulations. I therefore believe the UEFA will be able to enforce its rules, but the constraints on new players’ expenditures will be more significant on clubs relying on TV revenues (like Inter and most of the Italian clubs) than on those, like Barcelona and Manchester United, who are almost dependent on acquiring new footballers to keep their wealth constant. The results in the long run will be a transfer of the best players from leagues with lower attendance (like the Italian Serie) to leagues with higher attendance (la Liga , the Premier League , the German Bundesliga ).

This will create a virtuous circle for the Spanish, British and German clubs, able to raise the revenues and acquire top footballers while at the same time respecting the UEFA’s parameters (Graph 4).

Although the UEFA has no power over directly influencing the

humanities & social sciences way in which clubs raise their revenues, if the trend predicted by the virtuous circle is confirmed, Italian clubs’ owners will eventually be required to undertake supporter-friendly policies. Of the three main sources of income, Match Day Revenues have a positive impact both on the team’s finances and performances (positive effect of a full home crowd). The most of the English, German and Spanish clubs (including F.C. Barcelona and Manchester United) already own their stadiums, which are extremely welcoming environments for families and ensure high attendance to every home game. In Italy, instead, the majority of the stadiums are owned by the cities, which then rent them to the clubs. Inter for example pays €8 million per year to the municipality of Milan in order to play at the San Siro stadium. Most importantly, this interaction between public and private actors has resulted in an inefficient maintenance of the facilities, leading to cold and uncomfortable stadiums with low attendance and frequent episodes of violence. The UEFA’s requirement will force Italian clubs to invest in the improvement of their home fields in order to stay competitive, leading to the creation of “modern, safe and comfortable infrastructures (stadiums) for the supporters,” one of Platini’s main goals.

The second major puzzle this paper has tried to solve is analyzing the effect the UEFA’s Financial Regulations has had on team’s competitiveness.

Clinton R. Long claimed that the introduction of

these constraints will reduce the top clubs’ ability to stay competitive.

In order to see if that’s the case, the paper has compared the results of clubs out of the UEFA’s parameters in 2009 (A.C. Milan, Arsenal F.C. and Valencia C.F.) with more sustainably run teams (SS. Lazio, Stoke City F.C. and Granada C.F.). Based on the data, no significant reduction in the gap between these two groups has been observed.

The top, highly indebted teams were forced to sell some key players, but their competitiveness has not been reduced. In England both Arsenal and Stoke City registered minimum variations across the 4 seasons considered in terms of points achieved. Greater variability is observed in Italy. Notably, though, the highly indebted A.C. Milan’s best seasons occurred in 2010-11 and 2011-12, after the introduction of the financial regulations. The same trend is found in Spain, where Valencia, regardless of the forced sales of David Villa and David Silva in the summer 2010, has had its best season in 2010-11. Thus, there is no quantitative support for Clinton’s claim; the UEFA’s Financial Regulations have not had so far a significant negative impact on top clubs’ competitiveness. This study does not, however, exclude a future stronger effect of these rules on competitiveness. Clubs constrained to reduce their debts will eventually lose their power to acquire top players through the borrowing of more money. The paper’s suggestion to football clubs’ owners is to invest in young players’ development and, most importantly, to attract more supporters at the stadium. These two strategies will start the aforementioned virtuous circle, increasing, in the long run, the teams’ competitiveness in an economically sound way.

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18. Kennedy, Peter, The Football Industry and the Capitalist Political Economy: A Square Peg in a Round Hole?, Published on Critique: Journal of Socialist Theory, March 2nd, 2012.

19. The Swiss Ramble, Money’s Too Tight To Mention At Inter, Published on December 1st, 2011 on “The Swiss Ramble Blog Spot”. Accessed on 03-27-13 at: http://swissramble.blogspot.com/2011/12/ moneys-too-tight-to-mention-at-inter.html 20. Transfermarkt.com.uk, website used as a source of data for graph 2/3. For Inter’s transfers, I accessed on 04-25-13 the following link: http://www.transfermarkt.co.uk/en/inter-milan/transfers/verein_46_2012_default_default_alle_a_default.html . For Barcelona’s transfers, I accessed on 03-27-13 the following link: http://www.transfermarkt.co.uk/en/fc-barcelona/transfers/verein_131_1995_default_default_alle_a.html . For Manchester United’s transfers, I accessed on 03-29-13 the following link: http:// www.transfermarkt.co.uk/en/manchester-united/transfers/verein_985_2012_default_default_alle_a. html 21. Hamila, Sean and Waltersa, Geoff and Watsonb, Lee, The model of governance at FC Barcelona: balancing member democracy, commercial strategy, corporate social responsibility and sporting performance, Published on Soccer & Society, Volume 11, No. 4, July 2010, pages 475–504. Accessed on 03-27-13 at: http://ehis.ebscohost.com/ehost/pdfviewer/pdfviewer?sid=70f1f72d-a09b-49b4-9fea-0a33 686c9b40%40sessionmgr10&vid=2&hid=5 22. Deloitte, Captains of Industry Football Money League, Sport Business Group, January 2013. Accessed on 03-27-13 at: http://www.deloitte.com/assets/Dcom-UnitedKingdom/Local%20Assets/Documents/ Industries/Sports%20Business%20Group/uk-sbg-football-money-league-2013.pdf 23. ESPNsoccernet staff, Barca announce €45m budget, Published online at espn.com on June 9th, 2011. Accessed on 03-27-13 at: http://espnfc.com/news/story/_/id/926957/barcelona-announce%E2%82%AC45m-transfer-budget?cc=5901 24. Ozanian, Mike, Manchester United Becomes First Team Valued At $3 Billion, Published online at Forbes.com on 01-27-13. Accessed on 03-27-13 at: http://www.forbes.com/sites/mikeozanian/2013/01/27/manchester-united-becomes-first-team-valued-at-3-billion/ 25. Manchester United, Fanzone – For the Fans by the Fans, Subsection of the official website of Manchester United. Accessed on 03-27-13 at: http://www.manutd.com/en/Fanzone/Local-SupportersClubs.aspx 26. Cass, Bob, United moving down south as fanbase reaches 333 million, Published online at dailmail. co.uk on December 15th, 2007. Accessed on 03-27-13 at: http://www.dailymail.co.uk/sport/football/article-502574/United-moving-south-fanbase-reaches-333-million.html 27. BBC News, Glazer gets 98% of Man Utd shares, Published online at bbc.co.uk on June 28th, 2005.

Accessed on 03-29-13 at: http://news.bbc.co.uk/2/hi/business/4629401.stm .

28. Andersred Blog, Trying to make sense of the Glazers, debt and football finance, Published online on January 10th, 2010 at andersred.blogspot.com. Accessed on 03-29-13 at: http://andersred.blogspot. com/2010/01/half-billion-ii.html 29. Arsenal Football Club Holdings PLC, Statement of Accounts and Annual Reports 2009/10, Accessed on 03-29-13 at: http://www.arsenal.com/assets/_files/documents/oct_11/gun__1318505919_Arsenal_ Holdings_plc_Annual_Re.pdf 30. Arsenal Football Club Holdings PLC, Statement of Accounts and Annual Reports 2010/11, Accessed

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hurj spring 2014: issue 18 on 03-29-13 at: http://www.arsenal.com/assets/_files/documents/oct_11/gun__1318409853_Arsenal_ Holdings_plc_-_Annual_.pdf 31. Arsenal Football Club Holdings PLC, Statement of Accounts and Annual Reports 2011/12, Accessed on 03-29-13 at: http://www.arsenal.com/assets/_files/documents/oct_12/gun__1349166617_ARSENAL_HOLDINGS_plc_ANNUAL_RE.pdf 32. Barclays Premier League Official Website, Under Teams Profile – Statistics. Arsenal’s results taken from the following link accessed on 03-29-13: http://www.premierleague.com/en-gb/clubs/profile.statistics.html/arsenal . Stoke City’s results taken from the following link accessed on 03-29-13: http://www. premierleague.com/en-gb/clubs/profile.statistics.html/stoke .

33. Digby, Adamo, With Seven Sisters’ Return, Guys in Serie A Starting To Regain Respect, Published online at bleacherreport.com on August 5th, 2009. Accessed on 03-29-13 at: http://bleacherreport.com/ articles/230766-the-return-of-a-different-seven-sisters .

34. A.C. Milan, Relazioni e Bilancio al 31 Dicembre 2008 (translate to: Relations and Account Balance at December 31st, 2008). Accessed on 02-29-13 at: http://www.acmilan.com/uploads/bilancio/BilancioGruppoMilan_ACM_12-08.pdf 35. A.C. Milan, Relazioni e Bilancio al 31 Dicembre 2009 (translate to: Relations and Account Balance at December 31st, 2009). Accessed on 02-29-13 at: http://www.acmilan.com/uploads/bilancio/BilancioGruppoMilan_ACM_12-09.pdf 36. A.C. Milan, Relazioni e Bilancio al 31 Dicembre 2010 (translate to: Relations and Account Balance at December 31st, 2010). Accessed on 02-28-13 at: http://www.acmilan.com/uploads/club/bilancio2010/ pdf/Bilancio_Gruppo_Milan_10.pdf 37. A.C. Milan, Relazioni e Bilancio al 31 Dicembre 2011 (translate to: Relations and Account Balance at December 31st, 2011). Accessed on 02-29-13 at: http://www.acmilan.com/uploads/club/bilancio2011/ 38. S.S. Lazio S.P.A., Bilancio al 30 Giugno 2008 (translate to account balance at June 30th, 2008). Accessed on 03-29-13 at: http://syndication.teleborsa.it/Kataweb/documenti.aspx?ndg=225&titolo=S.S.+Lazio 39. S.S. Lazio S.P.A., Bilancio al 30 Giugno 2009 (translate to account balance at June 30th, 2009). Accessed on 03-29-13 at: http://syndication.teleborsa.it/Kataweb/documenti.aspx?ndg=225&titolo=S.S.+Lazio 40. S.S. Lazio S.P.A., Bilancio al 30 Giugno 2010 (translate to account balance at June 30th, 2010). Accessed on 03-29-13 at: http://syndication.teleborsa.it/Kataweb/documenti.aspx?ndg=225&titolo=S.S.+Lazio 41. S.S. Lazio S.P.A., Bilancio al 30 Giugno 2011 (translate to account balance at June 30th, 2011). Accessed on 02-28-13 at: http://www.sslazio.it/images/stories/documenti/pdf/investor_relator/Bilancio%20S.S.%20Lazio%2030-06-2011.pdf 42. S.S. Lazio S.P.A., Bilancio al 30 Giugno 2012 (translate to account balance at June 30th, 2012). Accessed on 03-29-13 at: http://syndication.teleborsa.it/Kataweb/documenti.aspx?ndg=225&titolo=S.S.+Lazio 43. Bernard, Ricardo, AC Milan in the Age of Financial Fair Play, Published online at grantland.com on September 18th, 2012. Accessed on 03-29-13 at: http://www.grantland.com/blog/the-triangle/post/_/ id/37305/ac-milan-in-the-age-of-financial-fair-play 44. Lega Serie A Italiana, Official Website. Statistics for S.S. Lazio found under Club History – S.S. Lazio section. Accessed on 03-29-13 at: http://www.legaseriea.it/en/serie-a-tim/squadre/storia-del-club/-/ squadre/Lazio/storia/1719 . Statistics for A.C. Milan found under Club History – A.C. Milan section.

