slide1 n.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
如何建構一個簡單的假說 PowerPoint Presentation
Download Presentation
如何建構一個簡單的假說

Loading in 2 Seconds...

play fullscreen
1 / 73

如何建構一個簡單的假說 - PowerPoint PPT Presentation


  • 142 Views
  • Uploaded on

如何建構一個簡單的假說. 賴明德 成功大學醫學院基礎醫學研究所 2004/10/01. Outline. Part I 科學家 Part II 假說 Part III 實驗設計 Part IV 研究計劃 . 成功科學家之條件. (1) motives: strong motive for science, or success. (2) diligence: hard working for competition (3) persistence: endure failure

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about '如何建構一個簡單的假說' - kipling


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide1

如何建構一個簡單的假說

賴明德

成功大學醫學院基礎醫學研究所

2004/10/01

outline
Outline
  • Part I 科學家
  • Part II 假說
  • Part III 實驗設計
  • Part IV 研究計劃
slide3
成功科學家之條件

(1) motives: strong motive for science, or success.

(2) diligence: hard working for competition

(3) persistence: endure failure

(4) careful observation: the detail determine

(5) intelligence: independent judgment, creativity

(6) collaboration: art is I, science is we.

scientist and business man
成功優秀的科學家(scientist)and 成功優秀的商業家 (business man)
  • 對事業的強烈熱情 (motive)
  • 認真努力不眠不休 (diligence)
  • 細心工作不輕忽細節 (careful)
  • 持之以恆不畏艱難 (persistence)
  • 仔細判斷掌握機會 (intelligence)
  • 與人合作共謀成功 (collaboration)
  • Then, What are the differences between…?
the difference
The difference
  • 對﹝科學發現﹞之熱誠或﹝經營商業﹞之熱誠之不同。
  • 能否在科學界成功或不成功,決定在科學界更細緻的操作流程。
a model for scientific process
A model for scientific process

Students

Post-doctor

Propose

hypothesis

?

Grant

funding

Perform

experiment

Principal Investigator

Analyze

result

Writing paper

1. 指導教授及學生在各部份所扮演之角色?

2. 研究假說及研究方向如何決定?

interactions between pi other pis and students
Interactions between PI, other PIs, and students

Students

Post-doctor

Propose

hypothesis

Other

PIs

Grant

funding

Perform

experiment

Principal Investigator

Analyze

result

Writing paper

part ii

Part II

假說

are all experiments based on hypothesis
Are all experiments based on hypothesis?
  • Hypothesis-based experiments
  • Non-hypothesis experiments
comments on some non hypothesis experiments
Comments on some non-hypothesis experiments
  • 研究科學不是拿一大堆數據作統計分析,認為這是素樸的實証分析。科學並不是一個水桶接受外面的水而後自然形成知識。科學如Karl popper所言是conjecture and refutation。所以科學應是反覆建構所形成的思想系統。
  • 但台灣的學者卻是複製外國的方法研究台灣類似之現象。無前因無後續。
  • 所以科學研究必須有一連串之理論基礎。

