Modelling Human Disease

Question 1

what is the clinical challenge that we are faced with? what do we need to understand?

Answer 1

• We are often presented with late or end-state
• We need to understand where, when, how, why diseases/dysfunction start
• We need to understand where, when, how, why diseases/dysfunction worsen

Question 2

what are the leading causes of death in the US?

Answer 2

Most of the mortality in the US is due to chronic diseases

• Heart disease
• Cancer
• Chronic lower respiratory disease
• Alzheimer’s disease
• Diabetes
• Suicide

Question 3

what diseases have been rising and which ones have gone down?

Answer 3

• cancer has gone down because we know more about it etc

- diabetes type 2 has been going up. causes a strain on economy of countries

Question 4

describe diabetes and what does it cause?

Answer 4

• a disease in which the body is no longer able to carefully control blood glucose, leading to abnormally high levels of blood glucose (hyperglycemia).
• Persistently elevated blood glucose can cause damage to the body’s tissues, including the nerves, blood vessels.
o Homeostatic blood glucose system would be imbalanced

Question 5

describe Chronic lower respiratory disease (CLRD) and what is causes

Answer 5

• 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 – important characteristic

Question 6

describe Heart disease

and what it can cause

Answer 6

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

Question 7

describe cancer and what can it cause if not controlled?

Answer 7

• 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 8

describe neurodegenerative diseases and what it can cause

Answer 8

• 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 9

is there much information on Anxiety and depressive disorders?

Answer 9

no. it is a poorly-characterised disorders characterised by a range of emotional, behavioural and physical symptoms

Question 10

describe cerebrovascular diseases and which ones are the most common types?

Answer 10

• conditions that develop as a result of problems with the blood vessels that supply the brain.
• Four of the most common types of cerebrovascular disease are: (brain not getting enough O2)
o Stroke
o Transient ischemic attack (TIA)
o Subarachnoid hemorrhage
o Vascular dementia

Question 11

describe dementia and what is it caused by and what can it cause?

Answer 11

• 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. As a result of the damage, neurons can no longer function normally and may die. The damage eventually impairs ability to carry out core body function.
o Note dementia can probably arise due to loss of vascularisation, loss of glial support cells, nerve degeneration

Question 12

what is a model organism?

Answer 12

• Any non-human species that is extensively studied to discover/understand a particular biological phenomenon with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms.

Question 13

what situations are there where it would be unsuitable to use a human model so instead use model organisms?

Answer 13

are widely used to research human disease when human experimentation would be unfeasible or unethical

Question 14

why is it possible to use model organisms to model human diseases?

Answer 14

• This strategy is made possible by the common descent of all living organisms, and the conservation of metabolic and developmental pathways and genetic material over the course of evolution.

Question 15

give some examples of model organisms

Answer 15

can include prokaryotes, plants, fungi, invertebrates, vertebrates, mammals – pretty much any non-human species.

Question 16

list 8 reasons to explain why we work on particular model organisms?

Answer 16

1. Sometimes, just chance – what was around at the time
2. Inbred populations within the species (useful in particular < rapid genome sequencing)
3. Outbred populations within the species
4. Costs, life-cycle time
5. Easy to induce mutations
6. Easy to score traits/phenotype
7. Easy to analyse disease traits
8. Easy to manipulate

Question 17

what of the model organisms are conserved that make it easier to model human diseases?

Answer 17

¥ We share a remarkable degree of gene conservation
¥ The basic mechanisms of gene action are conserved
¥ The way that our bodies are built and maintained are conserved
¥ Easy to interfere with genes in model organisms
¥ Can ‘see’ inside the body and watch it in real-time
¥ Model organisms = instrumental in identifying ‘lifecourse’ genes/events

Question 18

what are chronic diseases?

Answer 18

diseases that develop and get worse over a period of time (often years)

Question 19

what is an important advantage in terms of studying diseases/genetic mutations that model organisms have but humans don’t?

Answer 19

• Need to look at disease and dysfunction in relation to lifecourse
o Through the aging process
• Need models where it is possible to examine an individual over the lifecourse (to look at progression, systems, systems interactions)
o As we don’t have much information on a human over its lifetime
o Can look at the lifecourse of mouse, zebrafish and dorsophila

Question 20

in which model organisms is it the best to measure lifecourse genes?

Answer 20

mouse, zebrafish and drosophila

Question 21

what is meant by GxE and why is important to be studied in model organisms?

Answer 21

gene-environment interactions
- Genotype drives development and confers the potential for health and wellbeing across the lifecourse (and high likelihood of some diseases)
• Experience-dependent, environmental factors interact with genotype to trigger/exacerbate disease and dysfunction
o i.e Disease/dysfunction is increasingly understood to arise due to Gene x Environment interactions

Question 22

why are animal models useful in modelling GxE interactions?

Answer 22

1. Many animal models we work on are incredibly genetically-tractable (make a transgenic animal that has a particular mutation in a particular gene) – very precise
   a. To model human susceptibility genes
2. Can be examined in huge numbers (drosophila and zebrafish especially)
   a. Gives you the statistical power to confidently say an effect you are seeing is not just due to chance

Question 23

what is a new technique that the NHS adopted for the 1000,000 genome study that is used to sequence the genome?

Answer 23

genome wide association analysis
- can look for variants within the genetic sequence of those with a disease compared to healthy individuals and so then can focus attention on a variant that appears to correlate with disease susceptibility

Question 24

what is the disadvantage of GWAS?

Answer 24

doesn’t sow where and when the gene is expressed so doesn’t say id the protein coded by the gene triggers anything that contributes to r exacerbates the disease. this is why animal models are important in this field.

Question 25

what does the NHS 100,000 genome project entail?

Answer 25

• 2014, NHS set the ‘100,000 genome’ project and was set for 3 years
o idea was that this would lead to the way for personalized medicine
o initiative involved collecting and sequencing 100,000 genomes to enable doctors to have more info on specific conditions
o the long-term aim would be to transform the NHS by saying that for any individual, we understand the disease

Question 26

what techniques can be used to see which tissue/cell a gene is expressing? describe how 1 of these work

Answer 26

in situ hybridisation and immunohistochemsitry

- can make an antisense mRNA to a variant gene, do ISH and see where its been expressed

Question 27

how could you find the function of a cell/tissue controlled by the gene product?

Answer 27

• Conditional deletion of a gene

If you find a variant gene, can KO this gene in the area

Question 28

what is tissue specific KO?

Answer 28

Gene that you want to KO is flanked by lox sites and transgenic mouse 1 is made.
Where normal gene is replaced with the engineered gene
We refer to this as the ‘floxed’ allele (flanked by lox sites)

Question 29

describe how you would KO a specific cell in a gene

Answer 29

• Identify a promoter that governs tissue/time specific expression of a gene
• Whatever is downstream of that promoter will only be expressed in that tissue at that time
• Clone the coding sequence for an enzyme called ‘Cre-recombinase’ downstream of this promoter
• Make a second transgenic animal, Cre-recombinase expressed under a heart specific promoter
• Cre-recombinase acts on lox sites like a pair of scissors and recombinase at the ends (without your gene)
• Cross (i.e. breeds) the 2 mice together.
• Floxed allele will be excised where Cre-recombinase is expressed

Question 30

what is a technique of visualising cells in a live animal?

Answer 30

transgenic reporter line

Question 31

describe what transgenic reporter lines are including what they depend on

Answer 31

o These are animals whose genomes have been engineered to give a ‘colour report’ of expression of a particular gene
• They enable:
o Visualisation of cells/tissue in a living organism in real-time

♣ E.g. If looking for a population of cells in the liver, use transgenic reporter lines to label all cells in this population, extract the liver and sort through the cells. Those that fluoresce can be separated from the others using FACS sorting
• depend on the fact that genes are differentially transcribed as a function of the interaction of their promoter/enhancer and the cell specific transcription factors/activators

Question 32

how are labelled transgenic lines later isolated?

