matthias-schedl - Basic Science Partnership at Harvard Medical

Report
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.

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.
Syllabus

4 discussions/ lectures

Individual talks from different labs

Last session of course  your presentations
from your lab projects
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)
Outline for our course:

Session 1


Session 2


Book chapter 10, plus additional paper
Session 3


Book chapters 1-9
Book chapters 11-14
Session 4


15-19
Quiz
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.
CHAPTER 1-9
Key words:
Hypothesis
Problem/Question framework
System establishment
Model building
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/
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/
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
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.
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.
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.
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.
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.
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.
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.
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.

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.
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.
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.
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.
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.
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
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.
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.
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
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.
CHAPTER 4
Comparing the different approaches
Critical Rationalism





Decide on an
experimental project
Make a hypothesis
Subject the hypothesis
to falsification
Get a result
Determine whether the
result holds true in
repeating
Question/answer mode




Decide on an
experimental project
Ask a question
Get an answer
Ask the question again
and observe its
accuracy
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 ?
A system of sequential queries

You could formulate a
hypothesis:

“Walking on Boylston
towards Brookline will
take me to LHRRB in
the Longwood area.”
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?
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.

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
Discrete questions

Not all open- ended questions are all
encompassing
Project A
Which genes are
implicated
in glucose
metabolism
Project B
What is the
role of gene X
in glycose
metabolism ?
What is the
function of
gene X?
Discrete questions
Project A
Project B
Discrete question after
projetc study
Which genes are
implicated
in glucose
metabolism
?
What is the
role of gene X
in glycose
metabolism ?
What is the
function of
gene X?
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.
Inductive reasoning

Charles Darwin for example observed the
variety of Darwin finches on the Galapagos
islands and based on that build his theories.
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.
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’.
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.
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
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.
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.
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.
J. J. Thompson’s Cathode Ray-Experiment
Deductive 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
J. J. Thompson’s Cathode Ray-Experiment
Deductive 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
CHAPTER 6
How experimental conclusions are used to
represent reality
How experimental conclusions are
used to represent reality
How to build a model
What are the functions of proteins?
What is the
function of MuRF1?
What category does MuRF1 proteins
fall into?
Proteases
Transcription factors
Acetylases
Transcription factors
Phosphatases
Known versus unknown
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.
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
Model building
MuRF1 functions as an E3 ubiquitin ligase


The question: What is the function of MuRF1?
Is the framework question that can now be
further specified.
Refining the inductive space
What are the functions of proteins?
What is the function of E3 ubiquitin ligases?
MDM2
Skp2
CHAPTER 7
Establishing a System for
Experimantation
The value of positive, negative and sensitive
controls in experimental designs
Need to validate your system


What does it mean to validate a system?
Explain the metaphor of the car.
The use of controls
Control serves as reference point
 Validating controls

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
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

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…
The positive control
Source: http://www.microvet.arizona.edu/courses/mic419/ToolBox/elisa3.jpg
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?
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.
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
CHAPTER 8 and 9
Discussion Points
Designing the experiment



Definitions
Time courses
Experimental repetition
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?
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.
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
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
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
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.
Any more questions?
CHAPTER 10
Determining 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.
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
Designing the experimental project
-cutting DNA into pieces-
How does a restriction enzyme work?
http://employees.csbsju.edu/hjakubowski/classes/SrSemMedEthics/Human%20Genome%20
Project/DNA1.html
Designing the experimental project
-Inserting a DNA sample into a plasmid-
Source: http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Keogh/plasmids.html
Restriction enzymes

There are three different types of restriction
enzymes.
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.
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+
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
The restriction enzymes cut site:
At what sequence does EcoR1 cut DNA?
The process of ligation
Sticky ends are also known as cohesive ends.
What is a plasmid?

Independently replicating extra- chromosomal
structures in bacteria.
Designing the experimental project
-A biological example

Can other cells get our modified DNA
plasmids too?
Two classes of plasmids:
Conjugative
Non-conjugative
Conjugative plasmids
Horizonal 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
The ORI site:

What is an origin of replication (ORI)?
Source: http://homepages.strath.ac.uk/~dfs99109/BB211/Plasmidnotes.html
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
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
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.
DNA uptake into bacterial cells

Natural




Transformation
Conjugation
Transduction
Artificial




Electroporation
Bacteriophages
Lac operon, blue/ white selection
Chemical through substances such as CaCl2
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/
Artificial techniques for DNA
uptake



Electroporation
Bacteriophages
Lac operon, blue/ white selection
How does DNA uptake happen in
the lab?