─黃光國

slide11
非假說型實驗之必要性
  • The basic study of E. coli system lead to the development of recombinant DNA.
  • Knockout mice experiments lead to important scientific developments.
  • Structural study or structure-function study are mostly non-hypothesis based.
  • Assay of MAPK, JNK, ERK pathways, systematic analysis.
hypothesis versus descriptive
Hypothesis versus Descriptive
  • It would be wrong to make too much of discoveries. The greatest advances in molecular biology have grown out of the intent and unrelaxing study of characteristics of a single biological phenomenon or a single biological “system”, such as the genetic in E. coli and human.
when and how to set up a hypothesis
When and how to set up a hypothesis
  • Philosophical considerations:
  • Keep the importance of your experiments in mind: it is important to remember why you are doing what you are doing.
  • Be a critical thinker: The theories behind experimentation are not carved in stone, and there are several ways to explain how and why experiments should be done.
  • Remember that you must publish the results of your experiment: it is very easy to drift into other areas, and part of research is following new avenues. But exploration must be done rigorously, and not by default.
how to set up a hypothesis
How to set up a hypothesis
  • 科學研究必須有理論基礎。
  • 假說必須根據 Our preliminary data AND other’s published results.
  • 假說是由想像力而來但並非 純想像
  • 假說並非憑空單獨存在之理論,而是衍繹過去之理論並發展未來之理論。
how to find an idea
How to find an idea?
  • Observation is the key element.
  • 醫師:病人,病人家族,病歷,文獻報告,參加研討會,和同儕討論;基礎醫學教師合作。
  • 基礎學者:實驗觀察,文獻報告,參加研討會,和同儕討論;和醫師討論。
a creative idea
A creative idea?
  • It is hard to discuss how to have a creative idea.
  • Creative idea is for the “prepared mind”, the mind of scientist must be on the right wavelength.
  • Creative Idea: (1) create a new concept, for example, apoptosis, (2) create a new research approach, for example, PCR, (3) create a new question, smoking and immune.
  • We will discuss how to have a normal idea in scientific research.
normal sciences puzzle solving
Normal Sciences: Puzzle-solving
  • 科學認識的發展過程,是把原有的理論吸收到範圍更大內容更多的理論之中。科學有如寄居蟹,定期脫掉一層殼,然後換上一層更大的殼。
  • 事實可能是事實,但是知識的主要工具是模式。模式把諸多的事實結合在一個結構裡。設想一片有些散亂黑點的平面,看不出圖案,但再多增加些點,便有人能提出模式,預測其他更多點之位置。
  • —Thomas Kuhn 之說法
how to construct a hypothesis
How to construct a hypothesis?
  • 自然界通常用相似的方式達到相似的結果,例如:similar structure between neuromuscular junction and dendritic-cell-T-lymphocyte junction (agrin in the lymphocyte and NMJ)
  • 自然界之絕大多少結構或生命現象,都有其﹝功能﹞存在,例如:RNA is single-strand, replication always start with a primer, the endoplasmic reticulum structure.
  • 從較簡單的型式的生物體或問題去了解,例如:Drosophila, slime mold, yeast.
correlation or cause effect
Correlation or Cause-effect?
  • Hypothesis-based idea: to establish (a) correlation or (b) cause-effect relationship. Most ideas belong to this type.
example 1
Example 1
  • Oncogene. 2004 Jun 17;23(28):4856-61
  • Epidemiological studies indicate that vitamin D3 deficiency is linked with increased risk of development of several types of cancer including breast, prostate and colon.
  • Inhibition of cell growth by vitamin D3 are mediated through p21Waf-1 and p27Kip1
  • The mechanism by which vitamin D3 activates p21Waf-1 has been characterized. A functional VDRE was identified in the p21Waf-1 promoter and was shown to be involved in the induction of p21Waf-1 by vitamin D3.
example 1 1
Example 1-1
  • Recent study showed that vitamin D3 stimulated transcription of p27Kip1 via Sp1 and NF-Y transcription factor binding sites localized within the -555/-512 region of the promoter. Since no canonical VDRE was identified in the p27Kip1 promoter, the authors concluded that VDR was not required for the induction of p27Kip1 by vitamin D3.
example 1 2 rationale
Example 1-2 Rationale
  • Recent studies demonstrate that nuclear receptors, instead of direct binding to the response element, may interact with other transcription factors to activate gene transcription. For example, 17-estradiol (E2) induces the formation of estrogen receptor (ER)/Sp1 complex and activates gene expression via the GC-rich Sp1 site.
example 1 3 hypothesis
Example 1-3: Hypothesis
  • Because most of vitamin D3-sensitive cancer cells express functional VDR, we hypothesize that vitamin D3 may stimulate the interaction between VDR and Sp1 to activate p27Kip1. In this study, we test this hypothesis and our results support the notion that VDR is involved in vitamin D3-induced p27Kip1 expression.
example 1 4
Example 1-4

重要性:

  • Vitamin D deficiency  cancer
  • Vitamin D  turn on p21 and p27 cell cycle inhibitor
  • Vitamin D  p21 and p27  repress cancer cell growth

問題:

