BMS336 Modelling Human Disease and Dysfunction

Question 1

What are the top ten leading causes of death in the US?

Answer 1

1. Heart disease
2. Cancer
3. Chronic lower respiratory disease
4. Accidents
5. Stroke
6. Alzheimer’s disease
7. Diabetes
8. Influencer and pneumonia
9. Kidney disease
10. Suicide

Question 2

What is diabetes mellitus?

Answer 2

Diabetes mellitus is a disease in which the body is no longer able to carefully control blood glucose, leading to abnormally high levels of blood glucose (hyperglycaemia). Persistently elevated blood glucose can cause damage to the body’s tissues, including the nerves, blood vessels. This means that the core homeostatic blood glucose system would be imbalanced. This is required for respiration.

Question 3

What is chronic lower respiratory disease?

Answer 3

Chronic lower respiratory disease (CLRD) is a collection of lung diseases that cause airflow blockage and breathing-related issues, including primarily chronic obstructive pulmonary disease (COPD) but also bronchitis, emphysema, and asthma. Inflammation plays a key role in CLRD

Question 4

What is heart disease?

Answer 4

Heart disease is a term used to describe several conditions, many of which are related to plaque build-up in the walls of the arteries. As the plaque builds up, the arteries narrow, this makes it more difficult for blood to flow and creates a risk for heart attack or stroke.

Question 5

What is cancer?

Answer 5

Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. If the spread is not controlled, it can interfere with essential life-sustaining systems and result in death

Question 6

What are neurodegenerative diseases?

Answer 6

Neurodegenerative diseases are a group of diseases characterized by the loss of nerves. There are many different types of neurodegenerative disease, including Parkinson’s disease and motor neuron disease. As their loss increases, this results in death.

Question 7

What is anxiety and depression?

Answer 7

Anxiety and depressive disorders are poorly-characterised disorders characterised by a range of emotional, behavioural and physical symptoms

Question 8

What are cerebrovascular diseases?

Answer 8

Cerebrovascular diseases are conditions that develop because of problems with the blood vessels that supply the brain. Four of the most common types of cerebrovascular disease are:

• Stroke
• Transient ischemic attack (TIA)
• Subarachnoid hemorrhage
• Vascular dementia

Question 9

What is dementia?

Answer 9

Dementia is an overall term for diseases and conditions characterized by a decline in cognitive function that affects a person’s ability to perform everyday activities. Dementia is caused by damage to nerve cells in the brain. Because of the damage, neurons can no longer function normally and may die. The damage eventually impairs ability to carry out core body function.
Dementia can probably arise due to loss of vascularization, loss of glial support cells, nerve degeneration

Question 10

What is a model organism?

Answer 10

A model organism is a non-human species that is extensively studied to understand biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms

Question 11

How are model organisms able to be used to study humans?

Answer 11

This is made possible due to the common descent of all living organisms and the conservation of developmental pathways and genetic material over the course of evolution

Question 12

What do model organisms allow to study?

Answer 12

Using animal models enables a ‘whole organism’ or ‘systems’ analysis. An understanding of disease and deterioration requires a comprehensive understanding of the whole organism, and the interaction between its different components

Question 13

Give example of model organisms

Answer 13

• Drosophila
• Zebrafish
• Chick
• Mice

Question 14

Why do we use model organisms?

Answer 14

• It is ethically appropriate
• It is now very easy to generate a transgenic animal that has a specific mutation in a specific gene which can be used to model human susceptibility disease
• They can also be examined in large numbers to ensure that the outcomes are statistically significant and don’t just occur by chance

Question 15

What is needed in a model organism?

Answer 15

• Need models where it is possible to examine an individual over the life course to look at progression, systems, systems interactions
• Need models where it is possible to examine Gene x Environment interactions.

Question 16

How can genotype of an organism effect likelihood of disease?

Answer 16

• Genotype drives development and confers the potential for health and wellbeing across the life course and therefore the likelihood of some diseases
• E.g. BRCA1 gene increases the risk of breast cancer
• Disease/dysfunction is increasingly understood to arise due to Gene x Environment interactions

Question 17

What are GWAS studies?

Answer 17

Genome wide association studies

Question 18

How many protein coding genes are in the human genome?

Answer 18

The human genome consists of 3 billion DNA base pairs and carries about 20000 protein coding genes

Question 19

What is the 100,000 genome project?

Answer 19

NHS started the 100,000 genomes project

• Thought that it would lead the way in personalised medicine
• They would sequence the genome of 100,000 genomes to allows scientists and doctors to understand more about specific conditions

Question 20

What do GWAS studies identify?

Answer 20

GWAS studies identify variants that correlate with disease susceptibility. However, it does not tell us where or when the gene is expressed. Therefore, need model organisms.

Question 21

How can we use model organisms to see where a protein is expressed?

Answer 21

Use immunohistochemistry to tell us where and when a gene is expressed or a protein is synthesised

Question 22

How can we use model organisms to see the function of a gene?

Answer 22

Gain or loss of function approaches to tell us the function of the gene product. We can now do this in a conditional manner so that it is a tissue specific deletion of a gene

Question 23

How do you produce a tissue specific knockout?

Answer 23

• Add lox sites either side of gene that wants to be knocked out This is referred to as a floxed allele as it is flanked by lox sites
• Put this gene back into the mouse so that the normal gene is replaced with the engineered construct
• Identify a promotor that governs a tissue specific gene. Whatever is downstream of this promotor will only be expressed in that tissue at that time. Engineer coding sequence for the enzyme cre recombinase downstream of this promotor and make a second transgenic animal.
• Combine the mice and the cre recombinase acts on the lox sites and causes the gene to be excised wherever cre recombinase is activated. It is only activated where the tissue specific promotor is activated meaning only knocked out in specific tissue

Question 24

What are transgenic reporter lines?

Answer 24

Engineered a transgenic animal so that it gives a colour report when a gene is active (fluorescence). This is used as a way of identifying and following a cell, tissue or subcellular organ

Question 25

What are the uses of transgenic reporter genes?

Answer 25

• Can then use these transgenic animals to visualise the cells and tissues in real time
• Can also be used to isolate specific tissues using FACS sorting. This machine sorts fluorescent cells from non-fluorescent cells so can have a pure sample of the tissue that is being investigated

Question 26

How do you make a transgenic reporter line?

Answer 26

• Specific DNA sequence that governs gene expression in tissue of interest
• Take the coding sequence and engineer it so it is upstream to a fluorescent protein
• Make a transgenic mouse in which this gene is incorporated meaning that the reporter is only expressed in that particular tissue

Question 27

Why would we want to study cell behaviours in vivo and in vitro?

Answer 27

• Development of new therapeutics
• Experiments that can’t be done in vivo – e.g. a recording, cell culture without influence of the body
• Used to grow organoids

Question 28

What is diabetes?

Answer 28

A condition in which the body can’t regulate its blood glucose levels. This happens as a consequence of not producing insulin or by exhibiting a resistance to the effects of insulin

Question 29

What is the difference between type 1 and type 2 diabetes?

Answer 29

• In type 1, the pancreas doesn’t have the cells required to make insulin meaning blood glucose builds up. This is a genetic condition.
• In type 2, the pancreas can make insulin which then enters the blood stream. However, the cells of the body are insulin resistant meaning blood glucose cannot be up taken into the cells and builds up in the blood vessels. Over time this results in a second event: the cells of the pancreas become so damaged that they become depleted meaning that the condition is exacerbated. This is a chronic progressive disease.
• Both types of diabetes have a depletion of beta cells

Question 30

How is obesity related to type 2 diabetes?

Answer 30

Obesity is highly associated with type 2 diabetes

• If your waist circumference is less then 34 inches then there is a low risk
• Obesity is linked to the accumulation of excess fat in ectopic sites such as the liver and skeletal muscle, instead of its accumulation in adipocytes. This ectopic fat accumulation in the liver and skeletal muscle is associated with insulin resistance and type 2 diabetes.

Question 31

What contributes to hyperglycaemia?

Answer 31

Three major metabolic defects contribute to hyperglycemia in patients with type 2 diabetes: increased hepatic glucose production, impaired pancreatic insulin secretion, and peripheral tissue insulin resistance. These all act on completely different tissues in the body

Question 32

What is the link between hyperglycaemia and obesity?

Answer 32

It is unclear if obesity triggers the changes that lead to hyperglycaemia and which change occurs first

Question 33

How does obesity increase risk of other diseases such as stroke?

Answer 33

• Central obesity leads to increase in free fatty acids and inulin resistance but it is not clear whether the free fatty acids lead to insulin resistance or the other way around.
• These all lead to an increase in LDLs and a decrease in HDLs. This leads to a build-up of plaque

Question 34

How was the role of adipocytes in type 2 diabetes investigated?

Answer 34

To test whether adipocytes are essential in stopping type 2 diabetes. Genetically modified mice with a lack of adipose tissue are characterized by hyperphagia (eat too much), hyperglycemia, insulin resistance and type 2 diabetes.
However, because of the lack of adipose tissue, the mice are lacking leptin. Is this the primary cause linked to brain dysfunction?

Question 35

How were transgenic reporter lines used to investigate causes of diabetes?

Answer 35

• In zebrafish and mice, made transgenic reporter lines that look at specific tissues that govern glucose homeostasis so can visualise what is happening in space and time.
• This allows us to analyse the tissues In healthy animals, In animals with a genetic mutation (to understand the impact of a human genetic variant) and in animals exposed to different environmental conditions (e.g. high-fat diet).
• Would need to cross two transgenic lines: a knockout and a reporter
• Could cross three reporter lines so that the pancreas, liver and adipose tissue could be visualised and then feed the fish a high fat diet and wee which of these three tissues shows the first fault

Question 36

How can double transgenic animals be used to study diabetes?

Answer 36

Double transgenic animals can be made eg if the fish is exposed to UV light, the endocrine pancreas fluoresces red and the exocrine pancreas fluoresces green. Another use of reporter animals is to look at specific populations within a tissue. The Tg (ptf1a;GFP) is a transgenic line which reports the entire exocrine pancreas; the Tgb(ins;Kaede) is a transgenic line which reports just the b-cells of the exocrine pancreas

Question 37

How can you measure cell proliferation?

