Lecture 3

Question 1

What are growth factors?

Answer 1

Peptides that are released bye signalling cells to impact on the fate of target cells.
Several growth factors have been shown to be instrumental in controlling differentiation of neural cells.

Question 2

How do you identify genes involved in changes in gene expression?

Answer 2

Candidate approach
Global analysis

Question 3

Describe candidate approach.

Answer 3

Informed analysis of genes you think may change in response to growth factor treatment using quantitative PCR.

Question 4

Describe global analysis.

Answer 4

RNA-sequencing allows all gene expression changes to be interrogated to identify cohorts of genes involved in differentiation.

Question 5

What are the 6 principles of RNA sequencing?

Answer 5

1. isolate RNA from samples
2. fragment RNA into short segments
3. convert RNA fragments into cDNA
4. ligate sequencing adapters and amplify
5. perform NGS sequencing
6. map sequencing read to the transcriptome/genome

Question 6

What do we get out of RNA sequencing?

Answer 6

Can identify all transcripts that are expressed in our cells - the transcriptome.
Can compare transcriptomes between populations to give a global indication as to what genes are changing.
Heat maps indicate how highly or lowly the genes have been transcribed in response to treatments.

Question 7

What techniques can be used to assess protein level changes?

Answer 7

Western blotting
Immunofluorescence
ELISA
Flow cytometry

Question 8

What are the advantages and disadvantages of indirect immunofluorescence?

Answer 8

Advantages - allows multi-colour imaging. Proteins are in physiological state.
Disadvantages - protein of interest may not have a suitable antibody. Expensive and time consuming.

Question 9

What are the advantages and disadvantages of direct immunofluorescence?

Answer 9

Advantages - quick, multi-colour imaging, cheap.
Disadvantages - signal variation between samples, relies upon cells being transfectable, fluorophore may effect normal protein activity.

Question 10

What does ELISA stand for and what does it measure?

Answer 10

Enzyme Linked ImmunoSorbent Assay
Quantifies the level of a specific protein in a complex protein mixture (cell lysate).

Question 11

What are the 5 key stages of an ELISA?

Answer 11

1. Cell lysate added to vessel containing a capture antibody.
2. Target protein (analyte) specifically binds to the capture antibody.
3. Vessel is washed and primary antibody is added which binds to the target protein.
4. Secondary antibody is added and selectively binds to primary antibody.
5. Secondary antibody-conjugated enzyme is activated to generate a colour change.

Question 12

What is the colour change at the end of an ELISA experiment proportional too?

Answer 12

The quantity of target protein in the mixture.

Question 13

What is optical density directly proportional too when ELISA plates are read using spectrophotometers?

Answer 13

Optical density is directly proportional to protein concentration.

Question 14

What are the aims of clinical trials?

Answer 14

To improve current treatments available to patients.
To better screening and diagnostic techniques for diseases.
To prevent disease.

Question 15

What is the aim of a clinical trial using a new drug?

Answer 15

To determine if the new drug is:
1. safe and effective for a defined disease.
2. more effective/safer than any existing treatment.
3. effective for additional diseases.

Question 16

What information can be gained from animal models being treated with the new drug?

Answer 16

Give information on whether the new compound is effective against disease in a physiological context.
Allows predictions to be made for dosing and scheduling in man.
Provides information on toxicities that may be encountered.

Question 17

When would someone be in a position to conduct a clinical trial?

Answer 17

When a drug has shown promise in mammalian cell culture and animal model systems.

Question 18

What 3 questions should be asked before conducting a clinical trial?

Answer 18

1. what research questions are being addressed in the trial?
2. why should the trial be conducted?
3. is the trial adequately designed to answer the questions it addresses?

Question 19

Provide an example of concept development and experimental design for a clinical trial.

Answer 19

A clinician treats patients with disease X.
Talking to a subset of patients that had favourable outcomes it is noticed they are heavy coffee drinkers.
This raises the question ‘does caffeine intake improve the survival of patients with disease X?’
The experimental design that is used to test this hypothesis is articulated in a clinical trial protocol.

Question 20

How can you control bias in clinical trials?

Answer 20

1. randomisation
2. blinding

Question 21

Describe randomisation in controlling bias in clinical trials.

Answer 21

Assigns patients to treatment arms by chance.
Eg. if it is a trial with an experimental and control arm only then patients will be assigned to a single group by tossing a coin.

Question 22

Describe blinding in controlling bias in clinical trials.

Answer 22

Ensures that neither patients, doctors and researchers know to which group each patient has been assigned and is important to prevent false interpretation of data or patients receiving different care.

