ACT 1 – VIRAL GENE THERAPY
molecules are delivered into human
 What's an advantage of using viruses
as a vector for gene therapy?
 They deliver genetic material into
cells.
 Why do you think it's important to use
mice in the development of a virus for
gene therapy?
 To test the effectiveness and safety
of the new drug
 Prof. Shepherd: Which part of the
heart is affected in heart failure?
 Heart wall.
 If we talk about knock-out mice, what
does knock-out describe?
 The deletion of a specific gene
 What is the first knock-out mouse
experiment you'd like to perform to
test if SERCA2a is involved in heart
failure?
 Perform a fitness test with the mice
 What's your conclusion from the first
experiment with the SERCA2a knock-
out mouse?
 SERCA2a is potentially involved
in heart failure
 In which cell type is SERCA2a
mainly expressed?
 Cardiomyocytes
 In which cellular organelle does
SERCA2a reside?
 Sarcoplasmic reticulum
 Why is the contractility of the heart
muscle impaired in the absence of
SERCA2a?
 Myofilament relaxation is
impaired.
 In gene therapy, which type of
cells to correct for a genetic defect or
provide a new therapeutic function?
 RNA or DNA
 What will be the final result of the co-
transfection?
 Recombinant AAV carrying the
SERCA2a gene
 How do we make sure that the
plasmids efficiently enter the cells so
that virus can be produced?
 By adding transfection reagent
 Does recombinant AAV cause disease
in humans?
 No
 How is packaging of the therapeutic
gene into the virus ensured?
 By packaging signals flanking the
therapeutic gene
 Considered that the genome of AAV
is 4.8 kb long. What is the maximum
size of the therapeutic gene that can
be delivered by AAV?
 4.8 kb
 How would you proceed to test the
efficiency of recombinant AAV-
SERCA2a?
 infect a mouse with induced heart
failure
 In theory, the virus could spread to
neighboring tissue to express
SERCA2a there also. However, this is
not desired and could potentially
cause side effects. How is it ensured
that SERCA2a carried by recombinant
AAV is only expressed in heart
muscle cells?
 By using a gene promoter specific
to heart muscle cells
 What effect does AAV-SERCA2a
have on the heart performance in mice
with induced heart failure? Scroll
down to select the correct answer.
 A positive and specific effect
 If the mice have offspring, would they
also carry the new therapeutic gene?
 No, because the gene is not
integrated into the genome
 What could be a potential problem for
the widespread application of AAV-
SERCA2a as a heart failure drug?
JAAV neutralizing antibodies in the
patient
LABSTER ACT 2: PROTEIN
SYNTHESIS
 What do A and B represent in the
image?
-A is transcription and B is
translation.
 Which statement is true?
DNA has no hydroxyl group on the
second carbon.
 Which image has the circle in the
correct position?
-C, the next base of mRNA is attached
to the hydroxyl group on the third
carbon of the ribose sugar.
 DNA and mRNA synthesis proceeds
in the 5' to 3' direction. What do 5'
and 3' refer to?
-The position of a carbon atom in the
sugar backbone.
 To which site does tRNA add its
amino acid to the polypeptide chain?
-P (polypeptide) site
 Where do eukaryotic cells synthesize
proteins and which number represent
this organelle in the image?
-Ribosomes, 2
 What is the name of each sequence of
the three nitrogen-containing bases of
RNA that codes for one amino acid?
-Codon
 Who or what does a level II biosafety
cabinet protect?
-The personnel, the experiment, and
the environment.
 The tRNA shown in the diagram will
be able to form a base-pair interaction
with which mRNA?
-5'-AGU
 Which type of RNA matches with the
A, B, and C descriptions,
respectively?
-tRNA, mRNA, rRNA
 Which strand is the template strand?
-Bottom
 Which is the correct mRNA sequence
resulting from the EPO gene
transcription process?
-5' - AUU ACA AUG GGG GUG
CAC GAA UGU CCU - 3'
 What is the correct amino acid
translated from the resulting mRNA?
-MGVHECP
 With this information, how many
nucleic acids are there in mRNA
including the initiator and terminator
codons?
-582
 Downstream means near the 3' end, so
notice that the first codon is no
methionine. What is the correct amino
acid sequence?
