Biophysics PhD申請心得分享

同文發表於PTT studyabroad

Program: Biophysics PhD Fall 2009

Admission: all have guaranteed funding throughout the years

UCSF iPQB (integrative program in quantitative biology) (2/20~21 on campus) stipend 27K/yr, tuition waived (supplement to MOE scholarship)

UIUC biophysics & computational biology (2/24 interviewed on Skype) stipend 22,660/yr, tuition waived (should be supplement also, but not mentioned in the offer letter)

UW Madison biophysics (no interview) stipend 23K/yr, tuition waived—my first admission (late January)

UC Davis biophysics (no interview) no words on funding (guess the admitted students will have guaranteed funding as claimed on the website)

Pending after interview: UC Berkeley biophysics (2/17~19 on campus)

Interview invite: Goettingen GGNB/MPI, 5/3~12 on campus (sent after initial evaluation through video interview @ Taipei 101, 2/12)

Rejection: Caltech Biology, Harvard, Rockefeller, WUSTL DBBS, JHU CMDB, UPenn, UMich Ann Arbor PiBS, Scripps, Dresden MPI (in Germany)

Decision:



Education:
BS in Life Science, NDHU (2004)
GPA 3.94/4.00, Class Rank 1/61 (2%)
MS in Biochemical Sciences, NTU (2006)
GPA 4.00/4.00, Class Rank 2/27 (7%)

Test Scores:
GRE: V480/Q800/A4.5 (Oct 2007 PBT)
TOEFL: 99 | R27/L28/S22/W22 (Jul 2008)

Work Experience:
Research Assistant at IBC Academia Sinica (Aug 2006~present)

Publications:
1 first author journal article published on NAR
5 international conference posters (4 are first author)

Select Honors:
書卷獎*6 & graduated with honor (NDHU)
International Travel Award from Biophysical Society (2004)
Outstanding Student Travel Grant from 傑出人才發展基金會 (2004) -- these two are for attending 2004 Biophysical Annual Meeting
NSC Research Creativity Award (大專學生參與國科會專題研究計畫, 研究創作獎)
Best Poster Award in 生物物理新知研討會 (2006)

Select Scholarships:
林熊徵公學田獎學金 (2003)
行天宮資優學生培育獎學金 (2005-2006)
佑生基金會 碩士論文獎學金 (2006)
教育部留學公費甄試獎學金 (2008)

Recommendation
1 from master thesis's advisor & current boss (Academia Sinica IBS distinguished fellow)
1 from research collaborator (deemed my second mentor, UMDNJ Biochem faculty)
1 from undergrad research advisor (now NCTU faculty)
1 from undergrad coadvisor (Academia Sinica Chem research fellow)

-- Quick note: 申請過程的十個重點 --
http://niwhu.myweb.hinet.net/share_list.pdf
這裡分享一下我的選校清單，以及本人認為的10 things you need to know before application…希望能幫助到接下來申請的學弟妹。

整理這些學校的資料至少花了我一個半月(最花時間的是faculty研究內容)，龜毛如我一定會針對學校寫不同的SOP，在寫customized paragraph之前，我一定會挑出2~4個有興趣跟的老師，把他們近期的paper看一看，因為有些faculty page已經很久沒更新(例如JHU)，make sure you're not writing something stupid

-- 背景/選校/聯絡教授 --
我是生物主修，從大二進實驗室，一直以來研究經驗都是與生物物理相關，雖然沒刻意去修另一個主修或輔系，但從修課歷史也可看出興趣所在。也因為磨了好一段時間，我的目標很明確: 過去我做的東西主要是生物熱力學，包括特殊核酸二級結構的形成(under)，以及DNA-binding protein對不同DNA sequence的專一性(grad)，用卡計跟low resolution方法，提供的資訊很有限(ensemble average)，所以我想學single molecular techniques。另外，我碩班研究的這枚AAA protein，引發我對這類巨型蛋白質的興趣，這是我選校的重點之一，UC Berkeley有很多老師的研究主題是AAA family members，UCSF則是有一個巨型分子結構研究團隊(MSG, http://msg.ucsf.edu/)。

