The Baldwin Review 2017 by The Baldwin School

The Baldwin Review A collection of individual research papers produced by Upper School students of The Baldwin School

Foreword Though Baldwin has always had a well-deserved reputation for its academically superb student body, the reason for this reputation came into sharp focus for me when editing and compiling last yearâ&#x20AC;&#x2122;s inaugural edition of The Baldwin Review. The journal was truly emblematic of the academic spirit and intellectual talent of the Baldwin Upper School student body. This second edition proves to be an even broader compilation of Baldwin studentsâ&#x20AC;&#x2122; independent research projects from the 2016-2017 school year and summer, with entries from the fields of history, economics and the basic sciences. Through their writing, Baldwin students have again shown themselves to be curious, intellectually rigorous and highly motivated to learn independent of the classroom setting. It is an honor to be able to bear witness to this academic talent, and to be part of the process of presenting it to the greater Baldwin community.

- Eliza Thaler, Class of 2018

THE BALDWIN REVIEW 2017

SPECIAL THANKS TO Ms. Laurie Cato, Mrs. Lisa Lopez-Carickhoff, Mrs. Christie Reed and Mr. Eric Benke for their help with this journal.

Table of Contents A1

ELIZA THALER ’18 The Great Recession: An Explanation of the 2008 Catastrophe From Neuroscience to Macroeconomics Perspective

SIMI BLEZNAK ’19 An Overview of Multiple Sclerosis

CAROLINE BUCHNER ’18 Increasing Fog2 Gene Expression in Cardiomyocytes Using a Plasmid

ANNA BUNTING ’18 Ulster Scots: The Life Cycle of a Language

SANJANA DIXIT ’18 Electrophysiological Signatures of Language Impairment in Autism Spectrum Disorder

ANOUSHKA GIDH ’19 Increased Cell Inflammation in CD40 Mice with Multiple Sclerosis and Optic Neuritis

MIANJIA (RHEA) LI ’18 Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene

HILARY LIU ’18 Virtual Screening and Drug Discovery: Finding Drugs that Inhibit the BCR-ABL1 Oncoprotein

NEELAM PANDYA ’18 Observation of the Self-Assembly of a Tripeptide Containing Three Hydrophobic Amino Acids

SYDNEY SILBERG ’18 Generation of a Self-Inactivating Cas9 System

CASSANDRA STECKER ’18 Uncovered from the Archives: Examining the Relationship Between the Pennsylvania Abolition Society and La Société des Amis des Noirs de Paris through the Abolition Experiment in Cap Français TAYLOR TRAPP ’19 Immunohistochemical Assessment of Phosphodiesterase 5A (PDE5A) Expression in Infant Right Ventricular Failure THE BALDWIN REVIEW 2017

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ELIZA THALER â&#x20AC;&#x2122;18 Eliza Thaler is a senior from Ardmore, PA, and has attended Baldwin since Kindergarten. She is the head of Abacus Club and Model Congress and is captain of the field hockey team. Eliza also participates in Lamplighters and tutors younger Baldwin students in reading and writing. In her free time, Eliza enjoys running and reading.

The Great Recession: An Explanation of the 2008 Catastrophe From Neuroscience to Macroeconomics Perspective By Eliza Thaler â&#x20AC;&#x2122;18

THE BALDWIN REVIEW 2017

The Great Recession: An Explanation of the 2008 Catastrophe From Neuroscience to Macroeconomics Perspective | Eliza Thaler ’18

Since the market collapse of 2008 nearly a decade ago, there has been much debate and analysis concerning what exactly precipitated the massive crisis. Answers have cited duplicitous bankers, eager homeowners, inadequate regulation, incompetent politicians, hardwired neurobiological mechanisms, and more. The formation of the housing bubble, a complex, nonlinear process, was the culmination of these factors. The purpose of this paper is to show how it was not simply one of these factors at work but rather the culmination of their synergism that formed the the housing market bubble. This paper explains the bubble and subsequent crisis starting from a microscopic perspective, the neural coding scheme behind bubble formations, and ending with a macroscopic perspective, how the government responded to the crisis. Conventional asset pricing theory posits that markets are “unintentional and nonstrategic”.1 This means that returns on investments are determined only by price, which an individual person cannot affect. However, researchers at CalTech have shown that “the explicit information carried by prices and fundamental values accounts for significantly less variance in choice behavior when subjects are trading in bubble markets.”2 In their paper, “In the Mind of the Market: Theory of Mind Biases Value Computation during Financial Bubbles”, CalTech researchers, Benedetto De Martino, John P. O’Doherty, Debajyoti Ray, Peter Bossaerts, and Colin Camerer explore the neural coding scheme behind the formation of bubbles in competitive markets. In their experiment, the researchers asked their subjects to perform trades in an experimental bubble setting, in which the assets prices were above their intrinsic values. While trading, the subjects were connected to scans that traced the flow of blood to different parts of the brain. The core of their research explores whether theory of mind is at work during market bubbles. As defined by the above researchers, theory of mind (ToM), is the “ability to infer intentions of other agents”.3 Essentially, this is the ability for humans to make inferences about the mental states of others, whether it be about their beliefs, intentions, emotions, wants, knowledge, or thoughts in general. In this study, the researchers found that two frontal portions of the brain, the dorsomedial prefrontal cortex (dmPFC), and the ventromedial prefrontal cortex (vmPFC) were activated and highly correlated in bubble markets. Although ToM is a complex phenomenon, involving several portions of the brain, the researchers in this study focused primarily on the dmPFC as the region that computed social signals. The vmPFC is primarily responsible for processing value computations. In bubbles, these two regions of the brain are correlated because humans are not only assigning an overvalued price for an asset, but they are also trying to predict the behavior 1 Benedetto De Martino et al., In the Mind of the Market: Theory of Mind Biases Value Computation during Financial Bubbles, [Page 1], September 18, 2013, accessed August 10, 2017, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3781325/pdf/main. pdf. 2 Ibid. 3 De Martino et al., In the Mind, [Page 2]. A2

The Great Recession: An Explanation of the 2008 Catastrophe From Neuroscience to Macroeconomics Perspective | Eliza Thaler ’18

and intentions of other players in the market. The researchers found that in market bubbles, people’s decisions are driven more by ToM processes, such as visualizing the path of future prices and the actions of other traders, rather than by explicit information available in the market. The two regions are correlated because when trying to infer the intentions of the other players in the market, individuals change their assessment of how valuable an asset is.4 As indicated in Figure 1, increased activity in the vmPFC was correlated with a higher bubble susceptibility index. This means that in market bubbles, individuals are more likely to over-value an asset. Figure 2 illustrates the functional coupling between the dmPFC and vmPFC during financial bubbles. Figure 3 shows the over-valuation of assets in bubble markets, compared to the pricing of assets in non-bubble markets, where the price of the asset remains closer to its fundamental value. According to De Martino, “In a bubble situation, people start to see the market as a strategic opponent and shift the brain processes they’re using to make financial decisions”.5 Thus, while ToM can be advantageous in a multitude of scenarios, it is actually maladaptive in the case of financial markets.6

4 Ibid. 5 De Martino et al., In the Mind, [Page 4]. 6 Ibid. 7 Ibid.

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The Great Recession: An Explanation of the 2008 Catastrophe From Neuroscience to Macroeconomics Perspective | Eliza Thaler â&#x20AC;&#x2122;18

Prior to 2008, several distinct groups in the finance sector made dangerous assumptions that contributed to the massive housing bubble and subsequent crash. For decades, there was an overwhelming consensus that investing in the housing market was a low-risk, high-return investment. This was the case because mortgages were typically 30-year fixed rate loans on top of a 20% down payment. If the down payment was not met, mortgage insurance was required.10 Thus, people had to demonstrate their earnings were adequate to pay the monthly mortgage, making investments in loans quite safe. However, in the 1990â&#x20AC;&#x2122;s, several governmental policies that aimed to increase homeownership among lower income families and generate more competition in the mortgage loan market were enacted. Because the Department of Housing 8 Ibid. 9 Ibid. 10 Jeff Holt, A Summary of the Primary Causes of the Housing Bubble and the Resulting Credit Crisis: A Non-Technical Paper, [Page 1], accessed August 10, 2017, https://www.uvu.edu/woodbury/docs/summaryoftheprimarycauseofthehousingbubble. pdf. A4

The Great Recession: An Explanation of the 2008 Catastrophe From Neuroscience to Macroeconomics Perspective | Eliza Thaler ’18

and Urban Development started to augment the number of mortgage loans to lower-income families, Fannie Mae and Freddie Mac, two government sponsored enterprises (GSE’s) were forced to relax the standards that mortgages had previously been required to meet. Income and down payment stipulations were minimized. Another cause of the relaxed mortgage standards was the introduction of the internet in 1990.11 Because people could more easily compare different mortgage deals on the internet, there was greater competition between companies. Thus, mortgage companies were incentivized to give the lowest prices as possible in order to offer a more appealing deal than their competitor. These relaxed standards translated to the increased securitization of subprime mortgages. Subprime mortgages are mortgages given to people with poor credit risk, and have foreclosure rates ten times higher than prime mortgages. These relaxed standards prompted greater speculation in the market. After the market crashed, however, this resulted in an enormous increase in mortgage defaults when housing prices stopped rising.12 Another factor that contributed to the massive housing bubble were low interest rates. The Federal Reserve set low interest rates for several reasons. Primarily, foreign countries such as China and Japan invested heavily in the United States’ bonds economy, as they desired low-risk, highreturn investments. Initially, the foreign countries invested in government securities; however, over time, they desired higher returns on their investments, so they began to invest in mortgage backed securities issued by Fannie Mae and Freddie Mac.13 The foreign countries viewed these investments as low-risk because if they failed, the United States government would bail them out. The foreign investors became even more enamoured by the idea of high-return investments, so they moved onto investing in mortgage-backed securities sold by Wall Street firms.14 Foreign investors this time believed that the investments were low risk because credible rating agencies such as Moody’s and Standard and Poor’s gave the mortgages top-notch ratings. Thus, interest rates were kept low in part because there was a steady flow of money into the US economy by foreign investors. In fact, according to Ben Bernanke, the former chair of the Federal Reserve, “The net inflow of foreign saving to US increased from 1.5% of GDP in 1995 to 6% in 2006.”15 The other reason why interest rates were kept low in the years preceding the 2008 crash was because the Federal Reserve wanted to stimulate the economy after the 2001 recession. Low interest rates allowed people to pay low monthly mortgage payments even as housing prices increased, thus contributing to the bubble. Furthermore, low short-term interest rates prompted utilization of adjustable rate mortgages (ARMs). Because household incomes could not keep pace 11 Holt, A Summary, [Page 3]. 12 "The Origins of the Financial Crisis: Crash Course," The Economist, September 7, 2013, [Page 2], accessed August 10, 2017, https://www.economist.com/news/schoolsbrief/21584534-effects-financial-crisis-are-still-being-felt-five-years-article. 13 Holt, A Summary, [Page 3]. 14 Ibid. 15 Ibid.

THE BALDWIN REVIEW 2017

The Great Recession: An Explanation of the 2008 Catastrophe From Neuroscience to Macroeconomics Perspective | Eliza Thaler ’18

with the rise in housing prices, fixed rate mortgages were out of reach for many prospective home buyers. ARM’s supplied homeowners with cheaper monthly payments at the outset of the loan because short-term interest rates began at a significantly lower rate than long-term interest rates.16 People with poor credit who should not have been granted mortgages therefore did indeed receive mortgages. The lower, more affordable monthly mortgage payments offered by ARM’s were conducive to the rising home prices. Figure 4 shows the declining average mortgage interest rates from 1982 to 2005. 17

There was a significantly heightened state of speculative fervor, also referred to as irrational exuberance, regarding the housing market prior to 2008. Because housing prices had consistently risen since the Great Depression in the 1930’s, people assumed that housing prices would continue to rise.19 This over-extrapolation proved to be especially dangerous considering that the stock market is volatile, with constantly changing conditions. Our brain is hardwired to detect patterns in order to make inferences about the future, which is an underlying reason why people assumed that housing prices would continue to increase. People were convinced that the mortgages were valuable investments, and that there would be a low mortgage default rate because housing prices continued to climb. The reasoning at the time rested solely on the faulty 16 Holt, A Summary, [Page 4]. 17 Ibid. 18 Ibid. 19 Nicholas Barberis, Psychology and the Financial Crisis of 2007-2008, [Page 12], accessed August 2011, http://faculty.som.yale.edu/nicholasbarberis/cp10.pdf. A6

The Great Recession: An Explanation of the 2008 Catastrophe From Neuroscience to Macroeconomics Perspective | Eliza Thaler â&#x20AC;&#x2122;18

belief that because the market acted a certain way in the past, it would act the same way in the future. The flaw in this inference is that stock markets do not behave in a consistent manner. This perfectly illustrates what the CalTech researchers found in their study: they discovered that in bubble markets, participants are more likely to make decisions based on inferences about what think the market or participants in the market will do, and that they are less likely to make investment decisions based on explicit information.20 The rational thing to do in this circumstance would have been to ignore the overextrapolation that housing prices would continue to increase. However, instead of following stock market principles, the majority of people continued to invest in the subprime mortgages since humans are hardwired to use social signals, which, in this case, were the high ratings and the fact that everyone seemed to believe the housing prices would continue to rise. People were making decisions on what they thought was true, but the mistake was that people were following an incorrect, ruinous signal. The government did not take action to control rising home prices, because they did not realize that a bubble existed. Mortgage lenders continued to give triple A ratings to subprime mortgages and adjustable rate mortgages. Banks reasoned that these mortgages would have low default rates if housing prices continued to rise. Thus, the bubble was further augmented because there was an oversupply of credit to homebuyers in the form of subprime loans.21 Rating agencies may have given overly high ratings to subprime and adjustable rate mortgages for several reasons. Primarily, when the banks ran out of prime mortgages, mortgages with low default rates, to sell, they started bundling together subprime mortgages, mortgages with a high default rate, and sold them with high ratings (AAA) to investors. Additionally, rating agencies were paid by banks that issued the mortgages, so it was in their best interest, monetarily, to give the banks what they wanted: high ratings for all mortgages, prime and subprime. Lastly, as mentioned above, the agencies simply over extrapolated the past growth in housing prices too far into the future, which resulted in them being unable to predict the large number of subprime defaults.22 Over time, people were unable to pay back their mortgages, so they started to default on their mortgages, and simply walked away from their homes, sometimes, without evening paying a down payment.23 Each time a homeowner defaulted his or her subprime mortgage, a part of the bundle cracked. Eventually, when all of the subprime mortgages in the bubble defaulted, the investment was lower in value. Thus, banks were stuck with a large number of houses for which they had offered loans, but that people could not pay back. Because few people were able to pay 20 De Martino et al., In the Mind, [Page 2]. 21 "The Origins of the Financial Crisis: Crash Course," The Economist, September 7, 2013, accessed August 10, 2017, https:// www.economist.com/news/schoolsbrief/21584534-effects-financial-crisis-are-still-being-felt-five-years-article. 22 "The Origins," [Page 3]. 23 Holt, A Summary, [Page 5]. THE BALDWIN REVIEW 2017

The Great Recession: An Explanation of the 2008 Catastrophe From Neuroscience to Macroeconomics Perspective | Eliza Thaler ’18

for houses without manipulated mortgage deals, the banks had to significantly lower the housing prices so that people could afford to buy them and so that the banks would stop losing money. Figure 5 depicts the drop in housing prices preceding the crash.

On September 16, 2008, the Federal Reserve made known that it planned to bail out the insurance company AIG, which no longer had available cash. AIG’s lack of liquidity was especially concerning because the company was struggling to remit credit default swaps that it had supplied against mortgage backed securities that were now collapsing. On the Friday, September 19, the Federal Reserve instituted an Asset-Backed Commercial Paper Money Mutual Fund Liquidity Facility, loaning banks $122.8 billion to purchase commercial paper from money market funds.25 This move on the part of the Fed made public that credit markets were in part frozen. Although the bubble had been building up for a long period of time, the stock market finally crashed on September 29, 2008, subsequent to House Republicans voting down the Bank Bailout Bill. The Dow Jones Industrial Average fell 777.68 points on the one day, the most precipitous drop in history. The market lost $1.2 trillion, Standard and Poor’s 500 lost 8.8%, and Nasdaq composite fell 9.1%. Earlier that month, on September 16, 2008, Lehman Brothers declared bankruptcy. Prior to the vote, stocks had started to fall due to the uncertainty of whether or not Congress would pass the $700 billion bank bailout plan.26 People were apprehensive that Congress would not deliver 24 Ibid. 25 Robert Rich, "The Great Recession," Federal Reserve History, accessed August 10, 2017, https://www.federalreservehistory. org/essays/great_recession_of_200709. 26 Kimberly Amadeo, "Stock Market Crash of 2008," The Balance, accessed August 10, 2017, https://www.thebalance.com/ stock-market-crash-of-2008-3305535. A8

The Great Recession: An Explanation of the 2008 Catastrophe From Neuroscience to Macroeconomics Perspective | Eliza Thaler â&#x20AC;&#x2122;18

a solution for the basically frozen credit markets. Frozen markets occur when banks stockpile cash, rendering it challenging for individuals and businesses to get loans. The Bank Bailout Bill, composed by the Secretary of Treasury, Hank Paulson, stimulated banks to mutually lend by the government purchasing bad mortgage debt. Additionally, Congressional lawmakers included stipulations that safeguarded taxpayers and allowed them to profit if the companies experienced success. However, the House Republicans shot down the bill and unleashed a massive wave of panic in markets. The other piece of bad news on that September morning was that Wachovia planned to sell its banking assets to Citigroup.27 Global markets were also in free fall due to their investments in the US economy, and oil prices plummeted.28 The Federal Reserve and central banks in Europe, Japan, and England stabilized the economy by doubling currency swaps to $620 billion. Liquidity for the frozen credit markets were provided by governments around the world. Despite passage of the Bank Bailout Bill in October, panic had already manifested, and the economy had lost 159,000 jobs in September. October would witness the loss of 240,000 more jobs. At the end of the year, the Dow ended at a horrifying 8,776.39, 34% less than last year. The economy started to turn around after Barack Obama was elected president. His stimulus plan stopped the economic free-fall, restoring investor confidence. The recession officially ended in June of 2009, but the US economy had lost in total 8.7 million jobs.29 There are several implications and lessons learned from this catastrophe. In a New York Times Op-Ed, David Einhorn and Michael Lewis emphasize the damaging role of the corrupt rating agencies. They argued that the sizeable bonuses given to the Wall Street Banks in return for giving faulty high ratings should have been more strictly regulated.30 The government did not step in to help alleviate the crisis until a point too late in time. Thus, a key lesson from this crisis is that the government should impose somewhat stricter regulation on the market. The patterns of neurological behavior that drive economic decision-making should be considered in market analysis, and must be monitored during periods of economic uncertainty. Rather than making reactive corrections in the face of crisis, prophylactic management of economic behaviors founded in neuroscience principles might prevent the next market crash. Lastly, the flexibility of an independent, apolitical Federal Reserve allowed the US economy to rebound from the brink of ultimate destruction much more readily than many other developed economies, including the European Union. Preserving this independence is crucial. 27 Amadeo, "Stock Market," The Balance. 28 Alexandra Twin to CNN newsgroup, "Stocks Crushed," September 29, 2008, accessed August 10, 2017, http://money.cnn. com/2008/09/29/markets/markets_newyork/. 29 Twin to CNN newsgroup, "Stocks Crushed." 30 David Einhorn and Michael Lewis to New York Times newsgroup, "The End of the Financial World as We Know It," January 3, 2009, accessed August 10, 2017, http://www.nytimes.com/2009/01/04/opinion/04lewiseinhorn.html. THE BALDWIN REVIEW 2017

The Great Recession: An Explanation of the 2008 Catastrophe From Neuroscience to Macroeconomics Perspective | Eliza Thaler ’18

REFERENCES: Amadeo, Kimberly. “Stock Market Crash of 2008.” The Balance. Accessed August 10, 2017. https://www.thebalance.com/stock-market-crash-of-2008-3305535. Barberis, Nicholas. Psychology and the Financial Crisis of 2007-2008. Accessed August 2011. http://faculty.som.yale.edu/nicholasbarberis/cp10.pdf. De Freitas, Will. Will De Freitas to Business Insider newsgroup, “New Study Suggests Our Brain Biology Is To Blame For The Financial Crisis,” September 18, 2013. Accessed August 10, 2017. http://www.businessinsider.com/neuroscience-and-the-financial-crisis-2013-9. De Martino, Benedetto, John P. O’Doherty, Debajyoti Ray, Peter Bossaerts, and Colin Camerer. In the Mind of the Market: Theory of Mind Biases Value Computation during Financial Bubbles. September 18, 2013. Accessed August 10, 2017. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3781325/pdf/main.pdf. Einhorn, David, and Michael Lewis. David Einhorn and Michael Lewis to New York Times newsgroup, “The End of the Financial World as We Know It,” January 3, 2009. Accessed August 10, 2017. http://www.nytimes.com/2009/01/04/opinion/04lewiseinhorn.html. Holt, Jeff. A Summary of the Primary Causes of the Housing Bubble and the Resulting Credit Crisis: A Non-Technical Paper. Accessed August 10, 2017. https://www.uvu.edu/woodbury/docs/summaryoftheprimarycauseofthehousingbubble.pdf. “The Origins of the Financial Crisis: Crash Course.” The Economist, September 7, 2013. Accessed August 10, 2017. https://www.economist.com/news/schoolsbrief/21584534-effects-financial-crisis-are-still-beingfelt-five-years-article. Rich, Robert. “The Great Recession.” Federal Reserve History. Accessed August 10, 2017. https://www.federalreservehistory.org/essays/great_recession_of_200709. Surowiecki, James. “What Precipitated the Stock Market Crash of 2008?” The New Yorker, January 5, 2009. Accessed August 10, 2017. http://www.newyorker.com/business/jamessurowiecki/what-precipitated-the-stock-market-crash-of-2008. Twin, Alexandra. Alexandra Twin to CNN newsgroup, “Stocks Crushed,” September 29, 2008. Accessed August 10, 2017. http://money.cnn.com/2008/09/29/markets/markets_newyork/. A10

SIMI BLEZNAK â&#x20AC;&#x2122;19 Simi Bleznak, a junior from Wynnewood, PA, has attended Baldwin since Pre-K. Outside of the classroom she is a junior head of Lamplighters, an Athletics Association representative and a member of Model Congress, the yearbook and Abacus. In addition, she is the co-captain of the basketball team and the goalkeeper for the soccer team. In her free time, Simi enjoys taking pictures.

