basic science partnership (bsp)
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Basic Science Partnership (BSP). Matthias S. Schedl Summer program 2010. Course reading. Text: GLASS, J. D. (2007): Experimental Design for Biologists. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. - PowerPoint PPT PresentationTRANSCRIPT
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Course reading Text: GLASS, J. D. (2007): Experimental
Design for Biologists. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Supplement reading: CERF, O. et al. (2009) : Tests for determining in-use concentrations of antibiotics and disinfectants are based on entirely different concepts: “Resistance” has different meanings. International Journal of Food Microbiology 136, 247–254.
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Syllabus 4 discussions/ lectures
Individual talks from different labs
Last session of course your presentations from your lab projects
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Independent Project/Presentation Each student should create a 15 minute
PowerPoint presentation on the current lab project
Explain: 1.basic biology behind the mechanisms being
researched 2.include techniques as a methods section Your results section (optional)
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Outline for our course: Session 1
Book chapters 1-9 Session 2
Book chapter 10, plus additional paper Session 3
Book chapters 11-14 Session 4
15-19 Quiz
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This time ... Since you did not have the book so far I will cover the
first chapters today and you should read them up until next time.
Additional literature: Supplement reading: CERF, O. et al. (2009) : Tests for
determining in-use concentrations of antibiotics and disinfectants are based on entirely different concepts: “Resistance” has different meanings. International Journal of Food Microbiology 136, 247–254.
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CHAPTER 1-9Key words:
Hypothesis
Problem/Question framework
System establishment
Model building
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Philosophy of Science by Sir Karl Raimund Popper
Why do we set up hypothesis in science today? Karl Popper:
Born in Vienna, Austria worked in London, UK Schools:
Analytic Critical Rationalism Fallibilism Evolutionary epistemology Liberalism
Source: http://www.nndb.com/people/164/000087900/
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Philosophy of Science by Sir Karl Raimund Popper How did Popper developed his ideas about
science and philosophy?
Source: http://www.nndb.com/people/164/000087900/
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Philosophy of Science Sir Karl Raimund Popper
Physical world
Subjective personal perceptions
Objective abstract products of the human mind
Source: http://www.knowledgejump.com/knowledge/popper.html
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Who was Popper? He attended the local Realgymnasium Went to the University of Vienna in 1918 In 1919 Popper joined the left-wing politics,
the Association of Socialist School Students, Soon abandoned it entirely because of the doctrinaire character.
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Karl R. Popper (1902-1994) He discovered the psychoanalytic theories of
Freud and Adler, and listened entranced to a lecture which Einstein gave in Vienna on relativity theory.
The dominance of the critical spirit in Einstein, and its total absence in Marx, Freud and Adler, struck Popper as being of fundamental importance.
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Karl R. Popper (1902-1994) For Popper the critical spirit in Einstein
theory had crucial and testable implications which, if false, would have falsified the theory itself.
The total absence of critical spirit in Marx, Freud and Adler, couched in their theories in terms which made them amenable only to confirmation.
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Karl R. Popper (1902-1994) The dominant philosophical group in Vienna
at the time was the Vienna circle, the circle of ‘scientifically-minded’ intellectuals.
The principal objective of the members of the Circle was to unify the sciences, which carried with it, in their view, the need to eliminate metaphysics.
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Karl R. Popper (1902-1994) Popper became increasingly critical of the
main tenets of logical positivism. He articulated his own view of science, and
his criticisms of the positivists, in his first work, published under the title Logik der Forschung in 1934.
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Karl R. Popper (1902-1994) The book attracted more attention than
Popper had anticipated. Popper was invited to lecture in England in
1935. The growth of Nazism in Germany and
Austria compelled him, like many other intellectuals who shared his Jewish origins, to leave his native country.
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Karl R. Popper (1902-1994) After a teaching position in New Zealand in
1937 he finally moved to England in 1946 to teach at the London School of Economics, and became professor of logic and scientific method at the University of London in 1949.
His ideas finally became so prominent that biological science almost always starts with formulating a hypothesis.
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What is a hypothesis? Definition:
A research hypothesis is the statement created by a researcher when they speculate upon the outcome of a research or experiment.
Why do we need hypothesis in science?
