HARVARD COLLEGE PRISE BLISS PRIMO SHARP SURGH ABSTRACT BOOK 2016 Harvard College PBPSS PRISE | BLISS | PRIMO | SHARP | SURGH

ABSTRACT BOOK 2016 PBPSS 2016 Abstract Book

Table of Contents

Director’s Letter ...... 4

Editors’ Letter ...... 5

Program Overview ...... 6

BLISS ...... 7

PRIMO ...... 17

SHARP ...... 29

SURGH ...... 38

PRISE ...... 47

Acknowledgements ...... 130

List of Fellows ...... 131 Director’s Letter

Dear reader,

On behalf of Rakesh Khurana, the Danoff Dean of Harvard College, and Jay Harris, the Dean of Under- graduate Education, I am pleased and privileged to once again introduce the research of the Harvard College Summer Undergraduate Research Village community, which comprises PRISE, BLISS, PRIMO, SHARP, and SURGH. These five programs together form a robust, active, and interdisciplinary residential community of scholars focused on research with Harvard faculty in formative and substantive projects over ten weeks in the summer.

2016 marks the eleventh year since the genesis of PRISE, and the Summer Undergraduate Research Village concept that continues to evolve and provide exceptional opportunities for our fellows to explore intellectual questions of merit (and personal interest!). These questions, to a high degree, inform their undergraduate and postgraduate scholarship trajectories. Beyond research, the engagement of the fellows is evident in the broad, creative (and fun!) range of activities they organize themselves at Leverett House that essentially define each of the program cohorts and our community more broadly.

The abstracts included herein speak for themselves: impressive in their considerable interdisciplinary range, the projects are a testimony to the fellows’ diligent effort and compelling sense of purpose. With gratitude to our faculty hosts, the abstracts further underscore the truly inspiring research happening across the Harvard universe.

I wish you—the fellows in PRISE, BLISS, PRIMO, SHARP, and SURGH—the very best of success in your academic endeavors going forward. I suspect the important relationships you have cultivated over the summer will continue long after the fact, and I am deeply gratified to have witnessed your enthusiasm, inclusivity, and heartfelt bonhomie.

Yours truly, Gregory A. Llacer

Director, Harvard College Office of Undergraduate Research and Fellowships (URAF) Director, Harvard College Program for Research in Science and Engineering (PRISE) Editors’ Letter

Dear PBPSS Community,

It is unbelievable how quickly the summer has passed by. In just ten short weeks, we have not only con- ducted amazing research, but also developed a close-knit community. We’ve heard from scholars at the forefront of their fields through the Distinguished Speaker Series and Faculty Chats, and witnessed the incredible talent present in our own community through performances at Coffeehouse. We’ve gotten motion sickness from both the waves of the Atlantic Ocean and the roller coasters at Six Flags. And throughout all of this, we’ve bonded with each other, whether over the complexities of research, the sweltering heat, or just life in general. These experiences are what makes the research village so unique.

But of course, a fundamental part of the research village is. . . research. This summer, our research spanned unique disciplines and showcased the diversity of academic interests in the village. From the darkest corners of distant galaxies, to the depths of the self; from communities across the globe, to those right here in Boston, our research has remarkable breadth, and it has been amazing to learn about each other’s research while putting together the Abstract Book. Serving on the Editorial Board has been an incredible honor.

It would not have been possible to compile the abstract book without the help of many people. We’d like to thank Felipe Flores for all the time and effort he put into compiling the book, and Gurbani Kaur for the encouraging words and emotional support; furthermore, we owe tremendous thanks to the peer editors, the proctors and PAs, the PBPSS staff, all the research advisors and mentors, and most of all, to the PBPSS fellows. We are thrilled to have an abstract from every single one of you published in this book.

We hope this summer was as wonderful for you as it was for us. Wherever we’ve gotten to know you, it’s been a pleasure, and we wish you the best in your future endeavors.

Sincerely, PBPSS 2016 Editorial Board

Editors-in-Chief: Audrey Effenberger, Leah Marsh, Leah Rosen, Soumyaa Mazumder Editorial Board: Audrey Effenberger, Elgin Korkmazhan Gulpinar, Ellen Zhang, Leah Marsh, Leah Rosen, Miruna Cristuş, Soumyaa Mazumder, Suproteem Sarkar, Yong (Daniel) Shen

Peer Editors: Design Ninja: Adam Frim, Alexander Jin, Andre Sanchez, Audrey Felipe Flores Effenberger, Carl Denton, Christopher Li, Daniel Um, Elgin Korkmazhan Gulpinar, Ellen Zhang, Felipe Design Team: Flores, Gerardo Castillo, Gui Zhen Chen, Holly Breuer, Audrey Effenberger, Soumyaa Mazumder, Jacob Scherba, Jason Li, Jessica Kim, Jiafeng Chen, Yong (Daniel) Shen Julianna Lee, Kristen Fang, Krystal Phu, Kushi Mallikarjun, Leah Marsh, Leah Rosen, Lingjun Xia, Cover Design: Madeleine Granovetter, Mara Coyan, Margaret Panetta, Audrey Effenberger Michael Giles, Michael Liu, Miruna Cristuş, Quinn Sluzenski, Rachel Oshiro, Sarah Rahman, Soumyaa Mazumder, Suproteem Sarkar, Theresa Clark, Thomas Culp, Yong (Daniel) Shen Program Overview

The Harvard Summer Undergraduate Research Village was initiated in response to a recommendation of the 2005 Harvard University Task Force on Women in Science and Engineering (WISE). The first program, initiated in the summer of 2006, was the Harvard College Program in Science and Engineering (PRISE). It was charged with developing a diverse, residential community of Harvard College undergraduate scientists conducting re- search with faculty over ten weeks during the summer.

Five years later, at the recommendation of the Dean of Social Science in the Faculty of Arts and Sciences and the Dean of Harvard College, PRISE was joined by the Behavioral Laboratory in the Social Sciences (BLISS), a joint project of Harvard College and the FAS Division of Social Sciences. The Program for Research in Markets and Organizations (PRIMO), a collaboration of the College and the Harvard Business School, was also founded in 2011.

2013 marked the first year of the Summer Humanities and Arts Undergraduate Research Program (SHARP), launched with the support of the Dean of Arts and Humanities in the Faculty of Arts and Sciences and the Dean of Harvard College. Most recently, in 2015, SURGH (Summer Undergraduate Research program in Global Health) joined the Research Village, filling out the complete interdisciplinary structure of the Summer Under- graduate Research Village.

Today, PRISE, BLISS, PRIMO, SHARP, and SURGH form a vibrant community of student researchers en- gaged in the pursuit of knowledge. We are proud to present their outstanding work in this abstract book.

PRISE SHARP The Program for Research in Science and Engineer- The Summer Humanities and Arts Research Pro- ing aims to build community and stimulate creativity gram aims to build community and stimulate creativ- among Harvard undergraduate researchers in the life, ity among a small cohort of Harvard undergraduate physical/natural, engineering and applied sciences. researchers in the humanities and arts, providing Fellows possess both a strong dedication to academic students with diverse research opportunities in an interest and excellence in scientific research and an exciting range of research settings. SHARP fellows enthusiasm for participating in a diverse residen- contribute to the rich, interdisciplinary intellectual, tial community of scholars. PRISE fellows indepen- social, and residential environment in activities that dently coordinate their research positions and work include roundtable lunch talks with distinguished on projects with Harvard-affiliated researchers. faculty speakers, pre-professional seminars, and op- portunities to explore Harvard and the New Eng- BLISS land region more broadly. SHARP fellows work on The Harvard College Behavioral Laboratory in the research projects with Harvard-affiliated faculty and Social Sciences aims to provide a formative and sub- researchers, and library and museum administrators. stantive research experience, to build community, and to stimulate creativity among a small cohort SURGH of Harvard undergraduate researchers in the social The Summer Undergraduate Research in Global sciences. BLISS fellows work on projects designed Health program aims to build community and stim- by Harvard-affiliated faculty and researchers. ulate creativity among a small cohort of Harvard un- dergraduate researchers in global health, and offers PRIMO Harvard undergraduates the opportunity to research The Program for Research in Markets and Orga- critical issues in global health under the direction of a nizations aims to build community and stimulate Harvard faculty or affiliate mentor. SURGH fellows creativity among Harvard undergraduate researchers work on research projects with Harvard-affiliated in business and related fields. PRIMO fellows are se- faculty and researchers. lected to work on one of the pre-designated PRIMO research projects which span diverse topics (e.g. fi- nance, organizational behavior, marketing), disci- plines (psychology, economics, sociology), and meth- ods (quantitative and qualitative). Fellows work on projects with faculty at Harvard Business School. Adapted from the URAF website. BLISS BLISS

Intervention to Turn Social Networks into Supports for Student Success I conducted in-person and phone interviews with students, parents, and supporters in both the control Many students have and treatment groups at the end of the 2015–2016 strong relationships with school year. Participants were asked questions about Anna Zannetos supportive adults, in- Sociology, 2018 their relationships and interactions with each other cluding mentors, fam- and the perceived impact of the program. These in- ily friends, and extended Harvard Kennedy terviews gave insight into the mechanisms driving the family members. Much School of intervention’s impact. Data from the interviews sup- Government research indicates that plemented quantitative data about student perfor- strong relationships with mance. Data were used to improve the pilot program Advisor: Todd non-parental adults are in preparation for implementation in approximately Rogers, PhD associated with greater four school districts in the 2016–2017 school year. success for students in Mentor: Kim school and other domains. Bohling, EdM However, the Family Ed- New Dynamics in Urban Politics ucational Rights and Pri- vacy Act (FERPA) pro- The skylines of our hibits schools from communicating with these adults country’s largest cities about students’ performance in school. The My Stu- Anthony Chen have grown and changed Statistics, 2018 dent’s Team pilot project is a randomized controlled tremendously. However, trial of a low-cost, scalable approach to inviting and the effects of an increas- Harvard University empowering non-parental adults in high school and ingly urbanized and con- middle school students’ lives to support their success Advisor: Ryan Enos, centrated population on in school. This project is grounded in the “nudge” PhD government and demo- approach, an emerging branch of psychology and cratic life are less obvious. economics that translates the science of human be- Mentor: Peter Professor Ryan Enos’s havior into simple strategies for motivating specific Bucchianeri team is studying how the targeted behaviors. cityscape is affecting pol- itics and policies, exam- One hundred ninety-one parents and guardians of ining a wide range of data, from commuter/traffic students attending school in mid-sized city out- statistics to mayoral speeches. My main role this side of Boston identified a non-parental adult who summer has been devising data collection strategies, cares about their child and authorized the district to as well as conducting secondary research. communicate directly with this named “supporter.” These supporters then received communications One of the largest projects I have undertaken so far about the student’s educational activities, includ- is parsing city council meeting minutes to pull out ing attendance and grades, during the 2015–2016 individual bills and their corresponding vote tallies. school year. Supporters also received actionable in- We are looking to use this data to create adjacency formation about how to support and engage with the matrices, which are essentially graphs that visually student and his or her school performance. Those in show the political relationships and “networks” of the randomized treatment group received more fre- city councilmembers, based on their voting history. quent, actionable, and personalized communications Similar studies, called network theory studies, have than those in the control group. been conducted on a national level with Congress and have been very revealing, showing increased party This intervention investigated whether communi- polarization over the past few decades. Network the- cating with non-parental adults improved students’ ory has yet to be applied to local government, which educational success. It was hypothesized that stu- is most likely due to the fact that city council voting dents in the treatment group will see greater im- records are not nearly as organized as Congressional provement in grades and attendance than students voting records. For the actual data parsing, I used in the control group. It also investigated whether the statistical programming language R, utilizing a and how communications from the school affected wide variety of new functions and packages includ- non-parental adults’ perceived responsibility, per- ing regular expressions to create “wildcard” search mission, and ability to provide educational support patterns. to the student. A second project looked into the State of the City BLISS 9 speeches of mayors around the country. State of the City speeches, similar to the State of the Union However, punishment is not the only option we have speech given by the President, serve as impor- when responding to bad behavior. An alternative tant updates to citizens about the legislative on- to punishing an uncooperative partner is deciding goings and plans of the government. I wrote an R not to interact with the offending partner again. In script and the Selenium module to crawl through the literature, this is called partner choice. Charac- Youtube and pull captions from the website to cre- terized by voluntary cooperative relationships with ate a transcript of the speeches. Selenium allowed alternative partners readily available, partner choice me to automate web browsing and programmati- differs from partner control in that one’s response cally click links, making the daunting task of down- to defection is motivated by the ability to leave the loading hundreds of speeches into a simple pro- current partner and choose new partners. As such, gram. We can use these transcripts of mayoral the goal of partner choice is not to change a partner’s speeches to create visual displays based on word behavior but rather to predict whether that partner frequency and analyze the vocabulary used. In will be cooperative in the future, and if not, to end addition to these two larger projects, I created a this relationship and start a new one. Thus, those variety of datasets on topics ranging from racial engaging in partner choice should be sensitive to dif- demographics of elected officials to inter-state mi- ferent signals compared to those engaging in partner gration tables. While many of these projects are control. While those who engage in partner control very much in their preliminary stages, the excit- should care about whether punishment leads to a ing work done here is sure to reveal new discover- change in their partner’s behavior, those engaging in ies about the impacts of urban life on government. partner choice should be indifferent to whether their previous partner’s behavior was changed as a result of ostracizing them, and if anything, may be less Partner Choice and the Pedagogical satisfied when ostracism causes someone to change Function of Punishment (because of missing the opportunity to interact with When a partner de- a now-cooperative partner). fects on us, whether in Chace Shaw business or personal life, In an online experiment, two participants will play Psychology, 2017 we can respond in a num- a game in which one decides how to divide a sum of ber of ways. One common money between the two, while the other chooses how Harvard University response is to engage in to respond. In the partner control condition, the Advisor: Fiery some form of punishment. player will respond by deducting money from the Cushman, PhD In research focusing on co- first player, while in the partner choice condition, operative behavior, pun- the player will respond by choosing whether to con- Mentor: Justin ishment falls under the tinue playing with the same player or to leave and Martin broader heading of part- play with a new one. The responder will then report ner control. In partner his or her satisfaction with the interaction after re- control, individuals are ceiving a message from the other player indicating motivated to change the behavior of their partner. either a change in behavior or no change in behavior. This is accomplished through punishing the offend- ing partner for transgressing. Evolution favors the In this study, we aim to further highlight the unique tendency to punish offenders, because this ensures pedagogical function of punishment by contrasting more beneficial relationships with others. In this punishment decisions with partner choice decisions. way, the threat of punishment serves as a deterrent Following the evolutionary functions of these be- of future uncooperative behavior. One prediction haviors, we focus on their differential sensitivity to of this hypothesis is that successful punishment will feedback from individuals who have either been pun- change the offender’s attitude and behavior and, ful- ished or ostracized. Specifically, we predict that filling its pedagogical function, will leave the victim post-ostracism feedback indicating a moral in be- satisfied with the interaction. Thus, after punishing havior and attitude will actually result in a reduced the offender, the victim will be sensitive to indica- level of victim satisfaction in the partner choice tions of the successfulness of the punishment, such condition, but that post-punishment feedback indi- as post-punishment feedback from the offender. Pre- cating a change will result in higher levels of vic- vious research has established that victims are most tim satisfaction in the partner control condition. satisfied after punishment when the offender gives feedback to the victim, indicating that he or she understood that the victim was intending to punish and expressing a moral change of attitude and future behavior. 10 BLISS

Suicide Risk Factors and the RDoC Matrix To create the database, a team of abstract coders This summer, I have select from a broad literature articles that seem to been working on a study handle RDoC elements and outcomes related to self- Charlotte Anrig that examines past re- injurious thoughts and behaviors (SITBs). After English & search on suicidality in being identified and classified into an RDoC cate- Psychology, 2018 the context of the Re- gory (genes, self-report, etc.), the articles are sent Harvard University search Domain Criteria. to the article coders. These coders—myself and The matrix, commonly two other interns, as well as several other coders at Advisor: Matthew K. known as RDoC, repre- other sites—read, analyze, and input the papers into Nock, PhD sents an attempt to re- Qualtrics, a survey platform that has been adapted form the present classi- to use a series of predefined questions to allow for Mentor: Heather S. fication system for men- consistent data entry for this project; as a team, Pixley, MBA tal illnesses: the Diagnos- we decide which findings are relevant and figure tic and Statistical Manual out how to translate them for the RDoC system. (DSM) for Mental Disor- So far, we have been able to successfully build the ders, published by the American Psychiatric Associ- beginnings of a database. Data entry will hope- ation. The DSM treats mental disorders like somatic fully be finished in six months, with some sections disorders, framing each as a distinct, consistent en- potentially completed by the end of the summer. tity that can be easily identified through observable signs and symptoms—and thus each patient can be The Effect of Emotions in Anti-Smoking treated the same way as everyone else who has ever Public Service Announcements exhibited the same behaviors. Anti-smoking public In reality, though, brains are complex systems, and service announcements psychological disorders tend to be incredibly hetero- Gloria Yu (PSAs) often use a variety History and Science, geneous. Two people demonstrating roughly similar of methods to discourage 2018 symptoms may actually have completely different tobacco use. Some out- neurological problems, and they might need entirely Harvard Kennedy line the benefits of quit- different treatments as a result. The RDoC attempts School of ting, others illustrate the to account for this complexity by rejecting the idea Government horrendous health rami- of discrete disorders altogether, opting instead to ar- fications of smoking, and range a matrix of mental functioning as a whole. One Advisor: Jennifer still others provide tear- axis represents different domains of human psycho- Lerner, PhD jerking narratives of the logical ability while the other axis identifies levels of impact of smoking on biological functioning from genes to self-report, each Mentor: Joowon loved ones. Many of point on the matrix representing a different, highly “Jo” Kim, MA them also rely heavily specific piece of mental functionality that could po- on emotion, both positive tentially break down. This structure allows for more and negative, to motivate specific diagnoses, and its focus on underlying bi- changes in smoking behavior. Recent research by ology rather than subjective observation creates a Dr. Lerner, however, indicates that the use of cer- pathway towards more targeted and empirically- tain emotions in PSAs could backfire. Based on the supported treatments in the future. Appraisal Tendency Framework (ATF), a multidi- mensional theoretical framework for linking specific The Nock lab aims to identify RDoC elements that emotions to specific judgment and decision-making act as risk factors for suicidal behavior. By ex- outcomes, certain emotions lead to impatience and amining, translating, and organizing virtually all a higher desire to attain products. Hence, evoking relevant research, we hope to produce a comprehen- certain emotions in PSAs could ironically lead to sive database and data visualization tool to examine increased smoking behavior. suicide risk within the RDoC framework. Thus, the research depends on two basic sets of questions: Dr. Lerner’s research combines theories of public firstly, how can existing research about suicide be health, psychology, behavioral economics, and psy- adapted for the RDoC? Secondly, what are the most choneuroendocrinology to examine the mechanisms robustly justified risk factors for suicide, and what behind emotionally-evocative PSAs and their effec- are the gaps in the existing research? The answers to tiveness. We are in the beginning stages of this these questions should have significant implications tentative five-year research project. This summer, for large conceptual questions about classification in we have been working on analyzing survey and writ- the field as well as important clinical utility. ten data from the pilot study on anti-smoking PSAs BLISS 11 and how effectively they elicit various emotions. Us- While versions of these algorithms see plenty of use ing its findings we can fine-tune target stimuli and matching organ donors and patients, college appli- test parameters for the second round of studies. Ulti- cants and colleges, and hospitals and medical school mately, this program of research will provide theoret- students, our research has focused on the aforemen- ical advances in understanding the effects of emotion, tioned condition of stability, that no coalition of and practical advances in designing effective PSAs. participants could create a matching that would be more preferable for at least one of the members. Maciej Kotowski and Ivan Balbuzanov proposed a Matching Market Algorithms stricter definition of stability, where no coalition could strongarm other participants into giving its As opposed to usual members more preferred “houses” by threatening markets where con- not to sell others their houses. Hansenard Piou sumers have no prefer- Applied ences over what prod- In order to find properties of matchings under Mathematics, 2018 ucts they consume—there this definition, I coded a modified TTC algorithm Harvard Kennedy is no egregious differ- which creates a larger set of matchings than the School of ence between two apples, original TTC and coded functions which check Government for example—matching whether a matching satisfies this strongarm con- markets contain individ- dition. While we are very much working in the Advisor: Maciej uals who have prefer- realm of theory, determining the properties of sta- Kotowski, PhD ences of what they con- bility from exclusion and algorithms which produce sume. Moreover, while such stable matching will give future market de- the former markets can signers the tools to create more robust markets. be cleared by just having all participants buy and sell products at the optimal price, matching markets Memory and Imagination: Using the can only be cleared if certain conditions are met, Episodic Specificity Induction namely if no participants can create a better match- ing for themselves than the one they were given; This study looks to e.g., a set of men each with preferences over a set of demonstrate that an women whom they want to marry and vice versa. Harrison Tanzola episodic specificity induc- tion can enhance imagi- Psychology, 2019 During the early stages of my research, I learned nation of future events. about and coded in Python the two archetypal al- Center for Brain Prior studies have shown gorithms to clear a matching market and result in Science, Harvard that episodic memory a stable matching: the deferred acceptance (DA) University plays an important role and top trading cycles (TTC) algorithms. The first in imagining the future. algorithm is for instances when two groups of par- Advisor: Daniel This act of imagining fu- ticipants have preferences on who they want to be Schacter, PhD ture events, also known matched with. For this algorithm, members of one as episodic simulation, is group “propose” to their top choice of those in the Mentors: Kevin theorized to be a result of other group. Each “acceptor” then rejects all except Madore; Preston a process by which past their top choice of those who proposed to them, al- Thakral, PhD memories are recombined though they have the option of rejecting all of them in unique ways to produce and remaining solo. All of the rejected proposers an entirely new event. then propose their next top choice including them- selves, repeating the process until all proposers have An episodic specificity induction is a task that has been matched or have decided to go solo. been demonstrated to enhance episodic memory by increasing internal details, details pertinent to an The TTC algorithm is used when participants want event that is being remembered. Even more strik- to trade unique items amongst themselves. Each ing is the fact that the induction does not increase “agent” points to their top “house” of choice and external details, merely semantic or metacognitive each “house” to its owner. At least one cycle will statements; rather, it singularly affects internal de- form where a “preference” chain will begin and end tails, the details coming from episodic memory. And with the same agent. We clear all cycles by giving because episodic memory is said to have a much each agent their top pick, and the remaining agents larger contribution to imagining the future than is point to their top remaining choice. We repeat this semantic memory, this study theorizes that having process and keep clearing cycles until there are no subjects perform an episodic specificity induction more agents in the market. as opposed to a control induction will result in en- 12 BLISS hanced details in imagined events. accidentally causes harm and judge them nearly iden- tically to a person who caused no harm. However, For this study we used a within-subject control, when our cognitive capacities are occupied, we have so each subject received both an episodic specificity difficulty incorporating information about a harm- induction and a control induction, followed by math doer’s mental state into moral judgment. We fail to problems to serve as a control, and then a set of five inculpate agents with bad intent and fail to excul- cues for each. The subjects were asked to base a pate agents with good intent, judging them only on simulated future event on these cues. Both the in- their causal connection to harm. ductions and the cues were counterbalanced between subjects. Furthermore, all responses to cues were These results have obvious implications for Cush- self-timed and included a standardized set of general man’s two-process model: they suggest that there is probing to get as much detail as possible from the some step beyond the mere recognition of a harm- subject, without having the experimenter implant doer’s causal role and intent, and that this additional details that were not there. step requires cognitive effort; what Cushman’s model lacks is information on the point at which this addi- After all subjects have been run, and their inter- tional step occurs in our cognitive processes. views transcribed, transcripts of each subject will be scored using a standard autobiographical interview There are two ways we can interpret this. The first (AI) scoring protocol. If the hypothesis is correct, is that under cognitive load—when our mental re- the AI scores will reveal a statistically significant sources are taxed—people are sensitive to intentions increase in the average number of internal details but cannot seem to incorporate them into judgments in cue response following the episodic specificity in- of preference and wrongness. That is, participants duction, as opposed to those following the control are still aware that the accidental agent did not in- induction. Also, there should be little to no change tend harm and that the intentional agent did, but in the number of external details between inductions. somehow, because their cognitive capacities are oc- cupied, they fail to include this information into the final judgments made. The second interpretation is Moral Judgments and the Effortful Nature of that outcomes do have an influence on judgments of Forgiving Accidental Harm wrongness, and normally, we are capable of inhibit- In cases of moral judg- ing this influence. But under cognitive load, we are ment, people largely care unable to inhibit this influence and we judge perpe- Irene Limb about two factors: who trators of negative outcomes to be morally wrong. Psychology, 2018 caused the harm and The reason why previous results had shown an in- whether the harm was in- fluence of cognitive load on intent-based judgment Harvard University tentional. However, we is not because one’s ability to incorporate intentions Advisor: Fiery care about these factors was impaired, but because one’s ability to inhibit Cushman, PhD to different extents de- the influence of outcomes was impaired. pending on the type of Mentor: Justin judgment we make. Deci- To test this, we are in the process of running ex- Martin sions to punish the harm- periments aimed to distinguish people’s judgments doer depend upon both of punishment from their judgments of wrongness. the outcome and the in- Participants in our study will watch several video tent of the harm-doer, whereas judgments of wrong- clips showing a moral agent either accidentally or ness only depend on the intent. intentionally harming a second agent, either under cognitive load through a verbal shadowing task, or While Cushman (2008) articulated this idea at a not under load. We then ask participants such ques- coarse level by creating a two-process model that tions as, “Whose behavior was wrong?” and “Should draws connections between the inputs—causation he be punished?” in order to explicitly compare judg- and intention—and the judgments they influence, re- ments of wrongness and punishment. The pilot data cent studies have hinted at further delineations that has supported the second interpretation, that wrong- may be involved in our moral judgments. Specif- ness judgments occur because people have difficulty ically, it seems that people can seamlessly incor- inhibiting them when they observe a harmful action. porate causation into their moral judgments, but We predict that we will continue to find a significant taking intentions into consideration seems to require impact of cognitive load on judgments of wrongness, additional cognitive effort (Buon et al., 2013). That such that participants will be unable to inhibit the is, under normal circumstances, we judge someone influence of a negative outcome on their judgments with bad intent much more harshly than someone of how wrongly an accidental agent behaved. with neutral intent, and we forgive the person who BLISS 13

The Use of Historical Analogues in Foreign Third Party Intervention in Children Policy Decisions Past research has This question is part demonstrated that unin- Liana Yamin volved third parties who of the Applied History Human Evolutionary Krysianna project, led by Graham witness an unfair act are Papadakis Biology, 2017 Allison, Douglas Dillon willing to intervene to re- Social Studies, 2017 Professor of Government Harvard Lab for store justice, even when Developmental doing so is personally Harvard Kennedy at Kennedy School of Government. Diplomats Studies costly. Restoration of jus- School of tice can generally occur in Government and public officials fre- quently make use of his- Advisor: Felix two ways: punishing the Warneken, PhD perpetrator or compen- Advisor: Graham torical precedents in or- Allison, PhD der to evaluate possible sating the victim. It has courses of action, espe- Mentor: Young-eun been shown that adults Lee Mentors: Adam cially in cases where there reward third-party com- Siegel, MA; John is little information about pensators more favorably Masco a current problem. How- than third-party punish- ever, the use of historical ers in unfair distribution scenarios, but it is unknown analogies can be danger- whether young children share this preference for com- ous: it is often motivated by emotional responses pensators. to past experiences and tends to make people blind to the facts that distinguish present cases of con- This summer, I assisted with a study that evalu- flict from those in the past. Therefore, the Applied ated children’s perceptions of third party compensa- History project is partly aimed at encouraging a sys- tion versus third party punishment. We presented tematic approach to the use of historical analogues children ages five to nine with a story and visual in present decision-making. My role in this project is aids describing a group of children at a summer mainly to provide a broad literature review of schol- camp. One child, the “decider,” does not share arship on the uses of historical examples in (primar- candy with a peer. Two “watchers” witness this, ily) 20th century policy in international diplomacy. and one decides to take away candy from the de- cider while the other chooses to give candy to the This project began with reading the biographies of peer who received none. We asked participants ques- major statesmen like John F. Kennedy, whose biog- tions about each bystander to determine preference raphy was written by Kennedy’s “resident historian” and overall perception. Then, we provided them at the White House, Arthur M. Schlesinger. Histo- with an opportunity to punish, compensate, or do rians like Schlesinger are frequently present in the nothing in response to the same unfair division of rooms where foreign policy decisions are made, such candy that they saw in the story. Across all ages at the planning of the Bay of Pigs invasion and the tested, children preferred compensators over pun- nuclear escalations of the Cold War. Many of these ishers. When given a chance to intervene, a sig- historians then write retrospective analyses about nificant majority chose to compensate at a cost to how historical analogies influenced the decisions in themselves, rather than punish. These findings il- these cases. My role was to find and summarize luminate that a preference for promoting justice the work of such historians on how history has been through compensation emerges during childhood. used in the past and how it can or should be used in the future. My specific focus within this literature review is how the concept of “historical inevitabil- ity” is used, exploring how it often conceals implicit biases about what historical events are considered “good” or not, building off the work of E.H. Carr in his response to Sir Isaiah Berlin.

Finally, I have been assisting in the review of the appendix of Professor Allison’s forthcoming book, Destined for War: America, China, and Thucy- dides’s Trap. Specifically, I fact-checked the sixteen cases of stand-offs between rising and ruling powers in history, called the “Thucydides Trap Case File.” 14 BLISS

Political Implications of Class-in-Race Emotions and Morals Inequality There is a trolley Recent years have hurtling down the tracks, brought increasing class Quinn Sluzenski brakes broken and unable Sociology, 2018 Mara Roth disparities within racial to stop, about to smash Social Studies, 2019 groups. These disparities into five workmen who Harvard Kennedy are particularly interest- are stuck on the rails. Center for School of Government and ing to study, as from them Government You are standing next International Studies arises the conflict between to the track and see a maintaining group soli- Advisor: Jennifer switch—if you hit it, the Advisor: Jennifer darity and pursuing indi- Lerner, PhD trolley will divert to the Hochschild, PhD vidual interests. Because side track, saving the five a person’s socio-economic Mentor: Charles workmen. . . and killing status tends to determine Dorison the one workman on the where they live, the quality of their life, and their other track. What do you political beliefs and preferences, this increasing class- do? in-race inequality has the potential to create signif- icant political divisions within racial groups. So, Most people choose to pull the switch in this classic when does an individual choose his or her own in- philosophical question. However, when you change terests over standing in solidarity with their racial the scenario to you standing on a footbridge above group, and why? the track, when the only way to save the five work- men is to push a man over the side of the bridge to This research project attempts to resolve these ques- collide with the trolley, most people say that they tions by investigating policy disputes in four different wouldn’t push him. U.S. metro-regions. The examined policies and cities include pension reform in Chicago, educational pol- What would you do? Would you choose to be icy in Los Angeles, re-gentrification in Atlanta, and utilitarian—calculating the greatest net value and policing in New York City. Because all of these policy choosing five lives over one—or deontological— disputes have both class and race dimensions—some valuing the inherent ethical obligation to not cause clearer than others—they provide the perfect window direct harm? It may be that your emotional state through which to examine class-in-race dynamics. will impact your answer. Psychologists have argued that emotion stops you from saving five lives in the The research methods for this large scale project face of one, whereas deliberative reasoning leads you include reading news reports, analyzing legal pro- to save the five and act in a utilitarian manner. ceedings and census data, contacting subject experts, and traveling to the cities to conduct event/site vis- That research has been extended to show that in- its and interviews with union members, political ducing positive emotions makes people more likely figures, academic experts, and community organiza- to be utilitarian, while inducing negative emotions tions. The project is ongoing and this information makes people more likely to be deontological. How- is being used to analyze and explain patterns in the ever, as Jennifer Lerner’s lab has shown again and behaviors of African American, Hispanic, White, and again, different emotions have differing psychologi- Asian American people belonging to different socio- cal effects, even if both are “negative” or “positive.” economic classes. This summer, we studied how sadness and anger can affect our moral decision-making in scenarios like the While the current evidence seems to point to the idea trolley dilemma, in which we must choose whether that the well-off are more likely to abandon racial to cause harm or let greater harm occur. In order solidarity in favor of pursuing self-interest, more re- to study this question, we are surveying a wide vari- search remains to be conducted to further expand ety of people, inducing sadness or anger, and asking upon the complexities of class-in-race dynamics. The them to judge the morality of a variety of these kind results of this investigation and further inquiry into of dilemmas. We analyze their responses and their class tensions within racial groups are important for thought processes to build an idea of how people understanding inclusivity in America, and the future respond to these kind of moral situations. of economically disadvantaged Americans of all races. Ultimately, we hope to determine what effect that sadness and anger have on these decisions, poten- tially presenting evidence that sadness and anger have divergent effects. This exploration of morality BLISS 15 can help us understand how and why we make deci- book What We Owe Iraq: War and the Ethics of Na- sions, and could someday even have implications for tion Building in conversation with other academic public policy—as self-driving cars take to the streets, texts of a similar subject matter. the trolley dilemma transforms from a thought ex- periment to a real programming question—should we Subsequent to my work on this research paper, program utilitarian cars, or cars that protect their I am producing five multimedia blog posts on drivers? What is the appropriate trade-off? This a variety of topics on which the center has fo- study hopes to further the field of emotions in moral cused throughout the years: equality, govern- decision-making. When we better understand how mental corruption, ethics and the medical pro- we make decisions, we can make better decisions. fession, critiques of capitalism, and diversity.

Political Legitimacy and Nation Building Representations of Personal Identity in the Political Space The Edmond J. Safra Center, currently led by The Edmond J. Safra Sebastian Reyes Professor Danielle Allen Center focuses on teach- Social Studies, 2019 of the Government De- Susan Wang ing and researching eth- Social Studies, 2017 partment, is one of the ical issues as they re- Edmond J. Safra world’s preeminent cen- late to a variety of dis- Center for Ethics Edmond J. Safra ters for research on ethics Center for Ethics ciplines ranging from Advisor: Danielle and its practical applica- bioethics to the the- Allen, PhD tions. The center brings Advisor: Danielle ory of ethics. As together undergraduates, Allen, PhD research fellows with Mentors: Jess Miner, graduates, postgraduates, the Center, our job PhD; Tomer Perry and others in academia Mentors: Jess Miner, has two components— in a unique collaborative PhD; Tomer Perry archival work and inde- setting. pendently pursued re- search projects. At the time of its founding in 1986, then known as the Program in Ethics and the Professions, aca- The Center is approaching its 30th anniversary, demic inquiry into practical ethics was relatively with this year’s focus being “Diversity, Justice, and rare. Since that time, the center has become world- Democracy.” As part of the archival portion of our renowned, and those associated with it are among research, our research team is looking at the past the top scholars in a variety of disciplines, ranging thirty years of recorded events hosted by the Cen- from political theory to bioethics. ter to record highlights from past lectures that can be used for anniversary celebrations, and to create As the 30th anniversary of the Center nears, part a usable research database for future work. This of my work this summer involved going through the involves listening to and sorting through all past lec- center’s extensive archives, composed of hundreds of tures, conferences, and symposiums to pick out par- hours of public lectures, symposia, seminars, confer- ticularly interesting excerpts. These recordings are ences, and similar events recorded on cassette tapes, then marked with tags by topic, speaker, and date, CDs, VHS, DVD, and online. My first task was and entered into a catalogue so future researchers to develop a cataloguing system for all these ma- can access the Center’s materials. Highlights include terials. Subsequently, I delved into the lectures by talks from Amartya Sen on identity, Richard Rorty taking notes, writing down key quotes, and identify- on justice, Henry Louis Gates, Jr. on ethnicity, and ing “gems,” lectures that stood apart as exceptional Frances Kamm on high and low theory, to name a in some regard. few.

After this initial phase was completed, I began work We are also working on independent research projects on a research paper based on some of the material based in the Center’s archival materials. I will be I discovered in the archive. In particular, I am fo- compiling a data visualization of the Center’s work cusing on a lecture given in 2005 by Professor Noah over its history, looking at metrics such as demo- Feldman of Harvard Law School entitled “Iraq and graphics of speakers, topics of focus, and change in the Ethics of Nation Building.” I am exploring the language use, among other things, and compiling a concept of political legitimacy, both how Profes- comprehensive quantitative look at the Center’s past sor Feldman understands it and how it functions in work. The aim of this project is to produce an easily nation-building more generally. In addition, I will be digestible quantitative overview of the nature of the putting Professor Feldman’s lecture and his related Center’s work and focus, and how that has changed 16 BLISS over time. many who relied on support from the upper class to stimulate the local economy may find their prospects I am also working on a separate, longer research pa- adversely affected as funding is diverted overseas. per that examines representations of identity within the political sphere, and questions of how notions This project has attempted to provide empirical of personal identity translate into political stances evidence to support this claim. One aspect of the in developed, well-functioning, liberal democracies. project I have worked on looked at the public ser- Using Amartya Sen’s lecture on identity as a starting vice work of American Rhodes Scholars, who have point, I examine the transition of personal identity long been held as symbols of the elite of the United into political identity. In particular, how do aspects States’ education system. Using LinkedIn, a prior of our personal identity—such as ethnic background, registry of Rhodes Scholars, and other sources, two religion, gender—translate into coherent political distinct decades’ worth of scholars, 320 each from the positions? When those identity-based political po- 1950s and the 1980s, were examined to compare how sitions oppose each other, how can we successfully many years of their careers were devoted to public resolve conflict and reach compromise? The aim of service, non-profit and charitable organizations, resi- this project is to propose a way to respect plurality dence abroad, and a variety of other categorizations. in politics while still being able to reach effective Another of my studies examined the philanthropic consensus. tendencies of the largest American non-profit foun- dations to observe how charitable giving’s focus has Thus, our summer research aims at helping the Cen- evolved domestically and internationally over time. ter effectively catalogue its variety of resources for The fifteen largest organizations by asset size in 1969 future research, as well as developing specific projects and 2014 had their grants from those years broken that fit within the Center’s 30th anniversary theme. down by international or domestic focus, as well as through their specific fields of impact. Given the longstanding tradition of wealthy Americans giving Domestic and International Affinities of back to the community through private foundations American Elites embodied by men such as Andrew Carnegie and The United States has John Rockefeller, it will be interesting to see if this long given rise to some tendency has continued or whether global initiatives Will Strang form of elite upper class, spearheaded by organizations such as the Bill and History, 2019 whether this group be de- Melinda Gates Foundation have drowned them out. fined by their wealth, ed- More analysis in other topics, potentially including Harvard Kennedy School of ucation, or occupational study abroad, political opinions, and business prac- Government prestige. Many of these tices, should help to continue to clarify the trend individuals devote much towards cosmopolitanism amongst American elites if Advisor: John of their energy to improv- a trend even exists at all. Donahue, PhD ing conditions for others who are less fortunate through public service, philanthropy, and other means. With the growth of globalization and the increasing interdependence of the United States with other nations, many Amer- ican elites, especially the younger cohort, seem to have eschewed nationalist principles for a more cos- mopolitan ideal. Even as these Americans seem to remain roughly similar in terms of their desire to improve the lives of others, they now seem to be broadening their ambitions internationally. Anec- dotal evidence seems to bear out this hypothesis, as it seems that more students at top institutions such as Harvard are going into occupational fields with a more global focus. If this phenomenon can be observed on a broad scale, it will have far-reaching implications for the future development of American society. Shifts in opinion amongst political decision- makers may result in a more expansive and inclusive role for the United States in international issues at the expense of the American middle class. Similarly, PRIMO PRIMO

An Exploratory Study of Retirement The Resurgence of Independent Bookstores According to the Pew The independent Research Center, ap- bookstore has met with Alexander Rohe proximately 10,000 baby Angela Yang many threats to its sur- Psychology, 2017 Economics, 2018 boomers will turn 65 ev- vival in recent years. ery day until the year The rise of major chain Harvard Business Harvard Business 2030. In other words, School School bookstores like Barnes large numbers of people and Noble and Borders, Advisor: Teresa M. are reaching the age at Advisor: Ryan the founding and rapid Amabile, PhD which people tradition- Raffaelli, PhD growth of Amazon, and ally retire in the United the advent of the e-book Mentors: Jeff States. However, the ex- Mentor: Kathy Qu, have all led to many ana- Steiner; Andrew isting literature regarding EdM lysts’ prediction that the Brodsky people’s retirement ex- independent bookstore periences is surprisingly would soon become ob- sparse; researchers have solete. However, in recent years, the number of not yet deeply examined the ways in which various independent bookstores has increased substantially, factors affect people’s retirement decisions and re- despite all reasons to expect the contrary. This un- tirement transitions. In order to address this gap expected recovery gives rise to the questions of how in the literature and to gain new insights into the independent bookstores can manage to survive, or nature of retirement, Professor Teresa Amabile and even thrive, in the midst of these unfavorable con- her colleagues in the Retirement Research Group ditions, and what factors contribute to their resur- (RRG) are conducting an exploratory study in which gence. they are collecting both survey and interview data from participants at several companies. Participant This summer, I have been participating in mixed groups include millennials, employees approaching methods research with Harvard Business School pro- retirement, those going through the transition, and fessor Ryan Raffaelli to examine the resurgence of in- people who retired from the company in the past five dependent bookstores and, more broadly, to explore years. how organizations and industries redefine themselves in response to technological disruptions. The quali- This summer, I assisted Professor Amabile by an- tative component of the research includes literature alyzing qualitative data and by working with Re- review and rigorous analysis of articles, interviews search Associate Jeff Steiner to develop preliminary with bookstore owners, notes, and other sources. research presentations and reports to participating The quantitative component involves data collection companies and individuals. I also assisted doctoral and analysis of macroeconomic trends in the past candidate Andrew Brodsky, who is collaborating few decades, such as the performance of competi- with Professor Amabile, with his projects regard- tors like Barnes and Noble and Amazon. Similar ing online communications and idle time at work. phenomena have been observed in such industries as the watch industry, where Swiss watchmakers were forced to adapt to a technology shock in the form of the quartz watch. More recently, similar trends may be observed in the increasing popularity of street- cars, fountain pens, and vinyl records.

The case of the independent bookstore has impli- cations for many other industries, especially with the rapid advancement of modern technology. The ability of an industry to change its identity to adapt to changing economic conditions or of social movements like localism to drive significant mar- ket change may be deeply relevant wherever an in- dustry may be confronted with the need to adapt. PRIMO 19

Improving the Value of Health Care Delivery Employees and Startup Success

As the payment As startups grow and structure of the Amer- prosper, or wither and Antonia Chan Benjamin Cohen die, how do the roles ican health care sys- Statistics & Economics, 2018 and responsibilities of em- tem changes from fee- Computer Science, for-service to value-based 2019 ployees change? How Harvard Business do employees affect firm School payment plans, such as shared-savings capitation Harvard Business performance? In tradi- and bundled payments, School tional startup folklore, Advisor: Robert S. employees—especially Kaplan, PhD providers increasingly need to understand and Advisor: Rory M. early employees—and McDonald, MBA, founders are considered Mentors: Derek control their costs. Cur- PhD Haas, MBA; Mahek rent methodologies, such to be vital to startup suc- Shah, MD as using cost-to-charge cess. Traditionally these Mentor: Alicia ratios and relative value roles have been evaluated DeSantola units, produce cost esti- using qualitative meth- mates that have little relationship to the cost of the ods, making patterns that resources actually used to deliver care to patients. only exist in the aggregate Time-driven activity-based costing (TDABC), based hard or impossible to identify. on bottoms-up measurement of actual resource con- sumption over a patient’s cycle of care, offers a more Our research seeks to remedy this problem by ap- accurate form of cost measurement. plying quantitative methods to a novel data set from a widely used career networking site. The dataset TDABC requires that providers know how long includes an entry for employees of recently founded and how much of each resource—whether person- American startups and their experiences before and nel, equipment, space, supplies, or technology—is after working at a startup. After using natural lan- required during a cycle of care for a variety of med- guage processing techniques and other traditional ical conditions. Our research centers on the use of cleaning methods that translate employees’ input TDABC to help healthcare providers better measure into standardized forms, quantitative methods will and improve the value of care they deliver (out- be used to systematically identify patterns to un- comes relative to cost). A key component of this derstand how employee selection relates to startup research is working with clinicians to understand success. By aggregating the data on the startup and then redesign care delivery processes to improve level, a timeline of the firm’s employees can be cre- their efficiency and effectiveness. In a project with ated, helping answer questions about firm evolution Massachusetts Eye and Ear Infirmary, I observed a and venture capital pressures. By analyzing these same-day optometry clinic’s operations and designed startup timelines, we can understand how different a process map to better understand the breakdown roles change over time within a startup. Finally, of work, resource utilization, and time. These ob- when this timeline is combined with data on venture servations will facilitate standardized data collection capital funding from crunchbase, we can determine for further TDABC research in same-day optometry. how startups react and adjust to the venture cap- Our ultimate goal is to understand how to opti- ital process. Understanding the answer to these mize cost and quality when delivering health care, questions will contribute to a growing discussion translating our research into better outcomes for pa- about why some startups succeed while others fail. tients and providers, and reducing the cost to payers. 20 PRIMO

