BMS336 Modelling Human Disease and Dysfunction Flashcards

Question

What are the uses of transgenic reporter genes?

Answer 1

- 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

Answer 2

- 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

Answer 3

- 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

Answer 4

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

Answer 5

- 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

Answer 6

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.

Answer 7

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

Answer 8

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

Answer 9

- 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

Answer 10

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?

Answer 11

- 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

Answer 12

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

Answer 13

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

Answer 14

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

Answer 15

- 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.

Answer 16

- 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

Answer 17

- 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

Answer 18

- 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

Answer 19

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

Answer 20

- 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

Answer 21

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

Answer 22

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

Answer 23

The regeneration of pancreatic beta cells in vivo

Answer 24

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

Answer 25

- 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).

Answer 26

- 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.

Answer 27

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

Answer 28

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.

Answer 29

- 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).

Answer 30

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

Answer 31

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

Answer 32

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

Answer 33

- 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

Answer 34

- 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

Answer 35

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

Answer 36

- 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

Answer 37

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

Answer 38

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

Answer 39

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

Answer 40

Leptin inhibits NPY neurons

Answer 41

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

Answer 42

Leptin stimulates Pomc neurons

Answer 43

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

Answer 44

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

Answer 45

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

Answer 46

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

Answer 47

- 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

Answer 48

Iselt1 is a transcription factor that upregulates Pomc neurons

Answer 49

Nasif et al, 2015

Answer 50

- 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

Answer 51

- 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

Answer 52

- 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

Answer 53

- 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

Answer 54

- 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

Answer 55

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

Answer 56

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

Answer 57

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

Answer 58

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.

Answer 59

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

Answer 60

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

Answer 61

- 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?

Answer 62

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

Answer 63

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.

Answer 64

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

Answer 65

Poor diet and smoking

Answer 66

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

Answer 67

Hyperglycaemia is thought to link directly to the development of atherosclerosis

Answer 68

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.

Answer 69

- Breast - Bowel - Kidney - Womb

Answer 70

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

Answer 71

- 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.

Answer 72

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

Answer 73

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.

Answer 74

- 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.

Answer 75

- 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

Answer 76

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

Answer 77

- 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

Answer 78

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

Answer 79

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

Answer 80

- 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

Answer 81

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

Answer 82

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.

Answer 83

Increasing activity of activators or decreasing activity of suppressors

Answer 84

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

Answer 85

APC and Axin

Answer 86

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.

Answer 87

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).

Answer 88

- 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

Answer 89

- 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

Answer 90

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

Answer 91

Involves the inflammatory pathway

Answer 92

- 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

Answer 93

- Animal model studies can help us understand mechanism | - Animal modeling can be used for pre-clinical therapeutic development

Answer 94

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.

Answer 95

Basal cell carcinoma - More common but also more treatable - These are caused by mutation in the patched receptor

Answer 96

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

Answer 97

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

Answer 98

- Age - Gender - Skin type

Answer 99

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

Answer 100

- 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

Answer 101

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%

Answer 102

- 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

Answer 103

- 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.

Answer 104

That all melanoma cells have constitutively activate MAPK pathway

Answer 105

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

Answer 106

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

Answer 107

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

Answer 108

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

Answer 109

Drosophila and C. elegans

Answer 110

Vertebrates: Fish or mice

Answer 111

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

Answer 112

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

Answer 113

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

Answer 114

Xenotransplants of human cell lines or tumors in nude mice

Answer 115

- 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

Answer 116

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

Answer 117

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

Answer 118

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

Answer 119

- Large scale - Optic clarity - Fast genetics

Answer 120

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

Answer 121

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

Answer 122

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

Answer 123

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

Answer 124

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.”

Answer 125

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

Answer 126

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

Answer 127

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

Answer 128

- 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

Answer 129

- 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

Answer 130

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

Answer 131

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)

Answer 132

- 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

Answer 133

- 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.

Answer 134

- 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

Answer 135

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

Answer 136

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

Answer 137

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

Answer 138

- 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

Answer 139

- 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

Answer 140

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

Answer 141

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

Answer 142

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

Answer 143

- 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

Answer 144

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

Answer 145

Teratomers are tumours that contain all the germ layers.

Answer 146

- 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

Answer 147

- Embryonic stem cells | - Induced pluripotent stem cells

Answer 148

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

Answer 149

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

Answer 150

Leukaemia inhibitory factor (LIF), BMP

Answer 151

FGF2, TGFB

Answer 152

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

Answer 153

- 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

Answer 154

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

Answer 155

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

Answer 156

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

Answer 157

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

Answer 158

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

Answer 159

- 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

Answer 160

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

Answer 161

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

Answer 162

- 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

Answer 163

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

Answer 164

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

Answer 165

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

Answer 166

- 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

Answer 167

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

Answer 168

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

Answer 169

- 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

Answer 170

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

Answer 171

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

Answer 172

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

Answer 173

A gene called Disrupted in Schizophrenia 1

Answer 174

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

Answer 175

Stress, lack of sleep and substance abuse

Answer 176

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

Answer 177

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

Answer 178

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

Answer 179

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

Answer 180

If DISC1 is involved in mental health conditions

Answer 181

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

Answer 182

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

Answer 183

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.

Answer 184

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

Answer 185

- 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

Answer 186

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

Answer 187

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

Answer 188

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

Answer 189

- Genetics - Social environment - Personality and personal history

Answer 190

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

Answer 191

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

Answer 192

- 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.

Answer 193

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

Answer 194

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).

Answer 195

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

Answer 196

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

Answer 197

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

Answer 198

- 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

Answer 199

- 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

Answer 200

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

Answer 201

- 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

Answer 202

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

Answer 203

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

Answer 204

- 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

Answer 205

- 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

Answer 206

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

Answer 207

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

Answer 208

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

Answer 209

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

Answer 210

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

Answer 211

- 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

Answer 212

Between 2 and 30%

Answer 213

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

Answer 214

Animal studies in drosophila

Answer 215

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

Answer 216

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

Answer 217

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

Answer 218

- 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.

Answer 219

- 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

Answer 220

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

Answer 221

- 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

Answer 222

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

Answer 223

Metabolic Immune system Neurodegenerative disease

Answer 224

- 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

Answer 225

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

Answer 226

- 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

Answer 227

Progressive, irreversible decline in organismal performance

Answer 228

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

Answer 229

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

Answer 230

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

Answer 231

- C.elegans - Drosophila - Mice

Answer 232

- 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

Answer 233

the age when 50% of the population have died

Answer 234

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

Answer 235

Clancy et al, 2001 | - Drosophila Chico mutants are shown to have a longer median lifespan

Answer 236

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.

Answer 237

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

Answer 238

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

Answer 239

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

Answer 240

- 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

Answer 241

Reduced nutrient intake extends lifespan

Answer 242

- 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

Answer 243

- 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

Answer 244

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

Answer 245

8-10 years after diagnosis

Answer 246

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

Answer 247

- 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

Answer 248

- 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

Answer 249

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

Answer 250

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

Answer 251

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

Answer 252

By different signalling pathways including Wnt, Hh and Notch.

Answer 253

- 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

Answer 254

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

Answer 255

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

Answer 256

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

Answer 257

- 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

Answer 258

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

Answer 259

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

Answer 260

- 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.

Answer 261

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

Answer 262

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

Answer 263

- 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