An Overview of Multiple Sclerosis Simi Bleznak â&#x20AC;&#x2122;19 Bar-Or Lab

THE BALDWIN REVIEW 2017

An Overview of Multiple Sclerosis | Simi Bleznak ’19

What is Multiple Sclerosis? Across the globe, more than two million people live with Multiple Sclerosis (MS),1 an autoimmune disease in which the sheaths around a person’s nerve cells in the Central Nervous System (CNS) are attacked by the immune system, impairing the flow of information to and from the brain and spinal cord.2 The disease, believed to be triggered by both environmental and genetic factors, varies in severity as well as long and short term effects and has been categorized into four types or phases based on its clinical features.3 The first, known as relapsing-remitting MS, occurs when a person experiences a flare-up such as tingling, bowel trouble, or loss of vision which can partially or fully resolve after days or weeks. This period of remission typically lasts for months or years before the next symptom presents itself. Relapsingremitting MS makes up about 85% of the cases, and tends to develop into secondary-progressive MS, which is characterized by increased attacks in which symptoms may not completely resolve and instead may continue to worsen.4 The third clinical course of MS is primary progressive MS and is the progression of symptoms without flare-ups or recovery time. This accounts for about 10% of MS cases. The final course of MS is progressive-relapsing MS in which a person’s symptoms are continuously increasing from onset in addition to having relapses.5 This most extreme type of MS is found in about 5% of patients.6

The Four Types of MS7

_____________________________________ 1 “Who Gets MS?” National Multiple Sclerosis Society, accessed July 10, 2017, http://www.nationalmssociety.org/. 2 “What is MS?” National Multiple Sclerosis Society, accessed July 10, 2017, http://www.nationalmssociety.org/. 3 “The 4 Types of MS,” Bayer Health Care - Multiple Sclerosis, accessed August 11, 2017, https://www.multiplesclerosis.com/us/treatment.php. 4 Ibid. 5 Amit Bar-Or, email, September 24, 2017. 6 Bayer Health Care, “The 4 Types of MS.” 7 http://www.clevelandclinicmeded.com/medicalpubs/diseasemanagement/neurology/multiple_sclerosis/images/ figure-1.jpg B2

An Overview of Multiple Sclerosis | Simi Bleznak ’19

Who is affected? MS is typically diagnosed in a person between the ages of twenty and fifty with pediatric MS being less common than MS in the adult population.8 MS has also been found to be more prevalent in women beginning after age twelve, indicating that puberty may play a role, although more conclusive research must be done to confirm.9 In addition, MS is found most often in Caucasians.10 It is now accepted that there are both genetic and environmental risks for MS. According to a study by the Canadian Collaborative Genetics Group, full siblings of people with MS have about double the risk of getting the disease compared to half siblings, suggesting that genetics do play a role.11 However, they are not the whole picture. People living farther from the equator are more prone to MS,12 with some speculating a connection to vitamin D levels, although more research must be done. Finally, migration is also important in understanding MS risk factors.13 The risk for those who migrate at a young age correlates with the risk of the region they travel to, whereas people who migrate later on retain the risk of their native land.14

MS Prevalence Map15

_____________________________________ 8 National Multiple Sclerosis Society, “What is MS?” 9 “MS Scientific Research Foundation-supported study highlights puberty as a significant factor in development of multiple sclerosis,” Multiple Sclerosis Society of Canada, accessed July 20, 2017, https://mssociety.ca/research-news/article/ms-scientificresearch-foundation- Supported-study-highlights-puberty-as-a-significant-factor-in-development-of-multiplesclerosis. 10 National Multiple Sclerosis Society, “Who Gets MS?” 11 A.d. Sadovnick, Dyment D.a., Ebers G.c., and Risch N.j, “Evidence for genetic basis of multiple sclerosis,” The Lancet 347, no. 9017 (1996): 1728-730, accessed July 20, 2017, https://www.ncbi.nlm.nih.gov/pubmed/8656905. 12 National Multiple Sclerosis Society, “Who Gets MS?” 13 Ruth Ann Marrie, “Environmental risk factors in multiple sclerosis aetiology,” The Lancet Neurology 3, no. 12 (2004): 709-18, accessed July 20, 2017. doi:http://www.direct-ms.org/sites/default /files/MarrieRiskFactors.pdf. 14 Amit Bar-Or, email, September 24, 2017. 15 https://multiplesclerosis.net/wp-content/uploads/2013/01/global_2x.png THE BALDWIN REVIEW 2017

An Overview of Multiple Sclerosis | Simi Bleznak ’19

So what is happening? The CNS, comprised of the cells in the brain and spinal cord, controls body functions by receiving and interpreting information. These nerve cells have many different components that each perform unique tasks, but it is the coating around axons known as the myelin sheaths that play a key role in MS pathogenesis.16 Typically, these sheaths facilitate the passage of messages smoothly through the nerve cell. In people with MS, however, immune cells are able to pass through the blood-brain barrier and damage these myelin sheaths (demyelination), which in turn damages the flow of information and can lead to a variety of symptoms or effects. Also, as a result to the harm done to nerve tissue, lesions begin to form in the brain and spinal cord.17

Brain Lesion in MS Patients18

Demyelination19

_____________________________________ 16 “What is MS?” MS International Federation, accessed August 11, 2017, https://www.msif.org/about-ms/what-is-ms/ 17 Ibid 18 https://img.webmd.com/dtmcms/live/webmd/consumer_assets/site_images/media/medical/hw/h9991221.jpg 19 http://drwilderman.com/wp-content/uploads/2013/01/demyelinating-disorder.jpg B4

An Overview of Multiple Sclerosis | Simi Bleznak ’19

What is causing MS? When a person catches a cold, their innate immune system responds quickly by attacking these foreign pathogens without specificity. In contrast, the adaptive immune system works to identify and remember specific pathogens so that each time they appear, the body can fight off the illness in a targeted and more efficient manner.20 Two types of cells found in the adaptive immune system are T cells and B cells, both of which work to fight off viruses. T cells attack these viruses directly, whereas B cells produce antibodies that do so.21 However, in MS, a person’s immune system attacks self-antigens. In other words, the body is fighting itself. Up until recently, researchers focused in on T cells as the main culprit for CNS tissue attacks because of their presence in experimental autoimmune encephalitis (EAE) models, mouse models used to learn about MS.22 Now, however, doctors and researchers are beginning to recognize the effects of B cell depletion therapies. In a study conducted by Dr. Amit Bar-Or and colleagues, 104 patients with relapsing-remitting MS were split into two groups: 69 were placed on rituximab, an anti-CD20 drug that targets and depletes B cells, and the remaining 35 were placed on a placebo. After forty eight weeks, patients on rituximab were found to have had a decrease in the number of brain lesions as well as relapses.23 This advancement in MS treatment further facilitated B cell depleting medicine and research. Ocrelizumab, a recently FDA approved drug directed primarily to those with primary progressive MS24 and relapsing-remitting MS has been shown to slow down symptom development, and is the first to do so in primary progressive MS patients.25 These two drugs are just one example of the great strides made in recent decades to learn more about MS as a disease as well as how to treat it, and with further research there is no doubt that more knowledge will be acquired. _____________________________________ 20 Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune System in Health and Disease, 5th edition, New York: Garland Science; 2001, “Principles of innate and adaptive immunity,” https://www.ncbi.nlm.nih.gov/books/NBK27090. 22 Amit Bar-Or, “The Immunology of Multiple Sclerosis,” Seminars in Neurology 28, no. 1, 29-30. 23 Stephen L. Hauser, M.D., Emmanuelle Waubant, M.D., Ph.D., Douglas L. Arnold, M.D., Timothy Vollmer, M.D., Jack Antel, M.D., Robert J. Fox, M.D., Amit Bar-Or, M.D., Michael Panzara, M.D., Neena Sarkar, Ph.D., Sunil Agarwal, M.D., Annette Langer-Gould, M.D., Ph.D., and Craig H. Smith, M.D., for the HERMES Trial Group, New England Journal of Medicine, February 14, 2008, “B-Cell Depletion with Rituximab in Relapsing–Remitting Multiple Sclerosis,” http://www.nejm.org/doi/full/10.1056/NEJMoa0706383#t=article. 24 “FDA approves new drug to treat multiple sclerosis,” U S Food and Drug Administration, March 29, 2017, https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm549325.htm. 25 Amit Bar-Or, email, September 24, 2017.

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An Overview of Multiple Sclerosis | Simi Bleznak ’19

CITATIONS: Bar-Or, Amit. Email, September 24, 2017. Bar-Or, Amit. “The Immunology of Multiple Sclerosis.” Seminars in Neurology 28, no. 1, 29-30. “FDA approves new drug to treat multiple sclerosis.” U S Food and Drug Administration. March 29, 2017. https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm549325.htm. Hauser, Stephen L., M.D., Waubant, Emmanuelle, M.D., Ph.D., Arnold, Douglas L., M.D., Vollmer, Timothy, M.D., Antel, Jack, M.D., Fox, Robert J., M.D., Bar-Or, Amit, M.D., Panzara, Michael, M.D., Sarkar, Neena, Ph.D., Agarwal, Sunil, M.D., Langer-Gould, Annette, M.D., Ph.D., and Smith, Craig H., M.D., for the HERMES Trial Group. New England Journal of Medicine. February 14, 2008. “B-Cell Depletion with Rituximab in Relapsing–Remitting Multiple Sclerosis.” http://www.nejm.org/doi/full/10.1056/NEJMoa0706383#t=article Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The Immune System in Health and Disease. 5th edition. New York: Garland Science; 2001. “Principles of innate and adaptive immunity.” https://www.ncbi.nlm.nih.gov/books/NBK27090. Marrie, Ruth Ann. “Environmental risk factors in multiple sclerosis aetiology.” The Lancet Neurology 3, no. 12 (2004): 709-18. Accessed July 20, 2017. http://www.direct-ms.or g/sites/default/files/MarrieRiskFactors.pdf. “MS Scientific Research Foundation-supported study highlights puberty as a significant factor in development of multiple sclerosis.” Multiple Sclerosis Society of Canada. Accessed July 20, 2017. https://mssociety.ca/research-news/article/ms-scientific-research-foundationSupported-study-highlights-puberty-as-a-significant-factor-in-development-of-multiple-sclerosis. Sadovnick, A.d, D.a Dyment, G.c Ebers, and N.j Risch. “Evidence for genetic basis of multiple sclerosis.” The Lancet 347, no. 9017 (1996): 1728-730. Accessed July 20, 2017. https://www.ncbi.nlm.nih.gov/pubmed/8656905. “The 4 Types of MS.” Bayer Health Care - Multiple Sclerosis. Accessed August 11, 2017. https://www.multiplesclerosis.com/us/treatment.php.

An Overview of Multiple Sclerosis | Simi Bleznak ’19

“What is MS?” MS International Federation. Accessed August 11, 2017. https://www.msif.org/about-ms/what-is-ms/. “What is MS?” National Multiple Sclerosis Society. Accessed July 10, 2017. http://www.nationalmssociety.org/. “Who Gets MS?” National Multiple Sclerosis Society. Accessed July 10, 2017. http://www.nationalmssociety.org/.

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CAROLINE BUCHNER ’18 Caroline Buchner is a senior from Wayne, PA, and has been at The Baldwin School since 8th grade. She serves as head of Modern Science Club and as captain of Baldwin’s Varsity tennis team. Caroline is a staff writer for the Hourglass newspaper and participates in Girls Learn International, Skill Foundation and volunteering at Hospital of the University of Pennsylvania.

Increasing Fog2 Gene Expression in Cardiomyocytes Using a Plasmid By Caroline Buchner ’18 Marie Guerraty, MD, PhD Jim Wilson, MD, PhD University of Pennsylvania, The Rader Lab and The Wilson Lab, Philadelphia, PA Gene Therapy Summer Program 2017

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Increasing Fog2 Gene Expression in Cardiomyocytes Using a Plasmid | Caroline Buchner ’18, Marie Guerraty, MD, PhD, Jim Wilson, MD, PhD

INTRODUCTION

Coronary Microvascular Disease (CMVD) is defined as destruction or dysfunction of the

heart’s smaller arteries and results in significant cardiovascular morbidity and mortality. While the mechanism of the disease is poorly understood, we believe that genes involved in coronary development are important in adult coronary microvascular function.

A transcription factor is a protein that controls the rate of the transcription of genetic

information from DNA to mRNA. Fog2 (Friend of Gata2), a 4974 base pair co-transcription factor that binds with the Gata4 transcriptional regulator, plays a major role in angiogenesis, the formation of new blood vessels. In a recent study, Fog2 was inhibited in the cardiomyocytes of adult mouse hearts. This resulted in decreased expression of proangiogenic factors, such as Vegf, and increased angiogenic inhibitors.

Figure 1: 2009 Study Results of Fog2 Elimination in Mice1

The purpose of this current project is to perform further analysis on Fog2’s role in coronary

development by increasing Fog2 expression. We hypothesize that increasing Fog2 expression across a culture of cardiomyocytes in vitro would render a significant increase in the expression of proangiogenic factors.

METHODOLOGY RNA Isolation and cDNA Synthesis

Six heart muscle tissue samples were extracted from mice. The RNA was isolated using

Invitrogen TRIzol Reagent and alcoholic phenol extraction. The RNA was precipitated, washed, and solubilized. Following RNA isolation, cDNA was synthesized from the single stranded RNA

Increasing Fog2 Gene Expression in Cardiomyocytes Using a Plasmid | Caroline Buchner ’18, Marie Guerraty, MD, PhD, Jim Wilson, MD, PhD

following the High-Fidelity cDNA Reverse Transcription kit. RNA and DNA concentrations were assessed using absorbance spectrophotometry (Nanodrop).

Fog2 Amplification To clone the Fog2 cDNA, a Polymerase Chain Reaction was performed with 2X Phusion Master Mix. Both UTR and Restriction Enzyme forward and reverse primers were used for this reaction. The UTR primers were located at the UTR sites of the coding sequence (3456 bp), while the Restriction Enzyme primers were located at the 5’ and 3’ end of the sequence. The forward primer introduced the Spe1 Restriction Enzyme site and the reverse primer introduced the Xho1 Restriction Enzyme site.

Figure 2: Fog2 Coding Sequence and location of UTR and Restriction Enzyme Primers https://en.wikipedia.org/wiki/Messenger_RNA

Restriction Enzyme Digest

Amplified DNA and plasmids were digested with Spe1 and Xho1 restriction enzymes in

Cutsmart buffer (NEB). We incubated the samples at 37˚C for one hour and purified them in a 1% agarose gel electrophoresis. Later, we extracted the samples using the QIA Quick Gel Extraction Protocol.

Ligation

DNA ligase buffer, vector plasmid, insert DNA, water, and T4 DNA ligase were all used in

the ligation reaction to bind the insert DNA to the vector plasmid, following the Ligation Protocol with T4 DNA Ligase (M0202). We incubated the ligations overnight at 16˚C.

Transformation of Cells

The One Shot Top10 Competent E. Coli Cells were transformed with the ligated plasmid

through a process of heating and cooling. One 50 μl vial of One Shot cells was used along with each ligation sample mixed into the vial. 250 μL of pre-warmed S.O.C medium was also added to

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Increasing Fog2 Gene Expression in Cardiomyocytes Using a Plasmid | Caroline Buchner ’18, Marie Guerraty, MD, PhD, Jim Wilson, MD, PhD

the vial. After one hour of shaking at 37˚C, the transformation was spread on LB agar plates treated with ampicillin using MicroBeads and incubated overnight at 37˚C.

Results

The six heart cDNA samples had their concentrations measured listed below. Mouse heart cDNA sample

Concentration ng/μL

H1 1924.9 H2 1990.6 H3 2070.5 H4 1914.1 H1A 1935.9 H3A 1751.6 Table 1: Concentration of cDNA

Following various PCR trials with each sample, the samples titled “HIA” produced successful

amplification results. At the end of cloning, the HIA measured to be 373.0 ng/μL and the reamplified HIA stock measured to be 490.1 ng/μL. The two plasmid samples, amplified HIA, and reamplified HIA were successfully cut in the enzyme digest and purified in a gel.

Figure 3: Digested DNA, Plasmid and Undigested Plasmid for Reference

Increasing Fog2 Gene Expression in Cardiomyocytes Using a Plasmid | Caroline Buchner â&#x20AC;&#x2122;18, Marie Guerraty, MD, PhD, Jim Wilson, MD, PhD

The concentrations of the cut DNA and plasmid were measured as listed in the table below. Double Enzyme Digest Products

Concentrations ng/ÎźL

Plasmid 1

19.3

Plasmid 2

25.2

HIA 15.6 HIA re-amplified

17.8

Table 2: Concentrations of Cut DNA and Plasmid

The four samples of plasmid were successfully ligated. After the transformation of E. coli

cells with the ligated plasmid, a few successful colonies were found in the two agar plates, with one plate having sterile colonies for the control.

Figure 4: E. coli Cell Culture Example

DISCUSSION

We successfully cloned the Fog2 gene from the mouse hearts and introduced the

Restriction Enzyme sites, Spe1 and Xho1. We also cut a plasmid at these Restriction Enzyme sites and inserted the amplified Fog2 gene. The plasmid was then ligated. E. coli competent cells successfully grew colonies transformed with the ligated plasmid.

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Increasing Fog2 Gene Expression in Cardiomyocytes Using a Plasmid | Caroline Buchner ’18, Marie Guerraty, MD, PhD, Jim Wilson, MD, PhD

The next step of this project is to build a viral vector so that the overexpressed Fog2 gene

can be transmitted to cardiomyocytes in vitro. Using the Competent Cells that we transformed with the plasmid, this can be done. Eventually, a virus, specifically a lentivirus, can transmit the overexpressed Fog2 genetic information to a culture of cardiomyocytes. We will then analyze the expression of the proangiogenic factors.

We expect that increasing Fog2 will upregulate the angiogenic program, including Vegf

expression. Regulating and increasing proangiogenic factors could potentially lead to treatment of heart conditions such as CMVD.

ACKNOWLEDGMENTS

I would like to thank my mentor Marie Guerraty, MD, PhD, for guiding me through this

project. I would also like to thank everyone in the Rader Lab who taught me laboratory techniques and provided me the materials for my project. I couldn’t have done this research without the Gene Therapy Summer Program for High School Students, Laura Richman, MD, PhD, Jim Wilson, MD, PhD, Mrs. Christie Reed, and my family’s support.

REFERENCES 1. Zhou, Bin et al. “Fog2 Is Critical for Cardiac Function and Maintenance of Coronary Vasculature in the Adult Mouse Heart.” The Journal of Clinical Investigation 119.6 (2009): 1462–1476. PMC. Web. 10 Aug. 2017.

ANNA BUNTING ’18 Anna Bunting lives in Lower Merion, PA, and is a senior at Baldwin. She has attended Baldwin since 4th grade. This is Anna’s ninth year at Baldwin, as well as her ninth year living in the United States since moving from London in 2009. Anna has been playing on Baldwin’s field hockey teams since 6th grade, and she is a member of Baldwin’s select a capella group, the B-Flats. Anna is also the senior head of SPECTRUM, Baldwin’s GSA and is a part of the peer counseling program.

Ulster Scots: The Life Cycle of a Language By Anna Bunting ’18

THE BALDWIN REVIEW 2017

Ulster Scots: The Life Cycle of a Language | Anna Bunting ’18

The language or dialect of Ulster Scots, also known as Ullans1, is believed to have originated as a hybrid of Scots, Scottish Gallic, Irish Gaelic and English2 in the early 17th century. The language is spoken exclusively in the north of Ireland, in the province of Ulster3. This ancient province lies one third in the Republic of Ireland and two thirds in Northern Ireland, a constituent state of the United Kingdom. An international border has run through the province since the Partition of Ireland in 19224, and has been the focus of civil unrest in the north for much of the last 100 years. The Good Friday Agreement of April 1998 sought to end the most recent period of unrest, known as the “Irish Troubles” by addressing the concerns and common interests of both sides of the conflict: those who wanted to remain loyal to the British Crown (“Loyalists” or “Unionists”) and those who wanted this last remaining part of Ireland to rejoin with its republican neighbor (“Republicans” or “Nationalists”). At the heart of the agreement was a statement of mutual understanding of how these two identities were formed. The Good Friday Agreement included recognition of Ulster Scots as the language of this Loyalist community, a controversial provision derided by some as a token gesture5. This paper seeks to provide a context to Ulster Scots language and culture and to explain its relevance to the Loyalist community and to peace in Northern Ireland. It is unclear when Ulster Scots was first spoken. The language is closest to Scots, or Lallans (“lowlands”), a language of the Scottish Border region spoken in the 15th century, with origins in Old English and Norse. Its lexicon includes recognizable Anglo-Saxon words and phrases consistent with Northumbrian Old English which predates the Scots language. It is reasonable to surmise that an earlier form of hybrid dialect between Scotland and Ulster may have existed prior to the 1500s as a result of migration and trade between the two islands. The most logical and widely-accepted explanation for its emergence dates back to the Plantation of Ulster, a plan enacted by King James I of England during the 17th century, that led to the arrival of Scottish and English planters to Ulster6.

1 Gregg, R. J. "The Scotch-Irish Dialect Boundaries in Ulster" in Wakelin, M. F., Patterns in the Folk Speech of the British Isles (1972) London: Athlone Press 2 Trudgill, Peter. “Colonial Dialect Contact in the History of European Languages: On the Irrelevance of Identity to NewDialect Formation.” Language in Society 37, no. 2 (2008): 241–54. doi:10.1017/S0047404508080287. 3 Ulster Scots Agency. "An introduction to the Ulster Scots Language." Ulster Scots Agency. Last modified 2017. http://www.Ulster Scotsagency.com/what-is-Ulster Scots/language/. 4 The Stormont Papers. (Volume 2 (1922) / Page 147-8). 5 BBC. “Good Friday Agreement.” BBC. http://www.bbc.co.uk/history/events/good_friday_agreement. 6 Bardon, Jonathan. The Plantation of Ulster. N.p.: Gill & Macmillan, 2011.

Ulster Scots: The Life Cycle of a Language | Anna Bunting ’18

17th century settlement map of Ulster: “Undertakers” were wealthy landowners who undertook to import tenants from their own estates and “Servitors” were veterans of the army who sought land of their own in the plantation. Source: http://www.bbc.co.uk/history/british/plantation/settlement/index.shtml

A 1610 book encouraging Englishmen to settle in Ireland http://www.bbc.co.uk/history/british/plantation/planters/es03.shtml THE BALDWIN REVIEW 2017

Ulster Scots: The Life Cycle of a Language | Anna Bunting ’18

Parts of Ireland had been a colony of the English Crown since the mid 1100s with the arrival of Norman descendants of William the Conqueror, who invaded England in 10661. However, Ireland proved to be an onerous place to govern in the following centuries. Ireland was then a Lordship (delegates of the monarch ruled different regions of the island) and of considerably lesser importance than the threat to England from the continent to the east and the Scottish border to the north. Despite attempts to govern the Lordship of Ireland on behalf of successive English monarchs, constant uprisings and rebellions stopped the English Lord Lieutenants from achieving control much beyond the “Pale”, a limited area radiating westward from Dublin on the East Coast2. The “Old Irish” (original people) and the subsequently assimilated or “gaelicized” Hiberno-Normans3 were considered unreliable threats. Eventually, Henry VIII, after the English Reformation and his abandonment of the Catholic Church, wanted to gain greater control of Ireland and convert its people to his new Protestant Church of England. He and successive monarchs suppressed the Irish language and culture and confiscated land to give to protestant settlers more loyal to the Crown. This strict approach almost certainly hardened resistance to accepting the rule of England in Ireland and the Protestant faith. Henry’s grandson, James I, noticing this unrest, embarked on an arguably more benign policy of colonization through plantation, which the King called a “civilizing enterprise.” This exercise transplanted some 80,000 Scotish and English settlers, most of whom were Protestant, into a predominantly Irish-speaking Catholic province. Planters were given land confiscated from the gaelicized noblemen who had fled the country during Henry VIII’s rule. These settlers came from mostly agrarian backgrounds, and their quality of life was not much improved on the plantation. The planters perpetuated the agrarian economy of the previous inhabitants, but they lacked the rigid class system familiar to the original Irish people whose land these settlers inhabited4.

1 Campbell, Kenneth L. (2013). Ireland's History: Prehistory to the Present. A & C Black. p. 59. 2 Connolly, S. J. Contested Island: Ireland 1460–1630 (Oxford, 2007), p. 29. 3 Frame, Robin, English Lordship in Ireland 1318–1361: Clarendon Press ,1982. 4 Bardon, Jonathan. The Plantation of Ulster. N.p.: Gill & Macmillan, 2011.

Ulster Scots: The Life Cycle of a Language | Anna Bunting â&#x20AC;&#x2122;18

Figure: The Island of Ireland in 1450 showing the relatively small area of land around Dublin, controlled directly by the English Crown, known as the Pale (n.b. in this map, Hiberno-Normans are referred to as Anglo-Irish Lords). Source: http://www.levindor.ch/burnatty/Images/Map/Ireland/Historic/1450.gif

Most planters lived in abject poverty. It was in these circumstances that these people, far from their home, in a country with a different language, forged their own identity in a siegelike mentality. In subsequent centuries, relationships between the planters and the native Irish populations were often affected by turmoil in England and Scotland as well as the divided loyalties that each population felt for their neighbors1. 1 Bardon, Jonathan. The Plantation of Ulster. N.p.: Gill & Macmillan, 2011.