Private Equity Today examining official governance codes, analyst reports, news articles, and academic papers to address the fol- The private equity in- lowing questions: What are the issues in corporate dustry is one of the rela- governance in Japan that have motivated reform? Brian Cornyn tively newer aspects of the Economics, 2018 How are the reforms being enacted? How have re- financial world. However, forms changed corporate behavior and practices? much has changed in re- Harvard Business School cent years around many This background information is essential to bet- of the largest private eq- ter understand individual cases of reform and ac- Advisor: Paul uity firms. Returns have tivism in Japanese companies. I have also been Gompers, PhD fallen dramatically from researching and helping write a case study on the ac- fabled levels, with no rise tivist American hedge fund, Third Point LLC, and Mentor: Lauren seemingly in sight. Ob- its involvement with the Japanese retail company Cohen, MBA, PhD viously, a myriad of stim- Seven & i Holdings Inc. I have collected informa- uli and factors are influen- tion including news articles, analyst reports, inter- tial in this outcome; how- view transcripts, and official company documents, ever, the reduction of information asymmetry in the to examine the effect of the hedge fund’s involve- industry seems a strong driving force. Regardless ment on Seven & i’s company practices. Seven of these recent falling returns, private equity firms & i manages the Seven-Eleven convenience stores, still offer a higher return than intelligent investors Denny’s Japan restaurant chain, and the Ito-Yokado can find elsewhere. For this reason, many impor- supermarkets, among many other subsidiaries. De- tant funds, most notably pensions, are heavily in- spite it being the most prominent retail company vested in private equity firms. This phenomenon in Japan, the company, according to Third Point, has led to a large increase in cash on hand for pri- suffers from inefficient capital structure and offers vate equity firms as they struggle to find places a low dividend for shareholders. My research ex- to invest it. I am going to attempt to collect amines the internal management struggle of Seven some data to shed more light on how private eq- & i and the influence Third Point has had on uity firms have ended up in this situation and what improving profitability and company management options they are exploring to expand their targets. practices. The case study should be used to ad- dress larger questions on corporate governance and Activist Investing shareholder activism: How does an activist investor deal with company management and vice versa? My research with Pro- What practices in corporate governance are effective? fessor Suraj Srinivasan fo- Carolina Brettler cuses on activist investing Economics, 2018 Digital Disruption and its role in corporate governance in Japan. Ac- In its simplest form, Harvard Business School tivist investing refers to marketing is the process situations when an in- Christina Chiu by which a company cre- Economics, 2018 dividual or a group of ates and keeps a cus- Advisor: Suraj Wellesley College Srinivasan, MBA, shareholders of a com- tomer. Classic market- PhD pany exercise their rights Harvard Business ing formulas dictate that as owners and attempt to School as long as a brand has actively influence a cor- the right product, place- poration’s behavior or management. The goal of Advisor: Sunil ment, price, and promo- shareholder activism is to reform a company’s pol- Gupta, PhD tion, then any good prod- icy or management in order to increase shareholder uct will sell itself. How- value. Hedge fund-driven shareholder activism is an ever, the rapid growth of important and controversial development in the U.S. technology has transformed how people live—and business world, but is much less common abroad. by extension—shop. Social media platforms have changed the way that companies communicate with Despite Japan being the third largest economy in their customers. Native advertising engines, such the world, it has been experiencing stagnation and as BuzzFeed and YouTube, allow firms to produce declining corporate productivity for the past twenty valuable content for their target audiences. The years. I am researching and writing a background wealth of consumer data available with the use of note on corporate governance reforms in Japan, fo- search engines allow firms to customize their adver- cusing on recent changes in corporate governance tising to target their desired audience at precisely and their effects on corporate practice. I have been the right moment. However, though firms are get- PRIMO 21 ting “smarter” with the amount of consumer data this yearly fee. These facilities currently, in addition available, consumer trends show that people are too. to emergency childcare, include nighttime and daily Consumers have become more keen on brand au- childcare, elder care, and extra meals. In a business thenticity and corporate social responsibility, and world where work-life conflicts cost Canadian com- generally have shorter attention spans regarding ad- panies an estimated $4.5 to 5 billion a year and in a vertisements in an on-demand world. childcare market plagued with long waiting lists and inflexible options, Kids & Company, which ensures With Professor Sunil Gupta, I am studying the adequate space for children, high-quality care, and changing relationship between firms and consumers no waiting list, is an attractive option for both em- in the digital age. Using both quantitative and ployers and working parents. qualitative aspects of marketing, we aim to deter- mine how marketing strategies differ between star- In addition to fulfilling working families’ needs con- tups and incumbent firms, across different indus- cerning childcare, Kids & Company has become a tries, and between different countries. We aim to remarkably profitable and fast-growing company understand how companies are evolving their cus- through aggressive growth strategies, which Sopik tomer management and marketing strategies as they describes as a “greenfield build.” The effect of this move to digital. Our methodology involves review- strategy was larger visibility within working fam- ing industry examples, case studies, and academic ily groups in Canada, which in turn attracted more literature to form an analysis of current and fu- corporate clients. This strategy of expanding where ture marketing trends. In addition to creating a the company sensed a need for childcare, however, great, usable product, filling a void in consumer also resulted in expansion into less strategic areas or demand, and setting a fair price, the shift to- overestimation of centers’ capabilities. Regardless, wards digital technology ensures that firms must aggressive expansion allowed Kids & Company to embrace an online presence that provides an addi- receive much recognition as a fast-growing business, tional valuable service to its target customer base. winning titles such as Best New Business from Win- nipeg Biz in 2013 and the prestigious Ernst & Young Entrepreneur of the Year Award in 2012. In 2014, Case Study on Kids & Company’s Expansion Sopik announced the company had “basically fin- from Canada to United States Childcare ished building [its] footprint in Canada” and opened Markets its first non-Canadian center in Chicago. This case study aims to examine the expan- Expansion into the United States childcare mar- Christina Qiu sion of Canadian child- ket may provide challenges for the company, as the Applied Mathematics care company Kids & United States industry may differ from Canada’s & Economics, 2019 Company into the United in terms of quality regulations, parental prefer- Harvard Business States childcare market. ences and needs, alternative childcare available, School Founded in 2002 by CEO and government subsidies. This case study aims Victoria Sopik and CFO to identify these challenges with regard to Kids Advisor: Boris Jennifer Nashmi, Kids & & Company’s expansion and assess the com- Groysberg, DBA Company conceptualized pany’s decisions in addressing these challenges. itself as corporate child- Mentor: Kate care, functioning as an Multi-Sided Platform Strategies Connally introduction of Ameri- can backup or emergency Platform businesses childcare models into the are rapidly transforming Canadian market. While emergency and backup Daniel the world economy in a Nightingale childcare models specifically target the needs of variety of business sec- Economics, 2018 working parents who accommodate unexpected in- tors by bringing together terruptions in childcare plans by taking sick days Harvard Business different market partici- or leaving their jobs to care for their children when School pants, enabling easy in- space in other facilities is unavailable, the Kids & teractions and organized Company business model remains “corporate” for Advisor: David innovation in the pro- other reasons as well. The company’s main clients Yoffie, PhD cess. Examples include are large Canadian employers or multinationals, who large tech giants, such as for $5,000 a year can purchase a company mem- Facebook, Google, Ama- bership that allows its employees to access Kids & zon, and Apple; unicorn startups, such as Uber, Company facilities across Canada. Parents can only Airbnb, and Snapchat; credit card companies; dating access these facilities if their employers have paid websites; OS developers; and online marketplaces, 22 PRIMO among many others. Uber, for example, works as Managing Patient Experience, Medical a platform by matching drivers with riders. While Outcomes, and Cost in Healthcare these companies have many attractive qualities, such Operations as the potential for rapid growth through the power of network effects, they also face many challenges. Patient experience Platforms often struggle with clearing hurdles to ob- plays a fundamentally im- taining critical mass, developing pricing strategies Grant Zhao portant role in health- Chemical and that promote growth, and effectively governing all care. A patient expects Physical Biology, platform participants. 2018 not only to receive proper treatment and positive This summer, I am working with David Yoffie, the Harvard Business medical outcomes, but Max and Doris Starr Professor of International Busi- School also to be treated with ness Administration at Harvard Business School, as respect, to have their con- well as with Michael Cusumano, the Sloan Man- Advisor: Ananth cerns listened to with agement Review Distinguished Professor of Manage- Raman, MBA, PhD meaningful communica- ment at the MIT Sloan School of Management, and tion, and to have their Annabelle Gawer, the Professor of Digital Economy wait times, room clean- at Surrey Business School. Yoffie, Cusumano, and liness, and noise levels be managed appropriately. Gawer are currently writing a book on platforms in However, despite this importance, hospitals have order to bring some balance and nuance into the historically only emphasized medical outcomes as discussion of platform companies. With the hype a priority, often at the expense of positive patient for platforms in recent years, they are attempting to experience. Furthermore, previous fee-for-service dispel some of the current misconceptions related to payment systems incentivized higher costs and vol- platforms, stress the challenges of creating a success- umes of patients over improving quality of care and ful platform, and illustrate the significant overlap patient experience. between the operation of platforms and traditional businesses. They also hope to classify platforms In recent years, hospitals have faced increasing pres- into categories of similar business models, as current sure to improve their patient experience. Most no- discussions of platforms combine companies that tably, the Center for Medicare and Medicaid Services function quite differently. (CMS) has adopted a pay-for-performance or value- based purchasing (VBP) payment system, basing I began my research this summer by focusing on hospital reimbursements partly on patient experi- the financials of platform companies. We are try- ence scores. By 2017, hospitals with low patient ing to answer basic questions about the profitabil- experience scores may have up to 2% of their annual ity, value, and growth patterns of platform com- reimbursements deducted, a critical policy in a time panies and the different factors that affect these of significant financial hardship for the healthcare metrics. I am using a variety of methods in or- industry. Furthermore, with increasing competition der to parse the data and look for significant find- between hospitals, patients are placing experience ings. Additionally, we are beginning to look more scores at a higher priority when choosing hospitals closely at the development of hybrid platforms, plat- and doctors. forms that combine elements of transaction and in- novation platforms. This has involved gathering This research project aims to explore the drivers of detailed financial information on Amazon, Google, patient experience, medical outcomes, and costs in Facebook, and Apple. I might eventually help with healthcare operations. Qualitatively, I look at cases case studies for specific chapters of the book as well. such as the Cleveland Clinic, which made significant efforts to improve patient experience and jumped from the 55th to the 92nd percentile in overall sat- isfaction from 2008 to 2012. Quantitatively, I study publicly available data from the CMS, particularly in the Hospital Consumer Assessment of Health- care Providers and Systems (HCAHPS), which is the patient satisfaction survey required and used by CMS to determine hospital reimbursements. I cre- ate specific datasets from the survey information, and by analyzing them, I look for trends in patient experience, both overall and in particular distribu- tions by geography, hospital type, hospital size, etc. Lastly, I hope to perform analyses on individual PRIMO 23 patient-specific data to discover patterns in patient Pharmaceutical Innovation: Economics, experience based on illness type and severity, treat- Incentives, and Regulation ment duration, age, sex, race, or other factors. As advances in med- By analyzing trends in patient experience, we may ical technology have al- gain a deeper understanding of its drivers and their Holly Breuer lowed for the discovery Molecular and relation to medical outcomes and costs in healthcare and development of new Cellular Biology, operations. These findings may provide broader 2018 drugs, millions of lives insights to hospital management and healthcare have been improved and providers into how best to improve and maintain Harvard Business even saved. However, a high quality of care while keeping lower costs. School pharmaceutical innova- tion has come with very high costs. Recent esti- Migration, Diasporas, and Business Advisors: Ariel D. Stern, PhD; Amitabh mates of the cost of de- Networks Chandra, PhD velopment of a new drug Overseas Chinese have have been pegged at $2.6 had a profound influence billion, while new preci- Hellary Zhang on the economic develop- sion medicines that promise to personalize treat- Applied Mathematics ment of China, even after ments and therapies for many diseases are expected & Economics, 2018 they have left their home to be even more costly. In the face of such high Harvard Business country. Although ex- costs, our healthcare system now faces an industry School isting academic research landscape vastly different from, and more challeng- has examined the in- ing to navigate than, that of ten years ago. In this Advisor: Meg fluence and importance context, the research of Professors Ariel Stern and Rithmire, PhD of the huaqiao (overseas Amitabh Chandra focuses on the economic incen- Chinese) community in tives that drive biopharmaceutical innovation and specific geographic areas, the development of new drugs, including regulatory and although the Chinese government has kept ex- dynamics like FDA programs that make it easier tensive records of the volume of emigration from and less costly for certain kinds of drugs to receive China, no one has compiled this information cap- approval. turing the Chinese diaspora. Many questions are unanswered: What does the entire Chinese diaspora In order to become acquainted with the current look like on a world map? How far-reaching are their biotech landscape, I have been researching and begun movements? How can we visualize the transactions writing two HBS cases for use in Professor Stern’s between huaqiao communities and their hometowns? class “Demystifying Data: Managing with Analyt- Professor Rithmire’s research seeks to build an open- ics.” Each case examines an up-and-coming player source website that maps the movements of mem- in the healthcare space: the first, Genepeeks—a bers’ hometowns to Southeast Asia, the United Cambridge-founded company that seeks to provide States, South America, and beyond. We will be an innovative, analytical approach to carrier screen- compiling data from a wide range of sources at vary- ing for genetic diseases; the second, PillPack—a mail- ing administrative levels—from local gazetteers on order pharmacy providing new packaging methods overseas Chinese to national statistical yearbooks to help the growing number of American patients collected from census data—to create a visualiza- managing multiple medications. Through company tion of the magnitude of the Chinese diaspora and interviews and further literature research later this its economic influence on China through foreign year, we’ll be able to focus each case on a central direct investment (FDI), remittances, and other operational, analytical, or technological issue that flows of capital and resources. The hope is that will allow Harvard Business School students to take this website will be easily accessible to academics on the role of decision-making company executives in and the general public as an educational tool, as the classroom. I’ve also used my background in biol- well as serve as a portal through which researchers ogy to assist Professor Chandra in the preparation of can easily contribute their data and findings on the a presentation explaining the dynamics of Hepatitis huaqiao community. We also hope that, upon build- C virus and translating the biology of current drug ing this database, we will be able to draw conclusions treatments for a non-science audience. about the Chinese diaspora’s business networks. In another project this summer, we’ll examine the role of public research funding in recent biophar- maceutical innovation. We are currently in the data collection phase of the project, which involves match- 24 PRIMO ing clinical trials for new drugs with any NIH grants earning abilities with a few parameters. We would that played a role in their discovery and develop- like the parameters to be endogenously determined ment with information from the Thompson Reuters by factors like government spending and technologi- Cortellis Competitive Intelligence Database. From cal progress—to be determined by some sort of “abil- understanding current industry innovations and in- ity production function.” Empirical analyses, prelim- centives to examining healthcare’s regulatory land- inary and speculative at this point, would then allow scape and high costs, our work seeks to shed light us to uncover how income-earning abilities change on the challenges that have come with improved with respect to these explanatory variables. These medical outcomes in the hope of understanding empirical evaluations would then enlighten optimal how modern society can adapt to modern medicine. tax theorists to sensible, endogenous-ability mod- els that better attack income inequality problems. Government Spending, Income Inequality, and Optimal Taxation Decision Making and Behavioral Economics Income inequality has Economic theories generated much interest generally employ the ra- Jiafeng Chen for both academic and Kevon tional human—Homo eco- Applied Edmondson policy economists. Since nomicus—who strives to Mathematics, 2019 Psychology, 2018 governments often redis- maximize utility. This Harvard Business tribute with taxation, Harvard Business common, albeit ideal- School optimal tax theory— School ized, convention allows the study of taxes that economists to create over- Advisor: Matthew maximize welfare gains Advisors: Alison arching theories in a com- Weinzierl, PhD and minimize efficiency Wood Brooks, PhD; plex world. It should losses—is especially rel- Ryan W. Buell, come as no surprise that evant. Marrying income DBA; Francesca our decisions are not al- inequality and optimal taxation, however, has two Gino, PhD; Leslie K. ways systematic and de- difficulties. On one hand, the literature on income John, PhD; Michael liberate, as these the- inequality usually analyzes summary statistics of in- I. Norton, PhD ories often necessitate. come distributions, such as the 90/10 ratio (the ratio This summer, I worked of income at the 90th percentile to that at the 10th) Mentors: Grant alongside researchers in Donnelly; Oliver or the Gini coefficient, but summary statistics do not the Harvard Business Hauser, PhD; easily lend to numerical simulations, an important Ximena School NerdLab. This analysis tool for optimal tax theorists. On the other Garcia-Rada, MBA; lab delves into the irra- hand, the literature on optimal tax traditionally Tami Kim; Ovul tionality in human deci- treats taxpayers’ income-earning abilities (abilities Sezer sions and judgments via for short)—both innate and acquired—as exogenous. psychological and eco- However, realistically, the trend of widening income nomic frameworks. In one inequality may partly come from widening inequality project, we explored the role of chance encounters in the distribution of abilities; thus, treating ability in the dictator game, where there is a dictator and as exogenous leaves a large hole in tax theorists’ a recipient. The dictator is tasked with allocating models. Indeed, factors like government policy and a sum of money between him or herself and the technological change can conceivably influence the recipient. In our study, participants were assigned distribution of income-earning abilities, hinting that to either the “recipient” or the “other” condition. ability may be endogenously determined. In both conditions, there was a chance that the participants would have to reveal their allocations. Our research attempts to attack these two problems. Dictators in the “recipient” condition would reveal We fit income quantile data (U.S. 1979–2013) to a their allocation to the recipient, while dictators in probability distribution that hybrids the lognormal the “other” condition would reveal to non-recipients. and Pareto distributions, with properties consistent In the experiment, dictators were prompted to al- with empirical observations in income. The fitting locate $5 multiple times between themselves and a allows us to generate simulated individual income recipient, each time the probability of meeting the data. We then feed the income data through a recipient or “other” increased. In the end, one of simple labor-supply model and back out the ability the allocations was randomly selected and the dicta- distribution. Once we simulate a sample of ability tors were told whether they needed to disclose that from a sample of income, we fit the ability data to a allocation.The aggregated allocations of the dicta- probability distribution, which allows us to capture tors do not appear linear. The amount given to the over-time shifts in the distribution of income- the recipient seemingly approached an upper bound PRIMO 25 that was lower than $5, notwithstanding the in- tom of a market and then relentlessly moves up mar- creasing probability of a chance encounter. In a ket, eventually displacing established competitors.” rational world, one would expect keeping all $5 to By examining what factors separate the firms that yield more utility when compared to only allocating stay at the bottom of the market from those that $3 to oneself. However, the potential for receiving rise and disrupt the traditional order and hierarchy, negative utility from keeping all $5 exists. People our research can hopefully help firms discern which do not make decisions completely independent of rising companies in these nascent markets are more others; instead, the actors may consider how an- likely destined for success (and should thus perhaps other person may feel as a result of their decision. It be bought) and those more likely destined for failure is possible that being selfish (keeping all $5) could (which they can leave to fizzle out on their own). result in negative utility, as the dictator would be viewed as selfish by his or her peers. This could lead The Economics and Psychology of Consumer a dictator to allocate him or herself less than $5. Behavior The growing field of Strategy and Innovation in Nascent Markets behavioral economics dif- Nascent markets are Lingjun Xia fers from traditional eco- Economics & defined by what is per- nomics by introducing a Psychology, 2018 Lauren haps one of the most heavy dose of psychol- Nicholson Wellesley College unsettling words in ogy. Indeed, the research Sociology & Government, 2018 business—uncertainty. Harvard Business in the NerdLab investi- There are many variables School gates psychological mech- Harvard Business that are difficult to fore- anisms underlying human School cast and, as such, they Advisors: Alison behavior, oftentimes ex- make it difficult for firms Wood Brooks, PhD; plaining irrational behav- Advisor: Rory M. to plan for success. When Ryan W. Buell, iors that cannot be fully McDonald, MBA, competing in a market DBA; Francesca accounted for by tradi- PhD where everything from Gino, PhD; Leslie K. tional economics. This profit opportunities to John, PhD; Michael psychological framework Mentor: Cheng Gao regulations are unclear I. Norton, PhD is particularly essential in and unpredictable, under- fields such as marketing Mentors: Tami Kim; standing how firms can and consumer behavior. Ximena achieve competitive advantage in these fledgling Garcia-Rada, MBA Two of the projects I par- fields is of paramount importance. ticipated in this summer study the decision-making For my project with Professor Rory McDonald, process from the perspectives of consumers. we study competition and strategy in the nascent biotech field of direct-to-consumer (DTC) genetics One project studies joint-consumption decisions. Ac- testing industry. We collect data through archival cording to Gorlin and Dhar (2012), consumer deci- research and interviews to create timelines for the sions can be roughly sectioned into four quadrants. top firms in the DTC genetics testing industry so Along one dimension, one could make a decision that we can compare their methods and performance for oneself (single-decision) or with others (joint- against our success metrics to help determine what decision); along the other dimension, one could con- exactly it is that separates the companies that thrive sume the product for oneself (single-consumption) or and ultimately dominate the market from those that with others (joint-consumption). While most con- ultimately flounder. We then use the timelines to sumer behavior research focuses on single-decision, engage in comparative case analysis to build theory single-consumption scenarios, our project exam- on strategic actions firms can take to win in nascent ines single-decisions that consumers make for joint- industries. In addition to the DTC genetic testing consumption occasions. Through an online sur- industry, we also hope to examine these competitive vey, our results show that single-decision, joint- dynamics in other nascent industries, such as fantasy consumption occasions are ubiquitous in real life sports. and thus it is important to understand the dynamics behind these choices. We will further analyze the The main application for this research is derived data to look for consumption patterns, and follow- from the wildly popular idea of disruptive innova- up lab experiments will dive into understanding the tion, a term coined by Clayton Christensen which psychological mechanisms that shape these decisions. “describes a process by which a product or service takes root initially in simple applications at the bot- The other project examines the influence of gender- 26 PRIMO

related typecasting on consumers’ willingness to pur- correct “x1.” Both firms have the correct “x3” shock chase certain products. This project shows that con- variable in their respective mental models. sumers tend to avoid products that evoke gender labeling: women may reject a pink pen labeled “pink The first step in our simulations involves generating for women,” whereas they may have purchased the a pre-history for each firm by randomly generating pen had there not been a label. Within this project, quantity choices and using each firm’s mental model we conducted a field study simulating a political to generate a series of price-quantity data points for campaign: we asked female participants to choose the initial calibration of the parameters of each firm’s between a sticker and a button, both having slo- mental model. We explore several combinations of gans supporting Hillary Clinton. We manipulated neutral, optimistic, and pessimistic priors for the the slogan on buttons to sometimes say “candidate mental models in this process. An optimistic prior for women,” and sometimes say “candidate for U.S.” entails a firm assuming demand to be inaccurately We hypothesize that participants will forego qual- flatter than in reality and thereby producing an un- ity to avoid choosing the gender-labeled product. profitably large quantity, and a pessimistic prior en- The result is close to a marginally significant level; tails the opposite. Next we play out the competitive however, since field studies involve many uncontrol- interaction. This step involves randomly generating lable variables, usually resulting in weak statistical values for all shock variables in each firm’s respective significance, the result is actually rather satisfac- mental model and each firm subsequently choosing tory. Going forward, we will conduct further stud- a quantity by maximizing profit with respect to the ies along this path to examine under what condi- parameters of its mental model, the observed shock tions people will avoid gender-related typecasting. variables, and the competing firm’s quantity—which This study has applications in marketing, provid- the firm assumes to be the same as its own quantity. ing firms a smarter way to label their products. A market-clearing price is then generated based on the the true market demand function. This process is repeated for twenty periods, and the parameters Mental Models and Strategic Interaction of each firm’s mental model are re-calibrated based Mental models are on its history of quantity-price points at the start simplified representations of each new period as per our regression framework. Osama Arif of the world. Many eco- The simulations are repeated over 10,000 iterations Economics, 2018 nomic models assume for consistency and statistical power. that all firms have the Harvard Business School same mental model of the One of our initial findings is that the firm with world. In our project we relatively optimistic priors generally generates larger Advisor: Dennis Yao, use computational meth- profits, but this outperformance only persists in MBA, PhD ods to relax this assump- the long run when the firm with the incorrect tion and explore strategic mental model has these relatively optimistic pri- interaction between firms ors. This finding can be explained by the fact with different mental models. Our line of research that the regression framework allows the firm with specifically involves studying competitive interac- the correct mental model to eventually adjust its tion between firms with different mental models parameters to the correct ones found in the true of the world. In this case, different mental mod- demand, but the incorrect mental model does els entail different views of the demand function. not allow for such an adjustment. Actors’ repre- We use a regression framework for mental models sentations of the world are a crucial element of and a series of simulations in Python to conduct all types of strategic interaction and our project our analysis. We focus on Cournot competition in seeks to shed light on how varying these men- the case of a duopoly, in which each of the two tal models held by firms would affect competition. firms chooses a quantity to produce and the mar- ket price is determined by the total quantity pro- duced by the two firms and the demand function. The true demand structure in our simulations is P _t = α + [β1x1 + β3x3]Qt. We introduce variation in the mental models of the firms by varying the “x’s” in this structure, which are random shock vari- ables that affect demand. One firm holds the correct mental model and has the same shock variables as the true demand. The other has an incorrect men- tal model and has a shock variable “x2,” which has no deterministic power for true demand versus the PRIMO 27

Understanding Task Specialization Versus continue leading the firm. However, studies done in Task Diversification Through Real-Life Denmark have shown that family successions have a Examples negative causal impact on firm performance as op- erating profitability on assets fall during CEO tran- On March 19, 2012, sitions. Moreover, employers tend to hire employees Apple disclosed the ini- not only for ability-based characteristics (e.g. grad- Rohan Reddy tiation of dividend and Applied uating from a top university) but also for affinity- share repurchasing pro- Mathematics, 2017 based characteristics (e.g. ethnicity), despite the gram: “We have used fact that such decision-making decreases overall pro- Harvard Business some of our cash to ductivity and success in the firm. From my research, School make great investments in I hope to use new venture capital data to specif- our business through in- ically address the increasing percentage of family Advisor: Mihir creased research and de- firms. I will distinguish family history by matching Desai, MBA, PhD velopment, acquisitions, surnames and information provided online. After- new retail store open- wards, I will run regressions and statistical analyses ings, strategic prepay- to determine whether family firms are performing ments and capital expenditures in our supply chain, as well as non-family firms. I will compare differ- and building out our infrastructure. You’ll see more ences in the number of trade deals and IPOs to of all of these in the future,” said Tim Cook, Apple’s measure performance and determine a difference in CEO. “Even with these investments, we can main- performance between family versus non-family firms. tain a war chest for strategic opportunities and have plenty of cash to run our business. So we are go- ing to initiate a dividend and share repurchase pro- Decision Making and Behavioral Economics gram.” In 2013, Professor Mihir I. Desai initially The study of decision detailed the execution of the capital return program making and behavioral in a Harvard Business School case study titled “Fi- Yilin Chen economics lies between Economics, 2018 nancial Policy at Apple, 2013 (B).” Since that time, the fields of psychology Wellesley College there has been a series of important additions to Ap- and economics. This sum- ple’s capital return initiative that may provide in- mer I worked with the sight into the company’s internal operations. The Harvard Business School NERD lab, which con- purpose of this study is to analyze the additions sists of a group of profes- to the initiative from 2013 to 2016 and determine Advisors: Alison sors and doctoral students overall changes to Apple’s shifting capitalization Wood Brooks, PhD; from different academic structure, balance sheet position, operational per- Ryan W. Buell, fields including market- formance, and stock performance. This information DBA; Francesca ing, organizational be- will be then used to update the original case study. Gino, PhD; Leslie K. havior, and management. John, PhD; Michael The research at NERD I. Norton, PhD Family Influence in Venture Capital uses fundamental psycho- logical and sociological The importance of Mentors: Karen theories to investigate hu- Huang; Ovul Sezer venture capital remained man judgments and deci- Steven Tan relatively nonexistent un- sions. I worked on several Statistics, 2018 til the late 1990s and the projects related to these rise of the Internet bub- fields throughout the summer. Harvard Business School ble. Since then, venture capital has become a driv- One of these projects analyzes how people engage Advisor: Paul ing force in providing en- in conversation. Conversation is a pervasive human Gompers, PhD trepreneurs the ability to experience, one that is necessary to pursue intrap- pursue startups and busi- ersonal and interpersonal goals across myriad con- Mentors: Lauren ness endeavors. Among texts, relationships, and modes of communication Cohen, MBA, PhD; established venture capi- (e.g. written, spoken). In a conversation, people Kanyuan Huang tal firms, one can see the make a series of decisions about what to say and increase of family firms how to respond. The researchers collected data both where the family mem- in lab and through MTurk (Amazon Mechanical bers become co-founders and CEOs. Because one Turk), an online platform. We paired participants has the closest relationships with family members into dyads to chat for fifteen minutes. All conver- in most circumstances, it is understandable for en- sations were captured on ChatPlat, a chat software. trepreneurs to entrust their siblings or children to For each set of conversations, we analyzed the turn- 28 PRIMO by-turn text in the conversation, as well as overall impressions of the people who engaged in conversa- tions. Specifically, we analyzed the number of types of questions (e.g. follow-up, switch, and rhetorical), and the degree to which each chatter felt heard and listened to. Investigating these questions will help us as researchers better understand the psycholog- ical and linguistic mechanisms underlying human conversational behavior. SHARP SHARP

“Reframing Reality”: Exploring Artistic The third and final object on my tour was a scroll Representation in the Harvard Art Museums painting of Courtesan and Attendant, by Miyagawa Ch¯oki(active c. 1716–1751). The ukiyo-e scroll cap- How can art lead us to tures an instance of the real world influencing art, see the world differently? which in turn influences the real world. Eighteenth- Aaron Suduiko I aimed to answer this Philosophy, 2017 century Edo was known for its pleasure district, a question in my tour, “Re- result of wealthy merchants being trapped at the bot- framing Reality,” at the Harvard Art tom of Japan’s class system, socially immobile and Harvard Art Museums. I Museums in need of ways to spend their money. Artists at this explored various ways in time began painting in a style designed to capture Advisor: David Odo, which art can represent their impression of reality, rather than a mimetic rep- PhD the world as different from resentation of the world around them. These artistic our ordinary perception impressions then influenced the standards by which Mentors: Correna of the world, thereby en- real people lived their lives. Thus, an artist would Cohen, EdM; Erin couraging museum-goers paint his idea of a courtesan and thereby generate Northington, MTS to challenge their precon- standards of the feminine ideal to which real courte- ceived notions of what the sans would aspire. This particular artwork especially world is like. evokes the idea that the artist represents a type of person rather than a specific person by represent- The first object I selected for my tour was Endless ing the courtesan and her attendant against a blank Surface in the Form of a Column (1958), by Max background, divorcing them from any broader con- Bill. The piece is a roughly nine-foot-tall, metallic text. I ended my tour with this object to suggest that Möbius strip mounted on a wood-block base. Bill art doesn’t just provide us with new ways of seeing was a pioneer of “concrete art,” an artistic movement the world: precisely by changing people’s perspec- in which artworks neither reference nor represent the tives on the world, art also changes the world itself. natural world. Concrete art thus generates an aes- thetic based solely on the art object’s intrinsic form and the viewer’s spatial and optical relation to that Accessible Podcasts at Poetry in America form. The Möbius strip, a surface with only one side, epitomizes this artistic aim: as a form not found in My research this sum- nature, the strip challenges viewers to reconsider mer has been with Pro- their relationship with space. Brieanna Martin fessor Elisa New’s mul- English, 2018 tiplatform digital project The second object I selected was Still Life with Poetry in America, which Poetry in America, Inkwell (1911–1912), by Pablo Picasso. The frag- edX aims to show poetry’s rel- mented, geometric painting, typical of Picasso’s an- evance and accessibility to alytic cubist period, challenges the viewer to locate Advisor: Elisa New, a wider audience through the objects it purports to represent: an inkwell, a PhD conversations with diverse book, a collection of papers, and some writing in- guests, from Bill Clinton struments. Around the turn of the twentieth century, Mentor: Leah Reis to high school students mathematicians such as Charles Howard Hinton pop- Dennis to Nas. I’ve been work- ularized the idea of a fourth spatial dimension, which ing with her team as they humans, existing only in three dimensions, could not develop and produce con- perceive. Cubist painters sought to represent this tent for several several online courses, K–12 teacher- higher-order space by painting objects from multiple training courses, and a public television series in perspectives at once, as Picasso did in this painting. production with WGBH Boston. One can see this especially well in the bottom right area of the painting, where we see both the outside I think of my work this summer as helping answer of a book and pages from inside the book. Though the following questions: How do we make poetry this fusion of perspectives aims to capture a greater and conversations about poetry interesting, accessi- reality about the represented objects, it also renders ble, and relevant to people they wouldn’t otherwise the objects virtually unrecognizable, thereby chal- touch? How can digital media be used to make lenging viewers’ belief that their perception reflects the range of poetry readers as diverse as the voices the world as it really is. speaking through the medium? SHARP 31

My work so far with Poetry in America has con- achieved great fame in their own lifetimes, but all but sisted largely in developing podcasts from educa- a few were forgotten by the 19th century, when men tional content that was previously available only such as Voltaire, Diderot, and Rousseau were be- through classes on the edX platform and within the coming almost mythical figures. Only in the second Harvard Extension School. I watch the raw con- half of the twentieth century did feminist scholars versations between Lisa and guests, then edit and begin to resurrect these brave women: Émilie du rearrange them so that they follow an intuitive nar- Châtelet, Françoise de Graffigny, and Olympe de rative arc, and make for thought-provoking, enjoy- Gouges finally appeared on university syllabi and in able listening for poetry enthusiasts and those with critical editions next to the men they had debated. no experience alike. I’ve been learning the technical Even today, however, many women, especially those skills necessary to edit and record audio, using Final in genres like theater and journalism, remain to be Cut and learning the proper use of audio equipment, discovered. as well as applying the skills I’ve learned in two years of English seminar classes to decide which content Foucault, reinterpreting Kant, describes the En- to feature and what to include. lightenment as a philosophical perspective rather than a period—“a permanent critique of our histor- I’ve also been assisting the team here in develop- ical era.” In this spirit, our project aims to uncover ing content for the 1945 to the Present module of the long-neglected women writers of the eighteenth one of their online courses, Poetry in America for century. Who were these women? What unique Teachers: “The City” from Whitman to Hip Hop. institutional and social prejudices constrained their I’ve spent some time in Lamont’s Woodberry poetry ability to write and publish their work in any genre? room searching for poems related to one another and Which philosophical issues did women writers raise brainstorming people whose lives they might relate or engage with? How did women writers navigate to so we could bring them in for interviews. In this a society that considered them destined for the pri- work, I’m exploring how poems can be accessible vate sphere and incapable of creativity? A survey and relevant without sacrificing complexity, beauty, of these women, both the greats and the practically or freshness of expression. unknown, is much needed as a reminder of their presence and as an attempt to identify the tactics By the end of the summer, I will have produced they used to approach an unfriendly society. a cohesive and interesting series of podcasts that are accessible to anyone in both content and form. I will Preliminary findings reveal that women did write in also have a few answers to the question of how po- all genres: epic poetry and metaphysics to vaudeville etry can be a mode of expression and communication and republican journals. While many women wrote on a more basic human level, enjoyed and appreci- without challenging the status quo, others advocated ated by many people outside the academic world. for greater gender equality, relying on subtle allusions or subversive genre shifts to contest the literary and social establishment. Even if they were overlooked Femmes Littéraires: Une Histoire Culturelle by the literary historians of the nineteenth centuries, In Enlightenment- these women contributed to French literature and the era France, women were Enlightenment with just the sort of critical mental- Caleb Shelburne widely considered natu- ity that Foucault identifies. Femmes littéraires: une History and rally subordinate to men histoire culturelle will finally provide the recognition Literature, 2018 in intellect, emotional re- they deserve and, in doing so, will complicate and ex- Harvard University silience, and imagination. tend our understandings of the period and its people. This bias influenced even Advisor: Christie the most “enlightened” McDonald, PhD thinkers, whose medi- cal articles, philosophical Mentor: Kylie Sago treatises, and fictional en- deavors perpetuated un- critical understandings of women’s physiology and mental capacity. Never- theless, despite biases in education, restrictions on publishing, and conventions that disdained female authorship, women did write, producing works in all genres that complemented and challenged the philosophical explorations and artistic productions of their male contemporaries. Many of these women 32 SHARP

Investigating Wuzhou Networks with CBDB alongside the flourishing of a Neo-Confucian ideology during the Yuan dynasty. The China Biograph- ical Database (CBDB) Edith Claire is a computer database Enright that aims to catalog all Comparative Literature, 2018 recorded biographical and relational information Fairbank Center of from pre-modern China; Chinese Studies currently, the database contains almost 400,000 Advisor: Peter Bol, individual biographical PhD entries from throughout Chinese history, and is Mentor: Hongsu an especially invaluable Figure 1: Network connections of Wuzhou residents Wang, MA resource for the quantita- in the Southern Song dynasty. tive investigation of schol- arly, official, and familial links in middle-period China (960–1400 CE). The China Biographical Database project at Harvard is headed by Professor Peter Bol and Wang Hongsu, and I am conducting research using the database for the first time this summer.

This SHARP project entails using the information recorded in this database to investigate the degree of local interconnectedness of intellectual, literary, and kinship ties between members of the literary elite of Wuzhou prefecture in China’s Southern Song (1127–1279) and Yuan (1271–1368) dynasties, a pe- riod coinciding with the emergence and spread of Neo-Confucian philosophy. By mapping these net- works, we can determine the response of these elite communities to the dynastic shift that occurred in Figure 2: Section of Wuzhou kinship network in the the late thirteenth century when the Mongol em- Southern Song. pire conquered China’s Song dynasty and founded the Yuan dynasty. With the help of the geograph- ical imaging software QGIS and the social network Femmes Littéraires: Une Histoire Culturelle mapping software Gephi, we have mapped the so- cial connections of Wuzhou residents across China This project aims to and analyzed the extensive social and kinship ties explore the literary pro- between members of the Wuzhou elite in order to Henry Shreffler ductions of French women History and determine the degree of local connectedness as well of the 18th century. While Literature, 2018 as the most important actors in the network—often historically neglected by members of major literati families who are related to Harvard University scholars until the second imperial degree holders. half of the twentieth cen- Advisor: Christie tury when scholars be- We are now investigating the social networks of other McDonald, PhD gan to recover the works Song and Yuan dynasty prefectures for purposes of of some of these writers, comparing their extent and demographic makeup Mentor: Kylie Sago women of the period cre- with the Wuzhou network. Moving forwards, we ated literary masterpieces plan to construct database queries that will allow us in all genres. Women of to determine the local origins of authors of major all classes wrote plays, novels, philosophical treatises, texts, as another way of mapping the geographic and more, engaging with men on complex issues of distribution of literary culture. We intend for the the time, all the while facing significant structural quantitative data yielded by CBDB to be useful in barriers to their participation in the literary sphere. evaluating the hypothesis that Chinese social and lit- Men and women alike perpetuated harmful false- erary connections became locally denser and richer hoods about the female condition, asserting that SHARP 33 women were unable to reason, should read but not along with early biographies and readers. Other col- write, and were best occupied distracting themselves lections within Houghton prove equally rewarding. with their makeup and idle nothings at their toi- The John Keats collection, for instance, not only lettes. While men were expected to be involved with offers a window into Romanticism, but also into the the public sphere, it was widely understood that tumultuous personal relationship between Audubon women were to remain in the private sphere. Works and the Keats family. I also work with secondary such as Rousseau’s Émile only accentuated this prej- sources in other libraries, as well as the more scien- udice, lauding women for their role as mothers. tific holdings of Harvard’s Museum of Comparative Zoology. Such collections mirror the variety and Nonetheless, some women wrote, and achieved suc- richness of Audubon’s own experience. In both my cess and fame through their literary works. However, personal research and writings, as well as a planned even as published authors, women often wrote anony- public exhibition, I put various figures and move- mously, and often faced having their work misat- ments in a dialogue with Audubon at the center. tributed to the men in their lives. Femmes littéraires: une histoire culturelle aims to highlight the impor- Decolonization and the Constitution of the tant contributions of French women of letters in their International Order respective fields, rewriting a male-dominant histor- ical narrative to better reflect the historical reality. The wave of decolo- nization that swept the Lev Asimow globe between the 1940s My Work of a Different Nature: John James Social Studies, 2018 and 1970s brought to Audubon and Nineteenth-Century America fruition the ideal of self- Harvard Law School In the first half of determination for up- th the 19 century, John Advisor: Samuel wards of a billion people Jess Clay James Audubon traveled across the Global South. History and Moyn, JD, PhD across America to paint But beyond redrawing the Literature, 2017 the birds that would make map, the collapse of Em- Houghton Library him famous. His expe- pire in Africa, in Asia, and in Latin America rendered riences took him from uncertain the foundational precepts of the post-War Advisors: Emilie the Everglades to Acadia, international economic and political order. While Hardman, MA, MLS; from riverboat journeys this paradigmatic break with the structuring logics Heather Cole, MA, to transatlantic passages, of Empire spurred the birth of nation-states through- MLIS and from the backwoods out the formerly-colonized realm, this result was by of Kentucky to the White no means foreordained. House dining room. He became the most celebrated naturalist in Amer- The first aspect of this inquiry, thus, is into the ica, and arguably one of the nation’s best-loved victory of nationalism as the dominant principle for artists. My research engages with the life and work political self-determination, and the fading of al- of Audubon as a reflection and fulcrum of the early ternative organizing models such as federalism and republic. As a Houghton Library–SHARP fellow, I departmentalization. Understanding this dynamic, undertake self-directed research in a wide range of by reference to the writings and speeches of anticolo- fields pertaining to Audubon. Beyond his enormous nial leaders, can provide unique insight into both the contributions as an American artist and scientist, development of economic autarky, or self-sufficiency, I study the more literary and political aspects of and the modern conception of the nation-state that Audubon and his work. In particular, I explore shaped policies such as 1974’s New International Audubon’s influences and those he influenced, his Economic Order. involvement with Romanticism and Transcendental- ism, and his contributions to subjects ranging from Secondly, this project seeks to conceptualize the scientific advancement to national identity. notions of equality, development, and social better- ment mobilized by diverse actors in the formerly My primary resource is Houghton Library’s John colonial world, and to investigate the varied politico- James Audubon collection, featuring his artwork, economic methodologies proposed for actualizing letters, journals, manuscripts, and published works. these ideals. It is thus jointly an exercise in political Among these are his early drawings and mundane and intellectual history. Given the complex conflu- business transactions, as well as first editions of his ences of Soviet-style socialism and American capital- magnificent Birds of America and a letter to Daniel ist globalization that exerted influence on decision Webster. The collection also features documents makers in the Global South, casting light on the ide- from family members which pertain to Audubon, ological conflicts and compromises that motivated 34 SHARP political change in the era of decolonization necessi- vated, refurbished, and relit, but they could not tates a better understanding of such phenomena as merely function as a beautiful building. The space Léopold Sédar Senghor, Mamadou Dia, and Sékou had to be activated with programming and outreach Touré’s African Socialism; Kwame Nkrumah’s Pan- in order to become a dynamic environment for cura- Africanism; and Julius Nyerere’s Ujamaa. Economic tors, students, faculty, and the public to interact with growth was a paramount concern for the historically each other and the vast resources at their fingertips. impoverished nations of the Global South, who col- The Museums are still in a process of introspection lectively desired a greater international parity with and self-evaluation. Now that they have moved past their former colonizers. But certain socialist sensibil- their renovation project, they will only continue to ities motivated these fledgling nations to adopt the transform and grow. more ambitious goal they termed “development,” de- noting an intra-nationally equitable contribution to This summer, I had the pleasure to play a small and division of the spoils of growth. When compared role in this growth as a guide in the Division of Aca- to the welfare state model that, in this period, com- demic and Public Programs. Through conversations mitted the governments of the metropolitan Global with staff and independent research in the curatorial North to establishing basic social minima domesti- files, I was able to craft a tour that sampled from the cally, the economic policy of these developing na- diverse collections featured in the Museums. Each tions can provide valuable insight into the politics object on my tour, Remembrance and Renewal, is and ethics of international governance, economic aid, the work of an artist who, in the process of creation, and political intervention. looked to the past for inspiration while simultane- ously reinventing it for new generations. I emphasize Third and finally, interrogating the politics of de- evolution over revolution. In both museum galleries colonization provides a unique vantage point for and the public consciousness, the history of art is theorizing about human rights and conceptions of usually a narrative of innovation and supersession. global justice that persist today. As decolonization My hope is that visitors come away from my tour stretched the Western category of “the human” to with a more critical eye and a willingness to see art- its breaking point, motivated by theories of race, works not as autonomous creations, but rather as existentialism, and Marxism, the governing logics of links of continuity between the cultural traditions rights, privileges, and distributive justice similarly that precede and follow them. My variegated tour were tested, expanded, and teased apart. By view- focuses on a fresco depicting police brutality during ing decolonization as a theoretical and philosophical the Great Depression, a portrait mask made for a intervention, this project aims to more capaciously mummy in Roman Egypt, and a monumental altar- conceive the origins of the purportedly neutral model piece from Renaissance Venice dedicated to lost love of liberal internationalist human rights. By bringing and heavenly hope. together these economic, political, and philosophical problematics, we hope to make a modest contri- My overarching concern is the story of the renovated bution to the genealogy of our decolonized world. Museums, caught at the crossroads of tradition and possibility. On one hand, I hope to demonstrate that the Museums offer a portal the past, with ob- Remembrance and Renewal at the Harvard jects that conjure up vanished cultures and faded Art Museums artistic movements. I also hope, however, to show In November of 2014, that the Museums are a gateway to the future, with the Harvard Art Museums new research and initiatives, such as the Student Rob Hopkirk reopened to great fanfare Guide program, to help make it a space that is more English & Religion, after an extensive six-year accessible and inclusive for visitors of all ages and 2018 renovation. It was the backgrounds. Like the objects they house, the Muse- Harvard Art culmination of a long ef- ums act as mediator between the past and the future, Museums fort to revitalize the Mu- as well as between the artist and the viewer. Each seums as an integral in- tour I give is a new opportunity to bridge the gap Advisors: Jessica stitution of teaching and that prohibits visitors from deeply engaging with the Martinez, PhD; learning for Harvard and art. The renovation may have entered into the realm David Odo, PhD the wider community. Af- of memory, but the renewal of the ever-evolving ter that initial burst of Harvard Art Museums begins again each morning. Mentor: Correna publicity, however, it was Cohen, EdM clear that hidden within this apparent ending was another project just be- ginning to take shape. The Museums were reno- SHARP 35

An Undiscovered Contribution to Egyptian Quine’s Edits Archaeology This summer, I was From 1905 to 1947, lucky to work with the the Harvard University– Theresa Clark archive of W.V. Quine Philosophy, 2018 Sarah Judd Boston Museum of Fine at Houghton Library Statistics, 2019 Arts Expedition enjoyed through the Houghton– Houghton Library spectacular success at SHARP Fellowship. Harvard University twenty-three archaeolog- Advisors: Emilie W.V. Quine is one of the Advisor: Peter Der ical sites along the Nile Hardman, MA, MLS; foremost philosophers of th Manuelian, PhD in both Egypt and Nu- Heather Cole, MA, the 20 century; he spent bia, now modern Sudan. MLIS several decades at Har- This success can be seen vard, from his time as a in the wondrous collection of Boston’s Museum of graduate student to his Fine Arts, at Harvard University’s Peabody Mu- passing in 2000. Broadly, the goal of my research seum, and in museums in Cairo and Khartoum. One was to use the archive to gain a fuller picture of the key source for presenting the important story of development of Quine’s philosophical commitments, the Expedition and its leader, Harvard archaeolo- with a specific focus on understanding the rationale gist George Reisner, is the Ashton Sanborn papers. behind Quine’s edits in some of his famous papers. The papers are a collection of hundreds of letters More specifically, the process of my research fell into by Ashton Sanborn (1882–1970), a member of the three interrelated categories. HU–MFA Expedition, and lay undiscovered in the archives of the Schlesinger Library until just this First, using Versioning Machine, a textual display past year. This collection includes descriptions of interface, and Text Encoding Initiative (TEI), I en- work at the sites of Dendera and Memphis for the coded several of Quine’s papers, including “Epis- University of Pennsylvania Expedition; famous dis- temology Naturalized” and “Natural Kinds.” The coveries at the Giza Pyramids and at Meroe (Sudan), encodings of these documents, which are to be pub- as well as comments on Reisner’s archaeological field lished online, will be available to researchers for methods for the HU–MFA Expedition. There are the purposes of comparing different iterations of also mentions of many great Egyptologists, impor- Quine’s papers, thereby tracking the development tant world events and visitors to the sites, as well of his philosophical ideas. A critical compilation of as descriptions of urban life in Cairo and rural life Quine’s edits has yet to be published; therefore, this further up the Nile. Other Sanborn letters, written encoding offers a unique opportunity for scholars to from the Museum of Fine Arts, Boston in 1925 to directly compare drafts of Quine’s works. his wife temporarily living in Bryn Mawr, illuminate the history of the MFA and the HU–MFA Expedi- Second, the encoding of these drafts has allowed tion from the Boston perspective. The collection me to evaluate the significance of Quine’s edits— is made up almost entirely of letters from Ashton from single word changes to the insertion or dele- Sanborn to his mother, Lillian, and his wife Agnes, tion of entire paragraphs. Using Quine’s notes and as well as their responses, along with newspaper and his correspondence with other philosophers—housed journal clippings from this same period. The col- both at Houghton and at the Harvard University lection presents a unique window into the history Archives—I was able to match parts of Quine’s edi- of archaeology, early 20th century Egyptian poli- torial changes to the rationales described in his notes tics, international relations, and the many exciting and correspondences I mapped out the evolution of discoveries of the Harvard University–Boston Mu- his papers, thereby gaining insight into the develop- seum of Fine Arts Expedition. It is the goal of this ment of Quine’s ideas over time. research project to gather and organize the collec- tion, to study these newly discovered papers, and to Finally, I have focused on exploring points of con- process them in a filemaker database, enabling the fusion regarding Quine’s writings, by comparing preparation of scholarly and popular publications— Quine’s drafts with later philosophical analyses of and perhaps even museum exhibitions—as a result. his work. Quine’s writings, and in particular his seemingly singular views, have led to difficulties in interpreting his position. By comparing points of confusion cited in these later analyses to a compila- tion of Quine’s edits, I hope to gain a better under- standing of Quine’s philosophical commitments and to resolve some of these points of contention. 36 SHARP

The techniques I have uses this summer represent Baker’s first English 47 class at Harvard. Intrigued the vanguard of modern humanities research; as Ver- by his early success as a playwright, I delved into sioning Machine and TEI have thus far been used Sheldon’s papers, which are also housed in Houghton mainly for older text and poetry, I hope that my Library, and found that he lived an extraordinary work will encourage their use in a broader range life and left a remarkable artistic and social legacy of humanistic disciplines, and enable philosophical despite becoming totally paralyzed and blind by writing to be accessible to a variety of audiences. his mid-thirties due to a rare form of arthritis. I realized that Sheldon’s life would provide fascinat- ing dramatic material, so I have begun the pro- Edward Sheldon, George Pierce Baker, and cess of writing a play inspired by the life of Ed- the Origins of the American Theatre at ward Sheldon. In this process, I am utilizing not Harvard only Sheldon’s correspondence with friends ranging As one of SHARP’s from playwright Thornton Wilder to the philoso- Houghton Library fellows, pher Krishnamurti, but also his personal papers, Thomas I am pursuing an inde- his library, and the materials compiled by his bi- Peterson pendent research project ographer. As Sheldon played an essential role in History and Literature & in the library, which holds blazing the trail for American realism, influencing Theater, Dance, and the Harvard Theatre Col- many of his successors in Baker’s class, this artistic Media, 2018 lection among its many endeavor goes hand in hand with the academic paper treasures. I began my I will produce. The paper will focus on Baker, Shel- Houghton Library project with an interest don, and O’Neill in explaining Harvard’s important in investigating Harvard’s role in the development of the American theatre. Advisors: Emilie theatrical history, both Hardman, MA, MLS; curricular and extracur- Animation and Realism Heather Cole, MA, ricular, with the goal of MLIS producing both an aca- For Ruth Lingford’s demic essay and a play film A Girl in Question, inspired by my findings. Zachary Wong I have been tasked with Visual and The logical starting point for this endeavor was with crafting several key scenes Environmental the papers of George Pierce Baker, the first Harvard Studies, 2017 through the time-honored professor to offer practical courses in theatre. In technique of hand-drawn examining Baker’s papers, which take up eighty-two Harvard University animation. This process boxes in Houghton’s collection, I discovered that he of producing hundreds of maintained extensive correspondence with a remark- Advisor: Ruth drawings to create an illu- able range of colleagues and students. Lingford, MA sion of continuous move- ment is as much construc- The first name to catch my eye among his archived tive as it is analytical. correspondents was that of Eugene O’Neill, the No- Hand-drawn animation forces the animator to an- bel laureate and four time Pulitzer Prize-winning alyze and internalize the appearance and physicality playwright who is widely regarded as the first great of real-world movement: gravity, inertia, accelera- American writer of plays and one of the inventors tion, etc. It demands a hyperawareness from the of a truly American drama. The folder contain- animator, as each individual frame is both a con- ing O’Neill’s correspondence with Baker begins with tinuation of its predecessor and anticipation of the O’Neill’s request to join Baker’s playwriting class, drawings to come. entitled English 47. O’Neill had received no prior theatre training, but only a few years after his gradu- Hand-drawn animation’s deep connection to tim- ation from Baker’s class he had garnered mainstream ing and real-world physicality prompted the funda- success. In examining Baker’s papers, I have discov- mental question of my research for Professor Ling- ered that Baker’s English 47 class, which began at ford. While working on this project, I wondered if Radcliffe in 1904 and was introduced at Harvard the animation’s connection to live-action at the funda- following year, trained many in the first generation mental level of movement could be extrapolated to of notable American playwrights—a group instru- the broader components of an animated film: com- mental in introducing the gritty realism and social position, design, and editing. Professor Lingford consciousness that would define the American stage encouraged me to ask myself if the cinematic lan- in the twentieth century. guage and aesthetics of live-action films could be translated into an animated film, and if so, what its One name that consistently appears in Baker’s impact would be on a viewer. papers is that of Edward Sheldon, an alumnus of SHARP 37

Aside from my daily animation responsibilities, I research the films of Roman Polanski, Stanley Kubrick, and Alfred Hitchcock, specifically those that deal with psychological horror. I have divided my time between animating at my desk and viewing live-action films at the Sever Film Studies Library. What I am beginning to realize is that these films seem to suppress the naturalism of their subjects in an attempt to create an atmosphere of fantastic horror. The symmetry of John Alcott’s cinematog- raphy in Stanley Kubrick’s The Shining upholds a visually stunning geometric order that seems to over- whelm the psychological chaos of the film’s charac- ters. Robert Burks’ use of green light in Hitchcock’s Vertigo seems to anticipate not the lighting logic of naturalistic photography but rather the fantastical color palette of an animated film or Mary Blair color study. The interplay between live-action film and fantastical aesthetics allows the films to heighten the abnormality of the filmed characters’ circumstances.

These examples of live-action filmmaking emulat- ing fantasy qualify my original research question. If hand-drawn animation is inherently unrealistic, in what ways can it be influenced by live-action horror, a medium that in many instances seeks to suppress its own realism? An answer to this question would arise in the form of a fully animated scenes that have been inspired by live-action cinema. I hope that through my exploration of live-action filmmak- ing and hand-drawn animation, I can uncover new ways to extend the verisimilitude of an animated film. SURGH SURGH

Understanding Attachment Styles of mixed methods approach, my current task is to clean Children of Female Former Child Soldiers and analyze key informant interviews that discuss and their Mothers in Sierra Leone the attachment styles of children of female former child soldiers. Initial read-throughs of the interviews During the Sierra reveal certain themes. For instance, children raised Leone Civil War (1991– by non-biological parents following the war exhibit Gita 2002), nearly 20,000 chil- Bhattacharya more antisocial behaviours compared to children dren were forced into Cognitive who could reintegrate with their biological families militia groups such as Neuroscience and post-war. According to initial read-throughs of some Evolutionary the Revolutionary United twelve key informant interviews, adoptive family Psychology, Front (RUF) to oppose and community members were more likely to abuse December 2016 the existing leadership’s these foster children compared to their own biologi- corruption. These chil- cal children. Grounded Theory and Thematic Con- Harvard T.H. Chan dren were often forced to tent Analysis will likely reveal additional reasons for School of Public commit violence towards this difference in parental treatment, as well as fur- Health their community mem- ther trends in attachment style between the children bers as initiation into the of female former child soldiers and their mothers. Advisor: Theresa RUF, and were also vic- Betancourt, ScD tims and observers of vio- lence. Following the Civil HIV Prevention and Education for Mentors: Katharine War, thousands of chil- Collet, MPH; Elyse Boston-Area Adolescents dren were displaced, and Thulin, MSc; Tia McGill Rogers, PhD the question of their rein- Few epidemics in tegration into society re- the U.S. have carried mained. To investigate Henna Hundal more stigma and incited Undeclared, 2019 the negative health consequences of war on these more public fear, but former child soldiers, the factors contributing to also inspired more com- Center for AIDS their mental health outcomes, and protective pro- Research, Harvard munity activism, than cesses and forms of psychological resilience, the Re- University HIV/AIDS. When the search Program on Children and Global Adversity epidemic became one of (RPCGA) began a longitudinal investigation in 2002 Advisor: Wanda A. the most prominent pub- with data collection points in 2002, 2004, and 2008. Allen lic health concerns in the A mixed methods approach was applied over the 1980s, posters urging peo- course of the study to first gain a qualitative under- Mentors: Sannisha ple to “Put a rubber on standing of how former child soldiers were behaving, Dale, PhD, EdM; it!” and outreach efforts feeling, and socially interacting, that would inform Valerie Earnshaw, underscoring the disease’s quantitative assessments of mental health outcomes. PhD fatality flooded the Amer- Two hundred sixty male and female former child ican consciousness. Fast- soldiers between the ages of ten and seventeen at forwarding more than baseline were recruited from an NGO-run Interim three decades later, the success of HIV combination Care Center in Kono District. Results from the first therapy in reducing AIDS-related deaths has ren- waves of the study show that both war experiences dered HIV/AIDS a chronic, manageable condition. and post-conflict risk factors—the stigma associated Nevertheless, ample challenges continue to accom- with being part of the RUF, post-conflict family pany life with the virus, including stigma, access abuse, and the death of a parent—contributed to to and affordability of healthcare, and social deter- long-term mental health effects of former child sol- minants of health. Moreover, knowing that STIs diers. Post-conflict protective factors such as family increase the likelihood of contracting HIV, and given reintegration mitigated negative mental health out- that STI rates are on the rise in the U.S., targeted comes. HIV outreach efforts are still much in order.

My current project involves the fourth wave of the Our research aims to revise and re-implement a RPCGA’s longitudinal study, in which I am examin- community-based HIV education and prevention ing the intergenerational effects of war on the female model to better address the changing needs in former child soldiers who are now becoming mothers HIV/AIDS awareness in the U.S. The model, Safety and forming intimate partnerships. Continuing the Net, was last updated in 1992 and bears resem- 40 SURGH blance to the “Tupperware model” of direct selling. high-priority health technology innovations; then, The original Safety Net called for dynamic, engaged it provides the resources, funding, and momentum social gatherings at a community member’s resi- to accelerate development and commercialization. dence, where Safety Net educators could facilitate Of over fifty digital innovations from clinicians and discussions and provide resources meant to invite researchers that the Accelerator has vetted, I am individuals to take charge of their own health. The involved with multiple high potential projects. program, though launched with a focus on women, was later expanded to include couples, mixed-gender, As part of the Accelerator team, my work involves and adolescent sessions. using a research-first approach to digital technology development. The method emphasizes the impor- Our updated Safety Net, designed primarily for ado- tance of understanding the pain point or unmet need lescents aged fifteen to twenty living in the Dorch- the technology is meant to address before jumping to ester, Roxbury, and Mattapan neighborhoods of technology deployment. For each of the projects that Boston, seeks to retain critical aspects of its prede- I am supporting, I take into account current clinical, cessor, including the emphasis on peer-to-peer advo- workflow, or process pain points as well as major cacy, safe spaces, self-efficacy, and motivation to ad- business, regulatory, or clinical requirements. These vocate for one’s health. However, our updated Safety considerations are critical to a project’s development Net differs in several key areas, including accents on and serve as a starting point for further research. new prevention strategies, expanded testing options, structural and institutional players in healthcare, In the continuum of technology development, the and greater integration of HIV education with other majority of my time is spent in the research and STI information so as to reduce stigma. The afore- design phase, which will then be followed by clini- mentioned amendments were selected based upon cal research validation to show health care impacts, interviews with key informants and local health per- or an efficiency study to alter processes and work- sonnel as well as probes into research literature avail- flows. Furthermore, through extensive clinical in- able on the subject. To this end, our updated model terviews and market analysis, I am able to under- intends to utilize technology for the streamlined de- stand and justify a product’s business value which, livery of information as well as welcome active user in health care, often comes from improving care participation to drive the sessions. We will facilitate outcomes and reducing costs. Doing so will ensure two installments of the updated model and conduct that these innovative ideas become effective solu- focus groups, gauging effectiveness in meeting the re- tions, widely adopted by patients and clinicians. We vised criteria via a pretest and posttest assessment. hope to extend the reach and impact of these health- care solutions beyond Boston Children’s Hospital. Driving Healthcare Innovation Through Building New Technologies and Deploying a Investigating the Role of FtsQ in Regulating Comprehensive Digital Health Platform Cell Division in Mycobacterium smegmatis Across the health- Tuberculosis (TB) is care industry, there’s caused by the bacterium Isaac Hung been a growing in- Jenna Zhang Mycobacterium tubercu- Chemical and Molecular and terest in develop- losis (Mtb), and is one Physical Biology, Cellular Biology, 2018 ing digital health- 2017 of the deadliest infectious care solutions for clin- diseases still present in Boston Children’s ical, research, pa- Harvard T.H. Chan every part of the world. Hospital tient, and general School of Public The disease is passed from consumer use. In Health person to person through Advisor: John 2015, venture capi- the air; once infected with Brownstein, PhD tal funding for digi- Advisor: Eric Rubin, the bacteria, individuals tal health companies MD, PhD have a 10% lifetime risk of Mentor: Matthew surpassed a stagger- falling ill with TB. How- Murphy ing $4.3 billion in the Mentor: Cara ever, this risk increases US. Boutte, PhD in people with compro- mised immune systems I work with Boston Children’s Hospital’s Innova- such as individuals liv- tion & Digital Health Accelerator (IDHA), whose ing with HIV, malnutrition, diabetes, and other long mission is to shape the future of healthcare by build- term illnesses. This leaves developing countries par- ing new technologies and deploying a comprehensive ticularly at risk, and over 95% of cases and deaths digital health platform. IDHA identifies and vets occur in developing countries in Southeast Asia and SURGH 41

Africa. About one-third of the global population has Understanding HIV Transmission Patterns latent TB. This means that they have been infected in Botswana: Using Evolutionary Analytics by Mtb but are not yet ill and cannot transmit the to Improve Global Health disease because Mtb cell division is a prerequisite to infection. The importance of cell division for infec- Since its emergence tion makes the study of cell division processes and in 1981, HIV has in- their regulation in Mtb vital for stopping the spread Jonathan You fected 78 million peo- Molecular and of TB. As such, understanding the molecular mech- ple and claimed 39 Cellular Biology, anisms of cell division during adaptation to infection 2018 million lives world- stress is important. Many of the factors mediating wide. The deadly cell division are largely conserved from the model or- Harvard T.H. Chan virus, first clinically ganism E. coli to Mtb, although regulation of these School of Public observed in Los An- factors is different between the two. Health geles, began rav- aging Sub-Saharan FtsQ is an essential cell division protein in E. coli Advisor: Max Essex, Africa in the 1990s. that associates with other key cell division factors DVM, PhD By 2000, an un- in a complex known as the divisome that helps link precedented 35% of together major events of cell division. FtsQ has Mentor: Vladimir adults were infected in been shown to be phosphorylated in Mtb, but has Novitsky, MD, PhD Botswana, a middle- otherwise not been extensively studied in mycobac- income country in south- teria. Phosphorylation can modulate growth either ern Africa. by directly controlling the level of protein activity or by affecting the interactions between proteins in a Today, Sub-Saharan Africa remains the region most complex, and previous literature on divisome players affected by HIV. No cure or vaccine has yet been suggest a possible role for FtsQ in growth regula- found for the disease. However, global health ex- tion under oxidative stress. Using Mycobacterium perts, in collaboration with national ministries of smegmatis, a model organism for Mtb, and based health, have begun to tame the HIV epidemic with on studies in E. coli, my work this summer uses nationwide programs for affordable antiretroviral biochemical and genetic approaches to investigate treatment (ART), a safe and effective means of the hypothesis that FtsQ plays an important role restoring normalcy to the lives of HIV-infected pa- in the division complex to regulate cell division and tients. that phosphorylation of FtsQ helps integrate envi- ronmental stress signals into the regulation of the The Essex lab is a key partner in one of these ART division complex. The techniques I am implement- programs. Dr. Max Essex, Chair of the Harvard ing include timelapse microscopy with fluorescently- AIDS Initiative and the Botswana Harvard AIDS tagged FtsQ, affinity pulldowns to identify interac- Initiative Partnership, has worked in Botswana to tor partners of FtsQ, and growth and kill curves to conquer HIV since 1996. Botswana, once the most identify growth defects in mutant strains express- HIV-affected country in the world, has adopted an ing constitutively phosphorylated and unphospho- extremely successful national ART program since rylated FtsQ. In doing so, I hope to clarify the 2002—so successful, in fact, that Botswana now role of FtsQ in cell division and the ways in which leads Sub-Saharan African countries in proportional phosphorylation affects and regulates FtsQ activity. ART coverage of HIV-infected individuals.

