THURJ Vol. 1 Issue 1

8/14/2019 THURJ Vol. 1 Issue 1

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Dear Harvard Community,

Let me begin by congratulating the editors of the Harvard Undergraduate Research Journal on the wonderful

have a critical place in science education, but a deep understanding of science and engineering requires hands-

University Planning Committee on Science and Engineering and the Harvard University Science and Engineering

The Harvard Undergraduate Research Journal demonstrates the fruits of such hands on learning and the ability

Sincerely,

Steven E. Hyman Provost

L e t t e r

w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8


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Dear Harvard Community,

It is our great pleasure to present to you the inaugural issue of The Harvard Undergraduate Research Journal

(THURJ), a biannual publication that seeks to showcase the extraordinary research performed by Harvard

undergraduates. The journal has been in the works since the past summer, and we are very excited to see it come to

fruition. The idea behind THURJ, however, originated even earlier when we noticed that many other universities

including Stanford, Columbia, and MIThad established journals that showcased undergraduate science research

at their respective universities. Surely, we thought, Harvard and its students deserved an undergraduate research

journal dedicated to the profound work being done here in Cambridge and Boston, MA.

Though the seed of THURJ grew from the good-natured rivalry we felt with the other universities, we

encountered many important reasons for having such a journal while putting it together. It truly is the case that

many undergraduates are passionate about the sciences and perform advanced research; we believe that such

undergraduate research has not been lost on Harvard, which has developed several programs to foster research.

THURJ also gives both student manuscript submitters and evaluators insight into peer reviewing, which is central

As with any large endeavor, we and THURJ owe thanks to many people and institutions. The peer review process

our Faculty Advisory Board, consisting of Harvard faculty, who volunteered their time and energy to support us.

THURJs Content, Business, and Design boards all made similarly tremendous efforts to bring you the publication

that you now hold. Certainly this inaugural issue would not have been possible without the gracious sponsorship of

who submitted their research to THURJ, who made our lives easier by doing such excellent work. Finally, thank you

for opening the pages and experiencing the fantastic research going on at Harvard. Research is meant to be shared

and we are glad that you can join us.

Sincerely,

John Zhou, Co-Editor-in-Chief Shoshana Tell, Co-Editor-in-Chief

THE HARVARD UNDERGRADUATERESEARCH JOURNAL

L e t t e r s



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C o n t e n t

In the creation of this publication,we have received guidance,funding, and even written andvisual contributions from thefollowing, without whom this

publication would not exist:

Provost Hyman, Dr. Steven Freedman,Dean Jeffrey Flier, Felice Frankel, DeanVenkatesh Narayanamurti, Dr. CarenSolomon [deputy editor of the NewEngland Journal of Medicine], Dr. Katrina

Kelner [deputy editor of Science],Dr. Edward Benz and Dana-FarberDr. Steven Gygi, Dean Judith Kidd,Dean Paul McLoughlin, Dean JeremyBloxham, Wei Hong, Janice Ahn

Jae Hur, Sirinya Matchacheep, FrancesChu, Ethan Karp, Jessica Tibbits,Radcliffe Institute, The UndergraduateCouncil, Harvard College

Thank you!

EXECUTIVE BOARD

CO-EDITOR-IN-CHIEF

John Zhou 10 ([email protected])

CO-EDITOR-IN-CHIEF

Shoshana Tell 10 ([email protected])

BUSINESS MANAGER

Xin Pan 10 ([email protected])

MANAGING EDITOROF CONTENT

Aditi Balakrishna 10([email protected])

MANAGING EDITOROF

PEERREVIEWAND SUBMISSIONS

Lisa Rotenstein 11 ([email protected])

DESIGN CHAIR

Justine Chow 10 ([email protected])

BUSINESS BOARD

Yi Cai 11Aubrey Huynh 11

Jessie Jiang 11Kevin Liu 11

Alec Pinero 11

CONTENT BOARD

Yi Cai 11Ada Lio 11

Alterrell Mills 10Fernando Racimo 11

Peyton Shieh 10Sophie Warton 11

Farhan Murshed 11

DESIGN BOARD

Ada Lio 11John Liu 11Kevin Liu 11

Yan Yan Mao 10

Kate Xie 10

PEERREVIEW BOARD

John Liu 11 - Head Copy EditorMeng Xiao He 11 - Copy Editor

Charlotte Seid 10 - Copy EditorAna Garcia 11

Daniel Handlin 11Iddosshe Hirpa 11

Kevin Liu 11Tim Markman 11Yan Yan Mao 10Alterrell Mills 10

Joe Pollard 11Abby Schiff 11

Sheng Si 11

Helen Yang 11

PRODUCTION ADVISOR

Roque Strew, Harvard employee

FACULTYADVISORYBOARD

Paul Bamberg, Ph.DSENIORLECTURERIN MATHEMATICS

Michael Brenner, Ph.D

GLOVERPROFESSOROF APPLIED MATHEMATICSAND APPLIED PHYSICS

Myron Essex, D.V.M., Ph.DMARYWOODARD LASKER PROFESSOROF HEALTH SCIENCESINTHE FACULTY

OF PUBLIC HEALTH

Jeffrey Flier, M.D.DEAN, HARVARD MEDICAL SCHOOL, AND GEORGE C. REISMAN PROFESSOR

OF MEDICINE

Steven Freedman, M.D., Ph.DASSOCIATE PROFESSOROF MEDICINE

David Haig, Ph.DGEORGE PUTNAM PROFESSOROF ORGANISMICAND EVOLUTIONARYBIOLOGY

Dudley Herschbach , Ph.DFRANKB. BAIRD JR. RESEARCH PROFESSOROF SCIENCE

Richard Losick, Ph.DMARIA MOORS CABOT PROFESSOROF BIOLOGY

Hongkun Park, Ph.DPROFESSOROF CHEMISTRYANDOF PHYSICS

Steven Pinker, Ph.DJOHNSTONE FAMILY PROFESSOROF PSYCHOLOGY

Pardis Sabeti, M.D., Ph.DPOSTDOCTORALFELLOW, THE BROAD INSTITUTEOF MITAND HARVARD

Zhigang Suo, Ph.DALLEN E.AND MARILYN M. PUCKETT PROFESSOROF MECHANICSAND

MATERIALS

The Harvard Undergraduate Research

Journal (THURJ) showcases peer-reviewedundergraduate student research from

publication, THURJ familiarizes students

with the process of manuscript submissionand evaluation. Moreover, it provides a

on the cutting-edge research that impacts ourworld today.

At its core, THURJ allows students to gaininsight into the peer review process, which

THURJ manuscripts are rigorously reviewed

by the Peer Review Board (consisting ofHarvard undergraduates), and the top

manuscripts that they select are furtherreviewed by Harvard professors. Thisprocess not only stimulates faculty-student

collaboration and provides students withvaluable feedback on their research, but also

promotes collaboration between the Collegeand Harvards many graduate and professional

schools. In addition to publishing originalstudent research papers, THURJ is also animportant medium for keeping the Harvard

community updated on science research-

related news and developments.

Contact

Please email: [email protected]

Advertising

Please email: [email protected]

SubscriptionsPlease email: [email protected]

SubmissionsPlease email: [email protected]

Website

http://www.thurj.org

Copyright 2008 The HarvardUndergraduate Research Journal.

No material appearing in this publication

may be reproduced without writtenpermission of the publisher. The opinions

expressed in this magazine are those of thecontributors and are not necessarily shared by

the editors. All editorial rights are reserved.

About us



6/96v w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8

Felice Frankel is a Senior Research Fellow in FAS and heads the

Envisioning Science Program at Harvards Initiative in InnovatingComputing. For her next book, No Small Matter, (Harvard Uni-

versity Press 2009), with Harvard University Professor George M.

Whitesides, she constructed an 8-inch long acetate cylinder printed

with a hexagonal pattern to represent the structure of a nanotube.

ONTHECOVER: NANOTUBESBYFELICEFRANKEL

Features

Book Review

Unraveling the neural

circuitry behind moral

judgments

neuroeconomics

the plays in cancer research6

9

14Using small tools to tackle a

big problem22

Studying abroad17

The ABCs of learning

your ABC

Proust and the Squid: the storyand science of the reading brain

28

Engineering: linked in

to almost everything24

Manuscript Summaries

Opinion/Editorial

C o n t e n t s

1


7/96w w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8

Manuscripts

Targeting TRAF proteins as an anti-

tumor strategy

Synthesis of a bioresorbable elec-trospun guide tube for spinal cord

regeneration

Biological, socioeconomic, and po-

litical aspects of the Nassau grouper

Turks and Caicos Islands

ENGINEERING

MEDICAL SCIENCE

PHYSIOLOGY

SUSTAINABILITY SCIENCE

CELL BIOLOGY

31

54

80

polymer shell

maneuvers

50

73

CELL BIOLOGY

Thrombospondin-1 switches nitric oxide function

from inhibitory to stimulatory for gene expressionof human endothelial tumor markers

37

Bright, luminescent silicon nanoparticles for bio-

logical applications

CHEMISTRY

44

NADPH oxidase-derived free radicals are required

for cortex-controlled motor activity

NEUROSCIENCE

62

PHYSICS

Probing non-linear rheology with free oscillations69

CHEMIST

CELL

BIOLOGY

ENGINEER

MEDICAL

SCIENCE

NEURO-

SCIENCE

PHYSICS

PHYSIOLO

SUSTAINABI

SCIENCE

Visit http://www.thurj.org for news,details about the organization, and other

information on science at Harvard.