Answer 32

o Isolation of labelled cells/tissues (by FACS sorting)

Question 33

what are vantages of reporter lines?

Answer 33

• Can follow transgenic reporter cells over time in health, in disease, after ‘insult’ (e.g. starving/extra feeding of the model organism) and after drug administration
• At the same time, can perform simple analyses in real time –
• E.g. analysis of cell behaviour (e.g. is cell proliferating?) or analysis of cell function (e.g. is cell secreting a hormone?; is cell responding to a signal?)

Question 34

what is the advantage of using an animal model that has lots of offspring at a time?

Answer 34

Large numbers of animals and reporter lines mean that animal models used to powerful effect in development of new therapeutics

Question 35

what is the main advantage of doing reporter lines in fish?

Answer 35

fish are small and permeable to drugs and so can be used in drug screens

Question 36

why is it advantageous that model organisms allow us to do whole organism/system analysis?

Answer 36

• Because our bodies operate through complex and dynamic interactions between different body systems, organs and cells
• An understanding of disease and deterioration requires a comprehensive understanding of the whole organism, and the interaction between its different components

Question 37

what technique would enable you to study whole organisms/systems? how can they be examined?

Answer 37

multiple transgenic reporter lines
cane examined in vitro or ex vivo
- Can be done through multiplex transgenic reporter lines
- Can be examined in vivo and in vitro or ex vivo (as these cells are still living)

Question 38

why is it useful to analyse how cells are operating in vivo and ex vivo?

Answer 38

• If you want to make a recording or know you want to culture jus 1 cell type without the influence of the whole body, these techniques would be useful

Question 39

what are organoids and how can they be used?

Answer 39

mini organs in vitro: which is a way of having a 3D structure that would be easier to manipulate and interrogate then when in vitro

Question 40

what has allowed to clearer and better recording of cells?

Answer 40

Microscopy has gotten better e.g. light sheet microscopes which allow you to have a better understanding of cell dynamics

Question 41

what disease is the fastest growing health threat of our times?

Answer 41

diabetes

Question 42

what has been the increase in diabetes occurane from 2005 to now?

Answer 42

50% increase
in2016 - ~3.6m
in2025 will be ~5m

Question 43

what is diabetes?

Answer 43

Diabetes mellitus (DM) is a group of metabolic diseases characterized by high blood sugar resulting from defects in insulin secretion, insulin action, or both.

Question 44

how many people with diabetes have T2D?

Answer 44

Nearly 90% of patients with DM have Type 2 diabetes

Question 45

what happens in terms of insulin in type 1 and type 2 diabetics?

Answer 45

• type 1 diabetic – cells don’t make insulin so don’t get any insulin in the blood stream so the glucose builds up (genetic)
• type2 diabetic – initially normal in terms of insulin and glucose. Insulin goes into the blood stream but the cells in the body are insulin resistance so glucose isn’t taken up as normal in cells of the body and so builds up. Over time, results in 2nd event – cells of pancreas which were originally fine, they become so damaged and respond to the feedback loops and become depleted. Therefore, despite early mechanistic differences, both type 1 and late stage type 2 diabetes feature depletion of B cells.
• Persistently elevated blood glucose can cause damage to the body’s tissues, including the nerves and blood vessels
• Elevated hyperglycaemia, over time, increases morbidity.

Question 46

what is a factor that is thought to trigger type 2 diabetes? is this factor definitely a factor that causes it?

Answer 46

• Obesity: highly associated with T2D
• Strong positive correlation between obesity and T2D
• Clinicians measure waist circumference, if its less than 30-32 inches, have relatively low risk. By 38 inches, this triples.
• Unclear on how obesity results in T2D
• Biggest problem is that so many tissues involved – each interacts with the other, so very difficult to establish cause vs consequence (and in all likelihood, probably different trigger events in different individuals….)

Question 47

why is there confusion in free fatty acids an insulin resistance in terms of obesity and diabetes? what can it cause?

Answer 47

• Central obesity – obesity around the waits
• Leads to increase in free fatty acids and so insulin resistance. Not sure which one of these come first (FFA or IR)
• These 2 can lead to increase in apolipoprotein B and hepatic lipase
  o These are bad for your CVD
  o Increase risk of getting a plaque
  o Leads to comorbidity (CVD and T2D)

Question 48

what 3 major metabolic defects does hyperglycaemia in type 2 diabetes result in?

Answer 48

• Impaired insulin secretion
• Insulin resistance
• Increased glucose production

Question 49

what is obesity linked to and how does this link it to diabetes?

Answer 49

• 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; ectopic fat accumulation in the liver and skeletal muscle is associated with insulin resistance and type 2 diabetes.
  o So maybe normal adipocytes are essential in stopping you from getting T2D

Question 50

what is the molecule that binds as a thymine in DNA replication instead of thymine? why is it useful in scientific studies?

Answer 50

EDU binds in DNA replication where T normally would. when this happens, using a computation ‘click’ method, can fluoresce these molecules and so can see whether or not the cell is dividing/proliferating

Question 51

what in the body is what mediates homeostatic processes?

Answer 51

the brain

Question 52

what in the body is what mediates glucose homeostasis?

Answer 52

neurones in the brain regulate glucose homeostasis

Question 53

what behavioural traits does T2D manifest as?

Answer 53

T2D manifests as dysfunctional eating and energy metabolism. this means that its highly likely that there is a major disruption in core homeostatic things happening in the brain

Question 54

what are the 2 reasons that tere could be incorrect co-ordination between the brain and organs?

Answer 54

could be that there is abnormal peripheral organ signalling to the brain
cold also be that there is abnormal signalling from the brain to other organs

Question 55

what cell secretes leptin?

Answer 55

adipocytes

Question 56

what is the role of leptin the body?

Answer 56

leptin signalls to the brain to alert it of the status of the bodys energy content.

Question 57

what can be seen in mice lacking leptin? what can be inferred from this?

Answer 57

mice lacking leptin are obese, diabetic, infertile and hypoactive
tells you that the hormone is leading to obesity

Question 58

what could misignaling in the brain to peripheral organs manifest as?

Answer 58

could manifest as dysfunctional eating, energy metabolism and/or autonomic activity

Question 59

how could dysfunctional eating in leptin KO mice lead arise?

Answer 59

via indications from the brain that insufficient fat is present, thus triggering increased food intake and consequent increased body fat
or
reduced signalling to the endocrine pancreas and liver

Question 60

howcan you see what Leptin does in each cell?

Answer 60

do tissue specific KO of leptin receptor, so 1 by 1 make condtional leptin receptor null in each organ and see what happens

Question 61

what is another term for a conditional KO?

Answer 61

a selective deletion

Question 62

what was found when a selective KO of leptin receptors in neurones was done? how did they know that the effect was not in the liver?

Answer 62

they found that leptin neurones causes obesity in neuorones

mice with leptin receptor KO in the liver were normal

Question 63

what were the 3 things that were found about leptin receptors in animal models?

Answer 63

1. mice lacking leptin receptor signaling are obese, diabetic, infertile and hypoactive
2. deletion of leptin receptors in neurones indicate obesity whereas expression of Leptin receptors in neurones of mice lacking leptin receptors led to an amelioration of their obesity
3. intracerebroventricular (icv) administration of leptin in mice lacking teh leptin receptor causes reduction of body weight andfood intake (done by infusing leptin into CSF and this is contained in the brain)

Question 64

how would you go about trying to KO a neurone?

Answer 64

go for a neurone pomoter and flox it here so that you can KO all of these neurones

Question 65

how would you go about trying to KO a neurone in the brain? how would this help you figure out where leptin is expressed?

Answer 65

go for a specific promoter eg hypothalamus specific promoter – so then you can go deeper into which particular class of neurones or cells are expressing the protein (leptin)
then •	gradually choose promoters till you KO leptin in each cell type till you find where the leptins primarily acts

Question 66

what did animal models help to understand in terms of the location of leptin receptors?