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Ulster Scots: The Life Cycle of a Language | Anna Bunting ’18

Another significant event that would shape the future of Ulster Scots was the partition of Ireland in 1921. From the time of the plantation to the beginning of the 20th century, the primarily Presbyterian population had become the majority in six of the nine northern counties which lay in the province of Ulster. Unlike most people in the remaining 26 counties of Ireland, these Ulster protestants wanted to remain a part of the United Kingdom, with which they strongly identified1. After the Irish War of Independence, the Irish Free State was formed in December of 1921, together with the State of Northern Ireland with an international border partitioning the island. Later, after the Second World War, the Irish Free State left the British Commonwealth and became a Republic while Northern Ireland remained a locally governed constituent state within the United Kingdom. The border dividing the two parts of Ireland was designed by the British to reduce the potential for conflict, a tool also used in Palestine and India later in the 20th century with similar outcomes2. In Northern Ireland, civil disturbances broke out sporadically in the 1950s and by 1969 became a gruesome feature of daily life in the country. Some 3,300 people lost their lives between 1969 and 1998 and the violence perpetuated the divisions between the Loyalist and Republican communities. Republicans rallied around the Irish flag and crucially, the Irish language, a symbol of the oppression which dated back to the earliest invasion of the English3. To mirror these cultural symbols for the loyalist community, the language of Ulster Scots became a crucial part of the Good Friday agreement as a political symbol of identity with which to participate in peace talks as an equal partner. Beyond the evidence of the role of the Ulster Scots language as a political keystone, there is significant evidence of a culture driven by the language. The Linen Hall library in Belfast has an extensive collection of Ulster Scots literature, mostly poetry from the 18th century4. In 1728, William Starrett started writing poems in Ulster Scots, making use of its more lyrical qualities than English to create unique rhythmic effects5. This approach caught the attention of Edinburgh poet Robert Fergusson, who began to write his poems in Scots, which in turn influenced Robert Burns, the most celebrated poet in Scotland. Burns’ poetry promoted a short-lived revival of the Scots language, although his poems were originally published in 1778, not in Scotland but in Belfast, Ireland, where the text was easily understood by Ulster Scots communities because of Ulster Scots and Lowland Scots’ mutual intelligibility. His poetry influenced many and helped develop 1 Bardon, Jonathan. A History of Ulster. Dundonald, Belfast, Northern Ireland: Blackstaff Press. 1992. 2 MFPP Working Paper No. 2, "The Creation and Consolidation of the Irish Border" by KJ Rankin and published in association with Institute for British-Irish Studies, University College Dublin and Institute for Governance, Queen's University, Belfast 3 Taylor, Peter. 1999. Loyalists: war and peace in Northern Ireland. New York: TV Books. 4 Linen Hall Library. "Irish and Reference." Linen Hall Library. https://www.linenhall.com/pages/irish-and-reference. 5 Erskine, John, ed. "A supplement to the Annotated Bibliography of Ulster Scots Language and Literature." Ulster Scots Academy. http://www.Ulster Scotsacademy.com/ullans/12/bibliography-of-ulster Scots.php. D6

Ulster Scots: The Life Cycle of a Language | Anna Bunting ’18

the modern ‘rhyming weaver’ style, a structure frequently used by Burns1. Each verse of a poem written in this style were formatted to fit “Habbie stanza”. This form was named after a poem titled “Habbie Simpson, the Piper of Kilbarchan” written in Scots in the early part of the 17th century. The stanza has six staves or lines, and includes four basic repeating rhythmic units, or metrical feet, on “a” rhymes, and two metrical feet on “b” rhymes. The rhyme scheme for each stanza follows an “a a a b a b” rhythmic scheme2: Welcome, my frien’s, — ye’re just in time, The kettle’s on, an’ soon will chyme; An’ gif, tho’ us’d to strains sublime, Ye’ll listen me, I’ll clear my throat, an’ rudely rhyme In praise o’ Tea James Orr, 1804, extract from Tea

The modern weaver style became popular in the working class communities of the counties Antrim and Down, during the 18th century3. These Ulster Scots poets embraced the rhyming weaver style most probably because they lacked a formal education, and didn’t need to know Standard English in order to pen it, and the Habbie stanza format was simple. The rhyming weaver poets were popular and met in ‘reading houses’ to gather in the community and share poems that addressed every day life and political ideas4. Ulster Scots poets like James Orr and Robert Huddlestone demonstrated that poetry could still be complex and thoughtful when not written in Standard English. Both were prolific writers though Orr was considered more masterful. His poems were often political, drawn from his experiences in the Irish Rebellion of 1798, in which he fought for the United Army of Ulster. Orr was a radical, and often stated his political opinions through his poetry, offering insight on England’s presence in Ulster and emphasizing the importance of the Protestant community supporting itself in times of political turmoil. His emphasis on the importance of a close-knit Ulster community is illustrated in much of his work and provides a valuable perspective on this historical period5. 1 Minding Our Language. BBC, 2015. 2 Hosbaum, Philip. Metre, Rhythm and Verse Form. London: Taylor and Francis, 1996. Digital file. 3 Heritage Lottery Fund. "James Orr." The Weaver's Trail. http://www.weavers-trail.co.uk/james-orr-ulster-weaver-poets. 4 Minding Our Language. BBC, 2015. 5 Heritage Lottery Fund. "James Orr." The Weaver's Trail. http://www.weavers-trail.co.uk/james-orr-ulster-weaver-poets.

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Ulster Scots: The Life Cycle of a Language | Anna Bunting ’18

By the mid 1800s, the Ulster Scots language began to decline due to pressure from the British government, which actively discouraged usage in schools. The Education Act of Ireland and Scotland declared that English was to be the accepted language and all books and lessons in schools were to be conducted in English. Children were punished for using the language in class and were told they sounded uneducated1. This likely contributed to the decline of Ulster Scots literature and dilution of the culture. Ulster Scots music emerged in much the same way as the language. Up until the 1800s, traveling harpists were popular with Anglo-Irish and Gaelic aristocracy. As the popularity of harp music declined in the British Isles, nobility began to favour the more classical European style of music. Out of work and suddenly excluded from the upper echelons of society, these musicians returned to Ulster2. Here, the Ulster Scots shared a similar standard of living and provided these harpists with the community that had managed to bring the working class together back during the Plantation of Ulster. This was the catalyst for the synthesis of the quintessential Ulster Scots sound. Harpists, fiddlers and pipers interacted in Ulster, producing an amalgamation of their separate genres. The result of this collaboration was a genre of music containing aspects of Irish, Scottish and English music from the 18th and early 19th century. Music in the Ulster Scots community was closely connected to their tradition of parading within fraternal groups including the “Ribbonmen” and “Orangemen”, and commemorated historic victories for the community over their rivals. The link between military-style marching and music was strengthened because of the emphasis on rhythm in Ulster Scots music. The Lambeg and Fife drums were used heavily in many marching songs, and are both specific to the counties of Antrim and Down3.

Lambeg drummers in Newtownards, Co. Down http://abalmoralperspective-hma.blogspot.com/2010/02/ ulster Scots-lambeg-drum-2.html

1 Ulster Scots Agency. "An introduction to the Ulster Scots Language”. Ulster Scots Agency. Last modified 2017. http://www.Ulster Scotsagency.com/what-is-ulster Scots/language/. 2 "Ulster Scots Music." Queens University Belfast. http://www.qub.ac.uk/sa-old/resources/Belfast_Project/Sites_2004/USFO/pages/Ulster ScotsMusic.html. 3 Ibid. D8

Ulster Scots: The Life Cycle of a Language | Anna Bunting ’18

The tradition of marching and rallying to celebrate a cause is not unique to Ulster, but it is one of the Ulster Scots culture’s most defining attributes. Marching brought the community together to celebrate traditions such as the Twelfth of July Parade, headed by the Orangemen. The ‘Twelfth’ marching tradition began in 1795, commemorating the victory of the (Protestant) William of Orange over the (Catholic) James II at the Battle of the Boyne (1690). This victory resulted in a Protestant English monarchy and the reign of William and Mary. Later, during “The Troubles”, the period of civil unrest in the late 20th, Republicans in Northern Ireland saw the marches, led by Loyalists, as disrespectful and provocative. The Loyalists, however, saw it as an expression of their cultural identity and a celebration of the marching tradition that holds their community together. The culture of Ulster Scots is derived from the emphasis the community had on unity, bringing its eclectic group of cultures together. The perpetuation of the Ulster Scots language and culture is regarded by the Loyalist community as an existential issue, and was therefore an important component of the peace negotiations which ended the Troubles. What the music style, marching traditions and language represent to the Ulster Scots people is a history of solidarity and the coalition of different and even feuding cultures. Ulster Scots finally became legally recognized as a language in the European Charter of 2000, which sought to create common laws that every country in Europe would follow. The Charter states that “The Parties undertake to promote...mutual understanding between all the linguistic groups of [Ireland] and in particular the inclusion of the respect, understanding and tolerance in relation to the Ulster Scots language...and encouragement of the mass media to pursue the same objective.”1 Examining the Loyalists’ decision to push the recognition of the Ulster Scots language in the context of its rich history gives credence to their logic and the importance behind the traction Ulster Scots received in the Good Friday Agreement. The Ulster Scots Agency or Boord o Ulster Scotch currently works to achieve the “promotion of greater awareness and use of Ullans and of Ulster Scots cultural issues, both within Northern Ireland and throughout the island,”2 and as of 2011, roughly 140,000 people on the island claim to speak the language3. In order for Ulster Scots to remain a relevant and legitimate language and a useful point of political leverage, it needs to remain important to the Ulster-Scots community. Today, Ulster Scots as a language is an important political tool for holding the peace in Northern Ireland, which is a worthy benefit on its own. However, the richness and depth of its impact on culture would suggest that investment in its development would yield even greater return. 1 European Union. 2000. Charter of Fundamental Rights of the European Union. Official Journal of the European Union C346/1 Article 7 Section 3. Brussels: European Union. 2 Ulster Scots Agency. "About Us." Ulster Scots Agency. Last modified 2017. http://www.Ulster Scotsagency.com/about-us/. 3 Ulster Scots Agency. "An introduction to the Ulster Scots Language." Ulster Scots Agency. Last modified 2017. http://www.Ulster Scotsagency.com/what-is-ulster Scots/language/. THE BALDWIN REVIEW 2017

Ulster Scots: The Life Cycle of a Language | Anna Bunting ’18

BIBLIOGRAPHY Gregg, R. J. “The Scotch-Irish Dialect Boundaries in Ulster” in Wakelin, M. F., Patterns in the Folk Speech of the British Isles (1972) London: Athlone Press Trudgill, Peter. “Colonial Dialect Contact in the History of European Languages: On the Irrelevance of Identity to New-Dialect Formation.” Language in Society 37, no. 2 (2008): 241–54. doi:10.1017/ S0047404508080287. Ulster Scots Agency. “An introduction to the Ulster Scots Language.” Ulster Scots Agency. Last modified 2017. http://www.Ulster Scotsagency.com/ what-is-Ulster Scots/language/. The Stormont Papers. (Volume 2 (1922) / Page 147-8). BBC. “Good Friday Agreement.” BBC. http://www.bbc.co.uk/history/events/good_friday_ agreement. Bardon, Jonathan. The Plantation of Ulster. N.p.: Gill & Macmillan, 2011. Campbell, Kenneth L. (2013). Ireland’s History: Prehistory to the Present. A & C Black. p. 59. Connolly, S. J. Contested Island: Ireland 1460–1630 (Oxford, 2007), p. 29. Frame, Robin, English Lordship in Ireland 1318–1361: Clarendon Press ,1982. Bardon, Jonathan. A History of Ulster. Dundonald, Belfast, Northern Ireland: Blackstaff Press. 1992. Taylor, Peter. 1999. Loyalists: war and peace in Northern Ireland. New York: TV Books. Linen Hall Library. “Irish and Reference.” Linen Hall Library. https://www.linenhall.com/pages/irish-and-reference. Erskine, John, ed. “A supplement to the Annotated Bibliography of Ulster Scots Language and Literature.” Ulster Scots Academy. http://www.Ulster Scotsacademy.com/ullans/12/bibliography-of-ulster Scots.php. Minding Our Language. BBC, 2015. Hosbaum, Philip. Metre, Rhythm and Verse Form. London: Taylor and Francis, 1996. Digital file. Heritage Lottery Fund. “James Orr.” The Weaver’s Trail. http://www.weavers-trail.co.uk/james-orr-ulster-weaver-poets. “Ulster Scots Music.” Queens University Belfast. http://www.qub.ac.uk/sa-old/ resources/Belfast_Project/Sites_2004/USFO/pages/Ulster ScotsMusic.html. Ulster Scots Agency. “An introduction to the Ulster Scots Language.” Ulster Scots Agency. Last modified 2017. http://www.Ulster Scotsagency.com/ what-is-ulster Scots/language/. Ulster Scots Agency. “About Us.” Ulster Scots Agency. Last modified 2017. http://www.Ulster Scotsagency.com/about-us/.

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SANJANA DIXIT ’18 Sanjana Dixit is a senior at Baldwin from Wynnewood, PA. She currently serves as a senior representative for Service League, is an active peer tutor in the Writing Center and is the head of the Skill Foundation service club that she has chartered. She is also a member of Model UN, Modern Science Club, Baldwin’s French magazine Florilège and writes articles regularly for Baldwin’s Hourglass newspaper. She enjoys Varsity rowing and playing tennis for Baldwin in addition to working at the Children’s Hospital of Philadelphia throughout the year.

Electrophysiological Signatures of Language Impairment in Autism Spectrum Disorder By Sanjana Dixit ’18

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Electrophysiological Signatures of Language Impairment in Autism Spectrum Disorder | Sanjana Dixit ’18

Children with Autism Spectrum Disorder (ASD) have many differences in the electrochemical compositions of their brain in comparison to children who are typically developing, as demonstrated by Magnetoencephalography (MEG) exams, an imaging technique that identifies brain activity and measures small magnetic fields produced in the brain typically used to further understand seizures and their location. These MEG exams have determined at this point that the M100 component of the superior temporal gyrus, located in the right hemisphere of the brain, indicates a short but significant delay in ASD patients in auditory response with tones and frequencies that are often present in human speech (between 300-500 Hz)1. From this information, two main hypotheses have been concluded to account for the M100 delay that distinguished autistic children from those that are typically developing: first is the immature white matter present in ASD patients that contributes to the poor conduction velocity of the thalamocortical acoustic radiation projections. Second is the diminished gamma-band phase synchrony in ASD patients that factors into abnormal superior temporal gyrus synaptic transmission2. If normally developed, white matter tracts help transmit information effectively and efficiently throughout the brain. The corpus callosum is involved in the processing and integrating of higher order information and connecting the two hemispheres of the brain, helping to facilitate interhemispheric information transmission3. Structural MRIs have been utilized to show that in the brains of children with ASD, the callosal and corticopontine pathways are thinner overall and terminal areas in the cortical gray matter are significantly smaller4. Autistic brains have more short‐ range u‐fibers in the frontal lobe compared to brains of those who are typically developing. Gray matter pathways are more disorganized with fewer coherencies in the ASD brain, specifically the lateral aspects of the middle part of the brain, including motor areas, and both medial and lateral surfaces of the anterior frontal brain regions5. These discrepancies result in a poor conduction velocity of the thalomo-cortical acoustic radiation projections that impact ASD patients’ ability to process auditory tones in speech correctly and without delay. In regards to the second hypothesis, gamma band phase synchrony is extremely important for adequate and effective communications between different hemispheres in the brain, and without it ASD patients lack the appropriate connections and quick synapses to respond aptly6. Gamma-band oscillations observed in EEGs and MEGs throughout numerous studies have been of significant interest in recent years in ASD 1 Dr. Roberts, Timothy P.L. “Electrophysiological Signatures of Language Impairment in Autism Spectrum Disorder Protocol”. CHOPCAR system pages 1-2. 2 Ibid, page 1-3. 3 Wilkinson, Molly et al. “White and Gray Matter Fiber Pathways in Autism Spectrum Disorder Revealed by Ex Vivo Diffusion MR Tractography.” Brain and Behavior 6.7 (2016): e00483. PMC. Web. 10 Sept. 2017 4 Wilkinson, Molly et al. “White and Gray Matter Fiber Pathways in Autism Spectrum Disorder Revealed by Ex Vivo Diffusion MR Tractography.” Brain and Behavior 6.7 (2016): e00483. PMC. Web. 10 Sept. 2017 5 Ibid. 6 Dr. Roberts, Timothy P.L. “Electrophysiological Signatures of Language Impairment in Autism Spectrum Disorder Protocol”. CHOPCAR system page 3. E2

Electrophysiological Signatures of Language Impairment in Autism Spectrum Disorder | Sanjana Dixit ’18

patients because of their capability to serve as biomarkers to detect dysfunction in the underlying circuitry of ASD brains, which heavily implicates GABAergic transmission, GABA-related enzymes, glutamate neurotransmission7.

The various effects of immature white matter and diminished gamma band phase synchrony on M100 latency within the superior temporal gyrus can be independently examined through MRIs that target and access acoustic radiations, and through MEG scans that measure neural oscillatory activity to determine durability and state of neural circuitry through synaptic transmission. The ASD group of this study are divided into four subgroups based on white matter deficit and “oscillapathy”, identifying a dominant deficit for each subject; white matter and oscillation deficit, white matter deficit and oscillation surplus, white matter surplus and oscillation deficit, and white matter and oscillation surplus. After the subset groups are determined, the results are utilized to determine heterogeneity in ASD patients through an assessment of between group phenotypic difference and within group reduction in phenotypic variance as well as identification of correlations between these deficits and clinical language impairment symptoms like standard processing and cognitive ability. The study also determines whether auditory evoked response abnormalities in ASD patients are specific to the auditory domain of the brain or if they are results of a more widespread phenomenon common in both ASD and typically developing patients8. There is an ongoing consensus that ASD is a heterogeneous disorder, meaning that each diagnosis and each treatment must be different for each patient who is diagnosed with ASD. Not every single ASD patient suffers the same symptoms nor is able to respond similarly to the same treatment, and as a result the treatment of this disorder must be specific to each individual. The purpose of this study is to better understand the biological basis of Autism Spectrum Disorder 7 Rojas, Donald C., and Lisa B. Wilson. “Gamma-Band Abnormalities as Markers of Autism Spectrum Disorders.” Biomarkers in medicine 8.3 (2014): 353–368. PMC. Web. 10 Sept. 2017. 8 Dr. Roberts, Timothy P.L. “Electrophysiological Signatures of Language Impairment in Autism Spectrum Disorder Protocol”. CHOPCAR system. Page 4. THE BALDWIN REVIEW 2017

Electrophysiological Signatures of Language Impairment in Autism Spectrum Disorder | Sanjana Dixit ’18

in regards to heterogeneity, specifically M100 latency heterogeneity, and to create biologicallydriven classifications to reduce this heterogeneity by identifying these differences in biologically similar populations9. The utilization of such biologically-driven stratification has the capability to achieve a reduction in this phenotypic heterogeneity10, but also opens a door to future criteria for various treatment studies and targeted subgroup analysis of individuals with ASD. Additionally, this investigation across a phenotypically heterogeneous Autism Spectrum Disorder population offers insight to general biological markers of ASD. Signatures of Language Impairment in Autism Spectrum Disorder study, conducted by CHOP at the Center for Autism Research, is an observational, cross-sectional study conducted with imaging/recording tools including MEGs and MRIs. The two main groups, an ASD group and a typically developing control group, ranges from 8-12 years of age and the study itself requires at least 150 ASD subjects enrolled and 40 typically developing subjects enrolled11. Each subject undergoes a neuropsychological evaluation coupled with an MRI and MEG scan. Before the neuropsychological scan, when the subject is in the early stages of recruitment, screening must be conducted to obtain verbal consent of the parent in addition to the completion of a phone screen, which asks the parent of the subject’s personal health information and other information like address, date of birth, legal name, etc. After the completion of this screening the subject is assigned a subject number, which is used to reference them throughout the duration of the study. Following the screening stage comes the pre-visit questionnaire, if the subject is determined eligible. After all of these are completed and the subject is still determined to be eligible, they arrive at the CHOP research building for the clinical part of the study, where a psychologist performs certain behavioral tests on them to confirm the ASD diagnosis, obtain measures of clinical symptoms, and to evaluate cognitive function. The following behavioral tests are used in order to complete this step of the process12: ● ADOS-2 (Autism Diagnostic Observation Schedule- 2nd edition) – looks at a child’s behavior and language capabilities ● ADI-R (Autism Diagnostic Interview-Revised) [parent interview] - looks at autism symptoms, child’s language, social skills and behavior. ● ASRS (Autism-Spectrum Rating Scales) [parent interview] - a nationally standardized questionnaire regarding ASD symptoms and associated features ● BRIEF (Behavior Rating Inventory of Executive Functioning) [parent report] - designed to assess a child’s functioning in the home environment. 9 Ibid. 10 Ibid, pages 4-5. 11 Ibid page 7. 12 Ibid pages 14-15. E4

Electrophysiological Signatures of Language Impairment in Autism Spectrum Disorder | Sanjana Dixit ’18

● CBCL (Child Behavior Checklist) [parent report] – looks at behaviors like attention problems, aggression, anxiety, and depression. ● CELF –V (Clinical Evaluation of Language Fundamentals- 5th edition) - evaluates a child’s language understanding and expression. ● CCC-2 (Children’s Communication Checklist-2nd Edition) [parent report] - norm referenced parent report measure of structural language skills in children ● CTOPP-2 (Comprehensive Test of Phonological Processing – 2nd Edition) - measure of phonological processing, highly correlated with language-based learning disabilities and rapid temporal processing ● Handedness Questionnaire - a measurement scale used to assess the dominance of a person’s right or left hand in everyday activities. ● Intervention History Form- captures information about past treatments. This information can be an important moderating influence on the developmental trajectory of each child. ● Medication Form – The medication form asks subjects to list out all current medication use, dosage and reason for use. ● SCQ (Social Communication Questionnaire-Lifetime version) [parent completed measure] – a scale that asks about both a child’s social and communication skills over the span of the child’s lifetime. ● SRS-2 (Social Responsive Scale-2nd Edition) [parent completed measure] - a scale that asks about a child’s social development. ● VABS-III (Vineland Adaptive Behavior Scales (3rd edition) [Parent/Caregiver Rating Form] – a measure used to support the diagnoses of intellectual and developmental disabilities. ● WIAT-III (Wechsler Individual Achievement Test – 3rd Edition) - The WIAT-III is a measure of academic achievement: math calculation skills, single-word reading, spelling, and “phonological decoding”. ● WISC –V (Wechsler Intelligence Scale for Children- 5th edition) - measures a child’s problemsolving and reasoning skills13.

13 Ibid, page 13-16.

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Electrophysiological Signatures of Language Impairment in Autism Spectrum Disorder | Sanjana Dixit ’18

Overall, this study entitled “Signatures of Language Impairment in Autism Spectrum Disorder” is a tool, once completed, that can be applicable to a broad range of ASD children, all who express M100 latency delays and as a result cannot communicate in the most effective way possible. With the completion of this study, CHOP can apply the knowledge gained to determine differences and associations with M100 latency delays in typically developing and autistic children, correlations between clinical symptoms and oscillopathy and diminished gamma phase synchrony, and the specificity of M100 latency delays in Autism to auditory output in comparison to other sensory outlets14. At this time, the CHOP Center for Autism Research is at the crux of completing this study and formulating the information into a cohesive set of data that can possibly yield a clearer insight on the heterogeneous aspect of ASD that makes it so difficult to diagnose and treat in the best way possible.

14 Ibid, page 5.

Electrophysiological Signatures of Language Impairment in Autism Spectrum Disorder | Sanjana Dixit ’18

WORKS CITED Dr. Roberts, Timothy P.L. “Electrophysiological Signatures of Language Impairment in Autism Spectrum Disorder Protocol”. CHOPCAR system.