C o n t e n t


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A FOOTBALL AFICIONADO

CALLING THE PLAYS IN CANCER RESEARCHDr Edward Benz is the president of the Dana Farber Cancer Institute. His life story follows a road

has the scoop.

BYFERNANDO RACIMO, THURJ STAFF

U

one of the most prominent

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F e a t u r e s

I got so angry that I

actually did something

that I had never done

in schoolI was a good

student but I wasnt very

diligent. I went out and

got outside reading.

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Both a passionate player

and an insightful student

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Crossing the boundaries be

tween molecular and medi

cal biology

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One of those Eureka moments

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Looking back, thinking forward

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You really have todevelop a sense of

whats important

in life. Dont sweat

the small stuff.

his son in the phone. Hes a sport



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Unraveling the Neural Circuitry

Behind

Moral JudgmentsBYSOPHIE WHARTON, THURJ STAFF

Can moral reasoning be mapped in our neurons?Josh Greene, an assistant professor of psychologyat Harvard, believes that studying the brain can indeed

reveal the mechanisms that underlie our moral decision-

making. Greene has begun to combine moral philosophy

with advanced neuroimaging technology, pioneering an

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A Psychological Philosopher

As an undergraduate at Harvard, Greene pursueda philosophy concentration and became inter-ested in the questions of where our moral intu-

itions come from and whether we can trust them.

His senior thesis was a philosophical explorationof psychological biases in moral judgment. Af-

skeptic went on to Princeton to pursue a graduate

degree in philosophy.

Green soon pitched the idea of coupling neuroimag-

ing with philosophical dilemmas. Teaming up with Jona-

than Cohen of Princetons Center for the Study of Brain,

neuroimaging studies of moral judgment in 1999 and is

now continuing his research here at Harvard.

What was Greene looking to discover about moral

judgment? The question of how humans should make

decisions about moral dilemmas has been the central con-

cern of moral philosophy. The German philosopher Im-

manuel Kant believed that decisions ought to be rational

and reasoned with the intellect, while Scottish philoso-

pher David Hume argued that moral judgment is actually

driven by emotion. Greene hypothesized that both reason

and emotion are critical to judgment and that different

brain processes might be involved when we use different

modes of judgment.

Moral Dilemmas Meet Modern Technology

Greene tested his hypotheses by presenting volun-teers with classic moral dilemmas and monitoringtheir brain activity using a functional magnetic resonance

imaging scanner (fMRI). One such classic moral question

is the trolley problem that had captivated Greene early in

his career:

A trolley is running down a track out of control. If it

hanging out on the track. You can prevent this disaster

by throwing a switch, redirecting the trolley onto a siding

where it will kill one person. Do you hit the switch?

Most people answer yes, making the utilitarian judgment

consider this slight variation on the same dilemma:

F e a t u r

by Justine Chow, THURJ Staff



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Youre watching the same impending situation from a

certain death is to push a large person next to you off

the bridge and onto the tracks. Hell be crushed by the

people will live, as he will havestopped the train.

Although logically this situ-

ation is identical to the previ-

trade-off, people are much less

-variation is what Greene labels

an impersonal moral dilemma, the second is a per-

sonal moral dilemma. People seem to have an emotional

aversion to the idea of pushing the large person off thebridge.

One important difference is intentionpeople feel it is

different harming another person as a means to end, ver-

sus as a side effect, as collateral damage, Greene says.

The other vital distinction between the two scenarios

is the close body contact in the latter case, what Greene

calls personal force. Responses to other versions of

the trolley problem have shown that intention or personal

force alone are not enough to elicit the emotional reac-

response.In his neuroimaging study, Greene tested subjects with

these two scenarios and other similar personal and imper-

sonal moral dilemmas, and found that the medial fron-

tal gyrus, the posterior cingulate gyrus, and the angular

gyrusall regions of the brain involved in emotional

when the variation to the original dilemma was brought

up.

Areas associated with working memory --the right mid-

less active in the second situation. Greenes research

appears to suggest that the integration of an emotional

component in personal moral judgments seems to inter-

fere with the functioning of other cognitive faculties and

affect a persons ability to reason with pure utilitarian-

ism.

The reaction times of subjects in Greenes study were

also revealing. When the occasional person answered,

Yes, you should push the fat man off the footbridge

in the personal moral situation, justifying the act as be-

ing for the greater good, they took a longer time to

respond, because, Greene hypothesizes, they had to over-

come their initial emotional reaction to the situation.

Further Investigation

Greenes current research moral dilemma:

Its wartime and youre hiding

in the basement with some fellow

villagers and your baby. The en-

emy soldiers are outside and they

have orders to kill all remaining

your baby, and everybody else. Your baby starts to cry

and if you dont cover your babys mouth, the soldiers baby will smother to death. Is it morally acceptable for

you to do this?

Greene hypothesizes that when presented with this ter-

rible choice, emotional and utilitarian impulses will com-

pete and people may take a long time to come to a moral

decision. He predicts that the anterior cingulated cortex,

a region associated with competing behavioral responses

of all types (not just moral), will be activated during the

decision-making process.

He is also currently conducting more research involving

the trolley problem, but is varying the

parameters of the scenario

(the number of peo-

ple you are saving,

the probability that

they will die, etc)

and studyingusing

economic-type calcu-

lationshow the ex-

pected moral valuechanges with the

variations. Greene

is also planning on

testing psychopaths

in order to study their

responses to the same

moral dilemmas.

e a t u r e s

One important differenceis intentionpeople feel it

is different harming another

person as a means to end, versus

as a side effect, as collateral

damage.




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The Purpose of Emotion

If, as Greenes research suggests, emotion sometimes in-terferes with our rational, utilitarian system of judgment,what then is the evolutionary purpose of incorporating the

emotional system?

the emotion leads to a plan for action quickly and without

Often, rapid, automatic and emotional impulses lead us to

make good moral decisions.

We expect people to have rapid, immediate moral be-

liefs, Cushman says. Imagine somebody who had to think

if he made the right decision in the end, we would ten

to think theres something wrong about even having to

reason about it. Instead, we expect people to have an

Cushman also points out that people with brain dam

age often lack those emotional impulses and thus endup acting in ways the majority consider inappropriat

or antisocial. Thus, emotion may have evolved to pla

a role in the decision-making of a normally functionin

individual because it leads people to value the lives of

others and make rapid decisions in times of peril.

Relevance to Philosophy?

From the trolley car study, Greene has found thamoral judgments are arrived at through complexoften competing neurological processes, involving both

rational and emotional systems.Will this research change our philosophical concep

tions of morality? Many philosophers, including And

Reath, visiting professor in moral philosophy and exper

on Kantian ethics, advise that we take caution befor

research.

While psychology reveals how w

actually think, Reath explains, phi

losophy is concerned with how w

ought to think, which is something

brain scanning cannot tell us.But is psychology research entirel

phy?

Psychology professor Steven

Pinker raises the Kantian argumen

t h a t

o u g h timplies can, and

thus psychologi-

cal research into

our intuitive morals can help delineate what is possi

ble, what we can morally aspire to given our biologica

makeup. Pinker also believes we can act more morall

by learning to disregard certain visceral emotional re

actions, such as disgust towards homosexual practices

which may have arisen thousands of years ago as a lega

F e a t u r

Hell be crushed by

the trolley and will

-ple will live.


carefully, deliberately

and rationally about whether it was best

to save his younger

brother from drown-

ing in a pool. Even


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cy of evolution, but are no longer appropriate emotionalresponses.

Cushman too believes that psychological research isconnected to the philosophical ought.

Im inclined to think that morality is something hu-

mans create, not something they discover, Cushmansays. And for that reason, a psychological understandingof morality is critically important to the work that moralphilosophers aspire to accomplish.

No Role Left for the Soul?

Findings like Greenes may disturb people who preferto attribute morality to a soul that is apart from thephysical entity. Some believe that dualisma mind/bodydistinctionis increasingly unfeasible, and that researchlike Greenes shows that moral reasoning is the interplay

of physical brain processes and does not necessitate aseparate, immaterial soul.While the purpose of Greenes experiments is not to

disprove ideas about the soul, discussions of these ideasinvariably arise as a side-effect of his research.Although he is wary of completely dismissing the pos-

sibility of the soul, Greene says his research challengespeoples intuitive assumptions of what a person is.