Answer 66

they are located in the hypothalamus in a specific region called the arcuate nucleus

Question 67

what are the types of neurones in the arc nucleus? - what do they do?

Answer 67

NPY - make a neurotransmitter

POMC - makes a long protein

Question 68

what kind of actions do POMC and NPY do with eachother?

do they have the same jobs?

Answer 68

• –both exert antagonistic actions on a common downstream pathway
• –do different jobs once there - they have opposite effects to each other

Question 69

what are the exact roles of NPY and POMC?

Answer 69

• NPY neurones stimulate food intake

* Pomc neurones reduce food intake

Question 70

how does deficient activity of hypothalamic neurons leads to T2D and obesity?

Answer 70

• primary causal factor in T2D lies in the interaction of the brain with peripheral tissues such as the gut, the liver, the endocrine pancreas, adipose tissue and others. This is typically manifest as dysfunctional eating, energy metabolism and/or autonomic activity.
• 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)
  or
  • by abnormal signaling from the brain to other organs (e.g., reduced signalling to the endocrine pancreas and liver).
  o Results in abnormal balance of the pomc and nyp cell types which has a knock-on effect of abnormal signaling from the brain to other cells/organs
  o This understanding comes from animal model studies

Question 71

describe a model of the hypothalamic regulation of hepatic glucose production

Answer 71

• 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 72

what kind of cells can make generate cells?

Answer 72

• progenitor cells

- stem cells

Question 73

where in the body are leptin responsive neurones?

Answer 73

in the arcuate nucelus of the hyporhalamus

Question 74

what does leptin inhibit?

Answer 74

NPY (neuropeptide y)

Question 75

what is the action of NPY?

Answer 75

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

Question 76

what does leptin stimulate?

Answer 76

o Leptin stimulates pomc neurones

Question 77

what are pomc neurones?

Answer 77

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

Question 78

why can it be considered that energy and stress go together?

Answer 78

Pomc is a pro-domain neurone so by itself acts like this but when cleaved off becomes various subunits – 2 = involved in energy metabolism, 1 = involved in ACTH which triggers your stress response
♣ Energy and stress responses go hand in hand

Question 79

what is the location of pomc and NPY?

Answer 79

lie very close to the 3rd ventricle

Question 80

what is the name of the region of the brain that does not have blood flow?

Answer 80

• Median terminus – do not have a blood flow (few regions of the brain) – instead have communication between the brain

Question 81

what are pomc and npy cells generated from?

Answer 81

• Pomc and NPY neurons are generated from hypothalamic stem cells.

Question 82

what is the appearance of the hypothalamic cells?

Answer 82

o hypothalamic stem cells have a radial glial-like appearance (cell body sits at ventricle and projects as a basal projection, with the cell body at the ventricle (lines it) and a projection that is used as a scaffold

Question 83

what are specialised radial glial like cells in the hypothalamus called?

Answer 83

tanycytes

Question 84

what technique can be used to test the idea that pomc and npy neurones can be generated from hypothalamic stem cells through anticipation and response to the changing needs of the body? how do we know this?

Answer 84

o evidence comes from genetic lineage tracing analysis where people have shown, using conditional lineage tracing strategies that an adult tanycyte can give rise to NPY in an adult mouse
• Through animal model studies that look at the generation of hypothalamic pomc and NPY neurones from stem and progenitor calls through life – i.e. both in development and in the adult
• Studies done in development and in the adult

Question 85

what are the 4 ways that experiments canbe done to trace/understand pomc/npy and hypothalamic stem cells?

Answer 85

1. Identify different populations that develop over time through markers
2. Lineage trace the stem cell (do this in a way to conditionally KO the gene – if you have the right construct, lets you follow the cell and all its decedents over time (genetic lineage tracing))
   a. Can identify key genes that maintain or cause development of a gene and check which means fail to form due to this knockout
3. Identify key genes that maintain each cell. Ask if daughter cells fail to differentiate when these genes are knocked out
4. Use the promoters of these genes to make cytotoxic transgenes to eliminate particular cell populations

Question 86

• How do you determine a gene is important?

Answer 86

• See if it is there at the right time and place

Question 87

what do fgf and shh promote?

Answer 87

o Fgf promotes proliferation
oFgf acts to maintain proliferation and the action of the cell cycle (survival factor)

o Shh promotes differentiation
oThe expression of Shh, this leads to the generation of arc progenitor cells which produce islet1

Question 88

what tf is expressed in the cell just before pomc?

Answer 88

islet 1

Question 89

what is the role of islet1? how is it expressed?

Answer 89

— Islet 1 – tf that intrinsically up-regulates transcription of pomc gene

islet 1 = releases pomc encoded melanocortins which are potent anorexigenic
neuropeptides and their absence in mice or human’s leas to hyperphagia and severe obesity
early expression of
If pomc – final differentiated marker (determines what the molecule will become), then you would expect that if there was a tf before this, that there would be expression of the tf before this

Question 90

describe the signalling of islet1

Answer 90

Islet 1 tf binds to islet 1 promoter (critical homeodomain binding DNA motif) to upregulate transcription of the pomc gene (in vivo and in vitro)

Question 91

what can happen in the event of a mutation in the islet1 promoter site?

Answer 91

Mutations of these sites completely disrupt the ability of these enhancers to drive reporter gene expression to the hypothalamic pomc neurones in transgenic mice and zebrafish

Question 92

what is islet 1 necessary for?

Answer 92

Isl1 = necessary for hypothalamic POMC expression during mouse and zebra embryogenesis

Question 93

describe the technique of lineage tracing for pomc/npy?

Answer 93

o Lineage tracing from an adult stem cell:
o Techniques is a variation on a theme of any conditional effect
o Have a cell specific promoter (from a gene of the cell you want to lineage trace)
o Promoter = cloned and also Cre enzyme that is fused to ERT2
♣ Means that cre recombinase is only activated when tamoxifen is injected into the mice as ERT2 requires presence of tamoxifen for activity
o Have a second reporter construct and make a transgenic animal where the reporter gene is downstream from a ubiquitously expressed promoter but is prevented from being transcribed because you have a little bit of DNA in there so can’t transcriptionally read through from the promoter as the stop sequences are there and are flanked by loxp sites
o Idea = if you recombine these mice (have single double transgenic animal), at whatever point in it’s lifespan that you add tamoxifen, the cre begins to work and cuts out loxp sites and you begin to activate the reporter (GFP) so initial cell and all its decendents have the GFP in them
♣ Only thing that should be green is in the stem cells (all of its decendents)
o If it’s a stem cell, can self-renew or differentiate
♣ After 9 months, expect to see GFP seeing in the arcuate nucleus – can confirm that the hypothalamic stem cells go here

Question 94

what is the standard approach for lineage tracing?

Answer 94

• Use this to lineage trace from a stem cell:
• Clone CreErT2 downstream of a stem cell promoter.
• Make transgenic
• Make a 2nd transgenic where a stable reporter is downstream of a constitutive promoter, but separated by a floxed STOP sequence
• Breed mice. Add tamoxifen at any stage in life to recombine out STOP.
• Now the reporter will be stably expressed in that cell and all its daughters

Question 95

what is the tanycyte specific promoter?

Answer 95

• The tanycyte specific promoter = GLAST::CreERT2 is a transgenic mouse line that reports, and can be used to lineage-trace a-tanycytes (radial glial like hypothalamic cells)
• Recombined n 2 different lines
o A blue reporter z line
o A green GFP reporter line

Question 96

what can immediate and long term sacrificing show about tanycytes?

Answer 96

• Immediate sacrificing shows that only a subset of tanycytes are labelled
• Long-term lineage tracing shows that alpha-tanycytes can self-renew, give rise to other tanycyte subsets, and to neurones of the Arc

Question 97

what kind of jobs do teh neuroens that tanycytes give rise to end up having?

Answer 97

neurones that are involved in energy formation

Question 98

are newneuros being made all the time by tanycytes?