Answer 37

Using bromodioxyuridine (EDU) incorporation

• DNA replicates via semi-conservative replication. Incubate the cells with EDU which is an analogue of thymidine.
• When DNA replicates, instead of replicating with thymidine, it uses EDU.
• Can use click chemistry to visualise where the EDU has been incorporated

Question 38

What was the aim of Hill et al, 2016?

Answer 38

It is thought that a potential therapy for diabetes is to restore beta cell function. Therefore, asked the question what controls beta cell numbers?

Question 39

What did Hill et al, 2016 show?

Answer 39

• Genes and chemical signals are known to shape the earliest phases of an embryo’s development, including the development of pancreatic beta cells
• More recently, researchers have started to appreciate that environmental cues, such as signals from nearby bacteria also shape animal development.
• This paper shows that certain gut bacteria (and specifically a protein that they secrete) are necessary for the pancreas to populate itself with a robust number of beta cells during development.

Question 40

How did Hill et al, 2016 investigate how bacteria affect beta cell development?

Answer 40

• Normally, the number of beta cells in zebrafish larvae increases steadily in the first few days after hatching.
• However, developing zebrafish that were raised in a microbe-free environment maintained the same number of beta cells as they had before hatching. These events could easily be monitored in a Tg (Ins:GFP) reporter line.
• Low numbers of beta cells means less insulin
• Exposing the microbe-free fish to certain bacteria restored their beta cell populations to normal levels

Question 41

What did Hill et al, 2016 discover about what the gut bacteria produce?

Answer 41

• Further investigation revealed that these bacteria release a protein called BefA that restores normal numbers of beta cells.
• They then exposed the fish that were grown in a germ-free environment and incubate them with BefA. This also restored beta cell populations
• They wanted to know whether these fish didn’t have beta cells because of not enough proliferation or too much apoptosis.
• They used EDU experiment and found that the protein BefA causes the beta cells to proliferate

Question 42

Did Hill et al, 2016 also investigate the role of bacteria in humans beta cell development?

Answer 42

• Some bacteria in humans produce proteins that are similar to BefA.
• Performed experiments that showed that these proteins also stimulate beta cell development in microbe-free fish

Question 43

What understandings have the discoveries of Hill et al lead to?

Answer 43

Long term antibiotics may affect this as this may leave the person with a pancreas that is more susceptible to diabetes

Question 44

Give evidence that the pancreas can regenerate?

Answer 44

• Experimental ablation of β-cells by chemical treatment or partial pancreatectomy in rodents is followed by significant recovery of the β-cell mass, indicating that the adult pancreas has the capacity to regenerate.
• This is the same in humans. Recovery is not as significant but there is still increased recovery

Question 45

How can the pancreas’s property of regeneration lead to new drugs?

Answer 45

This regenerative capacity could potentially be exploited therapeutically—if the underlying mechanisms were better understood
- For example, as a treatment of diabetes - regenerate the beta cells to allow insulin production again

Question 46

What would need to be more understood to be able to use the pancreas’s property of regeneration?

Answer 46

Although the transcriptional cascade that regulates β-cell formation is well characterized, the extrinsic signals that regulate β-cell regeneration remain unclear

Question 47

What were Anderson et al, 2012 investigating?

Answer 47

The regeneration of pancreatic beta cells in vivo

Question 48

What transgenic animals did Anderson et al, 2012 use?

Answer 48

Double transgenic fish

• In which beta cell numbers are reported through Tg(ins:Kaede)
• Also transgenic for ins:CFP-NTR. The pancreatic β-cells are conditionally targeted for ablation by using β-cell specific expression of nitroreductase [Tg(ins:CFP-NTR)], which converts Metronidazole (MTZ) into a cytotoxic product
• When MTZ is added, the beta cells are killed. Typically, only 3-7 cells are left.
• This transgenic fish is created by the crossing of Tg(ins:Kaede) and Tg(ins:CFP-NTR) fish
• They also used a control fish in which NTR is not expressed meaning the beta cells are normal

Question 49

What did Anderson et al, 2012 do?

Answer 49

• The double-transgenic fish, treated with MTZ, were placed into 100s of wells.
• Added a different compound from a library to each well
• They then asked if any compound can cause beta cells to regenerate
• After 2 days of recovery, β-cell regeneration can be easily quantified in double-transgenic larvae, Tg(ins:CFP-NTR);Tg(ins:Kaede).

Question 50

What did Anderson et al, 2012 find?

Answer 50

• They found that some compounds allowed for more than a two-fold increase in beta cell regeneration and these compounds converged on the adenosine signalling pathway.
• They then checked this by screening a library of adenosine signaling pathway activators and found that two of those compounds also increased beta cell regeneration.
• This then resulted in six compounds capable of increasing beta cell regeneration and all converging on the adenosine signalling pathway.

Question 51

How does Anderson et al, 2012 have the potential for new drugs?

Answer 51

The six compounds capable of increasing beta cell regeneration are potentially new pre-clinical compounds that can now be taken through to mouse/rodent studies and then through to phase II and III trials

Question 52

Why is it important to understand how the brain organises glucose homeostasis when investigating diabetes?

Answer 52

It is highly likely that a primary causal factor in type 2 diabetes lies in the interaction of the brain with peripheral tissues such as the gut, the liver, the endocrine pancreas, adipose tissue and others. This will typically manifest as dysfunctional eating, energy metabolism and/or autonomic activity.

Question 53

How could the dysfunctional interactions between the brain and body lead to diabetes?

Answer 53

• This could occur via abnormal signaling by peripheral organs to the brain (e.g., indicating that insufficient fat is present, thus triggering increased food intake and consequent increased body fat)
• By abnormal signaling from the brain to other organs (e.g., reduced signaling to the endocrine pancreas and liver).

Question 54

What is leptin?

Answer 54

Leptin is secreted by adipocytes and signals to the brain the status of the body’s energy content

Question 55

What occurs in a leptin knockout mouse?

Answer 55

In leptin knockout mice, the mice are obese, diabetic, infertile and hypoactive
- This tells us that this hormone somehow leads to obesity

Question 56

All of the peripheral tissues have leptin receptors so how are we certain that leptin is linked to the brain?

Answer 56

Selective deletion of leptin receptor in neurons leads to obesity (Cohen et al, 2001)

• Selectively knocked out leptin receptors in neurons and it still lead to obesity. The same phenotype as the leptin knockouts
• Mice that had leptin receptors knocked out in the liver still appeared normal
• This shows that leptin exerts its action via neurons

Question 57

How have animal model studies show that the brain mediates the majority of leptin’s action on energy homeostasis?

Answer 57

• Mice lacking leptin receptor signaling are obese, diabetic, infertile, and hypoactive
• Deletion of leptin receptors in neurons induces obesity (Cohen et al., 2001)
• Inducing expression of leptin receptors in the neurons of leptin knockout mice rescues the obesity phenotype
• Intracerebroventricular (icv) administration of leptin in mice lacking leptin receptor signaling causes reduction of body weight and food intake

Question 58

How do we know that leptin specifically affects neurons of the brain and not just the CNS?

Answer 58

• Identify a brain specific promotor so that leptin is only knocked out in the brain
• Could also do a hypothalamic specific promotor as that is where homeostasis occurs
• Slowly choose promotors that are more specific to isolate where leptin is exerting its action

Question 59

Where in the brain has it been shown that the leptin receptor is expressed?

Answer 59

The neurons expressing leptin receptor were in the hypothalamus, in a region called the Arcuate (Arc) nucleus

Question 60

How does the Arcuate (Arc) nucleus regulate food intake?

Answer 60

• Two main types of neurons within this nucleus. One type that produce the hormone NPY and the other that produce the hormone Pomc. These neurons act antagonistically on a common downstream neuron.
• NPY expressing neurons stimulate food intake. Pomc expressing neurons reduce food intake

Question 61

How does leptin signalling act on the Arcuate (Arc) nucleus?

Answer 61

It seems that abnormal leptin signalling results in abnormal balance of the two cell types which has a knock-on effect of abnormal signalling from the brain to other organs e.g. pancreas and the liver

Question 62

Give the current model for the hypothalamic regulation of hepatic glucose production

Answer 62

Leptin-sensing neurons in the hypothalamic ARC receive input regarding energy stores, and in response to this input, pathways that increase hepatic vagal tone to the liver are activated, increasing hepatic insulin sensitivity

Question 63

What is NYP?

Answer 63

NPY is a neuropeptide (Y) whose action leads to the brain to co-ordinate an array of activities that stimulate food intake and reduce energy expenditure

Question 64

What is the effect of leptin on NYP neurons?

Answer 64

Leptin inhibits NPY neurons

Question 65

What is Pomc?

Answer 65

Pomc is a neurohormone whose action leads to the brain to co-ordinate an array of activities that neurons reduce food intake and increase energy expenditure

Question 66

What is the effect of leptin on Pomc neurons?

Answer 66

Leptin stimulates Pomc neurons

Question 67

How are the energy pathway and the stress pathway related?

Answer 67

Pomc is produced as one peptide and then cleaved. One part of the peptide becomes ACTH (adrenocorticotropic hormone) which is the hormone responsible for the stress response. This shows that the energy pathway and the stress pathway are related

Question 68

What are the hypothalamic stem cells known as?

Answer 68

Tanycytes

Question 69

Describe tanycytes

Answer 69

Like other neural stem cells, hypothalamic stem cells have a radial glial-like appearance, with the cell body at the ventricle and a projection that is used as a scaffold. If these are stem cells then they can divide asymmetrically to give rise to a radial glial cell and a second daughter cell which will develop into a neuron and uses the radial glial scaffold to migrate which then project directly into the arcuate nucleus

Question 70

Where are Pomc and NPY neurons generated from?