如果要nail down感興趣的生物問題，則是粒線體的分裂融合動態，類核蛋白質如何重新組織mtDNA以配合粒線體的行為？為何某些常態下應座落於基質的蛋白質有時會被發現於類核？作這類研究且符合我上述需求的program，只有Caltech的David Chan，JHU的Blake Hill，跟Goettingen的Stefan Jacobs (哥廷根的申請我是直接向GGNB office申請，一年兩次的selection，而非route 2聯絡教授)。

我只有跟Caltech及JHU這兩位老師聯絡，我只禮貌性的問他們明年是否會收學生，JHU沒回，Caltech說他對我的背景很感興趣，要我拿到AD後跟他聯絡，感覺上沒辦法幫什麼忙，我要去加州面試前又寄了一封信給他(Caltech面試周在2/13，如能獲得面試機會，可以跟Berkeley與SF行程串在一起)，他說已幫我告知director，但仍然沒收到任何來自ad com的信件，sadly最後JHU跟Caltech都拒絕我了。有興趣參考我聯絡教授的信件怎麼寫的，
請見http://niwhu.myweb.hinet.net/share_contact.pdf

-- 如何整理學校/老師資料 --
國際學生申請的學校數目動輒十幾間，有些要中英文成績單各一份，有些要班排名證明，每間學校對admission essay的要求也不盡相同，有些要求你回答特定essay questions (e.g., WUSTL DBBS)，有些要求SOP之外還要personal history statement & research statement等等，字數限制也不同，宅宅如我一定會想找個筆記軟體，有效率地整理這些瑣碎的細節。

這裡我要推薦Evernote: remember everything! (http://evernote.com/about/what_is_en/) Evernote算是名聲很大的(功能也很強)的免費筆記軟體，網路上可以找到許多教學文件(check this http://www.youtube.com/watch?v=bSWSn4e3lOQ&feature=related)。

我的作法是，在瀏覽學校網頁的同時，用實體筆記本記重點(申請費，deadline，TOEFL要求等等)，看完後把頁面內容dump到Evernote裡，必要時可以編輯tag(在標示faculty研究領域時特別好用)，它的同步化特點，讓你不受地點限制，只要有網路即可存取學校資料。它的搜尋功能很強大，你只要輸入關鍵字，它便能幫你搜尋所有符合敘述的entry，例如我忘記了那個學校要求排名證明，search "rank"，就幫我找到UC Davis是要這個文件的，把學校真正要求的文件都彌封在一個信封裡(of course they should be from the same alma mater)，不需要的證明文件就用不著寄發，製造垃圾又浪費錢。

有Evernote的幫忙，最後你要用表格整理各學校的資料時就方便多了，像我這種有檢查強迫症的人，Evernote也便於你確認沒有mistype anything

-- Essays --
不要太迷信editing service可以把你的essay改得多好(而且我超討厭我找的editor一直往自己臉上貼金)，畢竟寫的人是你，最好先找一本書或是google一下，瞭解這些essay是怎麼回事，再來動筆，我找的參考書是"Graduate Admissions Essays" by Donald Asher。

另外就是及早動筆，修改的過程你會不斷地再次檢視你自己。我在二月時寫了一個版本(申請獎學金用的)，寫完還覺得超滿意的，過一段時間後再回頭看，發現文章暴露了太多負面的資訊，是ad com不需要知道的(可能是因為我邊喝酒邊寫的關係XD)，十月初申請學校時，全部重寫過。

我把所有essay跟CV都放到blog上了，有興趣的話可以參考一下
http://niwhu.blogspot.com/

-- 面試 --
我一月中收到UCSF跟UC Berkeley的on site interview邀請(1/16收到UCSF通知，UC Berkeley網頁上說1/15會做出invite決定，因此寫信去問秘書，1/17收到ad com chair來信，告知他們剛開完會，I'm on the short list!)。