Answer 98

no

eg in a caged animal, • not making new neurons because its in a homeostatic norm (optimum)

Question 99

what is observed when fgf is infused into the 3rd ventricle? what is seen in teh control vs with fgf2

Answer 99

high levels of fgf can induce proliferaton of the tancytes
• In control, (just brdu injection), cells in s phase and very few, when adding brdu with fgf2, see high amount of proliferation so cells in s phase

Question 100

what is the movement of new born cells from tancytes when fgf is added?

Answer 100

o Some of the new born cells are beginning to migrate out aling the tanycyte processes – daughter cells that come from tancytes that migrate along and presumably will differentiate into the new neurons of the arc nucleus
o Suggests that physiology signals interact with developmental signals to govern how a neural stem cell in the brain is going to respond

Question 101

why is it being a problem to test if a core part of control of energy homeostatsis = in hypothalamus is about the balance between pomc and npy neurons is disrupted in obesity/T2D?

Answer 101

o So, difficult to test as need subject to be dead to be able to test amount of pomc neurons they have
o Also, would have to section that bit of the brain where the pomc neuornes are and only few neuropathologists are able to do this part of the hypothalamus

Question 102

what is a suitabe animal study to test if pomc neurone count is changed by T2D/obesity? why cant it be done in humans?

Answer 102

zebrafish
♣ In human, cant count the amount of pomc neurones without killing them (no antibody against pomc neurones), so beneficial that in zebrafish, they have far fewer pomc neurones and these can be counted through life
♣ The human subject would need to be the control for themselves but the problem is that you aren’t able to kill the subject twice obv so shows why animal models are important

Question 103

what did marysia’s study on fgf and tancytes in the hypothalamus show?

Answer 103

¬ Marysia’s studies have shown that in mouse animal models, in the hypothalamus, have a set of neural stem cells that can be stimulated by fgf2 within the hypothalamus, to proliferate and give rise to neurons that can provide energy ie nyp neurons

Question 104

what did scwartz (USA) study that was based inn fgf single injection in 3rd ventricle show? where was this effect seen?

Answer 104

¬ Schwartz (USA) – looked at diabetes in mice and rats and injected fgf1 into 3rd ventricle and found that this 1 single injection induced a sustained remission of diabetic hyperglycemia in rodents (able to fully induce remission in diabetic model)
o Effect = not on a peripheral system, involves a novel mechanism – brain has an inherent potential to induce diabetes remission

Question 105

how did scwartz show that the effect of a single fgf injection on the 3rd ventricle was on the brain and no where else?

Answer 105

o Don’t see similar sustained diabetes remission after systemic (intraperitoneal) FGF1 infusion (shows that its primarily going on in the brain)

Question 106

what did the single fgf injection into 3rd ventrile by scwartz show?

Answer 106

♣ The single injection reduces blood glucose levels to norm

Question 107

what did scarlett et al show about the activity of tanycytes after infusion of a certain molecule? what was shwo about hsp25 and fuzz?
what does this prove?

Answer 107

¬Scarlett et al looked at tanycytes, Saw increased activity after FGF1 infusion
¬ Looked at hsp25 and said that tanycytes become more active due to the gene hsp25
¬ Looked at another marker (fuzz) and saw that it was upregulated in these
o Functional link between activation of tanyctes and induction of diabetes remission induced by fgf1

Question 108

what is the name of the diseases that are reaching epidemic proprtions worldwide? how many deaths do they accohtn for worldwide?

Answer 108

Chronic non-communicable diseases
• Account for 60% deaths worldwide
• account for 44% of premature deaths worldwide

Question 109

what is the name of the scheme to try and tackle chronic non communicable diseases? how does the scheme work?

Answer 109

grand challeneges
they have goals and the biomedical scientists can work on identifying root causes, risk and protective factors and also raising the awareness of the global burden.

Question 110

what er 2 things that are known to contrive to millions of preventable diseases each year?

Answer 110

• poor diet and smoking are 2 factors that contribute to the millions of preventable deaths that occur each year

Question 111

what specifically links diabetes and CVD?

Answer 111

• hyperglycemia is thought to link directly to atherosclerosis
o increasing ill health and probability of death

Question 112

describe the full relationship between obesity and insulin resistace and dyslipidemia. what does it lead to?

Answer 112

central obesity has an increased amount of free fatty acids (FFA) (could be director due to the increased insulin resistance)
then see increase in apolopoprotein B, increase in heatuc lipase which then increases the amount of triglycerides, small,dense LDLs in the body and decreases HDL.
In this state, this is a syndrome called the metabolic syndrome

Question 113

describe the metabolic syndrome

Answer 113

Characterized by increase in insulin resistance (in complex disregulation of lipids i.e. increase in ldls etc)
Insulin resistance is a precursor to a variety of metabolic abnormalities, including systemic inflammation, visceral obesity, and type 2 diabetes.
Insulin resistance is also a risk factor for cardiovascular abnormalities, including hypertension, dyslipidemia (increased triglycerides and LDL and decreased HDL), disordered fibrinolysis, and endothelial dysfunction.
All of these aberrations contribute to the atherosclerotic process.

Question 114

what is the link between metabolic disorders and cancer?

Answer 114

increased risk of certain cancers with metabolic disorders e.g. breast cancer, bowel cancer, kidney etc.

Question 115

how many ways are there that metabolic dysfunction could be causal to cancer? what are they?

Answer 115

3 main ways:
1. Oestrogen
After menopause, oestrogen made by fat cells can make cells multiply faster in the breasts and womb, increasing the risk of cancer
2. Insulin and growth factors
Excess fat can cause levels of insulin and other growth factors to rise, which can also tell cells to divide more rapidly
3. Inflammation
Cells in fat called macrophages release chemicals called cytokines, encouraging cell to divide (including cancer cells) – issues in inflammation resolution

Question 116

what was found out abut mice in terms of what cancers they were susceptible to?

Answer 116

• 1930s: Some particular inbred strains were highly susceptible to mammary tumours
o i.e. females developed mammary tumours with high incidence

Question 117

what was discovered about the development of tumours when next to to particular viruses

Answer 117

• 1983: It was discovered that tumours form when a particular virus integrates next to a so-called ‘proto-oncogene, turning on abnormally high levels of transcription of the gene

Question 118

how do retroviruses hijck the body’s machinery?

Answer 118

o in free living form – lives as RNA but hijacks the hosts machinery and cDNA copy of itself – makes itself a dsDNA which inserts itself into the host DNA and integrates with it
o does this in a way that it integrates specifically next to some sorts of genes
o promoter = feble and weak and so have a low number of transcripts produced at a low rate
o if RNA retrovirus reverse transcribes and then its cDNA form integrates upstream of the coding sequence of that gene (could just be in its frame), you read through the very strong vial promoter so end up transcribing ~100 molecules of this gene

Question 119

what is the normal role for oncogenes?

Answer 119

• oncogene normally works to make sure you have the right amount of genes being transcribed in the body

Question 120

what is the role of proto-oncogenes?

Answer 120

o a proto-oncogene differs in that if that gene is mutated or is affected in a way that you start making any more copies of the transcript, you turn that proto-oncogene into an oncogene
o whatever he gene is coding for, if expressed at very high levels, the protein will give selective growth advantages to that cell

Question 121

what is the name of the mouse gene that was transcriptionally upregulated was cloned?

Answer 121

int 1

Question 122

how often was it found that mmtv (RNA retrovirus) integrated next to int1? where was it when it wasn’t next to int1?

Answer 122

¬ Found that in about 30% cases, MMTV integrated next to Int1 ( in the other cases, it often integrated next to Int2 and Int3, subsequently revealed to be FGF3 and Notch).

Question 123

how do you know that there is a growth advantage for int1 oncogene?

Answer 123

the placement of mmtv next to int1 is random yet still had a high likelihood of being close

Question 124

what happened in teh initial attempts to purify int1? what was done in the meantime?