Wilkinson, Molly et al. “White and Gray Matter Fiber Pathways in Autism Spectrum Disorder Revealed by Ex Vivo Diffusion MR Tractography.” Brain and Behavior 6.7 (2016): e00483. PMC. Web. 10 Sept. 2017.

Rojas, Donald C., and Lisa B. Wilson. “Gamma-Band Abnormalities as Markers of Autism Spectrum Disorders.” Biomarkers in medicine 8.3 (2014): 353–368. PMC. Web. 10 Sept. 2017. Children’s Hospital Of Philadelphia Center for Autism Research (CHOPCAR)

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ANOUSHKA GIDH ’19 Anoushka Gidh is a junior who lives in Harleysville, PA, and has attended The Baldwin School since 9th grade. She is the vice editor of Baldwin’s yearbook, Prism, and is the head of Baldwin’s More Than Me Club as well as an ambassador of the Academy, which supports girls education in Liberia, Africa. She is involved in Model UN and Lamplighters, and she is on the tennis and swimming team for Baldwin. In her free time, she volunteers for her local hospital and practices playing the piano.

Increased Cell Inflammation in CD40 Mice with Multiple Sclerosis and Optic Neuritis By Anoushka Gidh ’19

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Increased Cell Inflammation in CD40 Mice with Multiple Sclerosis and Optic Neuritis | Anoushka Gidh â&#x20AC;&#x2122;19

INTRODUCTION: Multiple sclerosis is a disease that affects both the brain and the nervous system through the spine. With multiple sclerosis (MS), the immune system will attack myelin, the tissue protecting the outside of nerves (Mayo Clinic). When demyelination occurs, the myelin sheath wears off on the optic nerves, and the nerves become damaged, which impact the connections between the retina and the brain and can cause vision loss. This phenomenon is also known as optic neuritis (Mayo Clinic). Optic neuritis causes vision loss due to the inflammation of the optic nerve when the immune system attacks myelin, the protein surrounding the nerves. The cells observed in this study, macrophages or microglia, are located in the optic nerve. Macrophages are located in the central nervous system to act as the primary immune defense (Wiki). Microglia are located in some parts of the brain and the spinal cord, and their main purpose is to search the central nervous system for destroyed or unneeded neurons. In this experiment, the main goal was to understand how much more inflammation was present in the infected mice compared to the non-infected mice. In the Stellar-Chance Laboratories at the University of Pennsylvania, a similar experiment was conducted earlier but with a dose of a drug called SIRT1 deacetylase that mimics optic neuritis. It was proven that the peak of the viral infection was near day 5 of the experiment. During this experiment, optic neuritis was induced in two different groups of mice. Increased cell inflammation occurs in mice without a gene that produces a protein called CD40, known as CD40 mice, compared to control mice.

MATERIALS AND METHODS: Mice Euthanasia: Three separate infected mice were used in this experiment: infected mice (CD) and control mice (RS, CDR). Mice were euthanized on days 5, 10, 15, and 26 of the experiment to collect data. Mice were first euthanized by being perfused which is to filter out the blood from the bodies. A vertical incision was made to expose the heart, and a needle connected to 1% PBS solution was inserted into the left ventricle with the aorta snipped to drain out blood. This caused the mouseâ&#x20AC;&#x2122;s body to stiffen, and soon the body parts were dissected and put into test tubes. The test tubes were put into an ice box with dry ice until taken back to the lab. Data was collected from the eyeballs, optic nerves, brain, spinal cord, and liver. The eyeballs and optic nerves would be used for the optic neuritis portion of the experiment, and the

Increased Cell Inflammation in CD40 Mice with Multiple Sclerosis and Optic Neuritis | Anoushka Gidh â&#x20AC;&#x2122;19

other body parts were for the multiple sclerosis part. The data for the optic neuritis would consist of an examination of the change in inflammation in the optic nerves to determine a definite increase of the infection in the mice throughout the experiment. Sectioning: At the lab, the optic nerves were placed in cassettes. The retinas and optic nerves were separated from the eyeball and put into cassettes. The retinas and optic nerves were rinsed clean and then embedded in small blocks of paraffin wax. The cassettes were then placed onto a rotary microtome machine and sectioned off to study a single piece of the retina or optic nerve. The sections were cut parallel to the optic nerve in order to examine each section of the retina. Each string of retina sections and optic nerve sections were placed in a hot water bath and quickly positioned onto microscope slides. These slides were left on laboratory racks for drying. H&E Staining Procedure: Hematoxylin and Eosin (H&E) staining is essential for showing the inflammatory cells (macrophages and microglia) in the optic nerve. Hematoxylin is a purplecolored stain used to identify the inflamed cells. Eosin is a pink-colored stain used to determine the other tissues present in the optic nerve. While observing the sections under the microscope, the two stains help differentiate the multiple tissues of the retina or optic nerve. Before staining, slides are required to be deparaffinized around the tissues so as to only display the tissues on the slides. Removing paraffin is accomplished by putting the slides on a heat block and placing them onto a slide rack to be washed in several chemicals. The rack was washed twice in Pro-Par Clearant for five minutes. After the ten minutes, the tissues were dehydrated by being washed in ETOH 100%, 95%, 70%, 50% solution each for three minutes and then in distilled H2O for five minutes. The rack was promptly introduced to Hematoxylin for one minute to stain the optic nerves with a vibrant purple color. The rack was then placed under running

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Increased Cell Inflammation in CD40 Mice with Multiple Sclerosis and Optic Neuritis | Anoushka Gidh â&#x20AC;&#x2122;19

water until the water ran clear to ensure that the only tissues that were being stained in this step were the inflamed cells. Then the slides were dipped three times in 1% HCl solution and placed under running water for two minutes, and later the tissues were dipped four times in 0.2% NH4OH solution and placed under running water for four minutes. The tissues were dipped in 50% ETOH solution until the ETOH dripped clearly and then were placed into Eosin for three minutes. The Eosin stained the other tissues in the optic nerve with a pink color. Finally, the slide rack was placed in 95% ETOH solution for one minute, 100% ETOH solution for two minutes three times, and finally Pro-Par Clearant for five minutes three times. The tissues would be observed in optic nerves on days 5, 10, 15, and 26 after immunization. The H&E staining mentioned above was completed for the optic nerves and retinas of the mice euthanized on day 10 and day 15 of the experiment.

RESULTS:

In the day 10 model, the CD16 mouse optic nerve showed more inflammation than the

RS28 mouse optic nerve. In the image of the RS26 optic nerve, there is less inflammation visible compared to the image of the CD16 optic nerve, the infected optic nerve. This proves that the immune system did attack the optic nerveâ&#x20AC;&#x2122;s myelin and caused increased cell inflammation. A similar phenomenon occurred in the day 15 model. The RS32 and CDR19 mice optic nerves showed a significant cell inflammation increase compared to the CD16 mice on day 10. This proves that the immune system did in fact attack the myelin surrounding the optic nerve, which

Increased Cell Inflammation in CD40 Mice with Multiple Sclerosis and Optic Neuritis | Anoushka Gidh â&#x20AC;&#x2122;19

initiates inflammation.

CONCLUSIONS: The results found in this section of the experiment give information as to what will be found in the later days of the experiment. As previously mentioned, the peak of viral infection in this experiment was near day 15 as there was an obvious increase of cell inflammation in the CD40 infected mice on day 15 than on day 10. This information will be used to advance the experiment. The study suggests that CD40 is important for trying to fight the inflammation as there is more inflammation when CD40 is not present.

ACKNOWLEDGMENTS:

I would like to thank the following people for guiding me and supporting me throughout

this research: Dr. Kenneth Shindler, Dr. Reas Khan, Kimberly Dine, Dr. Jayasri Das Sarma, Abhishek Bose, Soumya Kundu, Dr. Ahmara Ross and my family.

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Increased Cell Inflammation in CD40 Mice with Multiple Sclerosis and Optic Neuritis | Anoushka Gidh ’19

REFERENCES: Most of the resources came from the information of Dr. Shindler’s lab. The published papers and sources I referred to are mentioned below. Fonseca-Kelly, Zoe, Mayssa Nassrallah, Jorge Uribe, Reas S. Khan, Kimberly Dine, Mahasweta Dutt, and Kenneth S. Shindler. “Resveratrol Neuroprotection in a Chronic Mouse Model of Multiple Sclerosis.” Frontiers. April 28, 2012. Accessed August 10, 2017. http://journal.frontiersin.org/article/10.3389/fneur.2012.00084/full. Khan, Reas S., Kimberly Dine, Bailey Bauman, Michael Lorentsen, Lisa Lin, Helayna Brown, Leah R. Hanson, Aleta L. Svitak, Howard Wessel, Larry Brown, and Kenneth S. Shindler. “Intranasal Delivery of A Novel Amnion Cell Secretome Prevents Neuronal Damage and Preserves Function In A Mouse Multiple Sclerosis Model.” Scientific Reports. January 31, 2017. Accessed August 10, 2017. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5282572/. Khan, Reas S., Kimberly Dine, Jayasri Das Sarma, and Kenneth S. Shindler. “SIRT1 Activating compounds reduce oxidative stress mediated neuronal loss in viral induced CNS demyelinating disease.” Acta Neuropathologica Communications. January 02, 2014. Accessed August 10, 2017. https://actaneurocomms.biomedcentral.com/articles/10.1186/2051-5960-2-3. Khan, Reas S., Zoe Fonseca-Kelly, Catherine Callinan, Ling Zuo, Mira M. Sachdeva, and Kenneth S. Shindler. “SIRT1 activating compounds reduce oxidative stress and prevent cell death in neuronal cells.” Frontiers. December 11, 2012. Accessed August 10, 2017. http://journal.frontiersin.org/article/10.3389/fncel.2012.00063/full. “Microglia.” Wikipedia. October 11, 2017 Accessed October 12, 2017. https://en.wikipedia.org/wiki/Microglia. “Multiple sclerosis.” Mayo Clinic. August 04, 2017. Accessed September 7, 2017. http://www.mayoclinic.org/diseases-conditions/multiple-sclerosis/home/ovc-20131882. “Optic neuritis.” Mayo Clinic. November 04, 2016. Accessed September 7, 2017. http://www.mayoclinic.org/diseases-conditions/optic-neuritis/home/ovc-20263583. Shindler, Kenneth S., Elvira Ventura, Mahasweta Dutt, Peter Elliott, Denise C. Fitzgerald, and Abdolmohamad Rostami. “Oral Resveratrol Reduces Neuronal Damage in a Model of Multiple Sclerosis.” Journal of Neuro-Ophthalmology. December 2010. Accessed August 10, 2017. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3312784/. Shindler, K. S., E. Ventura, T. S. Rex, P. Elliot, and A. Rostami. “SIRT1 activation confers neuroprotection in experimental optic neuritis.” Investigative ophthalmology & visual science. August 2007. Accessed September 7, 2017. https://www.ncbi.nlm.nih.gov/pubmed/17652729. Zuo, L., R. S. Khan, V. Lee, K. Dine, W. Wu, and K. S. Shindler. “SIRT1 promotes RGC survival and delays loss of function following optic nerve crush.” Investigative ophthalmology & visual science. July 26, 2013. Accessed August 10, 2017. https://www.ncbi.nlm.nih.gov/pubmed/23821198. F6

MIANJIA (RHEA) LI ’18 Mianjia (Rhea) Li is a senior from Beijing, China, and has been a student at Baldwin for four years. She is the senior head of Brain Exercise and Training Association (BETA) math club and Amnesty International human rights club. She is also a service league representative and an active member of Lamplighters and DECA. In her free time, she enjoys baking and watching movies.

Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li ’18, Haoyuan Tan Flint Hill School 3320 Jermantown Rd, Oakton, VA 22124, USA The Baldwin School 701 Montgomery Ave, Bryn Mawr, PA 19010, USA Department of Mechanical Engineering, University College London Gower St, Bloomsbury, London WC1E 6BT, UK Corresponding Author: szhang@flinthill.org

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Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li â&#x20AC;&#x2122;18, Haoyuan Tan

ABSTRACT Ultrasonic welding method has been widely applied for adhesion of polypropylene, so the improvement of welding quality and efficiency become critical for the application of this method on an industrial scale. This research focuses on how ultrasonic sensitizing agent and interfacial roughness influence the strength and efficiency of ultrasonic welding of polypropylene. In the experiment, short glass fiber was added to PP as the sensitizing agent, and test specimens with four levels of surface roughness were prepared. The results of dynamic thermomechanical analysis (DMA) show that the addition of 30% chopped glass fiber has an obvious effect on the increase of storage modulus and reduction of tan Î´ of the sample, suggesting that the stiffness of PP is improved. Thus, the addition of an ultrasonic sensitizing agent ensures a more efficient transmission of ultrasonic energy from the sonotrode to the welding interface. In addition, tensile test and fraction analysis of the welded specimens show that the addition of 30% chopped glass fiber greatly improve the weld strength. The results from tensile test and fraction analysis illustrate that specimens made of the same material with greater interfacial roughness have stronger welding joints and higher welding efficiency, indicating that rougher welding interfaces exert a positive effect on ultrasonic welding performance. Keywords: ultrasonic plastic welding, storage modulus, loss modulus, welding strengthďź&#x152;welding efficiency

1. INTRODUCTION With the rapid development of chemical engineering, thermoplastic composites have been widely used in various industries due to their excellent mechanical properties. They are gradually replacing traditional metal or wooden materials as a bearing component because of their lower density, smaller coefficient of friction, and easier shaping process. Under the low carbon economy, any weight reduction in aircraft or automobiles considerably reduces carbon emission and assists sustainable economic development through an environment-friendly approach. According to relevant statistics, on average, more than 1000 kg of thermoplastic composites are consumed in the production of each automobile vehicle. [1] Besides, public transportation manufacturers use huge amount of thermoplastic composites in producing seats, baggage holders, etc. In some developed countries, thermoplastic composites have extensive applications in manufacturing industry, which contribute to about half of the total thermoplastic composite output.[2] Despite the wide application of thermoplastic composites, restrictions also apply. Since thermoplastic composite products are normally manufactured through injection moulding, the dimensions of the products are limited by the mould size. In order to satisfy various requirements G2

Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li â&#x20AC;&#x2122;18, Haoyuan Tan

in different applications, joining of the composites is inevitable. Traditional joining methods of polypropylene include riveting, adhesive bonding (cementing), threaded connection[3], thermal welding, etc. However, riveting results in stress concentration which lowers fatigue strength and toughness. Adhesive bonding involves complex process and addition of a third component. Threaded connection has numerous limitations in practical application. Thermal welding exerts great influence on material properties, and the strength of the welding joint is not guaranteed. Most importantly, these methods require substantial support of auxiliary appliances and are rather inefficient for mass production. As a result, finding a clean, efficient, and practical way of connection to replace the traditional methods has become the focus of contemporary research. In the 1950s, scientists found that using an ultrasonic wave as a heat source can melt and weld thermoplastic composites. It functions by engendering local heating on plastic parts through pressure conducted by ultrasonic mechanical oscillation. The heating is a result of the integrated effect of the friction both between the surface and between the atoms. After years of research and development, it has been proven that this welding method yields high mechanical performance, which ensures good sealing property of the concealed joint. Additionally, this welding method is advantageous in that it is fast in mass production (ready realization of automation, effective implementation of product consistency, guarantee of low energy consumption), superior in quality (high strength of the welding seam, aesthetic appearance of the welding joint, few damages on the product surface), and low in pollution generated during the process. There are four main stages in ultrasonic welding. First, the welding head touches the plastic parts and asserts pressure while engendering mechanical oscillation. Then, the melting speed accelerates, causing an increase in welding displacement and the interface area between the two plastic parts. During the third stage, solution layer thickness remains constant, and a steady state of fusion occurs with the consistent temperature distribution on the welding surface. Lastly, after a certain period of time, energy output, power stage, or displacement, the energy source is cut, causing the ultrasonic oscillation to stop. In this stage, the pressure acting on the interface was preserved, and therefore accounted for spilth of the material outside of the interface. [4] This research aimed to study the effects of sensitizing agent and interfacial roughness on the strength and efficiency of ultrasonic welding. According to past research and data, it was believed that the addition of a sensitizing agent, glass fiber, in this case, was advantageous to the overall performance and strength of the welding product. According to Ultrasonic Welding of Thermoplastics by Shengyu Zhang, The addition of a sensitizing agent (glass, talc, mineral, etc.) can either suppress or ameliorate the welding quality, depending on the concentration of the sensitizing agent. Sensitizing agents generally increase the materialâ&#x20AC;&#x2122;s rigidity. As the concentration reaches 20%, the modification improves the ultrasonic waveâ&#x20AC;&#x2122;s overall ability to transmit energy. THE BALDWIN REVIEW 2017

Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li ’18, Haoyuan Tan

As the concentration reaches 35%, the lack of thermoplastic resin may occur at the hermetic sealing joints, causing a decrease in the overall welding strength and product quality. As the concentration further increases, the resin becomes even less available at the joint, and therefore causes a steeper decrease in the welding joint strength. Therefore, a concentration of 30% glass fiber filling was selected for this research, aiming to achieve the best welding joint quality. Furthermore, the increase in surface roughness (through sandpaper filing and energy director addition) was believed to increase the interaction of atoms near the welding surface, therefore increase the welding strength. [5]

2. MATERIAL AND METHODS 2.1 Material preparation The PP used in this experiment was a general-purpose homopolymer (grade T300) purchased from Sinopec Shanghai Petrochemical Co., Ltd (Shanghai, China). The short glass fiber was supplied by Tonghong Fiber Co., Ltd (Shandong, China). The sandpaper (Model GXK51, P80 and GXK51-B, P120) was obtained from Yancheng San Ling Co., Ltd (Jiangsu, China) 2.1.1 Preparation of PP and glass fiber reinforced PP The PP and the short glass fiber(SGF) were first dried in the oven at 80℃ for five hours to dehydrate the material. Then, the PP was melt-blended with SGF in the internal mixer (Changzhou Suyan science and Technology Co., Ltd. China Model SU-70) that rotated at 60 r/min at 180 to 190℃. The mixture was then extruded at 190℃ under a pressure of 15 MPa. The extruder (Wuhan Qi’en Technology Co., Ltd. China Model R-3201) has temperature zones of 190℃/195℃/195℃/190℃, and it extruded the material at a screw speed of 60rpm. After that, the lumpish extrudate was pelletized and molded into certain size. 2.1.2 Sanding & Energy director The test specimens with different composition and surface roughness were prepared as shown in Table 1. The PP and Glass Fiber reinforced PP composite test specimens were roughened with sandpaper of two different grit sizes (Model GXK51, P80 and GXK51-B, P120) obtained from Yancheng San Ling Co., Ltd (Jiangsu, China) and molded to create energy directors on one edge respectively. 2.2 Experimental procedures Ultrasonic Welding During the welding process, the tested weld time was set at 0.8s, 1.0s, 1.2s, and 1.4s, and 12 3-bar G4

Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li â&#x20AC;&#x2122;18, Haoyuan Tan

samples from each type of specimen were wielded under these 4 distinct welding times, with 3 samples for each welding time, on which the comparison of welding efficiency was based. The influences exerted by different factors (welding time, pressure, energy, etc.) were examined to find the optimized welding condition (evidence includes fine infiltration near the welding area, preservation of the welding structure, and the presence of little or no PP spilth). The test specimens all had dimensions of 100 15 4mm, which were welded into a flat lap joint of 10mm under a uniform pressure of 3 MPa on the ultrasonic welding machine produced by Dongwan Yixin Automation Equipment Co., Ltd (Model YX-1535, Guangdong, China). The specimens were held in a fixture and welded by a sonotrode that contacted and transmitted ultrasonic energy into the top specimen. Three of each type of specimen were welded together as shown in Figure 1, and substrates with the same interfacial roughness were welded together. The relatively longer welding joint between the second and the third bar ensured that the tested welding joint would separate first during the following welding tensile test. This design also facilitated the tensile test by aligning the forces exerted on the every two specimen bars. 2.3 Performance test 2.3.1 Shear strength testing According to national shear strength testing standard of HG/T 4281-2011, the dimensions of the test specimen were: 100mm 4mm 25mm, with sectional area of 12.5 10mm2. When tested on the microcomputer control electron universal testing machine (Shenzhen Reger Instrument Co., Ltd. China Model RG100-10), tensile velocity was set at 2 mm/min. Three samples from each type of test specimen were tested, and the final result was calculated by taking the arithmetic mean of the individual testing results. 2.3.2 DMA Dynamic thermalmechanical analysis test of each sample was performed using a DMA Q800 dynamic thermalmechanical analyser produced by PerkinElmer, Inc. (Model SII, USA). The dynamic thermalmechanical analysis test was done under the pressure of 1 Hz to measure the storage modulus, loss modulus, and loss factor of PP with and without the addition of the sensitizing agent-short glass fiber-to examine the modifying effects of sensitizing agent on the material. The DMA tests, a technique used to study and characterize materials, illustrate the correlation of temperature and storage modulus, loss modulus, and loss factor under certain frequency. The dimensions of the test specimens were 35 14 4mm, and they were tested under a frequency of 1Hz with the temperature rising at 5â&#x201E;&#x192;/min from 20 to 200â&#x201E;&#x192;. By considering DMA results, the mechanism of ultrasonic welding was further understood, and modification approaches to optimize the welding results were identified. THE BALDWIN REVIEW 2017

Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li ’18, Haoyuan Tan

2.3.3 Welding Strength Test The comparative test of these eight types of samples using microcomputer controlled electronic universal testing machine was then conducted so as to disclose how interfacial roughness influenced ultrasonic welding strength. Tensile tests were operated according the national standard of GB/1040-2006, and dumbbell-shaped samples were prepared in dimensions of 75mm×5mm×2mm, with gauge length of 30mm. While these specimens were tested on the microcomputer controlled electronic universal testing machine, the two vices exert tension on the welding joint of each 3-bar specimen by pulling from either ends at 5mm/min, and the maximum tension that a joint could withstand before breaking were measured. Thus, the ultimate tensile strength of each welding joint was calculated, which served as an indicator of the welding strength and quality. Three samples from each type of test specimen were tested, and the final result was calculated by taking the arithmetic mean of the individual testing results. By closely examining the welding fracture surface, the presence of bubbles on the welding area and the thermal effect on the non-welding area were analyzed. Taking both mechanical properties and physical appearance of the product into consideration, judgments were made on how the addition of sensitizing agent would influence the quality of ultrasonic welding. Analysis on the fracture sides was conducted using a Gaosuo electron microscope supplied by GAOSUOX Co,. Ltd (Model Gaosuo, China)

3. RESULTS 3.1 Effects of glass fiber filling on the mechanical properties of PP The effects of glass fiber filling on the mechanical properties of PP could be analyzed by through both the DMA testing results, which indicate the loss and storage factor of the material, and the tensile results, which indicate the average tensile strength that the testing specimens were able to endure. 3.1.1 DMA Results DMA testing, also known as dynamic mechanical analysis, is mainly used to study the viscoelastic behavior of polymers. During the DMA testing, a sinusoidal force (stress ) is applied to a material and the resulting displacement (strain ) is measured: Stress: σ = σ0 sin(tω + δ) Strain: ε = ε0 sin(tω)

The resulting storage modulus in the DMA testing measures the stored energy,

representing the elastic portion, while the loss modulus measures the energy dissipated as heat, representing the viscous portion. The tensile storage and loss modulus can then be defined as follows: G6

Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li ’18, Haoyuan Tan

Storage Modulus: E ‘ = (σ0/ε0 ) cosδ Loss Modulus: E “= (σ0/ε0 ) sinδ

Then, the ratio between storage moduli and loss moduli, tan could be calculated from

the above values. Tanδ = E “/E ‘ In the DMA Figure 2 to 5, the results monitored the change of storage moduli E’ and tan δ (the ratio between storage moduli and loss moduli) as temperature varies. In Figure 2 to Figure 5 , behavior of E’ and tanδ of both pure PP and PP with glass fiber was closely traced under the frequency of 1Hz, 10Hz, 20Hz, and 50Hz. Then, DMA Figure 6 displayed the relationship between storage modulus, temperature, and frequency respectively for samples made of pure PP. DMA Figure 7 shows the relationship between loss modulus, temperature, and frequency for samples made of pure PP. Similarly, DMA Figure 8 shows the relationship between loss modulus, temperature, and frequency respectively for samples made of PP with glass fiber (sensitizing agent). And lastly, DMA Figure 9 shows the relationship between loss modulus, temperature, and frequency for samples made of PP with glass fiber (sensitizing agent). In DMA Figures 2 through 5, the vertical axis monitors the E’(storage modulus) and tanδ (the ratio between storage modulus and loss modulus) of the thermoplastics, while the horizontal axis monitors the temperature change from 20℃ to 120℃. In all 4 Figures, the storage moduli of PP with glass fiber were significantly higher than that of pure PP, which shows that PP with glass fiber displayed a much better ability than pure PP to store energy. In DMA Figure 2, which represent the viscoelastic properties of pure PP and PP with glass fiber under the frequency of 1Hz, tanδ for pure PP is lower than that for PP with glass fiber when the temperature is lower than 35℃. As temperature increases over 35℃, the tanδ for pure PP experiences greater increase than that for PP with glass fiber, and the tanδ for pure PP becomes higher than that for PP with glass fiber. Similar results were found in all 5 cases under different frequencies, except for that the horizontal component (temperature) of the intersection where the two tanδ crosses on the Figure increases as the frequency increases: they were approximately 40℃, 45℃, 47℃, 50℃, and

60℃, respectively, when the test was conducted under the frequency of 2Hz, 5Hz, 10Hz, 20Hz, and 50Hz. According to figure 6 to 9, a combined sweep was conducted. Because glass transitions (the ability of chains to move past each other) and secondary transitions are seen both in frequency studies and temperature studies, a multidimensional study was conducted, where temperature sweeps were performed at a variety of frequencies to provide a broader characterization of the material. For both pure PP and PP with glass fiber, E’ (storage modulus) decreases as the temperature increases, while the thermoplastic tested with higher frequency demonstrated higher E’. Additionally, DMA graph 7 and 9 showed that tanδ, temperature, and THE BALDWIN REVIEW 2017

Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li ’18, Haoyuan Tan

frequency displayed a common positive correlation between the ratio tanδ and temperature independent of the addition of sensitizing agent, while tanδ also appeared to have a negative correlation with frequency. In other words, the higher the frequency, the lower the tanδ became. While taking the addition of the sensitizing agent into consideration, one distinction that became immediately evident is that the storage modulus E’ for PP with glass fiber is significantly higher than that for pure PP, which proved that the addition of the sensitizing agent increased the storage modulus by almost a factor of two. On the other hand, the ratio between storage modulus and loss modulus for PP with glass fiber was also considerably lower. This indicated that the addition of glass fiber to pure PP is more advantageous at storing and preserving energy, since it has a higher overall E’ (measures the stored energy) and lower overall tanδ (measure of damping in the material). This demonstrated the potential predominance of the addition of sensitizing agent. One other distinction worth noticing was that the distribution of data in the testings of pure PP and PP with glass fiber was quite different: the data in the DMA testing of pure PP appeared to be more equally distributed, while the data for the testing of PP with glass fiber was separated into two branches. The graph for E’ showed a potential pattern of two branches converging together, while the graph of tanδ showed an opposite pattern of one branch diverging into two separate branches.