我很幸運，UCSF跟UCB的面試剛好一前一後(UCB:2/17~19/20*; UCSF: 2/20~21)，兩間學校彼此有協調過，事實上有不少domestic student同時收到他們的邀請，而我的旅費是由雙方共同負擔(UCSF機票補助上限$400，UCB是$500)，所以這趟加州之旅幾乎不需花錢。

得到這個面試機會相當開心，這次旅程收穫很多， UCB除了一兩個on sabbatical或out of town沒辦法排到面試，我幾乎都有跟自己感興趣的老師談到話(填9個排到5個, all one on one)，UCSF排了7場one on one面試，6個faculty加一個現在學生(排到3個當初填在申請書上的)，這是schedule



每場間隔只有10分鐘，都排在同一天，講到後來喉嚨都啞了。我回來後有寫了一些心得，但是犯懶還沒寫完(分享文富堅化?)，基本上on campus interview的情形，大家可以參考bluejelly的文章(on PTT studyabroad)，他寫得非常好☺

在加州面試的時候，收到UIUC director的信，說要安排電話或是Skype口試，在諸多考量下，我還是要求回國後再口試。跟UIUC的對談經驗很有趣，那時已經超過約定的時間15分鐘，我看他還沒打來，只好用Skype搜尋他的名字(他沒給我Skype account)，然後打過去，他接到後說他正準備要打給我的，然後就聽到紙張簌簌的聲音(他在整理手邊的文件)。

一開始就說他覺得很抱歉，沒辦法提供國際學生on campus interview，然後問我對他們的學程有沒有什麼問題，接下來50分鐘都是我問問題他回答，因為他(Robert Clegg，也是我寫進SOP的老師之一)待過哥廷根很長一段時間，我又剛好收到哥廷根的面試邀請，就問他認為這兩邊對博士班訓練的差異，他就滔滔不絕連續講了約20分鐘，不時自問自答"你如果要問我在哥廷根的經驗好不好，我沒辦法欺騙你說他不好，我太太是德國人，我兩個小孩都會說德文跟英文…"

我問完問題後，他幫我這個人做了一個很正面的總結@@!
"基於我剛剛跟你的談話，你有很明確的研究目標，包括…，而我們有能力針對你的需求提供很好的訓練，包括…" 說到這裡，我知道應該是上了XD，這個口試經驗，加上對AD coordinator的印象很好(問的問題都很快就得到回應了!)，UIUC在我school list的排名瞬間躍升好幾格。

-- 最後選擇 --
在飛過去面試之前，個人是偏好UC Berkeley的，但真正參訪學校與面談過後，I'm totally hooked on UCSF!

SF有安排facility tour，不過讓我印象深刻的並不是他們設備有多fancy(很新是真的!)，是學生能使用這些儀器的自由程度，根據tour leader的說法，你的學生證能讓你用遍shared facility的所有儀器，包括NMR與EM等等，你需要做的就只是走進儀器房，請資深的使用者教你操作程序，確定你都會了，上網預約時間之後就可以使用了，沒有討厭的繁文縟節，他說這種事情只有在UCSF才遇得到，不知道會不會唬我們？(會嗎？)我還是覺得很不可思議…。有被官僚的管理體系搞得作實驗倍受束縛，或是遇到凡技術門檻高的儀器，就好像那些東西是你永遠碰不得也沒機會學習的，搞到非得要談合作的朋友們，應該了解我的感受。

我對SF的另一個印象是，他們非常照顧學生。早在受邀面試之前，在我還沒丟出申請文件時，因為跟老闆溝通不良，推薦函出現一些狀況(有興趣了解的朋友，請爬一下我以前的文章)，我寫信給有狀況的學校，詢問是否能withdraw推薦函submit新的版本，多數得到的回應都是，可以，但必須由推薦人寫一個聲明，把新版本寄到特定信箱，這等於是要老闆承認這是他的失誤，雖然老闆平常很好說話，但這種事情很可能觸怒他，所以告知這樣處理的學校，我都放棄不管了，只有UCSF不但很快就收到他的回信" Let me check and see if I can delete the letter and have you send the link again. Who is the recommender?"，然後他就真的幫我withdraw了，為了幫老闆留住面子，只好跟他撒了個善意的小謊，說SF沒收到推薦函，補寄新的連結，請他重新上傳(終於說出來了，希望老闆萬一知道不要生氣才好@@)。