Answer 124

• Initial attempts to purify the ‘int1’ protein unsuccessful (only achieved in 2003) – making standard biochemical assays impossible
• But in the meantime, developmental studies in drosophila are enormous contribution to our understanding of Int1

Question 125

who found the link between int1 and wnt- what was this link?

Answer 125

o 1980: nusslein-volhard and weischaus: mutagenesis screen in drosophila identified ‘patterning genes’, including segment polarity genes. One of these segment polarity genes was wingless
o sequence homology showed int1 = wingless. Therefore renamed – wnt1

Question 126

what genes linked to wnt1 were discovered in 2007?

Answer 126

2007: genes activated by wnt signaling include cell cycle regulators were discovered e.g. CmyC, Cyclin D1 were discovered
some of the key gees – those that directly govern cell proliferation – e.g. CmyC and Cyclin D1 (promotes cell cycle)

Question 127

is it the cells that make wnt or those that respond to it that causes the high instance of mammillary tumour?

Answer 127

it i sthe cells that resnd to wnt signallling

Question 128

how can wnt signalling be considered as an autocrine pathway?

Answer 128

o if you have cells side by side that both receive and make wnt, have an autocrine system where cell A triggers cell proliferation in cell B and cell B is triggering proliferation in cell A.

Question 129

what can abnormally high levels of wnt signalling in mice cause?

Answer 129

mammillary tumour

Question 130

how can you activate the wnt signalling pathway?

Answer 130

¬ studies on the basic signalling pathway components that exert negative/suppressive effects
¬ 2 ways of ‘activating’ the wnt signalling pathway:
1. increase activators
2. remove suppressors

Question 131

why is it significant that high wnt1 signalling upregulates cyclinD1?

Answer 131

cyclinD1 is a tf that works in the cell cycle so more of this = more G1 especially in the cell cycle so growth of cell numbers

Question 132

has there been any evidence in humans that overexpresson of wnt leads to cancer?

Answer 132

no, but mutations in Wnt signalling pathway components leads to many human pre-cancerous states and cancers

Question 133

what kinds of mutations of wnt can cause precancerous states?

Answer 133

The mutations fall into 2 categories –
a. those which are activating (i.e. meaning that beta catenin is always in nucleus)
or
b. more commonly, those where both copies of a repressor are lost – again meaning that beta-catenin is always in nucleus)

Question 134

what is the most common form of human cancer linked to abnormal wnt signalling?

Answer 134

the most common form of human cancer linked to abnormal wnt signalling is colon cancer/colorectal cancer

Question 135

why is it importnat to predict value of all pathways using animal models?

Answer 135

by establishing how genes control signaling (e.g. whether they act as repressors or activators) one can make educated guesses how these genes might contribute to human cancer.
A repressor can be a tumor suppressor gene and an activator a dominant oncogene.
The same principle applies to other pathways, such as the Hedgehog-Patched pathway and the TGF beta pathway

Question 136

what was found using reporter lines of the axin gene in the wnt pathway?

Answer 136

Led to the idea that activation of wnt signaling is not necessarily promoting the proliferation of all cell types in a random manor, but is actually promoting enhanced proliferation in tissue-specific stem cells in a way that cell stem cells would be proliferating/self-renewing in response to wnt in an uncontrolled manor
This uncontrolled self-renewal is a hallmark of oncogenesis (predisposition to cancer)
The cells that may lead to high BC or KO activator/repressors, if these are stem cells, more likely will lead to cancer

Question 137

where is the best most well defined stem cell nuces?

Answer 137

• One of the best-defined adult stem cell niches is in the mammalian gut crypt (at the base of the villi). The crypt is a tube of cells with stem-like cells in a niche at the distal end and differentiating/ed cells at the proximal end

Question 138

what can be detected in pre cancerous adenomas in terms of wnt signalling pathway?

Answer 138

o In pre-cancerous adenomas, detect elevated levels of nuclear BC in crypt stem cells

Question 139

what can you use inbred mice for in terms of cancer development understanding?

Answer 139

• Identify oncogenes/tumour suppressor genes
• Identify signaling pathways that lead to specific tumour types. Predict which genes might give rise to cancers, if mutated.
• Understand the basic biology at a cellular level (e.g. more stem cells, uncontrolled stem cells)
• Impact on: diagnostics and treatment

Question 140

what was found about treatment effectiveness using herceptin vs other things for breast cancer?

Answer 140

In breast cancer, it’s a mass of proliferated ells that respond to estrogen and if targeted with Herceptin, can get rid of these cancerous cells – till remission
Have a set of breast cancer stem cells that weren’t responding to this drug because what was controlling them = wnt, Hh, Wnt, NFKb etc
So, if you have a treatment that targets Herceptin and also all these genes, could turn out to be a more effective treatment

Question 141

describe the process/technique of single cell RNA sequencing

Answer 141

take a mass of cells from a tumour, separate/dissociate these into single cells, encapsulate every single cell in its own isolated oil bubble and can put into these oil bubbles, the machinery that would tell you which tf are being expressed in that cell. Then can use bioinformatics to see how many of each of these cells can you see

Question 142

what does the analysis of stem cell behaviour in vivo allow you to do?

Answer 142

• Correlate particular signalling pathways with tumour aggression
• Find interacting genes that accelerate/retard
• Impact on prognostics
• Cancer as chronic disease: follow over time
• Cancer involves multiple systems – can dissect/understand these in an in vivo model
• Test functional consequences of specific human mutations
• Therapies?

Question 143

what is an adenoma? what is more dangerous than this?

Answer 143

adenoma - a proliferating block of cells
not particularly dangerous in most cases
metastatic tumour here the cells are breaking off and traveling and seeding secondarily in other parts of the body
This happens over time and what kills you isn’t the adenoma, but these secondaries that travel to different parts of the body

Question 144

what is involved to make an adenoma into a full blown tumour?

Answer 144

In order to progress from an adenoma to a full blown tumour, the inflammatory pathway comes into it

Question 145

how does the inflammatory system link into tumour progression?

Answer 145

if you constitutively activate wnt signaling, this leads to increased active Rac1, leading to hyperproliferation.
Rac mediates this effect via ROS (reactive oxidative species) and NF-KB, indicating a role for inflammation in progression of a benign to a malignant tumour

Question 146

what is ROS and NF-kB? how are they linked to inflammation?

Answer 146

ROS and NF-kB = pathways known to be instrumental in the inflammatory pathway

• The nuclear factor NF-κB is a proinflammatory factor: it positively regulates the expression of proinflammatory genes including cytokines and chemokines.
• ROS are key signalling molecules that play an important role in the progression of inflammatory disorders

Question 147

what is the potential mechanism of action for a tumour to become a metaplasia including the influence of the imflammatory signals?

Answer 147

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

Question 148

Why do we use animal models to understand human diseases?

Answer 148

• Animal model studies can help us understand mechanism

* Animal modelling can be used for pre-clinical therapeutic development

Question 149

what is melanoma?

Answer 149

• Melanomas develop from melanocytes (moles)
• Black pigmented cells, if they go wrong, will start growing and then break through their usual location and when these cells start desemnitating, then its untreatable
• If the tumour is localised, it can be treated well but as soon as they metastasise, it is incurable
• Very difficult to spot a melanoma from a normal mole

Question 150

what is the occurance of melanoma?

Answer 150

• Is a skin cancer and is the 5th most common cancer in the UK (2014), accounting for 4% of all new cases, 2500 deaths/yr

Question 151

what is more lethal, BCC or melanoma? why?

Answer 151

• Basal cell carcinoma, sickle cell cancer ~20 fold more frequent, easily treatable so medically, not such a big problem… Patched (Hh activator) are initiator mutations in BCC
• Melanoma is a much more lethal disease – if you’re beyond a certian point, there is no treating you
• Melanoma, according to the 20 most common cancers in 2014,is on the increase and so will likely continue to be a problem in the future

Question 152

what is the most important risk factors of melanomas? why?

Answer 152

age

o As you age, you accumulate mutations and the more mutations you wull get that could cause melanoma

Question 153

how are gender, sunbun/UV and skin colour risk factors for melanoma?