If theres anything your soul does, its make moral de-

cisions, Greene says. When we are able not only to seebrain regions activate, but to go further with each newstudy in understanding what exact regions are used for -ness.

Cushman does not think dualism is dead quite yet, butagrees with Greene that the knowledge gained from advances in neuroscience may edge the concept out. He

cautions, though, that neuroimaging cannot truly dis-prove dualism.

Neuroimaging allows us to look at physical, chemi-cal reactions in the brain, Cushman says. Dualism pos-

its that there are non-physical causes of behavior. Sincethose causes are non-physical, theyre not going to showup in a brain scanso the fact that we cant see themdoes not provide direct evidence that they dont exist.Whatever the impact of neurophilosophy research on

our beliefs about dualism, it is evident that work likeGreenes is expanding our understanding of the physi-ological processes involved in moral reasoning. Perhapsas we gain more knowledge about our intuitive responsesand instincts, we will be better able change and improveour moral behavior.

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What makes us

decide what

we purchase?

A classical econo-

mist might say that

we choose based on

calculating expected

utility and maximiz-

ing our personal

gains, essentially by

predicting value

based on an economic theory. A

psychologist might claim that we

choose products that evoke pleas-

ant memories or conjure thoughtsfeelings of happiness. A neurosci-

entist might posit that goods that

activate our dopamine centers (an-

atomical centers associated with

reward and motivation) would in-

All of these scientists are correct,

in part. But a new individual to

the tablethe neuroeconomist

is able to integrate the theories of these

three scientists in order to make his pre-

diction.

Neuroeconomics is a relatively new

-

sideration several of the social

sciences that are involved in choice. Be-

havioral economics

which considers th

psychological ef

fects on economic

decisions, may belimited in tha

emotions and

motivation are

often qualitative

characteristics that cannot be ex-

plained but by the individual ex-

periencing them. As a result, eco

nomic predictions are divided into

normative and positive branches

Normative economics describes which economic decisions max

mize personal gain, while positive

economics describes which eco

nomic decisions humans actually

choose.

Neuroeconomics furthers the

understanding of human cogni

tion within the framework of eco

nomic and behavioral decision

Neuroeconomics

F e a t u r e



BY ALTERRELL MILLS,

THURJ STAFF


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making with brain science. Neuroanatomical measures of

preference, belief, and cognition are found using Positron

Emission Topography (PET) scans and Functional Mag-netic Resonance Imaging (fMRI) to shed light on which

areas of the brain are activated as an individual makes an

economic decision.

For example, Harvard Professor of Economics, David

I. Laibson 88, found in a 2004 study that brain activity in

the frontoparietal system and dopaminergic reward sys-tem predicted economic behavior. The experiment asked

participants to choose a smaller or larger award; the con-

dition for the larger award was receiving it in the future,

compared to the present. Participants exercised the im-

mediacy effect, which dictates that human choice is heav-

Why does emotion underlie such a deviation in consum-

ers actual economic choices, diverging from economic

theories and models? According to Laibson, Emotional

cognition is fundamentally unlike analytical cognition,both in terms of mechanism and goals. Hence it is natural

that economicsa theory of long-run, rational decision-

making (optimization)is very different from emotional

cognition. In essence, our emotions override rational de-

cision making. The impact of this obvious fact is that it

emotional state of the consumer.

Kahneman and Tveryskys Prospect Theory is a de-

scriptive model for positive economic decisions, as

it models the observed, not expected, behavior of

individual choice. The main tenet of the theory is thatconsumers are averse to losses, instead preferring gains

More importantly, consumers are averse to perceived

losses when compared to perceived gains.

In the 1979 study, participants were presented with the

following scenario: There is a viral epidemic with an ex-

pected death toll of 600. The participants are given two

program proposals. In Program A, 200 people will be

saved, and in Program B, there is a one-third chance that

600 people will be saved, and a two-thirds chance that no

one will be saved. Participants were then presented with

an alternate scenario. They were asked to choose betweenProgram A, in which 400 people will die, and Program

B, in which there is a one-third chance that no one will

die and a two-thirds

chance that 600

will die.

The research-

es found that

67% of par-

t i c i p a n t s

choose pro-gram A in the

where 200

people lived

and 400

died, and

only 33% of

F e a t u r e s



23/96

participants choose program A in the second scenario,

where 400 died and 200 people lived. In both variations,

program A kills 400 and saves 200, and program B kills200 and saves 400. The economic behavior performed

by the participants demonstrated that humans are adverse

to perceived loss, not simply actual loss.

The Prospect Theory is representative of an error in

judgment, where human perception precludes the most

-

nomics and psychology is key in understanding this ap-

parent contradiction.

In another classic experiment, Read and van Leeuwen

found that 74% of subjects are more likely to choose fruit

over chocolate as a snack when the food will be deliverednext week, but that 70% of subjects were more likely to

choose chocolate over fruit for that same day.

Findings such as thesewhich demonstrate consum-

er impatienceare the basis behind mail-in

rebates and credit cards. With an under-

can affect our decision, there comes

a need to discuss what is fair to

consumers. Is it fair to prime

consumers, using their

emotional state, to in-

decisions?

La ibson

s a y s ,

p e o -

ple use

ing makeup to attract attention or using celebrity endorse

ments. It is a fair assertion that an advertisers primingis no different than trying to attract desired praise with

rouge, , or other beauty-enhancing techniques, he says

adding that consumers are aware of advertisers manipu

lations. He further states that it would be nice to know

when we are being emotionally manipulated, but that in

practice, our cognitive systems do not need censors or

paragraphs of disclosures that we would never read.

Neuroeconomics works to predict economic de

cision making, not to regulate how it should be

used, by looking one step deeper than behavio

economicsat the physiological machinery that govern

our actions. The use of brain imaging and emotional cog

nition is a groundbreaking way to approach modeling

economic theory. If Plato is correct in that Necessity

is the mother Invention, then the problem of making

better models of economic choices may lie within neu

roeconomics.

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F e a t u r e s




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W

hile Jamie Greenwald 08 conducted research

and took classes at a Botswana university dur-

ing the 2007 spring term, many of her fellow

Harvard College science concentrators trekked through

the relentless Cambridge winter snow to their laborato-

ries and classes to receive their academic credit.

Students like Greenwaldscience concentrators who

venture hundreds of miles outside the gates of the Yard

to study or conduct researchare a minority in the Har-

International Programs (OIP), science concentrators are

vastly outnumbered by non-science concentrators when

participating in conventional study abroad programs (tak-

ing courses at a Harvard-approved institution overseas).Though science concentrators have the additional option

-

grams limit enrollment to only a handful of students and

many do not provide credit.

step up from the status quo only a few years ago. Many

of these research and study abroad opportunities just

sprouted up in the past two years, and plans are in the

works to expand these programs.

G a i n i n g N e w P e r s p e c t i v e o n S c i e n c e

Study abroad is increasingly viewed as a vital part of astudents education. Giorgio DiMauro, the associatedirector of the OIP, asserts that the purpose of studyabroad is to broaden the mind, challenge assumptions

our perspective, and become a responsible global citi

zen.

Studying or researching abroad has become more and

more relevant to an undergraduate science concentrator

Studying

A b r o a d

Scientific advances areincreasingly achieved throughcollaborative efforts betweenmultinational teams ofscientists


F e a t u r e s

BYYI CAI, THURJ STAFF


25/96

education in our shrinking world. Dr. Marie Dahleh,

assistant dean for academic programs at the School of

advances are increasingly achieved through collaborative

efforts between multinational teams of scientists, being

exposed to a different culture and learning how people

[of those cultures] think is important so that students can

learn how to work with other societies.

Research or study abroad may also provide a rich edu-

a different country. Professor Robert Lue, director of

life sciences education and senior lecturer in molecular

and cellular biology, says he sees study or research abroadas an opportunity to see how a different culture or so-

ciety approaches sciences. Study abroad may be able to

provide students with the perspective to combine differ-

ent approaches to science, leading to the development of

or research abroad are undeniable to those who have ex-

perienced time abroad. Greenwald, who participated in

the Botswana HIV in Africa research and study abroad

program, recalled several visits to nearby villages exposed

her to much of the culture in Botswana. The experi

ence also provided Greenwald with a world view and

perspective that allowed her to see the privilege student

at Harvard enjoy. Greenwald also studied and used Set

swana, the national language of Botswana, during he

time there.

Simply being abroad may play an important, if no

crucial role, in the program. For example, the Harvard

sponsored study abroad program, Fauna and Flora o

Hispaniola, is dependent on being set in the DominicanRepublic. The Botswana location of the HIV in Africa

in person.