另外一個有趣的經歷是，UCSF發給面試邀請後，寄了一份問卷請受邀人填寫，其中有一個是nickname，然後我不知道是那條神經不對勁，自以為我的中文名字對美國人來說可能不好發音，就填了我的nickname (mostly called by my coworkers and classmates)，到場看到我的badge時整個傻掉…



我的first name就被換成nickname…

"你好，我是丸子陳"

(大陸人一定以為我是怪咖，幸好沒有日本人，應該會笑翻吧)
面試回來後，跟coordinator通信，他都直接叫我Maruko。

至於Berkeley，跟老師們的面談經驗都很愉快(SF當然也是)，最想跟的Carlos Bustamante，他雖然在休假年，剛好面試周有幾天是在校園的，我很幸運被排到跟他面試，有些受邀者排在最後一天的，因為Carlos回秘魯去了，只能跟他的學生談到話。另外還有James Berger，而Eva Nogales值休假年，沒辦法跟她談到話(好可惜…這也是後來我會選SF的原因之一)。

其實在這兩間學校做出選擇，對我來說是很困難的，luckily (?) the ad com and coordinator has made it easier，我一回國就發現thegradcafe上，有人已經收到Berkeley的AD了，然後我一直等就是等不到，兩個禮拜過後我寫信去問，秘書說"We currently have your application on hold."，但問他他又說不是waitlist，只是要確認給國際學生的funding是沒問題的，大概要等這個月底。我又寫信問了一些問題，得到的回應是"I can't really answer most of your questions, as the admission process is confidential."，同樣的東西，UCSF都有問有答，而且都回超快的。

我還是會等到月底，不過現在看來變數不大就是了。

對哥廷根比較不好意思，因為在offer下來之前，五月初到德國的機票已經定了，還讓哥廷根安排了10天住宿(在MPI guest house)，除非哥廷根能讓我印象大好，打敗UCSF，不然應該還是去SF。

-- 結語 --

Don't victimize yourself as a biology applicant!

這篇分享文我寫了連續三天，該講的不該講的都講了XD，希望能給正在或即將申請生物領域的朋友們一些鼓勵，(特別是主修生物的，我知道跨領域正夯，很多其他major的也來搶飯碗，結果大家都愛他們，搞得好像大學念生科是錯誤的選擇@@)，很多人說生物領域很難申請，但每年其實還是有不少台灣同胞們進入了很好的學校，據我所知，所上有幾個學弟妹今年申請的結果都相當不錯，但是都在潛水的樣子…。

我最要感謝的就是我的老闆，吳世雄老師，如果你沒有放任讓我自己發展，我一定還很迷惘不曉得自己適不適合作研究，申請學校這段期間，沒辦法把心思完全放在實驗上，難免讓你有些失望，也謝謝你包容我這些日子沒有良心的行為(我稱研究是良心事業…)。還要感謝我的second mentor，Carolyn Suzuki，真的很幸運能遇見你(雖然一直以來只有通信…)，讓我重新認識一個科學家應有的態度。謝謝中研院福利社前賣茶葉的阿姨，沒有喝到好喝的茶，我essay一定寫不出來的，哈哈。還有我的爸媽，要出國唸書這件事，我從大四講到現在，今年終於要出去了，說服你們從反對轉為支持，可是費了我好大一番工夫耶！很高興看到你們從一開始的擔心害怕，變成現在臉上堆滿著笑容祝福我，謝謝你們~!!!

也祝福大家☺

Personal History Statement

I have a special reason for appreciating every opportunity I have to obtain an education and explore my talents: both of my parents had to leave school at the age of thirteen and work full-time to support themselves and my grandparents. This was at the very beginning of the period that eventually came to be known as the “economic miracle” of Taiwan. My father has at various times worked as a mechanic and as a worker in a rubber production facility, and my mother continues to work long hours as a spa therapist at a small beauty salon.