Accessed on 03-29-13 at: http://www.legaseriea.it/en/serie-a-tim/squadre/storia-del-club/-/squadre/ Milan/storia/1716 45. Rogers, Martin, Ranking the world’s best soccer leagues, Published online at yahoo.sports.co on September 19th, 2007. Accessed on 03-29-13 at: http://sports.yahoo.com/soccer/news?slug=ro-topleagues091907 46. Gibson, Owen, La Liga debts reach £3bn to leave Spanish game in crisis, Published online at theguardian.co.uk on May 19th, 2010. Accessed on 03-29-13 at: http://www.guardian.co.uk/football/2010/ may/19/la-liga-debt-passes-three-billion 47. Beard, Eric, How Udinese beat the system and stands at the gates of La Liga, Published online at afootballreport.com. Accessed on 03-29-13 at: http://afootballreport.com/post/6514153638/how-udinese-beat-the-system-and-stands-at-the-gates-of 48. Acedo, Diego, Granada, en Primera 35 años después, Published on marca.com on June 19th, 2011.

Accessed on 03-29-13 at: http://www.marca.com/marcador/futbol/2010_11/promocion_ascenso/final/ vuelta/elc_gda/indexCronica.html 49. Chesters, Heath, Healthy finances and transparency at Granada CF, Published on Insidespanishfootball.com on January 17th 2013, Accessed on 02-28-13 at: http://www.insidespanishfootball.com/ healthy-finances-and-transparency-at-granada-cf/ 50. Lowe, Sid, Valencia’s brilliant bluff pays off as they close in on a winning hand, Published on May 3rd, 2010 at: theguardian.co.uk. Accessed on 02-28-13 at: http://www.guardian.co.uk/football/blog/2010/ may/03/valencia-brilliant-bluff-pays-off 51. ESPN, Italian Serie A Stats 2011-12, Published online at espnfc.com under the section Europe. Accessed on 04-17-13 at: http://espnfc.com/stats/_/league/ita.1/year/2011/italian-serie-a?cc=5901 52. Soccerstats.com, La Liga, Published online at socerstats.com under the section Spain, 2012-13. Accessed on 04-17-13 at: http://www.soccerstats.com/attendance.asp?league=spain 53. ESPN, Barclays Premier League Stats: Team Attendance - 2012-13, Published online at espnfc.com under the section English Premier League. Accessed on 04-17-13 at: http://espnfc.com/stats/attendance/_/league/eng.1/barclays-premier-league?cc=5901 54. Deutscher Fussball-Bund, Number of Spectators, Published online at dfb.de under the section Bundesliga. Accessed on 04-17-13 at: http://www.dfb.de/index.php?id=380609 55. Lago-Peñas, Carlos & Lago-Ballesteros, Joaquin, Game location and team quality effects on performance profiles in professional soccer, Published on Journal of Sports Science & Medicine, September 2011, Volume 10, Issue 3, p. 465-471. Accessed on 04-28-13 at: http://ehis.ebscohost.com/ehost/pdfviewer/pdfviewer?sid=ebac346d-89b7-40bd-a4bb-fa76b47c222d%40sessionmgr10&vid=2&hid=101 .

56. Santucci, Gianni, Affitto di San Siro, per Inter e Milan: meno contanti e più interventi (Translated to: San Siro’s rent for F.C. Inter and A.C. Milan: less money and more intervention), Published on Il Corriere della Sera, on September 2nd, 2010. Accessed on 04-28-13 at: http://archiviostorico.corriere.it/2010/ settembre/02/Affitto_San_Siro_per_Inter_co_7_100902021.shtml .

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The Importance of Education and CommunityBuilding to Prevent Mental, Emotional, and Behavioral Problems in Refugee Youth Rebecca Rimsky, Class of 2016 Public Health Studies In numerous countries around the world, refugee children affected by trauma, war and conflict often experience mental, emotional, and behavioral issues. According to a 2009 report, 42 million people around the world are refugees or displaced persons, with 45% of refugees under 18 years old.1 A study published in The Lancet, a UK journal, stated that “[refugee youth] are at high risk of mental-health problems because they are likely to have been exposed to violence, which is the strongest predictor of poor mental-health outcomes”.2 These statistics indicate that failure to address the mental health of refugee children is a significant public health issue because it can lead to the detriment of such a large group of people worldwide.

I worked as a peer counselor at the International Rescue Committee’s Refugee Youth Summer Academy. The academy is a six-week program in which refugee youth are taught primary subjects, such as English literacy, math, and science, as well as valuable social skills and creative arts. Each week of the program is dedicated to certain learning and school readiness objectives. These include adult support networks, understanding school culture, self-confidence in an academic setting, attitude towards schools, and motivation to achieve in school. The academy sets out to achieve these goals in order to

prevent any mental, emotional, or behavioral issues in the students, who have all faced various traumas, such as political unrest or family deaths. There are also community groups led by guidance counselors that are aimed to illustrate healthy ways to express the fear, sadness, and depression that many refugee youth feel.

The World Health Organization explains: traumatic experiences such as killings, material losses, torture, and sexual violence, harsh detention and uprooting, all affect people’s behavior for generations. Life in overcrowded camps, deprivations, uncertainty over the future, disruption of community and social support networks lead to psychosocial dysfunctioning.3 During training for my experience, the academy provided common background stories for refugee youth, so that the counselors could get a sense of some of the experiences that the incoming students had faced. We were informed that regularly, a parent dies in conflict or each parent must move separately to a country such as America in order to receive a steady income. After several years of separation, with the children living in a refugee camp, the children are given two weeks’ notice and are then uprooted to live with the parent who had left prior and is then able to provide a home for the family. Years of separation can create tension because the children are meeting their respective parents after such a long period of time. If internalized, this tension can result in emotional or mental issues. Mental issues can be difficult

Courtesy of Sailesh Naidu/TheIRC via rescue.org

The students of the six-week 2013 International Rescue Committee’s Refugee Youth Summer Academy visit the Metropolitan Museum in Manhattan.

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to diagnose in refugee youth, but it is estimated that the prevalence of posttraumatic stress symptomatology varies from 10 to 90%, manifested by anxiety disorders such as posttraumatic stress disorder (PTSD) and other psychiatric morbidities.4 Aside from tension stemming from reunification with a parent, other sources of anxiety for refugees can be language barriers and social expectations of the host country, such as hygiene. Hygiene was strongly encouraged at the academy, as the children had come from drastically different backgrounds and cultures.

Without the commonality of a language, children can feel aloof from a community, and miss the opportunity to make friends and have a support system. Many children had not been taught to wash their hands after using the bathroom, and several had not learned to ask to use the bathroom at all, and instead had daily accidents. These issues can be sources of humiliation for refugee youth, and if not dealt with, may lead to self-isolation from groups.

I was surprised at the various ways that students expressed themselves as a result of their experiences. I noticed that the children usually fit into one of three groups; the first group of children refused to participate in any activities or attempt to develop their English skills, the children in the second group were happy, playful, and acclimating seamlessly into their new lives, and the children in the third group expressed themselves through violence and disrespect towards counselors and fellow students. All counselors were assigned as buddies to a couple of students, and were expected to provide more individualized support to their buddies. My buddies were two sisters from Guinea, one of whom fit into the first group and the other fit into the third. The former required constant encouragement in order to participate in activities, but during the six weeks, gained confidence and became a vital part of the classroom community. She worked hard to further improve her weak English skills, meticulously writing and rewriting words on worksheets. She realized that the ability to communicate with others in the classroom

Photos courtesy of Autumn Potter/IRC via thirteen.org

Students at the 2012 Refugee Youth Summer Academy in New York 20

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was practical in terms of making new friends, rather than only speaking to children who also spoke French and Fulani. I often witnessed the latter victimizing other children and asserting herself as superior. I recognized the need for individualized attention for refugee youth because this young girl was quickly developing an unhealthy behavioral problem as a coping mechanism to the trauma she faced, which could hinder later efforts in making friends or establishing a supportive community. I spent a lot of time helping her channel her energy and tendencies into healthier outlets. She learned to assist the teacher in handing out work or projects to students, which allowed her to feel active in the classroom in a rewarding way. The quiet children from the first group were challenging, because while they may simply need time to adjust to their new life in America, it is also essential for them to participate in activities and begin learning English so they can succeed in school and have the opportunity to thrive in their new country of residence. Education and the stability of school was an imperative solution for all three groupings of children, even though their potential issues were drastically different.

A question that often arose during my experience was how to integrate cultural and social tendencies from a refugee child’s native country into expectancies of their new country. This issue became apparent when dealing with reward and punishment for right and wrongful behavior. At the academy, the counselors made a big effort to celebrate small triumphs, which could be as simple as raising one’s hand in order to ask a question. Punishments for wrongful behavior often included talking privately with a teacher or the principal, a time-out, or asking the child to instead demonstrate respectful behavior. Corporal punishment, or physical punishment, is accepted in almost all of the native countries of the students, creating a difficult barrier for refugee families once they relocate to new countries.