Vit D  Vit D responsive element on p21 promoter  p21

Vit D  ? (No vit D responsive element)  p27

類比

Vit D  SP1 and NF-Y element in p27 promoter  enhance p27

Estradiol  estrongen receptor/SP1  on SP1 site  activate c-fos

Hypothesis: Vit D  vitD receptor/SP1  on SP1 site  enhance p27

example 2 endophilin b1 is required for the maintenance of mitochondrial morphology
Example 2: Endophilin B1 is required for the maintenance of mitochondrial morphology
  • J. Cell Biol. 2004 166: 1027-1039.
  • The dynamin family of large GTPases has been implicated in the regulation of membrane scission at several subcellular locations.
  • Additionally, several cofactors including the BAR (Bin-Amphiphysin-Rvs)-domain proteins, amphiphysin and endophilin I, are necessary for vesicle formation and completion of dynamin-mediated endocytosis
  • Inhibition of endophilin I leads to formation of invaginated membrane intermediates that are arrested before vesicle constriction and scission, suggesting a critical role of this protein in the vesicle fission step.
example 2 1 hypothesis based on location
Example 2-1 Hypothesis based on location
  • Endophilin B1 can directly interact with Bax, a proapoptotic protein that has been shown to translocate to mitochondria and coalesce into foci colocalizing with the proteins known to regulate mitochondrial dynamics, Drp1 and Mfn2. As mitochondrial activation of Bax is temporally linked to changes in OMM properties, as well as to the inhibition of the mitochondrial dynamics and mitochondrial fragmentation,
  • Hypothesis: We have speculated that endophilin B1 may participate in the regulation of the mitochondrial network maintenance.
example 3
Example 3
  • The Journal of Immunology, 2003, 170: 528-536
  • Der f may possess certain properties that are prone to induce allergic airway inflammation.
  • Dermatophagoides pteronyssinus binds to human lung surfactant protein A and D, which are members of the collectin family of the C-type lectins. The binding was abrogated after deglycosylation, suggesting that carbohydrate moiety may be involved in the interaction.
  • In asthmatic subjects, the allergic airway inflammation was associated with functional changes of Alveolar Macrophages (AMs), including an altered phenotype pattern.
example 3 1
Example 3-1
  • Because Alveolar Macrophages (AM)s express a large number of surface receptors, including those that have specificities for sugar residues commonly found in nonmammals such as bacteria and fungi.
  • Hypothesis: We assume that AMs may be involved in early recognition of HDM allergens after inhalation into the lung.
  • Der f  allergy; Der f  require sugar residue
  • AMs allergy; AMs have receptor for sugars.
example 4 1
Example 4-1

Pre-S1 Pre-S2 S

HBsAg

Observation and rationale:

Type II GGHs were characterized

by the clustering distribution of

Ground-glass hepatocytes.

Clonal proliferation

Pre-S2 region deletion is important

For carcinogenesis.

example 4 2 hypothesis
Example 4-2: Hypothesis
  • Deletion of Pre-S2 region may render HBsAg into a potential oncogene.
example 5 a b c type hypothesis
Example 5: A-B-C type Hypothesis

Over expression Her2/neu  Drug resistance

Her2/neu  Akt  Drug resistance

(A) (C)

Mutant p53  drug resistance

(B) (C)

Hypothesis: Neu  inactivate p53  drug resistance

(A) (B) (C)

HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2

Phosphorylation. Nat. Cell Biol. (2001) 3: 973-981.

example 6 a b c type hypothesis
Example 6: A-B-C type hypothesis

Endopasmic reticulum stress  carcinogenesis

(A) (C)

COX-2  carcinogenesis

(B) (C)

Endoplasmic reticulum stress  COX-2  carcinogenesis

(A) (B) (C)

example 7 a b c type hypothesis
Example 7: A-B-C type hypothesis

Endopasmic reticulum stress  NF-kB

(A) (B)

NF-kB  COX-2

(B) (C)

Endoplasmic reticulum stress  NF-kB  COX-2

(A) (B) (C)

rationale
Rationale
  • Should a good hypothesis have a strong rationale?
  • It depends on the following parameters: (1) the importance of the hypothesis (2) the expandable knowledge from the hypothesis
  • What is the basis for the judgment of the strength of rationale? The answer may be: The predictability of the hypothesis.
  • Compare Example 6 and example 7
weak or strong rationale
Weak or Strong Rationale
  • A hypothesis, which has strong rationale, is usually less creative.
  • Therefore,新概念之產生是靠想像力, 但最重要的是檢驗。
slide37
易受爭議之假說
  • 背景資料不完整:有些人不管已有理論,憑自己常識,﹝大膽假設,小心求證﹞,隨意編出幾條假設,便大作其實證研究。建構一些﹝前不見古人,後不見來人﹞的本土性裡論─黃光國、朱敬一
  • 論證不仔細,未全面討論所有可能性。
  • 若假說成立,其廣泛性可能有問題
for example 7
背景資料不完整: For example 7
  • Reviewer comment: Patel et al (J. Immunol. 163: 3459, 1999) actually showed that tunicamycin repress COX-2 expression. Zhang et al. (Biochem. J. 312:135, 1995) also showed that Cox-2 is downregulated when treated with tunicamycin, an ER stress inducer.
for example 4
論證不仔細: For example 4
  • The development of carcinogenesis may be due to either
  • enhancing cellular oncogenesis or
  • decreasing immunological response