Every true experimental design must have this statement at the core of its structure, as the ultimate aim of any experiment.
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Hypothesis Usually the hypothesis is the result of a
process of inductive reasoning where observations lead to the formation of a theory. Scientists then use a large battery of deductive methods to arrive at a hypothesis that is testable, falsifiable and realistic.
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Hypothesis If a research hypothesis, stands the test of
time, it eventually becomes a theory, such as Einstein’s General Relativity.
Even then, as with Newton’s Laws, it can still be falsified or adapted.
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Hypothesis The precursor to a hypothesis is a research
problem, usually framed as a question.
The research hypothesis is a paring down of the problem into something testable and falsifiable.
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What is Critical Rationalism?
Popper rejected the term of classical empiricism, and of the classical observationalist-inductivist account of science that had grown out of it.
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What is Critical Rationalism? Scientific ideas can only be tested
indirectly because scientific theories are abstract and human knowledge generally, is irreducibly conjectural or hypothetical, and is generated by the creative imagination of humans.
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What is Critical Rationalism? No number of positive outcomes at the
level of experimental testing can confirm a scientific theory, but a single counterexample is logically decisive: it shows the theory, from which the implication is derived, to be false.
Hence falisfication
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What does it mean a hypothesis is falsifiable?
The term "falsifiable" does not mean something is false; rather, that if it is false, then this can be shown by observation or experiment.
Popper's account of the logical asymmetry between verification and falsifiability lies at the heart of his philosophy of science.
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What does it mean a hypothesis is falsifiable? Falsifiability, as defined by the philosopher, Karl
Popper, defines the inherent testability of any scientific hypothesis.
Science and philosophy have always worked together to try to uncover truths about the world and the universe around us. Both are a necessary element for the advancement of knowledge and the development of human society.
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When is a hypothesis not practicable? Human Genome Project Why did scientists not set out a hypothesis
such as: “There are ten genes in the genome involved
in insulin production”
Source: http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml
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Difference between critical rationalist and question/answer framework Critical Rationalist : Here the scientist uses
prior knowledge to frame a hypothesis and formulate a conclusion about the unknown.
Question/answer methodology:
The scientist uses prior knowledge upon which to ask a question about the unkown.
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CHAPTER 4
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Comparing the different approaches
Decide on an experimental project
Make a hypothesis Subject the hypothesis to
falsification Get a result Determine whether the
result holds true in repeating
Decide on an experimental project
Ask a question Get an answer Ask the question again
and observe its accuracy
Critical Rationalism Question/answer mode
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A system of sequential queries Why don’t you set up a
hypothesis to walk to Harvard Medical School when you are at Boylston Street ?
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A system of sequential queries You could formulate a
hypothesis:
“Walking on Boylston towards Brookline will take me to LHRRB in the Longwood area.”
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A system of sequential queries You could formulate a
hypothesis:
“Walking on Boylston towards Brookline will take me to LHRRB in the Longwood area.”
This hypothesis can than be subjected to falsification.
Why don’t we do that?
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What is the inductive space? The background information that is already
existing about a specific question prior to the study
Example in Chapter 6: Why can the question be:
“What is the function of MuRF1?” given we know MuRF1 is a protein.
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Types of questions The open- ended question
“What color is the sky?” Define the scope of the problem
The close- ended question “The sky is red” Two analyses:
Red, not- red
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Discrete questions Not all open- ended questions are all
encompassing
Project A Project B
Which genes are implicated in glucose metabolism
What is the function of gene X?
What is the role of gene X in glycose metabolism ?
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Discrete questions
Project A Project B
Which genes are implicated in glucose metabolism
What is the function of gene X?
What is the role of gene X in glycose metabolism ?
?
Discrete question afterprojetc study
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Inductive reasoning Initial observation lead to the discovery of a
certain pattern. This allows a tentative prediction to be made
which leads to a general theory about how things work.
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Inductive reasoning Charles Darwin for example observed the
variety of Darwin finches on the Galapagos islands and based on that build his theories.
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Inductive reasoning After some thought and reasoning, he saw
that these populations were geographically isolated from each other and that the variation between the sub-species varied over distance.