Answer 70

In development and potentially through life, the Pomc and NPY neurons are generated from hypothalamic stem cells

Question 71

Why is it thought that Pomc and NPY neurons are generated from tanycytes?

Answer 71

Glucose requirements may change considerably throughout life e.g. pregnancy, adolescence. It is therefore thought that Pomc and NPY neurons can be generated from hypothalamic stem cells through life to anticipate, and/or respond to the changing needs of the body

Question 72

What techniques in animal models are used to investigate the generation of pomc and NPY neurons?

Answer 72

• Identify different populations that develop over time, through markers
• Lineage-trace the stem cell
• Identify key genes that maintain each cell. Ask if daughter cells fail to differentiate when these genes are knocked out.
• Use the promoters of these genes to make cytotoxic transgenes to eliminate cell populations

Question 73

What is islet1?

Answer 73

Iselt1 is a transcription factor that upregulates Pomc neurons

Question 74

Give a study into the generation of pomc and NPY neurons?

Answer 74

Nasif et al, 2015

Question 75

What did Nasif et al, 2015 show?

Answer 75

• Investigating if there was a transcription factor important for the induction of Pomc neurons
• Found that islet1 is expressed in the hypothalamus just before the onset of Pomc neurons and that wherever Pomc is seen so is iselt1
• Showed that iselt1 is a transcription factor that upregulates Pomc transcription by showing that islet1 binds to the promotor of Pomc. If these binding sites are mutated then transcription is not increased
• If islet1 is inactivated then it causes obesity and hyperphagia in animal models
• This is a continuous process that occurs throughout life

Question 76

What have hypothalamic stem cells been shown to produce?

Answer 76

• Express Fgf10 and Pea3 (allows them to respond to FGF) this is a survival factor that keeps them in cell cycle
• Also found that they express Shh which can promote differentiation

Question 77

Outline the steps in tissue specific lineage tracing

Answer 77

• Find a gene that is expressed specifically in the desired cell type and clone its promotor
• Downstream of the promotor clone the enzyme Cre fused to ERT2, which means that the Cre recombinase is only activated when tamoxifen is injected.
• Make a transgenic animal in which a reporter gene e.g. GFP is present downstream to the tissue specific promotor but is prevented from being transcribed due to the addition to a stop sequence. These stop sequences are floxed sites
• Recombine these two animals to produce a double transgenic animal.
• When tamoxifen is injected, the stop codons will be recombined out so that the reporter gene will be activated in a specific tissue at a specific time

Question 78

How have tissue specific lineage tracing been used to study tanycytes?

Answer 78

• Find a promotor that is only expressed in a tanycytes and fuse to creER2 and create a double transgenic mouse with a reporter gene
• Inject tamoxifen to activate reporter gene. If this was done straight away, then only the stem cells will be reported. This ensures specificity
• Do this again in a sister mice but this time allow the mice to live for a longer period. You would therefore expect to see reported neurons in the arcuate nucleus (Pomc) that have come from the labelled stem cells

Question 79

What did Robins et al, 2013 show?

Answer 79

• Used the tanycytes specific promotor GLAST to carry out tanycyte specific lineage tracing
• Immediate sacrificing showed that only a subset of tanycytes were labelled (alpha tanycytes)
• Further tracing showed that these tanycytes can either self-renew or give rise to neurons of the arcuate nucleus

Question 80

Why are tanyctes required?

Answer 80

Tanycytes are required as an anticipatory mechanism for when more neurons are needed e.g. adolescence or when pregnant. At other time in life, tanycytes don’t proliferate much as they are in homeostatic norm

Question 81

How did Robins et al, 2013 show that tanycytes increase proliferation at certain stages in life?

Answer 81

They injected FGF into the third ventricle of a mouse and ask if FGF causes proliferation of the tanycytes. This did lead to a huge increase in tanycytes differentiation. Can see the migration of the daughter cells
This suggests that the physiological and developmental signals are going to interact to govern how a neural stem cell will respond

Question 82

What is thought to cause obesity and type 2 diabetes in relation to Pomc/NPY neurons?

Answer 82

If the balance of Pomc/NPY neurons is disrupted in obesity and type 2 diabetes

Question 83

Why is the balance of pomc and NPY neurons hard to investigate?

Answer 83

However, this is difficult to prove as can’t study on live people and difficult to locate the correct part of the hypothalamus. It is also unclear what the normal number of pomc and NPY neurons are as this would vary between individuals. This is why animal models are required.

Question 84

What did Scarlett et al, 2016 investigate?

Answer 84

This paper injected FGF1 into the third ventricle of rodents. One single central injection of FGF1 induced sustained remission in a diabetic rodent.
This involves a novel mechanism involving the brain. T

Question 85

What did Scarlett et al, 2016 conclude?

Answer 85

They conclude that If inject FGF1 then it will stimulate tanycytes activity which give rise to new neurons that are resetting the glucose metabolism regulation

Question 86

What unanswered questions are there that still need to be answered to prove the relationship between type 2 diabetes and tanycytes?

Answer 86

• Are neurons that centrally regulate T2D lost, damaged, or altered in number in T2D?
• Are these neurons restored after FGF infusion, and are they restored from Fgf-responsive tanycytes?
• Is it clear that the action of FgF in the brain is limited to its effect on tanycytes/Arc neurons?
• Do similar cells exist in humans and could a similar mechanism operate to induce a sustained remission of T2D?

Question 87

Give examples of Chronic non-communicable diseases (CNCDs)

Answer 87

Cardiovascular conditions (mainly heart disease and stroke), some cancers, chronic respiratory conditions and type 2 diabetes

Question 88

What percentage of death do CNCDs account for?

Answer 88

These conditions account for 60% of all deaths worldwide. 80% of chronic-disease deaths occur in low- and middle-income countries and account for 44% of premature deaths worldwide.

Question 89

What is meant by multi morbidity?

Answer 89

The presence of two or more chronic medical conditions in an individual

Question 90

What are the main risk factors in CNCDs?

Answer 90

Poor diet and smoking

Question 91

What is the link between divers and CNCDs?

Answer 91

As you become diabetic, there is an increased risk of getting cardiovascular disease

Question 92

Why does hyperglycaemia increase the chance of getting CNCDs?

Answer 92

Hyperglycaemia is thought to link directly to the development of atherosclerosis

Question 93

What is metabolic syndrome and how does it occur?

Answer 93

It is unknown why central obesity leads to the increase of free fatty acids but it is clear that an increase in free fatty acids leads to an increase in insulin resistance which in turn increases apolipoprotein B and hepatic lipase which increases the number of LDLs. This is known as the metabolic syndrome.

Question 94

Metabolic disorders are increase the risk of which cancers?

Answer 94

• Breast
• Bowel
• Kidney
• Womb

Question 95

Where has research into the link between metabolic diseases and cancer come from?

Answer 95

A third of the research has come from animal models, a third from cell culture and a third from human data analysis

Question 96

What are the three main ways that metabolic diseases can lead to cancer?

Answer 96

• After the menopause, oestrogen made by fat cells can cause cells to multiply faster in the breast and womb
• Excess fat can increase levels of insulin and growth factors which causes cell proliferation. Hormones are very important in cell proliferation
• Macrophages (which are present in fat) release cytokines which encourage division in cells. Inflammatory cells usually disperse after reaching the site of inflammation (inflammation resolution). However, sometimes this does not happen properly.

Question 97

What is the insulin pathway?

Answer 97

The insulin pathway has been highly conserved and is responsible for regulating transcription factors that are important in healthy aging.

Question 98

What are ‘fancy mice’?

Answer 98

In china, mice were inbred to select for characteristics. These are called fancy mice. They are now kept in the Jackson labs in the USA.

Question 99

Why were some strains of the fancy mice susceptible to mammary tumours?

Answer 99

• They were all infected endogenously with an RNA retrovirus. This virus exists as an RNA sequence and then uses the host machinery to produce cDNA and inserts itself into the genome of the host and integrates next to some sorts of genes.
• The cDNA has a very strong promotor and if it integrates downstream to a host gene then it Can turn on and enhance its expression. This causes the host gene to be transcribed more readily.

Question 100

What genes in the fancy mice genome were the RNA retrovirus inserted by?

Answer 100

• If it is inserted next to a proto oncogene, it will be upregulated and provide a growth advantage to that cell
• A proto oncogene is a gene that usually regulates cell number but if it is mutated then it can become an oncogene

Question 101

What gene was was being upregulated to cause the mammary tumours in fancy mice?

Answer 101

Int1

Question 102

What suggested that Int1 was a growth factor?

Answer 102

The peptide chain starts with a signal sequence that suggests that Int1 is a secreted factor, a growth factor.

Question 103

What did studies in the drosophila reveal about Int1?

Answer 103

• Nüsslein-Volhard and Wieschaus were carrying out a mutagenesis screen in drosophila to identify patterning genes. One of the segment polarity genes they identified was a gene called Wingless.
• This gene was homologous to Int1 so was named Wnt1

Question 104

How was the Wnt signalling pathway discovered?

Answer 104

Was discovered through analysis if model organisms such as mice, drosophila, xenopus and mammalian cell culture

Question 105

Briefly outline the Wnt signalling pathway

Answer 105

Wnt1 is the ligand and in the canonical signalling pathway it binds to the receptor frizzled. This allows beta catenin to be stabilised and enter the nucleus which then binds to coactivators e.g. Groucho to activate target genes

Question 106

What genes are activated by Wnt signalling?

Answer 106

• Some of the key genes that are upregulated are genes that directly govern cell proliferation e.g. CmyC and Cyclin D1
• This provides evidence that Wnt is a growth factor

Question 107

How was the RNA retrovirus causing breast cancer in fancy mice?

Answer 107

In the mice that have mammary tumours, Wnt was being overexpressed due to the insertion of the cDNA of the retrovirus causing increased cell proliferation in the cells responding to Wnt

Question 108

What is epistasis?

Answer 108

Epistasis is a way of ordering genes in a pathway. For example, to work out if beta catenin is upstream or downstream of frizzled, you could knockout frizzled and then artificially activate beta catenin. If this rescues the phenotype then beta catenin is downstream.