In contrast, I was born during an era when my country was undergoing enormous change. Forty years of martial law came to an end when I was five, and a new government administration included a group of technical bureaucrats who successfully pushed for public funding of basic science research and education. I benefited from those decisions in high school and as an undergraduate; unlike students in many other developing countries, I am very fortunate in having several options for my future. My decision is influenced by recent advancements in the biological sciences that include animal cloning and human genome mapping.

One of my best adolescent experiences was attending the 2001 Wu Chien-Shiung Science Camp. I had some minor surgery just before the camp, but the physical pain was not going to stop me from meeting Nobel laureates Steve Chu, Douglas Osheroff, and Yuan-Tseh Lee. You can imagine my excitement when I was selected to explain my proposed solution to HIV on a stage in front of hundreds of experienced scientists, despite not having any laboratory experience. I was very nervous, but I was also running on adrenaline from an all-night brainstorming session that I had shared with my teammates. The source of my energy that night was the spirit of exploration, and I was hooked by the experience.

Every creative mind must find its niche, and mine is in biophysics. I enjoy working with the big picture of biomolecule binding energetics, structural dynamics, and motion in living cells. However, as my undergraduate mentor Dr. Chia-Ching Chang says, “Straight A’s do not guarantee a successful research career.” I try to keep in mind my commitment and the work ethic that my parents taught me. Accordingly, I was willing to work on my master’s thesis research into the early morning hours, excited with anticipation at reading data compilation and calculation results at 2 a.m. Moments like those have cemented my interest in becoming a biophysicist, using my energy and enthusiasm to explore the hidden rhythms of biological phenomena.

Course Content Information

http://niwhu.myweb.hinet.net/Course%20Content%20Info.pdf

Transcript & Certificate

http://niwhu.myweb.hinet.net/transcript&certificate.pdf

Proposal Summary

Dynamic Structures and Composition of Human Mitochondrial Nucleoids: Biochemical and Biophysical Studies

人類粒線體類核結構動態: 生化及生物物理之研究


This proposal is centered on: (1) the proteomics of mitochondrial nucleoid; (2) characterization of protein-protein interactions between the nucleoid components; (3) the composition of mitochondrial nucleoid in connection with changes of cellular metabolic state as well as oxidative pressure, and how it is related to the regulation of mtDNA replication/transcription; (4) the dynamics of mitochondrial metabolic enzymes found in the nucleoid.

Mitochondrion not only is the cellular powerhouse but also acts as a critical trigger of programmed cell death and maintains the homeostasis of metabolites and calcium. Mitochondrial abnormalities typically have manifestations in brain, eye and muscle, and are thus termed mitochondrial encephalomyopathies. For example, patients with MERRF syndrome (myoclonic epilepsy associated with ragged-red fibers) suffer from ataxia, epilepsy and myoclonus. On the other hand, LHON (Leber hereditary optical neuropathy) is related to optic atrophy. However, mitochondrial diseases have genetic and clinical complications, thus the symptoms of a particular disease may different between individuals even if they are members of the same family. This phenomenon can be partly explained by the differences in mitochondrial loads and threshold effects among different tissues. While there is hundreds of nucleoids located within a single mitochondrion, the exact number of mitochondria and nucleoids varies from tissue to tissue. A nucleoid possibly contains 2-15 mtDNA molecules, and pathogenic mtDNA molecules that contain mutations may be transmitted and accumulated in daughter mitochondria. As the number of pathogenic mtDNA goes beyond a threshold, mitochondrial dysfunction becomes apparent. Clarifying the dynamics of nucleoid composition may help us understand the fate of pathogenic mtDNA (e.g. transfer and repair) and its relevance to the onset of mitochondrial abnormalities.