Question 23

What are the 3 types of blind trials?

Answer 23

Single blind - patient only
Double blind - patient and doctor
Triple blind - patient, doctor and researcher

Question 24

What are phase I clinical trials for?

Answer 24

To evaluate safety - determines a safe dose range and identifies side effects.
Generally involve small groups of healthy volunteers.

Question 25

Why do phase I trials for anti-cancer drugs typically involve patients who have failed conventional treatments and not healthy volunteers?

Answer 25

Anti-cancer agents are generally too toxic to give to healthy volunteers.

Question 26

What is maximal tolerated dose?

Answer 26

MTD
Defined as the highest dose of drug that does not elicit significant toxicity.

Question 27

What us dose limited toxicity?

Answer 27

DLT
Where the patient experiences any number of unacceptable adverse reactions to the drug.

Question 28

What is the 3+3 model for identifying maximal tolerated doses of drugs?

Answer 28

Enrol 3 patients at a dose level.
If first 3 patients don’t have dose limit to toxicity, add another 3.
Progresses on until you have to stop escalating the treatment.

Question 29

What is monitored closely in phase I clinical trials to assess?

Answer 29

Drug levels in blood - info on drug kinetics in the body.
Predicted effects of drugs on patients - biomarker analysis.
Toxicity - unwanted side effects.

Question 30

What are pharmacokinetic studies?

Answer 30

What the body does to the drug.
Eg. what concentrations are achieved in the blood? How long are they maintained?

Question 31

What are pharmacodynamic studies?

Answer 31

What the drug does to the body.
Eg. measure change in enzyme activity to show the drug acts on planned target. Toxicity.

Question 32

What are phase II clinical trials for?

Answer 32

To see if the new treatment is effective using defined biomarkers/treatment response in patients.
Further evaluates safety.
Generally involve larger groups of patients (40-100).

Question 33

What are phase III clinical trials for?

Answer 33

To further determine effectiveness and side-effects of the new treatment.
Strict and well-defined eligibility criteria and patients enrolled must meet these criteria.
Generally involves more than 200 people for statistical validity.
Assesses whether the new drug is superior to treatment already available.

Question 34

What are phase IV clinical trials for?

Answer 34

Continues testing after the drug or treatment has been approved or marketed.
Collects information about their effect ins specific population and side effects from long-term use.

Question 35

What are the 7 ethical requirements for clinical trials?

Answer 35

1. collaborative partnership
2. social value
3. scientific value
4. fair subject selection
5. favourable risk-benefit ratio
6. independent review
7. informed consent

Question 36

Describe collaborative partnership in regards to ethics of clinical trials.

Answer 36

Must involve the community in which it occurs.
Requires community participation in planning, conducting and overseeing research and integrating results into the health system.
Avoidance of supplanting existing health care services and the sharing of rewards with the community.

Question 37

Describe social value in regards to ethics of clinical trials.

Answer 37

Research must lead to improvements in health or advancement in generalisable knowledge.
Must consider how the research will improve the health of participants in the research, community in which the research is conducted and patients worldwide.

Question 38

Describe scientific validity in regards to ethics of clinical trials.

Answer 38

Research must be conducted in a methodological rigorous manner that is practically feasible.
It must also produce reliable and valid data that can be interpreted.
Invalid research could include underpowered studies, studies with biased endpoints or studies with insufficient subjects.

Question 39

Describe fair subject selection in regards to ethics of clinical trials.

Answer 39

Scientific objectives of the study should guid inclusion criteria and targeted populations, NOT vulnerability or privilege.
Lowering the risk and enhancing generalisability can then be considered.
Groups must not be excluded without fair scientific reason.

Question 40

Describe favourable risk-benefit ratio in regards to ethics of clinical trials.

Answer 40

Research must be conducted in a manner consistent with the standards of clinical practice and evaluate the likelihood and magnitude of harm.
If potential benefits to the individual outweigh the risks to the individual then proceed.

Question 41

Describe independent review in regards to ethics of clinical trials.

Answer 41

Independent review of research minimises the conflicts of interest of the investigators of the research.
Also assure society it will not benefit from the abuse of subjects.

Question 42

Describe informed consent in regards to ethics of clinical trials.

Answer 42

Researchers must prepare an information leaflet about the trial for patients.
The research ethics committee must check that the info leaflet is clear and accurate and written in easy to understand English.

Question 43

What 4 elements does informed consent consist of?

Answer 43

Competence of the subject
Disclosure of information to the subject
Understanding or comprehension by the subject
Voluntariness of the decision