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Inductive reasoning He therefore proposed that the finches all
shared a common ancestor, and evolved and adapted, by natural selection, to exploit vacant ecological niches. This resulted in evolutionary divergence and the creation of new species, the basis of his ‘Origin of Species’.
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Inductive reasoning This was an example inductive reasoning, as
he started with a specific piece of information and expanded it to a broad hypothesis. Science then used deductive reasoning to generate testable hypotheses and test his ideas.
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Inductive reasoning In the lab nowadays you do not have to travel
to the Galapagos islands in order to expand your inductive space.
You would rather
consult a computer
and do a web search
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Deductive reasoning Deductive reasoning is what most scientists
recognize as the standard scientific method, where a researcher starts with a wider theory.
The researcher generates a testable hypothesis and designs an experiment to observe the results, and prove or disprove the theory.
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Deductive reasoning Deductive reasoning, starts with a general
principle and deduces that it applies to a specific case.
Inductive reasoning is used to try to discover a new piece of information while deductive reasoning is used to try to prove it.
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Deductive reasoning Example J. J. Thompson’s Cathode Ray-Experiment
was an excellent example of this process, where he had ideas about how electrons behaved and generated theories about their nature.
Therefore, Thompson generated hypotheses, designed experiments and tried to find conclusive answers to add credence and weight to his initial theory.
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J. J. Thompson’s Cathode Ray-ExperimentDeductive reasoning
He found that by applying a magnetic field across the tube, there was no activity recorded by the electrometers and so the charge had been bent away by the magnet. This proved that the negative charge and the ray were inseparable and intertwined.
http://www.experiment-resources.com/cathode-ray.html#ixzz0pFB6EBah
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J. J. Thompson’s Cathode Ray-ExperimentDeductive reasoning
Out of this deduction Television was developed
Thompson receiver the Nobel prize in Physics in 1906.
http://www.experiment-resources.com/cathode-ray.html
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CHAPTER 6
How experimental conclusions are used to represent reality
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How experimental conclusions are used to represent reality
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How to build a model
What is the function of MuRF1?
What are the functions of proteins?
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What category does MuRF1 proteins fall into?
Transcription factors
Phosphatases
Proteases
AcetylasesTranscription factors
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Known versus unknown
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Model building How does the scientist access the inductive
space in the example of MuRF1 function? The scientist uses bioinformatic tools such as
BLAST or FASTA to find out that MuRF1 belongs to a protein family referred to as
E3 ubiquitin ligases.
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Model building Now the scientist has to perform an
experiment to see whether MuRF1 is an E3 ubiquitin ligase.
If the experiment is repeated and the result still indicates MuRF1 is a E3 ubiquitin ligase then the model can be stated as followinf: MuRF1 functions as an E3 ubiquitin ligase
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Model buildingMuRF1 functions as an E3 ubiquitin ligase The question: What is the function of MuRF1? Is the framework question that can now be
further specified.
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Refining the inductive space
What are the functions of proteins?
What is the function of E3 ubiquitin ligases?
MDM2
Skp2
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CHAPTER 7Establishing a System for Experimantation
The value of positive, negative and sensitive controls in experimental designs
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Need to validate your system What does it mean to validate a system? Explain the metaphor of the car.
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Control serves as reference point Validating controls
The use of controls
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The use of controls What is a negative control?
What is a negative control good for? To appreciate the number of times that the positive
readout was achieved compared to the negative readout
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The use of controls What is a positive control? What is a positive control good for?
To ensure that the subject being surveyed can be detected
So the scientist is sure that this specific subject yields a result
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The positive control Tell me what happens to the scientist
validating null cells as a negative control to detect M –cadherin
But he/she skips the necessity of a positive control…
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Source: http://www.microvet.arizona.edu/courses/mic419/ToolBox/elisa3.jpg
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The positive control What does this mean now? The scientist proofed that there is no antibody
recognition by the M-cadherin. Then he/she performed the experiment and
discovers that there is no M-cadherin detection.
Why?
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The positive control Does it mean there is no M-cadherin
expressed by the cells? If the scientist now includes a positive control
he discovers a strong signal.
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The use of controls What is a sensitivity control?