Question 109

What are the two ways of activating a signalling pathway?

Answer 109

Increasing activity of activators or decreasing activity of suppressors

Question 110

What are tumours suppressor genes?

Answer 110

Genes that suppress the over activation of a signalling pathway that controls cell proliferation

Question 111

Give example of tumour suppressor genes

Answer 111

APC and Axin

Question 112

What must happen to a tumour suppressor gene for it to cause cancer?

Answer 112

For tumour suppressor genes to cause activation of a pathway, both copies of the gene would need to be inactive. Therefore, cancer often occurs at a higher age as both copies need to be mutated.

Question 113

How can the Wnt pathway lead to cancer?

Answer 113

Cancer is therefore commonly caused by the upregulation of beta catenin (activator of pathway or dominant oncogene) or the loss of both copes of APC or Axin (suppressors of the pathway or tumour suppressor genes).

Question 114

How have animal models help in discovering biomarkers being used in clinical practice?

Answer 114

• If can predict elevated levels of beta catenin is likely to expose an individual to enhanced proliferation, you can biopsy tissue to see if there are elevated levels of beta catenin. This can allow for the detection of early stage cancer
• This highlights the importance of animal model studies when discovering these biomarkers

Question 115

How was it discovered that Wnt lead to the overactive proliferation of stem cells?

Answer 115

• Can also use animal models to produce transgenic reporter lines to see if Wnt signalling is being activated in a specific cell. These experiments showed that the cells that are initially activated by wnt are tissue specific stem cells. This lead to the idea that Wnt is particularly promoting enhanced proliferation in tissue specific stem cells leading to uncontrolled self-renewal of these stem cells
• Particularly studies in the gut crypt

Question 116

How can cancer be a multistep disease?

Answer 116

Wnt signalling activation causes an adenoma which is a proliferating section of cells which can then develop into a metastatic tumour. This occurs over time and it is when the tumour becomes metastatic that it becomes lethal

Question 117

How does cancer progress from an adenoma to a tumour?

Answer 117

Involves the inflammatory pathway

Question 118

What mechanism of action of the involvement of the inflammatory pathway in cancer progression has been proposed due to animal model studies?

Answer 118

• As tumour grows, its demand for nutrients and oxygen outstrips its supply
• Cancer cells begin to secrete proinflammatory signals, including cytokines
• Macrophages invade tumour and begin to secrete
• Even more cytokines that kick-start angiogenesis – ingrowth of new blood capillaries
• Inflammatory signals and cells also help break down extracellular matrix, to promote metastasis

Question 119

Why should we use animal models?

Answer 119

• Animal model studies can help us understand mechanism

- Animal modeling can be used for pre-clinical therapeutic development

Question 120

What is the prevalence of melanoma?

Answer 120

Melanoma is a skin cancer and is the fifth most common cancer in the UK (2014), accounting for 4% of all new cases and for 2500 deaths per year.

Question 121

What is the most common skin cancer?

Answer 121

Basal cell carcinoma

• More common but also more treatable
• These are caused by mutation in the patched receptor

Question 122

Wha is a melanoma?

Answer 122

Melanoma develops from melanocytes, the cells that form moles. Moles come in all flavors and even for the most trained eye, it’s extremely difficult to distinguish a normal, or atypical but benign, from a malignant melanoma

Question 123

Are melanomas treatable?

Answer 123

When discovered early, it can be excised without change or recurrence but when it starts to spread into the dermis and lymph it is a very deadly disease with no cure

Question 124

What are the main risk factors for melanomas?

Answer 124

• Age
• Gender
• Skin type

Question 125

Why is age a risk factor for melanomas?

Answer 125

This is because you accumulate somatic mutations during your life. Not surprisingly, this is a risk factor for very many cancers

Question 126

Why is gender a risk factor for melanomas?

Answer 126

• It is unclear why this is but It could be that males have a higher somatic mutation rate:
• It could also be because of hormonal or other physiological differences.
• Finally, psychosocial factors may play a role, maybe males due to their behavior more exposed to carcinogens, alcohol, smoking, not bothered to use sunscreen

Question 127

Why is skin type a risk factor for melanomas?

Answer 127

If you have a very sensitive skin you have 24 fold higher lifetime risk in getting melanoma. If you have had blistering sunburn more than five times in your life, then the risk of melanoma increases by 50%

Question 128

How can you be genetically predisposed to melanomas?

Answer 128

• Mutations in genes that are responsible for the repair of DNA damage caused by UV predispose people to melanomas . Xeroderma pigmentosum genes are an example of these
• Another genetic factor is a mutated Melanocortin receptor. This is often results in people having red hair

Question 129

Give examples of mutations that could increase the chance of developing a melanoma?

Answer 129

• CDKN2A gene. This gene encodes for two proteins p14 and p16, both are involved in cell cycle control and help to prevent cells from entering the cell cycle when DNA damage is present. If these genes aren’t there then cells don’t enter apoptosis. These are examples of tumour suppressor genes
• Promotor mutations that activate TERT expression. This also causes cells that should have died to survive.

Question 130

What is common about mutations in melanomas?

Answer 130

That all melanoma cells have constitutively activate MAPK pathway

Question 131

What causes melanoma cells to have constitutively activate MAPK pathway?

Answer 131

These are caused by mutations in BRAFV600E, NRAS (part of the MAPK pathway) which are known as initiator mutations

Question 132

What mutations are seen in the progressing stages of melanomas?

Answer 132

• TERT - Telomerase
• SW1 – Chromatin remodelling
• CDKN2A – Cell cycle

Question 133

What mutations are seen in the late stages of melanomas?

Answer 133

P53, PTEN

Question 134

What s the initating event of a melanoma?

Answer 134

For melanoma, there is very strong evidence that inappropriate/constitutive MAPK pathway activation is the main initiating event

Question 135

What is the BRAF gene?

Answer 135

BRAF is a particular RAF gene that acts in the RAS pathway. Thus, most melanoma cells have an activated RAS pathway

Question 136

What model system would you use to study the core cell cycle machinery?

Answer 136

Yeast

Question 137

What model system would you use to study the upstream controls which tell cells to divide (like the RAS, Notch pathway, the Hedgehog and Wnt pathways etc.) or differentiation and cell death?

Answer 137

Drosophila and C. elegans

Question 138

What model organisms would you use to study cancer?

Answer 138

Vertebrates: Fish or mice

Question 139

Why are vertebrates required to study cancer?

Answer 139

The processes in cancer are dependent upon: blood- and lymph vessels, and an elaborate immune system.

Question 140

What are the advantages of using mice to study cancer?

Answer 140

• Close to humans
• Can do precision genetics and knockouts
• Xenotransplants

Question 141

Give an example of precision genetics that can be carried out in mice to study cancer

Answer 141

Produce tragic mice that drive expression of activated nRAS into melanocytes and combine this with the loss of PTEN to create a double knockout mice that can act as a melanoma model: Tyr:Cre-ERT2; BrafCA/+; PTENlox/lox

• Upon injection of tamoxifen, Cre-ERT2 will go into the nucleus and cause activation of BRAF and simultaneous inactivation of PTEN
• This will only happen in the melanocytes where the Tyr promoter is active

Question 142

What are xenotransplants?

Answer 142

Xenotransplants of human cell lines or tumors in nude mice

Question 143

What are nude mice and how can they be used to study cancer?

Answer 143

• These mice are mutants in the foxn1 gene. They have a defective immune system meaning that human tumours can be grown and accessed in these animals
• Can use this to assess tumour size and metastasis

Question 144

What are the disadvantages of xenotransplants?

Answer 144

It is sometimes difficult to get the human tumour cells to the correct location and using human cells means that the mice are immunocompromised

Question 145

Give an example of a study using mice as a model into the genes involved in melanoma formation?

Answer 145

Davies et al, 2002: Mutations of the BRAF gene in human cancer

• They looked at many melanoma samples and looked at the mutations present in them
• Found that BRAAD V600E mutation was very common

Question 146

Give a study in mice that has lead to a therapeutic opportunity for melanomas

Answer 146

Yang et al, 2010

• Found a specific inhibitor of BRAF V600E called Plx4032 and tested it in mouse models
• First looked in cell culture and tested the compound. Found that the melanoma cells with that mutation are very sensitive to the compound by melanoma cells with the NRAF mutation are not sensitive to the compound. Other cancers do not respond to the compound showing its specificity
• They then did a Xenotransplant model in nude mice. They used three different melanoma cell lines and looked to see whether the compound could inhibit tumour formation. Found that the treated mice have much higher survival than untreated mice
• Now entered clinical trials and has seen to be very successful

Question 147

What are the advantages of using fish to study cancer?

Answer 147

• Large scale
• Optic clarity
• Fast genetics

Question 148

Give an example a fast genetic technique used in fish

Answer 148

Crispr mutagenesis

• Very efficient in zebrafish
• Easy to create knockouts in fish and is much cheaper than knockout mice
• May be able to be used to do gene editing

Question 149

Give a study in fish that has lead to a therapeutic opportunity for melanomas

Answer 149

White et al, 2011

• The initial success but also the inevitable failure of PLX4032 as a drug against melanoma has led to a lot of research concerning the reason behind this relapse and what are the mutations that drive resistance
• Developed a transgenic fish - mitfa:braf(V600E) - to drive the activated form of BRAFV600E in melanocytes forming melanomas. Additional mutations are required to give large rapid tumors: often p53 mutants
• Compared the melanomas isolated from adults to melanocytes from embryos. They developed a signature of melanoma cells and found that they have neural crest progenitor cells
• They carried out a chemical screen for compounds (using known drugs) that inhibit the formation of neural crest progenitor cells. They looked at the phenotype of embryos after exposure to compounds. They did an in-situ hybridisation for Crestin gene (a marker for neural crest progenitors). They found a compound called leflunomide that blocks the Crestin positive cell formation.
• This would be impossible in mice because they did this on 2000 embryos
• Could this compound be useful in humans? Found that leflunomide can inhibit melanoma cells. When combined with the PLX4720 compound it enhanced the potency of the PLX compound
• Then did xenografts in nude mice and saw that the combination of the two compounds was the most effective in preventing the growth of the melanomas

Question 150

Give a study in fish that investigated the mechanism of melanoma formation

Answer 150

Ceol et al, 2011

• Many oncogenes are well studied, but genes that cause progression is still ill-understood. A large region was identified in chromosome 1 that promoted malignant progression of melanoma but which gene in this region is it?
• They used Transgenic mitfa:braf(V600E) zebrafish.
• To test which gene from the amplified region in chromosome 1 is driving tumor progression they made a series of plasmids where the mitfa was present in addition to one gene from the region of chromosome. They injected each plasmid into the tester line. The two groups with clearly reduced survival after injection contain the SETDB1 gene.
• SETDB1 is a methyltransferase and could now be a therapeutic target

Question 151

How did Ceol et al, 2011 produce viable transgenic fis capable of forming melanomas?