One goal of the academy is to introduce the refugee youth to nonviolent reactions and punishments. The parents of the students frequently contradict this method and use violence in their homes because violence is a legal and socially acceptable aspect of their cultures.

Several of the students I worked with informed me of small arguments with their parents that had led to a hitting or spanking. One student

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told me that both of her parents hit her numerous times a week as in symptoms of depression and PTSD for the top tier of participants”.7 a form of behavioral conditioning, but that their actions resulted in Due to conflict in native countries, refugee youth have either little bruises and lasting pain. I immediately told my supervisor, who set up to no education or disrupted education. For this reason, providing a a meeting with her parents because the parents of refugee youth usu- stable education environment allows for successful learning, and can ally do not realize that violent punishment is illegal and that they can improve the mental health of the youth. be reported to child protective services. While most of the children In recent years, there has been a debate over the importance of are accustomed to painful punishments, they are quickly taught not education for refugees. Education has not been considered a sector for to tolerate it in their new countries, and an outcome of this conflic- humanitarian assistance, and therefore organizations have not allocated tion between native culture and new culture can cause emotional is- funding in emergency situations. One humanitarian donor declared sues in the refugee youth. In 2005, Fatima, a 16-year-old female Iraqi that her agency did not “fund education because it’s not relief. It’s develrefugee, showed signs of abuse when she arrived at school. The school opmental activity”.8 However, positive results from studies have offered faculty called child protective services and Fatima was removed from reasons to oppose this view, and agencies such as CARE, the Internaher house, with a court order for no further contact with her parents. tional Rescue Committee, and Save the Children, have included eduFatima had serious adjustment problems in foster care, became severe- cation programs for children.9 An Australian study done on the topic ly depressed, and went so far as to attempt suicide.5 In this situation, states “research suggests that one of the major challenges for recentlymisunderstandings between cultures led to both mental and behavior- arrived young people is to identify with a community to which they can al issues for a refugee youth. Without a safely belong. For many young people, community, Fatima felt isolated in foster schools provide a primary place of beWithout the commonality of a care and did not have someone to turn longing”.10 Studies should continue to be to for help. Community building can be conducted to spread the significance of language, children can feel aloof a lifeline to refugee youth who are in a education and community building for from a community, and miss the new country and have not yet learned refugee youth. opportunity to make friends and the language or cultural expectancies.

The academy allowed me to gain a How does the idea of life in Amerdeeper understanding of the challenges have a support system. ica compare to the reality? There are facing refugee youth after relocation to an immense number of refugee camps a new country. I found the children’s throughout the world. The UN Refugee greatest obstacles to be language barriAgency reported that four fifths of the world’s refugees are being hosted ers, hygiene, participation, and learning a new set of cultural expecby developing countries; only about 17% of refugees live outside their tations. The children also faced difficulties such as reunification with region of origin, with most fleeing to neighboring countries.6 This infor- a parent, and finding healthy ways to express loneliness, depression, mation provides a greater understanding of what is known about refu- and fear. Similar to others who experience trauma, the refugee youth gee youth nationally and globally. The refugee and humanitarian orga- are prone to lasting effects that may affect their personalities and benizations in America that are dedicated to supporting refugee youth are haviors. With such a large representation of children in the refugee great resources. However their efforts only extend to the minority refu- community, it is important to be knowledgeable about potential isgee population that relocates to the United States. The students I inter- sues and act to prevent mental, emotional, and behavioral issues As a part of the Refugee Youth Summer Academy, I was able to acted with at the academy would get excited over commonplace American things such as snack-machines, outdoor playgrounds, and Chuck see firsthand the reactions of many refugees as they were introduced E. Cheese’s. As young children, they are delighted by fun activities and to a new setting, lifestyle, and community. Though reactions varied, games. However, in refugee camps, many refugees are disillusioned with I believe that the academy benefitted the refugees and their families life in America. Hollywood movies often portray Americans as wealthy because?. My applied experience taught me that providing education citizens with stable jobs, large houses, cars, and swimming pools. Once and community building opportunities to refugee youth is imporrelocated to the United States, refugees can be met with overwhelming tant as it helps prevent mental, emotional, and behavioral issues. disappointment and confusion when they are sent to live in a cramped apartment with a high rent. Without support from organizations or lo- References 1. Refugee 101; Bridging Refugee Youth & Children’s Services Web site; http:// http://www.brycs. cal communities, refugees can struggle to maintain a happy and healthy org/aboutRefugees/refugee101.cfm; Accessed August 19, 2013.

2. Reed R, Fazel M, Jones L, Panter-Brick C, Stein A. Mental health of displaced and refugee chillifestyle for themselves and their children. dren resettled in low-income and middle-income countries. The Lancet. 2012;379(9812):250-265.

What steps can host countries take to prevent or minimize men- 3. Mental health of refugees, internally displaced persons and other populations affected by contal, emotional, and behavioral issues in refugee youth? In early 2013, flict; World Health Organization Web site; http://www.who.int/hac/techguidance/pht/mental_ health_refugees/en/; Accessed August 19, 2013. a study was completed that proved the importance of education and 4. Shaw J. Children Exposed to War/Terrorism. Clin Child Fam Psychol Rev. 2003;6(4):237-246. community building for refugee youth. In the study, 30 Somali youth, 5. Morland L, Duncan J, Hoebing J, Kirschke J, Schmidt L. Bridging refugee youth and children’s A case study of cross-service training. Child Welfare. 2005;84(5):791-812. who had experienced violence and loss, participated in programs such services: 6. Population levels and trends; The Office of the United Nations High Commisioner for Refugees as community resilience building and school-based early interven- Web site; http://www.unhcr.org/516286589.html; Accessed August 21, 2013.

Ellis B, Miller A, Abdi S, Barrett C, Blood E, Betancourt T. Multi-tier mental health program for tion groups to help with acculturation and socialization. As a result 7.refugee youth. J Consult Clin Psychol. 2008;76(4):184-193. of the study, students across all tiers of the program demonstrated 8. Sommers M; Emergency education for children; Massachusetts Institute of Technology Web site; improvements in mental health and resources. The conclusion of the http://web.mit.edu/cis/www/migration/pubs/mellon/1_children.pdf; Accessed August 20, 2013.

9. Kirk J, Cassity E. Minimum standards for quality education for refugee youth. Youth Studies study states that “resource hardships were significantly associated with Australia. 2007;26(1):132-139. symptoms of Post Traumatic Stress Disorder over time, and the stabili- 10. Burnett A, Peel M. Health needs of asylum seekers and refugees. Br J Health Psychol. zation of resource hardships coincided with significant improvements 2001;322(7285):544-549.

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hurj spring 2014: issue 18

Are Science and Religion Incompatible?

Understanding Divinity as a Scientific Notion Helen Zhao, Class of 2016

world without divine revelation. Not only did it refuse to answer questions about the physical nature of Chaos, Earth, Heaven, Night, and Day, but it also found those questions to be irrelevant, so long as Nowadays, one is hard-pressed to find an unbeliever of science. some explanation existed whether complete or not.

In the past few centuries, science has managed to explain much of the On the other hand, one could characterize Presocratic philosophy natural world, from interstellar space to the cellular organelle, while by the systematic, critical thinking of its followers and its desire for producing technologies that have bolstered our standard of living. Nev- ordered explanations. This could be seen as early as in Thales when ertheless, it is hardly a surprise that unbelievers of science exist, largely he, attempting to understand the composition of nature, hypothesized within religious communities, no doubt the result of a conviction that the fundamental constituent of all things to be water. After all, “seescience and religion are irreconcilably opposed. One can guess that ing that the nourishment of all things is moist […] and the seeds of all this antagonism has arisen from the problematic consequences that things have a moist nature,” he reasoned that water must be the princertain scientific theories pose for theociple substance (Fragment 2, RAGP 9). logical doctrines. Nevertheless, I argue Rather than appealing to a god as HesDo our forces and thermodynamic that religion and science are not, in fact, iod did or offering ad hoc explanations incompatible, though there may be imof natural phenomena, Thales attempted principles—as invisible as gods balances in their respective burdens of to define the world as a complete system except through the effects they proof. Rather, I propose a categorical under which universal principles could cause—differ in context from the distinction between the concepts of dibe expected to hold throughout. This vinity and of the mythical, one that may was apparent when he, in extending his theories of divinity proposed by open scientific rationale to the inclusion theory to all things macroscopic, addiPresocratic philosophers? of religious claims. tionally described earth to rest upon waI will construct my argument ter “because it can float like wood and through an interpretation of Presocratsimilar substances” (Fragment 3, RAGP ic philosophers, whose theories are believed to mark the beginning 9). Hereafter, it became common if not necessary for philosophers to of Western philosophy. Many of their ideas involve elements of gods forego mythic explanations for what I will call active inquiry—investiand divine substances; and yet, they are often considered the earliest gation marked by reason, logic, and critical analysis that would remain Western scientists. How can this be? By answering this question, I a staple of both Presocratic and scientific thought. will illustrate the overlap between religion and science, demonstratI would also like to address here a possible hole in my undering ways in which the two fields may coexist. My argument will pro- standing of “scientific.” Most sources include in their definitions an ceed in the following order: (1) I will define the scientific thinking empirical design and/or desire to know the physical world. Howpresent in Presocratic thought, distinguishing it from mythical sup- ever, while these traits are present in some Presocratic thinkers, noposition; (2) I will develop an understanding of the divine as it is tably Heraclitus, who once said, “All that can be seen, heard, expeemployed in Presocratic theory; (3) I will unify the aforementioned rienced—these are what I prefer” (Fragment 32, RAGP 28), not all concepts in order to show how the divine is not only present, but Presocratics would agree on the importance of sensible data, such as also integral to Presocratic theories of the natural world; and (4) I Parmenides and Zeno for whom the physical world was of secondary will extend the conclusions made in (3) to present-day by drawing a value. Nevertheless, I would not preclude their ideas from scientific comparison between contemporary scientific practices and notions thought for the following reasons: 1) That which I have defined as acof the divine in Presocratic science. tive inquiry, even if not empirical, was clearly present in Parmenides’ work, particularly when he by way of the goddess’ voice urged others (I) to “judge by reason” the words of a divine spirit, and 2) in giving his Several features distinguish Presocratic thought from the super- account of what is, Parmenides also established what he considered stitions that came before it, the most important being its method of to be physically real, an interest universal to science (Fragment 7, reasoning. Previously, phenomena were understood to be expres- RAGP 38). While he offered a distinction between what is and what sions of divine will, as shown by Hesiod’s appeal to the Muses to il- “mortals have established, persuaded that they are true”—the senluminate the origins of the universe: asking, for instance, how “Gaia, sible reality—I do not think he aimed to dismiss nature altogether the Earth, came into being” and bore her first child “Ouranos, starry (Fragment 8, RAGP 38-39). Rather, like all scientists, he desired to Heaven, just her size, a perfect fit on all sides” (Theogony, RAGP┼ define the literal world, one that precluded sensory data, but through 2). This form of understanding in which facts about the world arose pure reason could still be perceived by the philosopher. In this way, from faith and which I shall call passive inquiry, neither demanded his ideas and the ideas of like-minded Presocratics remained scienevidence nor suggested that humans alone could understand the tific, even if not empirically so.

