训练成为独立思考的科学家-共识与“偏见”

1、复旦大学新视野系列研究生课程：科研论文的构思、撰写和发表 复旦大学讲座教授复旦大学发育生物学研究所所长科罗拉多大学教授、HHMI研究员韩 珉主讲人下一讲：10月24日 马兰教授 1982年北京大学毕业后入选CUSBEA留美项目，1983年赴美国加州大学洛山矶分校攻读博士学位。1988年获博士学位后在加州理工学院从事博士后研究。1991年到卡罗拉多大学执教，先后任助理教授、副教授、教授。97年被遴选为著名的休斯医学研究所研究员。发现Ras途径在发育中的作用，所领导的研究组在细胞信号转导和发育方面有诸多重要贡献，在Cell, Nature, Science等刊物上发表了多篇论文并获专利2项。199

2、9年以来在复旦大学讲授细胞信号转导和发育课程，2000年获国家杰出青年科学基金，2001年与许田、庄原教授一起创建了复旦大学发育生物学研究所。韩珉教授简介训练成为独立思考的科学家- 共识与“偏见”韩珉Why do give such a talk?- As an over-achiever, I appreciate more about the good training I have received. Getting old, and on the down slope in the career, I am more interested in talking. A class at

3、Fudan Medical School训练自己尽早成为一个全面的科学家- 读研究生时做学问的方法决定了你将来科学生涯的基调- 正确的训练将帮助你在竞争好的博士后或者其他职位时优势更加明显- 只有以正确的方式进行研究才能感到科学的乐趣训练的目标是什么？ 正确的科学道德 思考科学问题和解决方法的能力 合理利用时间高效完成各种任务的能力 阅读和分析文献的能力 口头表达和确立观点的能力 撰写研究论文和综述的能力- 与他人交流合作的能力 和同事愉快相处的能力如何评价学生 推荐信和电话交谈 发表的论文- 对科研兴趣的自我表述面试How do NIH panels evaluate postdoctora

4、l fellowship applicants? Confidential letters by the advisor and other professorsRanking following aspects (1-5): - Research Ability and Potential- Write and Verbal Communications- Perseverance in Pursuing Goals- Self-reliance and Independence- Laboratory Skills and techniques- Originality- Accuracy

5、- Scientific Background- Familiarity with Research Literature- Ability to Organize Scientific DataExample of the evaluation of a good studentExample of the evaluation of a weaker studentWhat are the pre-requisites to be a successful graduate student?High GPA? Great GRE score?Wealth of your family?Ty

6、pe of undergraduate college?Fudan vs others?Rao Yis argumentMore about your college gradesGood grades in high school and college are far from sufficient. In fact, they are not even necessary. There are other important traits.Tom Cech: 1989 Noble Prize in ChemistryPresident of Howard Hughes Medical I

7、nst Professor at the University of ColoradoIt isnt always true that the people who are the brilliant high school students, who get the highest grades on the exam, are the ones who do well as practicing, experimental scientists. There are a lot of skills in doing experimental science that cant be tes

8、ted on standardized exams Cech says. B students often are the ones who end up doing the really great work in research. Example: Graig Hunter- Professor at HarvardUndergraduateGPA Oregon State University (80-81): 3.3University of Oregon (81-84):3.1Graduate Student University of Coloradosuper starPost

9、doctoral trainingUCSFsuper starProfessorshipHarvard (main campus)One of the best young guysWhat besides the grades for him?Recommendation letters from the advisor: Craig has had complete freedom in designing his own experiments and deciding overall strategy in the research he has conducted in my lab

10、. His understanding and mastery of the techniques of molecular biology and his sound scientific judgment compares very favorably with that of some of our best graduate students. I have the utmost confidence that Craig Hunter would be extremely successful as a graduate student in any.More about grade

11、sDifference between US and China Changes in US high schoolWhat are the pre-requisites to have the potential to be trained into a good scientist ?2. Being hard-working is essential, but not sufficient3. Loving what you do is important. - Motivated, but for the right reason - Good scientists often sac

12、rifice other aspects of life.1. Need to be smart. Yes. But .5. Ethical4. Having the potential to be initiative and independentGraduate school is not for everyoneHow to make sure that is what your want?Have some research experience first.Learn to address good scientific questionsThis is a major objec