Answer 151

They used Transgenic mitfa:braf(V600E) zebrafish. In a p53 mutant background, tumors grow rapidly meaning the fish die before they can reproduce. However, if you remove the mitfa gene in such a background the melanocytes die and these fish are healthy again. They used these fish in their experiment as they are healthy bit preprogrammed to form a melanoma. When they wanted to induce a melanoma, they injected a plasmid with the mitfa gene. Some cells will have taken up the plasmid and others not. The cells that did will form a melanoma

Question 152

How and when was cyclopamine discovered?

Answer 152

1940’s-1950’s
- Sheep-herders observed that normal adult sheep were giving birth to one-eyed (cyclopic) lambs. US Dept. of Agriculture found this was due to the adult sheep eating corn-lilies
1960’s-1970’s
- USDA scientists identified the compound in corn lilies that causes abnormalities in developing sheep and named the compound “cyclopamine.”

Question 153

How was Hh discovered?

Answer 153

1970s-1980s
- Developmental biology researchers identified mutations in a gene that cause a fruit fly to develop abnormally. The fruit flies look curled up and spiky, which led the scientists to name the gene “Hedgehog

Question 154

How and when was the pathway of Hh discovered?

Answer 154

1980’s

• Hedgehog/ Shh was found to be conserved between invertebrates and vertebrates. Cloned and characterised vertebrate homologues of Hh, including Sonic hedgehog (Shh)
• Researchers working in Drosophila, chick, zebrafish and mouse identify and characterise the Hh/Shh signalling pathway. Show that Hh/Shh mediates its effects via the protein, Smoothened

Question 155

How was the link between cyclopamine and the Hh pathway discovered?

Answer 155

1990’s

• Shh knockout mice have cyclopia and holoprosencephaly
• Clinicians found that mutations in Shh/Shh signalling pathway correlate with cyclopia/holoprosencephaly
• They concluded that because cyclopamine leads to cyclopia and loss of Shh or Shh signalling leads to cyclopia than cyclopamine antagonsises Shh or a component in the Shh signalling pathway.
• They found that cyclopamine inhibits smoothened

Question 156

When is Shh important in development?

Answer 156

• Studies on animal model systems demonstrated the importance of Shh signaling to the development of many tissues/organs, including brain, muscle, pancreas and bone.
• Shh is often negatively regulated at the end stages of developmental
• E.g. At early stages, Shh in ZPA in limb bud but end at the development it is downregulated

Question 157

What idea about the role of Shh emerged in 2000’s using animal model studies?

Answer 157

• That although Shh was initially identified as a ‘patterning’ protein, many of the genes that are directly activated by Shh signaling promote proliferation and stem/progenitor cell renewal. Genes include are cyclin D1, BCL2.
• These genes promote proliferation, repress apoptosis and promote stem cell renewal.
• This would explain why Shh is present in early development when cells are proliferating and then is downregulated when cells start to differentiate

Question 158

What clinical discovery did the discovery of the Shh’s role in cell proliferation lead to?

Answer 158

Dysregulation of Shh and Shh signaling was found to be involved in a wide range of cancers

Question 159

How did the role of Shh allow for the prediction of cancers?

Answer 159

Cancers may develop in places where Shh is involved in a developmental manner and then downregulated or in places where Shh levels are normally held at particular levels, and in a careful balance (e.g. at a stem cell niche)

Question 160

Outline the skin stem cell niche

Answer 160

• Skin cells are continually generated through life from a skin stem cell.
• Shh is expressed in the skin stem cell niche.
• Its levels are carefully regulated (e.g. by Wnt, BMP) to achieve the correct balance of stem cells, proliferating progenitors and differentiating cells.
• Proliferating cells are present at the base of the crypt like structure in the deep dermis (red box) and express Shh signal pathway components
• The progenitors move up from the dermis base up the crypt as skin cell differentiation is required

Question 161

How is the role of Shh in the skin stem cell niche related to cancer?

Answer 161

• You would expect that if Shh signalling is not downregulated then there would be more proliferation.
• This was found as an activating smoothened mutation will lead to the upregulation of GliA.
• Abudant GliA in the deep dermis is found in a basal cell carcinoma tumour.

Question 162

Why is a patched a tumour suppressor gene?

Answer 162

• A loss of funcition mutation of patched1 will also lead to BCC in the same way that gain of function smoothened does. This is because patched usually represses smoothened.
• Patched is therefore a tumour supressor gene

Question 163

Give a paper investigating how these cancers causing mutations can be reversed

Answer 163

Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine (Taipale, J et al, 2000)
- Cyclopamine competitively antagonises Smoothened and causes the transcription factors Gli1 and Gli2 to remain inactive. This then prevents the expression of tumour-mediating genes (cell proliferation genes, e.g. Cyclin D1, BCL2

Question 164

How was a therapeutic treatment for BCC discovered using Shh signalling pathway?

Answer 164

2003

• Curis Inc collaborated with Genentech for the development of preclinical development of compounds that inhibit the Hh pathway.
• They made compounds with the same chemical structure as cyclopamine but with some alterations to remove the side effects.
• They then developed a drug using this method called Erivedge (previously called Vismodegib).
• It has a very similar structure to cyclopamine and acts as a cyclopamine competitive antagonist of smoothened

Question 165

When did Erivedge become clinical available?

Answer 165

2007-2009
- Phase I and Phase II studies performed in adults with locally advanced or metastatic BCCs that were refractory to standard therapy. Clinical trials were very successful
2011
- Submission of Erivedge as an FDA-approved drug. Approved under priority review programme (a programme that expedites approval in instances of major advance for treatment)
2012
- Erivedge: the first licensed medicine for patients with advanced BCC

Question 166

Why has the use of Erivedge for other cancers involving Shh not been investigated?

Answer 166

• The potential role for HH in such metastatic cancers in informed directly by developmental biology. But it is horrifically costly. One month’s supply of daily capsules costs $7500 or approximately $250 per capsule
• The national institute for health and care excellence determined that the clinically relevant benefits of Erivedge did not outweigh the costs
• Erivedge is therefore no longer funded by the NHS

Question 167

Does the high cosy of Erivedge put an end to researching treatments of Shh causing cancers?

Answer 167

• Even though there is a patent for using cyclopamine to target the Hh channel but there are other ways of targeting the Hh pathway so these are now being researched
• There are other inhibitors of smoothened and the Hh pathway which have been highlighted and can be used in the same way to treat BCC. This competition will eventfully bring the costs down

Question 168

Define pluripotency

Answer 168

A cell that can generate cells in all three germ layers and the germ line

Question 169

Define stem cells

Answer 169

A cell that can indefinitely self-renew and differentiate into specialised cell types. They often usually lie within a niche

Question 170

When are cells pluripotent in mouse development?

Answer 170

Between days 3.5 and 7.5 in mouse development, cells are pluripotent. Between days 6 and 7, gastrulation occurs

Question 171

How can you define that the cells you have a pluripotent?

Answer 171

• Pluripotency relies on the presence of certain factors which can be used to determine whether a cell in a lab is pluripotent. Factors include: Nanog, Oct4, Sox2.
• The second way to identify pluripotent stem cells is by teratocarcinoma formation. Pluripotent cells can be grafted onto the kidney of a host mouse and give rise to teratocarcinomas. If stain the tumour, you will see all the cell types that a normal pluripotent cell will give rise to in development

Question 172

Where are pluripotent cells found in the embryo?

Answer 172

The pluripotent cells and factors are found in the inner cell mast

Question 173

What is a Teratomer?

Answer 173

Teratomers are tumours that contain all the germ layers.

Question 174

Why do we want to capture pluripotency in vitro?

Answer 174

• The embryos are difficult to study
• There are a small number of cells and ethics to consider. Capturing pluripotent cells in a petri dish can provide a solution to this
• In vitro modelling of embryonic development can lead to the production of clinically relevant cell populations

Question 175

What are the two ways to make pluripotent stem cells?

Answer 175

• Embryonic stem cells

- Induced pluripotent stem cells

Question 176

How do you get embryonic stem cells in culture?

Answer 176

The embryo must be dissected and the pluripotent cells removed from the inner cell mass. Then plate these cells on a layer of feeder cells which provide the trophic factors that would usually be in the niche of the embryo
There are critical signals required to maintain cells in self renewing, undifferentiated state. These signals can replace feeders when the cells have been removed

Question 177

How do you distinguish between the embryonic stem cells and the feeder cells they are plated on?

Answer 177

Can distinguish the embryonic cells from the feeder cells as they have been modified to express the transgene GFP. This can be detected under a fluorescent microscope

Question 178

What critical signals are required to maintain cells in self renewing, undifferentiated state in mice?

Answer 178

Leukaemia inhibitory factor (LIF), BMP

Question 179

What critical signals are required to maintain cells in self renewing, undifferentiated state in humans?

Answer 179

FGF2, TGFB

Question 180

What is the third way you can check that embryonic stem cells are pluripotent?