My research expands upon an exciting foundational step of the fight against HIV/AIDS in Botswana. To establish a baseline and monitor progress of HIV prevention interventions, researchers in Botswana, including those from the Essex lab, have surveyed thousands of households over several years to de- termine rates of HIV incidence (new diagnoses) and prevalence (total cases), identify new infections for follow-up care, and collect blood samples from those infected.

Like all viruses, HIV-1C, the HIV subtype most prevalent in Botswana, contains a set of genes that encode instructions for building and maintaining itself. These viral genes can be sequenced from HIV- 42 SURGH infected blood samples collected in the household the red cell and may ultimately provide protection surveys above. HIV-1C evolves so rapidly that the from the malaria infection. spread of the virus through a population can be tracked by examining the accumulation of changes Here we aim to investigate the impact of red blood in its genes in HIV-infected individuals over time. cell physiology on the parasite, in particular Plas- The greater the extent of evolutionary relatedness modium knowlesi, one of the five species that cause between two samples of HIV captured from distinct human disease. Specifically, I have been working to individuals, the greater the likelihood that the two understand why P. knowlesi propagation is reduced individuals were infected within the same transmis- in cord blood red cells containing fetal hemoglobin sion chain, a string of related viral infections. (HbF) and in oxidized adult red cells. Other in vitro studies have shown that when Plasmodium My project analyzes genetic sequencing data from falciparum is cultured in cord blood, the levels of a household survey of hundreds of HIV-infected invasion increases; however, growth is retarded. By individuals in Mochudi, a village near the capi- the end of a baby’s first year of life, the HbF levels tal city of Botswana. By employing biostatisti- drop to a few percent compared to 80% at the time cal and phylogenetic analyses, my research inves- of birth. For normal adult red cells it is thought that tigates the evolutionary dynamics of locally circu- oxidative damage to the host changes the erythro- lated HIV-1C lineages. By cross-referencing these cyte cytoskeleton and thus affects the cell membrane patterns with demographic data collected in the and alters normal cell mechanical function in rela- same survey, my project can help identify vul- tion to the parasite. nerable subpopulations, establish factors that af- fect HIV transmission, and, perhaps, inform public I have used microscopy and flow cytometry to assess health interventions. For instance, treatment-as- P. knowlesi proliferation in the red cells I’ve been prevention—calming active infections with ART to studying. Thus far I have observed that P. knowlesi reduce risk of further transmission—is considered a proliferation is reduced in both cord and oxidized red front-line intervention in resource-limited areas, but cells. To identify the specific parasite factors respon- this method is best suited for regions where HIV sible for reduced proliferation in cord blood, I have transmissions generally occur within an isolated set been attempting to adapt P. knowlesi to cord blood of ART-treated communities. If transmissions more by continuously culturing P. knowlesi in cord blood. frequently occur between ART-treated and ART- When I observe normal proliferation of P. knowlesi untreated populations, then a more comprehen- in cord blood, I will sequence the adapted parasites sive, prophylaxis-based approach may be required. to identify genetic mutations responsible for normal proliferation. I have additionally been employing an osmotic fragility assay to investigate the hypothesis The Effect of the Host Red Blood Cell on that P. knowlesi osmotic stability may be compro- the Malaria Parasite mised in cord and oxidized red cells. Finally, to test Malaria remains a sig- our hypothesis that reduced growth may be caused nificant life-threatening by premature lysis of the Plasmodium-infected red Julianna Lee disease, affecting over two blood cells (RBC) as a mechanism of host medi- Molecular and hundred million individu- ated protection from the malaria infection I have Cellular Biology, 2019 als yearly. It is caused by been working with the parasites’ genetics. I can use the Plasmodium parasite, NanoLuc, a reporter protein, to help monitor RBC Harvard T.H. Chan which is transmitted from lysis as described in a previous assay. School of Public infected to uninfected in- Health dividuals by mosquitoes. As malaria continues to place a large burden on The blood stage of a public health, studies like these can help reach Advisor: Manoj malaria infection is re- the potential of revealing novel mechanisms of Duraisingh, PhD sponsible for all symp- red cell mediated protection against malaria infec- toms of disease. Previous tion. Such findings have the potential to ultimately Mentors: Martha studies have revealed that help reach groundbreaking therapeutic interventions. Ann Clark, PhD; naturally occurring red Brendan Elsworth, blood cell variants in the PhD human population such as the sickle cell trait af- fect the behavior of the malaria parasite. These variants change the para- site’s ability to propagate and cause disease in the host, either by blocking invasion or growth within SURGH 43

Chaperone Independent Type III Effector effectors is independent of the known chaperones. To Secretion in Shigella flexneri screen for any potential undiscovered chaperones, we took two approaches. First, we examined CI-effector Shigella flexneri, secretion in the Minimal Type Secretion System a Gram-negative rod- (minT3SS) that lacks all the unknown ORFs. The Kristen Fang shaped bacterium and minT3SS, coded for by the entry region (~31kb) on Molecular and the causative agent of the virulence plasmid, only consists of the Type III Cellular Biology, 2019 bacillary dysentery or Secretion Apparatus known chaperones, and a few shigellosis, affects an es- effector and regulatory proteins. The entry region Massachusetts timated 90 million annu- of the virulence plasmid was isolated onto a plasmid General Hospital, ally worldwide, with at referred to as pSER2 for Shigella Entry Region 2. Harvard Medical least 100,000 fatal cases. The introduction of this plasmid into Escherichia School S. flexneri contains a vir- coli results in T3S competent E. coli (mT3 E. coli). ulence plasmid (>200kb) Secondly, we obtained a collection of twenty-five S. Advisor: Cammie that codes for a Type III flexneri strains, each with an unknown ORF re- Faith Lesser, MD, secretion system (T3SS), moved. We ran high-throughput secretion assays PhD a complex, syringe−like on the mT3 E. coli strain and the S. flexneri ORF nanomachine that de- knockout strains. In both approaches, there was no Mentors: Nadja livers effector proteins significant difference in CI effector secretion, sug- Ernst into the cytoplasm of hu- gesting that there are no undiscovered chaperones. man gastrointestinal cells. Together, these two strategies add evidence to the There are 25–30 known ef- existence of T3S chaperone-independent effectors. fector substrates of this system that together enable S. flexneri to manipulate host cellular processes over Early Child Development in Rwanda the course of an infection. There is currently no vaccine available to prevent shigellosis, and little is Early childhood de- known about the secretion pathways of T3S effec- velopment (ECD) inter- tors. Increased understanding of the T3SS in S. Kristy Hong ventions can significantly East Asian Studies, flexneri is a step forward in the process of devel- impact the lives of vul- 2017 oping a drug that targets the secretion system not nerable children through only in Shigella, but in other T3SS species including Harvard T.H. Chan enhancing family envi- Salmonella (typhoid fever), Yersinia (plague), and School of Public ronments and improv- Chlamydia (sexually transmitted disease). Health ing nutrition- and health- related practices. The Past research suggests that T3S effectors are defined Advisor: Theresa Government of Rwanda by an N-terminal secretion sequence. Some effectors Betancourt, ScD (GOR) has identified also have a chaperone-binding domain (CBD) that is ECD services as a key recognized by a T3S chaperone that likely plays a role Mentor: Rose pillar of the country’s in the hierarchy of effector delivery. More than 50% Wilder, MA flagship social protec- of S. flexneri effectors do not bind to a known chap- tion strategy, Vision 2020 erone. For these putative chaperone-independent Umurenge (VUP); this (CI) effectors, it is unknown what defines them as mandate falls under the Ministry of Local Govern- T3S secreted substrates other than their N-terminal ment (MINALOC). The overall social protection secretion sequences. Our project focuses on the se- policy focuses on complementing income support cretion pathway of chaperone-independent T3S effec- with cross-sectoral collaboration to break intergen- tors. We took two strategies: first, we investigated erational cycles of poverty and invest in Rwanda’s if secretion of putative CI effectors is dependent future growth and prosperity. The recently revised on known chaperones; and second, we investigated and approved ECD policy in May 2016 also states whether any of the annotated virulence-plasmid en- cross sectoral collaboration to ensure Rwandan chil- coded genes (open reading frames, ORFs) of un- dren reach their full potential by focusing on the known function act as a previously undiscovered crucial years of childhood development; this policy is chaperone. implemented by the Ministry of Gender and Family Promotion (MIGEPROF). S. flexneri strains, each of which carry a chaper- one knockout (∆ipgA, ∆ipgE, and ∆spa15 ) were Dr. Betancourt’s team has developed the Family tested in a high-throughput assay for secretion of Strengthening Intervention for Early Childhood De- CI effectors. There was no significant difference in velopment (FSI-ECD), a home-visiting, parenting secreted levels, suggesting that the secretion of these intervention aimed at families with young children 44 SURGH living in extreme poverty. The program consists prevention, treatment, and testing efforts. However, of a community-based volunteer “coach” visiting a as the average prognosis of those living with HIV household to engage the family in a series of fifteen has improved, sexual health education has declined. structured modules. These include modeling positive It is frequently inadequate, outdated, or completely parenting, presenting alternatives to harsh punish- nonexistent. Currently, there is a chlamydia epi- ment against children, promoting the involvement of demic among teens in Boston, and rates of sexually fathers, and providing prescriptive knowledge about transmitted infections have been rising nationally brain development, nutrition, health, and hygiene. over the past decade. The increased STI rates among adolescents coupled with the disparities and stigma The team used mixed methods—qualitative and that still surround HIV point to a need for compre- quantitative data collection—to test the program’s hensive sexual health education that incorporates effectiveness in select districts in Rwanda with recent changes in HIV/STI treatment, prevention, bachelor-level home-visiting “coaches.” Currently, and attitudes, particularly for underserved youth. the team is gearing up for a full scale trial to test the added value of the FSI-ECD home-visiting program In response to these issues, our research aims to with VUP public works services by evaluating both develop and evaluate a modernized, relatively short relative effectiveness and cost effectiveness. With an HIV and STI prevention intervention for Boston array of tested metrics, they plan to measure the adolescents. Among other recent changes, the in- added value of the FSI-ECD model combined with tervention addresses the development of rapid STI Rwanda’s flagship VUP public works program com- testing, HIV prevention medication, and prevailing pared with the public works program alone. The societal myths, new and old, about HIV and STIs. team is currently adapting the original manual for Additionally, it aims to address mistaken perceptions use by lower literacy community based volunteers in that HIV is a disease of the past, is no longer worth order to scale the program. preventing, or that certain groups are destined to contract HIV. We developed our intervention after By working with the GOR and development conversations with key stakeholders in the Boston partners, the data have the ability to influ- area, including public health officials, educators, ence future policy that emphasizes the im- community health workers, and individuals living portance and relative cost of incorporating with HIV. We also conducted a literature review on ECD services in social protection policies. theories of health behavior change. Our intervention is based on the Social Cognitive and Information- Motivation-Behavior theories, which stress the im- Improving HIV Prevention and Sexual portance of increasing a sense of self-efficacy and mo- Health Education for Adolescents tivation among participants in health education. In Over the past two the next few weeks, we will carry out our intervention and a half decades, the with groups of Boston adolescents and use pretests Laura Kanji HIV/AIDS landscape has and posttests along with focus groups to analyze Undeclared, 2019 changed dramatically. An its effectiveness. This evaluation will hopefully re- HIV diagnosis is no longer veal whether our educational methods were effective Center for AIDS Research, Harvard a death sentence; instead, in improving participants’ knowledge, confidence in University with the rise of highly ac- their ability to make and carry out decisions related tive antiretroviral ther- to sexual health, and attitudes towards STI testing Advisor: Wanda A. apy, HIV has become a and HIV, and will inform future prevention efforts. Allen chronic and manageable condition. However, HIV Mentors: Sannisha is not yet a disease of the Dale, PhD, EdM; past. It still affects peo- Valerie Earnshaw, ple of color and LGBTQ PhD people disproportionately, and these disparities have not changed over the past thirty-five years. While HIV medication is highly effective when taken as prescribed, it can have seri- ous side effects, and systemic issues surrounding the health care system—including poverty, education, and race—pose obstacles for adherence. Finally, there is stigma attached to sexually transmitted in- fections, particularly HIV, that proves detrimental to SURGH 45

Purification of WD40 via IPTG, and nickel column purification. After the purified protein is obtained, we will be able to Artemisinins are a engage it in further study to analyze functionality. category of antimalarial If all goes well, by the end of this process, we will Michael drugs commonly thought better understand if, and how, changes in WD40 Kennedy-Yoon of as frontline treatment confer resistance, and will be able to recommend Visual and Environmental for the disease. Their use courses of action in cases of artemisinin resistance. Studies, 2017 is widespread, and gen- erally effective at coun- Quantifying Public Sentiment of Healthcare Harvard T.H. Chan teracting malaria. It is Sector Organizations School of Public therefore troubling that Health there is an observed re- Since the dawn of the sistance to artemisinins digital age, both the rate Advisor: Dyann in several strains of Patrick Tan of information production Wirth, PhD Statistics, 2018 the plasmodium parasite and information collec- more commonly known tion have grown expo- Mentor: Allison as malaria, and specifi- Boston Children’s Demas Hospital nentially, giving organiza- cally in Plasmodium falci- tions and individuals ac- parum, the deadliest of cess to vast amounts of the five most common Advisor: Carl Brinton, MBA data. Within this con- subspecies. It is this concern that motivates our text, there has been a study of several factors that may confer immunity corresponding need to de- to artemisinins, including several single nucleotide velop efficient methods to systematically parse and polymorphisms (SNPs), which are one-base-pair mu- summarize these large quantities of data. One tech- tations in the DNA code of an organism, in the nique of interest is the concept of “sentiment anal- WD40 region of the Pf Coronin protein. Coronin is ysis,” which involves the use of natural language thought to be an actin binding protein, and WD40 is processing and machine learning to extract qualita- a large region within coronin that is dubbed a “pro- tive information from a body of text. This tech- peller region.” It is our goal to synthesize and purify nique has proved effective in a wide variety of ap- this WD40 region, in both wild type and mutant plications, ranging from the analysis of social media forms, in order to better study and understand how posts to classifying consumer reviews. Within the these SNPs may confer artemisinin resistance. healthcare sphere, where the focus is primarily on healthcare sector enterprises and startups, there is We began by creating a bacterial plasmid that con- a particular interest in determining the general sen- tained the WD40 protein code. A plasmid is a piece timent surrounding these healthcare organizations. of circular DNA, specific to prokaryotes, into which Armed with this information, a host of potential we can add genes to be expressed by the prokary- interest groups in the healthcare sphere (investors, otes. We then inserted the plasmid into E. coli, in partners, customers, suppliers, etc.) would be able to order to facilitate protein expression of the wild-type make better decisions with regards to these health- WD40. Most recently, we have attempted to induce care sector organizations (which ones to invest in, protein expression via IPTG induction. Our current which ones to support, etc.) Thus, the goal of this efforts are centered around the verification that pro- project is to develop an efficient, quantifiable met- tein expression has occurred. To this end, we are ric to summarize this sentiment. To accomplish running gels of samples of cells taken at different this, we train a machine learning algorithm to rec- times during the induction process, which will al- ognize positive and negative sentiment surrounding low us to determine whether or not the protein was a healthcare organization of interest within news ar- successfully expressed. If this process results in the ticles about the organization. We gather news ar- affirmation of protein presence, we will attempt to ticles that have preceded significant movements in purify the protein using a nickel purification column. the stock price as training examples, since these In the protein there is a negatively charged histidine news articles are likely to contain the strongest posi- tag that will hypothetically allow for the selective tive or negative sentiments. Finally, the algorithm extraction of the expressed protein using the posi- then takes in the wealth of recent news about a tively charged nickel ions. certain company and outputs a “sentiment score,” which represents our desired quantifiable metric Given that all of these measures are successful, we of general sentiment surrounding the organization. will proceed to expression of the mutant WD40 pro- teins. The procedure here is largely the same, with transformation of the plasmid, induced expression 46 SURGH

Quality Improvement of Congenital Heart communicable diseases such as congenital heart dis- Surgery in Developing Countries ease in developing countries are accounting for a greater percentage of deaths in childhood and must The International be addressed in health initiatives. By comparing Quality Improvement development indexes to the outcome data of IQIC, Sarah Rahman Collaborative for Congen- I seek to discover the relationship between devel- Sociology, 2018 ital Heart Surgery in De- opment stage and surgical outcomes. This can help veloping World Countries tailor quality improvement and investment strategies Boston Children’s Hospital (IQIC) collects surgical for the sites. outcome data from forty- Advisor: Kathy seven sites in twenty- Jenkins, MD, MPH two countries worldwide. Congenital heart surgery Mentors: Kate in resource-deprived areas Doherty; Jennifer is difficult and has high Kupiec, MPH mortality rates. IQIC aims to improve surgery practices in these regions through collaboration with local sites. IQIC deter- mined three key drivers to reduce mortality: safe pe- rioperative practice, infection reduction, and team- based practice (nurse empowerment). I worked in many facets of IQIC, with the overall mission to im- plement these drivers in the participating sites and improve surgical outcomes. I prepared for monthly teaching webinars focusing on these key topics that are broadcasted to the sites and discussed among surgeons, nurses, and staff. In addition to webinars, IQIC connects with partner sites to develop regional learning sessions, which nearby sites can attend in person, participating in presentations, simulation sessions, and panel discussions on quality improve- ment.

IQIC conducts annual site visits to meet with the local teams, perform data source verification, and to reinforce IQIC goals. Each year, annual benchmark- ing reports are provided to sites, detailing their out- comes and comparisons to the overall collaborative. I contributed to these reports through describing each site visit and organizing data, which sites can then use to understand their outcomes and areas for improvement. In addition to the surgery dataset, I am assisting with new IQIC initiatives: creating a new database on cardiac catheterization aimed to gather information on a procedure that has not been well documented in developing countries, as well as developing a pilot infrastructure assessment survey designed to evaluate the equipment and physical spaces available for surgery at the sites.

Furthermore, I am working on a global develop- ment project with IQIC. I plan to link IQIC con- genital heart surgery outcome data to World Bank global development indicators to understand the role of congenital heart disease in the global health agenda. The global health landscape has been chang- ing: as poverty and infectious disease decrease, non- PRISE Astronomy and Astrophysics

Kinematic Modeling of Oxygen-Rich Searching for Earth-Like Exoplanets using Supernova Remnant N132D the Radial Velocity Method A supernova remnant The first confirmed ex- (SNR) is the resulting oplanet observation was Charles Law structure left behind af- Chris Dolliff recorded in 1988; since Physics, 2017 Chemistry and ter the explosion of a then there have been Physics, 2018 star in a supernova explo- 3,443 confirmed observa- Harvard-Smithsonian Center for sion. SNRs are bounded Harvard-Smithsonian tions of exoplanets. There Astrophysics by an expanding shock Center for are several ways to ob- wave and are composed of Astrophysics serve the presence of an Advisor: Dan ejected material expand- exoplanet orbiting a dis- Milisavljevic, PhD ing from the explosion as Advisor: David tant star, but because it is well as interstellar mate- Phillips, PhD extremely difficult to op- rial swept up in the pro- tically resolve these plan- cess. A small subset of young SNRs, known as ets, almost all methods in- “oxygen-rich” SNRs, in which interior fragments of volve measuring the host star’s behavior. In a solar the supernova progenitor star are exposed to direct system, all bodies are orbiting around the center of investigation, allow for an opportunity to make direct mass of the system. This means that a star with an tests of stellar evolution and nucleosynthesis models orbiting planet will move with respect to an observer of massive stars. We have used the Inamori Magel- on Earth as it wobbles around the center of mass of lan Areal Camera and Spectrograph (IMACS) on the its system. This wobble can be detected by mea- 6.5 m Magellan telescope to map the [O III] λλ4959, suring the Doppler shift in the star’s emission spec- 5007 Å, emission-line dynamics of the oxygen-rich trum. By measuring these oscillations, we can de- SNR N132D in the Large Magellanic Cloud, a nearby tect the presence of a planet and determine its mass. satellite galaxy of the Milky Way. From the ac- This technique is called the radial velocity method. quired spectra, we were able to reconstruct an in- This method is applicable to many planetary sys- teractive, 3D kinematic map of N132D, which al- tems. When combined with methods that can only lows for a detailed study of the remnant’s internal determine the planet’s volume (most commonly the structure, particularly ejected material in the form transit method) we can determine the planet’s gen- of oxygen-rich filaments and knots. While previ- eral structure (rocky, gas giant, ocean planet, etc.). ous attempts at reconstructing the 3D structure of N132D have been hampered by insufficient data qual- The idea of finding exoplanets by measuring radial ity, our observations allow for a detailed study of velocities has been successful for almost thirty years. the remnant’s kinematics. Since the trajectory of The method excels at finding heavy, quickly orbiting, ejected material in a supernova explosion is ballis- and nearby exoplanets. The method struggles, how- tic in nature, the spatial and kinematic configura- ever, to find planets with Earth-like mass and orbital tion of heavy-element ejecta in a particular SNR period with the current spectroscopic technology and provides a sensitive measure of its age. Thus, in stellar noise theory. The best radial velocity mea- addition to probing explosion dynamics and nucle- surements can detect signals with amplitudes as low osynthetic yields, an improved understanding of the as ~1 m/s, which is not good enough to find plan- inner structure of N132D will allow us to estimate ets with Earth-like masses and orbits. For that, the a more accurate age for the SNR, a quantity that theory predicts a ~10 cm/s signal amplitude. My lab has, to this point, remained somewhat uncertain. has spent the last nine years setting up a high preci- sion laser comb that can be used to calibrate a spec- trograph to reach the required level of spectroscopic error. The lab’s goal is to use its solar telescope in the Canary Islands to observe the transit of Venus around the Sun by measuring the Sun’s radial veloc- ity. We test this technique on the Sun because there are not currently good enough models of the noise in spectroscopic data from Sun-like stars, even with the best calibration, to get 1 cm/s signals from point source stars. Thus, we wish to use our knowledge of the Sun and ability to resolve its surface to minimize Astronomy and Astrophysics 49 the error in a way that can be applied to other stars. Exploring the Dark Matter Content of Nearby Elliptical Galaxies My role in the project has been to analyze possi- ble sources of error in our measurement of the so- Exploring the evolu- lar spectra. I used Python to analyze a number tion of galaxies through- Oliver Philcox out cosmic time is of possible sources of error including the mounted Astrophysics, 2017 paramount in modern as- guide that keeps our telescope centered on the sun, Emmanuel College some strange behavior in images that cameras on trophysics. It is believed either side of the telescope take every five min- Harvard-Smithsonian that galactic development utes, and high temperature variance inside the tele- Center for hinges on three major scope. As I do this, part of the goal of the de- Astrophysics components: the central velopment of these tools is to produce a more ro- supermassive black hole bust diagnostic infrastructure to detect malfunctions Advisor: Akos with a mass millions of of the telescope quickly and easily as they happen. Bogdan, PhD times that of our sun, the stellar body, and the dark matter halo. Dark matter is a hypothetical type of matter that does not in- X-Ray Emissions from L, T, and Y Type teract with normal matter, except through gravity, Brown Dwarfs and there is strong experimental evidence to sug- gest that it makes up 85% of the total mass of the Brown dwarfs are sub- universe. The specific symbiosis of these three com- stellar objects that are ponents likely results in the demography of galaxies Deanna Emery not massive enough for observed across the Universe, and a study of the dark Physics & the nuclear fusion of hy- matter content in nearby elliptical galaxies can shed Astrophysics, 2017 drogen, necessary in main new light into how they are linked together, which can be used to improve models of galactic evolution. Harvard-Smithsonian sequence stars; however, they are more massive Research of this variety has been attempted before; Center for however, it was on a much smaller scale, consider- Astrophysics than planets, exceeding at least thirteen times the ing only seven galaxies rather than thirty, due to the smaller number of images previously available. Advisor: Akos mass of Jupiter. Brown Bogdan, PhD dwarfs are separated into Thus, its utility was far more limited. spectral types M, L, T, and Y, with M being the warmest type, and Y being the coolest. The spec- tral classes L, T, and Y, specifically, are cool enough that they are only supposed to emit in low energy wavelengths. However, in recent years, these brown dwarfs have been observed emitting in high-energy X-ray flares. The mechanisms that cause these brown dwarfs to flare are not yet understood. For this project, I am going through XMM-Newton and Chandra X-ray point source catalogs of large sky sur- veys containing 396,910 and 241,074 sources, respec- tively, in order to determine if any of the observed X-ray sources might be from brown dwarf flares. This is done by using Python code to correlate the coor- dinates of X-ray emissions with the coordinates of known brown dwarfs, while accounting for the proper motion of the brown dwarfs. I will present the results Figure 1: Galaxy M49. of this cross-correlation between catalogs and at- tempt to offer some insight on the emission of X-rays This project has focused primarily on calculating the from brown dwarfs in spectral classes L, T, and Y. gravitating mass profiles of a large sample of nearby elliptical galaxies, through use of X-ray images from the Chandra satellite. Launched in 1999, Chandra is the most powerful X-ray telescope made to date, capable of over 100 times greater resolution than its predecessor and currently in orbit 50,000 miles above the Earth. Shown above is an X-ray image of 50 Astronomy and Astrophysics the galaxy M49, located 56 million light-years from of possible sources of external interference as well as the Earth, and the code used in this study was ini- a variety of instrumental effects. tially written to analyze this, before being extended to other galaxies. The first part of my research involves developing a data acquisition system to measure radio frequency In order to find the galactic mass profile, the im- interference from hand-held radios and satellite com- ages had to be analyzed using a variety of methods. munications at the South Pole. I write software to Firstly, data from solar flares, which occur at peri- acquire, visualize, and analyze the data from Signal- ods of drastically increased solar activity, had to be Hound spectrum analyzers which continuously mon- removed, as did the point sources seen across the im- itor the radio spectrum from 100 kHz to 12.4 GHz. age. I was then able to extract spectra of the hot These devices operate simultaneously with the tele- gas in the galaxies at a range of radii, and, by fitting scopes to help account for potential external inter- established models to this, could create gas density ference. Alongside my work on the data acquisition and temperature profiles for the galaxy. These were system, I create a publicly accessible web browser then used to compute the total mass profile as a func- which allows for the download and analysis of these tion of radius, due to both dark matter and stellar time-stream spectrum data. material. Having obtained these data, they could be combined with the readily available measurements The second part of my research includes develop- of stellar mass and supermassive black hole mass in ing a generalized suite of Linux-based motion con- order to probe whether supermassive black holes ex- trol software for positioning stages which are used in hibit tighter correlations with their stellar or dark various calibration apparatus. The software is gen- matter halo masses. This project will contribute sig- eral enough to be used for near-field beam mapping, nificantly to the development of astrophysical under- Fourier transform spectroscopy, and controlling a ro- standing of galactic evolution. tating polarized source. These tools allow for the processing of instrumental polarization data which are essential to the integrity of the sensitive mea- The Design and Implementation of Data surement of CMB polarization. Acquisition and Control Systems in the Detection of B-Mode Polarization in the Cosmic Microwave Background The cosmic microwave background (CMB) is the Thomas Culp oldest light in existence, Physics, 2019 the relic radiation from the Big Bang that birthed Harvard-Smithsonian Center for the universe. The rela- Astrophysics tive isotropy of the CMB supports the theory of in- Advisor: John Kovac, flation, an exponential ex- PhD pansion of the universe during the first 10−32 sec- Mentor: Kirit onds of its existence. In- Karkare flation predicts the pro- duction of gravitational waves, which would leave their trace on the CMB in the form of a B-mode (curl) polarization pattern, which could be detected if inflation took place at energy scales predicted by Grand Unified Theories.

The detection of B-mode polarization depends on in- strumentation capable of measuring signals on the scale of nanokelvin, making the detectors which do this work the most sensitive of their kind. BICEP3 and the Keck Array are microwave telescopes which observe CMB radiation at the South Pole in search of this inflationary signature. Ensuring accurate mea- surement of the CMB requires precise measurement Biomedical Engineering and Science

The Role of T Cell Phenotype on Endothelial each condition. To study the role of T cell phenotype Cell Sprouting and Skeletal Muscle Stem on muscle regeneration, T cell-conditioned medias Cell Proliferation and Differentiation were applied to satellite cells isolated from mouse hind limbs. Proliferation and differentiation assays Musculoskeletal dis- were performed, followed by immunohistochemistry eases affect more than staining and confocal microscopy. In the prolifera- Erica Budina six million Americans an- Engineering Sciences, tion assays, satellite cells were stained for EdU, a nually. To improve mus- 2018 nucleotide analog that is incorporated into the DNA cle function following in- when cells are undergoing mitosis. In the differentia- jury or disease, therapies Wyss Institute for tion assays, cells were stained for myosin heavy chain, Biologically Inspired must be developed that a mature muscle cell differentiation marker. Prolif- Engineering promote both angiogene- eration was assessed by determining the percentage sis (the formation of new of EdU-positive cells and by measuring the intensity Advisor: David J. blood vessels from pre- of the EdU signal. Differentiation was assessed by Mooney, PhD existing ones) and mus- determining the percentage of myosin heavy chain- cle regeneration. Mus- positive cells and by calculating the fusion index for Mentor: Brian Kwee cle regeneration involves each myosin heavy chain-positive cell. the activation of a quies- cent population of skeletal Preliminary results from the aortic ring assays sug- muscle stem cells called satellite cells; upon injury, gest that Th2 conditioned media promotes endothe- these cells proliferate, migrate to the site of injury, lial cell sprouting, while Th1 conditioned media in- and eventually differentiate and fuse, forming new hibits sprouting. However, results from the skele- myofibers. tal muscle stem cell proliferation and differentia- tion assays suggest that Th1-conditioned media en- Previous research in the Mooney laboratory has hances skeletal muscle stem cell proliferation and demonstrated that the combined delivery of growth inhibits their differentiation. Overall, these stud- factors known to promote both angiogenesis and ies suggest that a combination of pro- and anti- myogenesis has the potential to reverse muscle in- inflammatory T cell cytokines may be necessary jury caused by tissue ischemia. However, the ability to dually promote angiogenesis and myogenesis. of these therapies to promote regeneration in is- chemic injury has been shown to depend on the presence of immune cells. This research work inves- Fabrication and Testing of a Novel + tigates the role of CD4 T cells in modulating these Therapeutic Dental Material processes. In the long term, it seeks to understand whether enhancing the number and type of T cells Over one billion dental at sites of ischemia can dually promote angiogenesis fillings are performed an- and myogenesis. In the short term, this research Jacob Scherba nually, but presently-used Engineering Sciences, work explores how the cytokines secreted by mouse composite resin filling ma- 2018 T cells differentiated into Th1, Th2, Th17, and Treg terials are toxic to cells. phenotypes affect endothelial cell sprouting in aortic Wyss Institute for If a filling is unsuccessful, ring slices, as well as satellite cell proliferation and Biologically Inspired it could necessitate more differentiation in vitro. Engineering painful and more expen- sive procedures like root To address this research question, CD4+ T cells were Advisor: David J. canals or extractions. The isolated from adult mice and differentiated into Th1, Mooney, PhD introduction of a reliable, Th2, Th17, and Treg phenotypes. After T cell phe- biocompatible dental ma- notype was verified via FACS, conditioned media Mentors: Kyle terial, therefore, would be was collected from each cell type. To study the role Vining, DDS; Adam of great clinical signifi- of T cell phenotype on endothelial cell sprouting, Celiz, PhD cance. In this study, we T cell-conditioned medias were applied to an aortic aim to develop a material ring assay. Aortic ring slices were then fixed, fluores- that is not only biocom- cently stained, and imaged via confocal microscopy. patible, but also therapeutic, to restore teeth to Endothelial cell networks were quantified using An- health. In situ curing of bulk monomers results giotool software; specifically, the average vessel di- in a polymer that may prove able to induce dif- ameter, length, and lacunarity were determined for ferentiation of dental pulp stem cells (DPSCs) into 52 Biomedical Engineering and Science odontoblasts that produce dentin, the mineralized Engineering Probiotic Bacteria for In Vivo component of the tooth. DPSCs were cultured on Tracking in the Gut polymer-coated tissue culture plastic (TCP) in or- der to determine the effect of the polymer on cell The gut microbiome growth and viability. High-throughput screening us- is composed of bacteria Jessica Kim that have a symbiotic re- ing polymer microarrays identified these polymeric Chemistry and lationship with the hu- materials. The best-performing polymer facilitated Physics, 2019 cell growth levels comparable to the TCP control and man body, helping to was thus chosen as the lead candidate biomaterial Wyss Institute for keep the immune sys- for use in in vivo studies. A differentiation study was Biologically Inspired tem healthy. Because performed to evaluate the level of DPSC differentia- Engineering gut microbiomes differ for tion on our materials compared to controls. DPSCs all people, studying and were cultured in osteogenic differentiation media for Advisor: Neel Joshi, imaging the gut micro- three weeks on our material, and qPCR analysis will PhD biome has become cru- be used to quantify gene expression of key protein cial in developing person- markers associated with osteogenic and odontoblas- Mentor: alized medications. Thus, Noemie-Manuelle tic differentiation. A cell adhesion time-course ex- the goal of this project Dorval Courchesne, is to create engineered periment was conducted to explore the mechanism of PhD DPSC adhesion to our material. Cells were counted bacteria similar to those and imaged over a 48-hour time period, and RNA that already live in the was collected. We hope to identify differentially ex- gut that could be ingested pressed genes involved in adhesion to our materials and tagged, so that the bacteria can be tracked as compared to TCP-positive control and Bis-GMA, they move throughout the gut. the monomer of composite resin dental fillings, as a negative control. To study the in vivo performance of In this study, a probiotic strain of Escherichia coli our material, we mechanically injured rat maxillary was chosen as the candidate bacteria because it can molars in a dental pulp injury model and implanted colonize and survive the harsh gut environment. Our our polymer, compared to existing filling materi- task was to then engineer this microbe so that it als. The polymer will remain in the tooth for four could be tagged and targeted with an imaging probe. weeks, at which time the tooth will be extracted. Previously, protein tags, functional peptides, or bind- We will perform histology, immunohistochemistry, ing domains have been introduced as appendages to and micro-computed tomography imaging of these these bacteria. This study aims to track probiotic to reveal the biocompatibility of our material as well bacteria using a novel probing system. as quantity of dentin at the injury site. This would confirm our in vitro findings that our therapeutic Because the gut environment is highly competitive material allows for the growth and differentiation of and many microbes must share a limited amount DPSCs. Our material, therefore, could have con- of nutrients to survive, we wanted to ensure that siderable implications for therapeutic dentistry and these engineered microbes could survive in this envi- may enable the regeneration of decayed dentin. The ronment while simultaneously acting as an imaging insights of this study unlock numerous possibilities probe. To test for fitness, we performed growth rate in dental care, but so too does the mechanism of experiments for these engineered microbes and com- our material urge the consideration of applying sim- pared their growth to that of their non-engineered ilar materials in other body sites for regeneration. counterparts. We used doubling time, the amount of time required for the number of cells in a cul- ture to double, as a proxy for organismal fitness. In addition, our subsequent genetic engineering ef- forts suggested that our engineered microbes could be selectively tagged with an appropriate functional near-infrared dye. Overall, both the normal growth of our engineered microbes, and their ability to be tagged suggest that, in the future, these engi- neered microbes would be prime candidates to al- low for in vivo tracking of bacteria in the gut. Biomedical Engineering and Science 53

Methods for CRISPR Library Design and In sequence into the host genome. Furthermore, trans- Vivo Delivery of Gene Editing Proteins fection tools for mediating this delivery show lit- tle efficacy in vivo. Here we design several meth- The development of ods for in vivo delivery, providing a plasmid-free CRISPR (clustered reg- way to use CRISPR-Cas9 with reduced off-target Joshua Meier ularly interspaced short effects. We envision that our methods will fa- Chemistry & palindromic repeat)- cilitate new applications of in vivo gene editing. Computer Science, 2018 associated Cas9 nucle- ase has enabled new Broad Institute of methods for interrogat- Thericardium: A Replenishable Reservoir for MIT and Harvard ing gene function. With Targeted Cardiac Therapy just a simple alteration Advisor: Feng of the single-guide RNA Cardiovascular disease Zhang, PhD (sgRNA) or direct repeat (CVD) is one of the (DR) sequence, one can Keegan Mendez leading causes of morbid- Biomedical Mentors: Neville reprogram a nuclease to ity and mortality world- Engineering, 2017 Sanjana, PhD; Ian target different sites in the wide. Myocardial infarc- Slaymaker, PhD genome. Genome-scale Wyss Institute for tion, one of the main CRISPR-Cas9 knockout Biologically Inspired clinical presentations of libraries have emerged as Engineering CVD, is responsible for popular tools for unbiased, phenotypic screening, the death of over 120,000 but technologies for library design in specific appli- Advisor: Conor people each year in the cations have lagged behind. Furthermore, in order to Walsh, PhD United States alone. Cur- fully realize the therapeutic potential of gene editing rent therapies for myocar- proteins and study their function in living organ- Mentor: William dial infarction, such as isms, new methods for in vivo delivery are urgently Whyte coronary artery revascu- needed. larization techniques, are effective at restoring lost Here we describe CRISPR Library Designer (CLD), blood flow to the heart; however, they often cause a web application for automated, tissue-specific li- residual myocardial scarring that patients are left brary design, and report on delivery methods that with permanently. Elimination of myocardial scar- can efficiently deliver Cas9-sgRNA nuclease com- ring and restoration of full cardiac function after a plexes in vivo. We then apply gene editing methods heart attack could eliminate the cascade of events to study the development of the mouse brain in vivo. that eventually lead to heart failure. At present, there is an absence of effective clinical therapies in CLD provides a complete pipeline for the design of this domain. Simple cell delivery to the infarcted custom sgRNA libraries for use in a wide variety heart has demonstrated promising results, especially of applications. It uses fast algorithms to search in a laboratory setting. However, improved patient for genome-wide sgRNA binding sites across an as- outcomes are limited by suboptimal localization of sortment of genes. Furthermore, it integrates tissue cells to the pathological site, low viability and poor expression data from the GTEx Consortium and on- engraftment efficiency. Clinical translation of cell target scores from Microsoft Azimuth to optimize therapy is dependent on new strategies to enable cells guide selection for specific tissue-based experiments. to survive and integrate within the harsh environ- When possible, CLD targets functional protein do- ment of the infarcted heart and exert therapeutic mains based on Pfam data, as mutagenesis of func- benefit over extended periods. tional protein domains leads to a higher proportion of null mutations and enhanced severity of nega- To address these challenges, we previously devel- tive selection. CLD can design optimized libraries oped an advanced delivery system entitled “Theri- in seconds and provides intuitive customization fea- cardium,” which enables the direct and controllable tures at each design stage for fine-tuning of the administration of therapeutic agents (cells, small libraries. For instance, to design a library targeting molecules, proteins) to the heart via a therapeutic 500 chromatin-specific genes with 6 guides per gene reservoir. The reservoir is implanted on the surface requires 8 seconds for the initial algorithmic gener- of the heart and can be replenished in a non-invasive ation, followed by an optional 10 minutes of user manner through an implantable conduit connected interfacing to fine-tune the library. to a subcutaneous port. This approach shows great promise. However, an adaptive immune response and Plasmid-mediated delivery of Cas9 into cells can subsequent cell death create a toxic biomaterial mi- result in uncontrolled integration of the plasmid croenvironment, hindering further replenishments. 54 Biomedical Engineering and Science

In the present study, we refined the therapeutic reser- mesh, which could result in stroke or heart attack. voir to maximize transplanted cell retention and sur- Anticoagulants are generally prescribed to prevent vival, and to enable protection from the immune re- this, but such a therapy may cause adverse side ef- sponse. The proposed reservoir is composed of a bio- fects for patients with other health issues, like risk material core with a cellular payload, surrounded by for internal bleeding. an outer impermeable soft shell, and separated from the heart tissue by a synthetic semipermeable mem- A SLIPS stent coating could eliminate these side brane. The outer shell, made from thermoplastic effects by creating a slippery drug-infused layer over polyurethane, is designed to provide structural sup- the stent mesh so that clots are unable to form. port, while preventing cell loss caused by the mechan- Previous testing of SLIPS confirms their anticoagu- ical disruption of a beating heart. The semiperme- lant properties; however, it is still unknown whether able polycarbonate membrane is engineered to allow the roughened matrix of SLIPS is able to release for the bidirectional movement of oxygen, nutrients, drugs critical to preventing arterial clogging and and cellular proteins, but prevents the inward migra- maintaining blood flow. During my research, I will tion of immune cells and antibodies responsible for be investigating the kinetics of drug release from the immune rejection of allogeneic cells. The trans- a SLIPS anti-coagulant coating to ensure that it planted cells, protected against the host immune re- can deliver the same medicinal benefits as the cur- sponse, may then release the bioactive factors neces- rent generation of stents. At this initial phase, sary for cardiac regeneration over an extended period research involves developing a variety of nanopar- of time. ticle layer porous films and testing their ability to both function as a SLIPS and hold and release drug In summary, we present a therapeutic reservoir that over a period of time. Ultimately, these tests will can mechanically protect its fragile cargo during help craft a better generation of stents for patients. transplantation and delivery, form an immunologi- cal barrier in the harsh inflammatory environment Developing a Predictive Model to Identify of the incessant beating heart, and be fully retriev- High-Risk Head/Neck Patients with able if deemed clinically necessary. We demon- Vascular Anomalies for Early Intervention strate that this system allows the targeted, replen- ishable, and sustained presentation of cellular ther- Vascular anomalies apy. The potential applications for this platform are malformations or tu- delivery system to other diseased tissues are vast. Pooja mors that result from the Chandrashekar abnormal development of Biomedical blood vessels. Because Drug Delivery through Slippery Lubricant Engineering, 2019 these anomalies can of- Infused Porous Surfaces (SLIPS) Boston Children’s ten progress into com- SLIPS, slippery Hospital plex conditions that affect lubricant-infused porous multiple parts of a pa- Natasha surfaces, are materials tient’s body, they neces- Dhamankar Advisor: John that can effectively re- Brownstein, PhD sitate complicated treat- Biomedical Engineering, 2019 pel bacteria and fluids. ment protocols that vary With this property, they Mentors: Jared widely among patients. Hawkins, MMSc, Harvard John A. have the potential to im- Thus, being able to in- PhD; Salim Afshar, Paulson School of prove the performance tervene during the initial DMD, MD Engineering and and safety of existing stages of patients’ disease Applied Sciences medical materials. Stents, progression and identify which are tubes com- whether a patient is high- Advisor: Joanna monly placed in clogged risk is critical to minimizing the severe physical and Aizenberg, PhD arteries to allow for blood psychological impact later-stage vascular anoma- flow, are among such lies have on patients. We therefore sought to de- Mentors: Steffi medical implants that velop a method that makes use of electronic medical Sunny, M.S.; Ida could be enhanced by a record (EMR) data and physician notes to rapidly Pavlichenko, MS, SLIPS coating. In addi- and accurately identify high-risk patients with head PhD tion to providing a sup- or neck vascular anomalies early on in their dis- portive cage inside an ease timeline. Additionally, we sought to develop a artery, stents release med- predictive model capable of linking current patient ications to prevent the narrowing of the arterial wall. symptoms and diagnoses with possible outcomes, Nevertheless, because a stent is a foreign object in treatments that may be required later, and further the body, it prompts clot formation around its own complications that may arise. To do this, we worked Biomedical Engineering and Science 55 with data from the Vascular Anomalies Database, To further filter the list of differentially expressed a previously untapped database containing all of genes, we used the MATLAB gene ontology (an- the physician notes from all patients diagnosed with notation) package to computationally decide upon vascular anomalies at Boston Children’s Hospital. genes of relevance (e.g. typically found in viral infec- By linking this data with the EMR data describing tions/blood regulation). Using clustering analysis, their diagnoses and outcomes, we hope to first iden- we were able to visualize the differential expression tify key terms within the text that can be used to results. Using the open source tool Cell type en- label a patient as high- or low-risk and then build a richment analysis applet, we then grouped the gene machine learning classifier that learns the relation- expression data by characterizing the different cell ship between these key terms and patient outcomes. types from which the samples came. Thus, the eventual goal is to use a physician’s initial examination of a patient with head or neck vascular We will likely find differentially expressed genes that anomalies, their notes, and the patient’s EMR data are involved in tolerance mechanisms in specific cells. to determine if the patient is likely to require a par- Finding genes likely responsible for tolerance as well ticular procedure later on or to develop a particular as the cell types they are usually active in helps condition. From this information, the goal is for elucidate pathways that are involved in tolerance the physician to be able to make better-informed, mechanisms. Moving from the dry lab to the wet evidence-based recommendations for early interven- lab then allows drugs to be developed that can tion, treatment, or lifestyle adjustments that can lead take advantage of these tolerance mechanisms in or- to fewer complications and better patient outcomes. der to provide resistance and tolerance to sepsis.

Identifying Genes Involved in Sepsis Developing a Higher Throughput Resistance and Tolerance Using Instrumented Skeletal Muscular Thin Film Bioinformatics Analysis Device Previous literature on Despite the billions of viral genomics has shown dollars invested by the Sai Shanthanand the viability of diagnos- Sayo Eweje pharmaceutical industry Rajagopal Engineering Sciences, ing different viruses by annually for bringing new Biomedical 2019 Engineering & measuring gene expres- drugs to market, there is Studies of Women, sion levels in the blood of Harvard John A. a high failure rate in the Gender, and humans. From the data of Paulson School of late stages of drug de- Sexuality, 2019 patients’ viral loads and Engineering and velopment because of a severity of symptoms, it Applied Sciences, lack of screening meth- Broad Institute of is shown that some pa- Wyss Institute for ods that adequately repli- MIT and Harvard tients are tolerant to the Biologically Inspired cate a patient’s physi- virus (high viral load but Engineering ology. Organs-on-a-chip Advisor: Jim Collins, low symptom severity) or (OOCs) are a new tech- PhD are resistant (minimal vi- Advisor: Kevin Kit nology that can solve this ral load) to the virus. Parker, PhD problem by recapitulating Mentor: Sriram Using these preliminary the necessary structure- Chandrasekaran, data, we are interested in Mentor: Johan Ulrik function relationships re- PhD Lind, PhD understanding if there is a quired to mimic both genetic basis for viral tol- the healthy and diseased erance or resistance. states of a particular or- gan. By doing so, OOCs can provide the pharma- Blood was drawn on healthy control pigs and infected ceutical industry with patient-relevant drug testing samples (deceased and surviving) over regular inter- models to use in pre-clinical screening that will re- vals of time. After blood transcriptome sequencing, duce the cost and improve the efficiency of drug de- the lab received gene expression data from all sam- velopment. ples, which included sequencing from multiple cell types. Using the MATLAB Bioinformatics toolkit, One example of this technology is muscular thin film we pre-processed the data by filtering data with low (MTF) devices. These devices allow the contractile overall expression values and low entropy. Using a strength of engineered smooth, cardiac, and skeletal t-test from the MATLAB bioinformatics toolkit, we muscular tissues to be determined in vitro. The cen- compared the tolerant vs. sensitive, tolerant vs. re- tral principle of these assays is the bending of a thin sistant, and resistant vs. sensitive populations to de- cantilever substrate by the contraction of muscle tis- termine differentially expressed genes in the studies. sue attached to the cantilever surface. The cantilever 56 Biomedical Engineering and Science is composed of elastomeric materials such as poly- dimethylsiloxane (PDMS) or gelatin. The deflection of the cantilevers is determined by visual tracking of the cantilever edge, to give a direct measure of tissue contractility.

Recently, instrumented MTF devices that allow fast quantification of the contractility of cardiac muscula- ture have been developed. However, current devices have not been adapted for use on skeletal muscle tis- sues. My goal is to address this challenge by devel- oping an instrumented MTF platform that is com- patible with engineered skeletal muscle tissues. Such a device could be used in studies of human skele- tal muscle for developing treatments for diseases like Duchenne muscular dystrophy or myasthenia gravis.

Muscle responds to both topographical and chemi- cal cues when adhering to a surface. Skeletal mus- cle tissue and cardiac tissue rely on different extra- cellular matrix (ECM) proteins to promote cell ad- hesion. Presently, many scientists who work with skeletal muscle thin films use microcontact print- ing to add patterned lines of these proteins onto substrate surface, resulting in uniaxial tissues. Mi- cromolded grooves in the substrate surface are also used to guide the alignment and development of the cells. Experiments will be conducted to find the best width, depth, and spacing of these lines for cell ad- hesion, alignment, and growth, as well as to identify the proper ECM protein(s) to use. The stiffness and thickness of the substrate itself must also be opti- mized, as skeletal muscle tissues have been shown to have significantly lower contractility than cardiac tis- sues.

If the project is successful, the device can be used to study the development of skeletal muscle con- tractility over the course of several weeks. This device can be an important part of pharmaceuti- cal research as a means of quickly conveying in- formation about the contractility of the skeletal muscle tissues being tested during pre-clinical tri- als and drug dose studies, reducing the need for companies to rely on animals for these tests. Ul- timately, this device could provide new possibilities for studying the health and disease of skeletal muscle. Chemistry and Biochemistry

Design, Development, and Synthesis of Novel gram-negative and gram-positive bacteria in or- Macrolide Antibiotics as Tools Against der to evaluate the biological potential of devel- Resistant Bacteria oped drug candidates. Of these newly synthe- sized macrolide antibiotics, several have demon- With the rise of antibi- strated quite promising activity against macrolide- otic resistance, the reser- resistant bacterial strains, further illustrating the Andre Sanchez voir of effective drugs Chemistry and potential impact of this approach. More specifi- available for treatment Physics, 2018 cally, this work focuses on the diversification and of various bacterial in- construction of carbazate linkers in ketolide antibi- fections and diseases has Harvard University otics, as well as structural modifications in azake- become dangerously low. tolide antibiotics. Furthermore, this project aims Advisor: Andrew G. For decades, chemists uti- to develop novel ketolide and azaketolide antibi- Myers, PhD lized a tool known as otics with increased on-target binding affinity and semisynthesis in order to overall potency against macrolide-resistant bacteria. Mentor: Ziyang combat the threat of an- Zhang, PhD tibiotic resistance. A semisynthetic strategy re- The Molecular Basis of T Cell Exhaustion lies heavily on the isola- tion and fermentation of complex antibiotics from The immune system nature that are subsequently modified through chem- protects the body against ical synthesis. Although semisynthesis has proven to Ashavari Ghose countless pathogenic Biochemistry, 2018 be an extremely useful tool in antibiotic discovery, it agents, from viruses to Emmanuel College is inherently limited. That is, semisynthetic methods cancer. T cells are an es- are structurally constrained, as starting substrates Brigham and sential component of the are extremely complex with many competing func- Women’s Hospital adaptive immune system, tionalities that make certain chemical transforma- whose function is neces- tions difficult or nearly impossible. This limitation Advisor: Richard sary to specifically de- is further exacerbated with the growing evidence of Blumberg, MD fend against these agents. bacterial resistance to some of the leading macrolides However, T cells have obtained semisynthetically, including azithromycin Mentor: Walter Kim, recently been described and solithromycin. MD, PhD to enter a dysfunctional state of exhaustion, char- In an attempt to gain control over the impending acterized by altered tran- crisis of antibiotic resistance, a fully synthetic route scriptional profiles and upregulation of inhibitory re- towards macrolide antibiotics was adopted. Such a ceptors on the cell surface. As a consequence, T cell technique allows for facile diversification of both ap- exhaustion severely diminishes the T cell response pendages of current lead macrolide antibiotics and to both pathogenic invasion and tumorigenesis. Re- new structurally diverse scaffolds that were previ- versal of T cell exhaustion at the molecular level has ously inaccessible by other means. Drawing lessons therefore become a major focus in the development from recent crystallographic studies that elucidated of novel infections and oncologic therapies. the interactions of antibiotics bound to the bacterial ribosome, new chemical modifications of macrolide Carcinoembryonic antigen cell adhesion molecule 1 antibiotics can be rationalized to improve their on- (CEACAM1) and T cell immunoglobulin domain target binding affinity. and mucin domain-3 (TIM-3) are cell surface recep- tors that are known to be upregulated on the surface Here, a new synthetic route is presented to pro- of exhausted T cells during chronic viral infections vide scalable and rapid access to structurally di- and in specific cancers. CEACAM1 functions as a verse macrolide scaffolds. Through convergent chem- ligand for TIM-3 and mediates enhanced T cell inhi- ical transformations from simple modular building bition. Recent studies have shed light on the binding blocks, one may build chemical complexity quite mechanism of human CEACAM1 (hCEACAM1) and rapidly. The robustness and versatility of the syn- TIM-3 (hTIM-3), revealing the specific domains in- thetic strategy was evidenced by the successful con- volved. However, the fine molecular details of the struction of more than 500 new macrolides span- interaction remain unknown. ning a vast chemical space. The novel macrolide antibiotics were then tested against a panel of This project investigates the interaction of CEA- 58 Chemistry and Biochemistry

CAM1 and TIM-3, specifically aiming to determine Rational Design and Synthesis of three things: the oligomeric state of the binding re- Nicotinamide N -Methyltransferase (NNMT) action (that is, the ratios with which the proteins Bisubstrate Inhibitors bind); the binding kinetics; and the sequence de- terminants (specific amino acid residues that are Nicotinamide N - involved in binding). Previous studies have been methyltransferase (NNMT) complicated by the propensity of hCEACAM1 to Brandon Wright is an enzyme that cat- Chemistry, 2018 homodimerize (bind to itself), and I am therefore alyzes a simple methyl- taking an alternative approach by using the murine transfer from cofactor Harvard University variants of CEACAM1 (mCEACAM1) and TIM-3 S-adenosyl-L-methionine (mTIM-3). Co-immunoprecipitation studies have Advisor: Matthew D. (SAM) to a nicotinamide provided evidence for mCEACAM1–mTIM-3 bind- Shair, PhD substrate. Nicotinamide ing, and co-blockade of mCEACAM1 and mTIM-3 is an important pre- enhances anti-tumor immune responses in mouse Mentor: Rocco cursor in the forma- colorectal cancer models. The aim of this project Policarpo tion of NAD(H) and is to provide direct biophysical evidence for the NADP(H), two key co- mCEACAM1–mTIM-3 interaction. To achieve this, factors in metabolic reg- I have cloned a naturally-occurring splice variant ulation. NNMT’s expression is inversely related to of the mCEACAM1 (mCEACAM1 D1,D4) into a production of the GLUT4 sugar transporter that suitable prokaryotic expression vector, expressed the protects against diabetes. As such, NNMT knock- protein in E. coli, and isolated it through the devel- down has been shown to prevent obesity in mice on opment of a novel purification strategy. The purified high-fat diets. Additionally, NNMT is known to be protein is being studied to determine the oligomeric overexpressed in several types of cancer, including state of the mCEACAM1 D1,D4 construct through breast cancer, bladder cancer, and renal cell carci- a series of biochemical assays, including size exclu- noma. Although NNMT plays an important role in sion chromatography, native gel electrophoresis, and various diseases pathologies, no small-molecule in- analytical ultracentrifugation. hibitors currently exist for the purpose of studying NNMT’s biological function. The fact that NNMT Studies of mCEACAM1 D1,D4 will inform subse- has been shown to regulate metabolic processes in quent binding studies with both the murine and adipocytes and is overexpressed in several forms of human forms of TIM-3 to determine both species- cancer makes it a prime target for understanding specific and cross-species binding characteristics. and treating these diseases. Similar techniques will be employed to determine the behavior of the mCEACAM1–hTIM-3 complex Rational design of novel bisubstrate NNMT in- as were used for the mCEACAM1-mCEACAM1 hibitors began using rigorous in silico screening to complex. If mCEACAM1 D1,D4 also demon- determine candidates that were both biologically po- strates high-affinity homodimerization, as seen with tent and synthetically viable. These docking studies hCEACAM1, alternative binding assays will be em- were performed with Schrödinger Maestro computa- ployed, such as surface plasmon resonance (SPR) tional chemistry suite and assigned a “Glide score” and enzyme-linked immunosorbent assay (ELISA), to assess predicted compound efficacy. Bisubstrate where CEACAM1 homodimerization can be mon- inhibitors generally consist of two fragments cova- itored and controlled. These assays will provide lently attached, each fragment targeting a differ- valuable information regarding the interaction mech- ent binding site of a bisubstrate enzyme, therefore anism of TIM-3 to mCEACAM1. Ultimately, study achieving a tighter binding by sampling both en- of the murine proteins will provide insight into the zyme binding pockets at once. The initial proof-of- human system and aid development of specific and concept compound NS1 was made on a small scale efficient therapies for reversal of T cell exhaustion. (10 mg) using an asymmetric synthesis consisting of 18 steps from D-ribose. This compound was subse- quently evaluated for its ability to inhibit NNMT in an HPLC-based NNMT inhibition assay developed in-house. Lead compound NS1 was determined to inhibit NNMT with an observed IC50 value of 600 nM. A scale-up of the linear synthesis is currently ongoing to generate sufficient material for NS1 ana- logue synthesis and ultimately for optimized in vitro biochemical and cell-based NNMT assays.