13、tive of your training in graduate school. Easy to say, hard to do. Many researchers are not good at it. “small science” often demands more on this. - This is THE most important thing I learned from my thesis advisor. Learn to address good scientific questionsBig question 1: leading the field# of pap

14、ers/yeartimeABCDA: ground breakerB: leading the trendC: working in a hot fieldD: catching the tailWhat is your work?Level of the fieldsA major fielde.g. Developmental geneticsA specific areae.g. Wnt signaling in developmentA very focused fielde.g. function of wnt receptorsWhat can we learn from its

15、tradition? The “MRC”The Medical Research CouncilIn 1947 the Medical Research Council set up a Unit for “Research on the Molecular Structure of Biological Systems” to enable Max Perutz and John Kendrew to develop their work using X-ray diffraction to study proteins. The “Laboratory of Molecular Biolo

16、gy” became known simply as the“LMB”. The independence of the researchersFrancis Crick2nd studentJim Watson1st PostdocMax PerutzJohn Kendrew1st studentAll four won the Noble Prize Max Perutz John Kendrew Francis Crick James Watson Fred Sanger Cesar Milstein Sydney Brenner John Sulston Many others wer

17、e trained there: Sydney Altman, Bob Horvitz, Andy Fire .The “Culture” created by Perutz at MRC Students and Postdocs were independent. PIs did not take credit for their work. They addressed big time questions. The funding mechanism encouraged risk taking. It was an extremely stimulating environment:

18、 Eating, drinking, and talking- Many very smart and devoted people around Brenner & Benzers “ridiculous” exploratory visionSydney BrennerSeymour BenzerPhage geneticistsC. elegansDrosophilaMRCCaltechWhat was Brenners thought?In a letter to Max Perutz, June 1963“ .It is now widely realized that nearly

19、 all the “classical” problems of molecular biology have either been solved or will be solved in the next decadebecause of this, I have long felt that the future of molecular biology lies in the extension of research to other fields”“The new major problem in molecular biology is the genetics and bioc

20、hemistry of control mechanisms in cellular development”“.The great difficulty of these fields is that the nature of the problem has not been clearly defined, and hence the right experimental approach is not known” (= very risky)What did Brenner do?In 1965, Sydney Brenner chose a nematode Caenorhabdi

21、tis elegans, as a promising model animal He published his first paper in Genetics in 1974.Since then, knowledge has accumulated to the extent that C. elegans is now probably the most completely understood metazoan in terms of anatomy, genetics, development, and behavior. Sydney Brenner Bob Horvitz J

22、ohn SulstonBig Question Example 2Apply technology to study important problemsUsing temperature sensitive mutations to study how a phage is assembled step by stepFunctional map of T4 assembly genesBob Edgar addressed the question and isolated temperature sensitive mutants that disrupt each step of th

23、e assembly process in 1960s. Bob Edgar and Bill Wood collaborated on the project at Caltech, early 1970sLee Hartwell was a student at Caltech He heard about Bob Edgar Ts mutants. Harwell asked another big question: How is the cell division cycle regulated? He isolated TS mutants that disrupt various

24、 steps of the cell division cycle. Cdc mutants (1970s UC Irvine and UW)2001 Noble prize. A great genetic tool was used to attack a big time question.Common “ unattractive” projects Wanting to do gene knockout, but does not address a good scientific problem. Doing two-hybrid screen on a protein of wh

25、ich function is unknown and there is no vision about what is going to happen. Repeating studies on proteins that have been well-characterized in another organism and do not have a good idea about what are the new things to be learned. Applying an advanced technique to something for the sake of apply

26、ing the technique. Making interesting observations, but not sure how to study the mechanism or develop the story. Practice: - When reading research papers, question the questions addressed by the papers. When listening the seminars, question the questions addressed by the speakers. - Write a proposa

27、l on your research plan. “BIG” or “small”, the questions should besignificant, interesting, and solvable重要的， 你喜欢的， 实际的Writing a research proposalC. Experimental designsDescribe in detail how you would experimentally address the questions. You often need to justify what you would do. You need to cons

28、ider possible problems and alternative methods. Specific Aims1. A short paragraph to summarize the goal of the proposal2. List 2-3 specific aims. Clearly address the specific questions and state the approaches used to attack the problems.Background and SignificanceWhere the questions come from, why

29、the questions are important, and the rationale of the approach suggested. Science 1987 Oct 23;238(4826):542-5A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants.Trahey M, McCormick F.The role of guanine nucleotides in ras p21 function was determined by usi