Biophysics

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(II) Concepts of the “divine” varied greatly within Presocratic thought but differed from mythical suppositions in two ways: 1) They came to be understood by means of active rather than passive inquiry, and 2) they were thought to be systematic forces, principles, or causal laws of the natural world. To begin, let us examine what is arguably the most explicitly theological mention of divinity, found in the writings of Xenophanes. Well-known for his rejection of polytheism, he constructed a concept of God that was convincingly “not at all like mortals in body or thought,” but which lacked a compelling reason for God’s existence. As a result, one might consider Xenophanes’ understanding of the divine to have been like the traditions of Greek mythology— similarly unjustified in assuming God’s existence. However, while his claim may have been inadequately supported, Xenophanes himself was more scientist than priest, clear through the logic he employed when countering anthropomorphism: “horses would draw the shapes of gods to look like horses and oxen to look like oxen” if “oxen and horses […] were able to draw with their hands […]” (Fragment 5, RAGP 10; Fragment 6, RAGP 22). In this case, rather than blindly accept the convention of gods as having human traits, he questioned its reasoning, employing critical analysis that was both necessary and indicative of an active inquirer and thus a scientific mindset. Moreover, rather than relying on the gods to “reveal all things to mortals,” he asserted that a curious human mind could lend more reliable truth—that “by searching” instead of accepting, “[we] discover better” (Fragment 12, RAGP 22). Consequently, it seemed that Xenophanes’ image of God held more in common with the products of scientific inquiry than they did with unfounded mythical assumptions.

In addition, the divine entities invoked by the Milesians and others appeared to embody universal forces and principles. For instance, Anaximenes claimed air to be “a god […] without measure, infinite and always in motion”—a divine substance necessarily meant to unify the fundamental properties of all things (Fragment 19, RAGP 13). Likewise, Heraclitus painted a portrait of the divine as a giver of wisdom and insight, not only by means of logos, an objective reality superior to subjective appearance, but also by means of a literal god: “the wise […] unwilling and willing to be called by the name of Zeus” (Fragment 27, RAGP 27). The Pythagoreans propagated an image of the divine as numbers, said to “constitute the whole universe” (Fragment 12, RAGP 18). Meanwhile, Parmenides, in rejecting the sensible world, was led to construct a definition of ‘what is’ that mirrored the divine—imperishable, whole, unshaken, and complete; not unlike the God of our monotheistic religions today. In seeking to make sense of the natural world, the Presocratics formulated laws in accordance with divine principles. Thus didtheir ideas diverge from those of the superstitious, for whom the divine and the earth were related in no systematic and necessary way.

(III) It is here that I will answer the question: Were, then, these socalled “earliest western scientists” truly scientists if they believed in divine beings? To answer, let me describe the state of science as it exists today. Since Isaac Newton first formulated his law of gravity, humans have become obsessed with all manner of unobservable objects—electricity, magnetism, dark matter, the electron. In fact, when the atomic model was first presented to the scientific community, it was met with derision from many who did not consider experi-

humanities & social sciences

ments dealing with these unobservable phenomena to be science at all.

Nevertheless, today we have accepted the atomic model and the existence of objects far smaller than the atom to be true, due in part to their abilities to explain the natural world and, through their manipulation, produce technologies of varying kinds.

How then, I now ask, do our forces and thermodynamic principles—as invisible as gods except through the effects they cause—differ in context from the theories of divinity proposed nndb.com

by Presocratic philosophers?

Many Presocratic ideas of divine Hesiod of Ancient Greece substances and principles served as explanations of observable facts—such as that the mag-n e t “has soul, because it moves iron,” or that “breath becomes cold when compressed and condensed by the lips” (Fragment 5, RAGP 10; Fragment 21, RAGP 13). We can therefore reasonably conclude that divinity was simply a primitive scientific explanation for natural phenomena, but scientific no less. If that is true, one might further ask how we can justify the divine principles invented by Parmenides that did not arise from empirical phenomena. I reply that the use of pure reason does not denote a lack of science, but rather embodies an attempt to understand the natural world through modes other than sensible data, such as by logical deduction. Likewise, theories of divinity as proposed by Parmenides represented his attempts to understand the world through non-physical principles, which, while empirically unverifiable, he believed to follow necessarily given a rational and logically coherent cosmos.

(IV) In summary, one must remember that science is not defined by true theories. After all, if that were the case, we might never have science, for who could say that our theories today are certainly true?

Rather, scientific thinking is defined by how we analyze and solve a problem using our rational, human intellect. We can conclude that Presocratic philosophers were scientists given their methods of reasoning, which employed active inquiry and demonstrated curiosity for unified theories beyond a simple desire for explanations that satisfied the phenomena. As a result, we can further conclude that the possibility of an intelligent designer, if derived by analytical and logical means, would be no less scientific today than were the theories of divinity proposed by the Presocratics. No doubt a greater burden of proof lies upon he or she who wishes to assert rather than deny the existence of something unobservable. Nonetheless, even if discrepancies exist between the contents of religious scripture and scientific belief, we might still be able to understand religious theories through the principles that comprise today’s scientific theories, principles which once innervated Presocratic notions of the natural world.

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Lift Off of Dehydrated Aqueous Two-Phase Systems from Various Substrates Upon Rehydration Michael Saunders1,2, Joseph Labuz2, Chang Che2, Shuichi Takayama2

Johns Hopkins University, Baltimore, MD 2University of Michigan Department of Biomedical Engineering, Ann Arbor, MI

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Introduction Aqueous two-phase systems (ATPS) enable simple cell and chemical patterning on a variety of surfaces. An ATPS is formed when two immiscible polymer solutions are combined. The first layer of the ATPS is laid down in drops on a surface and dehydrated.

A second aqueous phase is then laid on top of the drops, rehydrating them. Droplets of each phase can be prepared containing unique chemical indicators or biological organisms that are attracted to the polymer in that phase. Elements in the second phase can cross the interface into the first phase via diffusion, allowing researchers to bring distinct substances or organisms into contact. Despite existing for over a century, ATPS have only recently been employed in research at the micro-scale. Their uses have included a number of devices intended to purify biological materials (Asenjo and Andrews, 2012; Hardt and Hahn, 2012a), deliver proteases to enable direct purification of stem cell colonies (Frampton et al., 2013a), and prepare patterned bacterial biosensors (Yaguchi et al., 2010). The goal of this article is to explore and characterize the unconventional printing of ATPS patterns on various substrates for future applications in ATPS reactions and diagnostics. This paper is intended to provide the reader with an outline of some of the benefits and restrictions involved in using these particular systems, which may be applied to a number of different projects.

Materials and Methods We used Dextran (DEX) (Pharmacosmos; Holbæk, Denmark) and polyethylene glycol 35K (PEG) (Sigma-Aldrich; St. Louis, MO) to make our aqueous two-phase systems because they are known to form stable ATPS at a wide range of temperatures, and do not require high polymer concentrations to form (Tavana et al., 2009).

In solution, because DEX has a greater density than PEG, it forms the bottom phase of a PEG/DEX ATPS. If PEG were laid down first, rehydration with DEX would cause the PEG to rise to the surface in a mushroom-cloud shape (Fig. 1a). This PEG-DEX ATPS was used to study pattern lift-off. Specifically, we deposited droplets of dye-infused PEG over a hand-printed design and dehydrated the system overnight. Submerging the design in a DEX solution rehydrated the dye-infused PEG, causing it to rise to the surface of the solution (Fig. 1b). Depending on the kind of dye used, the pigmenteither remained in the PEG droplets or slowly diffused into the surrounding DEX solution. This article will present introductory information regarding patterning, partitioning, and rehydration of dehydrated PEG spots on various substrates for researchers pioneering future applications of biochemical reactions and visual fluidic designs.

Dye Partitioning In order to create visual designs, it is necessary to use a dye that will provide as much contrast as possible with its background. This dye must be strongly attracted to the PEG phase of an ATPS solution so a clear partition is found. A total of 6 widely available dyes were selected including: 10% (v/v) green food coloring in PBS (Kroger; Cincinnati, OH), 10% (v/v) blue food coloring in PBS (Kroger; Cincinnati, OH), 0.7% (w/w) Trypan Blue (Sigma-Aldrich; St. Louis, MO), 0.05 mg/mL Bromphenol blue (Sigma-Aldrich; St. Louis, MO), 0.1 mg/mL Resazurin (Acros, NJ), and 2 mg/mL Brilliant Blue Coomassie (Fisher Biotech; Fair Lawn, NJ). There were also three possible DEX solutions: Dextran T3.5, Dextran T10, and Dextran T500. The optimal DEX solution would create an ATPS with a clear partition and allow the droplets of PEG to stay in solution without diffusing on an experimental time scale. DEX T3.5 was tested, but did not clearly partition into two phases. This is likely due to the fact that in order to make stable ATPS a very high percentage solution of DEX T3.5 is needed (Albertsson, 1971). DEX T10 and DEX T500 showed acceptable partitions.