Understanding the biological efficacy of NS1 is Chemistry and Biochemistry 59 essential for evaluating the success of our ratio- which was then targeted by catalytically active Cas9. nal approach to designing NNMT inhibitors us- By sequencing the remaining plasmid pool after cut- ing in silico screening. Once a <100 nM com- ting with Cas9, we can gain an understanding of the pound is discovered according to optimized assays, target sequences that the Cas9 can target. In ad- it will be subjected to methyltransferase counter- dition, both plasmid and genomic target sites were screening to evaluate NNMT selectivity. The com- chosen in mammalian cells to test. Cell culture as- pound will then be evaluated for its ability to says were conducted and the results were processed reduce 1MNA levels in cells known to express either through flow cytometry or high-throughput se- high levels of NNMT. From these efforts we hope quencing. to arrive at a potent, selective, patentable drug- like NNMT inhibitor for preclinical development. Ultimately, in engineering a Cas9 capable of cut- ting a broader set of target sites, we hope to take steps in the advancement of genetic engineering. Directed Evolution of Cas9 Hopefully, this will eventually lead us to evolve The ability of Cas9, and harness a Cas9 capable of targeting and cleav- a nuclease, to target and ing any DNA. This Cas9 variant may potentially Christina Zeina edit DNA sequences has be very important and beneficial for the future of Chemistry, 2018 made it an invaluable both science and medicine as a gene-editing tool. tool for studying biology Harvard University and curing human dis- Discovering the Biosynthetic Origin of N-N eases. The most com- Advisor: David R. Bonds Liu, PhD monly used Cas9 is found in S. pyogenes as a de- Although they are Mentor: Johnny Hu fense mechanism against rare, natural products viruses. Using a single- Christine Zheng that contain nitrogen- Chemistry, 2018 stranded RNA guide se- nitrogen (N-N) bonds quence (the gRNA), Cas9 can be programmed to rec- have long been of inter- Harvard University ognize any DNA sequence with the only restriction est to medicinal and syn- being a protospacer-adjacent motif (PAM) sequence Advisor: Emily thetic chemists alike, due that is required to be present. Balskus, PhD to their unusual struc- tures and potent biologi- This summer, my project involved evolving Cas9 to Mentor: Tailun Ng cal activities, particularly recognize a broader set of target sequences. In order in the treatment of vari- to do this, we utilized phage-assisted continuous evo- ous cancers. Many FDA- lution (PACE) as a means to evolve Cas9. Developed approved drugs with indications for oncology contain in Professor David Liu’s lab, PACE allows for the N-N bonds, as do many of the top 100 most pre- evolution of proteins tied to the production of a nec- scribed drugs in the United States. However, despite essary phage protein, pIII. Its advantage over other their usefulness, N-N bonds are difficult to synthe- evolution methods lies in its ability to complete many size in a laboratory setting because their precursors more rounds of evolution without researcher inter- tend to be unstable, and very little is known about vention, allowing for the quick and precise evolution the biosynthetic origin of these compounds. of macromolecules. We cloned the catalytically-dead dCas9 along with a transcription activator into the Members of the Balskus group have identified various phage genome, moving the pIII gene into a separate natural products that contain N-N bonds as subjects plasmid. Upon dCas9 activity, pIII production is ac- of further study to determine how these bonds are tivated. The system was optimized and was shown made biosynthetically. I am working on discovering to be able to activate pIII production 100-fold over the biosynthetic pathway for the molecule alanosine, background levels. made by the bacterium Streptomyces alanosinicus, which has been used in Phase I and Phase II clin- After evolution, we submitted phage from PACE for ical trials for the treatment of methylthioadenosine sequencing to analyze the frequency of mutations. phosphorylase (MTAP)-deficient cancerous tumors. As Cas9 activity is tied to the production of new To study how this molecule is made, we must first phage, the most prevalent phage mutations should find the genes that code for the enzymes that are also contain the most active Cas9 mutants. A num- responsible for its synthesis. Previous work in the ber of assays were then conducted to characterize the Balskus group has already identified a strong candi- Cas9 and profile the target sites that can be targeted date gene cluster, named the ala gene cluster, in the by both the wild-type Cas9 and the evolved Cas9. S. alanosinicus genome. We have since created and A library of target sites was encoded on a plasmid, screened a genomic library of E. coli colonies contain- 60 Chemistry and Biochemistry ing different segments of the S. alanosinicus genome, bound together and coordinated to a metal atom, and have successfully isolated this cluster of interest. are capable of exhibiting oxygen-dependent fluores- Our current work is focusing on definitively linking cence. Put simply, the porphyrin glows strongly in the ala gene cluster to the production of alanosine an oxygen-deficient environment, while its lumines- through heterologous gene expression—transferring cence is quenched when placed in an oxygen-rich en- the ala cluster into host cells that do not natu- vironment. As luminescence is far simpler to mea- rally produce alanosine in order to see if alanosine sure than oxygen concentration, the porphyrin thus production then occurs—and gene knockout exper- simplifies the process of elucidating oxygen concen- iments—deleting various genes in the ala cluster in tration. Moreover, the porphyrin is stable and long- alanosine-producing strains to see if alanosine pro- lasting, allowing for continual oxygen measurement duction is halted. over time.

Notably, the ala gene cluster contains two enzymes My project involves further improving the oxygen- that are homologous to enzymes found in the biosyn- sensing properties of these porphyrins. In order to thetic pathways of other N-N bond-containing com- cause the porphyrin to glow, the electrons within its pounds, and therefore we believe that these two structure must first be excited with a laser emitting enzymes could be ultimately responsible for the for- photons of the proper wavelength. The current por- mation of the N-N bond itself. We are also currently phyrin’s excitation wavelength does not match the working on purifying these enzymes in order to bet- wavelengths of commercially available lasers, mak- ter understand their functions. ing it less than ideal for widespread use. Excita- tion wavelength is related to the size of the aromatic It is our hope that studying the biosynthetic structures that make up the porphyrin’s backbone; mechanisms for N-N bond formation in alano- my goal is to enlarge these aromatic structures in sine and other molecules like it will pro- order to decrease the porphyrin’s excitation wave- vide insight into this unique class of natural length, pushing it into commercially viable ranges. products and aid scientists in developing new methods of synthesizing them in the future. Previous attempts at synthesizing these larger por- phyrins involved first synthesizing larger aromatic structures and subsequently fusing them together; Oxygen-Dependent Phosphorescent such methods result in low yields due to the unfa- Porphyrins for the Measurement of Blood vorable entropic loss associated with the fusion step. Oxygenation Levels My approach is to take the already formed precursor Oxygen, despite its vi- porphyrin and grow their aromatic structures. This tal role in the proper is a two-step process, involving first the oxidation of George Qiao function of the human alcohol groups present on the ends of each of the aro- Chemistry, 2018 body, is notoriously diffi- matic structures into ketones, and second the attach- cult to measure and quan- ment of small carbon chains to the newly formed ke- Massachusetts General Hospital tify in the bloodstream. tones. The entropic loss associated with these steps While electronic methods is small; the end goal is to produce the improved Advisor: Conor L. exist for measuring atmo- porphyrin in larger yields. Attempts at oxidation Evans, PhD spheric oxygen content, have already yielded results, with the Swern method such methods are frus- in particular showing particular promise. The next Mentor: Emmanouil trating and tedious to steps are the verification of successful oxidation Rousakis, PhD translate into medical use. and the search for a carbon attachment method. Those crossover methods that do exist are unreli- able and moderately invasive; moreover, they pro- vide only one-time measurements, and are incapable of monitoring changes in blood oxygen concentration over time. An ideal oxygen sensor would be mini- mally invasive, finely calibrated, devoid of toxicity, and time-sensitive.

Recent work in the Evans lab has focused on the production of such a sensor utilizing a liquid ban- dage consisting of oxygen-sensitive porphyrins (an organic compound) suspended in oil. These por- phyrins, consisting of four cyclic aromatic structures Computer Science

Automated Placement of Components in increase enough within the groups. Printed Circuit Boards We are hopeful that the overall program will au- The design of elec- tomate the process of component placement, re- tronic chips has advanced ducing the cost and time of designing new sys- Artidoro significantly over the past Pagnoni tems and improving their overall performance. thirty years, with the Computer Science & Physics, 2018 number of transistors on a chip nearly doubling ev- Improving Sequence Handling in Deep Cadence Design ery year. Integrated Cir- Learning Systems cuits (IC) techniques can now fit over seven bil- Recent years have seen Advisor: Taylor lion transistors on a sin- an explosion of interest in Carl Denton Logan gle piece. Resources were the field of deep learn- Computer Science & fruitfully placed in the de- ing, a branch of machine Physics, 2019 Mentor: Gu-Yeon velopment of IC circuitry, learning that uses to- Wei, PhD leaving out the optimiza- Harvard John A. day’s immense computa- tion of the connections Paulson School of tional resources and mas- among components at the Engineering and sive datasets to train higher level of printed circuit boards. Applied Sciences models at a scale that would have been unimag- This question is becoming of greater importance to- Advisor: Alexander inable just a few years day in the design of large electronic systems. The Rush, PhD ago. Deep learning is placement of components is still done manually for particularly appealing be- the most part, even when the number of components cause it requires far less is in the tens of thousands. human intervention. Traditional approaches to ma- chine learning rely heavily on domain-specific knowl- High complexity has increased the price of design- edge, inserted by hand as a set of “features” in the ing new chips. Specific purpose devices are progres- data; in contrast, deep learning simply uses raw data sively being replaced with larger groups of standard- and allows the models to recognize patterns for them- ized chips. The number of components in complex selves. designs is therefore increasing and their high level of optimization makes other factors come into play. Deep models have seen considerable success, find- ing widespread application in fields including com- My work has focused on finding algorithmic tech- puter vision, speech recognition, and natural lan- niques to optimize the placement of components on guage processing (the focus of this work). Never- printed circuit boards. Our project goes beyond the theless, there are a number of tasks in which tradi- partial solution proposed in the late 80s by Profes- tional models still outperform other methods. Con- sors Sangiovanni and Newton at Berkeley University ditional random fields (CRFs), a type of probabilistic for equal-sized components. We intend to propose a graphical model, are widely used for sequence predic- truly practical solution that takes into account con- tion (i.e. predicting sequences rather than individual nectivity constraints but also magnetic fields, heat tags) and set the state of the art in tasks including dissipation, and multilayer design constraints. part-of-speech tagging and named entity recognition. Whereas deep models often rely on a huge number of To include these numerous constraints in our opti- parameters for their modeling power, CRFs exploit mization, we adopted the well-known genetic algo- the sequential structure of the data they process. rithms, which generate heuristic solutions simulat- ing the biological process of evolution. The opti- The goal of this work is to integrate CRFs and deep mization of parameters in our evolutionary frame- models. A number of current techniques in deep work and our representation of individuals (solutions learning make use of sequence predictions, often with to the placement problem) will be done by drawing far less sophisticated modeling techniques than those techniques from machine learning. In addition, much used by CRFs. In machine translation, for example, effort is being put in the clustering of strongly con- the model is given a source sentence in a source lan- nected components. We cannot rely on traditional guage and is expected to produce an equivalent tar- clustering algorithms because connectivity does not get sentence in a target language. Many of the most 62 Computer Science successful approaches to the task from deep learning structures without the concern for transactional cor- process the source sentence by using some notion of rectness. We look to combine these research ideas to “attention”—that is, they weight some parts of the produce data structures that both perform well and source sentence more highly than others in produc- simplify parallel programming by providing transac- ing the translation. However, most models ignore tional guarantees. the sequential structure of the input in producing these distributions. By applying a CRF, we hope to We first experimented with new, hybrid concurrency obtain attention distributions that more accurately control designs for high-contention data structures. reflect the structure inherent in the input. Contention is a performance problem that occurs when many threads have to wait for one another. More broadly, we hope that by integrating CRFs Most data structure libraries use either pessimistic with deep models, we can capture both the se- locking, which behaves well under contention, or op- quential modeling power of CRFs, and the flex- timistic concurrency control (OCC), which is faster ibility of deep models to create models which in all other cases. STO generally uses OCC, but we can more effectively learn from sequential data. introduce hybrid designs that use pessimistic locking for naturally-contentious operations, such as “pop” on queues and priority queues. With this simple Concurrency in Transactional Data Types change, our naive transactional queues gain perfor- Parallelism, where a mance nearly equal or even better than the cutting- program runs simultane- edge research on concurrent data structures. This Lillian Tsai ously on multiple CPUs, is a great result, because naive algorithms are much Computer Science, is increasingly critical for more likely to be implemented correctly. We there- 2017 performance in computer fore achieve close to optimal performance with a Harvard John A. software systems. This greater likelihood of correctness. Paulson School of is because CPUs are be- Engineering and coming more numerous We did find one strategy, Flat Combining, that per- Applied Sciences per machine, rather than forms better than any of our STO implementations. more performant individ- However, when we place the Flat Combining queue Advisor: Eddie ually. However, parallel into the STO transactional framework even without Kohler, PhD programming is also in- ensuring transactional correctness, we discover the herently difficult because inherent overhead of the STO system. The STO- the programmer must co- Flat Combining queue performs worse than even the ordinate the execution of multiple CPUs. This has original STO queue, and we had not altered the Flat driven a search for better programming tools. The Combining algorithm in the slightest. most promising is software transactional memory (STM), that allows programmers to write concurrent This result leads us to think that reasoning about code using sequential programming paradigms. Un- concurrent data structures is fundamentally differ- fortunately, STM is often very slow in practice and ent than reasoning about transactional data struc- rarely considered practical. Recently, a major per- tures. We have already discovered the benefits of pes- formance improvement has been discovered at Har- simistic aborts in transactions, in which a transaction vard with the development of a novel type of STM, will assume that conflicts will occur and therefore ei- STO (software transactional objects). The key in- ther lock the data structure for the entirety of the sight was to implement the STO system at a higher transaction, or abort other transactions immediately. level—data structure operations—than most previ- Because high-concurrency data structures have no ously developed systems, which work at the level of notion of transactions, this idea would not be found individual memory words. With STO transactional in high-concurrency research. Perhaps the high- data structures, programmers can now easily write concurrency algorithms have inherent properties that correct concurrent code and avoid the performance make them unsuitable for conversion to transactional overhead of a word-STM. data structures, and the specification for transac- tional data structures must be weakened in order The STO framework relies on libraries of data types to make them performant. As we investigate these built for concurrent operation. These data structures questions further and come closer to maximizing the are currently the limiting factor of STO’s perfor- performance of the STO data structures, we develop mance; although they ensure correctness and trans- a tool unmatchable in its ability to both simplify par- actional properties, they do not come close to max- allel programming and speed up program execution. imizing scalability or performance. However, there has been significant research done by others on max- imizing scalability and performance of these data Computer Science 63

Effectiveness of Multi-Task Peer Prediction Characterizing Predictive Neural Networks on Google Local Guides Deep learning is a We often hear about branch of machine learn- the “wisdom of the Mingu Kim ing that has been gain- Applied Matthew Leifer crowd,” but can we trust ing attention in the past Applied Mathematics, 2018 it? Moreover, if we try couple years. It has es- Mathematics, 2019 to exploit this “wisdom” Harvard John A. pecially been shown to Harvard John A. by paying people for their Paulson School of be effective in the field Paulson School of opinions, can we ensure Engineering and of computer vision. Us- Engineering and that they will invest ef- Applied Sciences ing a biologically-inspired Applied Sciences fort and not make up un- neural network architec- informed opinions? In Advisor: David Cox, ture, our lab has created Advisor: David essence, can information PhD a “PredNet” that is de- Parkes, PhD be crowd-sourced and re- signed to accept video se- liable? Consider, for ex- Mentor: William quences of objects and Mentors: Debmalya ample, as Google Local Lotter, MS continually predict future Mandal, MEng; Guides now does, ask- frames in a novel manner. Victor Shnayder, ing participants about The PredNet is able to ac- PhD the qualities of a par- curately predict sequences of events that are both ticular restaurant such synthetically and naturally created, and has a better as whether it is “noisy,” understanding of the physical nature of object rep- “family-friendly,” “romantic,” or “wheelchair acces- resentation than other network architectures. There sible.” While questions may have correct answers, de- are two main questions we wish to address. First, termining those answers ourselves could be near im- we would like to understand how biologically related possible, especially if we wish to learn about restau- the network is to our own brains. Second, we want rants around the world. To solve this problem of to investigate the generalization capabilities of the eliciting truthful information, a number of proto- network, and whether it has truly learned physical cols for evaluating people’s responses, known as peer transformation properties. For example, if the net- prediction mechanisms, have been developed. These work is only trained on video sequences of human mechanisms rely on comparing responses from two or faces rotating and animals moving, how well can it more participants on a particular task and perhaps predict a bottle spinning? These questions can help their responses to other tasks, and pay a participant show whether the PredNet is actually a functional if his report is predictive of other participants’ re- hybrid that possesses desirable characteristics of deep ports. Ultimately, the protocols are designed such learning, and the natural methods by which people that investing effort and telling the truth is an opti- learn. mal strategy. To answer the first question, we analyzed the fir- The focus of this research project is to apply ret- ing rates of each neuron in each convolutional layer rospectively a number of these mechanisms (Robust to see whether we can find any evidence for posi- Peer Truth Serum, a mechanism designed by Kamble tional invariance. This would correspond well with et al., and a generalized form of Correlated Agree- known neuroscience literature. We also wanted to see ment) to the response data gathered by Google Local whether our network can be fooled like our brains Guides. We will compare the size of the payouts of can, by specifically using an optical illusion known these mechanisms to the current system, which pays as the flash-lag effect. We can do this by measuring users according to the number of questions they an- the firing rates of the neurons for static sequences swered. We are going to determine how much these and comparing them to dynamic sequences at vari- mechanisms would have paid, had they been em- ous angles. Either a positive or negative result would ployed, as well as quantify how large of an incentive be useful, as we could see the degree to which our they would create for people to provide truthful in- network has similar characteristics that can be mea- formation. We also plan to calculate what fraction sured quantitatively. of the participants would have to be truthful in order for these mechanisms to be effective. We also hope For the second question, we can train the net- to learn if these mechanisms are any more effective work on video sequences for various different cate- on factual questions, such as those that ask about gories of objects and test this network on objects wheelchair accessibility, than questions that seek to that it has never seen before. We can quantita- elicit an opinion, such as whether a place is “noisy.” tively measure performance by using metrics such as the structural similarity (SSIM) index, and per- 64 Computer Science forming such tests allows us to analyze what fea- up a state in the cache and see what it would end up tures the PredNet might be using. We can also doing if it ever entered that state. Therefore, if the see if the PredNet can generalize different speeds main thread of the program ever realizes that its cur- of movement, by training on a given range of rota- rent state is identical to one of the predicted states tion speeds, and then determine if it can accurately in the cache, it can instantly update its current state predict an object rotating at a speed greater than to the speculatively executed state corresponding to it has been trained on. We hope to see that the that predicted state, “fast-forwarding” itself into the network can demonstrate a deep understanding of future and taking advantage of computation done on general physical properties of object representation. an auxiliary core. By continuously repeating this cy- cle of prediction, speculative execution, caching, and fast-forwarding across multiple cores, ASC can allow Automatically Scalable Computation a single-threaded program to take full advantage of I am working on multiple cores. the Automatically Scal- Peter Kraft able Computation (ASC) The goal of my project is to enhance ASC in two Computer Science, project, a collaboration related ways: improve the quality of its predictions 2017 between Harvard and and expand the class of programs for which ASC Harvard John A. Boston University, the can provide significant speedup. The computations Paulson School of goal of which is to speed that ASC has to perform during operation, like ex- Engineering and up single-threaded com- tracting state, making prediction, and doing cache Applied Sciences putation by exploiting ad- lookup, are not free. Achieving ASC’s goal of speed- ditional cores. Almost all ing programs up requires that these be made as fast Advisor: Margo modern computers con- and efficient as possible, otherwise overhead from Seltzer, PhD tain several execution en- them will outweigh the benefits of automatic paral- gines, called cores. In the- lelization. As ASC is made smarter and faster, the Mentor: Amos ory, most computation- set of programs it can speed up will hopefully grow Waterland ally expensive programs as well, making it a more general and useful tool. can be run on multiple cores simultaneously, and Swarm Locomotion: Biologically Inspired will run linearly faster as they use more cores. How- Rules for Self-Organized Bridge Building in ever, in reality, writing programs that use multiple an Army Ant-Inspired Soft Robot cores efficiently is hard—much more difficult than writing a similar program that uses only a single Natural systems ex- core. Most programs that people write are single- hibit complex behavior threaded, meaning they can only use a single core. Suproteem while employing individ- Sarkar This is extremely inefficient as it means the auxiliary ual agents with limited Computer Science & cores are simply wasted, their power completely un- Mathematics, 2019 information. Understand- used. Given the prevalence of multicore computers, ing emergent behavior in it is crucial to develop ways to transform programs Harvard John A. nature can help inform written for a single core into ones that take full ad- Paulson School of the engineering of artifi- vantage of multiple cores. Engineering and cial systems that operate Applied Sciences, at scale. Eciton burchel- ASC addresses this problem in an entirely new way. Wyss Institute for lii (commonly known It relies on the fact that most programs are deter- Biologically Inspired as the army ant) cre- ministic. Deterministic programs are always in one Engineering ates complex static and of some number of distinct states, and will always dynamic structures, in- do the same thing after they enter a particular state. Advisor: Radhika cluding bridges on for- The state of a program is the individual 1s and 0s it Nagpal, PhD aging trials and living manipulates in memory. ASC works by monitoring nests, which consist of the running programs and extracting their states. It then organisms’ own bodies. uses machine learning to guess potential future states Bridges form spontaneously without any apparent of the program. Next, ASC has auxiliary cores spec- central planning, and though their assembly hinders ulatively execute from the predicted future states. the movement of specific ants that enter the struc- What this means is that additional cores pretend the ture, the shorter paths created by these structures program has entered the predicted state and figure increase the efficiency of raids and prey collection for out what it would do from that state. ASC then cre- the colony as a whole. Prior work has found these ates a cache that maps between predicted states and bridges to be robust under changing conditions, sen- speculatively executed states. A program can look sitive to traffic flow, and favorable to paths that in- Computer Science 65 crease colony movement efficiency. The implications arm that she has pulled, but sacrifices information of E. burchellii’s transport methods were studied in about all other arms. relation to a soft robot that is capable of locomoting horizontally, vertically, and upside-down. A model of If we consider the $0 and $1 reward in the context the robot in a swarm was examined in physics simu- of content recommendation, $1 becomes a “click” lation, given varying sets of rules for locomotion and on a link, and $0 becomes “no click.” Thus, MAB attachment to other robots in the system. Condi- algorithms learn more about a system’s content as tions were varied across randomly-generated terrain users click, or do not click, on the content. Thus, the types, leading to the formation of robot bridges and average “payoff” becomes the average “click through ramps. Bridge formation and dissolution was studied rate” (CTR) of a link. in the presence of terrain with valleys, given variable conditions for traffic and valley dimensions. The Today, websites have more information about a video simulation provided insight into which terrain types than its CTR. Indeed, videos have tags and users and traffic levels would lead to self-organization of have accounts, which means that we don’t start with structures and which rules would lead to efficient a blank slate when recommending content. For ex- collective locomotion across rugged terrain. Future ample, a video might be tagged as “funny” and its work will further examine the formation and disso- title might contain the phrase “Donald Trump”; a lution of these structures and account for differing user might have an account, where she has stated rules and initial conditions. By drawing inspira- that she is 24 and from Chicago. If female users tion from nature and computationally simulating over 21 from urban settings have historically enjoyed self-organization this work aims to help understand funny videos about Donald Trump, perhaps so will collective movement in natural and artificial systems. this user.

The goal of my research is to improve content recom- MAB Algorithms and Content mendation and increase videos’ CTRs by developing Recommendation an algorithm that uses these features—video tags Websites recommend and user information—in conjunction with the MAB content to their users, framework. Val Leifer from news articles to Applied videos. In doing so, they In practice, however, observing features is Mathematics, 2019 face a trade-off between costly—assigning tags to a user or video takes time Harvard John A. recommending old links, and effort. Further, in reducing a user or video Paulson School of which they know a lot to a set of tags, certain features are not observed. Engineering and about, and new links, Maybe when our 24-year-old female user watches Applied Sciences about which they want a video about Donald Trump, it’s not because she to learn more. The multi- likes funny videos about Trump, but because she Advisor: David armed bandit (MAB) al- likes John Oliver, who made the video. When the Parkes, PhD gorithms are a class of set of features for a given user-video combination algorithms that are useful is incomplete, the algorithm can make false connec- Mentors: Nicole for such content recom- tions between known features. Immorlica, PhD; mendation systems. Greg Stoddard Prior work on content recommendation algorithms In machine learning, there assumes full access to relevant features. We are ex- is a trade-off between ex- ploring the efficacy of existing algorithms when faced ploration and exploita- with missing information, and working to develop an tion—between choosing an action that yields a cur- improved algorithm that performs well even in the rent high reward (exploitation), and one that sac- absence of relevant features. rifices maximizing current reward for the chance of obtaining better rewards in the future (exploration). The multi-armed bandit problem is a scenario in which a player at a row of slot machines, also known as “one-armed bandits,” must choose which machine (or arm) to play. If each arm returns a $0 reward or a $1 reward with a certain probability, then after in- finitely many plays, that arm’s average payoff equals the arm’s probability of returning $1. At each trial, the player faces a trade-off between exploration and exploitation; at each trial, she learns more about the Engineering and Applied Sciences

Test Bed for Minerva, a Deep Neural thin tethered wires, while researchers explore suffi- Network (DNN) Integrated Circuit, and ciently light-weight alternatives. The challenge with RoboBee Power Electronics using tethered wire is its high resistance (ranges from 20 Ohms to 30 Ohms), which when coupled Test Bed: with the 30mA-100mA variation in current drawn by the robot, results in up to a 3V voltage drop. Bahlakoana Machine learning has be- Mabetha My project involves developing a power supply sys- come more popular be- Electrical tem that will power chips on the RoboBee with con- cause of its ability to Engineering, 2018 stant output voltage independent of the variations solve problems that seem in the current drawn by the robot BrainSoC. This is Harvard John A. challenging (or impossi- achieved by implementing a tethered wire feedback Paulson School of ble) to solve using cur- loop that adjusts the output voltage. The analysis of Engineering and rent traditional comput- the regulator output voltage will enable characteriz- Applied Sciences ing algorithms. Ma- ing different responses of power supply feedback loop. chine learning implements Advisors: Paul pattern recognition tech- Whatmough, PhD; niques to solve problems Harvard iGEM 2016 Mario Lok such as poor facial recog- nition and poor handwrit- iGEM is a premier in- ing analysis. The Brooks ternational competition and Wei Lab developed an Daniel Um to advance synthetic biol- Integrative Biology, application-specific integrated circuit (ASIC) that ogy and develop collabo- 2019 computes deep neural networks (DNN), which, like ration in this field. This most machine learning approaches, uses a large set of Wyss Institute for year’s Harvard team con- input and output examples to learn about complex Biologically Inspired sists of eight undergradu- function approximations. The input/output dataset Engineering ates, four graduate school is used as training for data fitting, thereby enabling mentors, and one profes- relatively accurate predictions. My project focuses Advisor: Neel Joshi, sor. After three months on designing a test bed for the ASIC designed us- PhD of brainstorming and re- ing Minerva, a co-design framework for optimal de- searching, we narrowed sign of deep neural network hardware. The test Mentors: David Lips; our search to four ideas bed needs to flexibly power the ASIC from multi- Kevin Hoff; Bom to present to the team. ple sources, namely, USB, bench-top power supply, Pichet Subsequently, we decided and AC mains. Other specifications of the test bed Praveschotinunt; on our project, “Break- include portability and compatibility with USB to Anita Chandrahas ing PET,” that would enable interfacing with laptops to demonstrate the break down polyethylene ASIC. The end goal is to make the test bed as com- terephthalate (PET)— pact as possible, ideally the size of a small memory the plastic molecule that makes up one-sixth of the stick. world’s plastic products—with enzymes produced by a recently-discovered microbe. The additional goal RoboBee: of the project is to use the degradation products to generate electricity in a microbial fuel cell. Aerial vehicles and microaerial vehicles, commonly known as drones, have recently seen increased appli- Project “Breaking PET” is derived from the Oda cations. Several research groups at Harvard SEAS Group’s research conducted at the Kyoto Institute and beyond joined forces to develop RoboBees. of Technology. Their paper, “A bacteria that de- RoboBees are biologically-inspired microaerial vehi- grades and assimilates PET,” outlines the research cles that can fly using flapping wings driven by bi- conducted on a bacteria (I. sakaiensis) found at morph piezoelectric actuators. RoboBees have the a bottle recycling plant. We plan to use two en- potential to expand the application of aerial ve- zymes, PETase and MHETase, to break down this hicles to crop pollination, search and rescue mis- substance into its constituent parts. PETase breaks sions, and environment monitoring. To this end, down PET into the compound MHET, or mono RoboBees are meant to be autonomous and capable (2-hydroxylethyl) terephthalic acid. MHETase fur- of self-contained, self-directed flight. Currently the ther breaks down MHET into terephthalic acid and RoboBees are powered off of external sources through ethylene glycol. These two enzymes degrade PET Engineering and Applied Sciences 67

120 times faster than other PET-degrading enzymes. extremely difficult to recover the nanoparticles from From the Oda et al. paper, we found the DNA se- the purified water. Thus, people end up drinking the quences for the enzymes PETase and METase, which photocatalyst, and the TiO2 cannot be reused. we plan to insert into E. coli, a commonly used bacte- ria with a faster reproduction rate than the original The overall objective of this research is to develop I. sakaiensis. Using this process, we hope to op- and evaluate a viable method of using photocatalysis timize production of PETase and MHETase in E. to purify water. It is hypothesized that a novel pho- coli by characterizing the necessary conditions to tocatalytic sand could be synthesized, and would be degrade PET most efficiently. We will create Bio- more effective in removing bacteria and degrading Bricks (DNA constructs that can easily be swapped organics than a SODIS control. A simple method around) for PETase and MHETase and characterize of synthesizing photocatalytic sand will be devel- these parts against enzymes used in previous PET- oped, in which TiO2 nanoparticles are coated on degrading iGEM projects. Additionally, we hope to the surface of uniformly graded sand. The TiO2 engineer a secretion system for these enzymes. nanoparticles will not wash off into the water, and the sand will be easy to recover once the water is Following the breakdown of PET, we plan to use purified. one of the degradation products, terephthalic acid, in a microbial fuel cell to generate electricity. Al- For preliminary studies, several ratios of TiO2 to though microbial fuel cell technology is still in its sand (0.5%, 1%, 2%, and 5% TiO2 in sand) will be nascent stage, and thus cannot generate a large evaluated. The color change of methylene blue, an amount of electricity, we are using our project organic indicator dye, will be quantitatively deter- as a proof of concept to show that this tech- mined using UV-Vis spectroscopy in order to find the nology could potentially be used to recycle plas- minimum ratio of TiO2 to sand that is needed for tic bottles into something useful such as energy. maximum organic degradation efficiency. This op- timal ratio will be selected to evaluate the removal of E. coli that has been spiked in water to mimic Photocatalytic Sand for Removing Multiple drinking water contamination. Further tests will be Classes of Toxins from Water conducted to determine the largest volume of water Water is a substance a set amount of the photocatalytic sand can purify, that is essential for life, as well as determining what proportion of the TiO2 Deepika Kurup yet one-ninth of the (if any) washes off into the water. Neurobiology, 2019 world’s population lacks access to safe drinking It is expected that the photocatalytic sand will be Harvard University water. According to the able to degrade organics and remove bacteria at Advisor: Roy World Health Organiza- a much faster rate than traditional SODIS meth- Gordon, PhD tion, waterborne diseases ods. The photocatalytic sand is envisioned to have such as cholera, dysen- potential applications in point-of-use water purifi- Mentor: Marlene tery, and diarrhea are re- cation systems for individual users, water tanks for Wolfe, PhD sponsible for more than large communities, and even in wastewater treat- Candidate 2.2 million deaths a year. ment plants in developed countries. This project These diseases are by opens numerous possibilities for green, sustain- caused harmful pathogens able, and economically viable water purification. such as Escherichia coli and organics such as ben- zene, toluene, ethylbenzene, and xylene. Even in developed countries, natural disasters such as earth- quakes, hurricanes, and tsunamis can cause flooding and sewage overflow, resulting in water pollution.

Currently in developing countries, solar disinfection (SODIS) that uses ultraviolet radiation from the sun (UVA, λ = 315–400 nm) is used to purify water; however, SODIS is very slow and can take up to two days to purify water. In recent years, photocatalyst such as titanium dioxide (TiO2) have been used to accelerate the SODIS process through the creation of reactive oxygen species. While dispersing photo- catalytic nanoparticles in water is an effective way to remove bacterial and organic contaminants, it is 68 Engineering and Applied Sciences

Printed Sensors for Medical Devices ing the sensor for electrical insulation. Additional circuits and signal processing techniques are de- Medical devices veloped to condition and accurately read signals. equipped with sensors can Nicolas greatly improve medical Ontiveros Programmable Motion in Colloidal Systems care quality. Specifically, Electrical Engineering, 2018 medical practitioners are Traditionally, colloids, interested in measuring or suspensions in which forces exerted by devices Will Fried microscopic particles are Harvard John A. Mechanical and tracking a device’s dispersed within liquid Paulson School of Engineering, 2019 Engineering and location and history. Ex- media, are chaotic and Applied Sciences, erted forces are important Harvard John A. unpredictable. The sys- Wyss Institute for because certain human Paulson School of tem I study, where one- Biologically Inspired tissues must be dealt with Engineering and micron plastic spheres are Engineering delicately. Furthermore, Applied Sciences suspended in aqueous so- device tracking can ensure lution, is no exception to Advisor: Robert instruments are sterilized Advisor: Vinothan this rule of thumb. In Wood, PhD before a surgical opera- N. Manoharan, PhD this setup, particles are tion and no instruments driven to move randomly Mentors: Michael J. are left in the body after Mentor: Emily through the liquid as they Smith; Ruth Peña Gehrels Velasco surgery. For many med- continuously collide with ical instruments, one can passing water molecules. measure forces by equip- My project seeks to apply ping the device with a strain gauge, a sensor that chemical and thermodynamic principles to harness measures mechanical strain. Strain gauges are not these randomizing processes to program the colloid widely used in the medical industry because they to behave in a controllable manner. require adhesives, which cannot survive the steriliza- tion process and are at risk of failing during surgery. One method of achieving this controlled behavior in- Adhesives are also problematic for device tracking. volves creating a track of particles on which another Hospitals have begun using radio-frequency identifi- particle (called a dancer) is constrained to move, and cation (RFID) tags to track instruments, but some then manipulating the steps that the dancer takes by instruments would require significant modifications altering the environment of the system. To control and adhesives to be equipped with RFID tags. the interactions between the track and dancer parti- cles, we attach various sequences of single-stranded When compared to traditional sensors, printed sen- DNA to the surfaces of different particles such that sor technology offers several benefits: low-cost, low only particles coated with complementary DNA bind profile, and good adhesion to the device’s surface. together. We then control the motion of the dancer The goal of this project is to develop printed strain along the track by embedding different colors of dye gauges and RFID tags for medical devices. Med- into the track particles and sequentially exposing the ical devices under consideration include surgical system to different wavelengths of light. Each dyed staplers, surgical robot end effectors, forceps, and track particle should absorb light of a different color hemostats. The printing process involves advanced and dissipate the energy as heat. Provided that at microfabrication techniques, including chemical and a given moment the dancer is bound to a dyed par- physical vapor deposition. The surface of the de- ticle capable of absorbing the incident wavelength, vice is roughened with either sanding or abrasive the subsequent heat release should break the nu- blasting and cleaned with acetone. A thin layer cleotide bonds between the two particles and drive of Parylene-C, a biocompatible and biostable poly- the dancer towards a neighboring particle to which mer, is applied to create a conformal coating on the it is attracted. This process is then repeated to trig- surface of the device. The strain gauge or RFID ger more substantial movement. antenna is then deposited; this metallization process involves sputter deposition of the desired metals, However, before we are able to perform experiments which include Constantan and copper. Electrical using the dancer system, we must conduct several traces are also deposited and attached to the sen- preliminary experiments. First, we must verify that sor. For the RFID tags, a small chip is attached illuminating dyed particles at proper wavelengths to the antenna; this chip transmits information to can in fact locally heat the particles’ surroundings. an RFID interrogator when the antenna receives To confirm this hypothesis, we saturate a solution electromagnetic radiation from the interrogator. with dyed particles and add free-floating complemen- A final layer of Parylene-C is applied, encapsulat- tary strands of DNA to the medium. These DNA Engineering and Applied Sciences 69 strands are modified so that they fluoresce when issue by designing a novel auxiliary circuit to syn- excited by light of a certain wavelength, but only chronize the SCC frequency to a constant sampling when they are unbound from their complementary clock while still allowing for the immediate control strand. Consequently, the fluorescent signal is weak of the SCC frequency and thus the output current. at low temperatures, where the DNA is predomi- As a result, all voltage measurements can be taken nantly bound, and strong at higher temperatures, at the same phase of the SCC input, nullifying the where the increase in thermal energy causes the DNA influence of the ripple voltage on the measured sig- molecules to detach from their complements. There- nal. The SCC-based CMOS current injection circuit fore, when we illuminate our sample with two beams in combination with the newly developed auxiliary of light, one that excites the fluorescence and one circuit allows for the fabrication of tools with large that is absorbed by the dyed particles, we expect the arrays of stimulation and recording sites, which have fluorescent output to grow as heat is generated by immediate application in many biological research the dyed particles. fields including neurobiology and neurotechnology.

The other preparatory experiment involves charac- terizing the melting curves of DNA strands attached to the surfaces of dyed polystyrene particles. Us- ing a microscope to examine these particles, we can identify the wavelength, intensity, and duration of light exposure required to cause particles coated in complementary strands of DNA to detach from each other for a given dye and at a given tem- perature. We can then use this data to precisely and efficiently carry out our motion experiment.

A Novel Design for Compact and Parallelizable Current Stimulation and Recording Accurate micron-scale variable current sources Youbin Kim are extremely useful in Physics, 2018 the field of biotechnology and in improving the in- Harvard John A. Paulson School of terface between electron- Engineering and ics and living cells. Es- Applied Sciences pecially when electrically exciting cells, current- Advisor: Donhee based stimulation is pre- Ham, PhD ferred in comparison to voltage-based stimula- Mentor: Jeffrey tion, as the latter could Abbott compromise cell viabil- ity due to the nonlin- ear characteristics of the electrode-cell interface. Furthermore, complex com- munication between circuitry and biology requires multiple stimulation and recording sites, creating a desire for small-scale and parallelizable current sources. This project utilizes a switched capaci- tor circuit (SCC) as a micron-scale CMOS current source. Although the SCC-based design addresses the problems of size and parallelizability, it also in- duces a ripple voltage whenever the output switch is closed. The resulting voltage shift, although pe- riodic and based on the frequency of the SCC, can interfere with simultaneous stimulation and record- ing from the target cells. This project addresses this Genetics

X-Linked Dystonia-Parkinsonism bly modulate TAF1 transcription through a variety of different ways such as methylation of protein ag- Dystonia is a move- gregates. ment disorder character- Anne Cheng ized by sustained muscle Neurobiology, 2017 To further study the SVA retrotransposon inser- contractions causing ab- tion, I plan to study its downstream effects in normal, often repetitive, Massachusetts the absence of the other XDP-specific mutations. movements and postures. General Hospital To do this, I will isolate the SVA retrotranspo- These movements are typ- son from a patient line and clone it into a lu- Advisor: Cristopher ically patterned, twisting, ciferase vector to allow assessment of the SVA Bragg, PhD and may be tremulous, transcriptional activity. Understanding the SVA which is often initiated retrotransposon insertion and its effects on local Mentors: Christine or worsened by voluntary genes is crucial for both XDP and many other dis- Vaine, PhD; William action and is associated eases, including some types of cancers that incor- Hendriks, PhD; with overflow of muscle porate retrotransposon insertions as a mutation. Bjorn Brandl; Lilian activation. Cruz X-linked dystonia- Manipulating Innate Immunity Genes to parkinsonism (XDP), the Measure Their Regulation of the Microbiome only known form of dys- tonia that is inherited as an X-linked trait, is a neu- Host-microbiome in- rodegenerative movement disorder endemic to the teractions are recognized Philippine Island of Panay. Patients with XDP not Bonirath Chhay as a fundamental part Engineering Sciences, only experience dystonic symptoms but also exhibit of an organism’s biol- 2018 parkinsonism in their later years. XDP is associated ogy, closing gaps in un- with neuronal loss and astrocytosis in the caudate Rowland Institute at derstanding phenotypes and lateral putamen of patient brains. Because of Harvard left by genomics alone. the high rate of XDP in Panay and the genetically However, there are still non-lethal nature of the disorder, researchers believe Advisor: Robert many unknown mecha- that the disorder must have arisen from a genetic Brucker, PhD nisms that establish the founder effect. relationship between the Mentors: Jessica genome of the host and The disease locus, DYT3, affects the gene encoding Dittmer, PhD; that of the microbiome. the TATA-box binding protein-associated factor 1 Wenchao Gu, PhD; In particular, how does (TAF1). Within this region is a set of mutations Chris Stokes; the host regulate the mi- common to all XDP patients that is believed to sig- Winfield Hill crobiome and how does nificantly decrease TAF1 expression levels: four sin- the microbiome influence gle nucleotide polymorphisms, five disease-specific the host’s fitness? It is sequence changes, a 48-base pair deletion, and an widely accepted that the host’s innate immunity SVA retrotransposon insertion. Although these dis- can rapidly evolve and is likely a source of micro- ease specific mutations in the patient genome have biome regulation. Specifically, antimicrobial pep- been identified, their roles in the development of tides (AMPs) have been shown to have a role in XDP are not yet well understood. determining host species-specific microbiota commu- nities. One question, then, is that if the microbiome My goal in the XDP project is to further our under- is so vital to the characteristics of a species, how standing of the formation of the disease by studying would the microbiome of an organism be structured if the disease-specific SVA retrotransposon insertion native AMP genes were knocked out or even replaced located in the 32nd intron of TAF1. Retrotrans- with AMP genes from another species? Thus, we ex- posons comprise about 0.13% of the human genome perimentally test how host species-specific immunity and have the capability of influencing local tran- genes affect the composition of species-specific micro- scription. SVA retrotransposons are composed of a biomes in the model animal Nasonia, a genus of par- CCCTCT hexamer repeat, an Alu-like sequence, a asitic wasps. By utilizing the CRISPR-Cas9 genome GC-rich variable number tandem repeat (VNTR), editing system to manipulate the AMP genes of the a short interspersed element (SINE), and a poly-A Nasonia host, we measured phenotypic responses in tail. The SVA retrotransposon insertion could possi- the animal and its microbiome. Genetics 71

This experiment consisted of two steps. First, we Genome-Wide Analysis of Body Proportion developed a CRISPR-Cas9 genome editing system for Nasonia embryos. The CRISPR-Cas9 complex is Hundreds of common usually delivered via microinjection, but this method genetic variants have been Kristin Tsuo discovered to be associ- is technically difficult and time-consuming. We de- Organismic and ated with human height, veloped an electroporation device that is both sim- Evolutionary Biology pler to use and has higher transformation efficiency & Integrative but the genetics underly- than microinjection. Our device consists of two alu- Biology, 2017 ing body proportion have minum electrodes held on top of a microscope glass not been as extensively slide by silicone polymer. The device deviates from Boston Children’s studied. Genetic analy- the traditional electroporation cuvette to make it Hospital, Broad sis of sitting height ra- more suitable for handling embryos. Institute of MIT and tio (SHR), a measure Harvard of body proportion, has Second, we manipulated the immune genes to see yielded insights into the Advisor: Joel their effects on the microbiome. We started by biology of skeletal growth, Hirschhorn, MD, but only a few SHR- knocking down AMP genes of interest with apparent PhD induced expression and then observed the compo- associated variants have been identified. sition of the resulting microbiome via 16S rRNA Mentor: Rany sequencing, quantitative PCR, and fluorescent in Salem, PhD situ hybridization. We then knocked in AMP genes SHR is calculated by di- specific to other Nasonia species to directly study the viding the sitting height, aforementioned question of how changes in species- the length from a person’s specific immunity genes affect the host-microbiota head to the surface on symbiosis. which they are seated, by total (standing) height. To expand our understanding of skeletal growth, we The results of this research help us better under- conducted the largest (to date) genome-wide asso- stand vital host-microbiome interactions, which ciation study of SHR, performed on approximately can yield not only a fuller explanation of the 140,000 individuals of European ancestry. Broadly, a current phenomena in an organism, but also genome-wide association study aims to identify parts the mechanisms that have driven its evolution. of the genome that potentially affect the phenotype being studied, by testing for associations between individuals’ genotypes and their trait values. The first genome-wide association study of SHR found six regions of the genome significantly associated with SHR (Chan et al. 2015). In our study, we have identified more than one hundred novel genetic loci associated with SHR and will conduct further analy- ses to determine the biological pathways and tissues most highly associated with the loci. Together these loci can offer new insights into the genetic and bio- logical basis of body proportion and skeletal growth. 72 Genetics

Application of Open Source Machine Leveraging Large-Scale Databases of Genetic Learning Methods to Enhance Genetic Variation to Study the Effects of LRRK2 Variant Classification Accuracy Loss-of-Function Mutations on Health

The emergence of Parkinson’s disease next-generation sequenc- (PD) is a complex neu- Matthew ing (NGS) technologies Sahar rodegenerative disorder Aguirre Ashrafzadeh has enabled the cost- characterized by progres- Applied Molecular and Mathematics, 2017 effective expansion in Cellular Biology, sive loss of motor con- both number and scale of 2017 trol and cognitive im- Broad Institute of data-generating projects pairment. Approximately MIT and Harvard, that empower medical Broad Institute of 10% of patients have Massachusetts and population genetics. MIT and Harvard, known, disease-causing General Hospital Although the error rate Massachusetts genetic mutations. De- of NGS is very low at the General Hospital spite being the second Advisor: Daniel level of individual bases, most common neurode- MacArthur, PhD errors accumulate over Advisor: Daniel generative disorder, there large data sets, result- MacArthur, PhD are currently no preven- Mentor: Laura ing in false variant discov- tative therapies available Gauthier, PhD ery. Existing tools, such Mentor: Jamie to reduce the progression as the Broad Institute’s Marshall, PhD of the disease. Genome Analysis Toolkit (GATK), have been designed to generate a set of Studies indicate that rare high-quality genetic variants from short NGS reads mutations in leucine-rich aligned to a human reference genome. In the GATK repeat kinase 2 (LRRK2) greatly increase suscepti- pipeline, variant classification is accomplished via a bility to PD. Although the pathogenic mechanism of statistical model built on sequence- and alignment- LRRK2 is not fully understood, amino acid changes based scoring metrics. However, these models have due to missense mutations likely result in enhanced been fine-tuned using truth data from older projects kinase activity of LRRK2 on α-synuclein. In turn, consisting primarily of single nucleotide polymor- phosphorylated α-synuclein aggregates in regions of phisms (SNPs) in well-characterized regions of the the brain, contributing to the progression of PD. genome. They therefore have significant room for Thus, reducing LRRK2 activity in people with gain- improvement in classifying other types of genetic of-function mutations appears to be a promising variation as true data or technical artifact. For therapeutic approach against PD. Before seeking example, the accuracy of indel classification (i.e. a LRRK2 inhibitor, however, we must first under- insertions or deletions of sequence in the sample stand whether decreased presence of the protein has genome with respect to the reference) has histori- detrimental consequences on health. Fortunately, cally been subpar relative to that for SNPs. In this the large collection of human DNA sequencing data project, ensemble methods (e.g. boosting and ran- from relatively healthy individuals in the Exome dom forests), which make predictions based on a Aggregation Consortium (ExAC) provides an oppor- weighted average of the votes of weaker classifiers, tunity to gain insight into the function of LRRK2 were used along with other supervised learning tech- by studying the effects of loss-of-function variants on niques to develop new models for variant classifica- human health. tion. A thorough comparative analysis of these mod- els against those currently in GATK production was The goal of our project is to determine whether a also conducted. Preliminary results show compara- LRRK2 inhibitor may be a safe and effective ap- ble classification accuracy (~91%) for SNPs between proach to reducing risk and/or progression of PD in current GATK production and ensemble methods, individuals who have gain-of-function LRRK2 muta- and significant improvement in the accuracy of indel tions. For this objective, we will first use ExAC to classification (~90% vs. ~75%) under the new model. identify people with potential loss-of-function muta- tions in LRRK2. Our analysis is restricted to het- erozygous individuals since no individuals in ExAC are homozygous for loss-of-function LRRK2 vari- ants—an indication that complete loss of LRRK2 is likely detrimental to survival. Second, we will collect available patient cell lines and use CRISPR- Cas9 genome editing technology to develop cell lines with the mutations of individuals whose cells are Genetics 73 not available. Using these cell lines, we will study pression and function. The phenotype of the mouse protein expression by performing immunoblotting is expected to show abnormal joint development and analyses in order to verify that the candidate mu- possibly osteoarthritis at later lifestages. tations reduce the amount of functional LRRK2 protein present in human cells. Finally, we will This phenotypic change is important when assess- collect phenotypic data from patients and char- ing osteoarthritic risk, because subtle changes in the acterize the effects on health, if any, of loss-of- development of the joints could also increase the like- function mutations in LRRK2. Ultimately, demon- lihood of osteoarthritis since maintaining homeosta- strating that people living with reduced levels of sis of the skeleton becomes more difficult. Specific functional LRRK2 have no adverse health effects human mutations residing within the R37 enhancer will indicate that LRRK2 inhibitors may be a safe were identified that may alter its activity, i.e., drive therapeutic approach against Parkinson’s disease. significantly lower transcriptional activity than the non-risk allele. In order to test the transcriptional modulation of the variant, we use luciferase-based Investigating the Effect of Regulatory readout in mouse cell lines. First, risk and non-risk Genomic Sequences on Susceptibility to versions of the enhancer from human DNA were in- Osteoarthritis serted and amplified using bacterial vectors. Each More than three mil- vector has a built-in promoter region, which initiates lion individuals in the transcription of a nearby luciferase reporter gene. Siddharth U.S. are diagnosed with An active enhancer drives the transcription of the Yarlagadda osteoarthritis annually. luciferase reporter gene, which leads to a higher bio- Human Evolutionary Biology, 2018 Osteoarthritis is a dis- luminescent readout. Thus, if we test both enhancer ease in which the joint variants we should expect to see decreased lumines- Peabody Museum cartilage degrades, lead- cence with the risk allele, corresponding to lower ing to pain and immobil- GDF5 activity. Therefore, the remaining methods Advisor: Terence ity. While environmen- involve inserting the vectors into relevant mouse cell Capellini, PhD tal factors influence os- lines to see if the risk variant significantly affects teoarthritis, genetic fac- transcriptional activity. These experiments will help Mentors: Evelyn tors are particularly im- reveal novel human mutations that play a role in Jagoda, M.Phil.; portant. Growth differen- the etiology of human osteoarthritis, with the goal Jiaxue Cao tiation factor 5 (GDF5 ) of elucidating the mechanisms that lead to disease is the gene region most onset and progression. reproducibly associated with osteoarthritis, with mutation doubling risk. Specifically, risk variants show reduced transcrip- CRISPR/dCas9-VP64-SAM Screen of tional activity in bone and cartilage tissues when Cis-Regulatory Elements of the Cdx2 Gene compared to the activity of alternative alleles that have no association with risk or disease progression. We know to a great Importantly, GDF5 is expressed in key joint areas, extent how information such as the articular surfaces of bones and ligaments Yong (Daniel) stored in the genome as Shen that help stabilize joints. GDF5 mutations in mice DNA produces proteins, Human and humans reveal that the gene is required for Developmental and but we barely understand proper joint, long bone, and tendon development. Regenerative Biology, how this information flow 2019 is controlled by regulatory Given the importance of GDF5, the Capellini lab elements and the tran- has elucidated the regulatory control of the gene Brigham and scription factors that bind during embryogenesis: numerous on/off switches Women’s Hospital to these elements. How (enhancers) around GDF5 control the gene’s expres- do we find which regions sion in specific joints. Within these regions, there Advisor: Richard I. of the genome play an im- are sequences where proteins can bind in order to Sherwood, PhD portant role in gene reg- facilitate transcription of the gene. Each switch po- ulation? How do those tentially influences skeletal development in different Mentor: Budhaditya regions control gene ex- areas. One specific switch, the “R37 enhancer re- Banerjee, PhD pression? My research en- gion,” has been shown to modulate activity in the deavors to answer these knee joint. Currently, we are developing a knockout questions. version of the R37 enhancer in mice. This entails re- moving the region from the genome of the mouse and We are investigating Cdx2, a homeobox transcription observing how its loss leads to changes in GDF5 ex- factor that plays an important role in intestinal tissue 74 Genetics development and intestinal cancer development. Re- tory regions of the Cdx2 gene and offer insights cent studies suggest that the overexpression of Cdx2 into its regulation. We hope that our knowledge of transcription factor is required for the proliferation of the regulation of Cdx2 will contribute to our un- colorectal cancer cells. Cdx2 has also been identified derstanding of intestinal development and cancer. as a promising prognostic marker for intestinal can- cer. Though Cdx2 appears to be a vital regulator of intestinal cell development and cancer development, the mechanism by which Cdx2 is regulated remains largely unknown.