The fact that we were doing [HIV research] in Botswa

other level, says Greenwald.

Photo courtesy of Batsirai Chidzodzo and Catherine Rosseel


Students currently participating in the CIEE HIV in Africa program climb Kgale Hill, overlooking Gaborone. In

cluded are Sandy Bolm (09) in the yellow shirt, and Sarah Ashburn (09) in the background, with a blue bandana.

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D i v e r s e O p t i o n s

At present, there are a number of science-focusedprograms for study abroad available through the

OIP as well as several research-abroad programs through

the Harvard Summer School and Harvard departments.

The programs take students to a diversity of locales, such

as South Africa, the Dominican Republic, India, Germa-

ny, and Malaysia.

Harvard-sponsored study abroad programs include

Darwin and the Origins of Evolutionary Biology in

Oxford, England and The Biodiversity of Borneo in

Malaysia. The former program credits students with two

half-year courses while the latter program credits students

with a single full-year course. However, course enroll-

ment is limited.

Harvard-sponsored research abroad programs include

HIV in Africa in Gaborone, Botswana and RIKEN

Center for Allergy and Immunology in Yokohama, Ja-

pan. The HIV in Africas pilot program, held last spring,

allowed three students to spend a semester conducting

HIV research and take elective courses at the University

of Botswana. Thus, the students receive credit for a full

semester. Students participating in the RIKEN programfor immunology conduct immunology research in Yoko

hama while receiving Japanese language training. How

ever, no credit is awarded to the three students accepted

to the latter program.

Nevertheless, the number of science concentrator

study abroad remains quite low. Of the students who

studied abroad through OIP programs last academic yea

(2006-07), only 13% of the declared students were sci

ence concentrators.

The programs take students to a

diversity of locales, such as SouthAfrica, the Dominican Republic,India, Germany, and Malaysia.



International Students attend a University of Botswana orientation meeting, January 2008.

e a t u r e s


27/96

L i m i t a t i o n s a n d D i f f i c u l t i e s

So, why arent there more science concentrators going

abroad to study or research? One of the most ob-

vious reasons is cost. Although the OIP provides some

still be discouraged from going abroad because they may

still need to cover much of the expenses, according to Di-

Mauro. In some cases, there are not enough funding sup-

-

dents to receive adequate funding if they pursue research

abroad through individually-crafted programs (those that

are not already established through Harvard). AJ Garcia,09, who was a Herchel Smith Fellow, researched abroad

-

quired a tremendous amount of independent research

Services], [the David Rockefeller Center for Latin Ameri-

can Studies], and other Harvard organizations for infor-

mation, funding, and support.

Also, many of the established research and study abroad

programs available can only accept a handful of students.

In some cases, there are notenough funding support

programs for them. It isespecially difficult for students

to receive adequate funding ifthey pursue research abroadthrough individually-crafted

programs.



International students meet the Kgosi, or chief, at the Tlokweng Kgotla, a traditional tribal meeting place, early thi

January in Bostwana.

F e a t u r


28/961

The HIV in Africa program and the RIKEN programach only take three students.

Though language barriers may sometimes be a chal-enge for students who wish to go abroad, many of thehost organizations abroad are no strangers to the Englishanguage and many programs do not have language pre-equisites.Dr. Dahleh also suggests that some students avoid going

broad because they would not receive enough credit.For example, for the Bachelor of Science degree in

Engineering Sciences, there are 20 half-courses worth ofequirements and, in that case, the student may not havenough time, Dahleh says.Indeed, some research abroad programs sponsored

by Harvard departments do not award credit (Researchnternships in Science and Engineering in Germany,

RIKEN program in immunology, Cambridge UniversityHarvard Projects, among others). However, the study/esearch abroad programs (HIV in Africa, Darwin and

he Origins of Evolutionary Biology, The Biodiversityof Borneo, and more) through Harvard College SummerSchool and the OIP do award credit.Though none of these challenges are necessarily insur-

mountable, Dahleh says that only a handful of students

going abroad. She suggests that undergraduates may notbe fully aware of the available opportunities, as many ex-

sting programs are relatively new. Indeed, study abroadtself has increased in popularity of late. According tohe OIP, the number of students going abroad during

2006-07 was more than a 300 percent increase from thenumber in 2001.

It makes a great deal of sense to go abroad if youretudying international politics or economy, but may not

be so clear a trajectory for an inorganic chemist or a phys-cist, DiMauro suggesting another possible explanation.

Certainly, many science concentrators may think it un-necessary to go abroad to study or research when there is

wealth of resources here at Harvard. Greenwald recallshat in Botswana, she had to wait weeks for reagents torrive that [she] could have had shipped overnight in the

US. Garcia shared similar lab setbacks in Brazil, and thusecommends study and research abroad (especially in less

technologically-advanced countries) to students who are

L o o k i n g F o r w a r d

However, there are indications that more science con-centrators may take advantage of study or research

abroad in the future.Traditionally, science concentrators have seen [study

or research abroad] as orthogonal or contradictory to

what they need, Lue says. But students are realizing that[they] can combine the two [studying or researching withgoing abroad].And more programs are being formulated for science

concentrators. The RIKEN program in immunology wasjust crafted in the past year, and Lue says that a program

in Shanghai is in the works.DiMauro says that the OIP also hopes to make study

or research abroad a more prominent option for scienceconcentrators. The OIP has been working with Harvardscience departments to do so, and there are plans to ini-tiate more programs for both physical and life sciencesconcentrators, he says.



Students enjoy the view from the top of Kgale Hill in Bo-

tswana. From left to right, Nathan Leiby (10), Elizabeth

Loy (UPenn), Sarah Ashburn(09), Sandy Blom (09), and

e a t u r e s


29/96

C

ancer is the second most common cause of death

in the United States, according to the American

Cancer Society, second only to heart disease.

While traditional therapieslike chemotherapy and

radiotherapywork on a large scale, by attacking both

cancer and normal cells, a group of researchers at Har-

vard Medical School have advanced a small but powerful

new paradigm in cancer drug delivery.

The lab of Dr. Omid Farokhzad, an assistant profes-

sor at Harvard Medical School, develops functionalized

nanoparticles that target chemotherapeutic drugs and

diagnostic agents to diseased cells and tissues. When

ligands synthetically conjugated to these particles bind toantigens naturally found on membranes of targeted cell,

delivering drugs selectively to diseased cells. The nano-

particles come, they see, they conquer.

While these nano-Caesars are typically smaller than a

virus, they are providing therapy for a disease of diamet-

rically opposed magnitude. Nanotechnology utilizes ma-

terials on a scale of 1 to 100nm in sizethe head of a pin

is about the size of a millimeter, meaning it would have

to be divided a million times to bring it to the dimensions

of work being done in a nanomedicine lab. The size

according to Dr. Carolina Salvador-Morales, a memberof the Farokhzad lab, is just one factor in characterizing

nanoparticles.

The size of the particle, the charge, the densityal

-

plicable nanoparticles are to biological systems, Salva-

dor-Morales said.

-

useful applications for the technology.

The characteristics that endow nanoparticles with suchenormous potentialminiature size, large surface area to

volume ratio, solubility, and large drug-carrying capaci

tysimultaneously present advantages and challenges in

The question is how to control size or charge, Salva-

dor-Morales said. The main aspect of our work is con-

trolling these chemical and physical characteristics.

using small tools

to tackle aBIG PROBLEM

One labs application of nanotechnology in cancer research

F e a t u r e

BYFARHAN MURSHED, THURJ STAFF



30/963

Harnessing nanoparticles poten-

tial has been a trying task. In a paper

on cancer nanomedicine published

in the October 2007 issue of Clini-

cal Pharmacology & Therapeutics,

Dr. Farokhzad reports that research-

ers have been attempting to use the

nanoparticle liposome for targeted-

drug delivery for over 20 years, but

only a few particles have made it to

clinical trials, and none have been ap-

proved for use.

The Farokhzad lab, however, discov-

ered one promising bioconjugateknown as the nanoparticle-aptam-

erthat was able to successfully

target pancreatic cancer cells and de-

liver chemotherapy in vivo.

In a landmark paper in the April

2006 issue of Proceedings of the

National Academy of Sciences USA

(PNAS), the Farokhzad lab demon-

with an A10 RNA aptamera nucle-

ic acid ligandcould target and bind

-

gen (PSMA) on the surface of pros-

tate cancer cells.

These nanoparticles, which encap-

sulated the chemotherapeutic drug

docetaxel (Dtxl), would promote the

uptake of Dtxl into the cancer cells

and increase the drugs cellular toxic-

ity. After just one injection of Dtxl-

Nanoparticle-Apt bioconjugate, there

of seven prostate cancer-infected

mice, and all seven mice survived the

109-day study.