Answer 153

• Males have a higher risk than females
o True for many cancer
o Could be due to high somatic mutation rate
o Could be psychosocial?
• Skin colour
o Very light skin colour – 24xincreased likelihood of getting melanoma than if you are darker
• Sunburn/UV
o If you get sunvurnt, this significantly icreases the chances f you getting melanoma
o If you have had a blistering sunburn more than 5 times in your life, your risk of getting melanoma is 50% more likely

Question 154

what genetic mutations can be implicated in melanoma development? important

Answer 154

o DNA repair genes that repair damage due to UV irradiation of DNA are very well known to be very high-risk factors
o Xeroderma pigmentosumenes are involved here and f you get a mutation in this, you have skin that is very vulnerable/sensitive to getting UV sunburnt and so higher likelihood of getting melanomas
o Melanocortin receptor – if you are a mutant for this receptor which is seen in those wth red hair, you are also more vulnerable
o CDKN2A codes for 2 protein: p16 and p14 which are involved in sending cells to apoptosis when they have significant DNA damage. They protect the integrity of the cell. If these genes aren’t there/mutated, cells that should have died, don’t. loss of tumour suppressor function
o Rare promoter mutations that activate TERT expression. TERT is a gene that maintains our telomeres and so is able to immortalise cells and allow them to survive when they shouldn’t.

Question 155

what drive melanomas to cause mutations and keep alive cells with mutations in them?

Answer 155

• Melanoma cells all have constitutive MAPK pathway activation
• There are a number of mutations called initiator mutations that are always seen in melanomas
o 2 very important initiator mutations in the melanoma = BRAFV600E, NRAS
o both involved in the MAPK pathway
• often seen in progressing stages
o TERT … telomerase
o SWI/SNF … chromatin remodelling
o CDKN2A … cell cycle
• Often seen in advanced/late stages
o P53 and Pten
o P53 = important apoptosis mutation seen in many cancers

Question 156

how does a melanoma develop?

Answer 156

Melanomagenesis
• Moles already have an activated RAS pathway in general – but doenst immediately lead to a tumour but it is the first step
• Activating BRAF mutations are found in ~60% of nevi and more than 75% of melanomas have mutations in BRAFV600E
• Found in a study by Shain et al that at different stages of a melanomas progression eg benign lesion, intermediate, invasive and aggressive, there were different driver genes.
o At the benign lesion stage, BRAFV600E was the driver gene
o As you go further along, there are more mutations involved in this

Question 157

How do you model and study melanomas?
- specifically, core cell cycle machinery
what is a disadvantage to this model?

Answer 157

Yeast as their machinery = similar to that in humans

But yeast don’t have any upstream signals that tell the cells to divide – nlike in humans

Question 158

how would you study upstream signals that control cell cycle, division, differentiation and death?
-i.e. what pathway and in what model?
what are disadvantages of this?

Answer 158

Study the EGF/RAS/MAPK, Hedgehog, Wnt,, etc,
Use Drosophila melanogaster, Caenorhabitis elegans
However, there are some things that you want to study e.g. angiogenesis…. And this can only be done in higher model organisms because drosophila simply don’t have these

Question 159

why do you need to use higher order model organisms to study angiogenesis in tumours?
which models would be good here?

Answer 159

Tumor growth and metastasis: dependent on angiogenesis/lymph vessels, immune system and these things don’t exist in drosophila and C. elegans. So, use vertebrates to study these things in the same tissue context
Mice and fish

Question 160

why are mice good model organisms for studies in tumours? what techniques can be done in them?

Answer 160

• closest to humans
• Can do precision genetics
• Knockouts - you can make extremely complicated model systems using this
• Xenotransplants (mouse that take cells from other origins)

Question 161

why are fish good model organisms for studies in tumours?

Answer 161

• Can study in large scales (thousands of animals)
• Optic clarity as they are optically clear and so can study single cells in a tumour as you can use a high resolution and follow single cells over time.

Question 162

what are the benefits and negatives of using mice as melanoma models with xenotransplant studies?

Answer 162

• Xenotransplants of human cell lines or tumours in nude mice
(+) human cells
(-) location (sometimes don’t go to the exctlu rigjt location)
(-) immunocompromised (immune response is an important part of melanomageneiss and so camt really model their immue response when there immune system is compromised

Question 163

what type of mouse is used to study melanomas? describe them and why they are used

Answer 163

Nude mice – have mutation in the foxn1 gene and so have a defective immune system. Can grow human timour cells in these mice and then assess them in these animals. When grown, can study location, see speed of growth and whethr ot nor metastasis occurs

Question 164

describe the process of doing precision genetic engineered mice for melanoma studies.
ie the Tyr:Cre-ERT2; BrafCA/+; PTENlox/lox genotype mouse. what happens upon injection of tamoxifen?

Answer 164

• Drive melanocyte expression of activated N- RAS and normally, insufficnet to form a tumour and so need to irradiate with UV or co-inactivate CDNKN2A
• Drive melanocyte specific expression of Activated BRAF: nevi but insufficient in combination with loss of PTEN melanoma
• Need a doubleKO in order to form the tumours
• Created a mouse with this genotype:
  Tyr:Cre-ERT2; BrafCA/+; PTENlox/lox
  Contains a tyrosine that drives the expression of Cre-ERT2. This is activated by Tamoxifen, tyrosine promoter and only in melanoyctes
  BrafCA Cre-Activatable constitutive active from of BRAF
  PTENlox A “loxed” alelle of PTEN that can be inactivated by Cre mediated excision
  When you inject tamoxifen, Cre-ERT2 will go into the nucleus and cause activation of BRAF and simultaneous inactivation of PTEN
  This wil only happen in the melanocytes where the Tyr promoter is active

Have created melanosomes that not only have activated BRAF signalling but also noPTEN anymore

Question 165

what technique has now been developed which has allowed us to make null mutations in fish?

Answer 165

New developments – Crispr mutagenesis
Efficient in zebrafish
Ver easy to now make a null mutation in fish
Crispr could also be used in genome editing (not full developed technique yet)

Question 166

Can we replicate human disease using animals?

Answer 166

no because only humans get human diseases. we can model some disease processes i.e. the biological processes that we think are important in diseases

Question 167

how many people have heart defects? i.e. hole i the heart etc

Answer 167

1% of people have heart defects

10% have small holes in the heart that could predispose you to get various heart conditions

Question 168

describe the process of becoming a heart from day 15 to week 50

Answer 168

day 15 post fertilisation you don’t have a heart, just a region of cells that will become the heart
a week later, get a tube-like structure with ventricle at bottom and atria at top
week later, divided
week 50, get the sort of structure that you have now

Question 169

what are some weaknesses of using zebrafish as model organisms?

Answer 169

• Small size limits some techniques e.g. stents
• Single circulation
• Genetical less similar to humans than mammals/primates
o May not have all the homologues(only have70% - the rest doesn’t exist in these fish)
• Rapid development – moving baseline ; can affect this
• Don’t get CVD

Question 170

define pluripotent and stem cells

Answer 170

Pluripotent cells are able to generate the 3 germ layers including the germ line (so can make germ cells)
Stem cells are able to generate themselves for an infinite number of generations. They lie in a region called the ‘niche’ which is a place wherein they are provided with signals which gives them the capacity to make all these different cell types. So, have the capacity for self-renewal. And also to differentiate.

Question 171

what happens to stem cells at a point in their life?

Answer 171

At a point in their lifetime, stem cells are ether able to copy a version of themselves or they can differentiate and give rise to other cell types, therefore losing their ability to self-renew.

Question 172

how long does mouse gestation last for?

Answer 172

• Mouse gestation lasts 19-21 days (when the mouse ups are born)

Question 173

how long is pluripotency in mice? what process happens at day 6-7

Answer 173

• Pluripotency in mice only goes from day 3.5-7.5
  o Shows how pluripotency is a transient process – i.e. doesn’t last a very long time
  o Between 6-7, another critical process occurs – gastrulation (generation of all specialised cell types in the embryo

Question 174

are embryonic cels pluripotent or stem cells?