The tensile test conducted indicated the tensile strength of the material, in this case, PP

and PP with glass fiber. According to the data, the samples made of pure PP exhibited an average maximum tensile force of 1878 N and an average tensile strength of 31.3 mPa. PP with glass fiber, on the other hand, had an average maximum tensile force of 1782 N, and an average tensile strength of 29.7mPa. Both of the categories showed significant decrease as glass fiber was added to pure PP as a sensitizing agent. 3.2 Effects of glass fiber filling on welding strength

While comparing the effects of glass fiber filling on welding strength, the groups (pure PP

and PP with glass fiber) with the same surface roughness was compared: group No.1 is compared with group No.5 (smooth surface), No.2 with No.6 (surface sanded with fine sandpaper), No.3 with No.7 (surface sanded with coarse sandpaper), and No.4 with No.8 (surface with energy director). The independent variable in these data is the addition of glass fiber as a sensitizing agent. All groups were welded under the same pressure, while 4 welding times, from 0.8s to 1.4s each with the lapse of 0.2s, were used in the testing of each group. 3.2.1 group No.1 and group No.5 (smooth surface)

Group No.1 and No.5 compared PP and PP with glass fiber respectively. According to the

data, the maximum welding strength for both groups increases with welding time. All welded under a welding pressure of 3 mPa, the PP samples showed an overall lower product welding G8

Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li â&#x20AC;&#x2122;18, Haoyuan Tan

strength. This phenomenon was most obvious in this group, with a considerable average difference of 0.73 mPa between the sample group made of pure PP and the one made of PP with glass fiber. This pattern of increase when glass fiber filling was added was present in groups 2 and 6 as well, while the strength difference was fairly insignificant comparing to the result of these groups. This indicated that the original hypothesis that the addition of glass fiber filling would increase the welding strength did hold true under some circumstances, while other times the addition of glass fiber did not have a positive influence on welding strength of the product. 3.2.2 group No.2 and group No.6 (surface filed with fine sandpaper)

These two groups also exhibited the pattern present in groups 1 and 5: the addition of glass

fiber filling did increase the average welding strength. However, the increase was much less than that of the first two groups. On average, the welding strength increased by only approximately 0.26 mPa, comparing to the more significant increase of 0.73 mPa in the previous group. This difference was possibly caused by the change in surface roughness, which might have caused a difference in how the material near the welding interface interacted. When comparing these two groups, although both groups experienced increase in welding strength as the welding time increased, when the welding time was shorter (0.8s and 1.0s), the group of PP with glass fiber filling (group 5) was proved to be fairly superior in welding strength; however, when the welding time increased (1.2s and 1.4s), the group of pure PP was proved to be more advantageous in welding strength. 3.2.3 group No.3 and group No.7 (surface filed with coarse sandpaper)

Group No.3 and No.7 showed a very different pattern from the 4 groups discussed above.

In contradiction to our hypothesis, in this group (all four specimens filed with coarse sandpaper), the specimens with glass fiber filling demonstrated lesser welding strength for all four welding times. The average difference was considerable comparing to the group discussed above, and it was approximately 0.91mPa, suggesting that the samples without glass fiber filling were significantly stronger than those with the filling. Again, the change in the interface shape caused by the filing might have changed the pattern that atoms interacted on the interface, causing this unexpected phenomenon. 3.2.4 group No.4 and group No.8 (surface with energy director)

These two groups compared PP and PP with glass fiber equipped with energy directors.

Stunningly, these two groups showed significant similarity in welding strength: except for the data in 4-4 (with welding time of 1.4s), all other data displayed insignificant difference. In this group, the addition of glass fiber as a sensitizing agent did not seem to have a significant effect on the welding strength. However, 4-4 displayed great product welding strength when compared with other samples in its group. According to the welding status description, the melting of the energy director was rather thorough, and there was slight PP overflow. This thorough melding might be held accounted for the unexpectedly strong welding strength. THE BALDWIN REVIEW 2017

Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li â&#x20AC;&#x2122;18, Haoyuan Tan

3.3 Effects of interfacial roughness on ultrasonic welding 3.3.1 pure PP groups (groups No.1, No.2, No.3, No.4) The surface roughness was gradually increasing from sample No.1 to No.3. According to the test results shown in section 4, it could be easily found that the welding strength is positively proportional to the surface roughness. From sample No.1 to No.3, the welding strength has significant increase, the arithmetic average welding strength of sample No1 is 1.798Mpa, sample No2 is 3.073Mpa, sample No.3 is 5.088Mpa. With each increase in surface roughness, the welding strength increases 68.3% in average. (3.073Mpa-1.798Mpa)/1.798Mpa=70.9% (5.088Mpa-3.073Mpa)/3.073Mpa=65.58% (70.9%+65.58%)/2=68.3%1 3.3.2 PP+GF groups (groups No.5, No.6, No.7, No.8) The surface roughness is gradually increasing from sample No.5 to No.7. According to the test results shown in section 4, the welding strength is positively proportional to the surface roughness. From sample No5 to No7, the welding strength has relatively large increase, the average welding strength of sample No5 is 2.52Mpa, sample No6 is 3.33Mpa, sample No7 is 4.275Mpa. With each increase in surface roughness, the welding strength increases 32.24% in average. (3.33Mpa-2.52Mpa)/2.52Mpa=32.1% (4.275Mpa-3.33Mpa)/3.33Mpa=28.38% (32.1%+28.38%)/2=32.24% 3.4 Comparative analysis on the welding interface The welding interface conditions pointed towards a potential area that could be improved on for this research. According to the data recorded below in the illustration section, some of the condition of the welding interface was unaccessible because the cross section of test specimen fragmented during the tensile test before two welding specimens could be separated. 3.5 Comparative study of the welding efficiency For almost all the samples, longer welding time leads to higher welding strength. However, excess welding time will cause the melting PP flow out of the edge and affect the componentâ&#x20AC;&#x2122;s appearance.

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Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li â&#x20AC;&#x2122;18, Haoyuan Tan

For samples of same material, under the same welding time, the samples with higher surface roughness has higher welding strength. For example, for pure PP sample, under 1.2s welding time, the welding strength of: Smooth surface sample = 1.76Mpa fine sanded sample = 3.85Mpa coarse sanded sample= 4.41Mpa For samples with same surface roughness, under the same welding time, the samples with GF has higher welding strength. For example, for fine sanded sample, under 1.0s welding time, the welding strength of: Pure PP sample = 1.50Mpa PP+CF sample = 3.10Mpa As a result, higher surface roughness and addition of GF could both lead to higher welding efficiency.

4. Illustration Sample No.

Material Type

Polypropylene (PP)

PP filed with fine sandpaper(P120) GXK51-B

PP filed with coarse sandpaper(P80) GXK51 Yancheng San Ling

PP with energy director

PP+GF (Glass fiber reinforced PP composite)

Glass fiber reinforced PP composite filed with fine sandpaper

Glass fiber reinforced PP composite filed with coarse sandpaper

Glass fiber reinforced PP composite with energy director Table 1. Materials of test specimens used for ultrasonic welding THE BALDWIN REVIEW 2017

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Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li â&#x20AC;&#x2122;18, Haoyuan Tan

Maximum ower output (KW)

3.5

Welding time range (s)

0.01-5.00

Welding pressure range (MPa)

0.1-1.0

Hold time range (s)

0.01-5.00 Table 2. Ultrasonic welding parameters

Equipment

Test speed

Material

Shenzhen Reger Instrument Co., Ltd. China Model RG10010

5mm/min PP Glass Fiber reinforced PP (30%)

Tension (N)

Average Tension (N)

Ultimate Tensile Strength (MPa)

1892 1886 1856

1878

31.3

1789 1777 1781

1782

29.7

Table 3. Ultimate Tensile Strength of PP before and after the addition of glass fiber

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Sample number

Welding time (s)

Pressure (MPa)

Max. Welding strength (MPa)

Welding interface condition

1-1

0.8s

1.22

The condition of the welding interface is unaccessible because the cross section of test specimen fragmented during the tensile test before two welding specimens could be separated.

1-2

1.0s

1.76

The condition of the welding interface is unaccessible because the cross section of test specimen fragmented during the tensile test before two welding specimens could be separated.

1-3

1.2s

2.05

The condition of the welding interface is unaccessible because the cross section of test specimen fragmented during the tensile test before two welding specimens could be separated.

Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li â&#x20AC;&#x2122;18, Haoyuan Tan

1-4

1.4s

2.16

The condition of the welding interface is unaccessible because the cross section of test specimen fragmented during the tensile test before two welding specimens could be separated.

2-1

0.8s

2.66

The PP on the interface did not melt much

2-2

1.0s

1.50

The PP on the up and down edges of the interface slightly melted, but the center did not melt at all

2-3

1.2s

3.85

The PP on the four edges of the interface melted, the the center did not melt

2-4

1.4s

4.28

The melting PP from the edges of the interface flowed towards the center, but

0.8s

4.71

The PP melted and was tightly bounded

1.0s

5.69

The PP on the two interfaces melted and was tightly bounded.

1.2s

4.41

The PP on the interface did not sufficiently melt

1.4s

5.54

The melted PP and flow concentrates on one edge of the interface

0.8s

3.38

The energy director melted and the PP flowed towards the one edge of the interface

1.0s

3.50

The melting of energy director took place on the edges of the joint, and PP did not flow towards the center

1.2s

4.02

The melting mainly took place on the edges of the interface

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1.4s

5.02

The melting of the energy director was rather thorough, and there was slight PP overflow

0.8s

1.95

The welding interface is very smooth because the PP does not melt much

1.0s

2.36

The PP on the edges of welding interface melted, but the central region did not melt.

1.2s

2.78

The PP near the edges of welding interfaced melted, but it did not flow towards the center to cover the whole interface

1.4s

3.00

The condition of the welding interface is unaccessible because the cross section of test specimen fragmented during the tensile test before two welding specimens could be separated.

0.8s

3.30

The PP on 4 edges of the interface melted, but the central region of the interface did not melt

1.0s

3.10

The PP on 4 edges of the interface slightly melted, but the center was not welded together

1.2s

3.40

Only the region near the edges of the welding surface melted

1.4s

3.52

The PP on the edges thoroughly melted, but the PP on the center of the interface did not melt

0.8s

4.04

The joint was tightly welded, but the condition of the welding interface is not accessible because the cross section of test specimen fragmented during the tensile test before two welding specimens could be separated.

Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li â&#x20AC;&#x2122;18, Haoyuan Tan

1.0s

4.37

The condition of the welding interface is unaccessible because the cross section of test specimen fragmented during the tensile test before two welding specimens could be separated.

1.2s

4.13

The condition of the welding interface is unaccessible because the cross section of test specimen fragmented during the tensile test before two welding specimens could be separated.

1.4s

4.56

The condition of the welding interface is unaccessible because the cross section of test specimen fragmented during the tensile test before two welding specimens could be separated.

0.8s

3.95

The energy director melted thoroughly, and the melted PP flowed towards the back edge

1.0s

3.83

The edges of energy director melted, but the melted PP did not flow throughout the interface

1.2s

4.07

The PP from the energy director melted thoroughly, especially the region near the front and back edge of the welding interface

1.4s

4.15

The melted PP flowed towards the back edge of the interface and slightly overflowed

Table 4. Tabulation of welding strength and joint conditions

Figure 1. 3-bar-welding design

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Figure 3. Storage modulus and tan d of PP and PP/GF at 10Hz

Figure 2. Storage modulus and tan d of PP and PP/GF at 1Hz

Figure 4. Storage modulus and tan d of PP and PP/GF at 10 Hz

Figure 5. Storage modulus and tan d of PP and PP/GF at 20Hz

Figure 6. Storage modulus of PP at different time and frequency G16

Figure 7. Tan d of PP at different time and frequency

Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li ’18, Haoyuan Tan

Figure 8. Storage modulus of PP/GF at different time and frequency

Figure 9. Tan δ of PP/GF at different time and frequency

Figure 10. Storage modulus of PP and PP/GF at 1Hz

Figure 11. Storage modulus of PP/GF at 10 Hz

Figure 12. Storage modulus of PP/GF at 20 Hz

Figure 13. Storage modulus of PP/GF at 50 Hz

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Figure 14 Non-sanded PP

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Figure 15 Coarse sanded PP

Figure 16 Fine sanded PP

Figure 17 Energy director PP

Figure 18. Non-sanded PP+GF

Figure 19. Coarse sanded PP+GF

Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li ’18, Haoyuan Tan

Figure 20 Fine-sanded PP+GF

Figure 21. Energy director PP+GF

Figure 22 Bending of welding specimen

5. DISCUSSION 5.1 Interpretation of the DMA Results According the the DMA result, the addition of short glass fiber dramatically increased the storage modulus and loss modulus, and decreased the tan δ of the material. The storage modulus measures the amount of energy stored in the viscoelastic material as a result of the strain, while the loss modulus measures the amount of energy dissipated because of the viscous deformation. The tan δ is the ratio of loss modulus and storage modulus. The increase of storage modulus was caused by the high moduli of short glass fiber, which formed a strong interface with PP in the interior of the specimen. Results from the welding strength test illustrated that the joint strength of glass fiber reinforced PP specimens was generally greater than that of pure PP specimens. This could be

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explained by the increase in storage modulus, which prevented the dissipation of ultrasonic energy during transmission through the material, enabling more energy to reach the welding surface so as to generate the heat that melted the PP. Although the loss modulus also increased after the addition of glass fiber, the decrease in tan δ demonstrated that the addition of glass fiber had an overall positive effect on preserving the ultrasonic energy. Thus, the addition of glass fiber, an ultrasonic sensitizing agent, not only enhanced the welding strength by melting and coalescing the PP on the welding interfaces, but also improved the welding efficiency by curtailing the welding time needed to send a certain amount of energy to the welding surface. In fact, the study of Liu et.al [6] illustrated that unfilled polymer had higher loss modulus than filler polymer did, which was inconsistent with our experimental results. Despite this inconsistency, they drew a conclusion similar to ours, claiming that less energy transferred to the joint interface in unfilled polymer. However, as discussed in section 3.2, some of the results did not mesh with this explanation. For example, the welding strength of 4-4 is larger than that of 8-4 despite 8-4’s inclusion of glass fiber. This was probably caused by the decrease of tan δ after adding the glass fiber. In fact, after the PP entered the viscous phase and flowed through the interface, the viscous dissipation of PP started to contribute to the further heating welding interface. [7] At this stage, higher tan δ was favored, for further heat dissipation was needed to continue the melting of PP, which strengthened the welding joint. The power dissipated could be represented by the empirical formula below:

W=πfεo′E″=πfεo′E′tanδ [8] where E’ is the storage modulus (MPa), E’’ is the loss modulus (MPa), W is the power dissipation as heat (W), f is the strain frequency (Hz), and εo′ is the vibration amplitude (um). Therefore, finding a balance between the storage modulus and the loss modulus remained the very crucial problem in order to achieve an optimized welding efficiency. The two conditions mentioned above seemed rather contradictory to each other, but these moduli’s relationships with the temperature showed in figure 2 to 5 made such a balance attainable. According to the E’-Temperature, E’’-temperature, and tan δ-temperature graphs, E’ and E’’ of PP and glass fiber reinforced PP composites both decrease as the temperature drops, while their tan δ increases with rising temperature. This trend demonstrated that the viscous dissipation was relatively higher near the welding interface, where the storage modulus was lower, because of the higher temperature caused by friction in the interface. The farther away from the the welding joint, the lower the temperature was, which resulted in lower tan δ. Notably, the tan δ of glass fiber reinforced PP

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Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li ’18, Haoyuan Tan

is increasing with a steeper slope than that of pure PP, meaning that the gap between tan δ of PP and glass fiber reinforced PP was becoming smaller as temperature increases. As a result, even though the addition of glass fiber lowered the tan δ, potentially undermining the viscous dissipation on the welding surface, it did not make a huge difference under the temperature high enough to melt the PP. Further improvement could be achieved by attaching energy director made of a material with higher tan δ to the testing specimen, which would transfer ultrasonic energy into heat more efficiently, specifically in the interface. 5.2 Discussion on interfacial roughness The results from the weld strength test indicated that specimens with rougher welding interfaces formed stronger welding joints. In the ultrasonic welding process, the ultrasonic vibration caused dynamic friction between two interfaces that initially generated heat. As the surface roughness increased, the coefficient of friction also increased, so more and more frictional energy transferred to thermal energy, contributing to the melting the PP. The frictional heat flux produced during welding process can be expressed as the equation below:

where

is the frictional heat generation rate (W/m^2), δslip is the slip distance, Fap is the applied

force, μ is the friction coefficient, and ADz is the deformation zone area. In addition, the increase in surface roughness reduced the contact area between two welding specimens, concentrating the ultrasonic energy on specific contact points. This expedited the melting of PP and shortened the time it needed to flow throughout the interface, which improved the welding efficiency. Furthermore, the decrease in welding time also reduced the heat-affected zone, minimizing the effect of ultrasonic energy exerted welding specimens’ mechanical properties during welding process. Heat resulted in a decrease in acoustic impedance and an increase in energy flow density. According to the study of Hou [10], the decrease of heataffected zone also prevented embrittlement of welding specimens due to defibration, which ensured their relatively higher tensile strength. Hou also suggested that rougher welding surfaces improved welding strength by reducing the acoustic impedance and improving the energy flow density.

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5.3 Analysis of the fracture side

During the weld strength test on the microcomputer control universal testing machine,

some test specimens fractured near the joint while being pulled by the vices. Since the bonds of PP on the joint were very strong, two welding specimens started to bend near the welding joint as the tension increased. As shown in figure 22, welding specimens twisted so much on the edges of the welding joint that they finally broke before the interface was separated. Thus, the obtained tensile strength not only depended on the joint strength, but it was also affected by the toughness of the test specimens. Since the addition of glass fiber increased the stiffness of the PP specimen, pure PP specimen had greater toughness. Therefore, the bending strength of PP specimen was higher than that of glass fiber reinforced PP composites, causing the measurement of high tensile strength of PP specimen. This offered another explanation to those data (e.g. 4-4 vs 8-4) that contradicted with our hypothesis that the addition of glass fiber would improve the welding strength.

The evaluation of the fracture sides also showed that the center area of some welding

joints was poorly bounded, while the PP on the edges of the joint was tightly bounded or even overflowed. This was probably due to the heat from friction generated by horizontal displacement at the edges while the center area was static. According to Zhangâ&#x20AC;&#x2122;s study, the horizontal displacement and frictional stress of the welding interface generally decreases from the edge to the center. He also noted that greater height-to-width ratio increased the range of horizontal displacement. [11] Thus, the welding quality at the center could be improved by finding an optimized height-to-width ratio.

6. CONCLUSIONS AND FUTURE WORK 6.1 Conclusion Based on the work above, the following conclusions were made: 1. The addition of ultrasonic sensitizing agent such as glass fiber increased the storage modulus of polypropylene and improved the joint strength during ultrasonic welding process. 2. According to the experimental results, greater interfacial roughness of welding specimens increased heat generated from friction and led to stronger weld joints. 3. The addition of glass fiber and the increase of interfacial roughness both improved the welding efficiency by maximizing the transfer of ultrasonic energy to thermal energy for the melting and bonding of welding surface .

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Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li ’18, Haoyuan Tan

6.2 Future work 6.2.1 Future Improvements and additional work In this experiment, different surface roughness was obtained by sanding with sandpaper by hand. The defect of sanding by hand is that it is impossible to keep every sample with the same roughness or even shape. So samples of the same roughness group might actually have different surface roughness which could lead to inaccurate experimental results. Therefore, in future experiments, sanding machine should be adopted to control sample roughness to ensure same surface conditions in a group to achieve more convincing testing results. In addition, the storage modulus and tan δ were measured under low frequency during the DMA test, from which the general trend of their variation under ultrasonic frequency range was inferred. However, this inference might not be convincing enough due to the lack of exact data. Therefore, time-temperature superposition method is recommended in future experiments for determining the shift factor, from which exact dynamic moduli of PP and glass fiber reinforced PP under ultrasonic frequency range can be deduced. [12] Based on the experimental observations, excess welding time would cause the molten PP to flow out of the edge and affect the component’s appearance or even shape. On the other hand, insufficient welding time would cause the incomplete welding throughout the contact surface. As a result, find an optimized welding time though Taguchi’s method could be a continuation of our research. The measurement of the change of interfacial temperature and stress during the welding process may also facilitate the modeling for optimization of the ultrasonic welding. As this study only focused on the effects of glass fiber on the welding strength and efficiency of PP, the generalization of these effects to all other filler might not be convincing enough. In order to further reinforce the conclusion that the addition of ultrasonic sensitizing agents exerted a positive effect on welding strength and efficiency, comparative tests on other type of possible sensitizing agents are recommended. Other possible sensitizing agents might include Talc, Mica, and Calcium Carbonate. [13] 6.2.2 Future direction of ultrasonic welding research Improvement of welding strength has been the main focus of contemporary researches on ultrasonic welding of thermoplastics. Although welding strength is an important factor determining the welding quality, the capability of yielding strong welding joints is not the major advantage of ultrasonic welding over other welding methods. What distinguishes ultrasonic welding from other methods is its high efficiency and controllable welding quality, which makes it perfectly suitable for large-scale mechanized production. So the new focus of future researches should be the further improvement of ultrasonic welding efficiency and welding quality, which are possibly affected by the welding power, horn displacement, and energy supply.