In order to discover the regions important in the reg- ulation of Cdx2 (e.g., cis-regulatory elements), we are employing a modified version of the CRISPR- Cas9 genome editing system. In this genome edit- ing system, the Cas9 protein binds to a guide RNA (gRNA) targeting a region of the genome, and excises DNA at the region specific to the gRNA. Scientists have used CRISPR-Cas9 to introduce double strand breaks at desired locations to create mutations and allow the cell’s repair machinery to incorporate ex- ogenous genes into locations of interest. We have modified the CRISPR-Cas9 system so that transcrip- tion activator proteins could be brought to regions near Cdx2 to induce its expression. To do so, we knocked into mouse embryonic stem cells (mESCs) a catalytically inactive version of Cas9 protein (dCas9) that can no longer cut, fused with ten copies of the transcriptional activation domain VP64—a protein complex composed of four copies of a viral protein that activates transcription. We also designed the SAM activation module—gRNAs that bind strongly to MS2-P65-HSF1 activation helper proteins. To- gether, the VP64 proteins and SAM module (dCas9- VP64-SAM system) have the potential to induce gene expression. Once bound to a gRNA from a li- brary of thousands of gRNAs that cover the regions flanking the Cdx2 gene, the dCas9-VP64-SAM tran- scriptional activation domain fusion protein will tar- get a region of about twenty base pairs near the Cdx2 locus and attempt to activate the gene.

We first attempted to create a Cdx2:GFP mESC cell line for fluorescence-activated cell sorting (FACS). After sorting through cells that give high GFP flu- orescence, we would use next-generation DNA se- quencing to sequence the gRNAs in those cells. From an analysis of the gRNA sequences, we can infer regions important for the activation of Cdx2 gene. However, we were unable to create a Cdx2:GFP mESC cell line. We are now using real-time qPCR to identify cells with high Cdx2 expression and sub- sequently using next generation DNA sequencing to identify gRNAs and regions important in Cdx2 reg- ulation. We will also explore whether fusing dCas9 to a different activator protein module—p300—can identify the same or different set of important reg- ulatory elements. Our project will show regula- Linguistics

Developing a System for Testing the sentences by combining the phrases according Synchronous Tree-Adjoining Grammar to the rules. The system currently handles many Analyses of Linguistic Phenomena simple sentences as well as quantifiers and reflexive pronouns, and I hope to extend it to features such Understanding the as case, person, and number. interface between syn- Jennifer Hu tax (sentence structure) Mathematics & Automating these derivations will allow us to refine and semantics (sentence Linguistics, 2018 the MCTAG formalism to deal with complex lin- meaning) is one of the guistic phenomena and even extend across different most fundamental ques- Harvard John A. languages. By supporting a common derivation of Paulson School of tions in modern linguis- syntactic and semantic structures, MCTAGs could Engineering and tics, and our goal is to provide an elegant model of natural language that Applied Sciences unify the syntactic and will not only help us understand how humans com- semantic structures of municate, but also lead to exciting applications in Advisor: Stuart natural language despite machine translation, speech recognition, and other Shieber, PhD their surface-level incon- tasks in computational linguistics. sistencies. The sentence Mentor: Cristina “John hates every boy,” Aggazzotti for example, has “John” as a syntactic subject but puts semantic emphasis on “every boy.” Two sentences can even have the same semantics but different syntactic structures, such as “John likes Mary” and “It is Mary that John likes.” With incompatibilities like these in mind, lin- guists have struggled to model a parallel derivation for syntax and semantics without requiring extremely complicated rules. Our work offers a potential solu- tion to this problem, suggesting that these surface structures could be derived from the same underlying structure through a formalism called a synchronous multicomponent tree-adjoining grammar (MCTAG). Figure 1: A syntax tree.

Tree-adjoining grammars (TAGs) produce fully- formed sentences by combining trees that represent natural language expressions. To derive the sentence “Bill saw Mary,” for example, we would start with trees that represent the phrases “Bill,” “saw,” and “Mary,” and combine them using the appropriate op- erations specified by the grammar. A synchronous TAG simultaneously applies these composition op- erations across multiple trees, giving us a way to characterize relationships between languages. In fact, we can represent syntax and semantics as two languages defined by TAGs, which allows us to de- rive syntactic and semantic surface trees in parallel through a synchronous process.

To assess the power and limitations of the model, Figure 2: A semantics tree. I have been developing a compiler that converts a high-level description of a grammar into a program that can perform the corresponding MCTAG deriva- tions. The user provides a text-based representation of phrases and grammar rules, and my system pro- duces the final syntactic and semantic structures of Mathematics

Elliptic Curves Investigating the Symplectic Ellipsoid Embedding Function In 2000, the Clay Mathematics Institute fo- A symplectic man- Alan Bidart cused its attention on ifold is a smooth (dif- Computer Science, seven unsolved questions, Handong Park ferentiable) manifold 2018 Mathematics, 2018 which leading mathe- M equipped with a maticians considered of non-degenerate skew- Harvard University Harvard University key importance to mod- symmetric bilinear form Advisor: Joseph D. ern mathematics. These Advisor: Daniel ω, called a symplectic Harris, PhD problems became known Cristofaro-Gardiner, form. Understanding as “The Millennium Prob- PhD these manifolds has im- lems.” Of these seven portant physical applica- problems, six remain unsolved, and my project for tions, as symplectic geom- the summer is based around developing a better un- etry comprises the fundamental mathematics under- derstanding of the premises behind one of them, the lying Hamiltonian mechanics. “Birch and Swinnerton-Dyer Conjecture,” through the study of elliptic curves. To understand the properties of these manifolds, mathematicians often ask whether one manifold can An elliptic curve is a special type of order 3 poly- be embedded inside another. We say that a symplec- nomial on two variables which usually has the form tic manifold (M1, ω1) can be embedded into another 2 3 y = x + ax + b, where a and b are constants. For symplectic manifold (M2, ω2) if there exists an injec- −1 every different pair of a, b there will be different set tive map φ: M1 → M2 such that its inverse (φ ) is ∗ ∗ of solutions x, y to the equation. Moreover, for a par- smooth and its pullback (φ ) satisfies φ (ω2) = ω1. ticular pair, the set of solutions for the elliptic curve could have no elements, a non-zero finite number of Mathematicians haven’t uncovered the exact con- elements, or an infinite number of elements. The ditions under which a general symplectic manifold BSD Conjecture establishes a relationship between embeds into another; however, we can tackle the the structure of the set of solutions of a given elliptic problem for specific manifolds. One such specific curve with a, b belonging to the rational numbers manifold is the four-dimensional symplectic ellipsoid and the behavior of some special type of functions in R4 with axes of lengths a and b, denoted E(a, b). called L-functions. To better understand the general case, we can study the conditions that govern embeddings of these spe- The study of these sets of solutions is of particular cific ellipsoids. In this research project, we attempt importance not only to mathematicians, but also to study such ellipsoid embeddings by investigat- to computer scientists, because these sets have a ing the crucial ellipsoid embedding function C(x, y). group structure. In simple terms, a group is a set Given x and y, this function gives the smallest con- of elements that can be composed with each other, stant λ for which the ellipsoid E(1, x) embeds into under a given operation, with the certainty that the the ellipsoid E(λ, λy). result of the composition is still an element in the set. For example, the integers form a group: if we Previously, values for the ellipsoid embedding func- add two integers, the result will still be an integer. tion were only known when y is 1. It was known that Progress towards the proof of the BSD conjecture for x less than the critical point (17/6)2, C(x, 1) is would help predict the behavior of elliptic curves given by a piecewise linear function with infinitely over the rational numbers and lead to developments many horizontal pieces. In addition, researchers also in cryptography and computational algorithms. knew that for x greater than or equal to (17/6)2, the function is given precisely by the square root of x.

This project will determine such critical points for other y-values as well. By using computer-assisted computational methods to apply number-theoretic approaches to the problem, we can estimate C(x, y) for a range of x- and y-values. Using these estimates, we can attempt to find critical x-values when y 6= 1, and identify patterns among the shifting points. Mathematics 77

Throughout this project, we will attempt to prove We will discuss the question of when we can ex- such patterns, which provide new knowledge on the pect a hypersurface of a given degree d in n vari- nature of the ellipsoid embedding function C(x, y) ables to contain lines. We will find that when and could fuel new advances in solving symplectic 2n − d − 3 ≥ 0, a general hypersurface of degree embedding problems. Along with these patterns, d contains lines, but when 2n − d − 3 < 0, a we will also attempt to develop sharp estimates for general hypersurface of degree d contains no lines. several different points of the ellipsoid embedding function to better understand its overall behavior. Enumerative Problems in Algebraic In the future, these developments could eventually Geometry help us not only describe, but also determine an ex- plicit formula for the ellipsoid embedding function, Algebraic geometry which would improve our understanding of symplec- seeks to study, from a tic geometry and its various physical applications. James Hotchkiss geometric point of view, Mathematics, 2018 solutions to systems of multivariate polynomi- Lines on Hypersurfaces Harvard University als. Central to the field Algebraic geometry is Advisor: Joseph D. is preservation of num- the study of geometric ob- Harris, PhD ber—the idea that even if Hannah Larson jects, called varieties, that one varies the coefficients Mathematics, 2017 arise in the following alge- of polynomials in a sys- braic way: an (affine al- tem, the number of solutions to the system generally Harvard University gebraic) variety is defined remains the same. The desire to exploit this idea has Advisor: Joseph D. as a collection of points spurred numerous developments in algebraic geome- Harris, PhD that are simultaneous so- try and continues to propel the subject. lutions to a set of polyno- mials. When the variety Preservation of number gives rise to the possibility is defined by a single polynomial, it is called a hy- of solving an enumerative problem, a problem that persurface. The degree of a hypersurface is the de- asks for the number of geometric objects satisfying gree of the polynomial defining it, where the degree some conditions. For example, given three circles in of a polynomial in many variables is the largest to- a plane, one might ask how many circles one can find tal degree of all terms. An example of a degree 2 that are tangent to all three (where two circles are hypersurface we’ve all seen before is the solutions of said to be tangent if they meet at a single point). the polynomial x2 + y2 − 1: the collection of points Such problems are often classical; a variant of the (x, y) in the plane that satisfy x2 + y2 − 1 = 0 is above problem was first posed by Apollonius of Perga just the unit circle. A variety defined by multiple (262 BC–190 BC). Preservation of number suggests polynomials is the intersection of the hypersurfaces that the answer to Apollonius’ problem does not corresponding to those polynomials. generally rely on the original position of the three given circles. Given a variety, we might want to understand what varieties of a certain type are contained in it. The One branch of my research is focused on solving simplest such question is: when does a given hyper- enumerative problems, which proceed in five steps. surface contain a line? Trivial examples of hypersur- First, we try to find an appropriate parameter space, faces containing lines are lines themselves, planes, a geometric space with nice properties, the points of and more generally all degree 1 hypersurfaces. As a which correspond to the objects of interest. Second, more interesting example, consider the degree 3 hy- we calculate an algebraic invariant of the param- persurface defined by x3 +y3 +z3 −1, that is triples of eter space known as the Chow ring, whose points complex numbers (x, y, z) such that x3 +y3 +z3 = 1. loosely represent the objects satisfying additional It turns out that this hypersurface contains 27 lines: conditions. For example, in the Chow ring of the if ω = e2πi/3 is a complex number whose cube is parameter space of circles, there is a point repre- 1, then you can check that there are nine lines of senting the circles tangent to a single given circle. the form x = ωa, y = −ωbz where a, b ∈ {0, 1, 2}; Third, we identify each point in the Chow ring for nine of the form y = ωa, x = −ωbz; and nine of the each condition that one has imposed. Fourth, we form z = ωa, x = −ωby. In fact, this example is calculate the point in the Chow ring representing representative of a more general phenomenon: every objects satisfying all of the conditions. At this step, smooth hypersurface of degree 3 contains exactly 27 the point in the Chow ring becomes an enumerative lines. (Here smooth means that the partial deriva- formula, which, if the objects in question behave tives of the defining polynomial do not all simulta- nicely, yields the correct answer to the enumerative neously vanish at some point on the hypersurface.) problem. Fifth, we attempt to prove that the objects 78 Mathematics in question behave nicely.

A second branch of my research focuses on answer- ing subtler questions about the solutions to a given enumerative problem. For example, in Apollonius’ problem, can we solve for the equations defining the tangent circles directly from the equations of the given circles? This line of inquiry leads to a notion of the Galois group of an enumerative prob- lem, which encodes the geometric structure of the solutions to an enumerative problem, and we can solve for the solutions to our problem if and only if the Galois group satisfies an algebraic condition known as solvability. Finding a Galois group re- quires a delicate analysis of the problem’s geometry and is more difficult than the enumerative prob- lem. Indeed, there are many long-solved enumera- tive problems for which the Galois group is unknown. Molecular and Cellular Biology

Generating a Comprehensive Alveolar cleaving the genome at a later time in development Rhabdomyosarcoma Model in Danio rerio and allowing the chromosomes to reattach improp- erly, mimicking natural ARMS. The fish are injected Rhabdomyosarcoma is as single-cell eggs with CRISPRs designed to execute the most common pe- Alexander H. Jin this task and allowed to grow to adulthood. Upon diatric soft core tissue maturation, the injected fish are then screened for cancer, afflicting approx- Molecular and the insertion in two ways, the first being a simple imately 4.6 children out Cellular Biology, evaluation as to whether or not any of the cells in 2018 of every million in the the organism have taken the construct at all. For United States (Gurney et the individuals that pass this first screen, the next Massachusetts al., 1996). The ma- is a deeper sequencing to measure the frequency of General Hospital lignancy is divided into such insertions in the fish. two subtypes, alveolar Advisor: David (ARMS) and embryonal The current project has progressed up to this point. Langenau, PhD (ERMS), the former of While a few potential mosaic founders (fish that which is more aggres- have mutations in some of their cells, but not Mentor: Madeline sive and difficult to treat. Hayes, PhD all) have been identified, no genetically pure off- Molecularly, the two sub- spring have been identified and isolated, indicat- types are distinguished by ing that none of the insertions have taken hold a chromosomal transloca- in the germline cells of the injected fish thus far. tion in ARMS that increases tumor aggressiveness Newer and hopefully better variants of the sec- in comparison to ERMS (Barr et al., 1992). As a ond method are currently being tested as well, in survivor of Stage 4 alveolar rhabdomyosarcoma, this the hopes of generating the first pairs of mutant translocation and its consequences are of particular fish for breeding, testing, and, with luck, the first and poignant interest to me. artificially-induced alveolar rhabdomyosarcoma tu- mors in zebrafish ever, by the end of this summer. The Langenau Lab specializes in and is at the fore- front of developing novel disease models in Danio re- rio, or zebrafish. With low maintenance costs, high Modulation of Intestinal Epithelial rates of reproduction, and ease of genetic manipu- Homeostasis and Microbiota with Exercise in lation, zebrafish are ideal organisms for generating a Murine Model new models of genetically complex malignancies such as ARMS. With the continued development of the Inflammatory bowel incredibly powerful CRISPR-Cas9 genome editing disease (IBD) is a clas- technology, inducing desired mutations is also eas- Alexander sification of diseases char- Munoz ier than ever. acterized by inflamma- Applied Mathematics, 2018 tion of the colon and Additionally, the current field of research is lack- small intestines. Common ing in alveolar rhabdomyosarcoma animal models. Massachusetts symptoms of IBD include Of the rhabdomyosarcoma models in both zebrafish General Hospital abdominal pains, rectal and mice, only one is not of the embryonal subtype. bleeding, vomiting, and Therefore, this summer’s project is a continuation of Advisor: Richard diarrhea. These symp- work begun last year to generate a faithful and com- Hodin, MD toms often present in prehensive model of alveolar rhabdomyosarcoma in “flares,” where the symp- zebrafish by using CRISPR genome editing technol- Mentor: Sulaiman toms are suddenly acti- ogy. If successful, this will not only be the first ze- Hamarneh, MD vated for a time period. brafish and best animal model of ARMS, but will also In fact, IBD patients’ flare present a groundbreaking proof-of-concept regarding management can have a fusion gene models, which have not been attempted significant psychological and social impact on their in zebrafish and of which ARMS is only one of many lives, as frequent flares can decrease self-esteem and diseases that can be modeled using the techniques we isolate a patient from social spheres. Thus, finding are pioneering here this summer. a methodology to increase the time between IBD flares would not only be a somatic therapy for pa- The primary approach is to insert a construct at tients, but also a psychological and a social one. But the splice site that can be activated when desired, while nearly 400,000 Americans are newly diagnosed 80 Molecular and Cellular Biology with IBD each year, both the cause of the disease as in the exercise-induced colitis prevention pathway. well as preventative measures are still not entirely clear. Towards the Structure of an Outward-Facing Nramp Transition Metal Transporter Current research has shown that IBD patients have both decreased microbiotal diversity as well as in- Transition metal ions, creased intestinal permeability. In addition, obser- such as iron and man- vational research has shown that exercise has a pre- Brandon Lee ganese, are essential to Statistics & ventative correlation with intestinal inflammatory life, from enabling oxygen Molecular and disease; however, formal research has not yet shown Cellular Biology, transport to the catal- that relationship. As a result, we chose to investigate 2019 ysis of redox reactions. the effect of exercise on intestinal inflammation in a These ions are main- murine model. Mice were separated into two groups: Harvard University tained in a crucial bal- one that underwent two weeks of daily, rigorous ex- ance within organisms by ercise and one that was sedentary for the two-week Advisor: Rachelle various mechanisms, in- period. Within those groups mice were divided into Gaudet, PhD cluding specialized mem- two subgroups. One of the subgroups was treated brane transport proteins with one week of 3% dextran sodium sulfate (DSS) Mentor: Aaron Bozzi such as Nramp family in drinking water, while the other retained normal proteins. Nramps (nat- autoclaved drinking water. Because DSS causes ural resistance-associated chemically-induced inflammation of the colon, it has macrophage proteins) likely use an alternating access been established as a model for colitis. Following the transport mechanism, with the protein alternating exercise and DSS colitis phases, mice were sacrificed. between at least two states: one with the substrate- Intestinal permeability and gut microbiotal diversity binding site accessible to extracellular space, and were quantified. In addition, the colon was formalin- another with the binding site accessible to the cyto- fixed, paraffin-embedded, sectioned, and scored for plasm. The structure of an outward-facing confor- the degree of colitis via histopathology. Using this mation remains unknown, in addition to the mech- methodology of exercise and colitis induction with anism that alternates between the different facing DSS, we compared the exercised and DSS-treated states. I engineered Nramp proteins locked in an mice to the controls. We thus quantified the effect outward-facing state from which I now aim to obtain of exercise as a preventative therapy for the develop- an atomic resolution structure by crystallography. ment of increased intestinal permeability, decreased microbiotal diversity, and increased inflammation, I introduced individual mutations to tryptophan at which are all associated with colitis. Conclusive dif- eleven positions within the Deinococcus radiodurans ferences between the subgroups of mice (for example, Nramp (DraNramp), which I predicted would be ex- one where exercise induces increased innate immune posed in an outward-facing conformation. I reasoned regulation, thus preventing harsh acute colitis with that tryptophan, as the largest amino acid, may DSS treatment) could yield the potential for exercise force DraNramp into a locked outward conforma- as a human colitis preventative measure—and could tion. This strategy was previously successfully used have therapeutic value to patients attempting to in- in another transporter, lactose permease. This sum- crease time between IBD flares. mer I conducted preliminary screens of the mutants’ conformational preference to determine whether each Following confirmation of the protective role of ex- tryptophan mutation is outwardly-locking the pro- ercise in colitis, we chose to investigate the cause tein. of this relationship. To investigate this, we noted a significant exercise-induced upregulation of a brush- First, I used in vivo cobalt-uptake assays to quan- border gut enzyme—intestinal alkaline phosphatase tify the relative cobalt transport by my DraNramp (ALPI). We hypothesized that the upregulation of variants. I expected that the tryptophan mutants this enzyme, which is involved in regulating intesti- would exhibit varying degrees of cobalt transport, nal permeability, had a causative role in exercise’s with low activity supporting the hypothesis that the colitis prevention. We thus bred mice lacking ALPI mutant is indeed locked. Consequently, six of my and compared the phenotypes with wild-type mice eleven mutants demonstrated the lowered transport via a repetition of the exercise/DSS experiment. activity. Significant differences between the two groups (such as exercise serving a preventative role for colitis in I also used assays that take advantage of the unique the wild-type group but not in the group lacking reactivity of cysteine to form covalent bonds with ALPI) could demonstrate a necessary role for ALPI its sulfhydryl group. A cysteine must be solvent- accessible to chemical probes, such as the small Molecular and Cellular Biology 81 membrane-permeable N-ethylmaleimide (NEM), to Manipulating Cellular Morphology through be chemically modified. By collecting data from Light Activation of Membrane various single-cysteine DraNramp mutants, I can in- Curvature-Inducing Proteins fer the likely conformational state imposed by the tryptophan mutation (see Figure 1). After using Neurons have complex both inward- and outward-facing reporter cysteines, cellular morphology, with I have identified six mutants which are highly likely Catherine Li highly branched dendrites Chemical and to be locked in an outward-facing state; all six mu- and axons extending from Physical Biology, tants also displayed low transport activity. 2019 the cell body to receive or send electrochemical sig- I plan to use additional inward- and outward-facing Harvard University nals. Understanding the reporters in cysteine accessibility assays and mea- growth of these projec- sure binding data for my mutants with various metal Advisor: Adam E. tions could allow for the ligands in comparison with the WT DraNramp. Fol- Cohen, PhD manipulation of new func- lowing these studies, I will move forward to obtain tional synapse formation X-ray diffraction data of my mutants. I will em- Mentor: Hongkang and shed light on its role ploy membrane-protein specific techniques such as Zhang, PhD in neurological disorders. lipidic cubic phase (LCP), which has been success- fully employed in the crystallization and structure I sought to demonstrate a determination of two conformations of an unrelated light-inducible system for metal transport protein. As initial crystal hits often initiating growth of filopodial and neurite projec- diffract to low resolution, I will also attempt to opti- tions in HEK cells and neurons, respectively. We mize crystallizable constructs by truncating and/or harness the activity of membrane curvature-inducing mutating the loop regions that appeared disordered proteins belonging to the Bin/Amphiphysin/Rvs in our inward- facing structure. (BAR) domain protein superfamily. Each class of BAR domain proteins has a uniquely curved three- dimensional shape, and positive residues on one sur- face of the protein allow it to bind directionally to a cell’s negatively charged plasma membrane. Once bound, the curved BAR domain acts as a “scaffold” to which the membrane conforms, resulting in local membrane curvature that can trigger cellular out- growths. Depending on the class of the BAR do- Figure 1: L374W shows evidence of an main, the induced membrane curvature can be either outward-facing solvent-accessible vestibule.I inwardly (F-BAR, N-BAR domains) or outwardly (I- introduced each tryptophan mutation in an A61C BAR domain) oriented. Our work focuses on MIM background, serving as a reporter for that the (an I-BAR protein) and FBP17 (an F-BAR protein). outward-open conformation. I then applied different NEM concentrations to the resulting DraNramp vari- We coupled the membrane-bending activity of each ants, denatured the protein, and added a PEG5K- BAR domain protein to light signals through co- maleimide to mark any unmodified cysteines. Fi- expression with the plant photoreceptor Cryp- nally, I compared the amount of A61C modified tochrome 2 (CRY2) and its photodimerization part- by NEM vs. PEG5K-maleimide using a Western ner, calcium- and integrin-binding protein (CIB), in Blot. NEM will modify A61C at lower concentra- cultured cells. The BAR domain protein is fused di- tions in an outward-locked mutant, protecting it rectly to CRY2, while CIB is targeted to the plasma from PEG5K-maleimide modification. The WT pro- membrane by a genetically encoded CAAX motif. tein, sampling both the inward- and outward-facing Blue laser or LED stimulation excites CRY2, caus- conformations, requires a larger NEM concentration ing it to stick to membrane-bound CIB, thereby also to protect A61C from PEG5K-maleimide modifica- bringing the fused BAR domain to the membrane. tion. In contrast, L374W, an outward-locked candi- We hypothesize that the BAR domain will bind, in- date, is protected at a smaller NEM concentration. duce curvature, and promote filopodia or dendritic spine growth. By localizing the initial blue light stimulus to a certain population of cells, or to a spe- cific site on a single cell’s membrane, we predict that this light-inducible system will allow us to command the formation of membrane projections with spatial precision at designated locations and times. These structures may in turn develop into functional spines, 82 Molecular and Cellular Biology which interface and communicate with axons of other a middle fragment of RNase Y (to account for the cells at a newly-formed synaptic junction. This abil- possibility that binding might occur at residues in ity to induce outgrowths and construct new synapses the middle of the protein that span the first and between neurons using simple light stimulation would second halves), then introducing these to plasmids open doors to many experimental pathways in the and transforming bacteria with these plasmids. Ulti- study of neural networks and neurological disorders. mately, I hope to make successful constructs of both RNA polymerase and the regulatory region fused to the first and second halves of RNase Y. These will Investigating Interactions Between RNaseY be tested with constructs containing YlbF, as well and the Y Proteins as different controls. RNase Y, an endori- bonuclease in Bacillus If successful, this experiment can lead to further Eliza Llewellyn subtilis, is responsible for understanding of this important ribonuclease. Rep- Molecular and cleaving hundreds of tran- etitions of the search—by splitting whichever part of Cellular Biology, 2018 scripts, playing an impor- RNase Y interacts with YlbF again in half—can more tant regulatory role in the precisely identify the region of interaction between Harvard University cell. Strains without the RNase Y and the Y complex. Then, we can explore gene for RNase Y have de- the effect of amino acids substitutions at this region Advisor: Richard fects in biofilm formation, to see if RNase Y can cleave transcripts independent Losick, PhD competence, and sporula- of the Y complex. We will also use co-evolutionary tion, strategies important analysis to find candidate regions to test. This Mentor: Aaron for B. subtilis in stressful work will help us learn more about this impor- DeLoughery, PhD conditions. Bacterial two- tant ribonuclease and will elucidate details about its hybrid experiments have recently-discovered interaction with the Y proteins. shown that YlbF, YmcA, and YaaT (the Y proteins), required for biofilm for- The NLGN4X Gene as a Potential Target for mation, interact with RNase Y and may be necessary Clinical Therapy in Triple Negative Breast for RNA cleavage. My work this summer will inves- Cancer (TNBC) tigate the exact region where RNase Y and YlbF interact, using the concept of binary search. This in- Breast cancer is not a formation will help us learn more about the protein, single disease, but rather and it will serve as a basis for further experimenta- Felipe Flores a family of diseases with Human tion. Using information about where RNase Y binds distinct molecular sub- Developmental and to accessory proteins, we might ask whether or not Regenerative Biology, types. Among these it is able to function at all without these proteins. 2019 is triple negative breast cancer (TNBC) lacking The foundation for this experiment is a bacterial Dana-Farber Cancer the expression of estro- two-hybrid experiment, which identifies interaction Institute gen receptor (ER), pro- between proteins. One protein is fused to RNA gesterone receptor (PR), polymerase, while another is fused to a DNA bind- Advisor: Kornelia and human epidermal ing domain. The interaction will be tested in both Polyak, MD, PhD growth factor receptor directions by building plasmids that contain RNase 2 (HER2), hence the Y and YlbF fused to both RNA polymerase and the Mentor: Bojana name “triple negative” binding domain. These are expressed in a reporter Jovanovic, PhD (Foulkes, Smith, & Reis- strain in which the DNA binding domain is upstream Filho, 2010). Although of reporter gene lacZ. If there is interaction between TNBC represents only the proteins, they will bind to each other, which in 15–25% of all breast cancer cases in the U.S. (Foulkes turn will localize RNA polymerase to the binding et al., 2010), it nevertheless contributes to a signif- domain just preceding the reporter gene lacZ. lacZ icant fraction of breast cancer-related deaths, and will then be transcribed, and its activity can be mea- in general has a poor prognosis mainly due to lim- sured and ultimately used to quantify the degree of ited treatment options beyond chemotherapy (Hudis interaction. This project will involve searching for & Gianni, 2011). Hence, one important focus for residues or domains of RNase Y to find the exact TNBC research is the search for alternative TNBC region of interaction with YlbF, beginning by test- targets in order to find more effective treatments. ing the different halves of the protein, then searching within the half of the protein that shows a positive The focus of this project is to evaluate the role of interaction. Steps will include creating PCR prod- NLGN4X, encoding for a protein implicated in neu- ucts of the first and second halves of RNase Y and ral survival, in TNBCs. Specifically, we hypothesize Molecular and Cellular Biology 83 that NLGN4X is required for the survival and pro- Transuclear iNKT Mice Show that iNKT liferation of TNBC cells and may also play a role Functional Subsets are not Determined by in maintaining them in a poorly differentiated stem TCR Specificity, but Correlate with Tissue cell-like state. Polymorphisms in NLGN4X have of Residence been associated with autism and Asperger syndrome (Laumonnier et al., 2004), but it has not been studied Natural killer T in cancer. Based on our analysis of single nucleotide (NKT) cells are a sub- polymorphisms (SNPs) of somatic cell fusions de- Gui Zhen Chen set of T cells that play a Molecular and rived from luminal and TNBC cell lines (Su et al., crucial role in immunity, Cellular Biology, Cell Reports 2015), a SNP linked to NLGN4X was 2019 influencing the immune the only SNP that was associated with the inheri- response towards diseases tance of basal features (resembling another subtype Dana-Farber Cancer such as diabetes, inflam- of aggressive breast cancer). Institute matory bowel disease, and cancer. These cells Based on this interesting finding we decided to eval- Advisor: Stephanie recognize lipid antigens uate whether NLGN4X has any functional proper- Dougan, PhD presented through the ties in TNBC cells. Thus, we silenced the expres- MHC class I-like molecule sion of this gene in SUM159 TNBC cell line using Mentor: Eleanor CD1d, a set of surface doxycycline-induced shRNA system. The cells were Clancy-Thompson, proteins found on antigen- manipulated with lentivirus to introduce engineered PhD presenting cells. There fragments of RNA into the cells, and then treated are two main types of with doxycycline or kept untreated as a control, NKT cells; here we fo- providing us great power over NLGN4X’s expres- cus on type I, invariant NKT (iNKT) cells, which sion. A proliferation assay showed that silencing express a fixed TCRα (T cell receptor) chain that of NLGN4X significantly decreased cell prolifera- is complemented with a limited repertoire of TCRβ tion. We will replicate the experiment in a wider chains. Once activated, these cells rapidly produce spectrum of cell lines, representative of the different key cytokines, which are chemical signals that can TNBC subtypes. If the results are similar to those skew the subsequent T cell immune response. iNKT obtained in SUM159 cells, we will perform xenograft cells are classified into functional subsets that are dis- assays in mice, where cancer cell lines will be trans- tinguished by varying production of cytokines and planted into mammary gland of mice to evaluate signature expression of transcription factors during the effect of NLGN4X knockdown on tumor growth. thymic development. Among these subsets it has We will also characterize molecular and phenotypic been shown that a combination of the the transcrip- changes that may occur as a consequence downreg- tion factors Tbet, PLZF, and RORγt distinguishes ulation of NLGN4X in TNBCs including charac- NKT1, NKT2, and NKT17 subsets that secrete the terizing gene expression changes by RNA-seq and cytokines IFNγ, IL-4, and IL-17, respectively. assessing cell migration, invasion, and metastatic be- havior. The successful completion of the proposed The question remains as to what factors affect the project may link a gene implicated in neural progeni- lineage choice of iNKT cells. Therefore, our research tors to TNBCs, highlighting the epigenetic plasticity sought to determine 1) whether the specificity of the of this breast cancer subtype and identifying a poten- TCRβ chain impacts lineage choice and 2) whether tial novel therapeutic target in a subset of TNBCs. the tissue of origin impacts lineage choice. To address this, we used transnuclear mice generated through somatic cell nuclear transfer, using the nuclei of iNKT cells specific for different TCRβ chains to generate four lines of mice. One line contains poly- clonal mice that expresses iNKT cells with diverse beta chains, and thus, different TCR specificities. Each of the remaining three lines are of monoclonal specificity, meaning each line has one unique TCRβ chain, causing their TCRs to have the same speci- ficity. These mice have increased numbers of iNKT cells in their organs compared to wild-type mice, which allows for the study of rare populations of iNKT cells.

To determine whether the specificity of the TCRβ chain impacts lineage choice, we harvested thymus 84 Molecular and Cellular Biology and spleen from the transnuclear mice and analyzed tract pathogen. P. mirabilis colonies exhibit kin iNKT cells for expression of transcription factors recognition behavior while swarming on solid sur- and cytokines through flow cytometry, a method faces: two genetically identical swarms will merge, that detects cells successfully stained with fluores- while swarms of two different strains form a macro- cent antibodies that bind to the targets of interest. scopic boundary. Furthermore, when two different First, intracellular staining for IFNγ, IL-4, and IL- strains form a mixed co-swarm, one will territorially 17 assesses cytokine production by these iNKT cells. exclude the other from the swarm front. The Gibbs To determine lineage choice, we then stained intra- lab uses P. mirabilis’s behavior as a model for bac- cellularly for the transcription factors PLZF, Tbet, terial kin discrimination to study the social behavior and RORγt. Together, both stainings will allow us of bacteria and other microbes. to determine if skewing to a certain subset (NKT1, NKT2, NKT17) occurs in our monoclonal mice. If The Gibbs lab has identified a key gene cluster in- the TCRβ chain plays a determinate role in iNKT volved in P. mirabilis kin discrimination: Ids. Pre- cell lineage choice, we would expect to see skewing vious experiments indicate Ids works in a type VI of monoclonal populations towards one subset. secretion system (T6SS)-dependent fashion to intro- duce proteins into neighboring cells which mediate To determine whether tissue of origin impacts lin- kin discrimination behavior. The Ids cluster includes eage choice, we repeated the same analysis on iNKT six genes, two of which encode proteins that mediate cells from multiple organs in the transnuclear mice. kin identity: IdsD, an exported protein that signals If the tissue of origin plays a role, we would expect kin identity, and IdsE, which binds cognate IdsD and to see skewing of all iNKT cell subsets in a particular is thought to mediate the kin recognition response. organ. The Gibbs lab has produced an idsD deletion strain, an idsE deletion strain, and an exchange mutant in The central innovation of this project lies in the which the idsE is exchanged with that from another use of mice cloned by somatic cell nuclear trans- strain to study the mechanics of kin discrimination. fer as they provide an abundant source of iNKT The Gibbs lab has also created a strain deficient in cells to study. Expansion of patients’ iNKT cells type VI export, which continues to receive the IdsD is an approach currently in clinical trials for can- identity signal from other P. mirabilis cells yet can- cer, and the results have been modest, but promis- not export the signals itself. ing. As iNKT cells can rapidly produce cytokine in response to antigen, the knowledge of how to in- This summer, I used these idsD deletion, idsE dele- duce particular subsets would enhance the efficacy of tion, and idsE exchange mutants to study how mu- iNKT cell therapy. Therefore, we aim to determine tations of idsD and idsE affect kin recognition and which factors influence iNKT cell lineage choice to understand how these genes mediate kin discrimina- better harness them for future anti-cancer studies. tion, specifically in cases where liquid cultures of two strains are allowed to swarm together. I studied these mutations in a T6SS mutant background, thereby The Effects of Kin Discrimination on P. simplifying the two-way interaction of kin discrimi- mirabilis Swarming in Type VI Secretion nation and better isolate how idsD and idsE mediate Mutants kin discrimination effects on non-related bacteria. To When one thinks of so- study this behavior, I characterized the behavior of cial organisms, humans, T6SS mutants with idsD and idsE mutations when Ian Maynor other primates, or pack co-swarmed with the wild-type parent strain. In Linguistics, 2017 animals such as dogs come addition, previous experiments have found that the to mind. Yet even mi- starting ratio of the two strains inoculated together Harvard University crobes display fascinating in a co-swarm can affect Ids-mediated kin recognition Advisor: Karine social interactions, such behavior. I therefore also varied the starting ratios Gibbs, PhD as quorum sensing, altru- of each strain to investigate ratio-dependent changes ism, and kin discrimina- to kin recognition in these co-swarms. Mentor: Murray tion. Kin discrimination Tipping, PhD is the ability of individual Through these experiments, we hope to further our organisms to differentiate understanding of the exact mechanism by which other individuals in a pop- idsD and idsE allow P. mirabilis’s kin discrimi- ulation as genetically related or not and modify their nation system to affect swarming behavior. This behavior accordingly. knowledge will expand our model of kin discrim- ination in P. mirabilis and allow us to apply The Gibbs lab studies kin discrimination in the bac- these insights to social behavior in other microbes. terium Proteus mirabilis, an opportunistic urinary Molecular and Cellular Biology 85

creates a negative cross-feedback to Hsf1 activity that prevents expression, either basal and/or stress- Hsf1 Activity and Cell Fitness in Yeast inducible, of HDGs that are not targets of Msn2/4, Aging which I will call strict HDGs. In their folded states, proteins are the Since the basal and heat-induced expression of HDGs Jason Li workhorse of cellular ma- depends on Hsf1 in young cells, I will examine the Molecular and chinery. When exposed expression of HDGs during aging. I will fluorescently Cellular Biology, 2018 to proteotoxic stresses, tag endogenous HDG proteins, and track their ex- proteins misfold and ag- pression levels by fluorescence in both basal and Harvard University gregate, disrupting cell heat-stress conditions during various ages. Since function. One of the hall- Msn2/4 represses Hsf1 heat-inducibility in old cells Advisor: Vlad Denic, marks of aging is the ac- (measured by a transcriptional reporter), I expect to PhD cumulation of protein ag- see reduced HDG expression in old cells. Complicat- gregates in old cells. By ing matters, however, is the fact that some HDGs contrast, young cells re- are also regulated by Msn2/4, which becomes con- sist the accumulation of protein aggregates, a phe- stitutively active in old cells. My hypothesis is that nomenon known as protein homeostasis (proteosta- basal expression of strict HDGs will be reduced in sis). Exposure of young cells to heat shock revealed aging cells. I will also examine the effect of unin- a striking example of proteostasis: heat transiently ducible Hsf1 in old cells on tolerance to a variety induces the formation of protein aggregates that of genetic perturbations that normally induce Hsf1. become resolved over time due to transcriptional Genome-wide screening has identified gene deletions up-regulation of protein folding and disaggregation that induce Hsf1 activation but not Msn2/4. Some factors. of these mutations disrupt protein targeting and mis- folded protein degradation, suggesting that Hsf1 ac- Studies in the budding yeast have led to the discov- tivation is adaptive for these types of internal stress. ery of many longevity factors conserved across the I will call these strict Hsf1 stressors. My hypothesis eukaryote lineage, making it a good model system is that Msn2/4 activation by way of cross-feedback to study aging. In yeast, the heat shock response is inhibition of Hsf1 makes old cells uniquely sensitive mediated by activation of transcription factors Heat to strict Hsf1 stressors. To investigate this, I will shock factor 1 (Hsf1) and Msn2/4. By contrast, there induce degradation of strict Hsf1 stressor in both are numerous cellular processes that, if genetically young and old cells, and fluorescently track if Hsf1 disrupted, only activate Hsf1 but not Msn2/4. Hsf1 is no longer activated in old cells following strict is presumed to be essential for cell viability due to Hsf1 stress and how this affects old cell morbidity. its basal transcriptional program, the disruption of which is lethal to yeast. The Denic lab identified Subclonal Cooperation in Triple Negative the basal core transcriptional program of Hsf1 and Breast Cancer—A Study in Intratumoral termed these genes Hsf1-dependent genes (HDGs), Heterogeneity showed that restoring basal expression of just two HDGs encoding the folding chaperones Hsp70 and Cancer has recently Hsp90 enables young cells to live without Hsf1, and been established as a het- suggested that Hsf1 is critical for thermotolerance Jett Crowdis erogeneous disease com- Molecular and because it up-regulates expression of its basal gene posed of phenotypically Cellular Biology, targets. 2019 and genetically diverse cell subpopulations. This Preliminary work with fluorescent transcriptional re- Harvard Medical phenomenon, known as porters shows that in old yeast cells Msn2/4 is con- School intratumoral heterogene- stitutively active (i.e., in the absence of stress) while ity, is poorly understood, Hsf1 activity becomes uninducible by heat shock. Advisor: Joan but it has important im- Moreover, genetic experiments have suggested that Brugge, PhD plications for drug resis- Hsf1 inducibility may be repressed by Msn2/4 ac- tance, metastasis, and tu- tivity. My goal is to better define the phenomenon Mentor: Sabin mor development. The of deregulation of Hsf1 and Msn2/4 during yeast Dhakal population dynamics of replicative cell aging and test if it has consequences the tumor ecosystem have on cell aging. My working hypothesis is that Msn2/4 long been thought to be activation in old cells is a double-edged sword. On governed by competition for scarce resources, but re- the one hand, it is most likely helping cells respond cent studies have indicated that some subpopulations to some type of internal stress. On the other, it can communicate via secreted proteins, leading to co- 86 Molecular and Cellular Biology operation within the tumor. The exact mechanism we have seen decreased growth of some clones through which subclones crosstalk and the extent to in the coculture assay. Identifying which clones which this impacts tumor progression is largely un- can support other clones will be important, as we known. can later examine their genetic and proteomic pro- files to identify any active pathways that can ex- My lab has isolated thirty-one different populations plain the clonal interactions. Establishing posi- derived from single cells (SCPs) from the triple neg- tive interactions within these SCPs additionally of- ative breast cancer cell line MDA-MB-468. Prelimi- fers the scientific community a resource to study a nary studies have demonstrated that these SCPs dis- poorly understood and clinically significant topic. play remarkably different phenotypic properties, in- cluding resistance to cell death in suspension. In ad- An Age-Dependent Study of Innervation dition, some SCPs grow better when cultured with Patterns of Lymph Nodes the original cell line, indicating positive signaling in- teractions. The nervous and im- mune systems work to- Our research aim is to investigate clonal coopera- John Austin gether to help defend Molecular and tion within this triple negative breast cancer cell line. the body against harm- Cellular Biology, Demonstrating a mechanistic basis for clonal coop- 2017 ful pathogens. The pe- eration is an important step in understanding how ripheral nervous system intratumoral heterogeneity and cooperation between Harvard Medical (PNS) in particular is the heterogeneous subpopulations can support tu- School responsible for sensing morigenesis. and responding to stim- Advisor: Ulrich von uli. Thus, understanding To investigate clonal interactions, we are utilizing a Andrian, MD, PhD the PNS and, in partic- coculture assay, where subclones are cultured with ular, the sensory nervous other subclones and their growth is assessed after two Mentor: Siyi Huang, system is crucial to better weeks. In previous experiments, we have found that PhD understanding the process three clones display enhanced survival and growth of host defense. Recent when cocultured with the original cell line; these studies have shown that are our “dependent clones.” Other clones do well the innervation of various organs contributes to their in isolation and are resistant to cell death (strong function. Studying the innervation of lymph nodes clones). Fluorescently tagged versions of the depen- could lead to a better understanding of the role that dent clones are being grown with the strong clones the nervous system plays in immunity. in a 1:1 ratio, and their growth will be examined with a fluorescent microscope after two weeks. We Aging in mice produces significant anatomical and are examining thirty different pairwise combinations biochemical changes in lymphatic vessels. The lym- by mixing three dependent clones with ten strong phatic vessels’ close association with the surround- clones. If one strong clone proves able to stimulate ing capillary system means that the blood vascu- growth of all three dependent clones, this indicates lature as well displays some aging effects such as positive cooperation. vascular wall remodeling and changes in endothelial barrier integrity. Because lymph node innervating- To investigate the mechanism of clonal cooperation, sympathetic fibers and nociceptors physically asso- we are using a number of different approaches. Pro- ciate closely with the vessels, we believe that they teomic analysis of the MDA-MB-468 cell line shows will both exhibit age-dependent changes. We are that these cells express higher levels of the TGF- specifically looking at the changes in density of beta family of cytokines, protein factors known to lymph node-innervating neurons with regard to age. be involved in cell-cell signaling. To investigate if Any correlation could suggest an age-dependent re- these cytokines are responsible for mediating clonal lationship between PNS neurons and lymph nodes, interactions, we are currently performing knockdown in turn suggesting possible functional relationships studies with short hairpin RNAs, genetic overex- between the nervous and immune systems. pression studies, and synthetic cytokine/cytokine in- hibitor introduction with the coculture assays. To research this question, we will use mice of varying ages to look at the innervation density of the lymph We are currently waiting for data from the cocul- nodes over time. Popliteal lymph nodes will be har- ture analysis, but our biased approach to the cy- vested from the mice, and the nociceptors, sympa- tokine knockdown has been promising. Using Nog- thetic neurons, and lymphatic vasculature will be gin, an inhibitor of the BMP signaling cytokine, immunolabeled. Images generated via confocal mi- croscopy will help characterize the innervation pat- Molecular and Cellular Biology 87 tern and quantify the total amount of neuronal fibers. tially contributes to the differing virulence between these two clades of K. pneumoniae ST-258. Such a We expect that the data will suggest that nociceptors finding would have the potential to validate the cap- and sympathetic neurons retract with age, leading to sule/capsule biosynthesis genes as legitimate drug an overall drop in neuronal density. It is entirely pos- targets, focusing future drug development efforts on sible, however, that no relation with age will be seen. this particular virulence factor of K. pneumoniae. However, understanding how age affects the innerva- tion of the lymph node can help us understand the in- teraction between the nervous and immune systems. SID-1 Independent RNA Uptake in C. elegans Mechanisms of Virulence in Klebsiella SID-1 is a transmem- pneumoniae ST-258 brane protein that facil- Antibiotic resistance Julio Fierro itates import of extra- Molecular and is a global threat of cellular double stranded Cellular Biology, Julian Braxton rapidly growing signif- RNA (dsRNA) in C. ele- Chemical and 2017 icance, as more than gans, which initiates RNA Physical Biology, 2018 two million people in the Harvard University interference (RNAi), a United States alone are mechanism through which Broad Institute of infected with antibiotic- Advisor: Craig P. gene silencing occurs. MIT and Harvard, resistant bacteria ev- Hunter, PhD However, there is rea- Massachusetts ery year. Carbapenem- son to think embryos can General Hospital resistant Klebsiella pneu- Mentor: Eddie Wang also take up extracellu- moniae (CRKP) is one lar dsRNA independently Advisor: Deborah such species, and is a of SID-1. The SID-1- Hung, PhD major cause of hospital- independent RNA uptake requires the yolk protein acquired infections and (YP) endocytosis receptor RME-2. While SID-1 Mentor: Christoph infections associated with is known to facilitate the import of extracellular Ernst, PhD long-term care in the dsRNA, it is unknown if endogenous RNAs in adults United States. While in- depend on the YP-endocytosis pathway to gain ac- fections with CRKP are cess to embryos. Yolk protein is a vital instrument still relatively rare, they have dramatically increased that serves as a food source for animal embryos and in frequency in the last 10 years and have mor- is considered important for successful replication in tality rates of ~30–70%, making these bacteria a egg-laying animals, including C. elegans. Prior re- severe public health threat. Among CRKP, ST-258 search has led to the hypothesis that RNAs in C. is the most frequently isolated sequence type, and elegans could hitch-hike onto the yolk protein and can further be divided into two genetic clades, de- transmit from adults to embryos. noted 1 and 2. In a recent surveillance study at several Boston area hospitals, clade 2 appeared to To test the hypothesis that RNA-YP interactions be associated with more invasive infections than did are necessary for transmission of RNAs from adults clade 1. These clades differ mainly in the capsule to embryos, we will pursue three aims. The first polysaccharide synthesis (cps) region, which encodes seeks to confirm via RT-PCR and qPCR that a mu- the necessary proteins for the production of bacterial tant lacking ceh-60, a YP transcription factor, does capsule, a layer of densely packed polysaccharides on not express the vitellogenin precursors of yolk. If the outside of Gram-negative bacteria. the results are successfully replicated, the mutant will fail to express YP and be considered viable for The objective of this project is to compare the im- experiments requiring YP knockdown such as cre- pact of the capsule on the virulence of Klebsiella ating a ceh-60 ; sid-1 double mutant. This mutant pneumoniae ST-258 clades 1 and 2. My hypothesis can then be used to test for RNAi silencing signal is that the clade 2 capsule contributes to increased uptake defects as well as embryo uptake of endoge- virulence relative to the clade 1 capsule. To test nous extracellular RNA. Essentially, if we have C. this, I plan to delete the entire cps region in a clade 2 elegans who no longer have the means to generate strain, and then insert the corresponding region from yolk protein, we can then examine if the lack of yolk a clade 1 strain, generating a hybrid strain of clade protein inhibits transmission of RNA from adults to 2 genetic background but with a clade 1 capsule. the embryo. In assays of virulence, this hybrid strain should be less virulent than the corresponding wild type clade The second and third steps will investigate if endoge- 2 strain, suggesting that the capsule at least par- nous RNAs utilize the YP-endocytosis pathway to 88 Molecular and Cellular Biology gain access to the embryo. First, wild-type worms sions about which exons are included in the final will be transformed with tissue-specific mCherry- transcript—called alternative splicing—enables fine- tagged reporter constructs, DNA plasmids that allow tuning of mRNA expression levels and the produc- confirmation of expression of the tissue specific genes tion of a diverse array of proteins. via fluorescence imaging. Using only the progeny who express mCherry, we will monitor their lineage Previously, my lab characterized the role of alter- for loss of expression of reporter constructs in so- native splicing in the dynamic cellular response to matic cells, observing which specific somatic cells infectious agents. Using RNA-seq to quantify gene lose expression. Those worms will then be selected expression levels and isoform patterns in primary and their embryos taken to analyze what, if any, macrophages, they found significant global shifts in RNA is found. If the RNA corresponding to the splicing patterns in response to infection with Liste- reporter constructs is detected in the embryos of ria monocytogenes or Salmonella typhimurium bac- such animals, it suggest these endogenous RNAs use teria. However, the specific regulatory mechanisms RNA-YP interactions to gain access to the embryo. that regulate these global shifts are still unknown, If the hypothesized results are confirmed, the pro- and it is still unclear whether shifts in splicing are cedure will then be replicated in a double mutant part of the cell’s immune defense or a deleterious that knocks out both SID-1 and RME-2 with the result of the infectious pathogen. expectation that the RNA should not be able to be transmitted to embryos because both SID-1 and the Previous studies have shown that variation in gene YP-endocytosis pathway will be rendered inaccessi- expression amongst different conditions and individ- ble. uals can be informative of the regulatory networks that the cell utilizes to modulate expression. My The final step will continue investigating the path- goals are therefore to understand variation in splic- way endogenous RNAs take, this time using single ing levels between individuals before and after infec- molecule fluorescent in situ hybridization (FISH) of tion in order to elucidate regulatory mechanisms of tag-257, a protein kinase found in neurons. Sin- splicing that may play a role in innate immune re- gle molecule FISH uses fluorescent probes that bind sponses in the cell. I propose that an increase in the to a specific DNA or RNA sequence, in this case variance of splicing levels between individuals after the target sequences, to observe and localize the infection indicates that the normally tight regulation presence and abundance of the target sequence. of splicing has been disrupted, by either individual- Prior research identified tag-257 as a candidate to specific changes or stochastic variance after infection. be an endogenous RNA that uses RNA-YP inter- A decrease in variance would indicate that infection actions to go from the soma to embryos. Single targets splicing towards a single response regardless molecule FISH will allow us to visualize where in of interindividual differences. Little to no change in the body tag-257 RNA localizes to and evaluate variance would indicate a tightly controlled event not if it does indeed use these RNA-YP interactions. affected severely by infection. Preliminary results in- dicate that there are many genes that belong to all three of these categories, and each set of genes dif- Understanding Regulation of Splicing in fers in their associated functions and attributes. For Response to Infection instance, genes with an increase in splicing variance Eukaryotic organisms after infection are likely to be associated with in- have evolved remark- flammation, cell death, and phosphorylation-related Kimberley Yu able innate immune sys- processes, while genes with little change in variance Chemical and tems that allow cells to are often involved in essential housekeeping pro- Physical Biology, 2017 fight infection by foreign cesses. pathogens. These de- Massachusetts fense mechanisms are de- Currently, I am analyzing a measure of how effi- Institute of pendent on a cell’s abil- ciently cells from each individual were able to kill Technology ity to carefully regulate the foreign bacteria (bacterial clearance counts), to gene expression and pro- investigate whether ability to fight infection can be Advisor: Chris tein levels. One mecha- predicted by patterns of splicing variance amongst Burge, PhD nism by which cells can individuals after infection. Ultimately I hope to de- regulate gene expression termine, at a population-level, which splicing events Mentor: Athma Pai, is through mRNA splic- and associated genes, as well as per-individual fea- PhD ing, which involves ex- tures, may be important in determining an individ- cising introns from pre- ual’s susceptibility to infection and the efficacy of mRNA molecules and their innate immune responses. joining exons to produce mature mRNA. Deci- Molecular and Cellular Biology 89