Mitochondria constantly fuse and divide in response to cellular requirements, and the nucleoid may also change to adjust mtDNA distribution. Recent studies have found that mitochondrial nucleoid contains some metabolic enzymes in addition to conventional DNA transaction proteins. However, the physiological significance of these metabolic proteins locating at the nucleoid remains to be clarified. It is tantalizing to suggest that these enzymes may be involved in mtDNA regulation coupling with their metabolic functions. In this proposed project, we plan to characterize the protein components of mitochondrial nucleoid by using multidimensional liquid chromatography (MDLC) and ESI MS/MS. In addition, the protein-protein interactions between nucleoid components are analyzed by blue-native/SDS 2D PAGE in combination with MALDI MS analysis. The database of nucleoid composition and protein interaction network can be established from the above results (Figure 1). To further investigate the extent to which the metabolic proteins may affect nucleoid integrity, we (1) manipulate the expression of selected enzyme with siRNA, and then analyze the nucleoid constituents as well as the mtDNA stability. Alternatively, we (2) profile the changes in nucleoid composition and its protein interaction network under oxidative stress.

To examine the roles of nucleoid proteins with the relevance of mtDNA transaction, we analyze mtDNA fragments bound by the protein complexes. Digested by DNaseI, the mtDNA sequences in complex with nucleoid proteins are fractionated by sucrose gradient centrifugation and analyzed by PCR sequencing. Here we are interested in the fractions that contain consensus sequences, which may include the mtDNA non-coding region and G-quadruplex-forming sequences. We in turn analyze the nucleoid proteins in the fractions of interest by immunoblotting, and compare the results with those from 2D electrophoresis. We may then specify any metabolic protein involved in nucleoid regulation for further analysis. It is of special interest to identify members of AAA family (ATPase-associated with various cellular activities), a class of multifunctional proteins that possibly regulate mtDNA replication/transcription in response to cellular demands.

Finally, we may also choose a nucleoid protein of interest (identified from the above mentioned methods) for investigating its functional dynamics in a mitochondrion. Here we take hLon as an example. By expressing the plasmid containing hLon fused with fluorescence protein in cultured cells, the dynamics of the fluorescent hLon can be observed by using FCS (fluorescence correlation spectroscopy). We can compare the differences in diffusion coefficient and retention time among selected subensembles in mitochondria (Figure 2). As a motivation to study the structural dynamics of mtDNA-binding proteins, we label mtDNA with SYTOXR Blue and examine interactions between mtDNA and the fluorescent hLon in a separate experiment using FRET-FLIM (fluorescence resonance energy transfer coupled with fluorescence lifetime imaging microscopy). Taken together, this proposal is aimed to address genetic and age-related mitochondrial abnormalities by the way of better understanding the connections between the mt-nucleoid proteins dynamics and metabolic cues.





Figure 1. Expected result of nucleoid composition and the interaction network of these proteins



Figure 2. Scheme of human mitochondrial Lon (or other specified protein) dynamics in mitochondria. FCS stands for fluorescence correlation spectroscopy; D means diffusion coefficient and τdiff corresponds to diffusion time. FRET-FLIM is the abbreviation for the instrument of fluorescence resonance energy transfer coupled with fluorescence lifetime imaging microscopy, where τφ is fluorescence phase lifetime, τm is fluorescence modulation lifetime, Da is the diffusion constant of FRET acceptor, τr is its diffusion time, and the time constants are obtained by fitting of the relative quantity of donor (or receptor) along the time axis.

Selected Conference Paper

5th East Asian Biophysics Symposium, Okinawa, Japan (2006)
Chen SH and Wu SH, “Thermodynamic Characterization of DNA-binding Activity of Human Lon Protease”

Research Statement

SUMMARY OF GRADUATE RESEARCH AND CURRENT PROJECTS

The focus of my research in Dr. Shih-Hsiung Wu’s lab is a mitochondrial protein called Lon protease, a member of the AAA+ (ATPase-associated with various cellular activities) class of multifunctional proteins. To obtain a clone of human Lon (hLon) I contacted Dr. Carolyn Suzuki, an assistant professor at the University of Medicine and Dentistry in New Jersey. She has since become my second mentor, helping me organize my experimental results into a research paper that was published in Nucleic Acids Research early this year (http://nar.oxfordjournals.org/cgi/content/abstract/gkm1140v1?ck=nck).