This process is
called
Electroporation.
Designing the experimental project
-Ampicillin resistanceOrigin of replication
How can we grow the plasmids in
culture?
Source: http://picsdigger.com/keyword/ti%20plasmid/
DNA uptake by phages

Phage infection


Lytic cycle
Lysogenic cycle
Source: http://textbookofbacteriology.net/phage.html
Designing the experimental project
-A biological exampleThe Lambda phage
Electron microscopy image
Source: http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Keogh/plasmids.html
Designing the experimental project
-A biological example-
The mechanism of infection:
Adsorption
Irreversible attachment
Sheath contraction
Nucleic acid injection
Source: http://www.nsf.gov/od/lpa/news/02/pr0207images.htm
The lactose metabolism of E. coli

Another method of plasmid uptake screening
in bacteria.
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.
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.
Designing the experimental project
-A biological example
http://www.sumanasinc.com/webcontent/anim
ations/content/lacoperon.html
Designing the experimental project

Start the engine,
please!
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.
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.
Designing the experimental project

Blue/ white
selection
Source: www.absoluteastronomy.com/.../Blue_white_screen
Designing the experimental project
-The blue/white screeing
The hydrolysis of colourless X-gal by the βgalactosidase 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.
-The blue/white screening
Easy to pick the blue colonies from an agar
plate.
Summary of a cloning experiment





Cut
Paste
Insert
Grow
Purify
CHAPTER 10
Discussion Point
Bacterial resistance to antibiotics
Bacterial resistance to antibiotics

Antibiotics and disinfectants
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.
Designing the experimental project
-A biological example
Why are antibiotics not the magic bullet and
universal use is restricted and limited?
Reading material for next time


Session 3
Book chapters 11 until 14
Any questions?
CHAPTER 11
Experimental repetition: The Process of
Acquiring Data to Model Future Outcomes
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
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?
Biological example
Rat one
Control
Rat two
High-fat diet 12 hours
Does this rat represent the
General case?
So how many rats shall we take?
CHAPTER 12
The negative control
Discussion points
The negative control


Definition: A setting where the experimental
subject is not perturbed by the variable under
study.
 “Unperturbed by X”
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?
Does caffeinated coffee affect blood pressure,
and if so, is the caffeine responsible?

In this questions there are two variables
Coffee
(with all its ingredients)
Factor X
Caffeine
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
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.
Group settings
Group
Treatment
A
No treatment
B
Water
C
Decaffeinated coffee
D
Caffeinated water
E
Caffeinated coffee
F
Caffeinated cola
Group controls
Group
Treatment
A
No treatment: Unperturbed by any change
B
Water: Unperturbed by additional ingredients in coffee
C
Decaffeinated coffee: Unperturbed by coffee
D
Caffeinated water: Unperturbed by coffee
E
F
Caffeinated coffee: The actual test case
Caffeinated cola: Assumption control
The negative control
Why is it not enough to have only two groups
caffeinated and Decaffeinated coffee?
Group A
Decaffeinated coffee
Group B
Caffeinated coffee
The negative control
Group A
Decaffeinated coffee
10% increase in blood pressure
Group B
Caffeinated coffee
30% increase in blood pressure
The negative control- study design
Decaffeinated coffee
10% increase in blood pressure
Caffeinated coffee
30% increase in blood pressure
Caffeinated water
10% increase in blood pressure
The negative control- study design
Decaffeinated coffee
10% increase in blood pressure
Caffeinated coffee
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?
The negative control-study design
Decaffeinated coffee
10% increase in blood pressure
Caffeinated coffee
30% increase in blood pressure
Caffeinated water
10% increase in blood pressure
Water
5% increase in blood pressure
Caffeinated cola
10% increase in blood pressure
Nothing
0% increase in blood pressure
The negative control
Decaffeinated coffee
10% increase in blood pressure
Caffeinated coffee
30% increase in blood pressure
Caffeinated water
10% increase in blood pressure
Water
5% increase in blood pressure
Caffeinated cola
10% increase in blood pressure
Nothing
0% increase in blood pressure
The negative control
Decaffeinated coffee
10% increase in blood pressure
Caffeinated coffee
30% increase in blood pressure
Caffeinated water
10% increase in blood pressure
Water
5% increase in blood pressure
Caffeinated cola
10% increase in blood pressure
Nothing
0% increase in blood pressure
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
CHAPTER 13
The requirement for the positive control
Discussion Points
The positive control


Is the system capable of detecting the
experimental readout?
A demonstration that a measuring system is
operational positive control
The positive control
Treatment
Water
Caffeinated water
% Increase in blood pressure
10
10
The positive control
Treatment
Water
% Increase in blood pressure
10
Caffeinated water
10
Hypertensive drug
30
The positive control
Treatment
Water
Caffeinated water
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
Hypertensive drug
% Increase in blood pressure
10
10
12
15
20
30
The experimental design
Group
A
B
C
Treatment
No treatment
Water
Noncaffeinated coffee
D
Caffeinated water
E
Caffeinated coffee
F
Caffeinated cola
Why can the “caffeinated cola” group not be a positive control?
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?
The experimental design
Group
A
B
C
Treatment
No treatment
Water
Noncaffeinated coffee
D
Caffeinated water
E
Caffeinated cola
F
Caffeinated coffee
G
Hypertensive drug
More positive controls…