Enabling Natural Killer Cell Tumor target for NK cells. Immunotherapy Using CRISPR-Cas9 Deletion of Inhibitory Receptor Genes In order to create these genetically modified NK cells, I first designed and cloned CRISPR guide RNAs Cancers, highly prolif- (gRNAs) into lentiviral expression vectors to knock erative and prone to mu- out our target genes in both immortalized and pri- Kruti Vora tation, evade the body’s mary NK cell lines. Next, I characterized the nor- Molecular and immune system by escap- mal inhibitory NK cell receptor expression using Cellular Biology, 2017 ing both T and natural fluorescence-activated cell sorting (FACS). Once ex- killer (NK) cells. In 2016, pression is established, I can compare the degranula- Harvard Stem Cell it is estimated that over tion and killing activity of the modified NK cell lines Institute 1.6 million new cases of against native 721.221 cells or 721.221 cells that have cancer will emerge while been reconstituted with the corresponding inhibitory Advisor: Chad the disease will claim ligands. I will also include a negative control cell line Cowan, PhD around 600,000 lives. It in the killing assays that should not be susceptible is thus of pressing ur- to modified NK cell attack. Mentor: Torsten gency and importance to Meissner, PhD find new methods to help I hypothesize that my genome-edited NK cells will patients’ immune systems be resistant to the inhibitory effect of these lig- recognize and eradicate ands, showing greater degranulation and killing ac- malignant cells to prevent unchecked growth of tu- tivity. If my hypothesis is correct and we are mors. able to enable NK cells to recognize and kill can- cer cells, we could create new treatment options to Normally, circulating T cells act as a primary de- fight cancers using NK cell tumor immunotherapy. fense mechanism against malignant cells, scanning peptides presented on major histocompatibility com- Artificial Antigen-Presenting Cells for CAR plex class I (MHC-I) molecules on the surface of cells T Cell Activation as potential markers for destruction. However, can- cer cells escape T cell surveillance by down-regulating Harnessing the body’s MHC-I. own immune system to Leah Marsh fight diseases such as can- Molecular and As a backup system, NK cells recognize aberrant cer is an idea that has Cellular Biology, cells without the aid of MHC-I molecules, using the 2019 been around for many release of cytotoxic granules to eliminate cancerous years, but in the past and virus-infected cells. But certain cancers fight Massachusetts decade the field of im- back, expressing various inhibitory ligands that bind General Hospital munotherapy has seen to corresponding receptors on NK cells to prevent tremendous advances. their activation. Advisor: Marcela V. One recent immunother- Maus, MD, PhD apy innovation is the NK cells play an especially large role in targeting chimeric antigen recep- cancers with low mutation rates, such as pancreatic Mentor: Felipe tor (CAR)-modified T cancer, as T cells cannot recognize these tumor cells. Bedoya, PhD cell. In CAR T cell can- However, prior research has shown that pancreatic cer therapy, the chimeric cancer can inhibit NK functioning, and it currently antigen receptor targets remains the fourth most lethal cancer. an antigen that is expressed on cancer cells, and then causes the CAR-modified T cells to traffic to My research goal is to use the CRISPR-Cas9 sys- the cancer cells and destroy them. To create a CAR tem to target the KLRC1 inhibitory receptor, its ac- T cell, T cells are genetically modified to express cessory chain (KLRD1), and inhibitory KIR family the CAR, which is made of parts from the T cell’s genes in NK cells to test the resistance of these mod- own antigen receptor as well as parts from antibodies ified cells to the effects of various inhibitory ligands. (proteins the immune system uses to recognize anti- gens). The CAR recognizes a specific antigen based Deletion of these inhibitory receptors should enable on the type of antibody used to create it, and CAR NK cells to recognize and kill malignant pancreatic T cells will traffic to cells expressing that antigen. tumor cells, even in the presence of inhibitory lig- ands. We use 721.221 cells as our target cell line In order for T cells to proliferate in the body, they since these MHC-I negative cells are able to bypass must be activated by the T cell’s receptor recogniz- the T-cell immune response and act as an optimal ing and binding to a complex on the surface of an 90 Molecular and Cellular Biology antigen-presenting cell. In the laboratory, one can Studying Epithelial Polarity in the engineer artificial antigen-presenting cells (aAPCs) Nematode C. elegans to activate T cells in vitro. These can be used for normal T cells, or CAR T cells. The goal of my Epithelial cells have a project was to create and evaluate aAPCs that acti- crucial role as barriers vate CAR T cells recognizing the antigens CD19 and Leah Rosen and transporters in a di- Applied BCMA. These are antigens expressed on the cells of verse range of complex or- Mathematics, 2019 multiple myeloma and other blood cancers. ganisms, from humans to Harvard University C. elegans (roundworms). First, we created lentiviruses carrying the genes for In the intestine, for exam- CD19 and BCMA that insert this antigen DNA into Advisor: Susan ple, epithelial cells keep the genome of “blank” cells, causing the blank cells Mango, PhD toxins out while trans- to become aAPCs expressing CD19 or BCMA on porting nutrients in. Ep- their surface. To make the virus, we used a pro- Mentor: Stephen ithelial cells need to be cess called transfection to introduce the viral DNA, Von Stetina, PhD able to differentiate be- which coded for the virus’s physical structure, and tween their different sides, the CD19 or BCMA antigen DNA into a packaging and for this reason they cell line. After this, the viruses assembled themselves are polarized. They polarize by differentiating be- in the cytoplasm. We then collected the viruses and tween the proteins contained in their membrane on used them to transduce the blank cells as described different sides. The junctional domain separates the above, in which the viral vector delivers the anti- two main sides of the cell (the basal from the api- gen DNA to the genome. After transduction, the cal membrane), and connects the epithelial cells to- blank cells became aAPCs, expressing either CD19 gether so that they form a continuous barrier. Many or BCMA on their surface (they do not express any diseases arise when epithelial polarity is disturbed, viral proteins). and so it is crucial that we understand the pathways and molecules that give epithelia their polarity. We also used this process to create the CAR T cells, first creating lentivirus that carried DNA for the The C. elegans pharynx is an ideal system in which to CAR, and then transducing a T cell line with the study epithelia due to how simple, well-studied, and virus so that those T cells expressed the CARs rec- well-conserved across organisms it is. The pharynx, ognizing CD19 or BCMA. also known as foregut, is at the front of the digestive tract, just behind the mouth. C. elegans are both By culturing the aAPCs and CAR T cells together, simple and transparent, so that many things can be we can observe whether the CAR T cells become observed in vivo, and we have a lot of context while activated and begin proliferating. We can also test studying them. Despite the simplicity of C. elegans, different ratios of aAPCs to CAR T cells to see the molecular nature of epithelial cells is very similar which ratios work best; we hypothesize that there is in C. elegans and in mammals. Specifically, PHA-4 a specific ratio of aAPCs to CAR T cells that will (FoxA in mammals) is a transcription factor that has induce high levels of activation and proliferation, been found to be essential for pharynx development and we hope to find this ratio. Using these aAPCs in all organisms tested. will facilitate the study of CAR T cells as treat- ment for multiple myeloma and other blood cancers. The Mango lab has found a pathway of conserved molecules, culminating in the expression of the junc- tional protein DLG-1. The Mango lab has shown that in embryos lacking pha-4, DLG-1 protein is re- duced in the foregut. Surprisingly, the RNA for dlg-1 is still abundant, which is unexpected as PHA-4 is a transcription factor. The first aim of my project is focused on discovering why there is still abundant dlg-1 RNA in pha-4 mutants. We are looking at two possibilities. Firstly, we are studying the gene lin-26, which is known to regulate dlg-1 expression in the skin, and is found in the pharynx in mutants that lack pha-4. The hypothesis we are testing is that lin-26 is normally inhibited by PHA-4. It fol- lows that in the absence of PHA-4, lin-26 would be expressed and responsible for the activation of dlg-1 RNA. In the second possibility, we are investigating Molecular and Cellular Biology 91 whether a PHA-4 interacting protein and its homolog gests that other FXR1 isoforms may regulate mi- are involved in dlg-1 regulation. The lab previously croRNA levels. FXR1d is the other isoform present observed a phenotype only when both the protein in the cancer cells studied in our lab. Previous re- and its homolog are absent. search has found that unlike the a isoform, FXR1d does not promote translation; however, the role of In the second part of the project we are study- FXR1d in microRNA levels is not known. The goal ing another part of the pathway. A protein called of my research is to investigate the roles of FXR1d in ZEN-4 regulates DLG-1 protein production based regulating microRNA levels and activity. I am inves- on the dlg-1 RNA that was regulated by PHA-4. tigating the effects of the overexpression of FXR1d The molecular process of how ZEN-4 controls this on microRNA levels and their regulators. My re- transition is completely unknown. The Mango lab search will therefore provide insights into the role of performed an immunoprecipitation followed by mass FXR1 in regulating these important RNAs in cancer spectrometry to identify ZEN-4 interacting proteins. and clinical resistance. We are looking at these and seeing if they have an impact on DLG-1. A typical experimental procedure I use is to transfect different types of cancer cells with GFP as a con- We are studying all of these hypotheses by inacti- trol (to show that the transfection was successful), vating the relevant genes and analyzing their effects as well as with FXR1a, FXR1d, and forms of the in worms of different backgrounds, either by using FXR1a gene with mutations that prevent its ability mutant worms, or by performing RNAi, in which to interact with RNA. The proteins expressed have a the worms are fed bacteria that produce a molecule small tag called Flag that allows me to detect them that interrupts the gene expression pathway. We separately from endogenous FXR1 protein. The cells then analyze the effects in embryos, when the mu- I use are MCF7 (breast cancer cells) and H520 (lung tants are still viable, using antibody staining to see cancer cells), which express FXR1. I provide serum protein expression and single molecule fluorescent in to half of the samples, and serum-starve the other situ hybridization (smFISH) to see RNA expression. half so that I can make a comparison between prolif- erating cancer cells and cancer cells that are quies- cent—or in the G0 state—due to serum starvation. I Elucidating the Role of FXR1d in MicroRNA perform western blots with Flag antibody to detect Activity and Cancer Development the FXR1 I transfected, as well as with various anti- Our lab studies gene bodies to see whether overexpression of these FXR1 expression in quiescent forms affects the levels of microRNA regulators. Ul- Madeleine cancer cells, a subpopu- timately, I will use qRT-PCR to measure and com- Granovetter lation of cells in cancers pare the roles of FXR1a and FXR1d in microRNA Molecular and Cellular Biology, that resist clinical therapy regulation. I can consequently see which isoforms of 2019 and give rise to cancer re- FXR1, or which mutations of FXR1a, are involved currences. FXR1 is an in microRNA regulation. Massachusetts RNA binding protein with General Hospital multiple forms, and is as- Given that my experiments run successfully, I hy- sociated with aggressive pothesize that overexpression of FXR1d causes an Advisor: Shobha cancers, such as leukemia, alteration in microRNA levels by either promot- Vasudevan, PhD lung cancer, and breast ing the activity of oncogenic microRNAs or re- cancer. The lab previ- ducing the activity of tumor suppressor microR- ously uncovered that one NAs. Ultimately, I strive to understand how isoform, FXR1a, is overexpressed in quiescent can- FXR1d is contributive to cancer development in cer cells and promotes translation by microRNAs. both proliferating and clinically resistant G0 cells. MicroRNAs are small, non-coding RNA regulators of gene expression that play critical roles in cancer, either as tumor suppressors or as oncogenes. In the cancers with which it is associated, FXR1 is overex- pressed and promotes translation of important tumor invasion genes.

Research in the lab has found that knockdown of endogenous FXR1 (all isoforms) alters levels of mi- croRNAs and their regulatory proteins, and reduces clinical resistance in cancers; however, FXR1a does not interact with microRNA regulators. This sug- 92 Molecular and Cellular Biology

Role of Hexamer Repeats in Pathogenesis of Validating Mouse Models of Transient XDP Lubricin Deficiency

X-linked dystonia- Increased participa- parkinsonism, referred to tion in competitive sports Mai-Linh Ton as XDP, is a rare neu- Mara Coyan has led to the rising inci- Molecular and Molecular and rodegenerative movement dence of traumatic joint Cellular Biology & Cellular Biology, Visual and disorder. It combines 2017 injuries. These injuries Environmental symptoms of dystonia, can have lasting conse- Studies, 2019 such as sustained and Boston Children’s quences on joint health; repetitive muscle contrac- Hospital the mean arthritis-free in- Massachusetts tions and twisted pos- terval after an anterior General Hospital tures, and parkinsonism, Advisor: Matthew cruciate ligament tear is which includes the slow- Warman, MD only about fifteen years. Advisor: Cristopher ing of movements. Those Thus, young athletes with Bragg, PhD affected can have their Mentor: Yajun Cui, this injury are at high maternal ancestry traced PhD risk for being burdened back to Panay in the with disabling joint dis- Philippines, with individuals passing down the XDP- ease at a relatively young specific mutations in an X-linked recessive pattern. age. Effective clinical interventions that prevent joint injuries from progressing to joint failure are XDP involves the gradual loss of medium spiny neu- needed. Lubricin (encoded by Prg4 ), the principal rons; however, the mechanism behind this loss is not boundary lubricant in articular cartilage, plays a key fully understood. Additionally, it is not known why role in preventing cartilage wear. Genetic lack of the age of onset can vary so greatly for those with lubricin causes precocious joint failure, and acquired XDP. While some are affected in early adulthood, transient deficiencies of lubricin occur in patients others may only show symptoms late in life. following acute joint injury. It is not known if tran- sient lubricin deficiency in a previously normal joint The XDP-specific variants are believed to affect the causes joint damage and, if so, whether this damage gene encoding for TATA-box binding protein asso- is irreversible and progressive. ciated factor 1 (TAF-1). These variants include five disease-specific nucleotide changes, DSCs 1, 2, 3, 10, The objective of the present study is to determine and 12; a 48 bp deletion; and an SVA retrotranspo- whether and how cartilage becomes damaged in the son insertion in the 32nd intron of TAF-1. context of acquired lubricin deficiency. To address these questions, I am using mice with a conditional Genotyping individuals has revealed that all affected lubricin knockout allele (Prg4 F rt); the first two ex- individuals contain all markers for the disease. How- ons of Prg4 are flanked by flippase recognition (Frt) ever, the length of the SVA retrotransposon can vary sites, enabling the allele to behave like a wild-type among individuals. One region within the retrotrans- allele until flippase activity renders it nonfunctional. poson that varied the most among XDP patients Another mouse allele (Rosa26 F lpER) permits the was the region containing the CCCTCT hexamer re- temporal induction of flippase activity; the protein peats. My project will concern the SVA retrotrans- is not active until it is bound to tamoxifen. I expect poson, with added focus on these hexamer repeats. mice with the genotype Prg4 F rt/−; Rosa26 F lpER/+ The length of these repeats could help elucidate to have adequate lubricin expression until they re- why the age of onset for XDP can vary so greatly. ceive tamoxifen, after which lubricin expression will cease. I intend to administer a ten-day course of ta- moxifen to these mice when they become skeletally mature (i.e., two months old) and then monitor their development and progression of cartilage damage.

The Warman lab has already confirmed that the Prg4 F rt allele functions as a wild-type allele in the absence of flippase and as a knockout allele after flippase-mediated recombination. Critical to my experiments is that the Rosa26 F lpER allele is not enzymatically active in the absence of tamoxifen, and that it is highly efficient at recombining the Prg4 F rt allele when tamoxifen is present. I am as- Molecular and Cellular Biology 93 sessing the “leakiness” of the Rosa26 F lpER allele by sulin, and creatinine levels, and completed an exten- taking 2-month old Prg4 F rt/+ Rosa26 F lpER/+ mice sive lifestyle questionnaire. To initiate a large pi- that have not received any tamoxifen, and perform- lot analysis, we selected 400 of these participants ing droplet-digital PCR on DNA extracted from at random for metabolomic profiling. We used a their joints to measure recombination at the Prg4 liquid chromatography-tandem mass spectrometry locus. I will repeat this process for mice that have (LC-MS/MS) platform that measures approximately received a 10 day intraperitoneal injection program 80 organic acids and phosphorylated metabolites in of tamoxifen to assess the the efficiency of flippase the negative ion mode. Data was reviewed and activity. If I demonstrate that the Rosa26 F lpER al- normalized to correct for instrument drift over the lele is not “leaky” in the absence of tamoxifen and course of the run. After correction, metabolite lev- highly efficient when it is present, then I will be els were log transformed and compared to clinical in an excellent position to temporally inactivate lu- phenotypes using a Pearson correlation test. Us- bricin expression in juvenile and adult mice, and ing metabolomic data previously gathered from the then determine the consequences on joint health. Framingham Heart Study, we performed a similar correlation of metabolites to clinical phenotypes to what was done in JHS. My primary contributions Metabolomic Profiling of Chronic Kidney over this summer thus far have been to prepare Disease and Type 2 Diabetes in the Jackson the samples, extract the metabolites, perform LC- Heart Study MS/MS, generate the data on the mass spectrome- Chronic kidney dis- ter, and integrate the metabolite peaks to translate ease (CKD) is caused them into quantitative information. Mary Wan by various factors in- East Asian Studies, cluding type 2 diabetes We hope to find associations of metabolites to phe- 2019 (T2D) and high blood notypes in JHS that are consistent with associations Beth Israel pressure, which also con- previously made in the FHS cohort. As an example, Deaconess Medical tribute to cardiovascular we have previously shown that uric acid is highly Center disease. Although studies associated with a variety of metabolic risk factors in- have shown that African cluding BMI, glucose, and HOMA-IR. Additionally, Advisor: Robert E. Americans exhibit higher we hope to find novel metabolite correlations with Gerszten, MD risk of developing CKD the estimated glomerular filtration rate (EGFR), than caucasians, the bio- a measurement of kidney function performed in Mentor: Jordan logical basis of CKD de- JHS, that may be inconsistent with FHS, indicating Morningstar velopment has yet to be an African American-specific metabolic risk factor. fully understood. Occur- rence of CKD and T2D depends on a combination of numerous genetic and lifestyle variations such as body mass index (BMI) and blood glucose levels; the large number of possible factors complicates research in the field. However, we hypothesize that metabolomics, which is the study of small molecules, or metabolites in human cells, tis- sues and biofluids, may help to highlight novel mech- anistic underpinnings of CKD and its connection to T2D as well as to find novel biomarkers of both dis- eases. In order to do so, we will profile metabolites in human plasma from participants in the Framing- ham Heart Study (FHS) as well as the Jackson Heart Study (JHS) and, because the samples were obtained in 2002, we will identify metabolites that presage the clinical diagnosis by over a decade. Additionally, we hope that a metabolomics approach will provide in- sights on the high rates of CKD and T2D in African Americans.

The Jackson Heart Study comprises 5,302 African American participants from the Jackson, Missis- sippi area. Participants underwent a series of clin- ical measurements including cholesterol, glucose, in- 94 Molecular and Cellular Biology

Directed Evolution of Ferritin for the the healthiest colonies of bacteria from these plates Biomineralization of Heavy Metals and sequenced their ferritin genes to find the mu- tations of the most successful survivors. We verified Ferritin is a cage- that our method produced the desired outcome using shaped protein that con- mass spectrometry, as well as four novel biosensors, Michael Giles trols the concentration of which were constructed using promoters that have Applied free iron ions in the cell by been previously shown to be responsive to our four Mathematics, 2019 oxidizing them into iron metals of interest. We designed plasmids including Wyss Institute for oxide crystals in its cen- these promoters so that they drove the expression of Biologically Inspired ter. Ferritin’s structure is green fluorescent protein (GFP), allowing us to quan- Engineering relatively simple and well- tify the intracellular concentration of these metals by characterized, and the fer- measuring fluorescence. Advisor: Pamela ritin family of proteins Silver, PhD is well-conserved through- Successful mutants will be ones that sequester sig- out an extensive lineage nificantly more metals than the wild type. With Mentor: Leon Liu of both eukaryotes and future development, these could be useful in reme- prokaryotes. These fea- diating water that has been contaminated by these tures have made ferritin a metals, as well as in mining. The biosensors we de- promising platform for bioengineering and bionan- veloped, provided they are reasonably sensitive and otechnology, and many in vitro applications of the specific, could also be useful as a cheap and re- protein have been explored. silient way to test for heavy metal contamination.

Less has been done with ferritin in vivo, and this Synergy Between Myc and PI3K Inhibitors summer we attempted to engineer E. coli by modify- ing the ferritin that the cells expressed. Previous ex- My thesis research is periments performed in the Silver lab have created E. being done in the Koch coli possessing ferritin that crystallizes a larger num- Muhammed Ors Institute of MIT in the Molecular and ber of iron ions than wild-type ferritin. Interestingly, Koehler lab. The lab Cellular Biology, these mutant bacteria have been shown to better sur- 2017 studies dysregulated tran- vive poisoning by the toxic metals arsenic, cobalt, scription in cancer such as cadmium, and nickel. These metals react well with David H. Koch c-Myc overexpression, one iron oxide in an inorganic setting, and we surmised Institute for of the most common aber- that something similar could be occurring within the Integrative Cancer rations in human cancers, cell—that the modified ferritin proteins were able to Research having been linked to up- biomineralize these metals, co-crystallizing with the wards of 70% of cancers. iron oxide inside of the modified ferritin more than Advisor: Angela Being able to inhibit aber- in the wild-type ferritin. Koehler, PhD rant transcription factors would be instrumental, as We attempted to take advantage of this observed ef- Mentor: Francisco overexpression of even one fect by engineering ferritin to extract ions of these Caballero, PhD transcription factor, espe- metals from the cells’ environment. We approached cially a master regulator this problem using a technique called directed evolu- like c-Myc, leads in turn tion: organisms are mutated and placed in a selective to overexpression of any number of the proteins environment that favors the survival of a particular whose codes are downstream to the transcription fac- trait. Mutants possessing favorable traits reproduce tor. Therefore, they make a smart therapeutic tar- more than less fit individuals. Over one or more get in cancer research. Importantly, mouse mod- rounds of selection, the most successful traits are fa- els have shown that c-Myc inactivation stereotypi- vored, and the mutations that led to this increased cally results in the proliferative arrest, differentia- survival can then be characterized. tion, and/or apoptosis of tumor cells. Thus, I be- lieve any step forward in selectively modulating c- We applied directed evolution to ferritin by using a Myc expression is synonymous with a step forward technique known as mutagenic PCR to create many in tackling cancer. Unfortunately, for the most part, copies of ferritin gene, each with an average of one transcription factors lack typical binding pockets and DNA base changed. We forced many E. coli cells have been considered “undruggable.” Of course, there to express a copy of these mutant ferritins, and is hope: the Koehler lab has been finding and opti- plated them on agar supplemented with one of the mizing small molecule inhibitors of c-Myc, allowing four metals of interest at a concentration lethal to me the incredibly unique opportunity to experiment bacteria expressing unmodified ferritin. We picked with something completely novel. Molecular and Cellular Biology 95

My research is focusing on the synergistic effect in tubal pregnancies. Whole exome sequencing (WES) breast and brain cancer cell lines between the in- analysis followed by array-CGH and homozygosity hibitors of c-Myc and PI3K, a family of kinases mapping revealed two candidate genes in which the (phosphoinositide 3-kinase) which are referred to as affected sisters carried novel homozygous mutations: PI3K in shorthand. Previous research using RNA OSR1 and HS1BP3, of which Odd-Skipped Related interference (RNAi) of c-Myc has shown that resis- 1 (OSR1 ) is the more likely candidate based on in tance to PI3K inhibitors is dependent on c-Myc in- silico analysis. OSR1 encodes a zinc finger transcrip- duction. Being unable to inhibit c-Myc directly, an- tion factor which has previously been studied for other group decided to do so indirectly by inhibiting its role in the development of the heart and kidney. BRD4, a protein with a role in the same pathway as It is expressed in the intermediate mesoderm dur- PI3K and c-Myc, using a BET inhibitor called JQ1. ing embryogenesis. Our project focuses on the role The synergy of BET and PI3K inhibitors produced of OSR1 protein in the Müllerian duct, the embry- a sustained inhibition of PI3K and tumor cell death onic structure which differentiates from intermediate in twenty cell lines. mesoderm into the female reproductive tract (FRT), forming the fallopian tubes, uterus cervix, and the My methods for research can be partitioned into upper vagina. Our hypothesis is that OSR1 plays three phases. The first aim is to use co- an important role in differentiation of the interme- immunoprecipitations and western blotting to estab- diate mesoderm into the Müllerian duct, such that lish and verify that the c-Myc inhibitors were inhibit- mutations to OSR1 could explain the FRT anomaly ing the Myc/Max/DNA interaction. The second aim seen in our patients. We are using both in vivo and is to do cell viability assays in multiple cell lines using in vitro studies to investigate the role of OSR1 in the PI3K, BET, and the Myc inhibitors individually intermediate mesoderm differentiation. in serial dilutions from 40 µM to 0 µM/DMSO in or- der to generate dose-dependent curves, and addition- Global Osr1 knockout mice have previously been ally to do western blotting and confirm a reduction shown to die early during embryogenesis. Therefore, in protein expression due to increasing concentration to investigate OSR1 protein in the development of of inhibitors. Finally, the third aim will be to use the FRT, our mouse model had to be a conditional yet-to-be-developed synergistic cell viability assays Osr1 knockout, where Osr1 is only knocked-out in to contrast between the conditions of PI3K/BET in- the Müllerian duct and is expressed normally else- hibitors and the PI3K/Myc inhibitors. where. To generate our conditional mouse, we used the Cre-Lox recombination system. LoxP-flanked My hypothesis is that the synergistic effects of the Osr1 genes generated a homozygous Osr1 -Floxed PI3K and c-Myc inhibitors are better than those female mouse. In males, the Cre gene was inserted of the PI3K and BRD4 inhibitors. Namely, this into the promoter of a Müllerian-expressed Wnt7a would be a smaller IC50, the concentration of the allele. OSR1 knockout was thus limited to the Mül- drug required for there to be 50% inhibition in vitro. lerian duct in F2 generation individuals homozygous in Osr1 -Floxed and hemizygous in Cre allele. Odd-Skipped Related 1 in Female We are also looking at the interactions of the OSR1 Reproductive Tract Development protein with other proteins, such as HOXA10. We Our project began transfect human embryonic kidney (HEK 293) cells with three Brazilian sis- with plasmids containing the sequences for human Naomi Asimow ters, ages 33, 27, and 26, OSR1 and HOXA10 respectively. Using western blot Chemical and born of a consanguineous and co-immunoprecipitation experiments, we ana- Physical Biology, 2018 marriage, each present- lyze interactions of OSR1 with HOXA10 and with ing the same novel pheno- other proteins. Brigham and type. All three displayed Women’s Hospital primary amenorrhea, but Finally, we aim to examine expression patterns of otherwise normal sec- Osr1 in female mouse embryos using immunohisto- Advisor: Ursula ondary sex characteris- chemistry. We use 13.5-day-old mouse embryos with Kaiser, MD tics. MRI revealed thin a TY1 tag on OSR1. We bind the anti-TY1 antibody endometrium and uter- to OSR1 protein and then a fluorescent secondary Mentor: Adriana ine hypoplasia. The sis- antibody to anti-TY1 so that we have fluorescence Lofrano Porto, MD, ters had normal hormonal anywhere OSR1 is expressed. We are looking for flu- PhD levels, but endometrium orescence in the Müllerian duct and/or surrounding that proved unresponsive mesenchyme. to sex steroids. Intrigu- ingly, two of the sisters had been treated for ectopic 96 Molecular and Cellular Biology

Female reproductive tract abnormalities are one test this, we will overexpress these genes in human of the primary causes of infertility. Understand- melanoma cell lines and perform T cell cytotoxicity ing the role of OSR1 and its interactions with assays. Using CRISPR technology, we will knockout other proteins in FRT development may provide these genes from the melanoma cell lines and perform insight into many types of FRT abnormalities, similar assays. Finally, we will study the mechanism such as Mayer-Rokitansky-Küster-Hauser syndrome. of action of genes that are shown to be mediators of resistance to T cell killing by utilizing techniques of molecular biology and protein analyses. Mechanisms of Tumor Resistance to TIL Therapy and PD-1 Inhibition in Melanoma The second part of the project will analyze an- In the past few other highly promising immunotherapy, anti-PD1 years, immunotherapies (an immune checkpoint receptor), which has been Ramtin Talebi such as tumor infiltrat- recently approved for both melanoma and lung can- Molecular and ing lymphocytes (TILs) cer. Despite its promise, many patients still fail Cellular Biology, 2018 and checkpoint receptor to respond to anti-PD1 therapy, and some research blockade (such as anti- suggests the cause to be an up-regulation of alter- Dana-Farber Cancer PD1) have demonstrated native immune checkpoints like TIM-3 or LAG-3 + Institute that durable responses in CD8 T cells. However, many mechanisms of can be obtained in pa- tumor resistance to anti-PD-1 therapy remain un- Advisor: Levi tients with advanced can- known. Thus, using anti-PD-1 antibody in vitro, Garraway, MD, PhD cers. However, the major- we will improve the cytotoxicity of TILs on the ity of patients still fail to melanoma cell lines and develop this system for Mentor: Shruti respond or progress after large scale genomic screens to identify novel resis- Malu, PhD an initial response, creat- tance mechanisms for the joint therapy. In the long ing the need for combina- term, these findings may lead to the discovery of tion therapies. Moreover, combination therapies designed to anticipate tumor understanding the resistance mechanisms that the resistance and create a more durable and positive tumors impose can help us define better biomarkers patient response for those with advanced melanoma. of response to immunotherapy. This will be ben- eficial in predicting which patients might respond Characterization of Intron Elements that to immunotherapy as well as guiding them into the Control Splicing in Unc-16 Gene effective combination therapies for their own tu- mor. Thus, we have recently performed whole ex- In eukaryotes, the pro- ome sequencing and RNA sequencing of tumors from cess of precursor mRNA melanoma patients that underwent TIL based im- Santiago Vargas (pre-mRNA) splicing in- Chemistry and munotherapy and are using this data to understand volves the removal of non- Physics, 2019 novel resistance mechanisms to T cell based thera- protein coding segments pies. Harvard University called introns and the stitching back together In TIL therapy, autologous T cells that have in- Advisor: John of coding segments called filtrated a patient’s tumor are removed from the Calarco, PhD exons. These segments harsh tumor microenvironment, cultured and grown are cut and stitched by in large numbers in vitro in presence of interleukin-2 Mentors: Adam a piece of cell machinery (IL-2), a cytokine essential for specific growth of T Norris, PhD; Xico known as the spliceosome. cells. These cells are then re-administered to the Gracida, PhD Additionally, exons them- patient, leading to an anti-tumor immune response. selves can be selectively The first part of this project will focus on studying included or skipped in a the genes that appear significantly enriched in tu- process known as alternative splicing, which allows mors of patients who do not respond to TIL therapy for a greater degree of protein diversity from a more as compared to the tumors of patients who respond limited repertoire of genes. A gene can code for vari- well. In order to effectively identify the genes poten- ous different proteins depending on splicing patterns tially responsible for tumor resistance to this adop- and this allows for more diversity without necessar- tive cell therapy, we have performed whole exome ily increasing the genetic complexity of an organ- sequencing and RNA sequencing the patient tumor ism’s DNA. Splicing complexity has been tied to the samples. The hypothesis is that overexpression of complexity of an organism itself. Humans display these genes will lead to resistance to T cell-mediated a large amount of alternative splicing and this al- killing and knockout of these genes will make the cell lows for complicated and differentiated cell function lines more sensitive to T cell-mediated death. To within our bodies without the need for exceedingly Molecular and Cellular Biology 97 large sets of genes. Understanding splicing events is Identification of C3Orf59/MB21D2 as a important. For example, splicing characterizes how Novel Cancer Gene certain human mental disorders are caused on a ge- netic level. Recent genomic se- quencing efforts by the We know that certain DNA motifs upstream or Scott Xiao Meyerson lab of lung Chemistry, 2019 downstream of alternatively spliced exons can serve adeno and squamous cell as up-regulators or down-regulators of splicing carcinomas have identi- Broad Institute of events. These elements are known as cis elements MIT and Harvard, fied several novel onco- and they are believed to be recognized by trans fac- Dana-Farber Cancer genes and tumor suppres- tors within pre-mRNA and this will either increase Institute, Harvard sors. Part of the ef- or decrease the likelihood of splicing. Trans factors Medical School forts determined common are proteins that are coded for elsewhere in the DNA neoepitopes, or recogniz- that are made specifically to identify these splicing Advisor: Matthew able antigen segments, of control sequences. The model organism we are study- Meyerson, MD, PhD lung cancer cells, which ing is C. elegans, as it has a simple, well-characterized included well-documented nervous system, has known splicing, and has DNA Mentor: Douglas cancer genes. This anal- that is relatively easy to manipulate with exogenous Wheeler, MD, PhD ysis found that C3orf59 fragments. (the 59th open reading frame of Chromosome 3), Two interesting cis elements had been previously also known as MB21D2, is frequently mutated at found in the unc-16 gene that stimulate exon inclu- residue 311 from a glutamine to a glutamate (also sion in neuronal cells. To search for more sequences known as p.Q311E). Interestingly, we find that this that can regulate splicing, my work this summer mutation frequently and significantly co-occurs with started with analysis of high-throughput sequencing activating mutations in the oncogene PIK3CA in data of cDNA collected from animals expressing a li- lung cancers and other tumors. Our goal is to explore brary of splicing reporter transgenes containing ran- the potential of MB21D2 as a novel cancer gene and dom 6-base pair sequences (testable candidate cis el- to identify the function of this gene in tumorigenesis. ements). I developed efficient computational tools in order to sift through this data and to quantify MB21D2 is an unstudied gene which is tightly con- splicing patterns and test if splicing ratios changed served from zebrafish to humans. The MB21D2 pro- from wild-type and mutant benchmarks depending tein contains a Mab-21 domain, which is also found on what hexamer sequence was inserted into a given in a number of other proteins involved in develop- reporter. Positive results would include finding hex- mental processes but whose function is poorly un- amer (6-base pair segments) sequences that greatly derstood. Although the function of MB21D2 is un- affect the ratio of splicing in the animal. Other possi- clear, phosphoproteomic studies exploring the impor- ble results could be the characterization of other, pre- tance of MB21D2 phosphorylation have identified it viously uncharacterized splicing regulatory elements. as a substrate of the AGC kinase family, a group I then used this data to create oligomer sequences which includes protein kinase A, C, and AKT. These (DNA segments of interest) that I later inserted into data suggest that this protein may be phosphorylated C. elegans to experimentally confirm our computa- downstream of cellular signal transduction pathways. tional data. This data could then be used to cre- ate an evolutionary comparison to detect identical To study this uncharacterized gene, we first identified regulatory elements in other species of nematodes cell lines with high and no expression of MB21D2 . and possibly other clades of organisms. My work By immunoprecipitating MB21D2, we found that the this summer also included the adaptation of a fluo- cell lines HeyA8, BT-474, and NCI-H2009 expressed rescent reporter into a format that could be easily high levels of MB21D2 while the cell line NU-DUL-1 used in C. elegans. This reporter glows a different did not express MB21D2 at all. We have used these color depending on the splicing pattern occurring in cell lines to validate antibodies and cDNA reagents a certain tissue. I used this reporter in the exper- towards MB21D2. Immunoprecipitation of MB21D2 imental trial of our hexamer sequences of interest. from high expression and no expression cell lines has revealed several interacting proteins, which indicates that MB21D2 likely exists in one or more protein complexes. Interestingly, two cancer cell lines, BT- 474 and NCI-H2009, contain the p.Q311E mutation.

Because MB21D2 is a phosphoprotein, we asked whether it was downstream of signal transduction 98 Molecular and Cellular Biology pathways. We treated high expression cells with a One metric by which cells can be simply grouped is variety of kinase inhibitors, and found that inhibi- through their mRNA expression profiles, since these tion of protein kinase A (PKA) reduced the phos- transcripts in large part dictate the cells’ function. phorylation of MB21D2, which indicates that this Large-scale classification following this method has protein lies downstream of PKA signaling. We also been limited in the past by the fact that RNA-seq used the clustered regularly interspaced short palin- at the single-cell level can only be achieved for small dromic repeats (CRISPR-Cas9) system to disable batches of cells, as transcripts must be sequenced in- the MB21D2 gene in several cell lines and asked dividually for each cell. Recently, however, Macosko whether loss of MB21D2 function altered cellular sig- et al. introduced the Drop-seq method of massive naling in any way. We found that functional inac- single-cell mRNA expression profiling, in which in- tivation of MB21D2 diminishes phosphorylation of dividual cells are encapsulated in nanoliter droplets certain PKA substrates such as glycogen synthase ki- containing a bead bearing a distinct molecular bar- nase 3 (GSK-3) and cAMP response element-binding code, allowing all transcripts to be sequenced in par- protein (CREB). These data further suggest that allel and subsequently matched to their cell of origin. MB21D2 may have some regulatory function within With transcriptional profiles now available for tens or the PKA signal transduction pathway. even hundreds of thousands of neural cells, it is pos- sible to use computational methods to group cells Finally, to define the role of MB21D2 in onco- into distinct classes based on biologically significant genesis, we plan to manipulate this gene in vivo factors. and in vitro. We are in the process of con- structing vectors to conduct these experiments. We are currently developing a tool that will use In the meantime, we are also trying to bet- Drop-seq data from brain tissue to help discover pre- ter understand the connection between PKA and viously unknown subtypes of known cell classes in MB21D2, and whether such a link would have im- the nervous system; subsequent identification of en- plications for MB21D2 as a novel cancer gene. riched genes for each of these subtypes both can serve as markers and can provide an idea of their func- tional diversity. We use principal component analy- A Computational Tool for Classifying sis (PCA) on mRNA expression data from a single Nervous System Cell Types Using mRNA cell type to explain variation in the data through Expression Profiles a small number of statistically significant variables, In healthy organisms, then map data points in two dimensions using the t- genes are expressed at dif- distributed stochastic neighbor embedding (t-SNE) Victoria Lin ferent levels in different method of nonlinear dimensionality reduction. Fol- Molecular and cell populations; it is this lowing dimensionality reduction, we will cluster and Cellular Biology & Statistics, 2017 property that allows for classify the cells into subpopulations using the k- the functional specializa- nearest neighbors (k-NN) algorithm, in which the Harvard Medical tion of various cell types class of a cell is determined by a majority vote of School, Broad to form tissues and or- its k nearest neighbors; then extract, through differ- Institute of MIT and gans. When an organ- ential expression analysis, enriched genes from each Harvard ism is afflicted with a group, both to serve as markers and to provide an genetic disease, another idea of the function of the subtype. These results Advisor: Steve kind of differential gene from such a systematic examination will contribute McCarroll, PhD expression may appear, as to a more thorough understanding of the diversity disease-risk alleles can re- of cell populations in the nervous system, lend in- Mentor: Evan sult in abnormal expres- sight into how cell types specialize by tissue, and al- Macosko, PhD sion only in certain sub- low for the pinpointing of both healthy and disease- sets of cells. As such, a related phenotypes to more specific classes of cells. comprehensive characteri- zation of cell types in the nervous system would not only provide insight into the function of a complex network, but also contribute to a greater understand- ing of the pathology of neurological disease. De- spite the ongoing categorization of such cells by lo- cation, morphology, target specificity, electrophysio- logical properties, and other molecular markers, how- ever, the complexity of the brain has thus far limited attempts to fully identify the diversity of cells that it comprises. Molecular and Cellular Biology 99

The Role of PDK4 in Regulating Th17 Cell pathogenic or non-pathogenic Th17 cells. Further- Pathogenicity more, I chose to culture some of the cells with PUFA to investigate how PDK4 knockout affects Th17 cells T helper 17 (Th17) with lipid environmental cues. Specifically, I mea- cells are CD4+ T cells sured the changes in the expression of genes that were Zhuang Miao that produce the cytokine previously identified to play a critical role in regu- Chemical and interleukin-17 (IL-17) and lating Th17 cell pathogenicity by using the following Physical Biology, 2019 can exhibit distinct phe- techniques: 1) flow cytometry to measure protein ex- notypes. Th17 cells pression at the single cell level, 2) ELISA (enzyme- Brigham and are found in the intes- linked immunosorbent assay) to measure cytokines Women’s Hospital tine of healthy mice and secreted by Th17 cells, and 3) qPCR and nanostring man, maintaining mu- to quantify to determine gene expression at the tran- Advisor: Vijay cosal barriers and defend- scriptome level. Kuchroo, PhD ing against extracellular pathogens. However, Because the function of PDK4 to upregulate gly- Mentor: Chao Wang, Th17 cells are also found colysis is consistent with increased glycolysis in PhD at sites of inflammation, pathogenic Th17 cells, I initially hypothesized playing a pathogenic role PDK4 −/− cells would be less pathogenic. However, in inducing autoimmune my earlier experiments showed higher expression of diseases such as multiple sclerosis, inflammatory some pro-pathogenicity proteins in the PDK4 −/− bowel disease, and rheumatoid arthritis. These find- cells, suggesting PDK4 plays a more nuanced reg- ings raised an interesting hypothesis that not all ulatory role in Th17 cells. I find these unexpected Th17 cells are created equal. Research on the mecha- results especially interested and will further investi- nisms controlling the fate of Th17 cells will allow pre- gate the function of PDK4 in Th17 pathogenicity. cision targeting of pathogenic Th17 cells and block their differentiation in autoimmune diseases, while sparing the non-pathogenic Th17 cells that are ben- Elucidating the Resistance Mechanism of eficial for health. Medulloblastoma to BET Bromodomain Inhibition A recent study in my lab shows that Th17 cell pathogenicity is correlated with changes in cellu- Medulloblastoma is a lar metabolism. Analysis of the Th17 cell tran- high-grade, fast-growing scriptome revealed that genes involved in central Bluyé DeMessie tumor, and is the most Molecular and carbon metabolism show differential expression in prevalent malignant brain Cellular Biology, pathogenic and non-pathogenic Th17 cells. Fur- 2019 tumor in children. Sur- thermore, our metabolome study shows that non- gical removal, chemother- pathogenic Th17 cells have a higher ratio of polyun- Dana-Farber Cancer apy, and radiation are in- saturated fatty acids (PUFA) to saturated fatty acids Institute, Broad effective and damage the (SFA), suggesting altered lipid metabolism. These Institute of MIT and adjacent tissue. A phar- observations lead to some questions: what is the key Harvard macological approach regulator of these metabolic changes, and can it drive to target overexpressed Th17 pathogenicity? We hypothesize that metabolic Advisor: Rameen genes is being explored by genes at the junction of glycolysis and fatty acid Beroukhim, MD, our lab. The gene MYC is metabolism could contribute to Th17 cell function. I PhD overexpressed in patients chose to focus on a mitochondrial enzyme, pyruvate with a poor prognosis dehydrogenase kinase 4 (PDK4), which inhibits the Mentors: Pratiti and the transcription of conversion of pyruvate to acetyl-CoA, promotes gly- Bandopadhayay, MYC is regulated by the MBBS, PhD; Ryan colysis, and inhibits fatty acid synthesis. Moreover, bromodomain and extra- O’Rourke PDK4 is a nutrient sensor: its expression is turned on terminal domain (BET) in response to alterations in lipid metabolism, which family of proteins (e.g. might then drive Th17 cell pathogenicity. BRD4). The BET bromodomain regulates gene transcription by binding to acetylated histone tails. To study the role of PDK4, I used mice with targeted deletion of PDK4 (PDK4 −/−) and determine the ef- JQ1, a BET bromodomain inhibitor, has demon- fect of the loss of PDK4 on Th17 cell pathogenicity strated high activity towards BRD4 by displacing with several approaches. I cultured naive T cells iso- it from acetylated histones. Medulloblastoma cells lated from wild-type and PDK4 −/− mouse spleens treated with JQ1 have a sharp reduction in viability with different cytokines to differentiate them into and proliferation, but eventually develop resistance 100 Molecular and Cellular Biology to JQ1 and other BET bromodomain inhibitors. The aim of this work is to understand the mecha- nism by which cells become resistant to JQ1, and to determine the role of BRD4 as an essential gene in resistant models.

To determine the JQ1 resistance mechanism, it is necessary to identify the proteins and overexpressed genes in resistant cells which allow the cells to pro- liferate. We performed a genome-wide open reading frame (ORF) screen to identify genes that confer re- sistance when overexpressed. Medulloblastoma lines that are resistant to BET bromodomain inhibitors were generated by chronically passaging cell lines in drug until they exhibited growth. The in vivo behav- ior of resistant cells was studied using cell line based xenograft models. Western immunoblots were per- formed to study expression of candidate resistance proteins in resistant lines. Protein-protein interac- tion was studied by using immunoprecipitation to extract proteins bound to BRD4. These proteins were then identified using mass spectroscopy.

We generated resistant cell lines by chronically passaging sensitive cells in BET bromodomain in- hibitors. We next confirmed that the resistant lines also exhibited resistance in vivo. Following flank injection of both sensitive and resistant cells, we observed the resistant xenografts to proliferate in both vehicle and JQ1 treated mice. Mice harbor- ing xenografts from the sensitive lines exhibited an initial response, however we observed the eventual development of resistance.

Affinity purification mass spectroscopy of the im- munoprecipitated BRD4, and its related proteins in resistant cells, revealed specific proteins binding to BRD4. These results demonstrate that BRD4 may contribute to resistance through this domain.

This study has identified a multitude of overex- pressed genes involved in developing resistance to BET bromodomain inhibitors. We have determined that BRD4 is an essential gene in resistance to BET bromodomain inhibitors. We have identified which proteins BRD4 binds to in resistant cells. Future work involves identifying targetable pro- teins in order to create a drug treatment for BET bromodomain inhibitor resistant medulloblastoma. Neuroscience and Behavioral Science

Early Biomarkers for Language Development at 18 months in the HRA and LRC groups. Inves- in Infant Siblings of Children with Autism tigation of the relationship between EEG response Spectrum Disorder variability at early ages and later developmental out- comes may establish language-specific biomarkers, Early intervention and may ultimately provide insight into the viability programs for autism spec- of variability as a biomarker for ASD. Adriana Mendez trum disorder (ASD) have Leal shown that earlier enroll- Neurobiology, Additionally, comparing these relationships to pat- ment in treatment is as- December 2016 terns between EEG response and eventual diagnosis sociated with better long- at 36 months will give the opportunity for differ- Boston Children’s term outcomes. Though entiation between markers of atypical development Hospital ASD can be diagnosed or delay and ASD-specific diagnostic markers. It’s in controlled clinical en- possible that EEG variability is tied to general dis- Advisor: Charles vironments in infants ruption of connectivity, rather than ASD itself, and Nelson, PhD as young as 12 months, examining the relationship of early EEG to behav- underlying neurological ioral outcomes will help elucidate any differences. Mentor: April Levin, changes likely begin be- MD fore symptoms emerge. Recent evidence suggests Investigation Into the Role of CA3c, CA2, there is altered brain de- and the Entorhinal Cortex in Pattern velopment in children 2–6 months of age who later go Separation and Completion in Spatial on to receive ASD diagnoses, pointing to an oppor- Memory Encoding tunity for the isolation of a predictive biomarker for ASD available well before diagnosis using behavioral Pattern separation is measures is possible. Such a biomarker could provide the computational pro- insight into the neuropathology of ASD, and might Alvar Faustino cess by which similar in- Paris also allow for treatment earlier in the progression puts are made more dis- Medicine, 2017 of the disorder, even before the onset of behavioral Emmanuel College tinct during storage so symptoms. as to minimize interfer- Massachusetts ence between them, while Because autism is a heterogeneous disorder, compar- General Hospital, pattern completion relies ative analysis of EEG data from children with ASD Harvard Medical upon neurons retrieving and typically developing children is frequently lim- School stored output patterns in ited by inter-subject variability in signal in the ASD a way that completes de- group. However, evidence that intra-subject vari- Advisor: Amar graded or partial input ability in response to a repeated stimulus is higher Sahay, PhD signals. In the hippocam- in children with ASD than in typically developing pus, these phenomena re- controls has led some researchers to suggest intra- Mentor: Kathleen spectively allow the brain subject variability may itself be a viable candidate McAvoy, PhD to differentiate between for a predictive biomarker for the disorder. Fur- similar spatial contexts, thermore, infant siblings of children with autism are and to evoke a memory of twenty times more likely to develop ASD than their an entire context from a more limited stimulus. typically developing counterparts, and as a result they are well suited to prospective cohort studies In the past, theoretical and lesion studies have been investigating potential neural markers of ASD. used in order to elucidate the roles of the subdivisions of the hippocampus. These studies have generally My project is a subset of the Infant Sibling Project, reinforced a model in which population-based encod- a study that compares neurological and behavioral ing in the dentate gyrus underlies pattern separation, development in a group of infant siblings at high risk and area CA3 may support pattern separation or for autism (HRA) with that of a group of low risk serve an autoassociation function to support pat- controls (LRC). Specifically, my project will compare tern completion. Meanwhile, other regions within intra-subject variability in EEG response to native the hippocampus remain understudied, partly owing and non-native language in 6 month old infants (as to their perception as functionally continuous with measured by inter-trial alpha phase coherence) to anatomically nearby areas. Such regions include receptive and expressive language behavioral scores CA2 (which has often been considered to be transi- 102 Neuroscience and Behavioral Science tional between CA3 and CA1) and CA3c (which has Understanding Microglial Activation in a commonly been grouped with the neighbouring area Multiple Sclerosis (MS) Model CA3ab). Multiple sclerosis Recently, however, re-examination of these areas has (MS) is a demyelinat- highlighted that they are likely to be functionally Audrey ing disease characterized Effenberger unique from their anatomical neighbors. For exam- by lesions in the central Neurobiology, 2019 ple, CA2 has been found to be heavily involved in nervous system (CNS). social memory, and studies have shown that CA3c- Boston Children’s These scars predomi- lesioned animals show greater deficit in spatial pro- Hospital nantly affect white mat- cessing than CA3ab-lesioned animals. Thus, legiti- ter, which is composed of macy has been brought to the hypothesis that CA3c Advisor: Zhigang He, myelinated nerve fibers, and CA2 may have distinctive roles regarding pat- PhD or axons, crucial to in- tern separation and completion. formation transmission. Mentor: Jing Wang, However, the underlying To test this hypothesis, we investigate these subre- PhD cause of these lesions re- gions using catFISH (cellular compartment analysis mains unknown, as does a of temporal activity by fluorescence in situ hybridiza- comprehensive treatment. tion). We do this in both control mice and in mice where adult hippocampal neurogenesis has been en- While the nervous system is naturally capable of hanced, which has been shown to improve pattern limited regeneration, MS lesions fail to heal over the separation. These mice are exposed to two spatial course of the disease due to dysfunction of a key class contexts in a temporally controlled manner before of supporting cells: oligodendrocytes (OLs). OLs being sacrificed. By visualizing the subcellular loca- build the myelin sheaths in the CNS that are crucial tion of cfos mRNA, for which transcription is upreg- to neuron function; however, they often die after in- ulated when a neuron becomes active, we can then jury, and are progressively depleted in MS. Though calculate the population overlap of cells active in the nearby oligodendrocyte progenitor cells (OPCs) can two separate spatial contexts. In turn, we use this as proliferate and migrate towards lesions, this endoge- a measure of similarity in encoding, which—in con- nous response is not enough to prevent net OL loss. junction with data from connected areas—provides Without a sufficient OL population, substantial re- information about whether pattern separation or myelination fails to occur. The exposed axons are completion is being supported. thus nonfunctional and vulnerable to further degen- eration. To isolate the subregions in question, we define the anatomical boundaries of area CA3c and study the Additionally, MS is characterized by inflammation, region within the blades of the dentate gyrus, be- an immunological response that may contribute to fore comparing it to CA3ab. Second, we isolate area further breakdown of myelin sheaths and axons. In CA2 by using in situ hybridization staining with the CNS, innate immune cells known as microglia RNA probes targeted against the locally expressed are partly responsible. Active microglia perform genes rgs14 and cacng5. We also expand our investi- phagocytosis, the uptake of particles, and are re- gation to the entorhinal cortex (the main input area sponsible for initial inflammatory response. How- to the hippocampus) in order to study the complete ever, not all active microglia are the same. Whereas pathway of spatial context encoding. M1 microglia promote “classical” inflammation and damage tissues, M2 microglia decrease inflammation Together, these findings will resolve in greater de- and promote tissue repair. Natural skewing of the tail the roles that precise anatomical regions have in M1/M2 balance may compound the inability of the pattern completion and separation, as well as pro- CNS to heal or impact OL response in unknown duce a more complete picture of information flow ways. through the entorhinal cortex and hippocampus. These features of MS pathology motivate our re- search in an optic nerve injury model that shares pathological similarities with MS: inflammation and remyelination failure. Because microglia have histor- ically received little scientific attention, we seek to understand their role in demyelinating diseases like MS, as well as their relationship to OLs.

To understand how OL response is influenced by mi- Neuroscience and Behavioral Science 103

croglia, we have created a mouse optic nerve injury 5-HT3AR cells change during periods of heightened model where the microglial response is dampened. plasticity. Our goal is to study the development of By virally introducing Cre-inducible diphtheria toxin 5-HT3AR cells within the mouse primary auditory receptor (DTR) into the eyes, we have generated cortex. There are two main areas of focus for our mice where the eyes can be specifically ablated by project: 1) we will determine how 5-HT3AR cell mor- treatment with diphtheria toxin. This localizes dam- phology and connectivity changes across postnatal age to the nerve fiber and foregoes more invasive in- development, and 2) we will examine how early ex- jury methods that would trigger a full inflammatory perience alters 5-HT3AR cells. response. Based on preliminary immunofluorescence staining of microglial cell markers, we know that mi- To examine the development of 5-HT3AR cell mor- croglia are present and active to a reduced extent in phology, we are employing Brainbow, a genetic label- DTR mice. ing technique using multicolor fluorescent proteins, to visually distinguish and reconstruct 5-HT3AR cells From this model, we can begin to see if there is in 5-HT3AR-Cre mice. Combining this with immuno- a causative relationship between microglial activity cytochemistry has allowed us to determine how de- and OPC proliferation or differentiation. We stain veloping and adult 5-HT3AR cells form synaptic con- tissue samples for Olig2 markers that correspond to nections with other major inhibitory subtypes (PV OL lineage cells, allowing us to determine if OPCs and somatostatin-expressing) and excitatory pyrami- are surviving and proliferating in the nerve one or dal cell somata. We will also take advantage of an two weeks after it has been killed. CC1 markers approach that makes brain tissue optically transpar- of mature OLs can further reveal if these cells are ent and light sheet fluorescence microscopy for three- differentiating. If microglia play a role in post-injury dimensional 5-HT3AR cell reconstructions. We are remyelination and neural repair, we hope to see sig- particularly interested in how morphological changes nificant differences in the number and distribution in 5-HT3AR cells correlate with transitions into and of OPCs and OLs between normal and DTR mice. out of critical periods.