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Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li ’18, Haoyuan Tan

REFERENCE [1]Craig, F., Beattie, L. A., O’Brien, L. M., & Millar, A. M. (2009). Preparation of 99mtc radiopharmaceuticals: the effect on radiochemical purity of using sodium chloride injection from plastic ampoules that have been exposed to light. Nuclear Medicine Communications, 29(7), 868871. doi: 10.1097/MNM.0b013e3283300ffa [2]Liu, Chuan. (2003). Experimental Study on Mechanism and Methods of Ultrasonic Welding for Plastics. (Doctoral dissertation, Dalian University of Technology). doi: 10.7666/d.y636626 [3]Wen, Gong. (2008). Joining Techniques for Plastics Parts. World Plastics, 26(2), 76-77. doi:10.3969/j.issn.1002-5219.2008.02.017 [4] Zhang, Shengyu. (2014). Ultrasonic Welding for Plastics (Part one). Plastic Packaging, 24(6), 50-54. [5] Zhang, Shengyu. (2015). Ultrasonic Welding for Plastics (Part two). Plastic Packaging(1), 51-55. [6]Liu, S., Chang, I., & Hung, S. (2001). Factors affecting the joint strength of ultrasonically welded polypropylene composites. Polymer Composites, 22(1), 132-141. doi:10.1002/pc.10525 [7]Tolunay, M. N., Dawson, P. R., & Wang, K. K. (1983). Heating and bonding mechanisms in ultrasonic welding of thermoplastics. Polymer Engineering and Science, 23(13), 726-733. doi:10.1002/pen.760231307 [8] Ao, Y., Sun, Y., Chen, G., Chen, L., Li, L. (2011). Study on polypropylene fiber enhancing polypropylene resin. Chemical Engineer, 25(4), 10-13. doi:10.3969/j.issn.1002-1124.2011.04.004 [9]ŞAHİN, Ö. S., KOELLHOFFER, S., GILLESPIE, J., ADVANI, S., & BOGETTI, T. (2014). Thermal modeling during continuous ultrasonic welding. Turkish Journal of Engineering and Environmental Sciences, 38, 79-96. doi:10.3906/muh-1402-13 [10] Hou, X. (2001).Study on Technical Parameters of Ultrasonic Welding for Plastics. (Doctoral dissertation, Harbin Institute of Technology).

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Effects of Ultrasonic Sensitizing Agents and interfacial Roughness on Ultrasonic Welding Strength and Efficiency of Polypropylene Shuhan Zhang, Mianjia Li â&#x20AC;&#x2122;18, Haoyuan Tan

[11] Zhang, C. (., & Li, L. (2009). A coupled thermal-mechanical analysis of ultrasonic bonding mechanism. Metallurgical and Materials Transactions B, 40(2), 196-207. doi:10.1007/s11663-0089224-9 [12] Cavaille, J. Y., Jourdan, C., Perez, J., Monnerie, L., & Johari, G. P. (1987). Time-temperature superposition and dynamic mechanical behavior of atactic polystyrene. Journal of Polymer Science Part B: Polymer Physics, 25(6), 1235-1251. doi:10.1002/polb.1987.090250605 [13]Sancaktar, E., & Walker, E. (2004). Effects of calcium carbonate, talc, mica, and glassâ&#x20AC;?fiber fillers on the ultrasonic weld strength of polypropylene. Journal of Applied Polymer Science, 94(5), 19861998. doi:10.1002/app.21102

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HILARY LIU â&#x20AC;&#x2122;18 Hilary Liu is a senior from Wayne, PA, and has been a student at The Baldwin School since 5th grade. She is the senior head of the Brain Exercise and Training Association (BETA) math club, an Arts League representative and an active participant in Modern Science, Lamplighters, Girls Learn International and AFAR. She enjoys drawing and painting in her free time.

Virtual Screening and Drug Discovery: Finding Drugs that Inhibit the BCR-ABL1 Oncoprotein By Hilary Y. Liu â&#x20AC;&#x2122;18 1, Nicolas A. Pabon2, Bentley M. Wingert2, Carlos J. Camacho, PhD2 1 The Baldwin School, Bryn Mawr, PA 2 Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA

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Virtual Screening and Drug Discovery: Finding Drugs that Inhibit the BCR-ABL1 Oncoprotein Hilary Y. Liu ’18, Nicolas A. Pabon, Bentley M. Wingert, Carlos J. Camacho, PhD

INTRODUCTION

ABL1 is a non-receptor tyrosine kinase protein involved in many biological pathways and

cellular processes linked to cell growth and survival, including cytoskeleton remodeling, cell motility and adhesion, receptor endocytosis, DNA damage response, and apoptosis and autophagy. When translocation occurs within the breakpoint cluster region (BCR) on chromosome 22 (Philadelphia chromosome), this causes for the creation of an unregulated BCR-ABL1 fusion oncogene, which encodes an unregulated BCR-ABL1 oncoprotein that is closely linked to chronic myeloid leukemia (CML), a slowly progressing cancer of the blood.1 Studies in patients with CML have shown that around 50% of patients who reach undetectable BCR-ABL1 transcription levels for at least two years, indicating the inactivity of BCR-ABL1 in their bodies, remain in remission after cessation of treatment.2

Figure 1. Cellular signalling network3

Figure 2. BCR-ABL1 cellular signalling pathway4

Several tyrosine kinase inhibitors, such as nilotinib, imatinib mesylate, dasatinib, and

sorafenib, have been found to inhibit BCR-ABL1. These inhibitors have improved clinical outcomes for CML patients, over 80% of patients treated with imatinib surviving for more than 10 years. Nevertheless, 20% of patients do not experience good long-term treatment due to the emergence of drug resistance. Therefore, it is still important to discover more compounds that can bind to and inhibit BCR-ABL1. 1 “UniProtKB - P00519 (ABL1_HUMAN)”. UniProt. http://www.uniprot.org/uniprot/P00519#family_and_domains 2 Andrew A. Wylie, et al. (2017). The allosteric inhibitor ABL001 enables dual targeting of BCR–ABL1. Nature Review. 733-737. https://www.nature.com/nature/journal/v543/n7647/full/nature21702.html. 3 Walter Kolch & Andrew Pitt (2010). Function proteomics to dissect tyrosine kinase signalling pathways in cancer. Nature Reviews Cancer. 618-629. http://www.nature.com/nrc/journal/v10/n9/full/nrc2900.html 4 ibid H2

Virtual Screening and Drug Discovery: Finding Drugs that Inhibit the BCR-ABL1 Oncoprotein Hilary Y. Liu â&#x20AC;&#x2122;18, Nicolas A. Pabon, Bentley M. Wingert, Carlos J. Camacho, PhD

Figure 3. Kinase domain of ABL1 (blue) in complex with nilotinib (red)

Through genomic-based screening in a previous study using the NIH LINCS program, a

database containing data on changes in gene expression and other cellular process when cells are exposed to a variety of perturbing agents, 232 out of about 10,000 potential compounds have been predicted to ABL1.5 The goal of this project is to refine those predictions through a novel approach called virtual screening, a computational technique used in drug discovery, to identify small molecules that, through molecular docking and structural analysis, are most likely to bind to a drug target protein.

METHODS 1) Look for representative structures of ABL1 to which to dock compounds on the RCSB Protein Data Bank (PDB), a large database containing tens of thousands of protein structures found by previous researchers, through x-ray crystallography and nuclear magnetic resonance (NMR) screening. 2) Convert small compounds from simplified molecular-input line-entry system (SMILE), a type of 2D string of letters and symbols representing small molecules, to structure-data file (SDF) format for docking using OpenBabel. 3) Dock compounds using dock-close pipeline that runs on Smina to catalytic and myristolic sites of ABL1 receptors that correspond to most similar co-crystal ligands for each of the predicted compounds. If there is a co-crystal structure ligand already known to bind to ABL1, it would be good to dock a similar compound to the same receptor due to the changes in the binding pocket that the binding of a compound might cause to the protein. 5 Nicolas A. Pabon, et al. (2017). Decoding drug-target interactions in mRNA perturbations. THE BALDWIN REVIEW 2017

Virtual Screening and Drug Discovery: Finding Drugs that Inhibit the BCR-ABL1 Oncoprotein Hilary Y. Liu ’18, Nicolas A. Pabon, Bentley M. Wingert, Carlos J. Camacho, PhD

4) Analyze docked compounds on molecular visualisation system Pymol. In determining which compounds to analyze, several key factors were considered: i)

Genomic ranking of the compound from the genomic screening

ii)

Binding affinity between the compound and the receptor (kcal/mol) — a value generated by Smina of the bond energy between the docked compound and the receptor (the more negative the binding affinity, the better of an indication of promising protein-ligand interaction)

iii)

Similarity score between the compound and co-crystal ligand — a value generated by Smina of the bond energy between the docked compound and the receptor (the higher the value, the more similar the docked compound and the co-crystal ligand, and thus the more likely there might be a good protein-ligand interaction)

Figure 4. Virtual screening process overview

Virtual Screening and Drug Discovery: Finding Drugs that Inhibit the BCR-ABL1 Oncoprotein Hilary Y. Liu ’18, Nicolas A. Pabon, Bentley M. Wingert, Carlos J. Camacho, PhD

RESULTS

There exist two primary mechanisms of ABL1 inhibition—competitive inhibition (binding to

the catalytic site, to which ATP usually binds) and allosteric inhibition (binding to the myristolic site, to which myristol usually binds, inducing a conformational chance in the catalytic site which makes it unable to bind compounds).

Figure 5. 5MO4 structure ABL1 protein showing active domains and binding sites

In determining the binding potential of docked compounds, several key docking factors

were considered:6 1) Electrostatic forces – attraction between positively and negatively charged regions of compounds, repulsion between regions of compounds with the same electrical charge 2) Hydrogen bonds – intermolecular force between two molecules resulting from attraction between an extremely electronegative nitrogen, oxygen, or fluorine in one molecule to a hydrogen in another 3) Hydrophilic and hydrophobic group proximity – hydrophilic (water-loving) and hydrophobic (water-fearing) groups cannot be extremely close together 4) Critical interactions between the ligand and the receptor – in several compounds already known to bind to ABL1, there exists critical interactions between the ligand and the receptor, or interactions that are present almost all the time between a docked compound and the receptor. It is important when analyzing docked compounds to keep these critical interactions.

By looking at interactions between ABL1 and these compounds, 232 predicted drugs were

effectively narrowed down to 16 that were very promising. 6 Caterina Bissantz, Bernd Kuhn, and Martin Stahl (2010). A Medicinal Chemist’s Guide to Molecular Interactions. Journal of Medicinal Chemistry Perspective. 5061-5084. http://pubs.acs.org/doi/pdf/10.1021/jm100112j.

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Virtual Screening and Drug Discovery: Finding Drugs that Inhibit the BCR-ABL1 Oncoprotein Hilary Y. Liu â&#x20AC;&#x2122;18, Nicolas A. Pabon, Bentley M. Wingert, Carlos J. Camacho, PhD

Figure 6. Sorafenib (magenta) docked to 3QRJ structure of ABL1 (green) with co-crystal structure ligand 919 (cyan). Nitrogen (blue), oxygen (red), polar hydrogen (white), and hydrogen bonds (yellow) shown. Because sorafenib is already known to bind to ABL1, using sorafenib as a control compound looks promising and is a good affirmation of the accuracy and effectiveness of virtual screening as a drug discovery method.

Catalytic Site Binding

The catalytic site is a relatively large pocket that is able to fit a variety of compounds, with a

nonpolar region in the back of the docking site and a polar region toward the front.

Figure 7. AZ-628 (magenta) docked to 2E2B structure of ABL1 (green) with co-crystal structure ligand 406 (cyan). Nitrogen (blue), oxygen (red), polar hydrogen (white), and hydrogen bonds (yellow) shown. The docked compound has great similarity to the co-crystal ligand and maintains many of the critical interactions between the co-crystal ligand and the receptor, while also creating some additional promising interactions such as hydrogen bonds that the co-crystal ligand could not.

Virtual Screening and Drug Discovery: Finding Drugs that Inhibit the BCR-ABL1 Oncoprotein Hilary Y. Liu â&#x20AC;&#x2122;18, Nicolas A. Pabon, Bentley M. Wingert, Carlos J. Camacho, PhD

Figure 7. Palbociclib (magenta) docked to 2G2H structure of ABL1 (green) with co-crystal structure ligand P16 (cyan). Nitrogen (blue), oxygen (red), polar hydrogen (white), and hydrogen bonds (yellow) shown. Although the docked compound does not appear very similar to the co-crystal structure ligand, it creates many of the same critical interactions. Additionally, the docked compound creates triple nonpolar carbon ring stacking, which is a very good indication of docking potential.

Myristolic Site Binding

The myristolic binding site is much smaller than the catalytic site and is able to bind smaller,

more nonpolar compounds.

Figure 8. CGS-15943 (magenta) docked to 3PYY structure of ABL1 (green) with co-crystal structure ligand 3YY (cyan). Nitrogen (blue), oxygen (red), polar hydrogen (white), and hydrogen bonds (yellow) shown. Compound and co-crystal ligand have great similarity and promising interactions.

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Virtual Screening and Drug Discovery: Finding Drugs that Inhibit the BCR-ABL1 Oncoprotein Hilary Y. Liu â&#x20AC;&#x2122;18, Nicolas A. Pabon, Bentley M. Wingert, Carlos J. Camacho, PhD

Figure 9. canertinib (magenta) docked to 5MO4 structure of ABL1 (green) with co-crystal structure ligand AY7 (cyan). Nitrogen (blue), oxygen (red), polar hydrogen (white), and hydrogen bonds (yellow) shown. Compound and co-crystal ligand have great similarity and promising interactions.

This study through molecular docking and structural analysis refined predictions of drugs

that may bind to and inhibit the ABL1-oncoprotein.

Figure 10. Comparison of top compounds after molecular docking and structural analysis, with conditional coloration from worst (red) to best (green) for each set of criteria

Virtual Screening and Drug Discovery: Finding Drugs that Inhibit the BCR-ABL1 Oncoprotein Hilary Y. Liu â&#x20AC;&#x2122;18, Nicolas A. Pabon, Bentley M. Wingert, Carlos J. Camacho, PhD

Figure 11. Narrowing down and refining compounds predicted to bind to ABL1 overview and success

FUTURE DIRECTIONS

As a next step, the compounds in this study could be refined using an align-close program,

which will generate and minimize conformers for each compound. Compounds could be docked to binding sites on the SH3 and SH2 domains, which have not been as extensively studied as the kinase domain. The efficacy of these drugs as ABL1 inhibitors and as potential treatments for CML and BCR-ABL1-linked cancer will be screened, experimentally tested, and validated.

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Virtual Screening and Drug Discovery: Finding Drugs that Inhibit the BCR-ABL1 Oncoprotein Hilary Y. Liu ’18, Nicolas A. Pabon, Bentley M. Wingert, Carlos J. Camacho, PhD

ACKNOWLEDGEMENTS Carlos Camacho, PhD., Nicolas Pabon, Bentley Wingert, and Samir Abdouni Joseph Ayoob, PhD., Timothy Lezon, PhD., Emily Furbee, PhD., David Koes, PhD. Leah Russell, Gengkon Lum Solomon Livshits, David Boone, PhD., Steffi Oesterreich, PhD. DiSCoBio, TECBio, UPCI Summer Academy

BIBLIOGRAPHY “UniProtKB - P00519 (ABL1_HUMAN)”. UniProt. http://www.uniprot.org/uniprot/P00519#family_ and_domains Andrew A. Wylie, et al. (2017). The allosteric inhibitor ABL001 enables dual targeting of BCR– ABL1. Nature Review. 733-737. https://www.nature.com/nature/journal/v543/n7647/full/nature21702.html. Walter Kolch & Andrew Pitt (2010). Function proteomics to dissect tyrosine kinase signalling pathways in cancer. Nature Reviews Cancer. 618-629. http://www.nature.com/nrc/journal/v10/n9/full/nrc2900.html Nicolas A. Pabon, et al. (2017). Decoding drug-target interactions in mRNA perturbations. Caterina Bissantz, Bernd Kuhn, and Martin Stahl (2010). A Medicinal Chemist’s Guide to Molecular Interactions. Journal of Medicinal Chemistry Perspective. 5061-5084. http://pubs.acs.org/doi/pdf/10.1021/jm100112j.

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NEELAM PANDYA ’18 Neelam Pandya came to Baldwin in 2006 when she was in 1st grade. She lives in Radnor, PA, and is a proud member of multiple organizations and programs at Baldwin. Neelam is the Lead Critic of the Cappies theatre and journalism program at Baldwin, a 4-year Masker, as well as a 3-year B-Flat. She continues to support the arts by performing in every single production as well as being an assistant director of the fall 2017 production of “Much Ado About Nothing.” Neelam also is a member of the Model UN Team, a Bryn Mawr Tutor, an Hourglass guest writer and the senior head of the club, Women on the Rise. In her free time Neelam enjoys reading, making or listening to music and creating webpages and websites through HTML and CSS.

Observation of the Self-Assembly of a Tripeptide Containing Three Hydrophobic Amino Acids Lehman College By Neelam Pandya ’18

THE BALDWIN REVIEW 2017

Observation of the Self-Assembly of a Tripeptide Containing Three Hydrophobic Amino Acids | Neelam Pandya â&#x20AC;&#x2122;18

ABSTRACT

This study was performed to observe and understand why hydrophobic tripeptides form

certain distinguishable structures. The original hypothesis predicted that the tripeptides would stack on top of each other to form beta sheets or amorphous structures. To test this prediction, coarse-grained models of 2-16 unit tripeptide structures were generated through multiple programs in the Linux open source software. These models were generated the fastest out of three possible options and provided the structural information needed about the self-assembly of peptides. Countering the hypothesis, the 3-16 unit tripeptides formed beta spherulites instead of beta sheets or amorphous structures. Additionally, the models that had a base unit of three tripeptides were more stable than the rest of the models that were generated. The significance of this conclusion along with why the hydrophobic tripeptides formed beta spherulites will be explored in future research.

ACIDS There has been a widespread interest in studying the structure of the self-assembly of peptides over the past few years. While multiple applications of the self-assembly of peptides has been previously researched, the study of the actual structure has not been as widespread. Various models, such as the all-atom or coarse-grained model can be used to show these structures, but the final formations of the peptides are still unclear. Over the past six weeks, the team at Lehman College has been generating different combinations of peptide structures that will assemble into different formations in order to solve why certain combinations of peptides self-assemble in specific ways. Researching the self-assembly of peptides process can be beneficial to many different disciplines. Pharmaceutical companies use the self-assembly process to insert a drug in the center of these peptides and then put that combination into the body1. Once there, the peptides will disassemble and release the drug2. Another use of this process is to grow STEM cells. STEM cells are unspecialized cells that do not have a specific purpose yet3. Under certain conditions, STEM cells can become tissue or organ specific cells4. They can be useful in this scenario because they have the ability to repair parts of the body since the cells do not have a specific purpose at the beginning5. Boxes of these self-assembling peptides are constructed and the STEM cells are placed in the middle. Similar to the drug insertion, the STEM cells are transported into the body via self-assembling peptides that will help release the cells. The peptides can also be used to help Alzheimer and Parkinson patients6. In those diseases, the proteins deactivate because another identical protein attaches to it. Certain self-assembling peptides can hook onto one of the healthy proteins in the patientâ&#x20AC;&#x2122;s body in order to prevent this deactivation. The self-assembly of peptides is when the peptides come together in an organized manner and depend on three factors: hydrophobicity, concentration, and temperature.

Observation of the Self-Assembly of a Tripeptide Containing Three Hydrophobic Amino Acids | Neelam Pandya â&#x20AC;&#x2122;18

Depending on these factors, peptides can assemble in a variety of ways such as into beta sheets or into beta spherules. This paper explores the stability of various configurations of tripeptides composed of three hydrophobic residues. The tripeptide considered ranged from the dimer species to 16-tripeptide structures. The energy, heat capacities, and ordered parameters were calculated and recorded for each configuration that was generated by the computer simulation. A possible trend was noted between the lowest energy level of a certain configuration coinciding with the highest ordered parameter. While this trend was not always proven true, it was seen to be more accurate as the tripeptide structures became larger.

METHOD Equations

In order to generate the various coarse-grained models, multiple mathematical formulas

were used to calculate the energies of the various configurations. The model developed by Sorenson/Head-Gordon was used to represent the intermolecular forces, given by V, between the residues.

where

represents the intermolecular forces between the bonds, represents forces between the angles, and

represents the forces between the dihedral angles, (which for our system is zero since we are working with a three amino acid peptide rather than a four amino acid one). Lastly, the LennardJones potential represents the hydrophobic-hydrophobic interactions with

order to prevent evaporation of any of the peptide, a constraining potential has been used, i.e.

In order for self-assembly to occur, the tripeptides must be close enough to see each other.

Once they notice each other, they can come together to form a new structure. In the constraining potential, Rc represents the constraining radius, Ri represents the position of the ith residue, and Rcm represents the center of mass of the system. The constraining radius is range of area in which the

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Observation of the Self-Assembly of a Tripeptide Containing Three Hydrophobic Amino Acids | Neelam Pandya â&#x20AC;&#x2122;18

peptides can see each other and self-assemble. If a randomly generated number is larger than this radius, then the movement of a residue within the structure will not occur. If the randomly generated number is smaller, then the movement will occur. Sampling of configurations were performed using the standard Monte Carlo technique, where the probability of accepting a movement of an amino acid or residue, p, is given by,

where b is 1/kBT, kB is Boltzmann constant, T is the absolute temperature, and _U is the change in internal energy between the sampled configurations. The lowest number between the one and the value will be accepted, which is why the latter number must be smaller than the given 1. 552 different configurations of tripeptides ranging from 2 units to 16 units were generated by these methods to better understand how the self-assembly of peptides works. Computer Simulations The coarse-grained models were generated to depict how the tripeptides self-assembled using open software programs, like Linux, written by Professor Gustavo Lopez at Lehman College1. There are various models besides the coarse-grained one that can be used to depict the self-assembly of peptides. The all-atom model is the most detailed model out of the three commonly used ones but, because of its detail, the computer simulation takes the longest amount of time. Another type of model or structure, the united atom model, is a slightly simpler version of the all-atom model. This one groups the atoms into units so the simulation takes a shorter time to run. The last type of structure, the coarse-grained model, was the one used in this study. The coarse-grained model takes the shortest amount of time to generate but still gives the structural information needed to study why the tripeptides form their specific type of structure. The hydrophobic interactions between the molecules are more easily seen in this type of model since each molecule is represented as an atom, making the structure clearer, as seen in Figures 1 and 2. Figure 1 represents the coarsegrained model of a 36-residue tripeptide structure created by Professor Gustavo Lopez while Figure 2 represents the all-atom version of the same structure. Figure 1.

Figure 2.

Observation of the Self-Assembly of a Tripeptide Containing Three Hydrophobic Amino Acids | Neelam Pandya â&#x20AC;&#x2122;18

RESULTS While the team at Lehman College originally predicted that the hydrophobic tripeptides would self-assemble into stacked beta sheets as the McGregor Lab had seen, all of the structures consisting of three tripeptides onwards formed more spherical structures known as beta spherulites. Instead of stacking, the tripeptides started to interlock in angular or straight shapes in order to increase their intermolecular forces. As seen in Figure 3, the yellow and teal tripeptides did stack side-by-side as originally predicted like a beta sheet. The change to a more spherical structure occurred when three tripeptides made a formation. The royal blue molecule in Figure 4 did not stack on either the teal or yellow molecule as it would have if its structure had been like a beta sheet. The interesting aspect of the 3 unit tripeptide structures was that they formed spherical beta spherulites rather than the beta sheets that were present in the dimer (twotripeptide structure). Figure 3.