 -TGDR
 Amino acids are linked by a specific
type of covalent bond. The reaction of
this bond produces one water
molecules. This bond links multiple
amino acids together, what is it
called?
-Peptide bonds
 This polypeptide chain represents the
primary structure of a protein. The
linear sequence of amino acids within
a protein is considered the ... structure
of the protein.
-Primary
 Which level of protein structure is
formed by the weak bonds between
oxygen and hydrogen atoms within
the polypeptide backbone?
-Secondary structure
 The main two secondary structures of
proteins are alpha helix and beta sheet
which are represented respectively by
which two letters in the image?
-B and A
 Based on the mass spectrometer
result, what is the mass difference
between rhEPO expressed in CHO
and E. coli?
-12 kDa
 Based on the mass spectrometry
results, rhEPO is glycosylated in
which expression system?
-CHO
 Based on the mass spectrometry
results, how many glycosylation sites
exist in rhEPO-CHO?
4 protein.
 Bacterial cells often cannot produce
recombinant proteins that are identical
to their wild type. Why is this?
Bacteria lack the machinery for
making post-translational
modifications.
 Recombinant proteins produced by E.
coli cells are not glycosylated. This is
because E. coli cells do not possess
which organelle.
Golgi apparatus
 Based on the mass spectrometry
results, the masses of rhEPO
expressed in CHO and E. coli are 30
kDA and 18 kDA, respectively. We
also know that the EPO proteins
expressed in mammalian cells
glycoslyated. Therefore, we can say
that weight percentage of rhEPO
expressed in CHO are
polysacchariders.
40%
 Are mammalian expression systems
better than bacterial system for the
production of biologically active and
stable erythropoietin.
No, erythropoietin is best expressed
in mammalian cells.
 Mass spectrometry identifies
molecules based on what?
Mass and charge
 MALDI is an abbreviation for what?
Matrix-assisted laser
desorption/ionization.
 The macromolecule ionization
method, such as MALDI MS, allows
us to obtain a peptide mass
fingerprint, or PMF, that can be used
to do what?
Predict the identity of an unknown
 If X, Y, and Z refer to the parts of a
mass spectrometer that perform
different processes such that:
X detects the number of different ions
 Y is where ion production from the
sample takes place
Z separates ions with different mass-
to-charge ratios
In which order do X, Y and Z occur?
Y, Z, then X
 Protein samples are mixed with a
matrix prior to analysis. What is the
function of a matrix in MALDI MS
analysis?
Ensures ionization of protein
molecules
 Which of these histograms are NOT
coherent with the mass spectrometry
principle?
II, III, IV
 Which athlete is using rhEPO? Use
the buttons on the computer screen to
analyze the graphs.
C All options: 6 CO2 molecules, 2 ATP
 EPO can increase production of
energy (ATP) by increasing
production of what?
Red blood cells
LABSTER ACT 3: CELLULAR
RESPIRATION
 Metabolism can be divided into catabolic
and anabolic reactions. How are these
connected?
 catabolic reactions generate energy
that anabolic reactions use.
 How many carbon atoms does one
molecule of pyruvate contain?
 three carbon atoms.
 How do atp molecules store energy?
 High energy of phosphate group.
 What action would you recommend to
the point guard?
 drink a maltose sports drink.
LABSTER ACT 4: CELLULAR
RESPIRATION: KREBS CYCLE
 After glycolysis, the pyruvate molecules
can follow two different metabolic routs:
aerobic and anaerobic respiration, what is
the main difference?
Anaerobic respiration does not need
oxygen to occur and aerobic
respiration does.
 What is the net result of one glucose
molecule going through the Kreb's cycle?
molecules, 8 NADPH and 2 FADH2
molecules.
 Why is the Krebs cycle so important if it
only produces 2 ATP molecules?
It produces reducing agents for the
electron transport chain.
 Where exactly in a cell does the Krebs
cycle?
Matrix of mitochondria
 What must happen to pyruvate molecule
before entering the Krebs cycle
It has to be oxidized to form acetyl Co-
A, 1 and 1 NADH per molecule.
LABSTER ACT 5: CELLULAR
RESPIRATION: THE ELECTRON
TRANSPORTS CHAIN
 Congratulations on completing the
electron transport chain! There are many
details, but the overall concept is what is
 important. What is the primary role of the
electron transport chain?