Furthermore, we are investigating the impact of var- To determine how early auditory experience im- ious drugs on in vivo microglial subtype determi- pacts 5-HT3AR cells, we will passively expose 5- nation. By treating mice with compounds such as HT3AR-Cre mouse pups and mothers to continu- retinoid receptor agonists and staining for subtype- ous white noise, band-limited noise, or tones in specific markers such as CD206 and CD16/32, a sound-attenuating chamber. Continuous white we can determine if these drugs shift the balance noise has been shown to restore the tonotopic crit- of microglia towards the M2 state. Our results ical period. If 5-HT3AR cells are associated with should lay the foundations for future investigations the plasticity of cortical response properties, we ex- and applications, from more effective MS treat- pect them to be rejuvenated in accordance with the ments to more precise anti-neuroinflammatory drugs. restoration of the critical period. Previous studies have shown that repeatedly playing pure tones to mouse pups during a critical period enlarges the area The Role of 5-HT R Cells in Brain 3A of the auditory cortex representing the frequency Plasticity of that tone. Finding that this manipulation al- During development, ters the connectivity of 5-HT3AR cells during the the brain undergoes crit- critical period would suggest that the plasticity of Emma He ical periods of heightened these cells underlies the experience-dependent de- Neurobiology, 2019 plasticity, when the neu- velopment of auditory cortical circuits. Together, ral circuits are most sen- these studies will increase our understanding of how Harvard University sitive to external stimuli. 5-HT3AR cells contribute to neural development. Advisor: Takao Ongoing work in the lab Hensch, PhD has shown that a group of inhibitory interneurons Mentor: Anne that reside in cortical Takesian, PhD layer 1 (L1) and express ionotropic 5-HT3A recep- tors (5-HT3AR cells) play an important role in the enhanced plasticity during early life. These 5-HT3AR cells inhibit parvalbumin- expressing (PV) cells, another inhibitory cell sub- type that is known to underlie experience-dependent circuit rewiring. However, it is unknown whether 104 Neuroscience and Behavioral Science

Investigating Age-Related Changes in the sory processing is different in younger versus older Neuronal Circuits Underlying Multisensory mammalian systems, the mechanistic details remain Processing in Caenorhabditis elegans largely unknown. Therefore, we are currently test- ing younger larval stage C. elegans in the integrated Humans are con- behavioral response to elucidate these mechanistic stantly experiencing mul- differences. Preliminary data suggest that younger Gaia Linfield tiple stimuli that must be wild-type larval worms have a reduced capacity to Integrative Biology, processed and integrated leave the food lawn when paired with an aversive 2018 within their brains. The stimulus, despite moving away from the aversive cue Center for Brain ability to integrate infor- in a similar manner to adults. After testing various Science, Harvard mation obtained from dif- mutants in younger stages, it appears that the neu- University ferent sensory modalities ronal circuit regulating this behavior in adult worms is central to daily func- is already present in younger worms. This result sug- Advisor: Yun Zhang, tion, affecting the deci- gests that a separate pathway in younger worms is PhD sions we make and our suppressing the integrated behavior of which adults perception of our sur- are fully capable. Further dissection of this pathway Mentor: Gareth roundings. Some indi- in larval C. elegans could shed light on the man- Harris, PhD viduals who suffer from ner in which multisensory processing in mammals neurodevelopmental dis- differs from one developmental stage to another. orders, such as autism Such information is particularly important to under- spectrum disorders, are impaired in their ability stand since some neurodevelopmental diseases with to perform multisensory processing. To better un- deficits in sensory processing present at a young age. derstand the complex signaling pathways and neu- ronal circuits that regulate multisensory behav- Investigating the Ontogeny of Instinctive iors in mammalian systems, we use the nematode Behavior in Mice Caenorhabditis elegans as a model system. C. ele- gans has a well-characterized nervous system, and Mice rely heavily on advanced genetic tools allow us to study the effects of olfactory cues to de- manipulating the activity of a single neuron, which Helene Lovett tect social chemosig- Integrative Biology, is very difficult to achieve in more complex systems. nals to execute sex- 2019 appropriate social behav- Previous work in the Zhang lab has identified a net- Harvard University iors. The vomeronasal or- work of sensory neurons and molecules that regulate gan (VNO) in the mouse an integrated response to multisensory stimuli. To Advisor: Catherine nose detects pheromone map out this network, we used a behavioral assay Dulac, PhD cues secreted by other that challenges worms to choose between two con- mice, a signal that acti- flicting stimuli: a food lawn of E. coli, an attractive Mentor: Dhananjay vates neural circuits that stimulus, and a droplet of the chemical 2-nonanone, Bambah-Mukku, drive instinctive behav- a repellent stimulus. Using this assay, we generated PhD iors such as parenting, a time course quantifying the percentage of worms mating, and aggression. that leave the attractive food lawn in the presence of The gene trpc2 is required the aversive chemical over 60 minutes. After testing for the vomeronasal system to function, and its in- worms with ablated sensory neurons and mutated activation has been shown to cause deficits in social genes encoding neuropeptides and other molecules, behavior and sex discrimination. Mice lacking func- we discovered that certain mutant strains of worms tional TRPC2 protein appear unable to distinguish were “fast” and “slow” lawn-leavers, when paired between male and female mice and display a loss of with the aversive chemical, in comparison to wild- conspecific aggression. However, little is known as to type worms. This has allowed us to map out a circuit how the neuronal circuits that drive these behaviors of neurons, neuropeptides, and molecules that com- develop. Early in life, input from the VNO to the municate via inhibitory and stimulatory signals in neural circuits involved in instinctive behavior may order to modulate the processing of multiple stimuli. play an instructive role in shaping the behaviors. Alternatively, these circuits may be to a large extent We now aim to explore whether the same neurons, hardwired, and develop independent of input from molecules, and genes involved in the adult pathway the VNO during development. are implicated in younger stages, in order to bet- ter understand how age affects the wiring, function, To answer this question, the Dulac lab has devel- or plasticity of neuronal circuits involved in mul- oped a transgenic mouse using a genetic construct tisensory processing. Although we know that sen- adopted from bacteria that allows for the selective Neuroscience and Behavioral Science 105 activation of trpc2 expression only when mice are fed The Effect of Affective Priming on Reward a diet containing the derivative doxycycline (dox). Responsiveness in Individuals with High vs. The aim of my project is to optimize the reversible Low Depressive Symptoms expression of the trpc2 gene in order to understand the effect of trpc2 expression at certain time points Individuals with ma- in development on adult social behaviors. I am geno- jor depressive disorder typing the transgenic mice to identify those positive Isobel Green (MDD) demonstrate a Cognitive for all the necessary genes in the construct and then blunted response to re- Neuroscience and will attempt to selectively activate trpc2 only at cer- Evolutionary wards in the environment. tain intervals during development by feeding mice Psychology, 2017 Behavioral studies have dox at those times. quantified this blunting McLean Hospital by showing that depressed To determine if trpc2 was selectively activated, the individuals do not prefer- VNO is dissected to determine relative levels of trpc2 Advisor: Diego entially make choices that expression. If trpc2 expression can be rescued in mice Pizzagalli, PhD have previously elicited after inactivation at an earlier time point in devel- reward. Throughout the opment, then I will be able to reversibly control the Mentor: Poornima 20th century, behavioral activation or inactivation of the VNO and its ability Kumar, PhD researchers conducted ex- to detect pheromone cues. I will put the adult mice periments demonstrating through a number of behavioral paradigms to test that this pattern reliably the effect of selective VNO activation. If the mice set individuals with MDD apart from their healthy show no behavioral deficits associated with a loss counterparts. In the past few decades, neuroimaging of VNO function despite VNO inactivation during has emerged as a potent tool for linking patterns development, it will lend support to the idea that of neural activation to specific behaviors, including hardwired circuits are sufficient to execute instinc- reward processing in both healthy and depressed tive social behaviors. If deficits are observed, this people. One consistent finding is that individuals would indicate that sensory input from the environ- with depression show decreased reactivity in a re- ment early in life plays a critical role in programming gion called the ventral striatum during reward-based these behaviors. decision-making. The ventral striatum is the part of the limbic system and is key to linking motivational In addition to this reversible gene activation project, drives to behavioral actions. Aberrations in striatal I am assisting with another project aimed at un- activity have been linked to key symptoms of MDD: covering neural circuits deeper in the brain, which decreased sensitivity to positive affect (anhedonia) drive social behavior based on VNO input. I and increased negative affect (low mood). am scoring videos of various social behaviors and using a technique to compare the neurons acti- There exist two possible models of reward processing vated during consecutive behavioral assays in a in individuals with depressive symptoms. The “clas- brain region hypothesized to be downstream of the sical” model posits that individuals with depression VNO pathway. These experiments will lead to a have a baseline impairment in reward processing clearer understanding of the neuronal populations that causes them to experience a chronic blunting involved in the behaviors, and how neural activity of reward responsiveness, regardless of their stress along specific pathways drives instinctive behaviors. level. However, studies have shown that the aberrant profile of reward processing seen in individuals with depressive symptoms can also be induced transiently in healthy individuals by putting them under acute stress. This context-dependency of reward respon- siveness in healthy individuals suggests another pos- sibility: individuals with depressive symptoms exist in a state of heightened stress compared to healthy controls, and that their impaired reward processing is context-dependent, reflective of a stressful external state rather than a baseline impairment in reward processing. Distinguishing between these two pos- sibilities holds clinical importance as it points to different treatment routes for the disease.

My project this summer takes a next step towards identifying which of these two frameworks best rep- 106 Neuroscience and Behavioral Science resents reward processing in individuals with depres- tron microscopy counterparts. To address this chal- sive symptoms. Specifically, the work will investigate lenge, we test a dual-labeling method in which we reward processing in individuals with high vs. low mark neurons of interest in both light and electron depressive symptoms at three different time points: microscopy, making them immediately distinct from before a positive mood induction, after a positive their neighbors. In light microscopy, such labeling is mood induction, and after a delay following a posi- achieved by expressing fluorescent protein tags, while tive mood induction. Therefore, the study will allow peroxidase-derived tags can be used to increase the for investigations of first, baseline differences in re- contrast during EM visualization. The latter requires ward processing between individuals with high and a staining process in which the peroxidase catalyzes a low depressive symptoms; second, the acute effect reaction between peroxide and 3,3’-diaminobenzidine of a positive contextual prime on reward processing; (DAB), such that the resulting precipitate attracts and third, the extent to which an emergent increase osmium. This makes the region containing the per- in reward-related activation persists over time. oxidase appear dark in EM images. Finally, to co- express the two labels in the same neurons, we ge- Based on findings of context-dependence in neu- netically insert a construct with genes for both the ral activation during reward processing tasks, my fluorescent and peroxidase tags under the same pro- team hypothesizes that although high-depressive in- moter in the zebrafish genome. dividuals will show significantly lower reward re- sponsiveness before a positive mood induction, they For this project, we develop a protocol in zebrafish will normalize to a level near healthy controls af- for visualizing a recently available peroxidase tag, ter acute positive mood induction. We further APEX (“enhanced” ascorbate peroxidase). To test hypothesize that this increase in reward activa- staining protocols, we established a transgenic line tion will not last; following a time delay, reward with APEX and a red fluorescent protein (TagRFP) responsiveness in individuals with high levels of under the isl3 promoter, such that both are ex- depressive symptoms will again fall significantly pressed in a subset of the trigeminal neurons. These below that seen in healthy controls, represent- are mechanosensory neurons, which are located in ing a trait-like inability to sustain positive affect. the trigeminal ganglion behind the fish eyes and branch out to innervate the entire fish head. The stereotyped locations and long processes of these Fluorescence and Electron Microscope Image neurons facilitate the testing of staining protocols Registration using EM Dense Labels in and help us evaluate how reliably these can be re- Zebrafish constructed and matched to light microscopy images. The zebrafish (Danio So far, we have seen visual confirmation of DAB rerio) is an emerging stains with light microscopy, and are expecting suc- Jaden Freeze model organism for un- cessful EM labelling under the electron microscope. Chemistry and derstanding how neural Physics, 2019 circuits underlie animal Dopamine Neurons and the Execution of behavior. Recent techno- Harvard University Sequential Behavior logical advances in light Advisor: Florian microscopy allow us to Parkinson’s disease is Engert, PhD record the activity of the most common move- large neuronal popula- Jennifer Bi ment disorder, and the Neurobiology, 2018 Mentor: Mariela tions with single-cell res- second most common neu- Petkova olution in order to func- rodegenerative disorder. Center for Brain tionally identify potential Science, Harvard People affected by Parkin- neural circuits involved in University son’s have difficulties ex- the behavior of zebrafish larvae. The spatial res- ecuting sequential be- olution of light microscopy, however, is insufficient Advisor: Naoshige haviors, many of which to visualize individual connections between neurons. Uchida, PhD are habitual tasks (e.g. Therefore, to verify hypothetical circuits, we use elec- driving, brushing teeth, tron microscopy (EM) as a complementary imaging Mentor: Mitsuko etc.). Though symptoms technique. EM provides structural images of the Watabe Uchida of the disorder are asso- fish at subcellular-level resolution, which allows us ciated with low levels of to reconstruct neuronal processes and identify con- dopamine neurons (DA) nections. in the brain, the connection between the function of DA and symptoms of Parkinson’s disease is currently The challenge to this approach is to reliably match not well understood. The goal of our research is to neurons from light microscopy images to their elec- gain a more detailed understanding of the impor- Neuroscience and Behavioral Science 107 tance of DA in the execution of sequential behavior. of vision is becoming increasingly popular, as more and more studies demonstrate their ability to dis- Broadly speaking, our experiment is an odor-reward criminate between objects. Their neuroanatomy also behavioral assay in which mice are trained to per- has many parallels with human cortex; in humans form a sequential task by reacting to specific odors. the ventral “what” stream is responsible for deter- Each odor tells the mouse which one of two water mining “what” objects are present in an image, and ports to poke. The mouse is rewarded with a small the rodent’s LM, LI and LL regions are thought to drop of water (30 ms) upon choosing the correct wa- play a similar role. Therefore investigation of the ter port to the first odor, and a large drop of water roles of these higher-level processing regions in ob- (200 ms) upon choosing the correct water port to the ject recognition may help further our understanding second odor. After the second odor, the next trial of the function of human visual areas. starts with a random odor after a short interval of time, and the mouse repeats the task. I have trained rats to discriminate between two three- dimensional objects, using water-pump levers to pro- Once the mice are able to stably perform the task, vide both the method of object indication and to de- we then inactivate specific subsets of neurons within liver water as reward. The rats are trained over the the brain and examine changes in behavior. Our course of several weeks, with the difficulty of the task experiment examines inactivation in three sets of increasing in phases. The difficulty ranges from dis- neuronal populations: 1) dopamine neurons within tinguishing the objects face on, with horizontal sepa- the lateral substantia nigra pars compacta (SNC), 2) ration of the object also prompting the correct lever, neurons within the posterior striatum, and 3) neu- to recognising the objects transformed by a combina- rons within the anterior dorsal striatum. Temporal tion of rotation and size variation. The rats progress inactivation of these neuronal populations is per- to the next phase once they consistently perform sig- formed by a DREADD method involving the kappa nificantly above chance, with a correct response rate opioid receptor, KORD, which is introduced with a above 70%. The levers are wired such that the com- virus, AAV. KORD hyperpolarizes and inactivates puter automatically logs a range of data on the rats’ neurons when activated by its ligand, salvinorin-B responses, including the number of successes, failures (salB). Thus, by injecting AAV-KORD into target or ignores. I used Python to extract and interpret the neuronal populations, we can perform temporal in- data from the experiments. activation of these specific neurons by then injecting salB. Through inactivation of these neuronal subsets, The ultimate aim of the project is to selectively le- we hope to better understand the importance of DA sion the higher visual areas of the rats, under the in the execution of sequential behavior, and use our guidance of functional imaging techniques. These results to further understand Parkinson’s disease. lesions will damage the neurons solely in the se- lected region, causing the cells to die, while leav- ing other regions intact. Comparison of the rats’ On Object Recognition in Rats task performance before and after lesioning will al- Your ability to iden- low us to create hypotheses about the role of these tify the object that you brain regions in object recognition in rats. We be- Josh Breedon currently hold in your lieve that a detailed understanding of the cortical Medicine, 2020 hands as your cherished processing in this model system may also provide in- Emmanuel College abstract book is one that sight into the intricacies of the human visual system. Harvard University you most likely take for granted. As conditions Advisor: David Cox, in illumination, orienta- PhD tion, and degree of oc- clusion vary, a particular Mentor: Javier Masis object can project a po- tentially infinite number of patterns of light upon your retina. And yet, seemingly effortlessly, your vi- sual system is able to untangle this image to allow object recognition. While our knowledge of the pe- ripheral, parallel processing that an image undergoes is well developed, our understanding of the details of visual cortex processing is still nascent.

The use of rodents as a model system in the study 108 Neuroscience and Behavioral Science

Relations between Sleep Spindles and White The Neuronal Basis for Motor Skill Learning Matter Pathways in Schizophrenia and Execution in Rats

One of the core fea- Every time we tie our tures of schizophrenia shoes or practice our ten- Julia Versel (SZ) is cognitive deficits Kushi nis serve, we make use of Neurobiology, 2017 Mallikarjun including learning and precise motor skills. We Molecular and memory. Converging are not born with these Massachusetts Cellular Biology, General Hospital evidence suggests that 2019 abilities; rather, we learn sleep is critical for mem- them gradually until we Advisor: Dara ory consolidation. Sleep Harvard University are able to execute them Manoach, PhD spindles, which can be consistently. How our recorded and detected us- Advisor: Bence brain learns and executes Mentor: Bengi ing electroencephalogra- Ölveczky, PhD these motor skills with Baran, PhD phy (EEG) methods, are nearly perfect consistency short bursts of 12–15 Hz Mentor: Steffen is an intriguing question oscillations during stage Wolff, PhD in neurobiology. Though 2 of non-REM sleep. These oscillations, generated studying this question in in the thalamic reticular nucleus of the brain, are humans can be rather dif- known to play a role in sleep-dependent learning ficult, rats serve as a viable model organism to iden- and memory consolidation. Individuals with SZ tify the mechanisms the mammalian brain uses to have a specific reduction in sleep spindles which pre- learn and execute complex motor skills. dicts sleep-dependent memory consolidation deficits. Therefore, understanding the neural underpinnings The lab has developed a paradigm in which rats of sleep spindle deficits may help in finding new tar- learn a precise motor skill that mirrors how humans gets for treatment of cognitive deficits in SZ. learn complex motor skills. We make use of a high- throughput system in which the rats have to press a The primary focus of my research project is investi- lever twice with a distinct time interval and develop gating the structural integrity of brain regions (white a complex sequence of movements to fill this time matter pathways) critical for sleep spindle genera- gap. Thus, we have a model system for a motor task tion and propagation in SZ. I will quantify structural in hand that allows us to study the neuronal basis integrity using diffusion weighted imaging. Specif- of motor skills. With this system, we try to find out ically, my research question is whether there is a which parts of the brain are involved in the circuit, difference in white matter integrity between individ- how they communicate, and what roles they play. uals with SZ and matched controls that may be as- sociated with sleep spindle deficits and subsequently Previous experiments in the lab have shown that a impaired memory consolidation. lesion of the motor cortex, an important player in motor control, before the learning of this precise mo- For that purpose, I am using diffusion weighted tor skill, prevents the rat from learning. However, imaging (DWI), a neuroimaging technique that if the motor cortex is lesioned after learning has al- quantifies integrity of white matter tracts in the ready taken place, the rat is still able to execute this brain by measuring the molecular diffusion of wa- motor skill with precision and consistency, indicating ter in brain tissue. I hypothesize that patients that the motor cortex is important for the learning will show abnormalities in thalamocortical tracts but not the execution of this motor skill. Thus, an- which will correlate with spindle activity. By re- other region of the brain must be responsible for stor- lating sleep spindle activity from EEG data with ing and executing this motor skill once it has been white matter pathway integrity from DWI, I hope learned, and the motor cortex might act as a tutor to clarify the structural mechanism involved in for this region during learning. One potential tutor- sleep spindle deficits in patients. This investiga- ing target is the striatum, a subcortical area of the tion will hopefully illuminate the mechanism of basal ganglia involved in motor control. Further ex- sleep dependent memory consolidation deficits in SZ. periments in the lab have shown the striatum to be necessary for both the learning and the execution of the motor skill.

We want to find out if the striatum is really tutored by the cortex. To address this question, we use a viral silencing technique that allows us to prevent neurons in the cortex from communicating with the striatum. Neuroscience and Behavioral Science 109

A crucial aspect of these experiments is to determine This ability to single out proprioceptive neurons will the precise injection site of the viruses used for si- open up a world of research possibilities. Novel ter- lencing, the number and the exact location of the minal endings could be investigated; further classifi- infected and silenced neurons. We can correlate the cation of the broad “proprioceptive neuron” could be extent of the silencing with the impairment of learn- detailed; in vivo and behavioral experiments could fi- ing and conclude whether already small perturba- nally be accomplished—the enigmatic depths of the tions of the connection between cortex and striatum nervous system will be explored like never before. are sufficient to impair the learning. This approach will provide important information about the com- The Function of the Cysteinyl Leukotriene munication between different parts of the motor sys- Receptor 2 in Itch Pathology tem and how they interact to allow for the learn- ing and execution of motor skills. This knowledge Itch defines the irrita- will help us to understand how this essential part tion prompting scratching of our lives is actually implemented in our brains. Lin Ni that accompanies many Neurobiology, 2019 skin conditions such as atopic dermatitis. Orig- Classifying Proprioceptive Neurons of the Harvard Medical inally classified as a mi- Peripheral Nervous System School nor subtype of pain, itch Proprioception is the Advisor: Isaac Chiu, is now perceived as a sensory ability to per- PhD sensation different from Lauren ceive body position and pain, a separate entity in Sweetland movement. Made possible an organism’s physiolog- Neurobiology, 2018 Mentor: Tiphaine through proprioceptive Voisin, PhD ical pathway. Itch, al- Howard Hughes neurons and their accom- though a highly pertinent Medical Institute, panying terminal endings, sensation, was previously Harvard Medical such as the well-known an understudied phenomenon. In the last decade, School Golgi tendon organ and however, the study of itch has gained much more muscle spindle fibers, pro- interest. Focused research in this area has led to Advisor: David prioception is integral the discovery of itch-specific receptors and distinct Ginty, PhD to many locomotive or- molecules separating itch from other senses, and re- ganisms. Dysfunctional cent genetic profiling of somatosensory neuron pop- Mentors: Brendan proprioception is associ- ulations has identified specific neuron populations Lehnert, PhD; Yang ated with a wide range of showing high transcript expression levels for itch re- Zheng neurological and muscu- ceptors. There are, however, still many facets of itch lar impairments such as sensation that remain targets for future research. Ehlers-Danlos syndrome. Many aspects of proprioceptive neurons remain un- We focus our research on one of the target receptors, known, however. As a part of the peripheral sen- Cysteinyl leukotriene receptor 2 (Cystlr2), which has sory system, proprioceptive neurons have remained been identified in a neuron population implicated in largely indistinguishable from other sensory neurons, itch. Though the Cystlr2 receptor has an established preventing definitive research on properties such as role in the immune system, particularly during in- anatomy and central nervous system connections flammation, the role of the receptor in itch is still un- unique to the proprioceptive classes. clear. It is suspected that the Cystlr2 receptor could potentially mediate the interaction between Cystlr2 Through ongoing research at the Ginty Lab, we hope receptor ligands produced by immune cells and the to find a strategy to label subtypes of propriocep- consequent itch response. tive neurons. Recently, we have been using knock-in mouse models that utilize fluorescent proteins, such Experiments were performed to evaluate if a partic- as GFP, to visualize specific neuronal classes. Fur- ular subset of neurons could be activated by one of thermore, we use these mice to perform intersectional the endogenous Cystlr2 ligands, LTC4. Activation in situ and immunofluorescence strategies, allowing of a specific population of neurons in wild-type mice, us to qualitatively assess the expression of multiple but not Cystlr2 knockout mice, would identify the genes within one tissue sample. These approaches Cystlr2 receptor as a potential target in alleviating will help to identify gene expression levels unique to atopic dermatitis and other chronic skin conditions proprioceptive neurons, effectively cracking the bio- characterized by excessive itch. logical code necessary to distinguish proprioceptive neurons from the billions of other peripheral neu- We approach identifying the function of the Cystlr2 rons. receptor in itch through both in vitro and in vivo 110 Neuroscience and Behavioral Science experiments. In vitro, dorsal root ganglion (DRG) regulate leg movement send axons from the medial neurons are cultured from a wild-type (WT) mouse motor cortex to the lumbar spinal cord while CSMNC and a knockout (KO) mouse for the Cystlr2 gene. that regulate hand/arm movement send axons from Calcium imaging is then used to determine the re- the lateral motor cortex to the cervical spinal cord. sponse of neurons from the mice to capsaicin (as a During development, the proper pruning and speci- control, to eliminate dead neurons) and to LTC4. In fication of these long axon collaterals are necessary vivo, differing concentrations of LTC4 are injected for the establishment of mature connections and a into both WT and KO mice. Mice are then eval- functional central nervous system (CNS). Aberrant uated, in five-minute increments for thirty minutes, pruning of axons has been implicated in several neu- for the number of observed scratching bouts. rological disorders.

Preliminary results have shown some activation of KFM1 —a small leucine-rich repeat keratan sulfate neurons in response to LTC4 in DRG neurons in proteoglycan—is a prominent protein in the extra- vitro. In vivo, preliminary results indicate that KO cellular matrix (ECM). Microarray data from our mice tend to have a slightly diminished itch response lab have shown that KFM1 is expressed selectively to LTC4 injections as compared to WT mice, but the by CSMNC and not CSMNL. Biotinylated dex- results have yet to be consolidated into a definitive tran amine (BDA) and fluorescence imaging have finding. both revealed significant increase in axonal branch- ing of the CSMNL population within the cervical If follow-up studies show that there is indeed cords of KFM1 knock out (KO) mice. This is an a difference in responsiveness between the neu- intriguing observation, because KFM1 is expressed rons of knockout mice and the neurons of wild- only in the CSMNC population but the phenotype type mice to LTC4, we can establish an interac- was observed in the CSMNL population. How- tion between immune response and itch response ever, it remains unclear whether CSMNC -derived as mediated by the Cystlr2 receptor, making the KFM1 affects CSMN branching through direct or receptor a future target for treatment research indirect mechanisms. Previous studies have shown on chronic skin disorders and atopic dermatitis. that KFM1 is capable of binding and promoting the activation of macrophages. Therefore, we asked if the mechanism of specification by KFM1 was me- Investigating the Role of KFM1 and diated by microglia—the resident macrophages of Microglia in the Segmental Specification of the CNS. Our initial immunohistochemistry stain- Corticospinal Motor Neurons (CSMN) ing demonstrated a decrease in the ramification and In order to better expression of activation markers of microglia within understand the target- the cervical cords of KFM1 KO mice at P7 and P10, Michael Liu ing of corticospinal mo- corresponding to the fact that the axons that form Human tor neurons (CSMN), we the CST are pruned by the second week of post- Developmental and Regenerative Biology, are investigating the role natal development. To further elucidate the role of 2019 of KFM1 and microglial microglia and KFM1 in CSMN targeting, we will pruning in the develop- perform AAV rescue experiments and interrogate Harvard Stem Cell ing spinal cord. The the impact of microglial ablation on axon collateral Institute central nervous system branching. This project will also be accompanied by (CNS) features a tremen- an optimization of recently developed clearing tech- Advisor: Jeffrey dous diversity of neu- niques to obtain high-resolution three-dimensional Macklis, MD rons along a spectrum images of axon branching in the developing spinal of function, morphology cord. Mentors: John and molecular expres- Hatch; Yasuhiro sion. One particular sub- An understanding of the molecular mechanisms that Itoh, PhD type—CSMN—originates mediate axon branching is crucial to decoding the from pyramidal cells in intricate process of CNS development and to de- layer V of the neocortex signing treatments for aberrant circuit formation that descend through the corticospinal tract (CST). that underlie numerous pathologies. Furthermore, CSMN are the primary subpopulation of neurons the exploration of axon branching and pruning will that degenerate in ALS, hereditary spastic para- open an entirely new realm of therapies that may plegia (HSP), primarily lateral sclerosis (PLS) and be leveraged for regeneration and repair of the CNS spinal cord injury. The axons that project through after traumatic spinal cord injury and dysfunction. the CST arise from different anatomical regions of the motor cortex. Furthermore, CSMN are a het- erogeneous population of neurons. CSMNL that Neuroscience and Behavioral Science 111

Patterns in Odor Object Recognition Understanding Formation of Mirror Neurons in Humans Perception is the ba- sis of our understanding Mirror neurons are a Miruna Gabriela of the world. Thus, we class of neurons which Cristuş ask: how is sensory in- Mohamed Ebied have been identified in the Applied Biomedical formation encoded in the ventral premotor cortex, Mathematics, 2019 Engineering, 2019 brain and how does it re- the primary somatosen- Center for Brain late to behavior? In our Harvard Medical sory cortex, the sup- Science, Harvard experiments, we are try- School plementary motor area, University ing to answer this ques- the primary somatosen- tion in mice, by seeing Advisor: John Assad, sory cortex, and the infe- Advisor: Venkatesh how olfactory informa- PhD rior parietal lobule. Since Murthy, MD, PhD tion is represented in the their discovery in the brain and how it impacts Mentor: Antonino early 1990s, mirror neu- Mentor: Elizabeth their ability to identify Casile, PhD rons have become a crit- Shtrahman, PhD odors. Because natural ical consideration in our odors are mixtures of dif- broader understanding of ferent chemical species, neurological systems, sparking significant debate in we want to analyze whether odors are perceived as the scientific community due to their highly unique the sum of their individual components (elemental response properties. Mirror neurons are unique in processing) or as something with different holistic their ability to respond to the execution as well as qualities relative to the mere components (configural observation of goal-directed motor actions. Initially or synthetic processing). There is no current consen- observed in monkeys, the later discovery of mirror sus in the literature regarding these two outcomes; neurons in humans is considered to be one of the therefore, we aim to thoroughly characterize the pat- most important discoveries in systems neuroscience. terns behind these results. Specifically, we want to quantify what odor inputs and properties of certain Naturally, much research on this subset of neurons odor mixtures correspond to certain outcomes. has followed, and the literature is dense with at- tempts to understand their role from an evolution- In order to analyze these features, we have designed ary as well as functional perspective. However, the a behavioral task to test odor object recognition. available literature on precisely how they form is rel- The odor objects in our experiment are represented atively sparse. My research attempts to delineate by odor mixtures of three different components. The how mirror neurons form in response to new motor mice are thus trained to recognize such a mixture and stimuli. In order to accomplish this, we have built a differentiate it from other mixtures of three compo- virtual reality platform which allows us to carefully nents that they have never encountered before. We train human subjects on highly specific motor tasks, are testing a variety of target mixtures, chosen for minimizing the input from external stimuli. The use the chemical likeness of the components or similarity of a virtual reality platform also ensures that the vi- of their glomeruli responses in the brain (initial sites sual associations with the motor task remain highly of neural responses). In order to measure their per- specific, and are not influenced by surrounding vi- formance, we train water-restricted mice to respond sual distractions. Based on previous experiments, it to stimuli by licking “right” on a lickport for the has been shown that four days is the approximate target odors and “left” for the nontarget odors. This amount of time required for the brain to physiologi- experimental setup also allows us to see the type of cally respond to new stimuli. Hence, the “training” mistakes mice make and how their mistakes evolve period for subjects is set to four days. Subjects are during the training sessions. scanned using fMRI before, during, and after the training phase of the experiment in order to assess Preliminary results show that mice are able to the changes which are occurring in response to the learn this task well and we are further test- new motor stimulus. ing a variety of odor object stimuli. We ex- pect our results and future work to enable us It is our hope that garnering a greater understand- to find the link between these stimuli and their ing of how the brain actively rewires itself to generate perception as configural versus elemental odors. mirror neuron systems will help forward our under- standing of their function as a whole. Since fMRI data would allow us to quantitatively understand the cascade of events which lead to the formation of these neurons, we can begin applying our understanding 112 Neuroscience and Behavioral Science of this process to the functions that mirror neurons aberrant slow oscillations. The calcium transients of have been hypothesized to allow—namely empathy, the wild-type mice were observed to be as expected, intention, understanding, and even complex linguis- rhythmic and oscillatory, while in APP mice, they tic development. We do not yet have enough data were sporadic and of lower power than those of the to determine what extrapolative conclusions can be wild-type mice. Data analysis is ongoing to con- drawn, but we have taken the first step in develop- firm these promising results and better our under- ing an accurate model of mirror neuron formation. standing of AD, slow waves, and memory formation.

Disruption of Memory-Consolidating Slow Use of a Novel HEK 293 Culture Model to Oscillations and Calcium Transients by Examine the Role of Neuropilin-2 in Drd4 Amyloid in Alzheimer’s Disease Retinal Ganglion Cells Alzheimer’s disease I am investigating the (AD) is a progressive neu- role of the receptor pro- Shenyece rodegenerative disorder Vimal Konduri tein neuropilin-2 (Nrp2) Ferguson Molecular and characterized by the pres- in retinal ganglion cells Neurobiology, 2019 Cellular Biology, ence of amyloid plaques, 2017 (RGCs), which transmit Massachusetts neurofibrillary tangles, visual information from General Hospital and loss of neurons in the Harvard University the retina to the brain. human brain and is a ma- Neuropilin-2 has been im- Advisor: Brian jor cause of dementia, or Advisor: Joshua R. plicated in axonal guid- Bacskai, PhD memory loss. Slow oscil- Sanes, PhD ance, dendrite develop- lations are responsible for ment, angiogenesis, and Mentors: Ksenia consolidation of memories Mentor: Yirong the formation of tumors Kastanenka, PhD; during sleep. Peng, PhD such as melanoma and Guillaume Pagnier, glioblastoma. Neuropilin- MS To determine whether 2 is known to colocalize aberrant slow oscillation with the plexin receptor PlexA3 in the presence activity is part of the dis- of class-3 semaphorins, and with VEGFR-2 and ease progression, we used VEGFR-3 in the presence of vascular endothelial a mouse model overexpressing a mutant amyloid growth factor. The role of neuropilin-2 in the retina, precursor protein (APP) and a mutant presenilin which is part of the central nervous system, is cur- (PS1). Amyloid, a cleavage product of APP, is re- rently being studied in a collaborative effort between sponsible for disrupting slow waves in the cortex by the Sanes lab and the lab of Alex L. Kolodkin at possibly altering electrical activity in inhibitory in- the Johns Hopkins University. Previous studies by terneurons. To test whether interneuron activity is our lab in mice have shown that Drd4-RGCs, a altered, mouse brains with interneurons that could specific type of direction-sensing RGC (which sense be targeted in the context of AD were imaged with movement in the nasal direction and normally make multiphoton microscopy. A fluorescent calcium indi- up roughly 5% of the total RGC population), are cator, GCamp, was targeted to interneurons of APP greatly reduced in number when Nrp2 is knocked mice and wild-type littermates. Calcium imaging out. However, a mechanism of cell death has not was performed using multiphoton microscopy to im- been established. age calcium transients in interneurons. I will study neuropilin-2 by examining systems in I performed analysis by selecting regions of interest which its ligands, the semaphorins Sema3C and (ROIs) which corresponded to cell bodies of the in- Sema3F, and VEGF, are overexpressed. I will terneurons using ImageJ software. The change in use cells from the HEK 293 cell line transfected fluorescence over the average fluorescence was cal- with plasmids to constitutively produce these lig- culated for each selected cell over a 500 frame (ap- ands in order to simulate an overexpression, and proximately 215 second) timelapse. In MATLAB, will plate them in a co-culture with retinal gan- a Fourier transform was performed to identify the glion cells. After co-culture, I will assess cell sur- amplitude and frequency of the slow oscillations of vival and neurite (axon and dendrite) growth using each cell. In wild-type mice, we expected to observe live/dead and beta-III-tubulin staining, respectively. calcium transients that were similar to slow oscilla- I will carry out this assay for both RGCs in gen- tions of interneurons and at the same frequency as eral and Drd4-RGCs (which can be isolated using slow waves. However, in APP mice, we predicted FACS sorting) specifically. I will be able to assess the calcium transients to be disrupted by the pres- the proportion of RGCs that survive in culture and ence of APP, which could be responsible for the the average length of RGC neurites, and compare Neuroscience and Behavioral Science 113 each to a control in which RGCs are co-cultured with unmodified HEK 293 cells. The results of these studies can help shed light on how neurons in the central nervous system, such as RGCs, make key decisions related to survival and development. Organismic and Evolutionary Biology

Development and Evaluation of Diagnostic capable of optimizing the identification of highly con- Tools for Zika Virus served nucleotide regions in the Zika genome and de- signed new primers and probes to target these ho- New cases of Zika mologous regions. The specificity of the new assay virus are confirmed ev- design was evaluated using BLAST against the NCBI Cynthia Luo ery day in the Ameri- Organismic and nucleotide database. We are currently evaluating the cas, and the current out- Evolutionary performance of the assay by assessing assay robust- break has reached pan- Biology, 2019 ness, sensitivity (limit of detection), reproducibility, demic levels in parts of and cross reactivity to ensure that it will be highly Harvard University, Central and South Amer- specific and sensitive to detecting Zika virus in pa- Broad Institute of ica. Zika virus, an tient samples. MIT and Harvard RNA Flavivirus spread by Aedes mosquitoes, causes In parallel, we are using similar methodology to de- Advisor: Pardis the infectious disease Zika velop a multiplex RT-qPCR diagnostic test for Zika, Sabeti, MD, PhD fever. Between humans, Chikungunya, and Dengue fever. This will allow the virus can be sexually medical professionals to use one test to differentiate Mentor: Mary Lynn transmitted and passed Baniecki, PhD between all three clinically similar diseases, greatly from mother to child, reducing the costs and time associated with per- which may lead to brain forming these diagnostic tests. We hope that these malformations such as mi- tests will be directly transferable in diagnosing Zika crocephaly. More severe infections of Zika virus in virus in the field and will have profound impacts adults can also lead to Guillain-Barré syndrome, a on improving patient treatment for Zika infection. life-threatening autoimmune disease that damages the peripheral nervous system. Low viral titers in Zika-positive patient samples and the similarity of Developing a SNP-Based Barcode for initial symptoms to other infections such as Dengue Babesia microti fever and Chikungunya present multiple challenges to properly diagnosing Zika virus infection. Thus, Babesia microti is a extremely sensitive and accurate diagnostic tests for malaria-like parasite that Zika virus are needed in order to provide patients Jade Moon infects red blood cells and History and Science, with proper diagnosis and appropriate medical treat- is spread by Ixodes scapu- 2017 ment. laris ticks. Because the Harvard University, parasite is endemic to dif- Reverse-transcriptase quantitative polymerase chain Broad Institute of ferent regions across the reaction (RT-qPCR), a low-cost, robust technique MIT and Harvard world, the ability to iden- that requires minimal training, is an ideal technique tify the geographic ori- to deploy in field settings. RT-qPCR diagnostic Advisor: Pardis gin of a strain can help tests for Zika have been previously developed but Sabeti, MD, PhD us locate sources of out- lack specificity to the current outbreak. Previous as- break, monitor the pat- says were developed using whole genome sequencing Mentor: Mary Lynn terns and distributions of (WGS) data from an African strain of Zika, shown Baniecki, PhD infections, and ultimately to be 11% genetically divergent from strains isolated aid in elimination efforts. in the Americas during the current outbreak. A lack of strain specificity in current diagnostics could lead To distinguish between to improper patient diagnosis, which delays appro- strains, we look to the genetic variations that ex- priate treatment and worsens medical prognosis. ist between populations; for example, a single nu- cleotide polymorphism (SNP) allele common in one We are developing a RT-qPCR assay that is robust geographical group may be rarer in another. Thus, and highly specific to Zika virus strains across the genotyping SNPs provides a promising means of de- globe. To design our assay, we first identified the veloping an inexpensive and rapid assay that can most conserved region of the Zika genome by using uniquely identify the geographic origin of a given all 80 available whole genome sequences from the Na- strain. In Babesia. microti where SNPs have been tional Center for Biotechnology Information (NCBI) determined through whole genome sequencing, we and by creating multiple sequence alignments with can select a small subset of SNPs that will identify Geneious software. Next, we used in-house software parasitic infections and differentiate geographic ori- Organismic and Evolutionary Biology 115 gins. This set of SNPs will serve as unique genetic bryo’s life and is subsequently restricted, or it begins signatures or barcodes for Babesia. microti. expression later on specifically in the cell lineage that gives rise to neoblasts. We will also compare piwi- We identified informative SNPs by considering two 1 expression to expression of other neoblast markers criteria. First, the subset of SNPs must main- with possible related functions such as vasa and tu- tain clustering after performing principal compo- dor. We are generating a staged series as a reference nent analysis (PCA). PCA ensures that our selec- for embryonic development in Hofstenia. We are also tion of SNPs captures high degrees of population di- developing methods to use in situ hybridization to versity and differentiates between geographically dis- determine the expression pattern of piwi-1 mRNA tinct populations. Second, to avoid positive selec- in embryos at different stages. tion, we must only consider SNPs resulting in syn- onymous mutation. SNPs were genotyped using high As a new model organism, Hofstenia miamia of- resolution melting analysis (HRM). HRM takes ad- fers a unique opportunity to study the basis of re- vantage of the differences in melting temperatures of generation in bilaterians. Whole-body regeneration amplicons with different base pairings. could be the ancestral state: most early lineages of metazoa are capable of performing it. Thus later Developing a rapid, sensitive, and specific tool to diverging lineages of bilaterians seem to have re- identify parasitic origins will not only aid in cor- stricted regenerative capacities. By documenting rect diagnostic and treatment of infections, but the previously unstudied embryonic development of also allow for examination of the phenotypic dif- Hofstenia miamia, and studying the expression of ferences of different infections. Here, we de- piwi-1 in early stages of this development, we can veloped a 40-SNP barcode consisting of 20 mi- explore the genetic mechanisms and cellular path- tochondrial SNPs and 20 nuclear SNPs. Due ways necessary for maintenance and regulation of to the inheritance patterns of mitochondrial and a successful adult pluripotent stem cell population. nuclear DNA, the combination of mitochondrial In an evolutionary context, we can use these ob- SNPs and nuclear SNPs allows efficient identifica- servations to compare to gene expression during tion of geographic heritage and genomic information. the embryonic development of later diverging lin- eages, and potentially explore what changes occurred that led to the restriction of regenerative capacity. Embryonic Origins of Adult Stem Cells in the Acoel Hofstenia miamia The evolutionary basis and developmental mech- Silvia anisms for regeneration in Golumbeanu animals are not well un- Integrative Biology, 2017 derstood. Planarians (e.g. Schmidtea mediterranea) Harvard University and acoels (e.g. Hofste- nia miamia) are distantly Advisor: Mansi related animals both ca- Srivastava, PhD pable of whole-body re- generation; while they are evolutionarily distant, they both possess populations of pluripotent adult stem cells called neoblasts. However, the develop- mental origins of neoblasts are unknown.The acoel Hofstenia miamia is particularly useful in address- ing the question of neoblast origin, as it produces abundant embryos that are amenable to experimen- tal investigations.

We seek to understand the origins of the stem cell population by studying the embryonic develop- ment of Hofstenia using known adult neoblast mark- ers—primarily piwi-1. Our data will allow us to dis- tinguish between two alternative hypotheses for the developmental origin of pluripotent stem cells: either piwi-1 is expressed from very early on in the em- Physics and Biophysics

Slipping and Sound in the Motion of the Constraining Dark Matter-Electron Euler Disk Scattering For over a century, In the 1970s, as- physicists have studied tronomers noticed a dis- Adam Frim the mechanics of a coin David Gonzalez- crepancy between the Physics & spinning on a tabletop Dysinger Mathematics, 2018 measured total kinetic en- or a larger version of the Physics, 2018 ergy of galaxies and the Harvard John A. same problem known as energy that theoretical Paulson School of the “Euler disk.” Re- Harvard University models predicted given Engineering and cent analysis has tended the total mass of all the Applied Sciences to focus on this system’s Advisor: Cora stars and other visible unique feature of a finite- Dvorkin, PhD matter in the galaxies. In Advisor: Ariel Amir, time singularity; that is, order to account for this PhD in a finite amount of time, discrepancy, cosmologists the coin’s rate of preces- proposed the existence of dark matter, an undiscov- sion diverges. Put simply, ered form of matter that does not emit or interact the coin naturally “wobbles” at a rate that increases with light except gravitationally, and is made up without bound, which is easily observed by listening of particles outside the Standard Model of particle to the coin as it reaches the final seconds of its mo- physics. tion as a whirring sound is produced. This unique feature has led to a number of publications with Dark matter particles could possibly scatter with particular focus given to studying different energy baryons, such as protons and electrons. If elastic dissipation mechanisms. scattering between dark matter and baryons really occurs, it would affect the development of the uni- During the course of the coin’s motion, the coin can verse in detectable, quantifiable ways. In particular, roll with or without slipping. The case without slip- it would create observable changes in linear den- ping is far more easily approached; therefore, the goal sity fluctuations in the early universe. By analyzing of the current project was to build a mathematical data from the cosmic microwave background and the model that incorporates both the rolling and slipping Lyman-alpha forest, we can place constraints on the of the disk. Due to nonlinearity in the governing dif- strength of dark matter-baryon scattering. This will ferential equations for both the rolling and slipping allow us to narrow down the range of masses that regimes, the problem was approached numerically. dark matter particles can have in scattering models. The resulting numerical model can independently transition from rolling to slipping when certain con- I am studying the elastic scattering between dark ditions are met. Such a model can provide useful matter and electrons in the early universe, from right simulation while remaining experimentally verifi- after the Big Bang up until recombination, when able. Moreover, this numerical model should more electrons combined with protons to form hydrogen accurately suggest the rate at which slippage occurs, atoms. Using theoretical models, I compare the mo- a question that has not yet been sufficiently stud- mentum exchange between dark matter and electrons ied. Finally, the model could be used to suggest a to that between dark matter and protons to see if mechanism by which sound is repeatedly made dur- there are any regimes of dark matter mass and red- ing the coin’s motion. In particular, if this model shift in which the electron momentum transfer domi- predicts that the frequency of slippage aligns with nates over the proton momentum transfer. Studying experimentally observed sound spikes, this could the interactions between dark matter and low-mass be a strong indicator that the sound arises from a particles require the modification of the equations stick-slip phenomenon in the motion of the disk. used to study scattering with higher-mass particles. I am looking for a region in the parameter space where the dark matter-electron scattering dominates, predicting the effects of the scattering on observ- ables, and using data to constrain this scattering. Physics and Biophysics 117

The Statistical Physics of mRNA Dynamics the thermodynamic variables (temperature, concen- tration, etc.) they are related the most to, and the The universe would interplay between key players such as repressors, ac- be a very boring place tivators, and catalyzers. Our mathematical machin- Elgin if it were in equilib- ery involves simulations to reproduce what happens Korkmazhan rium, or more precisely, in real cells and theoretical analysis in order to under- Gulpinar Chemistry and in thermodynamic equi- stand them, to see how we can obtain a “big picture” Physics, 2017 librium. This is what of the network and perhaps interfere with it. is called the heat death Harvard University of the universe, and it Currently, we are working on explaining the localiza- would simply mean that tion of an mRNA onto the cell membrane of E. coli, Advisor: Erel Levine, energy would be evenly one of the most diverse bacterial species. Though PhD distributed throughout all mRNAs themselves cannot attach to the cell mem- of space. With no imbal- brane, the domains of the proteins they help syn- Mentors: Hamid ance to “drive” systems, thesize can. During protein production, the syn- Teimouri, PhD; Neil processes such as infor- thesis machinery moves along the mRNA and holds Peterman, PhD mation transfer, compu- the mRNA and the nascent protein together. Thus, Candidate tation, and life would be the existence of membrane binding domains on the impossible. Indeed, sys- already made part of the protein makes it possi- tems in equilibrium only ble for the mRNA to anchor to the membrane, account for a minuscule amount of what surrounds until protein production is done and the machin- us. Yet, we still do not know much about non- ery lets go of the mRNA. This mechanism sug- equilibrium systems. In fact, non-equilibrium sta- gests that membrane localization depends on factors tistical physics, the field trying to answer questions such as the collision rate with the membrane, the about the behavior of such systems, has been de- “stickiness” of the protein’s membrane binding do- clared as “one of the greatest challenges of 21st cen- mains, the size of the remaining part of the pro- tury physics.” A relatively new field, it has been de- tein to be produced, and the availability of more veloped and utilized in the studies of topics ranging free “anchors” on nascent proteins being simultane- from black hole formation to quantum device tech- ously made by other synthesis machinery progress- nology. ing along the same mRNA. Our aim is to under- stand these factors as comprehensively as possible. What we are interested in is what non-equilibrium statistical physics can tell us about processes in living Magnet Design for a Nuclear Fusion Reactor cells and how those processes can reveal important physics. Namely, we analyze the dynamics of mR- One of the biggest NAs. Proteins are molecules functionally and struc- problems humanity faces turally crucial for cells, and mRNAs are DNA-like Jacob Meyerson is producing electricity Physics, 2019 molecules mainly responsible for providing the pro- without fossil fuels. The tein production machinery with the necessary code ability to do so on a MIT Plasma Science for protein synthesis. It has been shown that the and Fusion Center large scale would have amounts, positions, and gradients of particular mR- widespread benefits, from NAs within and across cells are responsible for both Advisor: Joseph helping combat climate smaller-scale events such as the localization of pro- Minervini, PhD change to helping increase tein production and larger-scale changes such as the the independence of the differentiation of stem cells into cells of tissues such Mentors: Franco United States. Nuclear as those of the brain. Mangiarotti, PhD; fusion is an appealing op- Taylor Sorenson; tion that could help solve Recently, there have been experimental observations Aaron Hamilton this problem. Unlike so- of non-uniform gradients of specific mRNA involved lar or wind, it would be in antibiotic resistance pathways in bacterial cells. a constant source of elec- The surging medical and societal cost of the failure tricity, and unlike nuclear fission, it would not pro- of antibacterial drugs against resistant bacteria, or duce long-term radioactive waste nor pose the risk of “superbugs,” makes such systems very important to a nuclear meltdown. study. We seek the understanding of the reasons behind such mRNA gradients, their role in bacte- Fusion energy is produced when two hydrogen iso- rial cells, the effective ways in which they can be topes’ nuclei fuse to form a helium nucleus, a reaction perturbed, the information transfer needed for their which releases energy. For these reactions to occur, emergence, the means of this information transfer, hydrogen isotopes must be heated up to very high 118 Physics and Biophysics temperatures—hundreds of millions of degrees—to family of crystalline 2D materials, among them an form hot plasma. Since the plasma is so hot, you insulator boron nitride (BN) and transition metal cannot contain it in a box—it would melt through dichalcogenides (TMDCs). Although TMDCs have any material. However, one possible way of confin- been known for decades as inorganic semiconduc- ing the plasma is through the use of high magnetic tors, the ability to isolate their single layers opened fields. The magnetic field acts as an invisible force up new opportunities in nanoelectronics, optoelec- that suspends the plasma in vacuum. tronics, and photonics.

Producing these high magnetic fields is a chal- lenge. A method of doing so is through the use of superconducting materials. At extremely cold temperatures—at around 5–20 K, near abso- lute zero—superconducting materials have zero re- sistivity. Therefore, the materials can handle large amounts of current, consequently creating a large magnetic field. Creating these magnets is challeng- ing. They must produce the appropriate magnetic field, they must be able to withstand the stress due to the force from the magnetic field, and they must remain cold while million degree plasma floats a me- ter away.

Researchers at MIT’s Plasma Science and Fusion Center are working on designing these magnets. Yet, their in-depth simulations of various aspects of the magnet (for example, the magnet’s stress con- centrations) take weeks to complete. Along with two other students, I am working on a code that will calculate first-order approximations of the over- all magnet design—the stresses of the magnet, the magnet’s geometry, the cooling of the magnet, the structural composition of the magnet. By using the code, the researchers will hopefully be able to narrow down the areas in which they investigate.

Towards Electrically Controlled High Temperature Interlayer Exciton Superfluids The concept of re- duced dimensionality in In conventional semiconductors, electrical transport Kateryna physical systems has long is realized with either negatively charged electrons or Pistunova been studied as a purely positively charged holes (an absence of an electron in Physics, 2018 abstract although mathe- a place where it could exist in the crystal structure). Harvard University matically interesting idea. Due to electrostatic interactions, these oppositely Such systems were pre- charged carriers can form excitons, bound states with Advisor: Philip Kim, dicted to exhibit unique particle-like behavior. A characteristic feature of ex- PhD electronic and optical citons is their lifetime, the time it takes for electron properties, making them and hole to recombine. Normally, the close proxim- Mentors: Luis completely distinct from ity of electrons and holes in conventional semicon- Jauregui, PhD; their 3D counterparts. ductors results in very short lifetimes, such as a few Andrew Joe However, the success of picoseconds in GaAs. However, spatial separation of graphene, a single-atom- an electron and a hole, which form a so-called “space thick layer of carbon, has indirect” exciton, leads to much longer lifetimes due proven it possible to realize 2D systems experimen- to the increased distance between oppositely charged tally. Held together by weak electrostatic interac- carriers. Our project aims to form spatially indirect tion, often called van der Waals bonding, graphene excitons localized in vertically stacked monolayers layers can easily be cleaved from bulk graphite. The of n- and p-type TMDCs (that is, with high elec- rise of graphene has led to the emergence of a whole tron or hole concentration, respectively). Due to the Physics and Biophysics 119 reduced dimensionality of TMDC monolayers, elec- Heat Shock on C. elegans trons and holes are confined to a plane, resulting in an enhanced electrostatic interaction between them. Stress responses Consequently, the two-dimensional nature of mono- across organisms have layer TMDCs facilitates the formation of spatially Myles Ingram many similarities. The Biophysics, 2018 indirect interlayer excitons with prolonged lifetimes nematode Caenorhabditis predicted to be longer than microseconds. The ex- elegans is a simple model Harvard University tended lifetimes provide a platform for exploring ex- organism with relatively citon Bose-Einstein condensation (BEC) phenomena, Advisor: Erel Levine, predictable responses to a state of matter in which a large portion of particles PhD various stresses. Their re- occupy the same energy state and behave as a single sponse to heat shock is entity, exhibiting macroscopic quantum phenomena. Mentor: Kyung Suk especially useful to study Such exciton BEC is expected to form at record-high Lee, PhD because it involves stop- temperatures and has never been realized in TMDCs ping the unfolding of pro- before. teins. Researching the heat shock response pathway, along with other stress response pathways, has led to a deeper understand- ing of the biological mechanisms involved in com- plicated human diseases such as Alzheimer’s and Parkinson’s.

The heat shock response of C. elegans has been well-characterized by subjecting these worms to un- comfortable amounts of heat and subsequently mon- itoring their hormesis (positive response to low stress exposure) and their regulation of heat shock proteins (HSP).