In order to find the ideal dye and solutions of DEX, 500 µL 20% PEG 35K in PBS, 500 µL of 20% DEX solution (either T10 or T500) in PBS, and 5 µL of each of the dyes were combined to make a 10/10 % DEX/PEG solution by weight. Each was then vortexed, placed in a micro-centrifuge for 3 minutes on 300 RCF, and examined.

Pictures were taken of every Eppendorf tube after it came out of the micro-centrifuge (Fig. 2a). The mean color content of each phase and the background of the pictures were calculated using ImageJ64 software. The color content of the top phase minus the color content of the background was divided by the color content of the bottom phase minus the color content of the background to give the partition coefficient of each type of dye and DEX solution. An optimal dye would have a partition coefficient not equal to 1 to be visible.

Trypan Blue in DEX T10 and Trypan Blue in DEX T500 had the greatest partition coefficients (Fig. 2b) suggesting that in both solutions the dye was attracted to PEG. The total concentration of the PEG and DEX ATPS was also investigated. Stock solutions of 20% PEG, DEX T10, or DEX T500 were mixed with PBS in appropriate ratios to form 10/10 or 5/5 percent solutions by weight. However, as predicted by the binodal curve of DEX T10 and PEG 35K, when DEX T10 was used in a 5% solution, an ATPS did not form (Albertsson, 1971). When DEX T500 was used in a 5% solution, an ATPS did form, but its partition

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Figure 1a: Trypan-infused drops of PEG 35K rise in a DEX T10 solution, producing a mushroom-cloud effect over time. Pictures i-iv were taken at 10 seconds intervals.

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Figure 2a: (i) A 10% Green dye in PBS mixture added to a PEG 35K/DEX T500 solution lacks a clear partition. (ii) A 10% Trypan blue in PBS mixture added to a PEG 35K/DEX T10 solution has a clearly defined partition between the two phases. Note that the dye is almost entirely in the PEG phase on top. (iii) A 10% Neon Washable Paint in PBS mixture added to a PEG 35K/DEX T10 solution has a clearly defined partition. Note that the dye is almost entirely in the DEX phase on the bottom.

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Figure 3a: Trypan-infused PEG 35K laid out in lines in a DEX T500 solution at t = 0 and t = 10 seconds.

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Figure 3b: Drops with spacing of (i) 2 mm, (ii) 4 mm, and (iii) 8 mm were tested. A spacing of 4 mm was chosen because it allows for a large array of drops in a small area without allowing the drops to touch after rising.

Figure 2b: Different concentrations and types of dye yield different partition coeďŹƒcients, which were quantified using ImageJ64 software.

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Figure 2c: (i) Trypan-infused PEG 35K rising in a DEX T500 solution. (ii) Trypan-infused PEG 35K rising in a DEX T10 solution.

Figure 3c: Diameter when dry as a function of drop volume.

was not as clearly defined as either the DEX T10 or the DEX T500 10% solutions. Based on these observations, use of 5% solutions was abandoned.

lengthen the time it took for the droplets of 0.1% Trypan in PEG to diffuse. As can be seen in Fig. 2c, the PEG droplets in the DEX T500 rose slowly and diffused as soon as they reached the surface, while the PEG in the DEX T10 rose in less time and was much more stable once it had risen. While the PEG in DEX T10 rose in a single mushroom cloud per droplet within a similar timeframe, the PEG droplets in DEX T500 rose at varying times, and often times in more than one cloud per droplet. DEX T10, when pooled on top of PEG droplets, allowed the droplets to rise quickly and neatly, without causing the PEG droplets to immediately diffuse. Based on these results, DEX T10 was selected as the ideal DEX solution to print ATPS patterns.

PEG Droplet Detachment and Floating Because the results for Trypan in DEX T10 and T500 were so similar, we decided to complete further testing to determine the optimal DEX solution. To compare Dextran of differing molecular weights, we measured the time it took for PEG droplets to rise to the surface in each, as well as how long the dye took to diffuse into the solution. 20% DEX T10 in PBS and 20% DEX T500 in PBS, both pre-equilibrated with 20% PEG 35K in PBS were prepared. By combining and vortexing PEG and DEX, pre-equilibrated solutions of DEX were created and the solution was allowed to separate into two phases. As the phases do not separate completely, the bottom phase was not pure DEX. This phase was saved for future use. Droplets of 20% PEG 35K in separate dishes were laid down and dehydrated overnight. These droplets were then submerged in the pre-equilibrated 20% DEX T10 or T500. Pre-equilibrated DEX was used to

Effect of Interfacial Tension Dictates Types of Patterns That Can be Generated The first question in coming up with viable printing involved the kind of patterning we could use. We dehydrated dishes of Trypan-infused PEG, patterned with lines or droplets, to see if they would preserve their shapes after rising. We then rehydrated them with pre-equilibrated 20% DEX T10 (Fig. 3a). While droplets

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science & engineering worked well and rose in the design they were patterned in, the lines would clump into droplets on the bottom of the dishes before rising as droplets of varying volumes. This may be the result of surface tension effects and interfacial energy minimization between the two phases. This data led us to focus on how best to control dots as a design pattern.

Droplet Spacing The spacing of droplets of Trypan-infused PEG was important to prevent interactions upon rehydration. Using Autodesk CAD software arrays of dots with diameters of 2 mm and spacing of 2, 4, and 8 mm were created to use as a guide for patterning (Fig. 3b).

We experimented with dye-infused PEG droplets with volumes of: 0.2 µL, 0.5 µL, 1.0 µL, 2.0 µL, and 5.0 µL. The results of these experiments can be seen in Fig 3c, which shows the relationship between diameter and volume of the dehydrated droplets, and Fig 3d, which shows the relationship between diameter and volume of the droplets at the surface. We wanted to maximize droplets per unit of area while still leaving room for expansion after rising. From the data, we determined that the ideal combination was 0.5 µL droplets with 4 mm spacing. Conveniently, the 2 mm dots that we had been using held 0.5 µL of liquid. This is important because in order to lay down the droplets and avoid interactions, as in figure 1b, the Trypan-infused PEG must be contained within the printed dots of the array.

Substrates The effect of substrate material on PEG rehydration and rising in an ATPS was the next topic of inquiry. In order to apply ATPS to other technologies such as paper microfluidics, the effect of surface properties such as porosity or thickness of the paper on ATPS must be known. We tested our procedure on glossy photo paper (200 g/ m2), chromatography paper (185 g/m2), transparencies (135 g/m2), construction paper (90 g/m2), and standard printer paper (80 g/m2).

Initial experiments involved laying down 0.5 µL droplets of 20% DEX T10 on dot grids of 4 mm spacing on each type of paper. The best results came from transparencies, as the droplets were not absorbed. For construction paper, the size of the dehydrated droplets fluctuated due to the absorption. This led us to the conclusion that the porosity of a surface affects rising times. Due to the similarities between transparencies and petri dishes, we were not surprised that the dots rose at similar rates on both surfaces. In order to quantify our results, we developed a way of determining if a droplet had successfully risen. We counted how many droplets had fully risen after 10 minutes, calling them “successful.” The difference between a suc-

Figure 3d: Diameter after rising as a function of drop volume.

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hurj spring 2014: issue 18 cessful and unsuccessful rise can be seen in Fig. 3e. The success of chromatography paper was intermediate most likely because of the material’s surface composition, which is paper coated with a glossy finish. The success rates for all types of paper tested can be seen in Fig. 3f.

Discussion We have discussed the interactions and experimental properties of a number of substrates and their potential for use in aqueous twophase systems. Through experiments involving the production of patterns in ATPS solutions, we have demonstrated the ideal concentrations of different types of PEG-DEX solutions, and their relations to possible surfaces for future use. Our results are relevant to the use of ATPS in future technologies.

ATPS technology has great potential for use in diagnostic devices based on biochemical reactions. For example, fluorescently labeled protein substrates are used to detect proteases in patient samples in hospital laboratories (Farhath et al., 2013; Ozmen et al., 2008). It may be possible to use paper microfluidics to apply ATPS to detect proteases outside of medical laboratories. For example, a fluorescent protein substrate could be combined with patient samples in a biochemical reaction in an ATPS device (Farmer and Yuan, 1991). Fluorescent products caused by proteolytic activity could be detected with an inexpensive UV light source in the field.

If diagnostic tests cost less, more can be provided to the consumer at lower prices with significant implications for patients. Paper microfluidic devices currently under investigation include projects to test for hemoglobin (Chitnis et al., 2011), complete bioassays (Coltro et al., 2010), and separate whole blood into its major com-

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Figure 3e: Drops were laid down in a square grid, with the outlines of the dots printed onto the substrate. (i) Drops that have successfully risen to the top of the system, originally laid down on transparency. (ii) Drops that were not successful in rising to the top of the system after being laid down on construction paper.

Figure 3f: Dependence of successful rising on type of paper. To be successful, a drop needed to fully rise in 10 minutes or less.

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ponents (Tsukamoto et al., 2009). Combining paper microfluidics and ATPS offers a significant opportunity for the creation of a wide array of new and cheaper diagnostic devices. Looking towards the future, we have established a basis for uses of rehydrated aqueous two-phase systems. Since use of ATPS in diagnostic tests has only recently begun, relatively little about their properties is known.

The applications of aqueous two-phase systems are many, and they must all be explored in order to use the technology to its fullest potential.

References Albertsson, P.-A. (1971). Partition of cell particles and macromolecules; distribution and fractionation of cells, mitochondria, chloroplasts, viruses, proteins, nucleic acids, and antigenantibody complexes in aqueous polymer two-phase systems. (New York: Wiley-Interscience).

Asenjo, J.A., and Andrews, B.A. (2012). Aqueous two-phase systems for protein separation: phase separation and applications. J. Chromatogr. A 1238, 1–10.

Chitnis, G., Ding, Z., Chang, C.-L., Savran, C.A., and Ziaie, B. (2011). Laser-treated hydrophobic paper: an inexpensive microfluidic platform. Lab. Chip 11, 1161–1165.

