如何建構一個簡單的假說

1. 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

2. Outline • Part I 科學家 • Part II 假說 • Part III 實驗設計 • Part IV 研究計劃

3. 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.

4. 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…?

5. 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。

6. 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？

7. Interactions between PI, other PIs, and students Students Post-doctor Propose hypothesis Other PIs Grant funding Perform experiment Principal Investigator Analyze result Writing paper

8. Part II 假說

9. Are all experiments based on hypothesis? • Hypothesis-based experiments • Non-hypothesis experiments

10. 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

11. 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.

12. 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.

13. 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.

14. 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。

15. 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。

16. 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.

17. 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

18. 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.

19. Correlation or Cause-effect? • Hypothesis-based idea: to establish (a) correlation or (b) cause-effect relationship. Most ideas belong to this type.

20. 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.

21. 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.

22. 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.

23. 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.

24. 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

25. 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.

26. 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.

27. 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.

28. 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.

29. Example 4: HBs Ag and Hepatoma

30. 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.

31. Example 4-2: Hypothesis • Deletion of Pre-S2 region may render HBsAg into a potential oncogene.

32. 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.

33. 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)

34. 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)

35. 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

36. 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。

37. 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

38. 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.

39. 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.

40. 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?

41. 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.

42. 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. • .

43. 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.

44. 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

45. Part III 實驗設計

46. 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.

47. 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.

48. 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.

49. 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.

50. EXCESSIVE TRUST IN AUTHORITIES AND ITS INFLUENCE ON EXPERIMENTAL DESIGN Tung-Tien Sun