30、ng the ability of p21 protein to induce maturation of Xenopus oocytes as a quantitative assay for biological activity. Two oncogenic mutant human N-ras p21 proteins, Asp12 and Val12, actively induced maturation, whereas normal Gly12 p21 was relatively inactive in this assay. Both mutant proteins wer

31、e found to be associated with guanosine triphosphate (GTP) in vivo. In contrast, Gly12 p21 was predominantly guanosine diphosphate (GDP)-bound because of a dramatic stimulation of Gly12 p21-associated guanosine triphosphatase (GTPase) activity. A cytoplasmic protein was shown to be responsible for t

32、his increase in activity. This protein stimulated GTP hydrolysis by purified Gly12 p21 more than 200-fold in vitro, but had no effect on Asp12 or Val12 mutants. A similar factor could be detected in extracts from mammalian cells. It thus appears that, in Xenopus oocytes, this protein maintains norma

33、l p21 in a biologically inactive, GDP-bound state through its effect on GTPase activity. Furthermore, it appears that the major effect of position 12 mutations is to prevent this protein from stimulating p21 GTPase activity, thereby allowing these mutants to remain in the active GTP-bound state.Cite

34、d 1000 timesExample 3, A more specific questionRASGDPRASGTPPiGTPGDPtargetActiveInactiveDiscovery of GTPase activating protein (GAP)Observing or reading the literatureGTPase of Ras体外weakt 1/2 30 min体内strongt 1/2 3 hours, 1000XQuestion: What is it in cells that stimulates the GTPase activity?RASGDPRAS

35、GTPPiGTPGDPtargetActiveInactiveXoncogeneGAP was discoveredGAP- Read a lot about the subjects to gain good senseHave good discussion with your advisor - make him think hard. Do not always trust your bosss initial thoughts. - Talk to other senior students (or postdocs) in the lab.Picture this: If all

36、the experiments worked without any problems, would a paper resulting from the work be good enough to be published in a significant journal? In reality, only a limited percent of projects will get the most desirable results. However, if you know from the very beginning that the best results of your w

37、ork would have no chance, dont even start!Learn to judge the significance of a problemYour major work should be published in a journal with a rigorous review process that warrants respect Do not take the impact factor too seriously.Great papers are not always published in the big journals. Not all p

38、apers in big journals are good ones. At the same time:Great papers in non-top journals - S. Brenners first worm genetics paper: Genetics (1974, 2500X) Nobel Prize 2002Sulstons worm lineage papers: Dev. Biol. (1983, 1200X; 1977, 1100X)Nobel prize 2002Jack Dixon: PTEN is a lipid phosphatase: JBC (1998

39、, 500X)About secondary citations. Question yourselfWhy are we working so hard in the lab?1. We will contribute to the science of China and world2. We are getting the training for our future.Without addressing good questions, you will accomplish neither.And you might have .Push your advisor?PIs need

40、to constantly learn (read, listen, discuss, etc) to improve themselves so that they can address important questions in their current research programs. Three factors in choosing a lab/advisor Advisor: fame, reputation, personality, training philosophy Laboratory: other people in the laboratory, fund

41、ing Projects: questions addressed, potential project for you At your university, how do students choose their labs?In US: a decision is made by both students and PIs after rotationsHow did I select my thesis laboratoryChoices:Michael Grunstein: genetics on histone functionsNice guy but unpopular lab

42、Histones were considered extremely boringSteve Clark: Protein methylation- Super nice and smart- Pure biochemistry- Significance of the work was not clear then.Arnold Berk: Mechanism of transcription regulation- the most popular professor at UCLA- Super nice and brilliant - All three invited me to j

43、oin their labs. I was the first Chinese student that ever worked in these labs.Went with the questions on histones Michael Grunstein had good questions but was still in the stage to find effective genetic approachesWhat are the fundamental cellular functions of histones and nucleosomes?Are histones

44、and their modifications relevant to transcription regulation?Why should we do genetics on histones? Biochemical roles already well known- Giants in the transcription field did not believe that histones or nucleosomes have anything to do with transcription regulationSpecific Question: What are the ce

45、llular consequences if you conditionally shut down the production of a histone gene and disrupt nucleosome formation? Gal10 promoterHistone H2B or H4Galactose: promoter onGlucose: promoter off 1000 xSimple ResultsG2 arrest, chromosome segregation disrupted. Dramatic transcription de-repression even