Coltro, W.K.T., de Jesus, D.P., da Silva, J.A.F., do Lago, C.L., and Carrilho, E. (2010). Toner and paper-based fabrication techniques for microfluidic applications. Electrophoresis 31, 2487– 2498.

Farhath, S., He, Z., Saslow, J., Soundar, S., Amendolia, B., Bhat, V., Pyon, K., Stahl, G., Mehta, D., and Aghai, Z.H. (2013). Detection of pepsin in mouth swab: correlation with clinical gastroesophageal reflux in preterm infants. J. Matern.-Fetal Neonatal Med. Off. J. Eur. Assoc. Perinat.

Med. Fed. Asia Ocean. Perinat. Soc. Int. Soc. Perinat. Obstet. 26, 819–824.

Farmer, W.H., and Yuan, Z. (1991). A continuous fluorescent assay for measuring protease activity using natural protein substrate. Anal. Biochem. 197, 347–352.

Frampton, J.P., Shi, H., Kao, A., Parent, J.M., and Takayama, S. (2013a). Delivery of Proteases in Aqueous Two-Phase Systems Enables Direct Purification of Stem Cell Colonies from Feeder Cell Co-Cultures for Differentiation into Functional Cardiomyocytes. Adv. Healthc. Mater.

Hardt, S., and Hahn, T. (2012b). Microfluidics with aqueous two-phase systems. Lab. Chip 12, 434–442.

Ozmen, S., Yücel, O.T., Sinici, I., Ozmen, O.A., Süslü, A.E., Oğretmenoğlu, O., and Onerci, M. (2008). Nasal Pepsin Assay and pH Monitoring in Chronic Rhinosinusitis. The Laryngoscope 118, 890–894.

Tavana, H., and Takayama, S. (2011). Aqueous biphasic microprinting approach to tissue engineering. Biomicrofluidics 5, 013404–013404–8.

Tavana, H., Jovic, A., Mosadegh, B., Lee, Q.Y., Liu, X., Luker, K.E., Luker, G.D., Weiss, S.J., and Takayama, S. (2009). Nanolitre liquid patterning in aqueous environments for spatially defined reagent delivery to mammalian cells. Nat. Mater. 8, 736–741.

Tsukamoto, M., Taira, S., Yamamura, S., Morita, Y., Nagatani, N., Takamura, Y., and Tamiya, E. (2009). Cell separation by an aqueous two-phase system in a microfluidic device. Analyst 134, 1994–1998.

Yaguchi, T., Lee, S., Choi, W.S., Kim, D., Kim, T., Mitchell, R.J., and Takayama, S. (2010). Micropatterning bacterial suspensions using aqueous two phase systems. Analyst 135, 2848–2852.

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Soft-Bodied Robotics: Challenges Ahead, Review of Current Simulation Methods and Materials used in Actuation Rohil Malpani

Johns Hopkins University, Baltimore, MD, USA, National University of Singapore, Singapore

Abstract Background & Aims Soft-Bodied robotics is a bourgeoning field with widespread applications ranging from search-and-rescue in hostile environments to minimally invasive surgery and prosthetics. Soft-Bodied robots are constructed from soft materials with designs inspired by living organisms such as starfish, earthworms, slime molds, insects, and primates. They are programmed to be deformable and flexible and are therefore made of soft and elastic materials. Their actuators (any component that uses energy to change the state of the device) are usually made of artificial muscle fibers, unlike the metallic electromagnetic or mechanical actuators found in conventional robots.

In conventional robotics, the metrics that matter are speed, precision and controllability since the environments that these robots function in are carefully controlled. Soft-Bodied robots, in contrast, are designed to function under significant strain in varied environments, have an infinite number of degrees of freedom (DOF) and work well in unstructured environments [1]. Table 1 provides a definite guide of what constitutes a soft-bodied robot.

This article seeks to look closely at each material and simulation method to ascertain the qualities of each and to suggest their potential uses.

Challenges faced The inherent soft nature of these robots which gives them their unique capabilities also proves problematic during their design and construction. John Reiffel and Barry Trimmer, in their article “Body/ Brain Co-Evolution in Soft Robots” clearly present some of the problems associated with soft-bodied robotics [2].

The two main challenges facing soft robotics are problems with design and problems with control [2]. Because of the force distributive effects of soft materials, there are no “established principles” for mechanical control of such robots [2].

In brief, the issues facing the field of soft robotics are: problems with designing control systems and actuators, issues with simulating soft objects precisely, difficulty engineering the device to deal with its non-linear status along with infinite degrees of freedom, and problems deciphering the biomechanics of soft-bodied organisms. These issues are discussed further below.

The most obvious problem is that any relatively large soft-bodied object requires some hard material to support its own weight [3]. The hard material can be metallic wires that surround the structure (as used in the Meshworm) or an internal support structure similar to a skeleton [4]. In addition, since soft materials are energy-absorbing, the lack of rigid components does not allow amplification of forces from the actuators and prevents application of large inertial forces [5].

Sensing and controlling the shape of soft robots is difficult because of their ability to function under high strain. Additionally, as their structure is a continuum, obtaining exact coordinates of the shape is difficult, unlike hard robots which measure the position of each joint with an encoder [6]. Compounding this issue, kinematic trajectories of under-actuated systems e.g. soft robots, are only partially controllable making control over non-linear dynamics difficult to achieve [7].

Analysis of the methods used in Simulations

Courtesy of Tufts University via cacm.acm.org

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Models of soft-bodied objects have advanced exponentially over time and researchers have developed many biomechanical methods to simulate deformable bodies. Most

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algorithms develop simulations by breaking down a body into constituent discrete particles and then modeling changes in the shape and size of the particles based on the forces acting both internally and externally. At a macro scale, all of the combined changes observed in the constituent particles result in a model of the overall change in the state of the body. The non-linear nature of their materials and geometry makes modeling soft robots a complex task [8].

Various approaches to modeling deformable bodies are detailed below.

1. Mass-Spring method In the Mass-Spring method, springs are used to represent a material’s rigidity. A body is conceived as a set of nodes interconnected in various configurations by a number of massless, completely elastic springs observing Hooke’s law [9]. The net force on the nodes in this dynamic system is obtained by applying Newton’s second law to the point masses, taking into account the restoring force applied by the springs and other external forces (friction, gravity etc.), and then solving the resulting Ordinary Differential Equations (ODEs).

The Mass-Spring model is one of the simplest and most intuitive of existing models and this simplicity, combined with its ease of implementation is especially well-suited to tasks that require real-time simulations [9].

2. Finite Element Simulation Finite Element Simulation is a physically accurate approach to soft body simulation which involves the use of the Finite Element Method (FEM) to solve the partial differential equations that represent the motion of a deformable body. In the FEM, the model is rep-

Robot types

resented by a set of ‘finite elements’ connected at points of intersection known as nodes. Each of the elements is associated with certain physical properties such as elasticity, density etc. that give rise to its particular behavior during deformation. The finite elements can be one, two or three-dimensional. The nodes are usually placed at the vertices of the mesh and in three-dimensional elements; the meshes are usually in tetrahedral or hexahedral form. Because this approach is physically based, the models respond to external forces accurately, interact with each other naturally and react well to constraints like linkages [10].

FEM has already enabled researchers to simulate soft body deformation for an array of functions including surgery [5] and the movements of muscular hydrostats [8]. The major downside to using FEM techniques is the large processing cost it incurs. However, with further optimization of this approach and increasing applicability in newer scenarios, FEM is proving to be a very attractive tool for physically accurate computer based animations of soft bodies.

3. Finite Volume method (FVM) The Finite Volume method also uses the technique of splitting a body into a network of nodes on a mesh. In this method, the partial differential equations that result from trying to solve for the state of each node in the body during the simulation are represented in the form of algebraic equations [11]. The words “Finite Volume” refer to the small volume surrounding each node on the mesh that makes up the soft body. The values are computed at distinct positions on the mesh which provides an advantage since volume must be conserved locally to obtain realistic modeling of soft bodies [12].

Characteristics

Multi-section Hard Conventional (Rigid) robots Continuum robots

Soft Bodied robots

Mechanical properties: Resistance to deformations Consituents Degrees of Freedom Resilience to shearing forces Actuators

Very high Metals, Rigid Plastics Few Very high Few

High Rubber, Rigid Plastics Large High Many

Lowest Soft plastics, rubber, Infinite Low Throughout the robot

Regulated only Low Lowest Very High Highest Very Accurate Construction, Assembly Line Work Human Limbs

Regulated and Unregulated Medium High High High Accurate Bulk Transport, Object Retreival and Inspection Snakes, elephant trunks

Regulated and Unregulated High Highest Low Low Least accurate Minimally Invasive surgery, Prosthetics, Worms, eels, Octopus Arms, Soft appendages in general

Working properties: Functioning Environment Safety Ability to circumvent obstacles Gait and position control Simulation Accuracy Positioning precision Common functional possibilities Natural inspiration

Table 1: Contrasting characteristics of different types of robots

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science & engineering Since the FVM relies on a geometric rather than a variation network, it is generally found to be more intuitive than the FEM [13]. This intuitiveness not only eases implementation but also provides a boost for better optimization of the method. FVM is also superior to the Mass-Spring method since it can be applied to arbitrary constitutive models and unstructured meshes as well [13].

As a result, this method is used frequently in Computational Fluid Dynamic software.

Prominent Materials Used in Actuation Soft-Bodied robots have actuator different requirements to conventional rigid robots. Actuators that are a part of Soft-Bodied robots need to be deformable and so distribute stress over a large area, thus mitigating large forces concentrated over small areas. They must also have a high modulus of elasticity to change their shape as required. They should allow for accurate path control of the robot and have quick response times to stimuli. Some of the most prominent actuators currently being used for the construction of soft robots are detailed below: 1. Shape memory alloy (SMA) Shape Memory Alloys are alloys that can be deformed as required and can return to their original pre-deformed state upon heating. The two main types of SMA are Copper-Aluminum and Nickel-Titanium (Ni-Ti). An elemental SMA actuator consists of an elastic rod surmounted by two embedded SMA wires [14]. An SMA actuator is normally actuated with the aid of electrical energy, with the current causing Joule heating in the actuator. This heating converts the material from its martensitic phase (stable at low temperatures and high stress values) to its austenitic phase (stable at high temperatures and low stress values) [15]. SMA actuators have a high work density and under standard conditions, outperform hydraulic and magnetic actuators in the amount of force generated [16]. Most applications of SMAs make use of the pseudo-elastic effect displayed during their austenitic phase. This makes them ideal for soft robots since high deformations are common and actuators need to be resilient to such stress. The speed and power of SMA actuators also increase with miniaturization making them ideal for micro-scale systems.