In other studies, the Farokhzad lab

has shown the nanoparticles poten-

tial for carrying multiple types of

drugs (both hydrophilic and hydro-

F e a t u r e s

phobic), for triggering natural im

mune responses, and for diagnostic

imaging (nanoparticles are conjugated

with quantom dotsa type of semi

conductor nanocrystal with unique

optical properties). With such rapid

surprising that the NIH has deemed

nanomedicine one of the branche

of its Roadmap to re-engineer the

clinical research enterprise.

There has been a sharp increase

in funding regarding patients and i

has provided for research as a formamechanism, replied Dr. Farokhzad

when asked about NIHs Roadmap

commitment, There has also been

a tremendous increase from the in

vestment community and priva

sources- including those based on

An ultimate goal of the la

Farokhzad says, is to develop drugs

that would be available for oral de-livery. The translational potential for

nanomedicine promises to make a

Morales believes this is a critical time

for the lab.

The nanotech boom has been de

veloping for some years now, Mo

rales says. In the last seven or eigh

years, many phenomena have been

illustrated, and with ideas derived

from these phenomena, we are com-

ing closer to translate research into

medicine.

Even if Dr. Farokhzad and his army

of nanoparticles may not have con

quered Gaul, they represent a formi

dable threat to the disease of cancer.Scenes from the Farokhzad lab atHarvard Medical School.

by Farhan Murshed, THURJ Staff



31/96

One cold December morning, I walked into PierceHall, stepping around a piece of stray drywallfrom the ongoing construction of our sizable new engi-neering teaching lab. If things stay on schedule, sometimelater this spring, undergraduates will be busy delving intoCAD/CAM, mechanics, and photonics research.

-bowl I hope will become a haven for hands-on experi-

mentation and researchhighlights the renaissance

Engineering:

L!"#$% I"

& '() *&

E+$,-&.!"/

BY VENKATESH VENKY NARAYANAMURTI, DEAN, HARVARD SCHOOLOF ENGINEERINGAND APPLIE

SCIENCES; JOHN A.AND ELIZABETH S. ARMSTRONG PROFESSOROF ENGINEERINGAND APPLIED SCIENCE

PROFESSOROF PHYSICS

engineering and applied sciences has experienced at Havard, particularly during the past decade. Of course, o

future facility only tells part of the story.With the launch of the School of Engineering and A

plied Sciences in September, we are turning our attentioto implementing our ambitious plansenhancing eduction, advancing research, bettering societyand kickinup a bit of dust in the process. Our launch did more thsimply mark a moment in time; the event celebrated opast successes and highlighted the increasingly importa

role of engineering at Harvard and in the world.My mantra remains: You cannot do great scienc

whether it is physics, biology, or chemistry, without doingreat engineering. Likewise, you cannot solve the bigproblems, from global warming to clean water to betthealth for all, without making advances in technology antools. Granted, you might expect to hear this pitch fromdean of engineering. I think, however, with a look to hitory, I can readily convince you that everyone is linked

some aspect of engineering research.

You cannot do great

science...without

doing great engi-

neering.


O p / E


32/965

Engineering has come a long way since the timein the applied sciences at the famed Lawrence School in

tinkerers but is as thoroughly systematic, analytical, and

rigorous as the other sciences. In short, engineering re-

search leads directly to new knowledge, not simply to new

gadgets.

For example, understanding the behavior (the funda

mental physics and chemistry) of nanostructured ma

terials advances basic science through the creation o

new tools and processes (such as laser tweezers fo

manipulating individual atoms) and leads to innovativ


O p / E d



33/96

- developing novel delivery methods for common

vaccines; and

- devising solutions to controlling contaminatedwater supplies in the Dominican Republic.

Moreover, to reinforce engineerings fundamental role

we are working hard to create a more engaging, hands-on

curriculum by:

- considering broad based seminars on the world

of technology aimed at addressing questions likeHow

does a hydrogen car work?, What might the future of

electronic currency look like?, and Can we create de-

vices to meld nanomotors with biological ones?

- designing portal courses as multi-tiered entryways

into our concentrations as a way to guide those who wanto build new technologies as well as those who want to

manage them; and

- integrating design-focused projects (like how to

campuses) throughout our courses and weaving in more

research experiences (both term-time and summer), entre

preneurship opportunities (from business competitions

to on-campus incubators), and international exposure.

Bydoing so, our goal

the changing na-

ture of science and

society and to expose

more students from allareas to the interplay of

technology and society.

Further, because of our smaller scale, we have the op

portunity to make engineering more personal, more rel-

evant, and more directly related to the particular interests

and concerns of students. We believe that we have the

potential to be a connector and an integrator, a Schoo

that links to the most exciting activities across the whole

technologies such as solar photovoltaics. Taking this one

step further, such knowledge may then be channeled into

solutions for improving global health (new drug deliverysystems) or for sustaining a cleaner environment (alterna-

tive and green energy systems).

Todays technologies, the ultimate products of engineer-

ing and applied sciences, shape how we understand our

biological and physical world (from cells to CAT Scans to

quantum computing.) Thus, the timing of SEAS, a truly

21st century institution, could not be better for Harvard

In fact, I am most excited about the resulting educa-

tional and experiential opportunities that will come fromour transformation to a School. Already, we have quite a

few projects where undergraduates have worked along-

side graduate students, post-docs, and faculty from across

the sciences and engineering by:

- conducting research in nanophotonics and mi-

the Materials Research Science and Engineering Center

(MRSEC);

- working at the frontiers of quantum science to

implement a spin quantum computer;- proposing clever ways to improve Harvards ath-

letic facilities and make Maxwell Dworkin more energy

- using E. coli to manufacture a biologically-based

wire;

robotic soccer players that shoot and score thanks to el-

egant AI programming;

we have the

potential to be a

connector and anintegrator


O p / E


34/967

O p / E d

campus.

With our increased visibility, new courses, and expand-

ed research opportunities I anticipate that we will attract

more Harvard students to concentrate in engineering

and applied sciences, or at the very least, to check out

a few classes. By proving varied avenues to engineeringresearch, I think we can be one of the coolest kids on

the block.

Moreover, current students and alumni have told me

stories about how one great class, research project, or a

their entire experience at Harvard. As we go forward, all

of us at SEAS are keeping that in mind. After all, theres

a reason why our new teaching labs have glass walls: to

remind those looking out to always keep the bigger links

and connections in view; and to remind those looking inthat engineering can be a welcome surprise.

For more information on future plans for the School of Engi-neering and Applied Sciences visit: http://www.seas.harvard.edu/highlights/celebration.html

Elizabeth S. Armstrong Professor of Engineering and

Applied Sciences and Dean of the School Engineerin

and Applied Sciences at Harvard University. He is also a

professor in Harvards Physics Department. He served a

Dean of Physical Sciences at Harvard from 2003-2006.

About the author:


DANA-FARBERC A N C E R I N S T I T U T E

The Dana-Farber Cancer Instituteprovides expert, compassionate care

to children and adults with cancer.

4 4 B i n n e y S t r e e tB o s t o n , M A 0 2 1 1 5

Learn more at:

www.dana-farber.org


35/96

The ABCsof processing

your ABCsProust

andthe Squid:the

story

and

science

of

the

reading

brain

As we begin to look at the word

comprising this paragraph

we may ask ourselves whethe

reading the whole article is worth th

trouble, or if moving along to anoth

page may prove more worthy of ou

time. Seldom will any of us actual

ponder the very act of decodin

the individual words, of processinthe physiological effort involved i

the act of reading. Maryanne Wol

Director of the Center for Readin

and Language Research at Tuf

University, succeeds in doing so in he

book Proust and the Squid: The Stor

and Science of the Reading Brain.

Drawing a parallel between one o

the greatest writers of the 20tcentury and a marine cephalopo

may seem improbable at best, y

Wolf effectively uses them both

explaining the complexity of th

reading brain. To Proust, readin

was a sanctuary, a divine pleasure

that we learn to appreciate ove

time. Squids that couldnt swim wer

used during the 1950s to understan

neuronal activity in motor function


BYFERNANDO RACIMO, THURJ STAFF

B O O K R E V I E



36/9629

Similarly, the study of individuals that cant read may

provide important insights into the neurobiology of

reading, that special sanctuary of the mind. These two

literary images are Wolfs starting point, from which she

elegantly presents the latest insights into the cognitive

functions of the reading brain.

P writing systems, the second provides an account of the

acquisition of reading skills from childhood to adulthood,

and the third addresses dyslexic children and the causes

and implications of their differently-wired reading

machinery. Wolf takes the reader on a journey through

the cultural history of reading in human civilization and

the natural history of reading in the developing brain. This journey is easy to follow, even for the layperson,

but the book stops short of describing many cognitive

processes and events that could have further enriched the

otherwise comprehensive picture Wolf paints.

