Professor FUNG Ming-Chiu Department of Biology The Chinese University of Hong Kong

1. Scientific Research:Principlesand Methods Professor FUNG Ming-Chiu Department of Biology The Chinese University of Hong Kong mingchiufung@cuhk.edu.hk

2. What is scientific research? Scientific research is an investigation of a phenomenon (addressing a question) by scientific method.

3. Discovery ? or Invention ?

4. Story of Vaccination

5. Smallpox Smallpox Cowpox

6. Edward Jenner - 1798 Vaccine - Vacca mean “Cow” “Mother of Vaccination” ?

7. Invention Discovery One B cell one antibody (Nobel Price in Physiology or Medicine 1984, Niels K. Jerne, Georges J.F. Köhler and César Milstein) Monoclonal antibody DNA double helix structure (Nobel Price in Physiology or Medicine 1962, James D Watson and Francis Crick) Southern Blot Northern Blot DNA microarray DNA replication (Nobel Price in Physiology or Medicine 1959, Arthur Kornberg) DNA Sequencing (Nobel Prize in Chemistry 1980, Frederick Sanger) PCR (Nobel Prize in Chemistry 1993, Kary B. Mullis)

8. What type of research are you going to do? Discovery? Invention? Addressing a question? A question is raised by yourself A question has not been answered for long time (every scientist want to get the answer)

9. Discovery Unexpected results Discovery of post-transcriptional gene silencing In 1990, two research groups, Richard Jorgensen and Joseph N.M. Mol, introduced CHS gene into petunia. Chalcone synthase (CHS): The key enzyme in pigment biosynthesis

10. Experimental expectation Introduce CHS (pigment enhancing gene)

11. Experimental result Introduction of a CHS transgene inhibits anthocyanin pigmentation

12. RNase protection analysis The expression of endogenous and introduced CHS genes was co-suppressed Introduced CHS Endogenous CHS Comparison of steady-state CHS message levels in Violet and white flowers from transgenote

13. Discovery Invention RNA interference (Nobel Prize in Physiology or Medicine 2006, Craig Mello and Andrew Fire) shRNA siRNA Who made the first observation of RNA interference?

14. RNase protection analysis Wrong analysis: allele interaction, gene methylation The expression of endogenous and introduced CHS genes was co-suppressed Introduced CHS Endogenous CHS Comparison of steady-state CHS message levels in Violet and white flowers from transgenote

15. Discovery -- Logical deduction Life cycle of Schistosome The host fails to elicit any marked tissue response against the skin-residing schistosomulae. This reduced tissue response is evident only in live parasites infecting native host. Dead parasites can elicit a marked inflammatory response. A bird schistosome (T. ocellata ) often results in severe dermatitis in humans.

16. ES depleted with Sm16 Sm16 The discovery of Sm16 in S. mansoni Figure.intracellular levels of IL-1ra in human neonatal keratinocytes 72 hr after in vitro stimulation with: ES, Sm16 (a), ES depleted with Sm16 (b), a + b, media (control) Sm16 should be responsible for the anti-inflammatory effect of ES products of S. mansoni. Ramaswamy, B, et al. Journal of inflammation. 1996,46:13-22

17. Discovery By accident Wall paint (乳膠漆) Spoiled milk was accidentally pour into calcium oxide

18. Logical deduction 1 2 3 4 5 8 9 6 7 10 11 12 13 14 15 16 17 18 19 20

19. Logical deduction 1 2 3 4 5 8 9 6 7 10 11 12 13 14 15 16 17 18 19 20 Scientific Research needs Curiosity Keen observation Correct interpretation Imagination Luck

20. RNase protection analysis Wrong analysis: allele interaction, gene methylation Biased by the central dogma / past experiences The expression of endogenous and introduced CHS genes was co-suppressed Introduced CHS Endogenous CHS Comparison of steady-state CHS message levels in Violet and white flowers from transgenote

21. Against the central dogma

22. Mad cow disease found in Plurenden Manor farm, April 1985 1986, found in other far away farms

23. Bovine Spongiform Encephalopathy BSE (mad cow disease)

24. Scrapie-associate fibrils under electron microscope Patricia Merz, 1978

25. Stanley B. Prusiner glutamate metabolism research scrapie research 1982, purified the prion protein Proposed scrapie is caused by a protein but not a virus Nobel Prize in Physiology or Medicine 1997

26. Key discoveries Prion protein -- cause scrapie disease The infective agent (Prion) is a protein alone The prion protein sequence  leading to find the prion gene

28. Break for 5 minutes

30. Major Components of Science • Inquiry: Why? How? What? • Research: how to get the answer • Experiments, observations, data analyses, reasoning, etc.

31. Four Bold Claims of Scientific Investigation • Rationality • I hold belief X for reason R with level of confidence C, where inquiry into X is within the domain of competence of method M that accesses the relevant aspects of reality. • e.g. “I believe what my physics teacher taught is correct because I like him/her” versus “I believe what my physics teacher taught is correct most of the time because the contents are coming from the most current edition of a physics textbook. I have read the book and compared it with my notes….”