Answer 180

Create chimera mice

• Could put the pluripotent stem cells produced that express GFP back into the embryo. If the whole mouse expresses GFP then the cells are pluripotent
• This can’t be done in IPS cells because they are human

Question 181

How do you produce induced pluripotent stem cells? (IPS)

Answer 181

• Adult somatic cells can be reprogrammed back to a pluripotent fate
• Take a somatic cell, transgenically introduce a set of transcription factors (the pluripotency factors) that will programme the somatic cell to a pluripotent fate
• The discovery of this ability occurred in 2012 and resulted in Nobel prize

Question 182

How can we direct pluripotent stem cells to differentiate into specific lineages?

Answer 182

Take the stem cells out of the self-renewal niche and culture them in signals that we know developmentally lead to certain tissues and structures

Question 183

How do we define the correct cell type has been produced from pluripotent stem cells?

Answer 183

• Is it functional? E.g. does a neuron fire an action potential, does it synapse
• The expression of right cell markers

Question 184

What are the two ways to direct pluripotent stem cells to differentiate into specific lineages?

Answer 184

1. 3D – Remove signals that keep in an undifferentiated state and grow them in aggregates (balls of cells). They remember their ability to self-organise in an embryo and they begin to differentiate into the cell type of interest.
2. 2D or adherent – Plate a defined number of cells on the right substrate/extracellular matrix. Remove the signals that keep cells in a self-renewing state and grow them in an adherent state with signals that would act on pluripotent cells to differentiate into a specific cell type. However, we do not allow them to culture in 3D

Question 185

What are the advantage and disadvantages of culturing IPS cells in 3D when producing specific cell types?

Answer 185

This method replicates more accurately what occurs in an embryonic environment but it is difficult to observe a role of individual signals

Question 186

What are the advantage and disadvantages of culturing IPS cells in 2D when producing specific cell types?

Answer 186

This method is more tractable (for live imaging) and easier to test the roles of specific signals but we lose the cell interactions that may occur in vivo

Question 187

Give examples of culturing IPS cells in 3D when studying stem cells

Answer 187

• Axin2 is a marker of Wnt signalling and is expressed in the posterior embryo and its expression is expanded later. This is seen by the expression of LacZ that is under the control of axin2. Differentiate the cells using the 3D approach and after 3 days, regional expression Axin2 occurs. This proves that these cells have the ability to self-organise into an anterior and posterior structure within a ball of cells
• Organoids – Aggregates that have been directed to a cell type. For example, cerebral organoids are formed by culturing the cells under the conditions of only the neuroectoderm germ type. If leave these cells, they develop tissue that resembles tissue in the outer brain

Question 188

Give examples of culturing IPS cells in 2D when defining pluripotency

Answer 188

Brachyury which is a maker for mesodermal cells. The adherent pluripotent stem cells have been followed through live imaging and can see that GFP, under the control of brachyury, is expressed as the cells differentiate. This shows there are cells that develop into different cell types as some don’t express GFP

Question 189

How can IPS cells used in disease modelling

Answer 189

Take a biopsy of skin cells from a patient, add pluripotency factors to create IPS cells. Then put them in a petri dish and try to push them into the phenotype of the disease of interest

Question 190

What is Microcephaly?

Answer 190

• A neurodegenerative disorder in which infants are born with an abnormally - Caused by a variety of autosomal recessive mutations
• Causes neurological defects
• Mouse mutants fail to replicate the conditions so human model is required

Question 191

Give an example of using IPS cells to model a human disease

Answer 191

Lancaster et al, 2013

• They took skin biopsy from a patient with microscopy and one without. They produced IPS cells and produced cerebral organoid
• In the control, a cerebral organoid was successfully produced and it reassembled a mini brain. They saw this using the markers DCX and SOX2
• In the organoid created from the patient with microcephaly, the ordered structure is lost and there are less cells present. This phenotype is seen in the disease. Therefore, have created a human model of the disease that cannot be done in other models

Question 192

What did Garcez et al, 2016 do?

Answer 192

Investigated the Zika virus which caused an increase in microcephaly in pregnant women

• Generated cerebral organoids from human IPS cells and added the zika virus
• Generated a cerebral organoid with the phenotype of microcephaly and cell death

Question 193

Give an example of how organoids have been used in drug screening

Answer 193

Xu et al, 2016

• Looking for treatment of Zika virus
• They stained the cells in the organoid with CAS3, a marker for dying cells. There were more dying cells in the organoid treated with the Zika virus
• They then did a drug screening to find a drug that reversed this phenotype and they found a compound called emricasan that blocked increased cell death due to Zika virus infection

Question 194

What are the challenges of disease modelling using IPS cells?

Answer 194

• Many of the diseases have multiple genetic causes (e.g. autism)
• Complex phenotypes
• Late onset diseases e.g. Parkinson’s disease. These organoids can only really resemble foetal phenotypes
• Lack of efficient differentiation protocols e.g. for blood

Question 195

What is Parkinson’s disease?

Answer 195

Affects 1 in 500 people and is caused by the progressive loss of dopaminergic neurons in the substantia nigra. It leads to a tremor, slowness in moving, rigidity, dementia and anxiety

Question 196

Give a study into cell replacement therapy to treat Parkinson’s disease?

Answer 196

Kirks et al, 2011
- Produced dopaminergic neurones in a petri dish. When transplanted into a Parkinson’s mouse model, it can allow for regeneration of dopaminergic neurons and improved motor function. This suggests that this replacement could be used as a treatment for Parkinson’s disease

Question 197

What are the challenges of cell replacement?

Answer 197

• Use progenitors or differentiated cells
• Immune reactions to the cells
• Tumour formation
• The mechanism is unclear
• Positional identity – for example it is easy to generate neural progenitors for the anterior CNS but differentiation protocols for the posterior CNS is unclear

Question 198

Define mental health conditions

Answer 198

Mental health conditions refer to factors influencing the health of the mind, brain, and nervous system.

Question 199

What percentage of the global burden of disease do mental health conditions account for?

Answer 199

10%

Question 200

Give examples of mental health conditions

Answer 200

These conditions include depression, anxiety disorders, schizophrenia, bipolar disorder, alcohol and drug use disorders, mental disorders of childhood.

Question 201

What are Genome-wide association study (GWAS)?

Answer 201

Look at the DNA sequences of patients with a disease and look for changes in the genome that correlate to the disease

Question 202

What is disc?

Answer 202

A gene called Disrupted in Schizophrenia 1

Question 203

How was disc1 discovered?

Answer 203

It was first identified through genetic analysis of an inbred family with high-risk mental problems
All members of the family with different mental illnesses had a mutated DISC1 gene

Question 204

What are known triggers of mental illness?

Answer 204

Stress, lack of sleep and substance abuse

Question 205

What is meant by GxE?

Answer 205

When there is a combination of genes and environment involved in a disease

Question 206

Why is it hard to study mental health illnesses?

Answer 206

• Because of the many variables that are likely to be Involved and because it is a consequence of both genes and environment

Question 207

What could the effect of GxE explain for the variability of mental health conditions?

Answer 207

The effect of GxE may explain why the same mutation in DISC1 can result in such a variety of mental illnesses from mild depression to schizophrenia

Question 208

Why are zebrafish and drosophila more useful to stay mental health than mice?

Answer 208

When there are so many variables, as in mental health conditions, it is important to analyse a high number of models. This is too expensive and time consuming in mice

Question 209

What did Eachus, H et al, 2017 study?

Answer 209

If DISC1 is involved in mental health conditions

Question 210

How did Eachus, H et al, 2017 find out where disc1 is expressed?

Answer 210

Used in situ hybridisation
- Found that its mRNA is expressed in the developing hypothalamus. Further analysis showed that it is expressed in hypothalamic stem and progenitor cells and is probably regulated by Wnt signalling

Question 211

How is the hypothalamus involved in regulation of stress?

Answer 211

The master regulator is the hypothalamus which sends corticotropic releasing factor to the anterior pituitary gland. Cell in the anterior pituitary gland, called corticotrophs, then release adrenal cortical trophic hormone (ACTH) which act on the adrenal glands. The adrenal glands then release cortisol in response to stress. This then acts negatively to the hypothalamus to dampen this affect

Question 212

How did Eachus, H et al, 2017 investigate the effect of disc1 mutants?

Answer 212

Created two lines of disc1 mutant zebrafish

• Two point mutations that were homozygous viable and resulted in the production of no disc1 protein or mRNA.
• They then asked if they saw any difference in the hypothalamic progenitor cells.
• They used a marker of hypothalamic progenitor cells and looked to see if there were any difference between the wildtype and disc1 mutant.

Question 213

What did Eachus, H et al, 2017 find using the disc1 mutants?

Answer 213

There were less hypothalamic progenitor cells in the disc1 mutant meaning that there would be less production of hypothalamic neurons. The hypothalamic progenitor cells also appeared abnormal and thought that they were differentiating prematurely

Question 214

What did Eachus, H et al, 2017 conclude from there being less hypothalamic progenitor cells in the disc1 mutant?

Answer 214

• They differentiated prematurely so that there are too many of some types of neurons that are born early and no progenitors left to differentiate into the later born neurons
• When they looked at hypothalamic neurons in both the wildtype and the dsic1 mutant, there were more neurons that are born early in the mutant then there are in the wildtype and less neurons that are born late.
• Therefore, there are not enough neurons born that are involved in stress homeostasis

Question 215

How did Eachus, H et al, 2017 answer if the abnormal development of hypothalamic stress axis nerves correlate with an abnormal stress response?

Answer 215

Behavioural assays

• To see if the zebrafish had a normal behavioural response to stress
• The zebrafish were stressed by exposing them to high levels of NaCl in the water as they are fresh water fish and by adding the pheromone Schreckstoff
• Wildtype fish try to swim away in response to NaCl or Schreckstoff whereas mutant disc1 fish don’t react to it. No behavioural response to stress
• They then checked if they were stressed by measuring cortisol levels – mutant disc1 still don’t show a stress response

Question 216

Which drug is most harmful?