46、without the enhancerUASGal GluPublicationsYoshinaga, S., Dean, N., Han, M. and Berk, A. J. (1986). Adenovirus stimulation of transcription by RNA polymerase III: evidence for an E1A-dependent increase in transcription factor IIC concentration. EMBO J. 5, 343-353.Schuster, T., Han, M. and Grunstein M

47、. (1986) Yeast histone H2A and H2B amino termini have interchangeable functions. Cell 45: 445-451.Han, M. Chang, M. Kim, U. and Grunstein M. (1987). Histone H2B repression causes cell cycle specific arrest in yeast: effects on chromosomal segregation, replication and transcription. Cell 48, 589-597.

48、Han, M., Kim, U., Kayne, P. and Grunstein, M. (1988). Depletion of histone H4 and nucleosomes activates the PHO5 gene in S. cerevisiae. EMBO J., 7, 2221-2228.Kim, U., Han, M., Kayne, P. and Grunstein, M. (1988). Effects of H4 depletion on the cell cycle and transcription of Saccharomyces cerevisiae.

49、 EMBO, J. 7, 2211-2219.Kayne, P., Kim, U., Han, M. and Grunstein, M. (1988). Extremely conserved histone H4 N-terminus in dispensable for growth but essential for repressing silent mating type genes in yeast. Cell 55, 27-39.Han, M. and Grunstein, M. (1988). Nucleosome loss activates yeast downstream

50、 promoters in vivo in the absence of UAS elements. Cell 55, 1137-1145.Grunstein, M., Han, M., Kim, U., Schuster, T. and Kayne, P. (1989). Histone and nucleosome function in yeast. In Molecular Biology of Chromosome Function. Ed. K.W. Adolph. Springer-Verlag, New York.Role of histones and chromatin i

51、n transcription# of people /$/ papers/yeartimeAbout taking riskMax Perutz On Seeking an interesting scientific problem“Look for the important problem and dont be detoured if it turns out to be difficult because the important ones always are difficult. And young people now are under great pressure to

52、 produce publications, to produce results. So, they are really pushed to take on only problems which are safe, which you can answer within the time of a grant of three years. And, I think thats a sad thing. You shouldnt worry too much about how long it might take and whether it will be possible to s

53、olve the problem. Just the same as in other walks of life, in science, if you want to win, you have to take risks.” About taking a riskRisky projects may not always be that risky. Be brave and exploratory. Things will most likely work out. To be exploratory and safe: do at least two projects. Is the

54、 risk factor is often proportional to the significance of the discovery? Well, not always. It depends on the questions addressed. Conflict with our system and society We are not in an ideal world to do science; we are making discoveries and we are not allowed to take risk. We need to the quality con

55、trol of Ph.Ds; everyone got to have the paper; so there is the risk. The granting agencies and institutions are counting beans (paper #) to determine you funding, salary and promotion HHMI push its investigators the other way High risk, high reward- Gerry Rubins deletion test five papers are the key

56、 for renewal. CELL 84 : 843-851 1996. (and others from David Allis) Tetrahymena histone acetyltransferase A: A homolog to yeast Gcn5p linking histone acetylation to gene activationBrownell JE, Zhou JX, Ranalli T, Kobayashi R, Edmondson DG, Roth SY, Allis CD“Discovery of histone acetyltransferase and

57、 its role in gene activation”Histone modification and chromosome remodeling by HAT and HDAC have been the most significant advancement in the past 10 years in the transcription field.I chickened out the project because of the risk- Grunstein lab started to idenentify HAT in 1981- My first thesis top

58、ic: identifying and cloning HAT- Tried a novel method for 6 months- Felt too risky and started on second project to back it up. As soon as the second project seemed promising, I chickened out on the first one. - Had I stayed with the project . you never know. David Allis2004 Wiley Prize Winner2005,

59、US National Academy MemberMajor player in Chromatin functionsJoy and Jack Fishman Professor at RockefellerHigh risk, high reward: Linda Bucks 8 year struggle “I had tried so many things and had been working so hard for years, with nothing to show for it. So when I finally found the genes in 1991, I

60、couldnt believe it! . That was very satisfying.” Richard Axel and Linda Buck.1991 Cell paper brought them fames and 2004 Noble PrizeShould we always focus our effort on one subject?Linda Buck: Yes, but not until you know you are working on an important programBuck L, Stein R, Palazzolo M, Anderson D