32. Four Bold Claims of Scientific Investigation • Objectivity • Knowledge on an object, not a subject or knower; e.g. Dr. Fung is the speaker of this talk (whether you like this talk or not will not change the identity of the speaker) • Verifiable; e.g. you have never heard the sound of tree falling in a forest, did it really happen? You can set a sound recorder etc.

33. Four Bold Claims of Scientific Investigation • Realism • The correspondence of human thoughts with an external and independent reality, including physical objects • The scientific method provides rational access to physical reality, generating much objective knowledge • Does not come in degrees, either yes or no

34. Four Bold Claims of Scientific Investigation • Truth • The property of a statement corresponds with reality • Truth claims may be expressed with various levels of confidence • The price of holding onto the truth; e.g. • The story of Heliocentric Model Giordano Bruno (burned at the stake) • The story of agricultural centers and Nikolai I. Vavilov [jailed as a defender of the "bourgeois pseudoscience" (genetics)] • The story of jumping genes and Barbara McClintock (received a Nobel Price more than 30 years after her important discovery)

35. Major Steps in a Scientific Investigation Observation Question Hypothesis Set Evidence Conclusions Presuppositions + [Archive]

36. Major Steps in a Scientific Investigation Observation Question Hypothesis Set Evidence Conclusions Presuppositions + [Archive]

37. Making Important Observations is the Essential First Step • Sensitivity • e.g. the story of penicillin and Alexander Fleming • Comprehensiveness

38. Major Steps in a Scientific Investigation Observation Question Answer from literatures (?) Hypothesis Set Evidence Conclusions Presuppositions + [Archive]

39. What Kind of Questions to Ask? Investigation of unknown (basic science) • Delineating concepts related to life and nature; e.g. what are the different life forms, how life functions, and how lives interacting with each other and the environment Applying known knowledge to modify/preserve natural environment or enhance human life (applied science) Scientific concept • e.g. Using penicillin as a medicine to kill bacteria Methodology • e.g. Applying DNA fingerprinting techniques in forensic sciences, etc. • e.g. Inventing new methodology to allow better observation of the world; e.g. invention of PCR

40. Applying Logic in Making and Testing Hypothesis Observation Question Hypothesis Set Inductive logic Deductive logic Evidence Conclusions Presuppositions + [Archive]

41. Inductive Logic • From actual data to get an inferred model • Strong if its premises support the truth of its conclusions to a considerable degree, and is weak otherwise • e.g. for 100 living bacteria observed, they all are capable of doubling its DNA content during cell division; conclusion: in all bacteria, they have a mechanism to replicate DNA • Deductive Logic • From a given model to predict expected data • The truth of its premises guarantees the truth of its conclusions, and is invalid otherwise • e.g. since our model that all bacteria can replicate their DNA, we should expect to see DNA replication in bacteria #101, #102, and etc.

42. Presuppositions • Science requires several common-sense presuppositions, including that the physical world exists and that our sense perceptions are generally reliable; e.g. if you are not sure if you are real or you are just a dreaming butterfly, no science research can be done • Archive relevant knowledge Irrelevant knowledge

43. Some Common Logical Fallacies • Fallacies of Composition and Division • Na and Cl2 are poisonous; Conclusion: NaCl is poisonous • Many horses are not white, a white horse is white; Conclusion: a white horse is not a horse • False Dilemmas • My opponent’s theory is wrong; conclusion: my theory is right • Circular Reasoning • I won’t be wrong because I am always right • Fallacies of will

44. The Prediction Power of a Hypothesis Determines Its Validity • e.g. there were 2 hypotheses explaining why the neck of giraffe is long • Darwinism: mutations naturally occurred in giraffe populations; when the environment changes (less leaves close to the ground), the mutants survived better and dominated today’s giraffe populations • Prediction: mainly two kinds of giraffe fossils, long neck and short neck • Lamarck: when there were less leaves close to the ground, giraffe needed to exercise their neck and gained more muscles; this acquired ability passed onto subsequent generations • Prediction: giraffe fossils should exhibit a graduate change of neck length

45. Major Steps in a Scientific Investigation Observation Question Hypothesis Set Evidence Conclusions Presuppositions + [Archive]

46. How to Collect True Evidences (Carefully Designed Experiments and Accurately Recorded Observations) • Proper instrumentation; e.g. I. Newton decomposes light by using a prism • Careful experimental design: controls or baseline (i.e. reference points) • Accuracy of data; e.g. the story of phlogiston (Johann Joachim Becher), oxygen and Antoine Laurent Lavoisier • How to handle quantitative data (errors occur by chance): statistics; e.g. if your hypothesis is that “man is taller than woman”, it may not be always true (but can you generalize?)

47. Major Steps in a Scientific Investigation Observation Question Hypothesis Set Evidence Conclusions Presuppositions + Remark: data-driven research in post-genomic era [Archive]

48. You have to know the objective / hypothesis (purpose) of each experiment Set up all essential controls Use more advance technology

49. References • “Hypothesis, Prediction, and Implication in Biology” by J.J.W. Baker and G.A. Allen • “Great Scientific Experiments” by R. Harre • “An Introduction to the Logic of the Sciences” by R. Harre • “Scientific Method in Practice” by H.G. Gauch, Jr.