Major limitations still plague SMA actuators however.

Energy inefficiency is a serious concern since as thermal actuators they show a maximum efficiency of only 1% [16]. Slow response time is also a concern, since slow actuation delays locomotion reaction time of the device to stimuli. Large hysteresis is an additional issue [15].

Most problematically, SMAs are vulnerable to structural failure, a failure mode in which a large number of deformation cycles eventually leads to severe compromise in the structural integrity and actuation abilities of the material.

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hurj spring 2014: issue 18 2. Electro-active Polymer (EAP) EAPs are polymers that exhibit a noteworthy increase in size upon stimulation by an electric field. The changes in shape are adjusted in order to enable bending, stretching or contraction. At present, EAPs are among the best imitations of biological muscles and are preferred over other materials for their durability, flexibility, low mass and low power requirements [6]. EAPs have succeeded traditional ceramic actuators and can sustain up to 380% strain, much more than any ceramic actuator [6].

There are two main types of EAPs: Ionic EAPs (involving mobility or diffusion of ions) and Electronic EAPs (driven by electric field or Maxwell forces [6]. a) Electronic EAPs: In Electronic EAPs, actuation is initiated by the application of an electric field and the action of Coulomb forces. They can hold displacement under the application of DC voltage, which makes them ideal for robotic applications. Electronic EAPs are further subdivided into Ferroelectric polymers and Dielectric EAPs. Some of the major advantages possessed by Electronic EAPs are that they can be operated for extended periods under standard room conditions, react rapidly and can induce relatively large actuation forces [6]. The major disadvantage of Electronic EAPs is that their operation requires high activation fields, close to the breakdown voltage of the material. i) Dielectric Electroactive Polymers (DEA) A sub-class of Electronic EAPs, they are basically capacitors, comprising of a thin elastomeric film surrounded on both sides by electrodes. Upon the development of a high potential difference between the electrodes, the electrostatic pressure (Maxwell stress) causes expansion of the actuator in planar directions which persists until the applied voltage is removed. DEAs provide large displacements reaching 200-380% of the strain area, have rapid response times and are inexpensive to produce [6]. They transform electrical energy directly into mechanical work with large strains. The main problem associated with DEAs as with all EAPs is their high working voltages. b) Ionic EAPs: In Ionic EAPs, actuation is caused by the migration of ions in-

Courtesy of DARPA/Harvard University via trendguardian.com

hurj spring 2014: issue 18

science & engineering

side the polymer. The main types of Ionic EAPs are conductive poly- developing soft actuators like EAPs, PAMs and SMAs might provide mers and Ionic Polymer Metal Composites. Their major advantage the accuracy, speed and strain resilience that further developments over Electronic EAPs is that they only require a few volts for actua- in soft robotics require. Developing computer simulation techniques tion. They are also naturally bi-directional [6] and typically yield a like the FEM, the FVM, and the Mass-Spring method might hasten magnitude of deformation greater than that generated by Electronic prototyping of soft robots, and enable accurate and rapid computer EAPs. However, since the actuation is driven by a movement of ions, modeling of soft robots, instigating improvements in path sensing external voltage is required to keep the object in a given state, in- and control systems. creasing energy demand. Furthermore, they must be operated in wet Whether soft components constitute conjugates with trastate or in solid electrolysis and often generate lower actuation forces ditional rigid devices [6] or constitute robots on their own, it is than electronic EAPs [18]. certain that soft materials are i) Ionic Polymer-Metal set to revolutionize fields rangComposites (IPMC) ing from remote exploration IPMCs consist of ionic to minimally invasive surgery. polymers like Nafion or FemBio-inspired soft robots may ion surrounded on both sides be the next significant area of by conductive metal sheets. An development in robotics and applied current will then cause might one day enable safe inthe actuator to bend depending teractions between robots and on its polarity. The magnitude humans on a day-to-day basis. of deformation depends upon Despite the challenges facing the magnitude of the applied future research, soft-bodied rovoltage. IPMCs hold great bots are here to stay. promise as artificial muscles; however, because IPMCs work References on the basis of a chemical re[1] Deepak Trivedi, Christopher D Rahn, William M Kier, and Ian D Walker, “Soft robotaction, contamination severely ics: Biological inspiration, state of the art, and hinders their efficiency comfuture research,” Applied Bionics and Biomechanics, vol. 5, no. 3, pp. 99-117, 2008. plicating use in non-standard [2] John Rieffel and Barry Trimmer, “Body/ Courtesy of George Whitesides via robaid.com brain co-evolution in soft robots,” in 12th Int. environments. The ease of Conf. on Artificial Life (ALIFE XII),(Odense, electrolysis in the actuator is A soft robot created at Harvard University can arch and stand up.

Denmark), 2010, pp. 257-258. also a major problem [6]. Fur[3] Sangbae Kim, Cecilia Laschi, and Barry thermore, IPMCs currently do Trimmer, “Soft robotics: a bioinspired evoluin robotics,” Trends in biotechnology, 2013. not have the ability to replicate the extremely high number of tion [4] S. Seok et al., Meshworm: A Peristaltic Soft Robot With Antagonistic Nickel Titanium Coil work cycles that can be achieved by muscle fibers.

Actuators, 2012.

3. Pneumatic Artificial Muscle (PAM) Pneumatic Artificial Muscles (PAMs) are actuators consisting of pressurized air inside an inflatable bladder. By varying the pressure inside the membrane, the position and gait of the device can be adjusted. The core element is a reinforced, closed membrane which is affixed to both ends of the actuator and transfers the mechanical power to the load. By changing the air pressure inside the membrane, a pulling force on the load is generated, resulting in a unidirectional and linear motion [19]. The compactness, high power/weight ratio and simplicity of PAM’s make them highly suitable candidates for soft-bodied robotic actuators [20]. PAMs are similar to skeletal muscles as “linear contractile engines having a monotonically decreasing load-contraction relation (although this is not always the case for skeletal muscle)” [19]. Also mirroring the structural organization of skeletal muscles, PAMs are placed opposite each other on a body so that their opposing forces can move the body in both directions about that axis [19].

Conclusion From the above it can be seen that Soft-Bodied robotics has considerable potential in an array of fields despite facing a host of challenges. Further progress can only be made if a ‘bottleneck’ effect is prevented, i.e. if all aspects of Soft-Bodied robots: simulators, actuators, control systems etc. are developed simultaneously. New and

[5] Morten Bro-Nielsen, “Finite element modeling in surgery simulation,” Proceedings of the IEEE, vol. 86, no. 3, pp. 490-503, 1998.

[6] Yoseph Bar-Cohen, “Electroactive polymers as artificial muscles-capabilities, potentials and challenges,” Handbook on biomimetics, vol. 11, no. 8, pp. 1-3, 2000.

[7] Fumiya Iida, “Biologically Inspired Motor Control for Underactuated Robots--Trends and Challenges,” in Robot Motion and Control 2009.: Springer, 2009, pp. 145-154.

[8] Y Liang, RM McMeeking, and AG Evans, “A finite element simulation scheme for biological muscular hydrostats.,” Journal of theoretical biology, vol. 242, no. 1, p. 142, 2006.

[9] Ben Kenwright, Rich Davison, and Graham Morgan, “Real-Time Deformable Soft-Body [10] Demetri Terzopoulos, John Platt, Alan Barr, and Kurt Fleischer, “Elastically deformable models,” in ACM Siggraph Computer Graphics, vol. 21, 1987, pp. 205-214.

[11] Randall J LeVeque, Finite volume methods for hyperbolic problems.: Cambridge university press, 2002, vol. 31.

[12] Geoffrey Irving, Craig Schroeder, and Ronald Fedkiw, “Volume conserving finite element simulations of deformable models,” ACM Transactions on Graphics (TOG), vol. 26, no. 3, p. 13, 2007.

[13] Joseph Teran, Silvia Blemker, V Hing, and Ronald Fedkiw, “Finite volume methods for the simulation of skeletal muscle,” in Proceedings of the 2003 ACM SIGGRAPH/Eurographics symposium on Computer animation, 2003, pp. 68-74.

[14] Kai Yang and Yanjue Wang, “Design, drive and control of a novel SMA-actuated humanoid flexible gripper,” Journal of mechanical science and technology, vol. 22, no. 5, pp. 895-904, 2008.

[15] Reginald DesRoches, Jason McCormick, and Michael Delemont, “Cyclic properties of superelastic shape memory alloy wires and bars,” Journal of Structural Engineering, vol. 130, no. 1, pp. 38-46, 2003.

[16] Jan Peirs, Dominiek Reynaerts, and Hendrik Van Brussel, “The ‘true’power of SMA microactuation,” in Proceedings of the Micromechanics Europe Workshop MME, vol. 1, 2001, pp. 16-18.

[17] Guggi Kofod, “Dielectric elastomer actuators,” The Technical University of Denmark, Master’s thesis 2001.

[18] Kwang Jin Kim and Satoshi Tadokoro, Electroactive polymers for robotic applications: artificial muscles and sensors.: Springer, 2007.

[19] Frank Daerden and Dirk Lefeber, “Pneumatic artificial muscles: actuators for robotics and automation,” European journal of mechanical and environmental engineering, vol. 47, no. 1, pp.

11-21, 2002.

[20] Darwin G Caldwell, Gustavo A Medrano-Cerda, and Mike Goodwin, “Control of pneumatic muscle actuators,” Control Systems, IEEE, vol. 15, no. 1, pp. 40-48, 1995.