What is a sensitivity control good for? To ensure that the subject being surveyed can be
detected
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CHAPTER 8 and 9Discussion Points
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Designing the experiment Definitions Time courses Experimental repetition
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Designing the experiment Definitions: What different colors does the
sky have? Time courses: How often do you have to
measure the sky color during the course of a day?
Experimental repetition: How often do you have to repeat an experiment in order to predict the future?
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Designing the experiment Representative conditions: When would you measure the color of the
sky? An experiment has to be designed to be
studied under representative conditions.
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Analyzing the data and interpreting the experiment
1/1/07 1/2/07 1/3/07 1/4/07 1/5/07 1/6/07 1/7/07
6:05 a.m. black black black black black black black
6:10 a.m. black black black black black black black
6:15 a.m. black black black black black black gray
6:20 a.m. black black black black black black gray
6:25 a.m. black black black black black gray gray
6:30 a.m. black black black black gray gray red
6:35 a.m. black black black gray gray red blue
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Why repeat an experiment ?1/1/07 1/2/07 1/3/07 1/4/07 1/5/07 1/6/07 1/7/07
6:05 a.m. black black black black black black black
6:10 a.m. black black black black black black black
6:15 a.m. black black black black black black gray
6:20 a.m. black black black black black black gray
6:25 a.m. black black black black black gray gray
6:30 a.m. black black black black gray gray red
6:35 a.m. black black black gray gray red blue
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The choice of experimental controls
1) Record data over a specific range of light wavelenghts
2) Position the instrument in a particular direction
3) Decide upon which time period you record 4) Regular and particular intervals during
time period 5) Repeat the time course a number of times
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Reading material for next time Session 2 Book chapter 10
Supplement reading: CERF, O. et al. (2009) : Tests for determining in-use concentrations of antibiotics and disinfectants are based on entirely different concepts: “Resistance” has different meanings. International Journal of Food Microbiology 136, 247–254.
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Any more questions?
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CHAPTER 10Determining EcoRI´s restriction site
Additional reading: CERF, O. et al. (2009) :
Tests for determining in-use concentrations of antibiotics
and disinfectants are based on entirely different concepts: “Resistance” has different meanings.
International Journal of Food Microbiology 136, 247–254.
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Chapter 10 Designing the experimental project
-A biological example-
Example of experimental design: Determining EcoRI´s restriction site
Source: http://www.djblabcare.co.uk/djb/data/image/14/0/Hettich_EBA20_Portable_Centrifuge.jpeg
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Designing the experimental project-cutting DNA into pieces-
How does a restriction enzyme work?
http://employees.csbsju.edu/hjakubowski/classes/SrSemMedEthics/Human%20Genome%20Project/DNA1.html
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Designing the experimental project-Inserting a DNA sample into a plasmid-
Source: http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Keogh/plasmids.html
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Restriction enzymes There are three different types of restriction
enzymes.
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Restriction enzymes Type I Recognise specific sequences·but then
track along DNA (~1000-5000 bases) before cutting one of the strands and releasing a number of nucleotides (~75) where the cut is made. A second molecule of the endonuclease is required to cut the 2nd strand of the DNA e.g. EcoK.
Require Mg2+, ATP and SAM (S-adenosyl methionine) cofactors for function.
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Restriction enzymes Type II Recognise a specific target sequence
in DNA, and then break the DNA (both strands), within or close to, the recognition site e.g. EcoRI
Usually require Mg2+
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Restriction enzymes Type III Intermediate properties between
type I and type II. Break both DNA strands at a defined distance from a recognition site e.g. HgaI
Require Mg2+ and ATP
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The restriction enzymes cut site:
At what sequence does EcoR1 cut DNA?
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The process of ligation
Sticky ends are also known as cohesive ends.
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What is a plasmid? Independently replicating extra- chromosomal
structures in bacteria.
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Designing the experimental project-A biological example-
Can other cells get our modified DNA plasmids too?
Two classes of plasmids:
Conjugative Non-conjugative
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Conjugative plasmidsHorizonal gene transfer: The tra genes encode some of
the proteins required for the manufacture of a pilus - macromolecular tube that joins one cell to another and allows the transfer of plasmids
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The ORI site: What is an origin of replication (ORI)?
Source: http://homepages.strath.ac.uk/~dfs99109/BB211/Plasmidnotes.html
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Designing the experimental project-A biological example- Still, do we know now if the cell “swallowed” the
DNA junk or not?