The Pre-S2 region contain CD8 T cell epitopes.

for example 6
廣泛性可能有問題: For example 6
  • Reviewer comment: Overexpression of P450 has been shown to induce ER stress. Does this mean that P450 is an oncogene?
slide41
研究假說之延伸及細緻化 (I)
  • 重要性之延伸
  • Cancer cell (2002) 2: 323-334
  • Deregulated b-catenin is involved in oncogenesis. In this study, we found that b-catenin can physically complex with NF-kB, resulting in a reduction of NF-kB DNA binding, transactivation activity, and target gene expression. Importantly, activated b-catenin was found to inhibit the expression of NF-kB target genes, including Fas and TRAF1.
slide42
研究假說之延伸及細緻化 (II)
  • Furthermore, a strong inverse correlation was identified between the expression levels of b-catenin and Fas in colon and breast tumor tissues, suggesting that -catenin regulates NF-kB and its targets in vivo. Thus,b-catenin may play an important role in oncogenesis through the crossregulation of NF-kB.
  • .
slide43
研究假說之延伸及細緻化 (III)
  • 延伸性知識增加
  • Loss of Bik, a BH3 domain protein, was observed in colon and breast cancer
  • Nonviral delivery of Bik had therapeutic effects on xenografts of human breast cancer cells.
  • Phosphorylation of threonine 33 and serine 35 to alanine are required for its full apoptotic function.
slide44
研究假說之延伸及細緻化 (IV)
  • In the light of these findings and because of our continuous effort to search for a more potent gene for cancer therapy, we have mutated the residues threonine 33 and serine 35 of Bik to aspartic acid to mimic phosphorylation to create potentially constitutively active forms. We found that these Bik mutants enhanced their association with Bcl-2 and Bcl-XL. Moreover, these mutants exhibited a greater apoptotic activity in vitro and in antitumor activity in an in vivo animal model than wild-type Bik.
  • Cancer Res (2003) 63, 7630–7633
part iii

Part III

實驗設計

how to design experimental approaches from an idea
How to design experimental approaches from an idea?
  • Define the question: an experiment should address a hypothesis by answering one ortwo specific questions. Be sure not to pick questions that it is not possible to answer.
set up an experiment
Set up an experiment
  • 將問題以明確陳述句表示。
  • 試用圖解表示問題。
  • No experiments should be undertaken without a clear preconception of the forms its result might take. (you will include adequate control and have well-designed experiment by this way)
  • Design the experiment: experimental variables and controls.
controls
Controls
  • There are two types of controls: experimental control and treatment control.
  • Treatment controls are positive and negative, and show you whether the experimental handling of the cells has elicited an effect.
  • Experimental controls tell you if the basic experimental procedure are working properly. For example, transfection efficiency measured by LacZ cotransfection, or E. coli transformation efficiency.
variables n n 1 rule
Variables: N + N-1 rule
  • Based on the tolerable marginal error to determine (1) how many variables will be tested. (2) how much you can trust authority.
  • Nat Rev Mol Cell Biol. 2004 Jul;5(7):577-81 . Excessive trust in authorities and its influence on experimental design.
excessive trust in authorities and its influence on experimental design
EXCESSIVE TRUST IN AUTHORITIES AND ITS INFLUENCE ON EXPERIMENTAL DESIGN

Tung-Tien Sun

refute hypotheses
Refute hypotheses
  • Experiments are often designed not in such a way as to prove anything to be true-a hopeless endeavor-but rather to refute a null hypothesis. -Karl Popper
hypothesis and experiment
Hypothesis and Experiment
  • Fact: In eucaryotic cell, the specific transcription factors can bind enhancer at a distance (may be 100 bp to several kb) to activate the transcription on the promoter of a gene.
  • What is the mechanism of enhancer binding protein-induced transcriptional activation?
  • Models: (1) bind and walk (2) topoisomerse binding site (3) direct bind by DNA looping
  • Experiment design to prove --> DNA looping and direct contact model?
refute a null hypothesis
Refute a null hypothesis
  • Basic knowledge: 10 bases as a turn in B-DNA --> the spacing intervals between promoter and enhancer will affect the fold of activation in short distance.
  • Design a promoter and an enhancer with a 5p distance interval.

enhancer

55 bp

60 bp

50 bp

100%

60%

98%

promoter

slide56
假設在科學研究中之角色
  • 不要不停的創造假設,倒是要努力發現新事實因為新事實是永遠存在的。
  • 英國哲學家卡萊爾說:給我一個事實,讓我在這個事實前面頂禮、膜拜吧!
part iv