Ttracing the development of written communicationformsbeginning with early symbolic systems, moving

on to Summerian cuneiform, and concluding with the

birth of the Greek alphabetthe author shows how the

human brain learned to rearrange itself as this process

occurred through a series of cognitive breakthroughs.

processing and freed up cortical space in order to maximize

the brains ability to process written information. Wolf s

views on this progression are eye-opening, though they

throughout history. Reading did not always follow a linear

evolution and was sometimes subject to regressions dueto shifts in power or military domination.

For Wolf, the transition from an oral to a writtenculture constitutes a landmark achievement of humansocieties, as it opened up space and time for the brain to

have deeper and more insightful thoughts. She remark

that Socrates was fearful that this transition would mark

the end of creative rhetorical thinking. Socratess fear

are paralleled by Wolf s own fears regarding the present

transition to a digital culture: How would Socrates

...the transition froman oral to a writtenculture constitutes alandmark achievement

of human societies, asit opened up space andtime for the brain tohave deeper and moreinsightful thoughts.


Courtesy of brainsciencepodcast.libsyn.com

B o o k R e v i e w


37/96

3

ntry in Wikipedia or to a screen clip on YouTube?

After this section, Proust and the Squid reaches itsmost compelling stage, as it makes the reader divento the intricacies of a childs brain learning to read.

ead: the pre-reader, the novice reader, the decoding

How would Socratesrespond to a filmed

version of a Socraticdialogue, to his entryin Wikipedia orto a screen clip onYouTube?

hey pass through these stages, ther brains adjust their

own processing areas to make reading faster, easier, and

memories from the author herself, which may evoke

ecollections of each readers own struggles with learning

o read at an early age. By this point, however, Wolf s

tyle of writing tends to get slow and repetitive. After a

while, the reader might get fed up of hearing words like

utomaticity, logosyllabary, and morphophonemic get

Tinto perspective by introducing us to an exceptiono the rule: the dyslexic brain. To Wolf, there is no single

dyslexic brain, but instead a range of differently-wired

brains that, for a variety of reasons, cannot learn to

ead the way most people do. The theories attempting

o account for this are numerous and complex, but

hey are presented in a manner that is concrete and

asy to understand. Wolf also draws from her personal

xperience with her dyslexic son, whom she reveals as

one of her motivations for uncovering the secrets behind

the reading machinery. This allows for a deep, personal

connection between Wolf and her reader, enriching the

message she conveys: the dyslexic brains alternate wiring

can prove more dexterous at activities other than reading,

like pattern recognition and artistic endeavors.

All in all, Proust and the Squid constitutes a richand complete account of a key feature of humancivilization and developmentpeoples ability to decode

may in fact lie in its initial appeal: the themes covered

are made overly easy to understand, sometimes bordering

perhaps be left wanting more than he or she expected

from the book, but the authors theories and conclusions

are sound, strong and informative. Her ability to expertlydraw relevant facts and evidence from history, psychology,

developmental science and cognitive neuroscience makes

her book particularly fascinating. If youre done reading

this article and wonder how it is that you did it, Proust

and the Squid will surely provide the answer.



B o o k R e v i e w


38/961

Targeting TRAF Proteins as an Anti-tumor

Strategy

YI CAIAND JILL SUTTLES

University of Louisville, Department of Microbiology and Immunology, 319 Abraham Flexner Way,

Louisville, KY 40292

Secondary tumors resulting from metastasis are major causes of death associated with human can-

cer. To proliferate and metastasize, cancer cells express many secreted and membrane-bound pro-

teins. Better understanding of mechanisms involved in the production of these proteins will be useful

for developing treatment strategies in cancer therapy. Many studies have shown that interaction of

CD40, a member of the tumor necrosis factor (TNF) receptor superfamily, and its ligand, CD154,

plays an important role in tumor proliferation and metastasis and this signaling interaction is medi-

ated by TNF receptor associated factors (TRAFs). This study examined the roles of TRAFs in pro-liferation and metastasis of Lewis lung carcinoma (3LLC) cells, as well as 3LLC production of the

monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases-9 (MMP-9), and vascular

endothelial growth factor (VEGF), which plays important roles in tumor proliferation and metastasis.

Flow cytometric analysis revealed that 3LLC express both CD40 and CD154, which makes 3LLC

a good model to study CD40-CD154 interaction in cancer cells. With cell-permeable TRAF bind-

ing peptides (TRAFBPs) that inhibit the interaction between CD40 and TRAF, we demonstrate that

TRAFBPs can act as effective inhibitors of 3LLC proliferation as well as MCP-1, MMP-9, and VEGF

production. Thus, the interaction of CD40 and CD154 is involved in the proliferation of 3LLC and

in the production of MCP-1, MMP-9, and VEGF. Therefore, TRAFs can affect multiple pathways

that contribute to cancer proliferation and metastasis and may be good targets for cancer therapy.

____________________________________________________

INTRODUCTION

Cancer is a class of diseases characterized by uncontrolledproliferation and spreading of abnormal cells. Normal cells areunder strict control of proliferation and are eliminated through

growth regulatory signals. However, cancer cells can avoidapoptosis and continue to proliferate without control. Severityof cancer symptoms depends on the affected site and the abilityof cancers to invade other tissues, either by direct growth intoadjacent tissue through invasion or by implantation into distant

sites through metastasis. The secondary tumors resulting frommetastasis are major causes of mortality associated with humancancer. To grow, migrate, and avoid the attack of the immunesystem, cancer cells express a wide variety of both secreted andmembrane-bound proteins such as growth factors, proteases,immunosuppressive proteins, cytokines, chemokines, andcytokine/chemokine receptors.

As primary tumors (original tumors) and secondary tumorsgrow beyond 2-3 mm3, they need to go through angiogenesis, aprocess to develop a blood supply, to support their nutrient andoxygen needs.1 They achieve this by secreting angiogenic growthfactors such as vascular endothelial growth factor (VEGF), which

is associated with more blood vessels to the tumor, metastasischemoresistance, and poorer prognoses. VEGF binds toVEGF receptor (VEGFR) on the endothelium of blood vesselsstimulating endothelial cell proliferation and migration into thetumor. It can also recruit endothelial progenitor cells from thebone marrow for endothelial vessel formation. The new bloodvessels also provide the tumor with a route by which tumor cellscan enter into the bodys circulatory system.1-3

Matrix metalloproteinases (MMPs) are a group of extracellularproteinases involved in degrading basal membrane and extracellula

laminin. They play an important role in invasion and metastasiby degrading ECM in tissue surrounding primary tumors and inthe new organ into which they migrate. In addition, MMPs arealso involved in regulating cell behavior by cleaving non-matrixproteins, thus playing a role in tumor angiogenesis, immuneevasion, growth, and progression.4 Among the human MMPsMMP-2 (gelatinase A) and MMP-9 (gelatinase B) are expressedabundantly in various malignant tumors. They are considered keyenzymes for tumor invasion and metastasis.

The chemokines represent about 50 small chemotactic proteinsThey interact with their receptors, which are G protein-coupledreceptors with seven transmembrane domains.5 Chemokine

M a n u s c r i p t s


CELL

BIOLOGY


39/96

can recruit nearly all classes of

leukocytes, which would be expectedto be detrimental to the tumor and

surprisingly, most invasive cancers

produce the broadest spectrum and

the highest levels of chemokines.

Therefore, the up-regulation of

chemokines and their receptors may

directing autocrine growth factors for

nodal or systemic metastasis at the time of diagnosis, while t

four CD40 negative cancer cell lines were stage I tumors, witho

any evidence of regional or distant metastasis. These resu

suggest that CD40 expression on lung cancer may play a role metastasis.