Plasmids are maintained in cells due to selective pressure - the ability to confer an advantageous phenotype
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What can plasmids do? Phenotypes that can confer advantage to a host cell
include Antibiotic resistance, Antibiotic production Sugar fermentation for energy Degradation of aromatic compounds for energy Heavy metal resistance Toxin production
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Designing the experimental project-A biological example- Why can’t we pour our
bacterial culture in the trash? Why not it is just bacteria? Bacterial resistance to
antibiotics.
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DNA uptake into bacterial cells Natural
Transformation Conjugation Transduction
Artificial Electroporation Bacteriophages Lac operon, blue/ white selection Chemical through substances such as CaCl2
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Examples of Horizontal gene transfer
So how does the modified DNA come into a cell?
Source: http://biogetopics.wordpress.com/2008/11/27/bacterial-resistance/
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Artificial techniques for DNA uptake Electroporation Bacteriophages Lac operon, blue/ white selection
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How does DNA uptake happen in the lab?
This process is called Electroporation.
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Designing the experimental project-Ampicillin resistance-
Origin of replication
How can we grow the plasmids in culture?
Source: http://picsdigger.com/keyword/ti%20plasmid/
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DNA uptake by phages Phage infection
Lytic cycle Lysogenic cycle
Source: http://textbookofbacteriology.net/phage.html
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Designing the experimental project-A biological example-
The Lambda phage
Electron microscopy imageSource: http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Keogh/plasmids.html
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Designing the experimental project-A biological example-
The mechanism of infection:
AdsorptionIrreversible attachmentSheath contractionNucleic acid injection
Source: http://www.nsf.gov/od/lpa/news/02/pr0207images.htm
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The lactose metabolism of E. coli Another method of plasmid uptake screening
in bacteria.
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Designing the experimental project-A biological example- Prokaryotes such as the bacterium E. Coli,
have an efficient mechanism for metabolizing lactose. Three proteins that are important in lactose metabolism are all encoded in a single expressible unit odf DNA, called the lac operon.
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Designing the experimental project-A biological example- The bacterium does not waste energy
expressing these proteins if lactose is not present in the growth medium. It only makes these proteins when lactose is available to be metabolized.
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Designing the experimental project-A biological example- http://www.sumanasinc.com/webcontent/
animations/content/lacoperon.html
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Designing the experimental project Start the engine,
please!
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Designing the experimental project-Explaining the lac operon- Can we see the gene
when looking through a a microscope?
Selecting for those plasmids which have the gene of interest integrated.
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Designing the experimental project
-The blue/white screening-
However bacterial colonies in general, are white, and so a bacterial colony with no vector at all will also appear white. These are usually suppressed by the presence of an antibiotic in the growth medium. A resistance gene on the vector allows successfully transformed bacteria to survive despite the presence of the antibiotic.
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Designing the experimental project Blue/ white
selection
Source: www.absoluteastronomy.com/.../Blue_white_screen
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Designing the experimental project-The blue/white screeing-
The hydrolysis of colourless X-gal by the β-galactosidase causes the characteristic blue colour in the colonies; it shows that the colonies contain vector without insert. White colonies indicate insertion of foreign DNA and loss of the cells' ability to hydrolyse the marker.
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-The blue/white screening- Easy to pick the blue colonies from an agar
plate.
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Summary of a cloning experiment
Cut Paste Insert Grow Purify
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CHAPTER 10 Discussion PointBacterial resistance to antibiotics
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Bacterial resistance to antibiotics Antibiotics and disinfectants
Supplement reading: CERF, O. et al. (2009) :Tests for determining in-use concentrations of antibioticsand disinfectants are based on entirely different concepts: “Resistance” has different meanings.International Journal of Food Microbiology 136, 247–254.
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Designing the experimental project-A biological example- Why are antibiotics not the magic bullet and
universal use is restricted and limited?
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Reading material for next time Session 3 Book chapters 11 until 14
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Any questions?