Answer 216

Alcohol is the most harmful drug both to others and the user
In 2013, there were over 8000 alcohol related deaths
The consumption of alcohol creates an enormous social, economic and healthcare burden

Question 217

What receptors do alcohol bind to?

Answer 217

Most drugs bind to a specific receptor but alcohol binds to multiple including GABA and NMDA
Most, if not all, drugs of abuse act secondarily through dopamine and the mesolimbic ‘reward’ system

Question 218

What are contributing factors to addiction?

Answer 218

• Genetics
• Social environment
• Personality and personal history

Question 219

Are flies a good model for addiction?

Answer 219

Yes
- Flies have catecholamine reuptake transporters, nicotinic AcR, ligand and voltage gated ion channels, and a G-protein activated potassium channel.
- 75% of human genes implicated in disease are present in Drosophila
No
- No opioid receptors, no cannabinoid receptors.
- Flies diverged from our common ancestor about 500 million years ago
- Environment and personality are hard to model in Drosophila

Question 220

What experimental approaches can be used to model addition in flies?

Answer 220

The booz-o-mat
- Flies kept in a chamber where there is an air supply and ethanol fume supply
- Then see how they respond to the ethanol containing air
- Can see that flies move more when intoxicated with ethanol
- This can be quantified using Locomotor Velocity Profile
The inebriometer
- Ethanol fumes are supplied to tubes of flies
- Ladder structure inside which the flies can move around in but when drunk they would fall to the bottom
- Can quantify this and count how many flies drop out of the tube at each time point

Question 221

How can the booz-o-mat be used to show flies are a good model for addiction?

Answer 221

• First starts with a startle response and then increased locomotion, leading to sedation
• Concentrations at which flies show increased locomotion and sedation are similar to the concentrations that elicit these responses in mammals including humans.

Question 222

Define sensitivity

Answer 222

Sensitivity is the inability to tolerate the adverse effects of a drug: start point

Question 223

Define the development of tolerance

Answer 223

Development of tolerance is the reduction in sensitivity when a drug is used repeatedly over time (higher amount is required to achieve the same level of response and adverse effects).

Question 224

What did Moore et al, 1998 do?

Answer 224

Identifies the cheap date mutant which are much more sensitive to ethanol. At all concentrations of alcohol, cheapdate mutants are more sensitive and become drunk quicker

Question 225

How did Moore et al, 1998 discover the cheap date mutant?

Answer 225

Irradiated the flies to cause random mutations in the genome and collected flies that are either oversensitive (drop out of inebriometer first) or under sensitive (come out last)
Then identified the genes present in the groups of flies

Question 226

What is the cheap date gene?

Answer 226

cheapdate is an allele of amnesiac, a neuropeptide that activates the cAMP pathway

Question 227

How does the cAMP pathway lead to gene transcription?

Answer 227

• Neuropeptide binds to receptor, which activates heterotrimeric G-protein complex.
• Gs-alpha subunit then exchanges GDP for GTP and is released from the complex.
• Then it binds to adenylyl cyclase, which converts ATP to cAMP leading to activation of PKA.
• PKA enters the nucleus and activate CREB by phosphorylation which binds to CBP protein.
• This binds to the CREB binding domain on a target gene and activate transcription

Question 228

How did Moore et al, 1998 find the link between caMP pathway and alcohol sensitivity ?

Answer 228

• Looked at the ethanol response of cAMP pathway mutants
• They looked at a adenylyl cyclase mutant, a PKA mutant and dunce mutant: cAMP phosphodiesterase (inactivates cAMP) mutant
• Found that adenylyl cyclase and PKA mutants lead to an increase in sensitivity but mutation in cAMP phosphodiesterase does not increase sensitivity
• This implies that activity of the cAMP pathway decreases alcohol sensitivity

Question 229

How can we measure the development of tolerance in flies?

Answer 229

Repeat the sensitivity experiment in the inebriometer in the same flies to see if their sensitivity has changed - does it take longer for the flies to fall out of the tube

Question 230

What did Scholz et al, 2000 find?

Answer 230

• Found that after second exposure of flies to ethanol, it takes longer for flies to fall off the tube
• This shows that flies do develop tolerance

Question 231

What are hangover mutants?

Answer 231

Hangover mutants do not develop tolerance as much as wild type flies. Even after long exposure, they do not develop as much tolerance proving that there is a genetic component in developing tolerance

Question 232

Wha is the hangover gene?

Answer 232

Encodes a Zn-finger protein which are proteins that usually interact with nucleotides

Question 233

What did Ruppert et al, 2017 find out about the hangover gene?

Answer 233

• Hangover binds Dunce RNA, increasing the amount of transcript
• Hangover mutants have increased cAMP, suggesting hangover and do not form tolerance
• Turning off the cAMP pathway is required in developing tolerance as dunce mutants (cAMP phosphodiesterase mutants) also do not develop a tolerance. This shows that cAMP phosphodiesterase function is required in developing tolerance

Question 234

What is the relationship between hangover and dunce?

Answer 234

• Hangover encodes an RNA binding protein that binds dunce RNA (cAMP phosphodiesterase that inactivates cAMP) and increases the amount of transcript
• The balance between hangover and dunce expression determines degree of tolerance as a reduction in cAMP level is required for tolerance
• A high level of dunce and hangover would mean that tolerance increases as there is less cAMP

Question 235

What is the current model on sleep/awake?

Answer 235

Current models suggest that sleep and awake states are decided by two interaction process: a sleep homeostatic drive and the circadian clock

Question 236

What is the circadian clock?

Answer 236

Circadian rhythms are 24 hour rhythms in physiology and behaviour generated by molecular clocks, which serve to coordinate internal time with the external world

Question 237

Why do most organisms have a circadian clock?

Answer 237

Most organisms have evolved circadian clocks that anticipate the regular 24hr environmental changes and establish endogenous 24 hour rhythms to ensure that physiological and behavioural outputs are optimised to the appropriate time window each day

Question 238

When is melatonin at its highest?

Answer 238

Every 24 hours, there will be an increase and decrease in melatonin – highest at the end and start of the day

Question 239

What regulates the circadian cycle?

Answer 239

Most cells in the body possess molecular clocks but are all maintained in synchrony by a master clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus

Question 240

How does the molecular clock regulate biological and physiological functions?

Answer 240

• There are thousands of clock control genes in the body that orchestrate the oscillation of tissue specific metabolic and physiological functions e.g. hormones.
• The proteins have very small half lives and their mRNA is regulated by clock controlled genes

Question 241

How much of the transcription of tissues display circadian rhythm?

Answer 241

Between 2 and 30%

Question 242

What core biological and physiological processes are under the control of clock genes?

Answer 242

Body temperature, Cortisol, blood pressure, melatonin, growth hormone, testosterone, prolactin

Question 243

How do we know about the circadian molecular clock?

Answer 243

Animal studies in drosophila

Question 244

Outline the mechanism underlying the circadian regulation

Answer 244

Cell autonomous transcription translation feedback loops.

• Transcription factors (CLOCK, BMAL1) drive the expression of other genes (Period and Cry1/2), whose protein products in turn feedback to inhibit CLOCK and BMAL1
• This process takes 24 hours

Question 245

How can external factors can entrain the molecular clock?

Answer 245

Sun light stabilises Cry and promote its binding to TIM, therefore affects the working of the clocks

Question 246

How do we know about the mechanisms underlying the circadian molecular clock?

Answer 246

This knowledge came from loss and gain of function studies and epistasis studies in drosophila and mice

Question 247

What is the link between mental illness and circadian rhythm disruption?

Answer 247

• Patients with neuropsychiatric diseases, such as bipolar disorder, schizophrenia and depression exhibit sleep and circadian rhythm disruption.
• These patients show dysregulation of multiple circadian outputs and of the core molecular clock.

Question 248

How was the link between mental illness and circadian rhythm disruption discovered?

Answer 248

• This was shown by isolating fibroblasts from schizophrenic patients
• It showed a loss of rhythmicity in CRY1 and PER1 expression, and their peripheral blood leukocytes have decreased and/or disrupted diurnal expression of CLOCK, PER1/2/3, CRY1 and a functional CLOCK homologue NPAS2 in comparison to healthy controls
• This can be shown using RT PCR, antibody staining or by adding a GFP construct into a mouse model for the disease and a control

Question 249

What evidence is there for the link between mental illness and circadian rhythm disruption?

Answer 249

GWAS and SNP analyses show that genes encoding the core components of the molecular clock have been associated (weakly) with schizophrenia, bipolar disorder and depression

Question 250

Is the disruption if the circadian rhythm cause or consequence of mental illness?

Answer 250

• Studies, including many animal model show that disruption of the molecular clock is not just a consequence of neuropsychiatric illness, but instead forms part of a bidirectional feedback loop with neuropsychiatric disease, whereby dysfunction in one exacerbate dysfunction in the other

Question 251

Give evidence for disruption if the circadian rhythm being a cause of mental illness

Answer 251

Roybal, K et al, 2007)
- Mouse models of clock mutants show manic behaviours, implicating mechanistic and causal links between circadian clock and disease pathophysiology
- Increasing a dopaminergic activity in the forebrain can also lead to this, therefore suggesting a causal link between the clock and disease
Landgraf D et al, 2016
- Knocked out clock in the suprachiasmatic nuclei of the hypothalamus
- This disrupted SCN circadian rhythms which resulted in anxiety like behaviour in mice, establishing them as a new animal model of depression
Wulff K et al, 2012
- Sleep and circadian rhythms are casual to mental illness

Question 252

What diseases have become more clear due to the understanding of the clock?

Answer 252

Metabolic
Immune system
Neurodegenerative disease

Question 253

What is the original function of melatonin?

Answer 253

• It is ubiquitous in nature (highly conserved) and its key function is a potent antioxidant to detoxify free radicles.
• A key idea is that these free radicles are built up in the day in response to UV light and then at night, melatonin is released and they are detoxified

Question 254

How is melatonin thought to be involved in mental illness?