Figure 4.

N=2

N=3

Both Figures 3 and 4 support the theory that tripeptide structures become more spherical as they grow larger. Figure 5 shows a 6-unit tripeptide structure while Figure 6 represents a 12-unit tripeptide structure. Figure 5.

N=6

Figure 6.

N = 12 THE BALDWIN REVIEW 2017

Observation of the Self-Assembly of a Tripeptide Containing Three Hydrophobic Amino Acids | Neelam Pandya â&#x20AC;&#x2122;18

Figure 7 below shows the stability of the generated structures. As the respective energies of the structures dropped, they became more stable. Figure 7.

Figure 8 depicts the coarse-grained model with the lowest energy from each tripeptide structure. The energy suddenly dropped between the dimer (two-tripeptide structure) and the three-tripeptide structure. The four and five unit tripeptide structures did not see a similar drop in energy. Instead, the energy rose, dropped, and then became even lower as the six-unit tripeptide energy was calculated. The cycle of having a large energy drop repeats every time three tripeptides are added to the existing structure. Figure 8.

Observation of the Self-Assembly of a Tripeptide Containing Three Hydrophobic Amino Acids | Neelam Pandya â&#x20AC;&#x2122;18

DISCUSSION The original intention of this research was to determine the structure of hydrophobic tripeptides. The original hypothesis stated that the hydrophobic tripeptides would form a beta sheet like structure or an amorphous one. An amorphous structure is one where there is no specific or defined shape. After generating the computer simulations depicting the coarsegrained models of self-assembly, the results showed a consistent spherical shape throughout the 3-unit and 16-unit tripeptide structures. The dimers (two-unit structures) seemed to form beta sheets but the three unit and larger structures seemed to form spherical structures. Per Figure 8, it is known that all of the structures were stable as seen from the lowering of the energies, but the reason why the tripeptides were interlocking instead of stacking was still unknown. At the recommendation of Professor Gustavo Lopez, ordered parameters were also generated for their respective coarse-grained models. An ordered parameter reveals the degree of solidity or liquidity of a certain structure. The original hypothesis indicated that the structure with the lowest energy and the highest ordered parameter would be the most stable and solid structure out of the generated coarse-grained ones. As the ordered parameters and energies were being generated, the prediction was found to be untrue. While the energies and ordered parameters of the larger structures had more correlation than the smaller ones, there was no definitive trend. It was concluded that each structure had about the same degree of solidity or liquidity. After looking at the structure of the models, it was seen that the tripeptides bond together through a mixture of stacking and interlocking in order to increase their intermolecular forces. The reason why this occurs could not be explored in this study due to time but it will be studied in the future. The most interesting result of this study was the cycle of how the energy suddenly drops every time three tripeptides are added to an existing structure. This leads to the conclusion that the 3-unit base tripeptide structure is a type of base unit for the self-assembly of peptides. A possible hypothesis is that two 3-unit base structures will form a 6-unit tripeptide structure and so on. Unfortunately, this exciting hypothesis will have to wait to be tested in the future.

CONCLUSION Further research needs to be done to see whether the hydrophobic tripeptides actually form beta sheets, beta spherulites, or another kind of structure altogether. As seen in Figure 8, the coarse-grained spherical models are all stable but the energy drops even lower when a 3-unit base tripeptide structure is present. The reason why these tripeptide structures are more stable than the rest of the structures needs to be further studied along with whether the 3-unit tripeptide structure can be a base for larger structures. 6 and 9 unit structures may be composed of 2 or 3 unit tripeptide structures respectively and further research can confirm or deny this hypothesis.

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Observation of the Self-Assembly of a Tripeptide Containing Three Hydrophobic Amino Acids | Neelam Pandya â&#x20AC;&#x2122;18

ACKNOWLEDGEMENTS I would like to thank Professor Gustavo Lopez for his expertise, time, teaching and models as well as Lehman College for its generous resources. Thank you to Catherine Tissot for her feedback on this paper. Thank you to Priyanka B. for answering countless questions about everything from careers in STEM to formatting scientific papers. Lastly, thank you to Professor McGregor, Sheila and the team at the McGregor Lab for their endless support.

REFERENCES

1 Lopez, Gustavo, Lecture Notes, August 8, 2017 2 Lopez, Gustavo, Lecture Notes, August 8, 2017 3 National Institutes of Health. (n.d.). Stem Cell Basics I. Retrieved August 13, 2017, from https://stemcells.nih.gov/info/basics/1.html 4 National Institutes of Health. (n.d.). Stem Cell Basics I. Retrieved August 13, 2017, from https://stemcells.nih.gov/info/basics/1.html 5 National Institutes of Health. (n.d.). Stem Cell Basics I. Retrieved August 13, 2017, from https://stemcells.nih.gov/info/basics/1.html 6 National Institutes of Health. (n.d.). Stem Cell Basics I. Retrieved August 13, 2017, from https://stemcells.nih.gov/info/basics/1.html 7 Wikipedia. (2-17, August 12). Linux. Retrieved August 14, 2017, from Wikipedia website: https:// en.wikipedia.org/wiki/Linux

SYDNEY SILBERG ’18 Sydney Silberg is from Lower Merion, PA, and has been at the Baldwin School since 7th grade. This past summer, Sydney conducted research at the University of Pennsylvania in the Gene Therapy lab. Outside of science, she runs on the Varsity cross country and track teams, and also swims on the Varsity team. Sydney is a peer counselor to the Middle School and is the head of the Kiva service club.

Generation of a Self-Inactivating Cas9 System By Sydney Silberg ’18

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Generation of a Self-Inactivating Cas9 System | Sydney Silberg â&#x20AC;&#x2122;18

ABSTRACT:

Crispr is a system used in gene therapy which helps scientists edit specific regions of

genomic DNA. Crispr stands for Clustered Regularly Interspaced Short Palindromic Repeats. Specific regions can be targeted by using guide RNAs which search the genome until they reach their sequence-specific targets and recruit the Cas9 nuclease. Here, Cas9 cuts the DNA to turn the gene expression off. While very useful, Crispr/Cas9 has shown to become less specific the longer it is present in a cell, and will begin to edit genes unintendedly. The goal of this project was to generate a self-inactivating Cas9 system to reduce off-target effects while retaining on-target editing capability of Cas9. To achieve this goal, we cloned and transfected HEK293 cells with two plasmids, one encoding SaCas9 (Staphylococcus aureus Cas9) flanked by two target sites (human PCSK9 and T1), and the other single guide RNAs against these two targets. The expected result was less Cas9 expression in the RNA of the cells transfected with both plasmids compared to controls without losing genome editing efficiency at human PCSK9 locus.

METHODS AND RESULTS:

To achieve the goal of this project, we sought to transfect four plasmids in different

combinations into HEK293 cells. I constructed three out of the four plasmids as illustrated in Figure 1: pHS07, pHS14, and pHS21. First, vector DNA was prepared by gel extraction following restriction enzyme digestion. Insert DNA was generated by annealing oligos. Then the vector and insert was ligated together and transformed into E.coli cells and plated to agar plates with Carbenicillin to allow the correct cell colonies to grow. On the next day, the colonies are picked and grown in TB medium supplemented with Carbenicillin. After the bacteria have grown, mini prep was conducted to extract the plasmids. The first plasmid completed was pHS14, a plasmid constructed by adding sgR.Pcsk9 and a U6 primer reversely into plasmid p0101 which contains GFP, which is a protein that causes live cells to light up under microscope if transfected properly. Next, pHS07 was cloned using a multiple step process. pHS07 is a combination J2

Figure 1: Schematics of DNA plasmids used in this project and expected results from transfection.

Generation of a Self-Inactivating Cas9 System | Sydney Silberg â&#x20AC;&#x2122;18

of pHS02, a plasmid containing sgR.T1 (a target for Cas9), and pHS06, a plasmid containing previously mentioned pHS14 and a Nhe1 cut site. When pHS06 and 02 was combined reversely at an Mlu1 site with a U6 primer, the final construct of pHS07 was made. Finally, pHS21 was made by inserting guide RNA target sites for hPCSK9 and T1 to flank SaCas9 in p4076.

Multiple tests were conducted to verify the correctness of the cloned plasmids. Briefly, plasmid

DNAs were incubated with restriction enzymes at 37C for 1-2 hours and run on an electrophoresis gel to check if the plasmid cuts in the right place. Other methods include sequencing the DNA by Sanger method and comparing the results to the reference sequence. If the two sequences match up, then the cloning is considered successful.

Once the plasmids were cloned, they can be transfected into HEK293 cells. In this experiment,

there were five different combinations of plasmids and one control well (Table 1), however all were transfected twice as technical replicates. The wells were as followed, each the same on plate A and plate B. Table 1: Experimental setup for HEK293 cell trasfection

The first two wells were transfected with pHS07 and pHS21 in different ratios. pHS21 is a

plasmid containing 2 target sites and SaCas9 while pHS07 contains two guide RNAâ&#x20AC;&#x2122;s. It was hoped that the guides in pHS07 cut pHS21 where the target sites were and therefore will cut out Cas9 and decrease the expression of Cas9 in the mRNA. Well number three contains pHS21 and pHS14. While pHS21 still has the target sites, pHS14 has only one guide so the Cas9 will not be cut out. In well number 4, p4076, a plasmid with Cas9 but no target, was combined with pHS14. Since there was only the guide in pHS14 in this transfection, Cas9 was not expected to be cut out. For both wells 3 and 4, the SaCas9 expression in the mRNA was not expected to decrease. Well 5 was the negative control and only contained pHS21 while well 6 was untransfected. Three days after transfection, fluorescent images were taken from these cells. pHS07 and pHS14 both contain GFP and caused the wells containing these cells to glow up green. This green fluorescence showed that the transfection was successful (Figure 2). The cells were then harvested for gDNA and RNA extraction. The gDNA was used for PCR using the primers spanning the

Figure 2: Representative GFP fluorescent image of HEK293 cells transfected with GFP containing plasmids THE BALDWIN REVIEW 2017

Generation of a Self-Inactivating Cas9 System | Sydney Silberg â&#x20AC;&#x2122;18

predicted cleavage site in human PCSK9 locus. The PCR products were run on a gel and all lanes had a single band at around 400 bp, the expected amplicon size. This means that the PCR was successful and a surveyor assay can be done to detect genome editing at human PCSK9 locus. Surveyor assay showed three bands in each lane that was run except lane 5 (negative control) and 6 (untreated), Figure 3: Surveyor Assay products run on an electrophoreses gel

demonstrating that the DNA has been edited. The RNA extraction was used to measure the amount of SaCas9 mRNA expression in the cells. To do

this, Dnase treated RNA were converted into cDNA by reverse transcription. Real-time PCR was then conducted using cDNA as template as well as pHS21 plasmid DNA as standards and H2O as negative controls.

CONCLUSION AND DISCUSSION: While the Crispr/Cas9 system is an important tool for gene editing, as it stays inside of cells it will begin to cut unintended genes. To fix this problem, we attempted to create a self-inactivating Cas9 system. To do so, plasmids were constructed and paired, one with SaCas9 and target sites, and another with guide RNAs to edit the plasmid with SaCas9. While the plasmids cloned successfully, it was found that pHS07 was missing part of the U6 primer used. In addition to successful cloning, transfections and the PCR were also fruitful. Furthermore, the Surveyor Assay using the PCSK9 primer cut correctly; however, when paired with the T1 primer, we did not observe surveyor digestion as expected, indicating inefficient editing of the T1 site. The final step of the project was to do a Real Time PCR to measure the amount of SaCas9 expression, but unfortunately this failed likely due to technical errors. Hopefully in the future the error in the real time PCR will be corrected, and the SaCas9 expression can be properly measured.

CASSANDRA STECKER ’18 Cassandra Stecker is a Baldwin senior from Bryn Mawr, PA, and a returning contributor to the Baldwin Review. An intern at the Historical Society of Pennsylvania and volunteer at the Lower Merion Historical Society, Cassandra was first introduced to historical archives through Baldwin’s collections. Separate from her historical endeavors, Cassandra serves as senior head of Lamplighters, Baldwin’s student tour guide and admissions ambassador program, as well as President of Model United Nations. She has positions on numerous Baldwin publications, including the Hourglass newspaper and the French literary magazine Florilège; she will be unveiling new platforms to share political views at Baldwin in her senior year.

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As a city deeply entrenched in Quaker ideology, Philadelphia has long been the hotbed for

the promotion of the abolition of slavery. Abolitionist thought in William Penn’s colony can trace its roots as far back as 1688, when a group of Quakers in Germantown drafted the Quaker Petition Against Slavery. The significance of this document is more symbolic than legislative or practical: it was the first written document in American history which advocated for universal equality. In this regard, the petition is a little-known predecessor of great American documents including the Declaration of Independence and the Constitution.1

It would not be until almost a century later, when The Pennsylvania Society for the Relief

for Free Negroes Unlawfully Held in Bondage (colloquially known as the Pennsylvania Abolition Society) was founded in 1775 by a group of ten men, mostly Quakers, at a Philadelphia tavern, that the United States saw the formation of its first abolition society.2 Interestingly, the impetus for the founding of a formal abolitionist organization arose over the case of an Indian slave. The slave, having been brought to Philadelphia from New Jersey by her master, claimed that she was free in the colony of Pennsylvania. Quaker activists, notably Israel Pemberton, an influential Philadelphia merchant, came to the defense of the slave in her trials. After two years of deliberation, the woman was refused her freedom; enraged, activists decided that a formal organization was needed to thoroughly and actively seek justice for slaves.3 The first president of the Pennsylvania Abolition Society was John Baldwin. However, with the chaos of the Revolutionary War and the controversy surrounding the Quakers’ wartime adherence to pacifism, the society fell into forgotten obscurity.4

The society was re-established in 1884 under the new name ‘The Pennsylvania Society for

Promoting the Abolition of Slavery, for the Relief of Free Negroes Unlawfully Held in Bondage, and for Improving the Condition of the African Race.”5 For the purposes of this paper, this organization will be referred to as the Pennsylvania Abolition Society (PAS), as used by most historians writing on the topic. This re-invigorated abolition society was bolstered by the membership of prominent public figures such as Thomas Paine, Benjamin Rush, and the society’s new president, Benjamin Franklin.

1 Germantown Protest Materials, Undated, MC-950.308, Box 3, Folder 4, Haverford College Library Quaker and Special Collections, Haverford, Pennsylvania. 2 Bacon, Margaret Hope. History of the Pennsylvania Society for Promoting the Abolition of Slavery: The Relief of Negroes Unlawfully Held in Bondage; and for Improving the Condition of the African Race.

(Philadelphia: Pennsylvania Abolition Society, 1959), 25-29.

3 Needles, Edward. An Historical Memoir of the Pennsylvania Society for Promoting the Abolition of Slavery; the Relief of Free Negroes Unlawfully Held in Bondage and for Improving the Condition of the African Race. (Philadelphia: Merrihew and Thompson, printers, 1848), 18-20. 4 Bacon, 41. 5 Ibid.

Although the word ‘abolition’ has garnered widespread use in contemporary times as

an umbrella term for all anti-slavery endeavors, there is a marked distinction between Abolition Societies, such as PAS, and anti-slavery advocates, notably William Lloyd Garrison. Abolition Societies sought to eliminate slavery and alleviate the problems facing blacks at a local level through quiet tactics. For example, the Pennsylvania Abolition Society did not usually see its role as advocating for the national abolition of slavery. In contrast, individual abolitionists, sometimes referred to as ‘secondary abolitionists,’ were vocally fervent and aggressive in their mission to emancipate all slaves across the country.

Before delving into specific endeavors of the Pennsylvania Abolition Society, an

understanding of the climate of slavery in Pennsylvania is necessary. Although some of the first Pennsylvania Quakers were slaveowners, by the turn of the 18th century, many Quakers had already freed their slaves, worried that owning another man or woman was incompatible with Quaker ideology. In 1758, slaveholding Quakers were excluded from Quaker leadership, and by 1774, this policy extended to membership in the Meeting of Friends. In 1780, the Pennsylvania Legislature passed the Act for the Gradual Abolition of Slavery.6 While this still made it possible for certain people to be held in slavery in Pennsylvania, the act envisioned a future Pennsylvania without slavery. Furthermore, the slave population in Pennsylvania in 1780 was not significant. In 1776, Delaware, an area which contained the majority of slaves in the Pennsylvania colony, became an independent state. Thus, only about three percent of Pennsylvanians were held as slaves in 1780.7 This is largely due to the fact that slavery was not a profitable endeavor in Pennsylvania as compared to the south; massive slave-run plantations were simply unsustainable. Some accounts claim that there were slaves in Pennsylvania until the Civil War, but Pennsylvania was nevertheless a largely free society throughout the 19th century. Many slaves were freed by manumission, and strict laws for the emancipation of most of the Pennsylvania slave population were passed by the state legislature in 1820 and 1847.8

The Pennsylvania Abolition Society was not a lawmaking body. Rather, it endeavored

to enforce laws surrounding slavery and to oppose any proposed discriminatory laws. With the revival of the society in 1784, freeing slaves was not necessarily the primary goal of its members. When the society added ‘Improving the Condition of the African Race,’ to the end of its name, it established that it sought to support all black Americans. While certainly better than a life in bondage, ‘free’ blacks were far from free. In Philadelphia, they were usually tried in a special court without a jury and were subject to strict laws regulating schooling, marriage, and interaction 6 Needles, 39. 7 Ibid., 34. 8 Ibid., 35-38. THE BALDWIN REVIEW 2017

with white people. The Pennsylvania Abolition Society provided legal support to the free blacks of Philadelphia, often in cases concerning a debt either owed by the free man or to the free man by his former masters Another variant of the legal situations addressed by the society were black men and woman ‘unlawfully held in bondage,’ as stated by the formal name of the society. With the complicated laws surrounding slavery in 18th and 19th century Pennsylvania, many masters manipulated the system through falsified manumissions, kidnapping, and a number of other devious schemes. Local witnesses to offenses of this sort often wrote to the Pennsylvania Abolition Society which, in turn, would address the case. Lastly, since the Pennsylvania Abolition Society was the first of its kind in America, it encouraged, guided, and assisted in the formation of other similar societies nationwide. PAS even supported abolition societies in in the south, where slave society was more central to the economy and the culture, such as the Alexandria Society for Promoting the Abolition of Slavery.9

Considering that the Pennsylvania Abolition Society was the seminal group in American

abolition activism, its members, efficacy, and endeavors have been long studied by notable historians. Nevertheless, many of the papers of the Pennsylvania Abolition Society have sat untouched in historical archives across the United States for much of their life. Included in these papers is a collection of letters sent to the society between the years of 1775 and 1852. Correspondence to the society came in two common forms: inquiries from freedmen seeking information regarding their family members and political letters from allies of the society, influential citizens, and state legislatures. In this collection of Pennsylvania Abolition Society papers recently obtained by the Historical Society of Pennsylvania, a private research library in Philadelphia, Pennsylvania, the alliance between “La Société des Amis des Noirs de Paris” (The Society of Friends of Blacks of Paris) and the Pennsylvania Abolition Society has been discovered through an initiative to make these letters accessible in a digital database. This paper seeks to discuss the relationship between these two integral abolition societies, striving for similar goals for two very different environments, and to bring to light supporting evidence only recently uncovered in the archives at the Historical Society of Pennsylvania.

La Société des Amis des Noirs de Paris was founded in Paris in 1788 to advocate for

the abolition of French colonial slaves. Although its operations came to a halt in 1793, the society resumed its political advocacy in 1776. The society disseminated abolitionist literature throughout France and addressed the National Assembly to argue for abolition. In 1794, the National Assembly freed colonial slaves with the Universal Emancipation Decree; it should be 9 Letter, Alexandria Abolition Society to Pennsylvania Abolition Society, October 1798, Box 15, Folder 2, The Pennsylvania Abolition Society Papers, The Historical Society of Pennsylvania, Philadelphia, Pennsylvania. K4

noted that this took place while the La Société des Amis des Noirs de Paris had stopped its functions. Similar to the abolition laws passed in Pennsylvania, plantation owners in French colonies did not always honor the decree and commonly found deceitful ways to maintain their slaves. This was an especially prevalent problem in Saint Domingue, a French colony on the island of Hispaniola in the Caribbean (modern-day Haiti). Saint Domingue was unparalleled in its wealth, often referred to as the “Pearl of the Antilles,” as it was a leader in exporting commodities such as sugar, coffee, and cotton. This contributed to the landowners’ hesitancy to change a system so economically beneficial to them.10

Saint Domingue was the site of Toussaint Louverture’s Haitian revolution, which lasted

from 1791 to 1804. To suppress Louverture’s fighting with Spanish forces against the French, the National Assembly recruited French abolitionists and members of La Société des Amis des Noirs de Paris Étienne Polverel and Léger-Félicitié Sonthonax to offer alternative, nonviolent abolitionist measures in Saint Domingue and to suppress the revolution. Polverel and Sonthonax concentrated their efforts on Cap Français, a commune in the northern area of Hispaniola which the French kept under their control throughout the revolution. Sponsored by La Société des Amis des Noirs de Paris, Sonthonax issued a General Emancipation Decree to abolish slavery in Cap Français in August of 1793. Throughout the Haitian Revolution, Cap Français, under the leadership of Sonthonax, remained a successful example of an experiment in the abolition of slavery. However, it would be misleading to surmise that the abolition of slavery in Cap Français translated to freedom for former slaves equivalent to the rights of Europeans on the colony. Slaves became low-paid ‘laborers’ on plantations, where their work was highly regulated by the French government.11 Nevertheless, it is extraordinary that general peace was maintained in post-slavery Cap Français even in the midst of the Haitian Revolution.

Citizen Giroud, a Frenchman who spent a significant amount of time in the United States,

was the central figure in connecting the Pennsylvania Abolition Society and La Société des Amis des Noirs de Paris. An ardent French abolitionist, Giroud was a member of both the Pennsylvania Abolition Society and La Société des Amis des Noirs de Paris. When Pennsylvania Abolition Society president Thomas Harrison discovered Giroud’s personal relationship with Sonthonax, a universally well-regarded abolitionist, Harrison extended an invitation of membership to the Pennsylvania Abolition Society to Sonthonax through Giroud. In responding to the offer in April of 1796, Sonthonax wrote to the Pennsylvania Abolition Society: 10 Bruno Benoit and Marcel Doringy, “La Société des amis des noirs, 1788-1799. Contribution à l'histoire de l'abolition de l’esclavage,” UNESCO/EDICEF, 1998. 11 Ibid. THE BALDWIN REVIEW 2017

Uncovered from the Archives: Examining the Relationship Between the Pennsylvania Abolition Society and La Société des Amis des Noirs de Paris through the Abolition Experiment in Cap Français | Cassandra Stecker ’18 Citizen Giroud, a member of your society, who has just arrived from the United States, has informed me that you have been so good as to admit me as a member of your society and to enroll my name amongst you. To acknowledge my lively gratitude for this flattering mark of distinction I now write. The immutable principles I have laid down in [my proclamation of August 29, 1793] are your ownwe have, my brothers and friends, devoted our time and labor to the propagation of these principles, and we shall continue to do so.12

It is clear that a strong sense of shared principles and a common goal of equality solidified the cooperation between La Société des Amis des Noirs de Paris and the Pennsylvania Abolition Society. Although Sonthonax had seen the implementation of more tangible abolitionist change in Cap Français than PAS had seen in the United States, Sonthonax still corresponded with the Pennsylvania Abolition Society with eagerness and excitement. This emphasizes that abolition was often seen as a global as opposed to a domestic necessity in the eyes of acclaimed abolitionists during the Age of Enlightenment, in accordance with evolving ideas about the fluidity and interconnectedness of the globe. Additionally, a letter from Giroud, written on Cap Français to PAS, offers insight into the global reputation of PAS: The Society has never forgotten that the first aims formed by humanity, the first acts of virtue exercised for the amelioration of the fate of unhappy blacks, stem from the philanthropists of Philadelphia…The Society of the Blacks in Philadelphia gave birth to those [societies] with which America and Europe are honored.13

Thus, although not involved in flagrant abolitionist activities like slave revolution, the Pennsylvania Abolition Society was revered as a ‘parent society’ not only by other American societies, but by Abolition societies everywhere.