Part IV. 研究計劃

Nancy Rothwell

Professor at University of Manchester

MRC Professor

slide58
研究提案
  • 重要性
  • 新奇性
  • 時效性
  • 可行性
  • 明確性(組織結構)
slide59
重要性
  • 把研究提案放在一個〝大場景〞去看。
slide60
假說型實驗或敘述型實驗
  • 一般而言,假說型實驗較受喜歡。假說型實驗之決定點在於〝新穎性〞及〝可行性〞。但這兩者常是相互矛盾,所以通常會用〝混合型〞。
  • 敘述型實驗,不管有些科學家喜不喜歡,是很重要,常是重要科學發現之基礎。敘述型實驗常是用來產生假說,所以在描述〝敘述型實驗〞,應再說明可能產生之假說及如何進一步深入探討。或在敘述型實驗中加入〝重要性〞及〝比較性〞。例如:N’-neu-IL-2, N’-neu-IL-4, N’-neu-GM-CSF.
slide61
時效性
  • 研究結果和最近科學文獻新知或社會事件之關聯性。
slide62
可行性
  • 不可因第一個假說被否定,便全盤皆墨。須有 Back-up hypothesis, back-up experimental approaches.
  • 技術之可行性:Some preliminary results are preferred.
slide63
明確性
  • 目標明確
  • 撰寫方式明確:可讀性及清晰度
references
References
  • “Advice to a young scientist” by Peter Medawar, 1981.
  • “Advice to a young scientist” by Santiago Ramon y Cajol, 1898. 中譯本by程樹德,究竟出版社。
  • “Who wants to be a scientist” by Nancy Rothwell. 中譯本:科學家的第一堂課,陳佳伶譯,早安財經文化有限公司,2004。
slide65
如何作好科學研究
  • 研究之成功應決定於如下之因子:
  • 強烈動機 所以

(1) 作自己有興趣的事

(2) 將每一件被指定的事作好

(3) 渴望成功

(4) 為國家民族人民之利益

slide66
如何作好科學研究
  • 實驗之目標正確

(1) 研究重要問題

(2) 此問題可被目前科學實驗系統解答

(3) 目標必須精確

(4) 大多數好的實驗是研究一個假說之真偽。

  • 研究重要問題
slide67
如何作好科學研究
  • 實驗之方法合理
  • 適當之positive control and negative control (experimental and treatment control);實驗之結果必須對假說之可否(yes or no)能區別。
  • Use multiple approaches to study a phenomenon: For example: western, immunohistochem, RT-PCR to prove the increase of the expression of the target gene.
  • 了解實驗步驟之原理:在幾個不同實驗方法中為何選擇此系統,又如細胞株之選擇,DNA轉染之時間 。
slide68
如何作好科學研究
  • 仔細注意細節,The devil is in the detail.
  • 實驗細節之精密度:精確的注意可控制之因子,例如試管溶劑之殘留或混合均勻。儘可能調整實驗條件使實驗在可重覆狀況下。
  • 仔細觀察(careful observation)及仔細分析數據(careful analysis of experimental data)
what is the unique point of one scientist
What is the unique point of one scientist
  • 在廣告用語中有 獨特性銷售主張法 (unique selling proposition, USP)
  • 在管理學中也有 SWOT (strength, weakness, opportunity, threat)
  • 尋求並設定自我在眾多科學家中之定位。
comment
Comment
  • 多我們不為多
  • 少我們不為少

─楊國樞、文崇一

some comments
Some comments
  • 有些人不管已有理論,憑自己常識,﹝大膽假設,小心求證﹞,隨意編出幾條假設,便大作其實證研究。建構一些﹝前不見古人,後不見來人﹞的本土性裡論。
  • 有些人乾脆連假設都不要了,同時測量一大堆變項,希望呈現﹝事實﹞,﹝讓數字自己說話﹞。有了統計軟體,更以為只要用複雜的統計數字,便可以從中浮現﹝理論模式﹞。

─黃光國、朱敬一

some comments1
Some comments
  • All science is concerned with the relationship of cause and effect. Each scientific discovery increases man’s ability to predict the consequences of his actions and his ability to control future events. – Laurence J. Peter
  • Science is facts; just as houses are made of stones, so is science made of facts; but a pile of stones is not a house and a collection of facts is not necessarily science. -- Henri Poincare
some comments2
Some comments
  • Science commits suicide when it adopts a creed. –Thomas Henry Huxley
  • No amount of experimentation can ever prove me right; a single experiment can prove me wrong. – Albert Einstein