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hurj spring 2014: issue 18

Purification and Characterization of Enterotoxigenic Escherichia coli CS1 Pili CooD Protein Camila Villasante1, Kathleen Dori2, Dr. Steven Poole2

Johns Hopkins University, Baltimore, MD 2Enteric Diseases Department, Infectious Diseases Directorate, Naval Medical Research Center, Silver Spring, MD

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Abstract Enterotoxigenic Escherichia coli (ETEC) is a primary cause of foodborne illness and bacterial diarrhea. ETEC uses long helical filaments, called pili, to attach to the wall of the small intestine and aid in colonization. In this study, we looked at the CooD minor pilin protein of the CS1 pili (Class 5b pili). Each CS1 pilus is comprised of two parts: the major subunit (pilin) CooA, which forms the majority of the body of the pilus, and the minor subunit (adhesin) CooD, which forms the very tip of the pilus body. Mature CooD has a molecular weight of 43.2-kDa. Despite the small ratio of CooD to CooA (~1:1800) in the pilus as a whole, studies have demonstrated that CooD is essential to pilus formation via the alternate chaperone pathway. Furthermore, studies have shown that CooD is crucial in mediating the attachment of CS1 pili to the small intestine.

In this study, we purified and characterized recombinant CooD (dscCooD), stabilized with a donated β-strand from the C-terminus of CooA, from insoluble and soluble lysate fractions. dscCooD protein was expressed in a recombinant E. coli strain and then, after cell lysis and centrifugation, purified from the clarified lysate (soluble) or resolubilized from the cell pellet (insoluble).

Introduction Enterotoxigenic Escherichia coli (ETEC) are gram-negative bacteria that colonize the small intestine, causing gastroenteritis. Hosts are infected through ingestion of contaminated food or water, often a result of poor sanitation and water quality. ETEC is particularly widespread in developing countries, which share an unequal burden of the disease. E. coli poses a threat to susceptible segments of the population who have lowered immune defenses, such as young children and the elderly.

The bacteria use long helical filaments, called pili, to attach to

Figure 1. Phylogenetic tree of the minor subunit proteins of Class 5 ETEC pili. Figure from Anantha 2004.

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the wall of the small intestine and aid in colonization. The different types of pili can be grouped according to their characteristics. In this study, we examine the CooD protein of the coli surface antigen 1 (CS1). CS1 is in subclass 5b of the eight-member Class 5 pili family (Fig. 1), which also includes CFA/I, CS1, CS2, CS4, CS14, CS17, CS19, and CF0711,3.

Figure 2. SDS-PAGE analysis of dscCooD (insoluble and soluble). Both proteins were determined to have a molecular weight of 41.3 kDa. After performing densitometry, the purity of the insoluble CooD was 100%, while soluble CooD was 95% pure.

hurj spring 2014: issue 18 The CS1 pilus is comprised of two parts: the major subunit (pilin) CooA, which forms the majority of the body of the pilus, and the minor subunit (adhesin) CooD, which forms the very tip of the pilus body2. At its full length, CooD has a molecular weight of 43.2kDa. Despite the low ratio of CooD to CooA in the pilus (1:1800), CooD is essential to the formation of the pilus via the alternate chaperone pathway, which utilizes a chaperone protein that eases formation of pilus subunits and helps to stabilize the protein1,3. An usher protein then assists in transferring the subunits to their final position on the outer membrane, and forms a pore in the bacterium’s outer membrane. Furthermore, studies have shown that CooD is crucial in the attachment of CS1 pili to the small intestine2. In this study, we purify and characterize the CooD minor subunit protein of the CS1 subclass.

science & engineering Figure 3a

Methods Bacterial growth conditions.

The dscCooD expression clone was grown for 12 hours in 100mL Alternative Protein Superbroth (APS) + 50μg/mL Kanamycin (Km) at 32°C, 200 rpm. 1L of APS + Km was inoculated with 10mL of the 100mL culture, and grown at 32°C, 200 rpm for 6 hours. Then, 10L of APS + Km was inoculated with 100mL of the 1L culture. The 10L culture was grown until it reached an OD600 of 5 and induced for 3 hours with 1mM IPTG. Cells were then harvested and stored at -80°C.

Figure 3b

Soluble protein purification.

The cell paste was thawed and resuspended in 20mM Tris, 250mM NaCl, and 5mM imidazole (pH 8.0), and left at 4°C overnight. The resuspension was then lysed via microfluidization.

The lysate was centrifuged twice at 10,000 × g and the supernatant was removed for the load. The remaining pellet was used in the insoluble protein purification method. The clarified lysate was Figure 3. HPLC analysis of the insoluble (Fig. 3a) and the soluble (Fig. 3b) CooD, the purity reported was 98.0% applied on a Ni-NTA SuperFlow 5mL column on for the insoluble and 98.2% for the soluble. Both had similar retention times of around 10.8 minutes. the AKTA Explorer FPLC machine. The dscCooD protein was eluted with an imidazole step gradient.

The fractions containing protein were pooled, diluted 20-fold, lysate (soluble) and the dscCooD protein purified from the peland applied to a 5mL HiTrap SP HP column. The dscCooD pro- let (insoluble) were concentrated to ~2.5 mg/ml (as determined by tein was eluted from the column with a linear sodium chloride BCA assay). gradient.

SDS-PAGE/Western Blot Analysis. dscCooD purified from both the supernatant (soluble) and the Insoluble protein purification. pellet (insoluble) were separated on 15% SDS-PAGE gels (stained by The pellet from the soluble extraction was resuspended in silver stain and gel code blue ) and characterized by Western blots 20mM Tris, 100mM NaCl, 5mM imidazole, and 2M Urea (pH 7.9), (α-his, α-csbD, α-CS1). incubated at 30°C, 200 rpm for 1 hour, and centrifuged at 10,000 × g. The supernatant was removed and loaded onto the two 2mL HisBind columns, prepared using two Bio-Rad econocolumns packed Circular Dichroism Spectroscopy (CD).

CD was performed on dscCooD (soluble and insoluble) to anawith Novagen His-Bind Resin Slurry. The protein was eluted with lyze their secondary structure and determine their melting points. an imidazole step gradient. Fractions containing dscCooD were Th e samples were subjected to a gradient of wavelengths (250 nmpooled and applied through a 5mL HiTrap SP HP column as de190 nm) at two temperatures (25°C, 90°C). The wavelength at which scribed above. The dscCooD protein purified from the clarified

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science & engineering there was the most difference between the readings at the two temperatures was chosen as the wavelength to perform the melting temperature determination. The samples were then subjected to a temperature gradient (10°C – 90°C) at the pre-determined wavelength.

Mannose-Resistant Hemagglutination Assay (MRHA).

MRHA was performed using the two forms of dscCooD to determine if the proteins could agglutinate erythrocytes after adsorption to polystyrene bead. In a ceramic tile well, 25 μl of proteinadsorbed beads and 50 μl 1.5% bovine erythrocytes were mixed.

The ceramic tile was rocked on ice for 20 minutes. The reactions were graded on from none (--) to +4 based on the strength of agglutination.

Results The soluble and insoluble preparations of dscCooD exhibited similar SDS-PAGE and HPLC profiles (Fig. 2 and 3). Both preparations reacted to anti-His, anti-CsbD (closely related Class 5b adhesin), and anti-CS1 (data not shown). However, the insoluble preparation was able to induce a weak, but observable mannoseresistant hemagglutination reaction(1+), while the soluble preparation could not (Fig. 4). As this result could indicate differences in folding between two dscCooD preparations, both the soluble and insoluble preparations of dscCooD were analyzed by CD to determine secondary structure and melting temperature. Surprisingly, only slight differences were observed, both proteins had an initial melting transition at 68-69°C and showed a CD spectra that was indicative of a protein whose secondary structure consists mainly of β-sheets (Figure 5 and 6). It appeared that dscCooD (soluble) had an additional transition at 81°C, which could indicate that it is folded differently or that another species is present in the sample. The CD spectra and melting temperature were similar to that determined for another Class 5b adhesin, dscCsbD. The two proteins also differed in the amount of material yielded from their purification methods, the yield of insoluble CooD was 22.65 mg, and the yield of soluble CooD was significantly less, at 2.92 mg.

hurj spring 2014: issue 18 Discussion In this study, we saw that the purification schema for dscCooD—particularly, the need to solubilize and purify material from the cell-debris pellet in order to maximize yield—was similar to that previously developed for dscCsbD, an adhesin in the same subclass as CooD. This suggests that the other class 5b minor subunit proteins (CosD, CsdD) act similarly to CooD and CsbD. Further studies need to be done in order to elucidate a method to increase the yield of CooD from the pellet and/or find a streamlined process for purifying CooD protein. In addition, more research must be carried out to explain the slight differences between the insoluble CooD and the soluble CooD during characterization, and to surmise the possible effects of these slight differences. Knowledge of the purification schema and basic characteristics of CooD provide information about potential drug targets as research in this field moves forward.

References 1. Anantha, R., et al. Evolutionary and Functional Relationships of Colonization Factor Antigen I and Other Class 5 Adhesive Fimbriae of Enterotoxigenic Escherichia coli. Infection and Immunity, 2004. 72.12.7190-7201.

2. Sakellaris H., et al. A conserved residue in the tip proteins of CS1 and CFA/I pili of enterotoxigenic Escherichia coli that is essential for adherence. PNAS, 1999. 96.22.12828-12832.

3. Sakellaris H., et al. The Level of Expression of the Minor Pilin Subunit, CooD, Determines the Number of CS1 Pili Assembled on the Cell Surface of Escherichia Coli. Journal of Bacteriology, 1999. 181.5.1694-1697.

Figure 5. Circular dichroism (CD) analysis reveals that both proteins have a folded structure that is characteristic of a protein dominated by β-sheet secondary structure.

Figure 4. In the MRHA assay, the insoluble CooD was weakly +1 positive, whereas the soluble CooD was negative.

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Figure 6. CD melting point determination for dscCooD (insoluble and soluble). The melting point for the insoluble dscCooD was determined to be 68°C. The melting point graph for the soluble dscCooD had two transitions, one at 69°C and another 81°C, indicating the presence of two melting points.

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