However, few fatal heat shock experiments have been conducted to describe the survival of C. elegans and little experimentation has been done to describe the In order to experimentally study BEC phenomena motility of C. elegans after a fatal amount of heat in TMDCs, high-quality electrical contacts need to shock. This manner of killing will expand upon be demonstrated to control the chemical potential what is already known about C. elegans and their and electric field between TMDC layers. However, heat shock response. due to TMDCs’ semiconducting nature, a potential energy barrier is formed at the metal-semiconductor In these experiments, we aim to characterize both junction, increasing the contact resistance, prohibit- the survival and motility of four C. elegans strains ing current flow, and lowering the device perfor- subjected to heat shock, leading to the death of mance. Our goal is to fabricate devices with the right a significant subpopulation. Survival in wild-type metal/TMDC combinations to achieve low-resistance worms after heat shock was compared to that of two contacts. We obtain monolayers of TMDCs and few- different mutants—one lacking the primary thermo- layer BN by mechanical exfoliation using the Scotch sensory neurons, AFD, and another lacking the tape method. Electron beam lithography, followed thermo-sensory interneuron, AIY—while monitoring by metal evaporation, is used to make pre-patterned the expression of hsp-16.2, an intestinal heat shock contacts on BN. The second BN is then picked up protein. Without AFD neurons, worms cannot sense with polycarbonate stamp on a glass attached to a extreme temperatures and without the AIY neuron, micromanipulator. The van der Waals interaction worms cannot sense extreme cold. We counted the makes it possible to pick up monolayer TMDC with number of dead, alive but not motile, and motile BN and place it on top of pre-patterned contacts. worms at five different time points to describe sur- After the device fabrication, the electrical and mag- vival and motility. We test for motility by tapping netic measurements are performed in a cryostat with the worms on the nose, a well-known C. elegans re- temperatures ranging from 300K to 1.4K. If suc- flex point. cessful, the low contact resistance would allow the study of high-temperature interlayer exciton BEC, as To deepen our understanding of extreme heat shock well as many other quantum transport phenomena. in relation to motility and survival, we also stressed the worms with mild heat shock and starvation be- fore the fatal heat shock. We stress the worms with 120 Physics and Biophysics mild heat and starvation to understand the effect Through electrically isolating graphene with boron of hormesis and to investigate the correlation be- nitride we are able to create gated samples where tween different stresses. In addition to evaluating changes in capacitance can be sensitively measured motility and survival, we also monitor the expres- since boron nitride can be made relatively thin and sion of hsp-16.2 in the two mutant strains (ERL35 still maintain its electrically isolating properties. and ERL36) and the reporter strain (TJ3001) after Measuring fluctuations in capacitance informs us of the fatal heat shock. With these experiments, we how the density of states varies as the chemical po- hope to demonstrate that the inactivation of cer- tential of graphene changes. This is useful since it tain neurons included in the heat shock response enlightens us as to the geometry of these unique elec- can have a positive or negative effect on both sur- tronic phases. By using amplifier circuits, thermal vival and motility in C. elegans exposed to a fatal oscillations can be converted to alternating voltages, dose of intense heat. Our work will broaden the which allows us to determine the fluctuations in dis- literature concerning the roles of heat shock pro- placement current across the capacitor. In using teins and regulation mechanism of their expression. this technique, we should theoretically be able to de- tect how thermal fluctuations influence displacement current, and in turn, how they influence changes Quantifying Thermodynamic Properties in in entropy. The end goal is to make repeatable Graphene Heterostructures measurements of entropy variations in graphene at ultra-low temperatures and be able to derive and Modern condensed predict how these variations take shape. matter physics has suc- Shaan Ajay ceeded in creating elec- Desai Whole Brain Imaging to Determine tronic devices of excep- Physics, 2018 Neuronal Dynamics in C. elegans tional purity, allowing for Harvard University the discovery of the frac- How is it that net- tional quantum hall ef- works of interconnected fect and electron hydro- Benjamin neurons enable animals Advisor: Philip Kim, Sorscher dynamics (Dirac fluid), to process sensory inputs PhD Physics & among other phases of Mathematics, 2018 and encode appropriate Mentor: Jonah matter. While these suc- behavioral outputs? Our Waissman, PhD cesses are attributed to Center for Brain own brains contain many electronic transport and Science, Harvard billions of neurons, yet the resistance measurements, University roundworm Caenorhabdi- many underlying properties of these phases remain tis elegans is capable of unknown and unverified. It is for this reason that Advisor: Aravinthan strikingly complex pro- thermodynamic measurements are crucial to the D.T. Samuel, PhD cessing with a mere 302 study of condensed matter physics. Quantities such neurons. C. elegans can as specific heat, magnetization, and thermal conduc- Mentor: Vivek translate a diversity of tivity can reveal fundamental properties underlying Venkatachalam, PhD sensory stimuli (tempera- material and electronic phase transitions that cannot ture, odor, electric field, otherwise be detected using transport systems. touch) into one of several mutually exclusive, stereotyped behaviors (forward The experimental challenge that lies in determin- and backward locomotion, left and right turns, and ing thermodynamic properties is the scale of nano reversals). By studying the dynamics of the C. el- devices. The specific heat of a two-dimensional elec- egans nervous system we may begin to understand tron system in which the quantum hall effect was the fundamentals of how this complex encoding of discovered, for example, is dominated by the 3D behavior takes place. crystal structure surrounding the electrons, making for low noise-to-signal ratios and hardly detectable Thanks to advances in confocal microscopy, it is now signals. However, the recent discovery of graphene possible to image the whole brain of freely moving and related two-dimensional materials such as hexag- C. elegans at single-cell resolution. Further, a pan- onal boron nitride now provides a new way forward. neuronal promoter enables us to express fluorescent These 2D materials can be stacked together simply calcium indicators in every neuron of the C. elegans via van der Waals attraction, which allows both the nervous system. Calcium ions play a crucial role in creation of clean electronic devices (as it eliminates intercellular signaling, and thus reflect the activity of our need to grow layers), and heat to be trapped in an individual neuron. Combined, these techniques an individual 2D material layer, eliminating the need afford an unprecedented peek at the whole C. ele- for multi-layered stacks. gans brain as it processes information. In order to Physics and Biophysics 121 study the global dynamics of neural activity, I record flakes of BSCCO. We will then chill these samples in whole-brain movies as the animal interacts with sen- a cryostat and supply a current, tracking the associ- sory stimuli and behaves in its environment. From ated voltage drop between contacts at varying tem- these movies, I extract a time series of activity for peratures and field strengths. Starting with thicker each neuron, and look for patterns of activity be- flakes, we will decrease their thickness over the course tween neurons. Using principal component analysis, of iterated device fabrication to explore what elec- I can reduce the dimensionality of the pan-neuronal trical contact to thin BSCCO is possible. Produc- activity data and identify dominant signals across tion of devices aims for the single-layer limit, which many neurons that correspond with specific behav- will be most useful for possible observation of a Bose ioral states. I can then employ statistical techniques metal state far below the superconducting transition. like clustering and empirical dynamic modeling to identify structure in the neural activity data and iso- late potential signaling pathways and sensorimotor circuits. By studying the whole-brain dynamics that translate sensory stimuli into the neural antecedents of behavior in C. elegans, I hope to begin to un- derstand how neural networks are able to solve the problem of encoding sensory information into behav- ior.

Exploring the Phase Diagram of Thin-Film BSCCO When thinned to a few atomic layers, su- Margaret perconductors behave dif- Panetta ferently than they do Physics & Astrophysics, 2017 in their bulkier forms. While electrons in thin Harvard University film superconductors still form “Cooper pairs” that Advisor: Philip Kim, normally condense into PhD a superconducting state, when a magnetic field is Mentor: Frank Zhao applied to these materials, their superconductivity is destroyed even at the low- est experimentally achievable temperatures. A two- dimensional “Bose metal” state, the existence of which is debated, theoretically lies between the insu- lating and superconducting regimes allowed for a ma- terial capable of superconductivity. We are using re- sistance measurements to investigate this transition for thin films of Bi2Sr2CaCu2O8+x, called BSCCO, a copper-oxide (cuprate) high-temperature supercon- ductor.

BSCCO consists of layers of copper oxide interca- lated with layers of its other constituent materials, al- lowing production of extremely thin films incorporat- ing only a few iterations of the fundamental repeating crystal unit. We will measure the resistance of a thin- film sample down to the millikelvin range under an applied magnetic field, seeking evidence of the quan- tum metallic state at one thousandth of the BSCCO superconducting transition temperature of 85K. To do this, we will use a novel stencil-mask technique to evaporate gold electrical contacts onto few-layer Stem Cell and Regenerative Biology

NEUROD6 as a Novel Therapeutic Target Investigating the Role of Non-Classical for Parkinson’s Disease Progesterone Receptors in the Regulation of HLA-G Expression in EVT Dopaminergic (DA) neurons play an impor- HLA-G is a major his- Alex Pai tant role in cognitive and tocompatibility complex Human motor function. In the Andrew (MHC) protein that, un- Developmental and Mazzanti midbrain, DA neurons like most other types of Regenerative Biology, Human 2019 are prominently found in Developmental and HLA, is not involved in the substantia nigra pars Regenerative Biology, peptide presentation to T Harvard Stem Cell compacta (SNpc) and the 2017 cells. Instead, it inter- Institute ventral tegmental area acts with NK cells to pre- (VTA). Of interest is the Harvard Stem Cell vent their cytolytic activ- Advisor: Lee Rubin, degeneration of DA neu- Institute ity. NK cells are innate PhD rons in the SNpc as a phe- immune cells that can tar- notype of Parkinson’s dis- Advisor: Jack get and kill cells infected Mentor: Tim ease (PD). Though DA Strominger, MD by virus or cells found to Ahfeldt, PhD neurons in the SNpc and be foreign or “non-self.” VTA share a common Mentors: Leonardo Suppression of the NK neural progenitor, VTA Ferreira, PhD; cell-mediated immune re- DA neurons are much less affected in PD. An RNA- Torsten Meissner, sponse is potentially dan- PhD sequencing experiment conducted using DA neuron gerous, and accordingly, populations extracted from different brain segments the expression of HLA-G of reporter mice identified Neurod6 as a gene that is is limited to specific tis- differentially expressed between DA neurons in the sues and cell types, including the placenta. Extrav- SNpc and the VTA. The role of Neurod6, which is illous trophoblasts (EVT) are fetal-derived cells that exclusively expressed in VTA neurons, is not well invade maternal tissue during the course of preg- understood outside its role as a transcription factor nancy. Though fetal-derived cells express paternal that regulates cell fate, but recent studies suggest antigens, and EVT is exposed to the maternal im- that Neurod6 plays a role in mitochondrial biogene- mune system, NK cells do not lyse EVT due to sis and maintenance that may explain the resistance the expression of HLA-G. Progesterone signaling has of VTA DA neurons from the cellular stresses asso- been shown to increase the expression of HLA-G in ciated with PD. EVT. However, EVT are devoid of the classical pro- gesterone receptors PR-A and PR-B. Non-classical To explore our hypothesis that in humans NEU- receptors must be investigated to better understand ROD6 has neuroprotective effects, we plan to use how progesterone regulates the expression of HLA-G an in vitro model utilizing a tyrosine hydroxylase in EVT. (TH) reporter line of human pluripotent stem cells (hPSCs) transduced with a lentivirus to allow for The approach to this research has three steps. First, inducible expression of NEUROD6. We will in- candidate non-classical progesterone receptors will duce expression of NEUROD6 in differentiated neu- be identified in consultation with the current litera- rons, which fluoresce red under the microscope, and ture. Candidate genes will be knocked out from JEG- use a cell stress and death assay to quantify sur- 3 cells, a cell line model of EVT, via the CRISPR- vival under various mitochondrial stresses. If NEU- Cas9 system. Next, HLA-G expression will be mea- ROD6 is found to be neuroprotective, targeted ex- sured at the mRNA and protein levels both before pression in SNpc DA neurons to confer resistance and after treatment with progesterone and various may represent a novel therapeutic avenue in PD. progesterone analogs to determine which if any of the gene knockouts impact the ability to respond to pro- gesterone. Finally, expression of knocked out genes will be restored via an expression vector construct to rescue the knockout phenotype. At the same time, overexpression of gene candidates can be assessed for any impact on HLA-G expression. Currently, two knockout lines are being screened via FACS and west- ern blot while two more knockout lines are being gen- Stem Cell and Regenerative Biology 123 erated in parallel. Overexpression experiments will of interest. follow confirmation of successful knockouts. I have taken fluorescent images of both untreated If a knockout line fails to upregulate HLA-G after cells and cells treated with various drugs, including progesterone exposure, that gene will be a likely proteasome and autophagy pathway inhibitors, and candidate for the non-classical progesterone recep- have worked with the CellProfiler program to create tor. Since progesterone-mediated upregulation of an analysis pipeline that can show whether or not my HLA-G is essential to the survival of the fetus and neurodegenerative disease model works. The value to successful pregnancies, better understanding the I use to analyze my results is the cytoplasmic flu- signaling pathway could lead to the development orescence divided by the nuclear fluorescence (C/N of treatments to reduce the incidence of pre-term value); thus, the higher the C/N value, the more birth and increase the rate of successful pregnancies. cytoplasmic TDP43 there is relative to the TDP43 in the nucleus of the cell. The analysis software helps me calculate the C/N value for each individual TDP43 Mislocalization in ALS and Related cell, and then I qualitatively evaluate the results via Neurodegenerative Diseases histograms and quantitatively evaluate them using TDP43 is a gene in- a 2-group t-test. The t-test can prove that there volved in ALS and other is a statistically significant difference between the Christopher neurodegenerative condi- untreated and treated cells’ C/N values, and can Cantrell tions. The protein prod- therefore prove that TDP43 mislocalization to the Human Developmental and uct of this gene, TDP43, cytoplasm is occurring. Regenerative Biology, is normally located in 2018 the nucleus of neurons, Preliminary experiments demonstrate that protea- but when one has a neu- some inhibition causes TDP43 mislocalization in Harvard Stem Cell rodegenerative disease, u2os cells, and the same seems to be true in neurob- Institute the protein is mislocal- lastoma cells. However, it does not appear that the ized into the cytoplasm use of autophagy inhibitors causes the same result. Advisor: Kevin of the cell. TDP43 has I will continue to test my model to ensure that it Eggan, PhD various functions, includ- effectively models TDP43 mislocalization under neu- ing mRNA transport and rodegenerative conditions via immunofluorescence Mentor: Daniel transcriptional repres- imaging and the CellProfiler analysis program. Also, Mordes, MD sion. This protein mis- my experiments will include the use of neurons I localization results from differentiate from human stem cells. If my analysis protein imbalances in the consistently shows that my model works, I will pro- neurons. My project is to build a model of this ceed with my project to conduct tests of association neurodegenerative condition and to then determine to attempt to discover which related pathways cause what causes the TDP43 protein to mislocalize from TDP43 mislocalization, as that would have implica- the nucleus into the cytoplasm of neurons. I have also tions for ALS and other neurodegenerative diseases. been developing methods to quantify the change in cellular localization using fluorescent microscopy and analytic software called CellProfiler. Additionally, I have been exploring if other pathways in neurode- generative diseases cause a similar TDP43 mislocal- ization.

My experiments involve the use of cell lines that are easier to work with than neurons, which is necessary in order to build my model of neurodegenerative disease conditions. For instance, I have used hu- man bone cancer (u2os) cells, human embryonic kid- ney cells, SHSY5Y neuroblastoma cells, and SKNFI neuroblastoma cells. I have gained experience in working with and growing stem cells, and have also become familiar with the methods to generate neu- rons from stem cells. I have also gained proficiency in the various techniques required to successfully con- duct experiments involving tissue culture, including methods that will allow visualization of the protein 124 Stem Cell and Regenerative Biology

A Cas9-Mediated Genome-Wide Screening lentiviral genome-wide gRNA library, we withdrew Approach to Identify Novel Regulators of growth factors necessary to sustain pluripotency. Primed Pluripotency in Epiblast Stem Cells Cells maintaining Oct4-GFP reporter expression are likely to carry knockouts for genes required for dif- Pluripotency is a fea- ferentiation, and subsequent isolation of these cells ture of embryonic stem and sequencing of integrated gRNAs will identify Christopher Li cells (ESCs) and de- the mutated genes of interest, which prevent EpiSC Human scribes the cellular po- commitment. Developmental and Regenerative Biology, tential for multi-lineage 2018 differentiation—the abil- This work will help to reveal novel regulators of ity to give rise to cell pluripotency and to dissect the mechanism by which Harvard Stem Cell types of all three embry- primed pluripotency is maintained in murine EpiSCs, Institute, onic germ layers. In vitro, and these studies will inform future efforts to gener- Massachusetts ESCs can self-renew in- ate human naive ESCs. Our findings should further General Hospital definitely while maintain- our understanding of the pathways that govern stem ing pluripotency. Recent cell pluripotency, and hold significant implications Advisor: Konrad investigation of the sig- for clinically relevant strategies to modulate stem Hochedlinger, PhD naling mechanisms that cell fate in regenerative medicine. regulate configurations of pluripotency has revealed two distinct states—naive and primed—each with unique epigenetic, molecular, and functional charac- teristics.

In mice, naive embryonic stem cells are derived from the pre-implantation blastocyst, while primed epi- blast stem cells (EpiSCs) are derived from the post- implantation epiblast. Intriguingly, human ESCs are thought to be more closely related to primed EpiSCs than to naive ESCs in terms of their developmen- Figure 1: GFP expression in Oct4-GFP ESCs, tal potential as well as their signaling requirements EpiSCs, and EpiSC-differentiated cells as measured and epigenetic properties, which therefore offers an by flow cytometry. These data validate our use of an invaluable model to study primed pluripotency us- Oct4-GFP reporter of pluripotency, which is rapidly ing transgenic tools readily available in mice but and completely downregulated upon EpiSC exit from not in humans. Although the molecular and sig- pluripotency. naling pathways that sustain naive pluripotency in murine embryonic stem cells (mESCs) are rather Effects of DNA Damage on Fibrotic Cell well described, the mechanisms regulating primed Cycle Arrest in Kidney Organoid Models pluripotency remain incompletely understood. We are particularly interested in the mechanism of exit Chronic kidney dis- from pluripotency in EpiSCs, as well as in the sim- ease (CKD) is an ill- ilarities and differences between the maintenance of Edgar Garcia ness affecting ten percent Human human and murine primed pluripotency. of the worldwide pop- Developmental and Regenerative Biology, ulation, resulting from The CRISPR-Cas9 system for genome editing is a 2018 continuous or repeated robust method to systematically assay loss of func- injury to the kidney. tion phenotypes and is a powerful tool in mammalian Brigham and Kidney fibrosis, a hall- genetics. To further elucidate the molecular mech- Women’s Hospital mark of CKD, is scar- anisms controlling pluripotency, we are performing ring that results from a comprehensive Cas9-mediated genome-wide screen Advisor: Joseph failure to regenerate kid- to identify novel regulators of the signaling path- Bonventre, MD, PhD ney tubules after in- ways that promote or permit exit from pluripotency jury. However, little in EpiSCs. We exploited the use of an Oct4-GFP Mentors: Ryuji is known about the fi- reporter of the pluripotent state, which is rapidly Morizane, MD, PhD; brotic pathway and the and completely downregulated when EpiSCs exit Navin Gupta, MD exact mechanisms by pluripotency, and derived a TetO-Cas9/Oct4-GFP which one could suppress EpiSC line from E6.5 post-implantation epiblasts. it. After Cas9 induction followed by infection with a Stem Cell and Regenerative Biology 125

Mouse models have related fibrosis to cell cycle ar- age, G2/M arrest, and fibrosis. With the rest in the G2/M checkpoint (Yang et al., 2010). results from the final phases of my experi- I believe that DNA damage from chemotherapeutic ments, I hope to fully determine the molec- treatments likely results in G2/M arrest and leads to ular relationship between fibrosis and CKD. fibrosis. The Role of the ARRDC3 in Fructose Current animal models are limited in their ability to Absorption in the Small Intestine effectively model human pathology, especially in the kidney where there is considerable heterogeneity of The intake of fruc- solute transporters. Therefore, kidney tissue devel- tose in modernized soci- oped from human pluripotent stem cells (hPSCs) is Ellen Zhang eties has increased dra- Human an attractive model to explore mechanisms of renal matically in the past Developmental and fibrosis linked to DNA damage in human tissues. Us- Regenerative Biology, decades. Excess fructose ing hPSC-derived human kidney organoids as high- 2019 may be a significant fac- lighted in Morizane et al.’s (2015) Nature Biotechnol- tor in metabolic disorders ogy paper, I hope to explore the associations between Harvard Stem Cell like diabetes and non- DNA damage, G2/M checkpoint arrest, and kidney Institute alcoholic fatty liver dis- fibrosis. ease. Therefore, a com- Advisor: Richard plete understanding of To do so I used cisplatin, a chemotherapeutic drug Lee, MD fructose metabolism has that causes proximal tubular injury and DNA dam- important implications age. I began by finding the concentration and inter- Mentor: Shannon for human health. val of cisplatin that best induces fibrosis and mini- Carroll, PhD mizes generalized nephrotoxicity. After differentiat- Following ingestion, fruc- ing the hPSCs, I treated the organoids with 5 µM tose is transported from cisplatin for 24 hours twice weekly. I collected sam- the small intestinal lu- ples, and their corresponding controls, after 1, 3, men into the enterocyte via glucose transporter 5 and 5 treatments. I then assayed for fibrosis at the (GLUT5). GLUT5 is a specific transporter for fruc- mRNA level using quantitative reverse transcription tose and is expressed exclusively on the apical mem- PCR (RT-PCR) and at the protein level using im- brane of the enterocyte (Douard & Ferraris, 2008). munohistochemistry (IHC). I saw an upregulation of Fructose exits the enterocyte and enters the portal fibroblast signaling (p<0.001) after treating with cis- bloodstream through the activity of glucose trans- platin at least three times. porter 2 (GLUT2), which transports both monosac- charides and is expressed on the basolateral mem- After showing the induction of a fibrotic-like state brane of the enterocyte. in the kidney organoids, I went on to characterize the cell cycle distribution within the treated sam- Previously, Dr. Lee’s lab found a protein called ples. I used phospho-histone H3 (HH3) staining to thioredoxin-interacting protein (Txnip) that inter- assay for cell cycle distribution within the cisplatin- acts with both GLUT5 and GLUT2. Utilizing a treated organoids. HH3 is differentially phosphory- Txnip knockout mouse, they found that Txnip is lated at the various stages of the cell cycle, giving necessary for fructose absorption in the small in- it distinct staining patterns for each of the different testine of mice. However, another protein closely stages of the cycle. Using this knowledge, it appeared related to Txnip called Arrestin domain containing that there was greater skew towards G2 arrest within 3 (Arrdc3) was also found to interact with GLUT5 the cisplatin treated samples when compared to their and GLUT2. This discovery suggests that Arrdc3 negative controls. may also regulate fructose absorption in the small intestine in a manner similar to Txnip; alternatively, Finally, I sought to modulate the fibrosis pathway Arrdc3 may have other effects on fructose absorp- and characterize the fibrotic response resulting from tion. these modulations. I did so by using a small molecule inhibitor that induces cell cycle arrest in G2/M. Fol- Little is known of Arrdc3’s function or expression lowing these experiments, I performed similar stain- pattern, although the gene for Arrdc3 is linked to ing and quantitative RT-PCR to assay for changes in obesity in men. If Arrdc3 has a role in fructose fibrotic levels. absorption in the small intestine, we hypothesize that Arrdc3 will be expressed in the enterocytes of These experiments thus far support the hypoth- the small intestine. We will test this hypothesis us- esis that there is a link between DNA dam- ing two different models. First, we will utilize an intestinal human cell line (Caco-2 cells) to study 126 Stem Cell and Regenerative Biology

ARRDC3 mRNA expression in vitro via quantita- known as hematopoietic cell E-/L-selectin ligand tive PCR. Secondly, we will utilize a ARRDC3/lac-z (HCELL). reporter mouse to track expression of Arrdc3 in the small intestine. This will require isolation and β- Creating HCELL on exosomes has many clinical ap- galactosidase staining of the small intestine followed plications; in therapeutics, stem cell transplants have by histological sectioning and microscopy. mixed results due to the variability rate of cells to transmigrate across the vascular endothelial to the This research will give valuable information about bone marrow. Glycan engineering to create HCELL Arrdc3 expression in enterocytes and the small in- expression allows the efficient vascular delivery of testine which is necessary for continued research on specific cells due to the nature of strong step 1 inter- the role of Arrdc3 on fructose absorption. This re- actions that HCELL can undergo with E-selectin. search will also provide a foundation for understand- ing Arrdc3 expression in the enterocyte is regulated. Here, we sought to analyze the effect of exofuco- sylation and the creation of E-selectin ligands on exosomes using α-(1,3) fucosyltransferases. The Enforced Expression of E-Selectin Ligand on current study involved the isolation of exosomes Mesenchymal Stem Cell-Derived Exosomes derived from hMSCs through differential ultracen- via Exofucosylation trifugation. Exosomes were bound to 4 µM alde- Exosomes are cell- hyde/sulfate latex beads overnight via passive ad- derived microvesicles sorption. Identification and characterization of ex- Gerardo Castillo which are secreted from osomes was performed through the use of a panel Molecular and many different cell lines, of cell surface markers, such as CD81 (a specific Cellular Biology, 2018 including human mes- biomarker of exosomes), CD63, CD73, and CD44 enchymal stem cells (hM- (specific markers of hMSCs) by flow cytometry. Brigham and SCs), and have been Exosomes were exofucosylated with α-(1,3) Fuco- Women’s Hospital, shown to play a role in syltransferase VI (FTVI) via GPS and HECA-452 Harvard Medical intracellular signaling. (mAb which recognize sLex structures) reactivity School, and Program Specifically, prior stud- was analyzed to identify the enforced expression of of Excellence in ies have shown that exo- sLex determinants on exosomes. This study sup- Glycosciences somes derived from hM- ports the utility of cell surface glycan engineer- SCs reduce myocardial is- ing to enforce the migration of hMSCs-derived ex- Advisor: Robert chemia, induce peripheral osomes to sites of tissue injury or inflammation. Sackstein, MD, PhD tolerance, trigger autoim- mune responses, and re- Designing a New High-Throughput Screen Mentor: Gisela pair acute kidney injuries. Pachon, PhD for Molecules that Contribute to Cell Fate Therefore, it is possi- Determination ble that isolated hMSC- derived exosomes can be Type 1 diabetes is a used as a therapy at specific sites and tissues. common disease in which Katie Kixmoeller the insulin-producing Chemistry and Prior studies have also shown that it is possible to beta cells of the pancreas Physics, 2018 direct certain cells to specific sites given the presence are destroyed by an au- of E-selectin and a potent E-selectin ligand, which Harvard Stem Cell toimmune response. Pa- allows for transmigration of the cell through the vas- Institute tients can’t produce in- cular endothelial wall. The multistep paradigm of sulin, so they can’t re- transmigration is dependent on tethering/rolling in- Advisor: Douglas spond properly to an in- teractions (step 1 of cell migration) which involves Melton, PhD crease in blood glucose. the binding of ligands expressed on cell surfaces, Regenerative medicine mainly E-selectin ligands, to selectins expressed on Mentor: Nadav presents the possibility for vascular endothelium. Research conducted in the Sharon, PhD a cure. A few years ago, Sackstein lab has data to suggest that hMSCs lack the Melton lab published E-selectin ligands that are important for transmi- a protocol for creating hu- gration. However, these cells do express native man stem cell-derived beta cells with the goal of pro- CD44 and could be modified via a process known viding rejection-free beta cell transplants to patients as exofucosylation through glycosyltransferase pro- with severe diabetes. The protocol mimics normal grammed stereosubstitution (GPS) to enforce E- pancreatic development, guiding stem cells along the selectin-binding sialyl Lewis X (sLex) decorations, process of differentiation into beta cells. It is an thereby creating the most potent E-selectin ligand astonishing accomplishment, but many challenges Stem Cell and Regenerative Biology 127 remain before these cells can be given to patients. Regulation of Cardiac Regenerative One significant difficulty is that the yield of beta cells Capacities remains low. Also, the hallmark of functional beta cells is glucose-stimulated insulin secretion (GSIS), Heart attacks are a and the cells produced can’t perform GSIS as well leading cause of death as normal beta cells. Krystal Phu in adults worldwide be- Human cause they lead to the Developmental and The goal at each stage of the protocol is to have Regenerative Biology, loss of heart muscle cells, a homogenous population of cells so that the final 2019 known as cardiomyocytes. product is homogenous beta cells. However, at one Cardiomyocytes in adult of the intermediate stages of the protocol, we iden- Harvard Stem Cell humans regenerate very tify two populations of cells, pancreatic progenitors Institute slowly, and the inabil- and more-differentiated endocrine cells. This hetero- ity to replace dying car- geneity lowers the yield of the protocol, so we need Advisor: Richard diomyocytes leads to de- to adjust the ratio between the two populations. Lee, MD cline of the pumping per- formance of the heart. In High-throughput molecular screening could help Mentors: Ahmed contrast, newborn mam- solve this problem by identifying molecules that af- Mahmoud, PhD; mals, or neonates, can fect this ratio. However, acquisition of cell fate is a Christian Shigley fully regenerate damaged gradual and complex process. It involves a combina- cardiomyocytes, though tion of molecular signals regulating the expression of this capability is lost hundreds of genes. So, it is extremely unlikely that a shortly after birth. This regeneration occurs by cell single molecule alone could cause a cell to acquire a division of other pre-existing cardiomyocytes and not particular fate. Traditional high-throughput screens by stem cells. It has been speculated that a critical are poorly suited to identify molecules that have only step in cell division of cardiomyocytes is disassembly a partial effect for two reasons. First, the output is of the rigid sarcomere structures that provide pump- generally binary (cell alive or dead, protein present ing function but could otherwise impede mitosis. or absent). Second, these screens are limited in the number of genes that can be tested at a time. One theory for the loss of regenerative capability in adult human hearts is that neonatal cardiomyocytes I set out to design a new high-throughput screen mature into high performance adult heart cells that to identify molecules that contribute to determining are less able to divide. Previous work in the Lee lab cell fate. Although each molecule alone wouldn’t de- has found transcription factors that are expressed termine cell fate, some combination of them could be differentially between the neonatal and adult car- used in the lab’s protocol to adjust the ratio between diomyocytes. Of these, several transcription factors cell populations. have previously been associated with sarcomere for- mation or disassembly. Although these factors have Recent developments in single-cell RNA sequencing other known functions, their capacity to regulate sar- technology allow analysis of multiple genes across comeres may be important for cardiomyocyte divi- multiple samples. My screen harnesses this technol- sion potential. ogy for high-throughput screening. Additionally, the screen’s output is sequencing data, allowing for more To evaluate the impact of these factors, we use sensitive detection of gene expression levels than tra- an N-1 combinatorial approach via lentiviral trans- ditional outputs such as fluorescence. My project is duction into adult rat cardiomyocytes, which nor- to design, test, and calibrate this new screen with mally are very resistant to cell division. Then the goal of deploying it experimentally in the fall. we immunohistochemically stain cardiomyocytes for sarcomere-related markers and utilize imaging tech- This screen could also be applied to other research niques to measure morphological differences in sar- questions, both in the Melton lab and beyond. comere structure between cells receiving the candi- We also plan to look at cells that have reached date factors in the experimental conditions and those the end of the lab’s protocol but do not perform receiving empty lentiviral vectors in the control con- GSIS as well as normal beta cells. This screen dition. We hypothesize that in conditions wherein has great potential to improve the differentiation key transcription factors associated with sarcomeres protocol, which is an important step toward clin- are necessary for cardiomyocyte division, those fac- ical applications of stem cell derived beta cells. tors cause sarcomeres to be disassembled prior to the mitosis event.

If our experiment performs as hypothesized, we 128 Stem Cell and Regenerative Biology will have identified factors capable of disassem- IL10Rα and IL10Rβ using CRISPR/Cas9. Clonal bling sarcomeres that can participate in cardiomy- lines with gene inactivation by frameshifts or by ocyte division. Future study will test the im- complete deletion of the coding region will be es- pact of expression of these factors on sarcom- tablished by single cell cloning. The knockout will ere assembly in the context of adult cardiomy- be confirmed by western blot and the ability or in- ocyte proliferation in vivo. This work could rep- ability to phosphorylate STAT3 in response to IL10 resent a step toward transiently reprogramming stimulation. Such a knockout model will be useful adult cardiomyocytes into a regenerative state. in testing lentiviral and alpharetroviral gene transfer vectors with different configurations to identify the most promising candidates for in vivo mouse experi- Hematopoietic Stem Cell Gene Therapy for ments. Furthermore, the knockout cells will provide Very Early Onset Inflammatory Bowel a comprehensive method to optimize a toolset for Disease Caused by IL10 Receptor Deficiency gene editing which will later be used to correct pa- Loss of function mu- tient derived iPS cells. This includes identification tations in IL-10 or the of the most effective Cas9 nuclease type, the specific Linda Qin IL-10 receptor lead to life- guide RNAs, and the size and location of the repair Human threatening very early on- template to achieve specific and efficient expression Developmental and Regenerative Biology, set inflammatory bowel of the IL10 receptor. These knockout cell lines will 2019 disease (VEO-IBD). Pa- eventually be used to test the species cross reac- tients with VEO-IBD tivity of components of the IL10-receptor signaling Harvard Stem Cell present with severe symp- pathway and to establish the required expression Institute, Boston toms from a very young strength of IL10R chains required for full signal- Children’s Hospital age and are largely un- ing competence, and will greatly facilitate the test- responsive to conven- ing and selection of gene therapy vectors and gene Advisor: David A. tional anti-inflammatory editing tools required to develop novel therapies. Williams, MD treatments. Allogeneic hematopoietic stem cell Towards Vascularizing Cerebral Organoids Mentor: Christian transplantation (HSCT) In Vitro Brendel, PhD is the only available cura- tive treatment option, but The creation of in the limitations of finding vitro organ models of hu- suitable HLA-matched donors and the underlying Lucy Nam man development is an Molecular and risks of acute toxicities and graft-versus-host-disease important and emerging Cellular Biology, (GVHD) are significant deterrents. 2017 area of study, with ap- plications in drug screen- However, preliminary data from our group have Wyss Institute for ing, disease modeling shown that, in mice, the transfer of the IL10R Biologically Inspired and tissue engineering. gene into IL10R-deficient bone marrow precursors Engineering In particular, induced followed by HSCT can prevent the development of pluripotent stem cells (iP- IBD. This suggests that lentiviral gene therapy of au- Advisor: Jennifer SCs) can self-organize tologous hematopoietic stem cells (HSCs) may carry Lewis, ScD into three-dimensional potential to be a safe and effective alternative. Addi- tissue-like architectures, tionally, recent data indicates that transfer of healthy Mentor: Mark or “organoids,” enabling anti-inflammatory macrophages may be sufficient to Skylar-Scott, PhD the real-time observation prevent colitis in IL10R-deficient mice. These data of complex developmen- suggest that a feasible therapeutic option may en- tal processes. If patient- tail Cas9-mediated gene editing in patient derived specific iPSCs are used in deriving the organoids, induced pluripotent stem cells (iPS) to correct the specific pathologies can be recapitulated and studied defective gene, differentiate iPS cells into mature in vitro. However, to date, existing organoids have macrophages in vitro, and subsequently inject cells been avascular, and subsequently develop necrotic into the patient. Both approaches require suitable cores that limit the size and complexity of the re- in vitro test systems to evaluate and optimize the sulting tissues. efficacy of gene transfer or gene editing in a simple system and in a time-effective manner, but currently To construct vascularized cerebral organoids, or cere- neither human nor murine cell lines with a complete brovascular organoids, iPSC cultures must be differ- IL10R knockout do exist. entiated into both neural (ectoderm) and vascular (mesoderm) tissues. However, generating pure pop- We will first create human knockout cell lines of both ulations of tissues from distinct germ layers using Stem Cell and Regenerative Biology 129 a single media condition is unlikely to be success- neurons, in which signals decay in milliseconds, many ful. Thus, we used genomic engineering to generate hypothalamic neurons have been shown to be capa- two separate populations of iPSCs: a wild-type pop- ble of retaining signals over extended timescales, al- ulation, and a second population that underwent lowing for integration of temporally distant events. differentiation into vasculature via a drug-induced Loss of this ability has been seen to cause weight gain upregulation of the transcription factor ETS-variant in the case of POMC and PVH neurons or loss for 2 (ETV2). AGRP neurons in mice. Understanding connectivity and mechanisms within the hypothalamus, and of its When these cells were co-cultured in proneural me- reciprocal interactions with the alimentary tract, are dia conditions in the presence of doxycycline, and fundamental to developing our knowledge of disor- implanted into a Matrigel extracellular matrix, the ders in weight such as obesity. wild-type iPSCs form cerebral organoids, while the dox-inducible ETV2 iPSC population differentiates Obesity is no longer seen simply within a paradigm into endothelium that express both CD31 (PECAM- of eating too much and exercising too little but in- 1) and vascular-endothelial cadherin (VE-Cadherin). stead as a complex interplay of factors. The project These endothelial cells further undergo vasculogene- centres on the use of human embryonic stem cells to sis and angiogenesis to generate a three-dimensional generate human hypothalamic neurons. The ability microvascular network that surrounds the developing to do this is central to developing an in vitro model cerebral tissue. of how the gut and hypothalamus communicate, and to identifying novel factors which may have an effect Using this method, we aim to perfuse the mi- on weight. I will stain the resultant cells to ensure crovascular network that surrounds the cerebral they are expressing elements indicative of hypotha- organoids to provide the requisite oxygen, nutri- lamic lineages and test their electrophysiology. ents and waste removal for the developing tissue to thrive. We will achieve this by embedding the Upon confirmation of their nature, I will begin cerebrovascular organoids in-between two pin-casted investigating the effect of secretions of enteroen- microchannels that are connected to an external docrine cells (produced in the lab through directed peristaltic pump for perfusing fresh, oxygenated re-differentiation) on their behaviour. The hormones media through the tissue. Once the ETV2 cell- secreted by enteroendocrine cells have a putative im- derived microvasculature connects across the two portance in regulating satiety and eating behaviours. microchannels, the media flow will be re-directed An in vitro model would allow for screening of com- through the endothelial network to nourish the pounds for their possible effects in the hypothala- tissue. We will monitor the subsequent growth mus; this could yield currently unknown endogenous and development of the tissue to identify whether molecules to pursue in mouse models for their ef- a primitive blood brain barrier (BBB) will de- fects on eating. Ultimately, we hope to identify puta- velop, and whether we can prevent the forma- tive targets which can be exploited pharmaceutically tion of a necrotic core as the tissue further grows. to either suppress appetite (in obesity) or stimulate it (such as in patients undergoing chemotherapy). In Vitro Modeling of the Gastro-Hypothalamic Axis to Elucidate Appetite and Satiety Signals Despite comprising a small percentage of the Luke Smith adult human brain, the Biomedical Science, hypothalamus is a com- 2017 Emmanuel College plex and essential neural element regulating a vari- Harvard Stem Cell ety of systems, including Institute energy homeostasis. It may regulate a “set point” Advisor: Qiao Zhou, for body weight. Neu- PhD rons in this region of the brain are well placed to sense neural and physio- logical signals from the alimentary tract via hor- mones and the vagus nerve, relaying this to high or- der structures that influence behaviour. Unlike other 130 Stem Cell and Regenerative Biology

Evaluation of Candidate Genes for in our system. Controlling Limb Size in Vertebrate Regeneration By using RNA in situ hybridization, we plan to validate where these transcripts are expressed in Worldwide, ten mil- mini-limbs and the controls. It can then be deter- lion individuals suffer mined what would happen if expression was stopped Rachel Oshiro from an amputation; cur- or increased allowing us to elucidate the transcripts Molecular and rently, the treatments functions within the regenerating limb and possibly Cellular Biology, 2019 available often struggle how appendage size is matched to body size. We to replace the lost limbs. also plan to examine internal tissue composition us- Harvard Stem Cell The ultimate goal is to ing immunohistochemistry allowing the visualization Institute, Brigham stimulate human limb re- of the different types of cells within the limbs. Us- and Woman’s generation. To begin to ing this technique, the mini-limbs can be studied in Hospital devise approaches to ful- terms of nerves as despite the fact that mini-limbs filling this goal, a thor- have fewer targets since they are smaller than regular Advisor: Jessica ough understanding of limbs, they should have the same number of nerve Whited, PhD what occurs at the molec- cells. ular level that enables Mentors: Donald animals to regenerate is In the end, we will compare the mini-limbs and Bryant, MS; Victor likely to aid in this goal. normal limbs in order to elucidate what de- Luria, PhD Unlike humans, axolotls termines and restricts size in regeneration and (Mexican salamanders) growth and have an increased understanding of can regenerate their limbs the function of nerves within the regrowing limb. after amputation. However, the process by which they do this is not entirely understood. We do know that the process involves the formation of a Using In Vitro Mouse and Human Spinal blastema which is thought to be composed of cells Muscular Atrophy (SMA) models to that have become “dedifferentiated” (lost their spe- Investigate the Effect of Survival of Motor cific function) but might also include contributions Neuron (SMN) Deficiency on Satellite Cells from stems cells, another type of undifferentiated cells. The new limb then grows from the blastema The leading genetic cells. Interestingly, nerves are necessary for proper cause of death for infants, regeneration to occur. Soumyaa Spinal Muscular Atrophy Mazumder (SMA) is a neurodegen- Molecular and The Whited lab has created animals with “miniatur- Cellular Biology, erative disease character- ized limbs” or mini-limbs by repeatedly cutting the 2019 ized by the progressive limb blastema to prevent proper growth, resulting in loss of motor nerve cells in a limb substantially smaller than normal in a large Harvard Stem Cell the spinal cord. Affecting fraction of animals. These can be used to understand Institute approximately 1 in 6,000 what drives limb regeneration and what mechanisms infants, SMA is caused by integrate limb size and body size. To discover the Advisor: Lee Rubin, a mutation in the Sur- genes which may control these processes, the lab PhD vival of Motor Neuron 1 performed mRNA sequencing, to determine expres- gene (SMN1 ). This mu- sion levels of mRNA, from mini-limb samples and Mentors: Rebecca tation leads to the insuf- control sibling samples. The data was analyzed to Gibbs; Tim Ahfeldt, ficient expression of the determine what transcripts were “downregulated” or PhD Survival of Motor Neu- decreased and what transcripts were “upregulated” ron (SMN) protein, which or increased in mini-limbs in comparison to the con- subsequently results in se- trols. The thresholds were made with expression vere muscle weakness and often death. levels increased by a factor greater than 2 and ex- pression levels decreased by a factor less than 0.5 but Although SMN is expressed by all cells, most thera- greater than 0. Several genes, notably those encod- pies focus on targeting motor neurons (MNs), as it ing the CATK (downregulated), FGFR1 (downregu- is thought that MNs are the most severely affected. lated), and SEM6D (upregulated) proteins, seemed However, there is considerable evidence from this lab promising as their expression levels varied greatly and others to suggest that in addition to the death between the control and the mini-limb axolotl; pre- of MNs, SMN deficiency may have deleterious, inde- existing studies from other contexts suggest testable pendent effects on other cell types, such as muscle hypotheses about the possible roles of these factors itself. Stem Cell and Regenerative Biology 131

Given these findings, as well as results suggesting Evolutionarily Conserved Role of C5aR1 in that muscle weakness in SMA mice may be in large Cardiac Regeneration part caused by postnatal muscle growth abnormali- ties, this project focuses on understanding the effect According to the of SMN deficiency on a population of cells respon- American Heart Associ- Yamen Abbas ation (AHA), heart fail- sible for postnatal muscle growth and regeneration: Human ure—a chronic, progres- satellite cells. During postnatal growth, satellite cells Developmental and (muscle stem cells) proliferate and differentiate to Regenerative Biology, sive condition in which form muscle fibers. Previous studies have suggested 2017 the cardiac muscle fails that the loss of SMN may diminish the ability of dif- to pump blood efficiently ferent cell types to proliferate. Based on my mentor’s Harvard Stem Cell enough to meet the preliminary experiments, this project aims to investi- Institute body’s need—is a grow- gate if SMN deficiency specifically affects satellite cell ing epidemic, with more proliferation. Our main hypothesis is that the loss of Advisor: Richard than 870,000 new cases SMN decreases satellite cell proliferation, resulting Lee, MD diagnosed annually in the in a decrease of myogenic precursor cells needed to United States. Heart fail- Mentor: Aysu Uygur, support postnatal muscle growth and a reduction of ure (HF) is characterized PhD the satellite cell population. by progressive loss of car- diomyocytes, which even- To test this hypothesis, we are using an in vitro SMA tually compromises the mouse model to identify SMN-dependent defects in contractility of the remaining cardiac muscle. Un- satellite cell proliferation. Using mouse embryonic like lower vertebrates such as salamanders and ze- stem cells (mESCs) from SMA and wild-type (WT) brafish, mammals fail to regenerate the heart follow- mice, I will differentiate mESCs into satellite cells ing injury. Thus, developing therapeutic methods and myofibers by adding myogenesis-promoting fac- that replace the lost cells is an attractive strategy tors at appropriate time points. While the entire dif- for the treatment of HF. Interestingly, one simi- ferentiation process takes place over several weeks, larity between regeneration-competent animals is a cells from both the SMA and WT line will be fixed weak immune systems. The link between the im- weekly and immunostained for markers of satellite mune system and regeneration has been investigated cells, myoblasts, myogenic precursor cells, and my- by many groups; inflammatory response, which is ofibers. In addition to immunostaining, western blot- tightly regulated by the immune system, has been ting and qPCR will also be performed to measure ex- shown to either drive or inhibit regeneration de- pression of key genes expressed by presomitic meso- pending on context. In our lab, C5aR1, a com- derm cells, satellite cells, and other types of muscle plement protein, has been identified to be highly cells. These techniques will allow us to track the dif- upregulated in the hearts of zebrafish, axolotls, and ferentiation of mESCs as they change over time. neonatal mice post apical resection injury. In ad- dition, our lab has shown that its activation is In addition to maintaining an SMA mESC line in necessary for neonatal mice cardiac regeneration. culture, we are developing an in vitro human SMA My work focuses on characterizing C5aR1, identi- model. One key project is to develop a PAX7 red fying downstream targets, and performing gain-of- fluorescent tdTomato reporter for satellite cells. The function experiments on adult mice. Thus far, my transcription factor PAX7 is a key marker of satel- work shows that contrary to what we would ex- lite cells, and thus such a reporter would be useful for pect, C5aR1 is mostly expressed in cardiomyocytes tracking human induced pluripotent stem cell (iPSC) and endothelial cells, rather than macrophages. differentiation and ESC differentiation into satellite cells. In particular, this reporter would be useful for developing future SMA patient-derived iPSC lines.

If our in vitro models support the hypothe- sis that SMN deficiency impairs satellite cells, this would suggest satellite cells as an impor- tant, novel therapeutic target for treating SMA. 132 Stem Cell and Regenerative Biology

Characterization of the Stem Cell Population in the Acoel Hofstenia miamia

Regeneration is a fun- damental feature of an- Andreé imal biology. Thomas Franco-Vasquez Hunt Morgan’s extensive Human Developmental and cutting experiments with Regenerative Biology, the planarian Schmidtea 2017 mediterranea first estab- lished it as a regenera- Harvard University tive organism capable of whole-body regeneration Advisor: Mansi worth studying further. Srivastava, PhD Since then, endless stud- ies have been conducted in order to completely characterize the process of regeneration in this species, which has turned planarians into the model organism for studying regeneration. Regeneration studies with Schmidtea, including the characteriza- tion of molecular signaling involved in the process, have revealed some mechanisms underlying regen- eration. However, the lack of additional model or- ganisms capable of whole-body regeneration has pre- vented conclusive comparative analysis of the mech- anisms involved in this process.

Hofstenia miamia, commonly known as the three- banded panther worm, is an acoel species capa- ble of whole-body regeneration and has been re- cently established as a new model system. Sev- eral parallels have been discovered between it and Schmidtea in terms of regeneration mechanisms, in- cluding the presence of pluripotent stem cells, called neoblasts. Neoblasts have been characterized ex- tensively in planarians but have been studied min- imally in Hofstenia. We aim to identify additional neoblast markers in the new organism by charac- terizing the expression profile of genes enriched in a neoblast-specific RNAseq dataset through fluores- cent in situ hybridization. Our preliminary studies have revealed several neoblast markers, some that are shared with Schmidtea and some that appear unique to Hofstenia, which we are investigating function- ally. The parallels in regeneration between the three- banded panther worm and planarians, two species separated by 550 million years of evolution, are nu- merous and will be crucial for the comparative study of regeneration. Understanding the similarities of the molecular mechanisms controlling this process in both species will lead to the ability to compare re- generation across phyla. Such evolutionary analyses could lead to the identification of currently unknown conserved pluripotency factors that could be essen- tial features of animal biology. Acknowledgements

We extend our deepest thanks to the following individuals, without whom the Summer Under- graduate Research Village would not have been possible.

Program Staff PRISE Faculty Advisory Board

Gregory Llacer, Director of Harvard College Scott Edwards, Professor of Organismic and Evo- PRISE; Director of the Office of Undergraduate Re- lutionary Biology search and Fellowships (URAF) Howard Georgi, Mallinckrodt Professor of Physics Jennifer Shephard, Coordinator of BLISS; Spe- cial Initiatives Program Manager in the Office of the Evelynn Hammonds, Chair, Barbara Gutmann FAS Dean for Social Sciences Rosenkrantz Professor of the History of Science; Professor of African and African American Studies Marais Young, Coordinator of PRIMO; Programs Operations Manager in Harvard Business School Jay Harris, Dean of Undergraduate Education; Harry Austryn Wolfson Professor of Jewish Stud- Jeffrey Berg, Coordinator of SHARP; Assistant ies Director of the Office of URAF Georgene Herschbach, ex officio, Former Dean Christy Colburn, Coordinator of SURGH; As- of Administration for Harvard College sistant Director of the Global Health & Health Policy Undergraduate Program Gregory Llacer, ex officio, Director of Harvard College PRISE Emily Maguire, Coordinator of SURGH; Assis- tant Director of the Global Health & Health Policy Robert Lue, Professor of the Practice of Molec- Undergraduate Program ular and Cellular Biology; Director of Life Sciences; Director of HarvardX; Director of the Bok Center Jozef Sulik, Coordinator of Undergraduate Re- for Teaching and Learning search in the Office of URAF Joan Reede, Associate Professor of Medicine; Dean Cammi Valdez, Assistant Director of the Office for Diversity and Community Partnership, Harvard of URAF Medical School

Program Assistant Fellows John H. Shaw, Dudley Professor of Structural and Economic Geology Justin Dower, Lead Program Assistant Fellow Joel Bateman Dona Lee Wong, Associate Professor of Psychia- Jennifer Chiang try, Harvard Medical School Vimal Konduri Hueyjong Shih House Staff Madeleine Snyder Kaan Yay Paul Hegarty, Leverett House Building Manager

House Proctors Dick Nerden, Quincy House Building Manager

Luke Pizzato, Lead Proctor Kaitavjeet Chowdhary Madeline Cooper Niamh Durfee Michelle Guo Jessica Hermann, Emmanuel Proctor Gurbani Kaur, Proctor/Administrative Fellow Josh Stallings List of Fellows

BLISS Fellows Caleb Shelburne Henry Shreffler Charlotte Anrig Aaron Suduiko Anthony Chen Zachary Wong Irene Limb Krysianna Papadakis Hansenard Piou SURGH Fellows Sebastian Reyes Mara Roth Gita Bhattacharya Chace Shaw Kristen Fang Quinn Sluzenski Kristy Hong William Strang Henna Hundal Harrison Tanzola Isaac Hung Susan Wang Laura Kanji Liana Yamin Michael Kennedy-Yoon Eun Jin Yu Julianna Lee Anna Zannetos Sarah Rahman Patrick Tan Jonathan You PRIMO Fellows Jenna Zhang

Osama Arif Caroline Brettler PRISE Fellows Holly Breuer Antonia Chan Yamen Abbas Jiafeng Chen Matthew Aguirre Benjamin Cohen Sahar Ashrafzadeh Brian Cornyn Naomi Asimow Kevon Edmondson John Austin Lauren Nicholson Jennifer Bi Daniel Nightingale Alan Bidart Christina Qiu Julian Braxton Alexander Rohe Erica Budina Angela Yang Christopher Cantrell Hellary Zhang Gerardo Castillo Grant Zhao Pooja Chandrashekar Rohan Reddy Gui Zhen Chen Steven Tan Anne Cheng Yilin Chen, Wellesley Fellow Bonirath Chhay Christina Chiu, Wellesley Fellow Mara Coyan Lingjun Xia, Wellesley Fellow Miruna Cristuş Jett Crowdis Thomas Culp SHARP Fellows Bluyé DeMessie Carl Denton Lev Asimow Shaan Desai Tez Clark Natasha Dhamankar Jess Clay Christian Dolliff Edith Enright Claire Mohamed Ebied Robert Hopkirk Audrey Effenberger Sarah Judd Deanna Emery Brieanna Martin Feyisayo Eweje Thomas Peterson Shenyece Ferguson List of Fellows 135

Julio Fierro Alex Pai Jaden Freeze Margaret Panetta Felipe Flores Handong Park Andreé Franco Vasquez Krystal Phu William Fried Kateryna Pistunova Adam Frim George Qiao Edgar Garcia Linda Qin Michael Giles Sai Shanthanand Rajagopal Silvia Golumbeanu Leah Rosen David Gonzalez-Dysinger Andre Sanchez Madeline Granovetter Suproteem Sarkar Isobel Green Jacob Scherba Elgin Korkmazhan Gulpinar Yong Shen Emma He Benjamin Sorscher James Hotchkiss Lauren Sweetland-Martin Jennifer Hu Ramtin Talebi Myles Ingram Mai-Linh Ton Alexander H. Jin Lillian Tsai Jessica Kim Kristin Tsuo Mingu Kim Daniel Um Youbin Kim Santiago Vargas Katie Kixmoeller Julia Versel Peter Kraft Kruti Vora Deepika Kurup Enxin Wan Hannah Larson Brandon Wright Charles Law Scott Xiao Brandon Lee Siddharth Yarlagadda Matthew Leifer Kimberley Yu Valia Leifer Christina Zeina Catherine Li Ellen Zhang Christopher Li Christine Zheng Jason Li Joshua Breedon, Emmanuel Fellow Victoria Lin Ashavari Ghose, Emmanuel Fellow Gaia Linfield Alvar Paris, Emmanuel Fellow Michael Liu Oliver Philcox, Emmanuel Fellow Eliza Llewellyn Luke Smith, Emmanuel Fellow Helene Lovett Cynthia Luo Bahlakoana Mabetha Kushanth Mallikarjun Leah Marsh Ian Maynor Soumyaa Mazumder Andrew Mazzanti Joshua Meier Keegan Mendez Adriana Mendez Leal Jacob Meyerson Zhuang Miao Jade Moon Alexander Munoz Lucy Nam Lin Ni Nicolas Ontiveros Muhammed Ors Rachel Oshiro Artidoro Roberto Andrea Pagnoni