In some cases, both CD40 and CD154 are expressed on canc

cells. The autocrine interaction between this receptor:ligand phas been shown to decrease drug-induced apoptosis on CD4

expressing non-Hodgkins lymphoma and breast carcinoma c

lines.14 This effect was abrogated by anti-CD154 antibody, indicati

CD154 was involved in the protective effect. Coexpression of CDand CD154 was found in breast tumor biopsies.15 Coexpression

CD40 and CD154 in the immobilized epithelial cell line increas

proliferation, mobility, and invasion in vitro. Interestingly, transie

stimulation of the parent cell line with recombinant solub

CD154 did not produce these changes. Therefore, CD40 nee

constitutive engagement to mediate these changes.15 Accumulatievidence suggests that high-dose transient stimulation of CD

may be favorable for apoptosis while constitutive engagememight favor proliferation.8

The mechanisms of CD40-CD154 signaling have not been fu

characterized. CD40 does not encode a catalytic domain in

cytoplasmic sequence. Upon ligation with CD154, CD40 form

a trimer and binds to TNF receptor-associated factors (TRAFTRAFs are a family of genetically conserved adaptor proteins a

18 Different TRA

have different biological functions17, but all share a commo

stretch of amino acids at the C terminal TRAF domain. The TRA

domain has been divided into TRAF-C and TRAF-N subregion

The C terminal TRAF-C region mediates both homo- and heter

dimerization of TRAF proteins and interacts with receptors threcruit them. At the N terminus TRAFs, with the exception

important for downstream signaling events. Among the six TRAF

TRAF4 is a predominantly nuclear protein and does not bind

cell surface receptors. The other TRAFs bind with the cytoplasm

domain of CD40 at two sites, a distal site for TRAF1/2/3/5 and

proximal site for TRAF6 (Figure 1). TRAF6 is not only involvin TNFR signaling, but also serves as an adaptor protein for th

IL-1/Toll-like receptor (TLR) family.17,18 Many of the biologi

effects of TRAF (also IL-1/TLR by TRAF6) signaling appear

extracellular signal-regulated kinase (ERK)1/2, and the activat

protein-1 (AP-1) family that leads to induction of cytokin

chemokines, proliferation, and rescue from apoptosis. Howev

Activation and blockade of CD40 have both been tested as

means of modulating tumor behavior. Inhibition of TRAF signali

might be an effective way to inhibit cancer cell proliferatio

because it may be able to block CD40 signaling as well as oth

pathways, such as interleukin-1 (IL-1)/TLR, that might favor cancproliferation. One way to inhibit CD40 signaling is to use TRA

binding peptides (TRAFBPs) that have similar sequences to t

TRAF binding sites on CD40. The presence of such peptides w

compete with CD40 to bind with TRAF, and thus disrupt CD

FIGURE 1. TRAF binding sites on

CD40. Binding of CD154 triggers

trimerization of CD40, which in-

Sequences of the binding sites on

murine CD40 and the corresponding

TRAFs are shown

M a n u s c r i p

cancer cells, providing paracrine growth advantages via angiogenesis,

enhancing invasion, enhancing metastasis by vessel entry, determining

the location of secondary tumors, and inducing immune evasion for6 Selected chemokine receptors, including CXCR4,

CCR7, and CCR10, are often up-regulated in a large number ofcommon human cancers. They may potentially facilitate tumor

dissemination at several key steps of metastasis, including adherence

of tumor cells to the vessel endothelium, the passing of cancer

cells through blood vessels, metastatic colonization, angiogenesis,

proliferation, and activation of key survival pathways.7

Cluster of differentiation 40 (CD40), a member of the tumor

necrosis factor receptor superfamily (TNFRSF), is expressed atuniformly high levels in carcinomas of the nasopharynx, bladder,

cervix, and ovary and at more variable levels in carcinomas of the

breast, skin, and lung.8 The natural ligand of CD40 is CD154. Itis a member of the TNF superfamily (TNFSF), which exists

both in a transmembrane-bound form and a soluble form. High-

levels of transient CD40 activation has been shown to inhibit cell

proliferation and/or increase apoptosis on human bladder, ovarian,cervical, squamous cell, lung, and breast carcinomas.9 Ligation of

CD40 on tumor cells can enhance immune responses and result in

anti-tumor activity independent of host CD40 expression.10 CD40

ligation has also been shown to stimulate host anti-tumor immune

response by activating dendritic cells to enhance the antigen-

presenting capability and by increasing immunostimulatory cytokineproduction.8,9 Treatments based on the growth- and immune-

regulatory features of CD40-CD154 interaction in normal cells havebeen explored to promote corresponding direct and indirect tumor

growth-inhibitory outcomes. These treatments used agonistic anti-

CD40 monoclonal antibodies, recombinant CD154, or CD154 gene

and autoimmune consequences of these treatments is a concern andthe success of clinical trials is limited.9,11

On the other hand, CD40 ligation may also increase proliferation

in many tumors, such as acute myelomonocytic leukemia, low-grade

B cell malignancies, Burkitts lymphoma, HIV-related lymphoma,

and Hodgkins diseases.9,12 In one study, 18 human lung cancer cell

13 Thirteen of fourteen

cancer cell lines that expressed CD40 on their surface had eitherw w w . T H U R J . o r g T H U R J V o l I I s s u e 1 S p r i n g 2 0 0 8


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signaling by dramatically decreasing the interaction between CD40and TRAF. To facilitate the permeability of the peptides to cells, asignal sequence is added to the peptides. It has been shown that theTRAF6 binding peptide was an effective inhibitor of CD40 induced

in human monocytes and macrophages.19 This project studied the

effects of TRAFBPs on proliferation of the Lewis lung carcinoma(3LLC) cell line, which expresses both CD40 and CD154.

MATERIALS AND METHODS

Reagents and antibodies. Phycoerythrin conjugated anti-

conjugated anti-mouse CD154 antibody were obtained fromBeckman and Coulter (Fullerton, CA). Anti-mouse CD16/CD32monoclonal antibody was purchased from BD Pharmingen (San

antibody was purchased from BioLegend (San Diego, CA). Methyl-tritiated-thymidine (3H-thymidine, 37 MBq/ml, 1 mCi/ml) usedfor proliferation assays was obtained from Amersham Biosciences(Piscataway, NJ). The liquid scintillation counting (LSC) cocktail was acquired from Packard Biosciences (Billerica, MA). Cellculture medium RPMI 1640 and fetal bovine serum (FBS) wereobtained from HyClone (Logan, Utah) and Atlanta Biologicals(Lawrenceville, GA), respectively.

TRAF2,3,5BP and TRAF6BP: TRAF2,3,5BP and TRAF6BPwere purchased from Invitrogen (Carlsbad, CA). The sequence ofmurine and human TRAF2,3,5BP is AAVALLPAVLLALLAPN-TAAPVQETLHGCQPV and the sequence of murine TRAF6BPis AAVALLPAVLLALLAPPAARRQDPQEMED YPG. A signal

was added to the TRAFBPs to allow for cell permeability. Theadditional amino acids represent a portion of the murine CD40protein that includes the TRAF binding sequences (shown as boldportions in red).

Cell culture. 3LLC cells were obtained from American TypeCulture Collection (ATCC) (Manassas, VA). They were cultured in100 mm tissue culture dishes from BD Labware (Franklin Lakes,NJ) in RPMI 1640 with 5% FBS, 0.01 M HEPES, and 50 !g/ml

Afterwards, they were removed using cell scrapers. The 3LLC cellswere plated in 96-well plates at either 5"103 cells/well for proliferation

assays or 1"104 cells/well for monocyte chemoattractant protein-1(MCP-1), MMP-9, and VEGF experiments.

Proliferation assays. The 3LLC cells were cultured in R5and plated in a 96-well plate in triplicates at 5"103 cells/well andallowed 0.5 hours to adhere. The 3LLC cells were either untreated

with TRAF6BP or TRAF2,3,5BP at 100, 200, or 300 !M. Theplates were incubated at 37 C, 5% CO2 for either 1, 5, or 18hours for the anti-CD154 antibody experiments or for 18 hoursfor the TRAFBP experiments. After incubation, 3H-thymidinewas added at 1 !Ci/well. The plates were pulsed for 5 hours, and

cocktail was added to each well and the amount of radioactivity each well was then counted on a TopCount scintillation counte(Packard Biosciences).

Flow cytometric analysis. 3LLC cells were scraped antreated with Fc Block (anti-mouse CD16/CD32 monoclon

antibody) for 15 minutes at room temperature and then labeled wiboth PE-conjugated anti-mouse CD40 and FITC-conjugated antmouse CD154 antibody in Dulbeccos phosphate buffered salinwith 5% FBS and 0.01% azide for an hour at 4 C. Afterward

activated cell sorter (FACS) (Becton Dickinson, San Jose, CA).Production of MCP-1, MMP-9 and VEGF by 3LLC cells

The production of these proteins was measured using enzymlinked immunosorbent assay (ELISA). Cells were plated in 9well microtiter plates in triplicates at 1"104 cells/well with non

hours incubation, supernatants were harvested. Murine MMPand VEGF were assayed with ELISA kits from R&D System(Minneapolis, MN) and murine MCP-1 was assayed with aELISA kit from BD Pharmingen. Absorption of the samplwas measured using an E-max Precision microplate reader froMolecular Devices (Sunnyvale, CA).

Statistical analysis. Data is presented as mean standar

sample t-test was used to compare treatments with the controls an

RESULTS

Coexpression of CD40 and CD154 on 3LLC cells. T

CD40 and CD154 on both murine and human lung carcinoma ce

cells were dual-stained for CD40 and CD154 (Figure 2). Th3LLC line has served as an important tumor model for metastatand angiogenic studies.20 Our study focused on the 3LLC cell linbecause the coexpression of CD40 and CD154 provided a goomodel for studying the autocrine interaction of CD40-CD154.