 To generate a proton gradient so the
protons can be pumped through ATP
synthase to generate ATP
 What two processes are coupled
throughout the ETC that together make
up oxidative phosphorylation?
 The oxidation of electron carriers and
the phosphorylation of ADP
 Which process involves the diffusion of
protons across the mitochondrial
membrane?
 Chemiosmosis
 Why is ETC considered an aerobic
process?
 The process requires oxygen.
 How are electrons transported to the
membrane at the beginning of the
electron transport chain process?
 By two electron carriers NADH and
FADH,
ACTIVITY 6 - BASIC CHEMISTRY
THERMODYNAMICS: SOLVE THE
CHALLENGE OF STORING
RENEWABLE ENERGY
 How is the energy for this process
stored? Electricity is used to pump the
water from the sea to the reservoir?
 Water flowing back down drives a
generator which makes electricity.
 Can you think of a reason why this way
of storing energy is not ideal for our solar
power plant?
 Because the solar power plant is
located in a desert, far from large bodies
of water.
 Lithium-ion batteries are not used for
long term storage of energy. Why do you
think that is?
 Because they lose charge over time
 How do you think we can determine how
much energy is stored in a chemical
compound?
 Burn it and measure how much heat it
releases.
 The unit of enthalpy is J. What is J a
measure of?
 Energy
 Which part of the equation stays constant
in this experiment?
 v (volume)
 What happens to the equation AH = AU
+ VAp if the change in pressure is very
small?
 Then AU >> VAP, so we can assume
AH = AU
 Which of these statements regarding the
storage materials is false?
 LP gas and gasoline have the same
specific energy and thus are equally well
suited as fuels for a vehicle
 The combustion of octane is an
exothermic reaction. Which statement is
true for an exothermic reaction?
 It releases heat because the reactants
have a higher energy than the products.
 What can you then say about the
formation of a fuel?
 The reaction for formation of the fuel
is the opposite, so it is endothermic.
 Do you know what entropy is?
 Entropy is a measure of the disorder
of a system

 Which of these reactions leads to an
increase in the entropy of the system?
 Ice melting
 Since the unit of AH is J, the unit of
temperature is K and the unit of AS is
JK, what is then the unit of AG?
Remember that all the equations are
available in the media tab.
 The unit for AG is Joules.
 The reaction at the holo-table is
spontaneous. It has a negative AH and a
positive AS. What will be the sign of AG
for a spontaneous reaction?
 We subtract a positive value from a
negative value, so AG will be negative.
 What happens to the equation AG = AH -
TAS when the temperature is OK?
 When T=0 then AG = AH
 Let us start with a familiar example: The
melting of ice. Where on the right screen
do you think this process belongs?
 Endothermic and increases entropy
(top right corner).
 But what happens when you increase the
temperature? Choose a hypothesis:
 AG will change sign when T gets high
enough
 Observe this graph by clicking on the
VIEW IMAGE button. At what
temperature does the melting of ice start
to be spontaneous?
 At 273 K (0 °C)
 Where on the right screen do you think
this process belongs?
 Endothermic and decreases entropy
(bottom right corner)
 Since photosynthesis is endothermic, the
reaction is not spontaneous at T=0. What
do you think happens when you increase
the temperature? Choose a hypothesis:
 AG will stay positive
 This one should be easy for you. It is the
reaction from the calorimeter: The
burning of a fuel. Where on the right
screen do you think this process belongs?
 Exothermic and increases entropy (top
left corner)
 The reaction you looked at in the
calorimeter will be spontaneous at T=0
because it is an exothermic reaction, but
will it stay that way? Choose a
hypothesis:
 AG will change sign when T gets high
enough.
 Let's look at one final case: The rusting
of iron. What do you think, where does
this belong?
 Exothermic and decreases entropy
(bottom left corner)
 So rusting of iron is spontaneous at T=0
because it is an exothermic reaction, but
what happens at higher temperatures?
Choose a hypothesis:
 AG will change sign when I gets high
enough
 Does this mean that cars don't rust in the
summer? Look at the graph. At what
temperature does the rusting of iron stop
being spontaneous?
 At 1499 K (1226 °C)
 Can you tell which one of the four
examples corresponds to the making of a
hydrocarbon fuel from co, and water?
 AH > 0 and AS < 0 - like
photosynthesis