Answer 174

Early embryonic cells are pluripotent

Question 175

what do the different germ layers of the embryonic cells become?

Answer 175

• ectoderm has cells e.g. surface, neural and neural crest. Becomes the skin, PNS and CNS
• mesoderm; axial, paraxial, intermediate and lateral cells. Becomes: blood, heart, muscle and kidney etc.
• endoderm: cells make gut and internal organ. Becomes the liver, pancreas and intestine

Question 176

describe the 2 ways that pluripotency can be defined

Answer 176

1. A way to define pluripotency could be by the cells that express the pluripotency factors that only these cells can express
   In a petri dish – this is important as you wouldn’t be able to see the exact cell it comes rom/makes in vivo. So, need a way to define it
   Have 2 definitions: a descriptive definition (defined by pluripotency cells it expresses) these transcription factors that only pluripotent cells express includes nanog, oct4 and sox2. These are used in reprogramming. These tf can be found in the inner cell mass (ICM) or blastocyst.
2. Another way to define pluripotent cells is functionally by a teratocarcinoma formation assay. This is by their ability to form a teratocarcinoma. Pluripotent cells can be grafted onto the kidney of a host mouse and give rise to teratocarcinomas (=type of tumours containing all cell types)
   Contain all the germ layers – appear in abnormal germ cells i.e. testis or ovaries
   You can graft the pluripotent cell on the kidney of a mouse and see it grows a large tumour. When studying this tumour, it’s expected to have all the cell types from the different germ layers in it. In contrast, non-pluripotent cells wouldn’t give rise to teratocarcinomas, instead would give rise to a small growth of cells that have a large mass.

Question 177

how an pluripotentc emrbyonic cells be studied?

Answer 177

since embryos are very difficult to study, can capture the pluripotent cells in a petri dish and do in vitro modelling of the emrbyonic developmenst to lead to production of a clinically relevant cell poplation

Question 178

what is the advantage of using pluripotent embryonic cells in invitro modelling?

Answer 178

• So here can generate the cell of interest in very high numbers

Question 179

what kins of cell type are embryonic stem cells? how were they initially cultured?

Answer 179

• Embryonic stem cells are a type of pluripotent cell
o Can put them on a plate with a feeder layer of cells (where there are irradiated stromal cells derived from later embryos which support ES cell growth)
o Once embryonic cells have divided a few times, disaggregate and re-plate
o ES cells expressing permanently a transgene encoding GFP
o This was how people initially cultured ES cells.

Question 180

what are pluripotent cell signals used for and what are they in mice and humans?

Answer 180

• Critical signals to maintain cells in self-renewing, undifferentiated state and can replace feeders
• Mouse: leukemia inhibitory factor (LIF)
• Human: FGF2, TGFb

Question 181

how can you capture pluripotent cell signals in vitro?

Answer 181

They can find that when putting ES cells in vitro, they express the same pluripotency transcription factors
Can be done through immunofluorescence (use right antibody against the pluripotency factor of interest in order to detect the expression in the embryonic stem cells
ES cells express the main pluripotency factors Oct4, Nanog, Sox2 like their counterparts in the ICM

Question 182

when capturing pluripotent cell signals, how can you tell that differentiation has happened?
what do ES cells form when transplanted in a permissive environment?

Answer 182

No expression of genes indicative of differentiation

And ES cells form teratocarcinomas when transplanted in permissive environments

Question 183

what is the chimera formation test and how can it test for functionality?

Answer 183

Chimera formation: mouse ES cells can be reintroduced to normal blastocysts and contribute to normal development
Can introduce the pluripotent cells with GFP back into the ICM
Put this back into the embryo ad this should lead to the formation of GFP embryos
The way to distinguish these from their wt counterparts is due to the presence of GFP in their cells.

Can have pluripotent cells by isolating them directly from a pluripotent embryo but can also generate them using reprogramming
Can re-programme adult somatic cells to a pluripotent cell fate
Can take somatic cell (skin cell) and transgenically introduce (using lentiviruses) transcription factors to re-programme the cell from somatic cell back an embryonic stem cell fate.
For the reprograming factors, usually use the pluripotent cells
In the mouse, use a set of 4 transcription factors: oct4, sox2, c-myc and klf4
Found that when these were put into cells that are no usually pluripotent cells, tit out them back into a pluripotent state.

Question 184

how can you test that cells have become pluripotent from mice to humans?

Answer 184

In the human, to test that these cells have become pluripotent = do the descriptive and functional tests
Make chimeras (in mice) or teratocarcinoma test
Take out of self-renewing neurones which would make them out of the self-renewal state (out of their niche), put the cells into a different state, i.e. into mesoderm and see if it makes axial mesoderm etc.

Question 185

what are the names of the 2 approaches used to target cell types in in vitro differentiation?

Answer 185

the 3D approach and the 2D or adherent approach

Question 186

describe the 3D approach of in vitro differentiation including its advantages and disadvantages

Answer 186

1. 3D
   a. Crude technique. Tests the ability of the cells to self-organise
   b. Remove the signals that keep them in a self-renewal self and allow them to differentiate. Grown in aggregates (=in embryoid bodies) in presence or absence of signals
   c. EBs contain many differentiated cell types
   Advantages: recapitulates more accurately embryonic environment
   Disadvantage: difficult to ‘observe’/dissect role of individual signals
   Often generate a mix of cells – difficult to get a very pure population of cells of interest

Question 187

describe the descriptive definition of knowing if signals are expressed and where

Answer 187

DESCRIPTIVE DEFINITION
In embryo, (5.5), cells have embryonic expression of a wnt signalling reporter Axin-LacZ in the primitive streak. At 6.5, axin2 is only expressed in the posterior side of the embryo (only 1 side), later on it expands and expresses

Make embryonic bodies and once you remove the signals that would normally give rise to axin2 and lacz, make these cells express and see regionalised expression of LIF.BMP
3 days after removing the signals that would give us the self-renewing cell fate (LIF/BMP), start getting regionalised expression of axin2
shows that these cells are capable of expressing regionalised tfs outside of the embryo – in aggregates

Question 188

describe the functional description of if you want to show that the heart is beating in a petri dish

Answer 188

FUNCTIONAL DEFINTION
If you want to make cardiomyocytes from EBs, ultimate proof is if you have a heart that is beating in the petri dish
Shows that this is functioning
- Also, can form cerebral organoids through this 3D approach. Here, you make organoids which are like EBs but are slightly more directed into making one cell type
- But the cells in an environment that promotes the expression of only that cell line
- For cerebral bodies, take EBs and grow them in the conditions that promote the formation of only one germ layer ie here, would be the neuroectoderm
- If you leave them long enough, you get a tissue that resembles the very outer tissue of the brain
o These are called cerebral organoids
o The cells in the organoids self organise into neurones in the inner and outer layers of the neural precursors
o So, shows that it was able to express the right thing at the right place

Question 189

describe the 2D/adherent approach of in vitro differentiation. including its advantages and disadvantages

Answer 189

1. 2D or ‘adherent’
   - plate a defined number of cells on the right substarte/extracellular matric
   - remove signals that keep cells in an ‘undifferentiated’ state (e.g. BMP/LIF for mouse ES cells or FGF2, TGFb for human ES cells)
   - grow in defined medium with appropriate amounts of signals (e.g. FGF WNT etc.)
   Involves removing the signals that keep the cells in their niche (in their self-renewing state). These cells are grown in an adherent fashion and supplemented with normally act on pluripotent cells to make our cells of interest
   But here, it’s in a 2D fashion, to let cells grow in a 3D structure
   Don’t grow in 3D way so easier to test the roles of the signals and can get more homogenous population of cells

Advantages: more tractable system (e.g. for live imaging), easier to test the role of specific signals
Disadvantages: loss of cell interactions that may occur in vivo