Answer 254

Animal model studies have been used to show that this is the function of melatonin and that mental health and neurodegenerative diseases can be caused by the inability to clear free radicles

Question 255

How has the understanding of the clock allowed advancements in treatments for cancer?

Answer 255

• The time of day may be critical in the bodies response to chemotherapy
• Clock evolved to help organisms protect against UV-induced DNA damage. Molecules that are involved in DNA repair are expressed at highest levels in late afternoon, lowest at night
• Chemotherapy works by promoting DNA damage. Idea is to give chemo when body is least-able to function optimally in repairing this damage
• Understanding of the clock function is therefore helpful when optimising chemotherapies when treating cancer

Question 256

What is ageing?

Answer 256

Progressive, irreversible decline in organismal performance

Question 257

What are the causes of death after the age of 65 in the UK?

Answer 257

• Malignant cancers
• Heart problems
• Lung problems
• Alzheimer’s
• Parkinson’s
  All diseases associated with ageing

Question 258

What is the maximum life span of humans?

Answer 258

125 years

Question 259

What factors influence ageing?

Answer 259

Genetics
- Genotype at birth and accumulation of mutations with age
Environment
- Diet, life style, exposure to external factors

Question 260

What qualities must model organisms of life span have?

Answer 260

• Small and easy to culture
• Short lifespan
• Genetically tractable: homogeneous, many tools available
• Controllable environment (food intake)

Question 261

What model organism are used to study ageing?

Answer 261

• C.elegans
• Drosophila
• Mice

Question 262

How can you measure ageing?

Answer 262

• There is no direct method of measuring ageing as ageing is an undefined loss of function. E.g. some people need glasses from a young age while others don’t need them until later life
• Usually measure age at death (life span). Can use model organisms to measure the age at death of large numbers of genetically identical animals
• Can use drosophila powerful genetic tool kit to find genes regulating life-span through genetic screens

Question 263

What is a median lifespan?

Answer 263

the age when 50% of the population have died

Question 264

Outline the insulin pathway

Answer 264

Insulin or IGF1 binds to the insulin receptor and the co receptor Chico will trigger the PI3 kinase signalling pathway which has effects on the transcription factor FOXO, which affects cell number, and S6K, which affects cells size

Question 265

How was the importance of the insulin pathway in lifespan discovered?

Answer 265

Clancy et al, 2001

- Drosophila Chico mutants are shown to have a longer median lifespan

Question 266

How does the insulin pathway effect longevity?

Answer 266

In mammals, C. elegans and drosophila, insulin or insulin like peptides act on the insulin receptor. The cytoplasmic component of the receptor then binds to Chico. This triggers the PI3 kinase pathway which activated PDK1, followed by Akt. Akt then represses FOXO. FOXO usually stimulates a repressor of growth and life span.

Question 267

What does an increase of transcription factor FOXO due to decreased insulin lead to?

Answer 267

A decrease in insulin, causes an increase in FOXO resulting in an increase in autophagy (removal of unwanted debris), an increase in DNA repair and a decrease in oxidative stress. This increases health and longevity

Question 268

What are the names for insulin receptor?

Answer 268

Insulin receptor is conserved and has different names in different species: Insulin receptor in mammals, daf-2 in C. elegans and dinR in drosophila

Question 269

What is the mammalian homologue of Chico?

Answer 269

IRS1-4

Question 270

What is Methuselah?

Answer 270

Gene that encodes the receptor which triggers insulin release in response to nutrients.

Question 271

What did Petrosyan et al 2014 do?

Answer 271

• Methuselah mutants are long lived suggesting that loss of function of this gene will increase life span
• A peptide antagonist of Methuselah was therefore added to drosophila and it was found to extend life-span. However, the functional abilities of the flies (measured by how long they can fly) was below the control level. Life span increased but healthy ageing decreased

Question 272

What is the effect of dietary restriction on lifespan?

Answer 272

Reduced nutrient intake extends lifespan

Question 273

What did Partridge et al 2005 do?

Answer 273

• Dietary restriction is the oldest known proven method for lifespan extension
• Drosophila under dietary restriction are shown to live longer. Control group had a median life span of 35 days while dietary restriction group had a median life span of 55 days, showing a significant increase in life span
• Even a modest dietary restriction increases life span

Question 274

What did Mair et al 2003 do?

Answer 274

• Does diet need to be restricted throughout life to increase life span?
• Flies who were fully fed and then dietary restricted after 14 and 22 days were shown to have a dramatic decrease in probability of death compared to those who were never restricted. The probability decreased to the same level of the flies who have been dietary restricted throughout their whole life
• If dietary restricted and then fully fed, the death probability increases to the same as the wild type
• Dietary restriction is not required throughout life as it reduced the mortality risk at any time

Question 275

How does dietary restriction increase life span?

Answer 275

Insulin pathway
- Fewer calories, less insulin released, more FOXO. This will result in an increase in autophagy and DNA repair and therefore increase health and longevity
Also, thought to involve other pathways such as the inflammatory pathway which impact on disease that impact health and longevity

Question 276

What is the average life expectancy of people diagnosed with Alzheimer’s?

Answer 276

8-10 years after diagnosis

Question 277

What is Alzheimer’s?

Answer 277

It is a Neurodegerative disease that leads to neuronal cell death and tissue atrophies. Also leads to accumulation of beta-amyloid peptides. It is not clear whether this is the cause or a consequence

Question 278

How can drosophila be used to study Alzheimer’s and promote healthy ageing?

Answer 278

• Overexpression of beta amyloid peptide in drosophila - This makes is possible to screen for genes that reduce
• This is done through the Gal4/UAS system

Question 279

How can the Gal4/UAS system be used to study Alzhiemer’s?

Answer 279

• Make two fly strains. One where Gal4 is expressed downstream to a promotor of a gene expressed in a tissue for example the brain. Another one where the desired gene is proceeded by UAS.
• Cross flies to create a fly that expresses Gal4 in all the neurons and expressed B amyloid.
• The Gal4 acts on the UAS to cause overexpression of beta amyloid in the brain. This will cause the flies to die early
• Can do this in combination with a mutagenesis screen to screen for genes that restore the fly’s lifespan

Question 280

What gene was found to restore life span using mutagenesis screens and the Gal4/UAS system in drosophila?

Answer 280

Insulin degrading enzyme (IDE)

• An evolutionary conserved metalloendopeptidase
• It increases life span and reduced neurotoxicity induced by beta-amyloid peptide
• This implies that the insulin pathway is not only involved in life span but is also involved in healthy ageing

Question 281

What did Piers et al,2016 review?

Answer 281

Targeting cellular signalling pathways in breast cancer stem cells and its implication for cancer treatment

Question 282

What are BCSCs?

Answer 282

Breast cancer stem cells are a small subpopulation of stem like cells within a tumour mass and are responsible for tumour development, recurrence and metastasis of breast cancer

Question 283

How are BCSCs gene expression driven?

Answer 283

By different signalling pathways including Wnt, Hh and Notch.

Question 284

Give evidence for the role of Wnt signalling in breast cancer

Answer 284

• Wnt signalling is involved in the maintenance of stem cell properties meaning BCSCs are regulated by Wnt.
• Inhibition of Wnt1 has therefore reduced tumour formation in vitro and in vivo.
• Injected Wnt1 knockdown cells into mammary fat pads of female mice

Question 285

Give evidence for the role of Hh signalling in breast cancer

Answer 285

Heet al observed positive correlation between the expression of SMO and GLI1 (components in Hh pathway) with BCSC markers CD44+CD24−

Question 286

How has the discovery of signalling pathways involved in BCSC formation changed how cancer should be treated?

Answer 286

Should therefore treat breast cancer with combination of anti-Oestrogen (to target progenitors) and an anti-Notch/anti-Wnt/ anti-HH to target BCSC)

Question 287

What did Meant et al, 2013 investigate?

Answer 287

ROS production and NF-kB activated triggered by RAC1 facilitate WNT-driven intestinal stem cell proliferation and colorectal cancer initiation

Question 288

What did Meant et al, 2013 find?

Answer 288

• APC gene mutation is common in most colorectal cancers and leads to the constitutively active Wnt signalling
• After APC loss, RAC1 is a critical mediator of tumorigenesis. RAC1 triggers ROS production and NF-kappaB activation
• The nuclear factor NF-κB is a proinflammatory factor: it positively regulates the expression of proinflammatory genes including cytokines
• ROS are key signalling molecules that play an important role in the progression of inflammatory disorders
• Their activation therefore indicates a role for inflammation in progression of a benign to a malignant tumour

Question 289

What did Zhang et al 2017 investigate?

Answer 289

Environmental perturbation of the circadian clock during pregnancy leads to transgenerational mood disorder-like behaviors in mice

Question 290

What did Zhang et al 2017 do?

Answer 290

Exposed pregnant mice to chronic circadian disturbance (CCD) and then looked at the offsprings phenotype

Question 291

What did Zhang et al 2017 find?

Answer 291

• Offspring in the F1 and F2 generations that also exhibited mood-associated behavioural phenotypes despite the lack of direct stressful experiences during their postnatal or adult period
• Core circadian clock gene expression was disrupted in the suprachiasmatic nucleus of the offspring. There were changes in hypothalamic protein expression with sex-specific differences. Particularly proteins that were associated with cellular activities, metabolism, development and diseases.

Question 292

What did Zhang et al 2017 conclude?

Answer 292

Conclude that maternal exposure to chronic circadian disturbance during pregnancy can lead to sex-specific mood disorders that persist for at least two generations

Question 293

Why does the rate of nucleotide excision repair exhibits circadian rhythmicity?

Answer 293

The essential nucleotide excision repair protein XPA is a first-order clock-controlled protein

Question 294

How does circadian rhythmicity of XPA expression help in cancer treatment?

Answer 294

• Chemotherapy works by causing DNA damage and excision repair is responsible for the repair of this damage.
• Excision repair shows circadian rhythmicity in most tissues, shown in mice, meaning administration of the drugs when excision repair is in the descending phase is expected to improve the therapeutic index by administering a less toxic dose