FIGURE 2. Surface coexpression of CD40 and CD154 on 3LLC

cells. The 3LLC cells were dual-stained for surface expression o

cells.

M a n u s c r i p t s


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BIOLOGY


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CD40-CD154 interaction contributes to 3LLC

proliferation. The CD40-CD154 interaction may lead to

proliferation or growth inhibition depending on cancer cell

types. In order to study the role of this interaction, 3LLC cells

were incubated with 0, 5, or 10 !g/mL of anti-CD154 antibody

for 1, 5, or 18 hours. The cell growth was then measured byproliferation assays. The anti-CD154 antibody inhibited 3LLC

proliferation in a concentration-dependent manner (results from1 hour incubation are shown in Figure 3), suggesting that the

autocrine interaction of CD154 and CD40 contributes to the

proliferation of 3LLC cells. At the 10 !g/mL level, anti-CD154

antibody decreased proliferation of the 3LLC cells by 36%, 24%,

and 9% respectively for 1, 5, and 18 hour incubation (5 and 18hour data not shown). This weakening in inhibition over time may

test this possibility, 3LLC cells were incubated with an anti-CD154

antibody for 3 hours and analyzed for CD154 surface expression,

was detected from the freshly stained 3LLC cells, but there was no

hours (data not shown). This indicates that there was not much

anti-CD154 antibody left outside the cells, most likely because the

antibody was engulfed and destroyed by the 3LLC cells.

Proliferation of 3LLC is blocked by TRAFBPs. The

results of the previous experiment suggested a role of the CD40-

CD154 interaction in 3LLC proliferation. However, treatment of

in reducing proliferation. Hence, we considered the possibility that

use of TRAFBPs may be a better alternative to the anti-CD154

antibody because they are taken up directly by cells and may be

less easily destroyed. Thus, we examined the effect of TRAF6BP

and TRAF2,3,5BP on 3LLC proliferation. The TRAFBPs containa KFGF signal sequence (to allow for cell permeability) and

sequences corresponding to those of the TRAF binding sites onCD40. Therefore, they can enter cancer cells and bind to TRAFs,

preventing TRAFs from binding to CD40. The 3LLC cells were

either untreated or treated with 100 !M, 200 !M, or 300 !M of

the TRAFBPs overnight and their proliferation was measured by

proliferation assay. As shown in Figure 4, 3LLC cell proliferation

was inhibited by both TRAF2,3,5BP and TRAF6BP in a dose-dependent manner. The proliferation of the cells decreased by 90%

and 99% with 300 !M of TRAF6BP and TRAF2,3,5, respectively.

TRAFBPs decrease MCP-1 production of 3LLC cells.

Cancer cells produce various cytokines and chemokines to recruitleukocytes to help them to grow and invade surrounding tissues.

MCP-1 is one such chemokine that can attract macrophages whicfacilitate tumor cell migration, invasion, and metastasis.21 Thu

we examined the effect of TRAFBPs on MCP-1 production

3LLC cells. The experimental design was similar to those of th

experiments examining the effect of TRAFBPs on cell proliferatio

except that ELISA was used to measure MCP-1 production. Hig

levels of MCP-1 were detected from the untreated 3LLC cel

TRAFBPs in a dose-dependent manner (Figure 5). These resu

indicate that MCP-1 production by 3LLC cells can be inhibited b

the TRAFBPs.TRAF6BP decreases production of MMP-9 by 3LL

cells. Since MMP-9 is produced by many carcinomas and pla

an important role in cancer invasion, metastasis, and angiogenes

the role of TRAF6BP in MMP-9 production was studied. T

same approach as for the MCP-1 experiment was used, exce

that MMP-9 was measured. Substantial levels of MMP-9 we

produced by 3LLC cells. Production decreased drastically by t

TRAF6BP effectively inhibited production of MMP-9 by 3LLC.

TRAFBPs reduce VEGF production by 3LLC cells.VEG

is a potent pro-angiogenesis protein. Its importance in tum

growth has been well documented.1 The effect of TRAFBPs o

VEGF production by 3LLC cells was examined. 3LLC cells we

incubated with or withoutTRAFBPs and the VEGF in the cell supernatants was measur

by ELISA (Figure 7).

A similar reduction of the level of VEGF production w

generated regardless of the concentration of TRAF2,3,5BP use

suggesting that TRAFs2,3,5 may play a partial role in induction o

VEGF production. However, production of VEGF was reducby TRAF6BP in a concentration-dependent manner and was mo

effective than the TRAF2,3,5BP. This may be due to the ability

TRAF6BP to inhibit multiple pathways or to inhibit a pathway

most critical to VEGF synthesis.

DISCUSSION

Lung cancer is one of the most common and malignant cance

notorious for its poor prognosis and high mortality, even wi

early detection. The deaths related to lung cancer are the mo

common among cancer-related deaths for both men and women

the United States.22 Better understanding of the causes of the hig

proliferation and metastatic features of lung cancer will be usef

The 3LLC tumor model is important for studies concerni

metastasis and angiogenesis because 3LLC is highly metastatic an

can metastasize locally and distantly.20 Our experiments indicat

that both CD40 and CD154 are expressed on 3LLC cells. Sin

autocrine interaction of CD40 and CD154 has been reported

contribute to proliferation and metastasis,15 we examined its roon proliferation and metastasis of 3LLC cells. The anti-CD1

but this effect decreased with the increase of initial incubatio

FIGURE 3. CD40-CD154

interaction contributes to

3LLC proliferation. 3LLC

cells were pretreated with

or without anti-CD154 Ab for

-

midine for 5 hours, and as-

Values and error bars are

means and SD of triplicate

determinations (**: p < 0.01

M a n u s c r i p t

**

**

0 0.5 1 1.5 2

10 !g/mL

Ab

5 !g/mL

Ab

Control

CPM/1000



42/965

experiment indicated that most of the antibody was engulfed anddestroyed within 3 hours by 3LLC cells. The anti-proliferativeeffects observed from 5 and 18 hour incubation experiments wereprobably caused by the slow recovery from the inhibitory effects

originating from the antibody at the early stage of incubation. The involvement of CD40-CD154 interaction in 3LLC

TRAFs are essential in the CD40-CD154 signal transductionpathway. Their activity can be blocked by TRAFBPs, which havethe same sequences of TRAF binding sites on CD40, throughcompetitive binding with TRAFs. Both TRAFBPs were effective ininhibiting 3LLC proliferation, especially the TRAF2,3,5BP, whichalmost completely blocked proliferation. Thus, the TRAFBPsare more effective than the anti-CD154 antibody in impedingproliferation. Reasons for different effects of TRAF6BP andTRAF2,3,5BP on 3LLC proliferation still need to be explored.

Cancer cells express a large variety of proteins to avoid attack bythe immune system and facilitate their proliferation and metastasis.MCP-1, a strong attractor of macrophages, is one such proteinsecreted by many cancer cells. Macrophages play an important rolein immuno-responses. They are known to have diverse activitiesdepending on their microenvironment.23 A tumors ability to recruitmacrophages and to create a microenvironment that promotes

angiogenesis, and metastasis.21,24 CD40 and TRAF6 were shown tobe involved in MCP-1 production in human renal proximal tubuleepithelial cells.25 Our results indicated that both TRAF2,3,5 and TRAF6 were involved in MCP-1 production in 3LLC cells. In

addition, the CD40-CD154 interaction also plays an important roleTRAFs may be good targets for cancer therapy.

Two additional important proteins to consider are MMP-9 and VEGF, which facilitate tumor invasion and metastasis. MMP-9is known to be up-regulated by the CD40 pathway in cervicalcarcinoma cells.26 Also, TRAF2 and TRAF3 were reported to beinvolved in induction of MMP-9 by latent membrane protein 1(LMP-1) in nasopharyngeal carcinoma cells .27 In both cases, the

Our results show that production of MMP-9 was inhibited byTRAF6BP. A role of TRAF2,3,5 in MMP-9 production by 3LLCis currently under investigation. In addition to digestion of basal

membrane and ECM proteins, MMP-9 is also involved in cleavagand activation of other proteins, such as VEGF. It was shown thVEGF was expressed in non-angiogenic, angiogenic, and contrislets as well as tumor cells. MMP-9 doubled the release of VEG

and induced angiogenesis, possibly by cleaving the ECM boun164-amino-acid VEGF isoform to the freely soluble 120-aminacid VEGF isoform.28

Our results show that production of secreted VEGF winhibited by both TRAF2,3,5BP and TRAF6BP. The effect

be more effective at higher concentrations. The different effecof TRAF2,3,5BP and TRAF6BP on VEGF secretion suggest thdifferent mechanisms might be involved in VEGF regulation. HoTRAF2,3,5BP and TRAF6BP produce different effects remains be investigated.

The effect of TRAFBPs was not due to cellular toxicit

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