Is this positive control enough?
What other benefits from an additional
positive control does the author point out?
CHAPTER 14
Method and Reagent Control
Discussion Points
Method and reagents controls


Is a single
methodology or
reagent causing an
effect?
Or is there some
unseen additional
mechanism that is
missed?
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?
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?
Method and reagents controls

The reagents (e.g.: small molecules, genetic
constructs, antibodies, detection tools...)
Method and reagents controls

 The methodology control
Method 1
Method 2
A second mechanism that controls
for the first method used
A ‘NON-Method 1 control”
Reading material for next time


BSP Session 4
Chapters: 15-19
Any more questions?
CHAPTER 15
Subject controls
Discussion Points
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?
Can we take all people for a weight
loss study?
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?
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
Double blinded studies

Why do scientists use
double blinded trials?
What is the difference between single and double blinded?
Matching subject controls in some
studies


Animal experiment
No chance of an placebo effect in animals
Matching study subjects

Great variation in the
experimental output
Matching study subjects
Do all mice have the same strenght?
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?
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.
Variables and genetic “Model systems”

Can we compare a fruit
fly to humans?
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.
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
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.
How to make a know- out mouse
Parents
1:2:1
F1
How to make a know- out mouse
+/ko
F1
+/ko
heterozygot
ko/ko
Only about 25% of the progeny in F2 will be homozygoze for the knock out.
This has to be phenotypically determined.
Variables and genetic “Model systems”
The genetic background of genes
What if all humans would be the same?
Could we then conduct genetic studies in humans?
Changing the genetic background
-Congenic inbred strains-
Heterozygous and homozygous
subject controls

It makes a difference
which genetic
background your
experimental animal
has?
The use of cell cultures

Why do we use cell
cultures in biological
experiments?
CHAPTER 16
Discussion Points
Assumption control
Does our drug help patients in the
advanced cancer stage too?
Good efficacy of drug X
In early stage cancer
patients
End stage cancer patients
Assumption control
Good efficacy of drug X
In early stage cancer
patients
End stage cancer patients
Shall we simply increase the does of the drug? Why not?
Assumption control
Good efficacy of drug X
In early stage cancer
patients
End stage cancer patients
How can we assume that late stage
cancer patients respond in the same way
as early stage patients?
Assumption control
The assumption control would be a group of subjects
with end- stage cancer.
Good efficacy of drug X
In early stage cancer
patients
End stage cancer patients
Because... ?
Assumption control
The early stage cancer group does not represent the end stage cancer group
Good efficacy of drug X
In early stage cancer
patients
End stage cancer patients
CHAPTER 17
Experimentalist Controls
Discussion Points
What is the “objective truth”?



Accessible from different angles
Intersubjective
Independent
Intersubjectivity

What if only one scientist observes something
and others can not confirm this finding is the
scientist then wrong?
Intersubjectivity

If you find one million people that believe
there are kangaroos in Austria does this testify
your observation to be true?
Intersubjectivity


Intersubjectivity relies on “Objectivity”
Objectivity does not require any “believe” or
any “help” from the person observing.
Establishing Objectivity
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?
Different evaluators

The principal investigator will interpret the
data  to prevent bias the PI will check with
different evaluators
Different evaluators

The structure of a science labs
Research assistant
Postdoctoral fellows
Graduate students
The principal investigator
Other laboratories
Computers
PhD students
Different evaluators




More than one scientist
Research is done in
groups
Remember method and
reagents control
Discussing results in
groups is important
CHAPTER 18
A Description of Biological Empiricism
Discussion Points
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.
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.
Finding causal links in biological
systems
Factor A
Specific phenotype
Factor C
Factor B
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
Finding causal links in biological
systems

A causes B
Eating fatty food
(A)
Heart disease (B)
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
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
Finding causal links in biological
systems

Aren’t there people suffering a heart attack
without eating fatty food too?
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.
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.
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
Determining causal links in biology


Statement one:
Neither Gene A nor Gene B is required or
sufficient for the phenotype.
Gene A
Gene B
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.
Nonuniversal truth

Lots of biological experiments are gigantic
and thus only a small nonreproducible result
might be yielded.
Nonuniversal truth

Critics will point out that the future case
might be different.
Remember the elephants metaphor!

So does this mean empiricism is flawed?
Thank you for your participation

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