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CHAPTER 11 Experimental repetition: The Process of Acquiring Data to Model Future Outcomes
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Categories of Experimental repeats Is it sufficient if the scientist measures the
color of the sky only once? Multiple measurements at a single time point Single measurements over the course of many
days Many measurments of the sky color during the
course of the whole day Multiple measurements at different time points
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Questions What does it mean to verify a model? Why is it important to find a representative of
the general case? What´s wrong if I take one rat and analyse the
mRNA expression and compare it with another rat?
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Biological example
Control High-fat diet 12 hours
Rat one Rat two
Does this rat represent the General case?
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So how many rats shall we take?
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CHAPTER 12 The negative control
Discussion points
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The negative control Definition: A setting where the experimental
subject is not perturbed by the variable under study.
“Unperturbed by X”
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The negative control- The caffeine/blood pressure example Control for relevant
variables Measure “X” and tell
from “not-X” Want to measure only
“X and only X” is varied
Caffeine or something else in the Coffee?
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Does caffeinated coffee affect blood pressure, and if so, is the caffeine responsible? In this questions there are two variables
Factor X
Coffee (with all its ingredients)
Caffeine
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Study design
Establish six groups each 50 people Determine the subjects` starting blood
pressure. Take initial blood and urine samples. Get only people with a blood pressure range
between 140/90 and 90/60. Match study subjects by gender
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Study design
Body mass index should be ranging between 19 and 40.
Match groups for average BMI, age, normal diet
Exclude people with anxiety disorders or hypertension.
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Group settings
Group Treatment
A
B
C
D
E
F
No treatment
Water
Decaffeinated coffee
Caffeinated water
Caffeinated coffee
Caffeinated cola
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Group controls
Group Treatment
A
B
C
D
E
F
No treatment: Unperturbed by any change
Water: Unperturbed by additional ingredients in coffee
Decaffeinated coffee: Unperturbed by coffee
Caffeinated water: Unperturbed by coffee
Caffeinated coffee: The actual test case
Caffeinated cola: Assumption control
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The negative control
Group A
Group B
Decaffeinated coffee
Caffeinated coffee
Why is it not enough to have only two groupscaffeinated and Decaffeinated coffee?
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The negative control
Group A
Group B
Decaffeinated coffee
Caffeinated coffee
10% increase in blood pressure
30% increase in blood pressure
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The negative control- study design
Decaffeinated coffee
Caffeinated coffee
10% increase in blood pressure
30% increase in blood pressure
Caffeinated water 10% increase in blood pressure
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The negative control- study design
Decaffeinated coffee
Caffeinated coffee
10% increase in blood pressure
30% increase in blood pressure
Caffeinated water 10% increase in blood pressure
Water 5% increase in blood pressure
Why is it necessary to have the just water control?
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The negative control-study design
Decaffeinated coffee
Caffeinated coffee
10% increase in blood pressure
30% increase in blood pressure
Caffeinated water 10% increase in blood pressure
Water 5% increase in blood pressure
Caffeinated cola
Nothing 0% increase in blood pressure
10% increase in blood pressure
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The negative control
Decaffeinated coffee
Caffeinated coffee
10% increase in blood pressure
30% increase in blood pressure
Caffeinated water 10% increase in blood pressure
Water 5% increase in blood pressure
Caffeinated cola
Nothing 0% increase in blood pressure
10% increase in blood pressure
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The negative control
Decaffeinated coffee
Caffeinated coffee
10% increase in blood pressure
30% increase in blood pressure
Caffeinated water 10% increase in blood pressure
Water 5% increase in blood pressure
Caffeinated cola
Nothing 0% increase in blood pressure
10% increase in blood pressure
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Intrasystem/intersystem control Difference between
The intrasystem negative control A negative control provides a point of contrast to
ensure unbiased measurement measures “not-X”
The intersystem negative control “unperturbed by X” control X is the system being
applied Ensuring the system is not in itself perturbing the
outcome
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CHAPTER 13 The requirement for the positive control
Discussion Points
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The positive control Is the system capable of detecting the
experimental readout? A demonstration that a measuring system is
operational positive control
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The positive control
Treatment % Increase in blood pressure
Water 10
Caffeinated water 10
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The positive control
Treatment % Increase in blood pressure
Water 10
Caffeinated water 10
Hypertensive drug 30
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The positive controlTreatment % Increase in blood pressure
Water 10
Caffeinated water
10
Hypertensive drug 30
Caffeinated water; caffeine equivalent Of one cup of coffee
Caffeinated water; caffeine equivalent Of two cups of coffee
Caffeinated water; caffeine equivalent Of three cups of coffee
Caffeinated water; caffeine equivalent Of four cups of coffee
12
15
20
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The experimental design
A
B
C
D
E
F
Group Treatment
No treatment
Water
Noncaffeinated coffee
Caffeinated water
Caffeinated colaCaffeinated cola
Caffeinated coffee
Why can the “caffeinated cola” group not be a positive control?