Previous project: Biophysical Characterization of Lon Protease and Its DNA-binding Activity

Lon has an indispensible role in mitochondrial metabolism: it degrades abnormal polypeptides as well as certain regulatory proteins and metabolic enzymes, acts as a chaperone, and binds to nucleic acids. However, our understanding of its physiological importance is obscured by its multiple duties. For instance, does Lon bind to mtDNA for replication regulation or for substrate targeting? To determine its sequence specificity for mtDNA binding, I conducted a study of hLon binding to G-quartet-forming oligonucleotides (GFOs) that are identical to the H-strand of mtDNA. A combination of electrophoretic, thermodynamic and spectroscopic data revealed that hLon is not only favorable to sequences that form G-quadruplexes (as compared to double helical and single stranded DNA), but also differentiates among GFOs through different levels of thermal fluctuation reduction. hLon binding to DNA is accompanied by little or no significant change in heat capacity for all except one GFO: a sequence that overlaps mtDNA regulatory region. In sum, our results indicate that hLon recognizes its cognate target(s) by sequence-dependent structural tightening.

Ongoing projects: Proteomics and Interactomics of Mitochondrial Nucleoid & Structural Profiling of Mitochondrial DNA

The fundamental question to Lon is whether it serves as a sensor of environmental stress: for example, a shift from degrading mt-matrix proteins to regulating mtDNA replication/transcription in response to metabolic cues. If true, we may expect its dynamic localization between the matrix and mt-nucleoids within a mitochondrion. My primary job is to investigate the involvement of hLon in mt-nucleods and to discover its regulatory partners as well as substrates.

The basic idea is to separate groups of protein complexes within mt-nucleoids (isolated from rat livers at different ages) in the first step of 2D blue native/SDS PAGE, and the protein components of each group are resolved in the second dimension. We use in-gel digestion and MALDI MS to analyze the resolved spots. In parallel, the purified nucleoids treated by DNaseI are fractionated by sucrose gradient centrifugation, and the bound mtDNA fragments in each fraction are identified by cycle sequencing using appropriate fluorescein-labeled primers. For comparison with the data obtained from 2D PAGE, the sucrose-fractionated protein complexes are analyzed by LC and ESI MS-MS. The experiments to this stage are designed to clarify the interaction network of the nucleoid proteins and their target sequences. As for the real-time observation of a specific nucleoid protein of interest in mitochondria, I invite you to read my proposal at http://niwhu.blogspot.com/2008/10/proposal-summary.html.

On the other hand, I went on to follow up G-quartet formation on mtDNA, a possible scenario that we proposed in the NAR article. I am particularly interested in the connections between mtDNA G-quadruplexes and mt-nucleoid organization, as well as their influences on mtDNA integrity and mitochondrial metabolism. Our experimental scheme is to analyze mtDNA replicative intermediates in association with G-quartet fluorescent markers (e.g. porphyrin or carbazole derivatives) using 2D agarose gel electrophoresis and blotting.

UNDERGRADUATE RESEARCH EXPERIENCE

I learned how to do hands-on research at Dr. Chia-Ching Chang’s molecular biophysics laboratory in my sophomore year at National Dong Hwa University. I was given the responsibility of cloning metallothionein and studied molecular dynamics using Insight II (Accelrys Software Inc.). During vacations I was an intern at the Institute of Chemistry of Academia Sinica under the supervision of Dr. Lou-Sing Kan. There I used thermodynamic and kinetic approaches to study physicochemical properties of triplex DNA formation. I wrote my own grant proposal, and received a research fellowship from the Republic of China National Science Council (NSC). This became my senior undergraduate thesis project: an independent investigation of pyrimidine motif triplex formation that earned an NSC Research Creativity Award. The conference papers that I wrote won financial support for my attendance at the 4th East Asian Biophysical Symposium in Taipei and the 48th Annual Meeting of the Biophysical Society in Baltimore.

Curriculum Vitae

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Curriculum Vitae SHChen

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