Despite the example set by the Pennsylvania Abolition Society, the year 1796 saw a

reversal in the roles of Pennsylvania Abolition Society and La Société des Amis des Noirs de Paris in their relationship. Since an experiment in abolition was possible on Cap Français, the French abolitionists had more opportunity to see the effects of liberty on society. To illustrate this, Giroud wrote to PAS: 12 Letter, Sonthonax to Pennsylvania Abolition Society, April 6 1796 (Adapted from French Republican Calendar), Box 15, Folder 3, The Pennsylvania Abolition Society Papers, The Historical Society of Pennsylvania, Philadelphia, Pennsylvania. 13 Letter, Giroud to Pennsylvania Abolition Society, April 1796, Box 15, Folder 3, The Pennsylvania Abolition Society Papers, The Historical Society of Pennsylvania, Philadelphia, Pennsylvania.

Uncovered from the Archives: Examining the Relationship Between the Pennsylvania Abolition Society and La Société des Amis des Noirs de Paris through the Abolition Experiment in Cap Français | Cassandra Stecker ’18 The society has attained the principle object of its aims, since the French Republic has announced the abolition of slavery in its colonies and has accorded by a constitutional law the title and rights of French citizens to all men black and colored that live here; this Society, I say, now concerns itself with making that liberty useful to the now French citizens of the colonies and in spreading among these instructions, knowledge and the moral maxims that belong to free and civilized men.14

Although the Pennsylvania Abolition Society still worked to aid freedmen, it did this on a caseby-case basis with the largest initiatives being freedmen schools, which educated only up to around 20 students in a classroom.15 Thus, the Pennsylvania Abolition Society did not oversee or implement liberty in such a mass scale. Furthermore, there was not a revolutionary movement in Pennsylvania (or in the United States until the Civil War), so American initiatives could not have been more widespread.

In their letters to the Pennsylvania Abolition Society, French abolitionists on Cap Français

habitually highlighted the economic benefits which followed abolition in addition to speaking of enlightenment and moral ideals. Sonthonax praises the free laborers of Cap Français as having, “a motivated vivacity,”16 and uses cordial language when discussing these laborers, although historical recordings show that life for these laborers, while better than slavery, was not the immaculate beacon of freedom Sonthonax depicted it as. However, since abstract enlightenment ideals about slavery could only support an argument to a certain extent, Sonthonax did not fail to provide evidence that his experiment was successful. He wrote to PAS: Planting has doubled in every part of the North of Saint Domingue. Everyday I am able to see very advantageous prices for the republic in the planting of coffee, sugar, and cotton.17

It was especially necessary to address the effect of abolition on the economy with American 14 Letter, Giroud to Pennsylvania Abolition Society, June 12 1796 (Adapted from French Republican Calendar), Box 15, Folder 3, The Pennsylvania Abolition Society Papers, The Historical Society of Pennsylvania, Philadelphia, Pennsylvania. 15 Minutes, Pennsylvania Abolition Society Education Committee, October 10 1800, Box 15, Folder 8, The Pennsylvania Abolition Society Papers, The Historical Society of Pennsylvania, Philadelphia, Pennsylvania. 16 Letter, Sonthonax to Pennsylvania Abolition Society, September 23 1797 (Adapted from French Republican Calendar), Box 15, Folder 3, The Pennsylvania Abolition Society Papers, The Historical Society of Pennsylvania, Philadelphia, Pennsylvania. 17 Ibid.

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groups, as this was the primary abolition concern in the United States. Nevertheless, the French correspondence was unfailingly saturated in praise of enlightenment ideals. This is especially prevalent in Giroud’s writing, such as when he wrote to PAS: [Cap Français] is totally comprised of our brave and good brothers the black Republicans, to whom France, enlightened by its philosophies and directed by the principles of justice and humanity, represented by Sonthonax, has given liberty.18

This devotion to such principles is a direct testament to the importance of the enlightenment in the culture of the French Republic. Giroud speaks candidly and and with zeal about the enlightenment ideals which formed Cap Français into a successful experiment much more so than about the economic benefits reaped by abolition. This favoring of the abstract over concrete evidence suggests that French culture was more adept to implementing change based on morality than the United States, where the conversation around slavery was more slanted towards the economy.

The French are stereotypically idealist and romantic, especially when compared to

their more logical and realistic American counterparts. When Giroud wrote to the Pennsylvania Abolition Society urging them to model American abolition after the Cap Français experiment, stating, “Such is the situation that the philanthropists of the American continent would find if they form an establishment like Saint Domingue,” he captured the discordance between the two concordant societies. To implement the Cap Français experiment, something developed in a small Island commune, across the United States is an illogical proposition riddled with issues. Still, ever the persistent optimists, the French encouraged this. The Pennsylvania Abolition Society and La Société des Amis des Noirs de Paris shared similar ideas about a world of abolition, but the ideals amongst these ideas were most well suited to the French colonial environment as opposed to the United States.19

18 Letter, Giroud to Pennsylvania Abolition Society, June 12 1796 (Adapted from French Republican Calendar), Box 15, Folder 3, The Pennsylvania Abolition Society Papers, The Historical Society of Pennsylvania, Philadelphia, Pennsylvania. 19 Letter, Sonthonax to Pennsylvania Abolition Society, September 23 1797 (Adapted from French Republican Calendar), Box 15, Folder 3, The Pennsylvania Abolition Society Papers, The Historical Society of Pennsylvania, Philadelphia, Pennsylvania.

TAYLOR TRAPP ’19 Taylor Trapp, a junior from Philadelphia, PA, has attended Baldwin since 1st grade. Taylor has been in the Black Student Union since 9th grade and DECA for two years. She participates in Volleyball, Swimming and Lacrosse.

Immunohistochemical Assessment of Phosphodiesterase 5A (PDE5A) Expression in Infant Right Ventricular Failure By Taylor Trapp ’19 Dr. Francis McGowan Children’s Hospital of Philadelphia Department of Anesthesiology and Critical Care Medicine STEMPREP 2017

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Immunohistochemical Assessment of Phosphodiesterase 5A (PDE5A) Expression in Infant Right Ventricular Failure | Taylor Trapp ’19

BACKGROUND: The heart is composed of four chambers--two atria and two ventricles--and four major blood vessels. A wall (called a “septum”) separates the two atria and ventricles from each other.1 Two of the major blood vessels, the inferior vena cava (IVC) and the superior vena cava (SVC), carry venous blood from the body back to the heart. The third major blood vessel, the aorta, distributes blood from the left ventricle of the heart to the body. Finally, the pulmonary artery carries blood from the right atrium and right ventricle to the lungs. 2

(Figure 1) Figure 1 shows the different parts of the heart and the direction of blood flow.

The heart is a muscular organ which pumps blood through the circulatory system providing oxygen and nutrients to the body. The two types of blood vessels, called arteries and veins carry blood through the body and back to the heart, respectively. Deoxygenated blood returns from the body to to the heart through the SVC and IVC, and enters the the upper chamber of the heart known as the right atrium. From the right atrium, the blood flows through the tricuspid valve into the right ventricle. From the right atrium the blood is pumped by the right ventricle through the pulmonary artery to the lungs which exchange carbon dioxide (exhaled during breathing) for oxygen (taken up from air by the blood as it travels through the lungs) . The oxygenated blood then returns to the heart by way of the pulmonary veins to the left atrium. From the left atrium blood flows through the mitral valve and to the left ventricle. Lastly, the left ventricle pumps the oxygenated blood through the aorta and out to the body where the cycle repeats itself in a continuous loop.3 1 “Congenital Heart Defects (CHDs).” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 25 June 2014, www.cdc.gov/ncbddd/heartdefects/howtheheartworks.html. Accessed 2 July 2017. 2 Congenital Heart Defects (CHDs).” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 25 June 2014, www.cdc.gov/ncbddd/heartdefects/howtheheartworks.html. Accessed 2 July 2017. 3 Congenital Heart Defects (CHDs).” Centers for Disease Control and Prevention, Centers for Disease Control and L2

Immunohistochemical Assessment of Phosphodiesterase 5A (PDE5A) Expression in Infant Right Ventricular Failure | Taylor Trapp ’19

Heart valves are another major part of the heart. The purpose of heart valves is to keep the blood flowing one direction with no backflow. There are two atrioventricular valves, the mitral valve and the tricuspid valve. The tricuspid valve is located between the right atrium and right ventricle and consists of three flaps (“cusps”). The purpose of the tricuspid valve is to keep backflow from the right ventricle to the right atrium when the ventricle contracts. 4The next main valve is the mitral valve. The mitral valve opens up to increased pressure as the blood flows from the left atrium to the left ventricle. As the heart contracts and forces blood to the aorta, the mitral valve closes. This valve is located on the left side between the left atrium and left ventricle and consists of two cusps. 5 Valves in the aorta and pulmonary artery similarly open when each of these chambers contracts to eject blood. The valves close when the ventricle relaxes so that blood from either the aorta or pulmonary artery does not flow backward into the corresponding ventricle. There are a number of different situations in which the heart can develop problems, or can have congenital problems. In situations where the heart develop problems over time in adults it usually affects the left ventricle. Heart attacks are an example of a heart problem caused by a lack of blood flow and oxygen to the heart. This lack usually results from blockages in one or more of the coronary arteries that arise from the aorta and supply blood to the heart muscle. As the name suggests, congenital heart defects are present at birth. Depending on the specific lesion, they can affect one or more of the cardiac chambers, arteries, veins, and/or valves. 6

(Figure 2) Figure 2 shows major overall types of congenital heart disease. Prevention, 25 June 2014, www.cdc.gov/ncbddd/heartdefects/howtheheartworks.html. Accessed 2 July 2017. 4 Katz A.M., Physiology of the Heart, Lippincott Williams & WIlkins, New York, 2001 5 Katz A.M., Physiology of the Heart, Lippincott Williams & WIlkins, New York, 2001 6 Congenital Heart Defects (CHDs).” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 25 June 2014, www.cdc.gov/ncbddd/heartdefects/howtheheartworks.html. Accessed 2 July 2017.

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Immunohistochemical Assessment of Phosphodiesterase 5A (PDE5A) Expression in Infant Right Ventricular Failure | Taylor Trapp ’19

Although there is no known cause of congenital heart disease, we know that there are likely to be genetic, environmental, and other factors. All cause abnormalities in the how blood moves through the heart and/or lungs. Defects can be relatively simple, such as hole in the septum between 2 of the chambers of the heart, e.g. an atrial septal defect or a ventricular septal defect. They also can be much more complex in terms of the anatomy and the resultant abnormal physiology. Tetralogy of Fallot is an example of a more complex defect. It has four specific features: 1) a hole in the septum between the left and right ventricles (“ventricular septal defect”); 2) narrowing of the pulmonary valve area and/or pulmonary artery (“pulmonary stenosis”); 3) abnormal growth and thickening of the muscle in the right ventricle (“right ventricular hypertrophy); and 4) abnormal location of the aorta as it arises from the left ventricle (“overriding aorta”) [Figure 3] The obstruction to pulmonary artery prevents blood from flowing normally to the lungs. Because of the septal defect between the right and left ventricles, blue (deoxygenated) blood flows instead to the left ventricle and out to the body. This causes the infant to appear cyanotic (blue). The levels of blood oxygen can become low enough to risk injury to the infant’s brain and the heart itself.

(Figure 3) Figure 3 shows a picture of a normal heart versus a heart which has tetralogy of fallot.

For a number of reasons that vary with the specific type of congenital heart disease, decreased function and eventual failure of the right ventricle is a very significant problem. In fact, RV failure is becoming the major cardiac cause of poor long term outcome in “repaired” congenital heart disease patients as they get older. Again, this is quite different from adults where the left ventricle is more often the problem. L4

Immunohistochemical Assessment of Phosphodiesterase 5A (PDE5A) Expression in Infant Right Ventricular Failure | Taylor Trapp ’19

The McGowan laboratory and many others have begun to learn that the right ventricle is very different not only in its shape but also how it functions under normal circumstances and when it is damaged or exposed to increased workloads such as occur when there is a ventricular septal defect or pulmonary stenosis. In addition, clinical research studies in congenital heart disease patients with heart failure have shown that drugs that can improve heart function in adults do not work (and sometimes can even be harmful) in patients with heart failure related to congenital heart disease. All of the current commonly prescribed drugs were developed for adult (left ventricular) heart failure using models thereof. The McGowan lab therefore created an infant animal model in which right ventricular hypertrophy develops leading to right ventricular failure due to the pressure overload of the right heart muscle. The factors that contribute to how the heart contracts normally and as it fails are many and extremely complex. Phosphodiesterase 5A, also known as PDE5A, is an enzyme. It primarily catalyzes the breakdown of cyclic GMP. Cyclic GMP is one important regulator (a “second messenger”) of how well the heart contracts and relaxes. cGMP is produced in response to a number of different primary stimuli and activates protein kinase G (PKG). The roles of cGMP and PKG in regulating heart function are not yet completely understood. PKG is believed to have a number of effects leading to decreased amount and activity of other enzymes that contribute to the development of heart hypertrophy and heart failure. It is therefore a regulator of these other enzymes and pathways. Increased PDE5 activity would be expected to decrease the amount of cGMP and therefore PKG. The expected result would be increased activity of these other systems that contribute to hypertrophy and heart failure. Conversely, reducing or inhibiting PDE5 might be helpful as it would lead to increased cGMP and PKG actions.

(Figure 4 ) Figure 4 shows an overview of the cGMP-PDE5 signaling pathway in the heart.

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Immunohistochemical Assessment of Phosphodiesterase 5A (PDE5A) Expression in Infant Right Ventricular Failure | Taylor Trapp ’19

RESEARCH DESIGN: The overall hypothesis for these experiments is that increased amounts and activity of PDE5A contribute to infant RV failure. Prior experiments in our lab found that there were in fact increased amounts of PDE5A RNA, protein, and enzyme activity in failing infant RV in this model compared to RV tissue from the sham (control) animals. In this experiment, the purpose was to define the amount and cellular location of PDE5A in the right ventricle normal rabbits (control group) and the experimental group in which RV failure had been induced by narrowing (“banding”) the pulmonary artery. There were thus two groups of animals, the control group and the treated group. In the treated group of rabbits, the left side of the rabbit’s chest was opened at 1 week of age (“left anterior thoracotomy”), and the pulmonary artery was narrowed to mimic pulmonary stenosis and to create right ventricular hypertrophy. In the control group of rabbits, the thoracotomy was performed but the pulmonary artery was not banded (this was a sham operation to create a control group).

(Figure 5) Figure 5 displays the pulmonary artery which was the part of the animal that was surgically narrowed in the experimental group of rabbits.

The animals exposed to RV pressure overload developed right ventricular failure after

6-8 weeks. The amount of RV failure was measured by echocardiography. At that time, the animals were anesthetized and the hearts were removed from the chest and fixed in 10% paraformaldehyde (Figure 6). Following washing in PBS, the tissues were exposed to 20% sucrose followed by 30% sucrose overnight. The sucrose treatment dehydrated the tissues, making the hearts easier to cut (section). To prepare the cardiac tissue for immunohistochemistry, the hearts were sliced into smaller pieces.

Immunohistochemical Assessment of Phosphodiesterase 5A (PDE5A) Expression in Infant Right Ventricular Failure | Taylor Trapp ’19

(Figure 6) Figure 6 displays the heart before it was sliced into smaller pieces to be sectioned.

The next step was to freeze the sliced tissue using tissue freezing medium to stabilize it for cutting (“sectioning”). The frozen tissues were sectioned using a cryostat and then placed on glass slides. Staining comes after the deparaffinization and rehydration, the slides were placed in xylene three times for five minutes each, followed by ethyl alcohol for one minute starting with 100% two times and 95% two times ,followed by 80%, 70% and 50% ethyl alcohol each one time. When rehydrating the slides, they must be kept in the fume hood due to the use of xylene and ethanol which produce potentially toxic fumes. After the rehydration process the slides were placed in a coplin jar containing PBS, pH 7.4 for 2 minutes at room temperature. This was followed by treatment with 0.01M citrate buffer pH 6.0 in a water bath at 95 degrees centigrade. The purpose of the citrate buffer is to unmask target antigens on the tissues allowing for better binding of the antibody to the antigens. After the slides incubated in the warm water bath for twenty minutes, they were placed at room temperature to cool for 30 minutes. The slides were then washed in PBS to wash twice for five minutes each. The slides were then treated with peroxidase blocking solution to block the endogenous peroxidases in the tissues at room temperature for ten minutes. After the slides were treated with the peroxidase blocking solution, they were washed in PBS, pH 7.4 for two minutes twice. The slides were then treated with 10% normal donkey serum (NDS) for 30 minutes to block nonspecific binding of the primary antibody to the tissue and reduce background interference. The blocking steps reduce non-specific binding of the antibodies to irrelevant proteins which increases the accuracy of the immunostaining. Once the tissues were blocked with NDS the next step was to treat the tissues with the primary antibody. The function of the primary antibody is to target the antigen of interest. The primary antibody was developed

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Immunohistochemical Assessment of Phosphodiesterase 5A (PDE5A) Expression in Infant Right Ventricular Failure | Taylor Trapp â&#x20AC;&#x2122;19

in goats against human PDE5A. Although it was known to react with PDE5A from a number of different species, it was no known whether it would react with rabbit PDE5A. This antibody was prepared as a 1-20 dilution using 10 microliters of mouse anti-PDE5A in 190 microliters of 10% NDS. One hundred microliters of the diluted goat anti-PDE5A was placed on the tissues on each slide with the exception of one slide in which no primary antibody was used. This serves as a control to detect non-specific binding of the secondary antibody (see below). The tissues were then placed in a humidified chamber overnight at room temperature. Another primary antibody that was used to determine viability of the tissues was mouse anti-desmin. Desmin is a protein specific for muscle tissue. The desmin slides were treated same as the PDE5A tissues with a peroxidase block, treatment with NDS and a 1-100 dilution using 2 microliters of mouse antidesmin and 498 microliters of 10% NDS. One hundred microliters of the antibody dilution was placed on each tissue in a humidified chamber overnight. Also, there was one slide which did not receive the desmin primary antibody to act as a control for non-specific binding.

To begin day two of the staining process, the slides were first washed with PBS pH 7.4

three times for two minutes each. After the washing process, the secondary antibody was added onto the slides. The secondary antibody that was used for the PDE5A was a donkey anti-mouse antibody conjugated to horseradish peroxidase (HRP). For the desmin slides the secondary antibody was also an anti-mouse antibody conjugated to HRP. The slides were incubated for one hour in the humidified chamber before preparing the HRP substrate for color development. The substrate used was 3,3â&#x20AC;&#x2122;-diaminobenzidine (DAB). The secondary antibody binds to the primary antibody; the HRP conjugate allows this binding to be visible under the microscope . HIGHDEF DAB CHROMOGEN (Enzo Kit) was the substrate added to the desmin and PDE5A treated tissue for 15 minutes which gave them a brownish color which was formed by the DAB and hydrogen peroxide in an enzymatic reaction with peroxidase. This step stains the tissue allowing target markers (PDE5A and Desmin) to become visible under light microscopy . After incubating with the HIGHDEF DAB Chromogen for approximately 3 to 15 minutes, the slides were washed with PBS in a coplin jar. The slides were then counterstained with HIGHDEF Hematoxylin for two minutes with enough solution to over the slides to cover the tissue. Hematoxylin stains the DNA within the nucleus which aids in localizing cells under the microscope. The slides were then rinsed again with water in a glass coplin jar which removed the excess hematoxylin. The presence of DAB and hematoxylin staining was examined using light microscopy at magnifications ranging from 4 to 40X.

Immunohistochemical Assessment of Phosphodiesterase 5A (PDE5A) Expression in Infant Right Ventricular Failure | Taylor Trapp â&#x20AC;&#x2122;19

(Figure 7) Figure 7 shows the slides before they were taken to be assessed under the microscope.

Finally, it should be noted that we initially tried to use secondary antibodies that used

fluorescence but there was a lot of background fluorescence which made us switch to using the peroxidase-DAB enzyme-substrate reaction.

RESULTS: In the first experiment, as expected there was a significant amount of desmin immunostaining in cardiomyocytes (Figure 8). By way of comparison, Figure 9 shows a control slide for the immunostaining where only secondary antibody-HRP-DAB were applied.

(Figure 8) Figure 7 desmin stained with HRP-DAB. The dark brown color is the desmin while the blue reflects hematoxylin staining of DNA (nuclei). Magnification = 20X

These results confirmed my overall ability to perform immunostaining on these specimens. Using desmin as the target was beneficial since it is present in large amounts in cardiomyocytes.

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In addition, our laboratory has used this anti-desmin antibody in rabbits in the past. Thus, these results served as a useful positive control for my overall methods. To test our methods for PDE5A, we first used tissue sections from normal rabbit right ventricle. Here, PDE5A immunostaining showed a deep brown stain which is consistent with significant amounts of PDE5A. This staining was present across the entire right ventricle, indicating that most of the cardiomyocytes in normal rabbit RV contained PDE5A (Figure 10). More important, this result demonstrates that we can use this PDE5A antibody to assess PDE5A in rabbit heart, including those from our sham and RV failure hearts.

(Figure 9) Figure 9 displays the immunostaining control slide which is the slide that did not receive the primary antibody. No immunostaining is evident, indicating the absence of non-specific binding of the secondary antibody.

(Figure 10) The image on the top is a low power (4X) view of the rabbit right ventricle immunostained for PDE5A, on the bottom the same region at higher magnification (40X).

Before these experiments began, it was hypothesized that there would be increased expression of PDE5A in the cardiomyocytes of failing infant RV. That hypothesis remains to be L10

Immunohistochemical Assessment of Phosphodiesterase 5A (PDE5A) Expression in Infant Right Ventricular Failure | Taylor Trapp ’19

tested by examining tissue from sham and failing RV obtained in our model. Based on the results from this preliminary experiment, we believe that we will be able to use these reagents and techniques to test this hypothesis. Earlier results from our laboratory showed increased amounts of PDE5A, RNA and protein in tissue from failing infant RV compared to normal infant RV. Finding that the increased PDE5A was localized to cardiomyocytes would advance those results in an important way. To confirm this, we would need to 1) examine a larger number of samples from failing infant rabbit RV, and 2) compare those results to immunostaining in sham (control) RV, where we would expect to see much less PDE5A in the cardiomyocytes confirmed, these results could lead to testing the effects of drugs that inhibit PDE5A activity (e.g. tadalafil, also known as Cialis) in this model and eventually in humans with right heart failure .

REFERENCES:

Figure 1: http://www.sciencekids.co.nz/images/pictures/humanbody/heartdiagram.jpg “How the Heart Works.” Today I Found Out. N.p., 27 Nov. 2012. Web. 6th July 2017. Figure 2: https://en.wikipedia.org/wiki/Tetralogy_of_Fallot “Tetralogy of Fallot.” Wikipedia. Wikimedia Foundation, 2nd July 2017. Web. 11 July 2017. Figure 3: https://www.gstatic.com/healthricherkp/pdf/congenital_heart_disease.pdf “Congenital Heart Disease.” Center for Disease Control. N.p., n.d. Web. 6th July 2017. Figure 4: http://emedicine.medscape.com/article/898075-overview “Pulmonary Artery Sling.” Background, Pathophysiology, Epidemiology. N.p., 20 June 2017. Web. 13 July 2017. Figure 5: Courtesy of my lab Figure 6: Courtesy of my lab Figure 7: Courtesy of my lab Figure 8: Courtesy of my lab Figure 9: Courtesy of my lab Figure 10: Courtesy of my lab

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