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The positive control Remember: The question to address was: Does caffeinated coffee affect blood pressure
and if so, is the caffeine responsible? A positive control has to be capable of
detecting the readout What is the readout in our case?
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The experimental design
A
B
C
D
E
F
Group Treatment
No treatment
Water
Noncaffeinated coffee
Caffeinated water
Caffeinated coffee
Caffeinated cola
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More positive controls… Is this positive control enough? What other benefits from an additional
positive control does the author point out?
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CHAPTER 14 Method and Reagent Control
Discussion Points
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Method and reagents controls Is a single
methodology or reagent causing an effect?
Or is there some unseen additional mechanism that is missed?
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Method and reagents controls The method applied might yield different results
individual scientists use uniform approaches. Is a cell biologist using the same methods as a
pharmacologist?
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Method and reagents controls Is a lawyer supposed to know how to measure
blood pressure? What was the point the author illustrated in
this chapter?
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Method and reagents controls The reagents (e.g.: small molecules, genetic
constructs, antibodies, detection tools...)
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Method and reagents controls The methodology control
Method 1
Method 2
A second mechanism that controlsfor the first method used
A ‘NON-Method 1 control”
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Reading material for next time BSP Session 4 Chapters: 15-19
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Any more questions?
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CHAPTER 15 Subject controls
Discussion Points
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Finding a responsive subject The study subject has to representative the
“typical” case? What is the typical case?
Do these boys represent a typical case?
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Can we take all people for a weight loss study?
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Subject control What example does the author give?
Is it suitable to choose only highly motivated people for a study on a weight loss drug?
Does it matter if a cancer drug can only help people with a certain gene mutation? As long as it helps someone.
Why control for a particular subject type?
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Randomizing study subjects After a screening process Patients do not know which group they are in The scientist is not able to pick study subjects
and assign them to a specific group
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Double blinded studies Why do scientists use
double blinded trials?
What is the difference between single and double blinded?
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Matching subject controls in some studies Animal experiment No chance of an placebo effect in animals
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Matching study subjects Great variation in the
experimental output
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Matching study subjects
Do all mice have the same strenght?
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Matching study subjects In order to yield results the scientist will
distribute individual animals into certain groups.
Alternatively one has to expand the group size manyfold, which can be problematic.
Why can it be problematic?
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Variables and genetic “Model systems” Clonal strains of animals were producted over
the last century that scientists use today. There are many identical inbreed mice strains,
fruit flies, worms, zebra fish frogs and even single-celled brewer´s yeast as genetic model systems available nowadays.
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Variables and genetic “Model systems” Can we compare a fruit
fly to humans?
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Variables and genetic “Model systems” Many findings in animals will eventually be
found to hold in humans too. But what if our gene of interest is under the
influence of another gene and alters its function?
We are missing effects induced by particular genetic variations.
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Who was Gregor Mendel? Gregor Johann
Mendel (1822 - 1884) was a member of an Augustinian order in Brunn, Austria.
http://kentsimmons.uwinnipeg.ca/cm1504/mendel.htm
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How can we study gene functions? One way to study the function of a gene is to
delete its function in the genome. This is called knock out. The mice are said to
be genetically null for this particular gene.
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How to make a know- out mouse
F1
1:2:1
Parents
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How to make a know- out mouse
F1
+/ko +/ko
Only about 25% of the progeny in F2 will be homozygoze for the knock out.This has to be phenotypically determined.
ko/ko
heterozygot
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Variables and genetic “Model systems”
What if all humans would be the same?
Could we then conduct genetic studies in humans?
The genetic background of genes
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Changing the genetic background-Congenic inbred strains-
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Heterozygous and homozygous subject controls It makes a difference
which genetic background your experimental animal has?
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The use of cell cultures Why do we use cell
cultures in biological experiments?
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CHAPTER 16Discussion Points
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Assumption control
Good efficacy of drug X In early stage cancer
patientsEnd stage cancer patients
Does our drug help patients in the advanced cancer stage too?
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Assumption control
Good efficacy of drug X In early stage cancer
patientsEnd stage cancer patients
Shall we simply increase the does of the drug? Why not?
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Assumption control
Good efficacy of drug X In early stage cancer
patientsEnd stage cancer patients
How can we assume that late stage cancer patients respond in the same way as early stage patients?
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Assumption control
Good efficacy of drug X In early stage cancer
patientsEnd stage cancer patients
The assumption control would be a group of subjectswith end- stage cancer.
Because... ?
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Assumption control
Good efficacy of drug X In early stage cancer
patientsEnd stage cancer patients
The early stage cancer group does not represent the end stage cancer group
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CHAPTER 17Experimentalist Controls
Discussion Points
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What is the “objective truth”? Accessible from different angles Intersubjective Independent
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Intersubjectivity What if only one scientist observes something
and others can not confirm this finding is the scientist then wrong?
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Intersubjectivity If you find one million people that believe
there are kangaroos in Austria does this testify your observation to be true?
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Intersubjectivity Intersubjectivity relies on “Objectivity” Objectivity does not require any “believe” or
any “help” from the person observing.
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Establishing Objectivity
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Different evaluators Why don’t just all scientist do some research
by themselves? Wouldn’t this accelerate the scientific
progress? More scientists can work on more questions
and more things could be discovered in a shorter period of time.
Why not?
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Different evaluators The principal investigator will interpret the
data to prevent bias the PI will check with different evaluators
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Different evaluators The structure of a science labs
The principal investigator
Postdoctoral fellowsGraduate students
Research assistant
PhD studentsOther laboratories
Computers
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Different evaluators More than one scientist Research is done in
groups Remember method and
reagents control Discussing results in
groups is important
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CHAPTER 18A Description of Biological Empiricism
Discussion Points
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What is Empiricism? It is a theory of knowledge that asserts that
knowledge arises from sense experience. Empiricism is one of several competing views
that predominate in the study of human knowledge, known as epistemology.
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What is Empiricism? Empiricism emphasizes the role of experience
and evidence, especially sensory perception, in the formation of ideas, while discounting the notion of innate ideas.
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Finding causal links in biological systems
Factor C Factor B
Factor A
Specific phenotype
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Assigning causality and assessing the requirements for necessity and sufficiency
A B A can be either necessary (=required) for B
or Sufficient to result in B
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Finding causal links in biological systems A causes B
Eating fatty food(A)
Heart disease (B)
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Finding causal links in biological systems However, heart diseases may only develop in
people with high cholesterol levels.
Eating fatty food(A)
Heart disease
+High cholesterol levels
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Finding causal links in biological systems So, does it mean eating fatty food causes
heart disease?
Eating fatty food(A)
Heart disease
+High cholesterol levels
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Finding causal links in biological systems Aren’t there people suffering a heart attack
without eating fatty food too?
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Finding causal links in biological systems Therefore eating fatty foods may neither be
necessary nor sufficient to result in heart attacks in individuals with high cholesterol levels.
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Finding causal links in biological systems Yet, the consumption of fatty food may
demonstrably cause heart attacks in those with high cholesterol levels.
Thus, a causal link may exist only in a particular context.
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Determining causal links in biology Gene A and Gene B have to be simultaneously
deleted in order to observe a change in the ear phenotype.
Gene A
Gene B
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Determining causal links in biology Statement one: Neither Gene A nor Gene B is required or
sufficient for the phenotype.
Gene A
Gene B
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Determining causal links in biology Statement two: In the absence of one gene both Gene A and
Gene B are necessary and sufficient for the ear phenotype.
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Nonuniversal truth Lots of biological experiments are gigantic
and thus only a small nonreproducible result might be yielded.
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Nonuniversal truth Critics will point out that the future case
might be different.
Remember the elephants metaphor!
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So does this mean empiricism is flawed?
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