Question 190

what disease has been modelled with its phenotype having been capture? describe how this was done

Answer 190

Took cells from someone who is a carrier of CDK5RAP2 gene. (has microcephaly)
They took the skin biopsy and added the transcription factors to cause pluripotency and developed the induced pluripotent stem cells
Then they made cerebral organoids with these cells and another with the control

In the control, get an organoid
In the ips induced mutant, the cerebral tissue wasn’t able to induce the tissue for the organoid. Also had a lot less cells – which is something that wasn’t able to be seen in the ‘mutant’ mice
Proves these can be used to capture the phenotype of disease
Can be expanded to use drug screening to see if the phenotype can be reversed

Question 191

which disease included drug screening? describe how this was done including the genes involved

Answer 191

Disease modelling and drug screening case study: Zika virus infection

• increase in microcephaly cases linked to Zika virus outbreak
• to recapitulate these cells, took embryonic stem cells, generated cerebral organoids and was able to infect the organoids and make them into smaller versions of these organoids
• Used ZIKV which is a zika virus infected cells and used CA53 which highlighted the dying cells. Found that when emricasan was added (a molecule that blocks cell death), this increased the cell death here due to the zika virus infection which was blocked by emricasan

Question 192

what are the challenegs in disease modelling?

Answer 192

• Multiple genetic causes (e.g. autism spectrum disorder)
• Complex phenotypes (e.g. cleft palate)
• Late onset (e.g. Parkinsons disease)
• Lack of efficient differentiaton protocols (e.g. blood cell lineages)

Question 193

what are the symptoms of parkinsons and what causes it?

Answer 193

• Affects 1:500
• Symptoms tremor(shaking), slowness of movement, rigidity (stiffness), dementia and anxiety
• Progressive loss of dopaminergic neurones (mDA Neurones)

Question 194

what protocols have been used to replace parkinsons disease cells?

Answer 194

Can make hES-cell derived mDA neuorns
When these are transplanted into the mouse, you can get regeneration of the mDA Neurones into the model organism. Also found evidence of improvement in motor function in these models.
Recently proceeded this into clinical trials

Question 195

what are the cell replacement challenges?

Answer 195

• Progenitors vs mature differentiated cells
• Immune reaction/tumour formation
• Mechanism: replacement or regeneration
• Positional identity: it’s important?

Question 196

why is it difficult to model mental health diseases in animal models? what do they study instead of the cortex and why?

Answer 196

• Trying to study the cortex in animals as model organisms is very difficult because animals don’t have a cortex that is as complex as our own
• So, in studies of mental health, scientists don’t try to compare and study the cortex, instead they study the hypothalamus, amygdala and basal ganglia because these are the parts that have been highly conserved through evolution because they are mediating core homeostatic mechanisms
o Including stress pathway
o Homeostatic pathway
o Feeding pathway
o Reproduction pathway

Question 197

what are mental health conditions and what is their occurrence?

Answer 197

• Mental health conditions – factors and disorders influencing the health of the mind, brain and nervous system
• Mental health conditions account for ~10% of the global burden of disease and as a group are a leading cause of disability worldwide

Question 198

what can biomedical scientists do to tackle mental health issues?

Answer 198

• identify risk factors for neuropsychiatric diseases across the life course
• identify social and biological risk factors that are modifiable
• try and understand how normally you can adapt and be resilient to a trigger i.e. stress

Question 199

how are GWAS used in mental health diagnosis and what was used prior to this?

Answer 199

GWAS studies ae used to identify ‘risk genes’ – i.e. genes that correlate with mental health
Prior to GWAS studies, risk factors were identified by analysing genomes of families in which in-breeding had occurred

Question 200

what gene was identified to have high association with mental health problems? how was it discovered?

Answer 200

Disrupted in Schizophrenia 1 (DISC1)
• First identified through genetic analysis of an inbred family with high-risk mental problems
• i.e. the Disc1 gene is a genetic risk factor for mental illness
• found that 12/17 of the 3rd generation offspring had neuropsychiatric disorders
• found that a=in all of these, Disc1 gene was mutated in a certain way

Question 201

what are well known triggers of DISC1?

Answer 201

•	but, as with many other mental health conditions, social and environmental factors appear to act as triggers
•	well known triggers:
o	stress
o	lack of sleep
o	substance abuse

Question 202

why can it be difficult to understand mental health conditions?

Answer 202

o manifestation of the mental health disorders is likely to be due to a consequence of both the mental and environmental risk factors
o so an external stress will be induced internally through the internal stress pathway
• when there are many variables, it can be hard to dissect the mechanisms in which things happen

Question 203

what makes small organisms like zebrafish and drosophila good models to study for mental health diseases?

Answer 203

• it is important to analyse high numbers so that robust conclusions can be drawn- so that statistical analyses can be done
• zebrafish and drosophila re useful in this respect as you can analyse high numbers
• in this way, mice are less useful than zebrafish

Question 204

what was found about where DISC1 is expressed and what can this mean?

Answer 204

• in the zebrafish embryo, using ISH, see that disc1 mRNA is first expressed in the developing hypothalamus
• furthermore, analysis of disc1 expression with that of other markers showed that disc1 is expressed in the hypothalamic stem/progenitor cells (and incidentally is probably regulated by Wnt signalling
• hypothalamus is an ancient, conserved central regulator of homeostasis and is in the ventral forebrain
• through other work, we knew that the hypothalamic stem/progenitor cells give risk to neurones that are involved in the ‘stress regulatory pathway’. These neurones include pomc, sf1 and crh neurones

Question 205

what are the key players in the stress pathway? describe what happens in the stress pathway

Answer 205

• core master regulator is the hypothalamus in the brain which sends releasing hormones that control the activity of the cells in the anterior pituitary gland
• hormones that are released from cells from endocrine glands in the anterior pituitary gland affect other glands in the body
o in the stress pathway, affects the adrenal gland which sites directly above the kidney
• cortisol is a hormone released from our adrenal glands in response to stress and is the major stress hormone in humans
• the cortisol Is released when the cells in the adrenal gland are stimulated to release it I they see ACTH
• ACTH is released from a set of cells called corticotrophs which are in the anterior gland
• How do corticophs know to release ACTH? When they are stimulated by corticotrophic releasing factor from the hypothalamus
• Pomc and steroidogenic factor neurones interact in this pathway to trigger the release of crf, acth and subsequently act on the adrenal cells to release cortisol
• There is a feeback loop as you can’t have ongoing stress response
• So, as soon as cortisol is released, it feedsback negatively to dampen this effect

Question 206

what did Wood found when he created 2 lines of DISC1 mutant zebrafish?

Answer 206

John Wood created 2 lines of disc1 mutant zebrafish
1 making transgenic line (L115 and the other was Y472
the fish were homozygous viable but made no full-length mRNA or protein
so, fish with this mutant did not make disc1 mRNA or protein

in the absence of disc1 function, there is a decrease of hypothalamic progenitor cells
this is problematic because we need hypothalamic progenitors to make the hypothalamic neurones
think that they differentiate prematurely
when you’re building the nervous system,
in some way in disc1 mutants, make too many of 1 class of cells born too rarely then don’t have enough stem or progenitor cells to make any kind of new neurones
not sure how disc1 causes this to happen
we think this because if you look at hypothalamic neurones in WT compared to the mutant, you don’t have the right balance of differentiated neurones involved in the stress homeostasis in the mutant. They differentiate abnormally

Question 207

how can the stress response of larvae and adult fish be measured?

Answer 207

1. Behavioural assays
2. Neuroendocrine function
   a. Upregulation of neurohormones after stress
   b. Upregulation of cortisol after stress
   A physiological readout of if the stress pathway is working normally would be measuring the levels of cortisol
   The behavioural readouts would be no response to stress trigger
   These things can be measured by zebrafish

Question 208

How can you stress zebrafish to measure its behaviour?

Answer 208

If you expose zebrafish to higher levels of sodium chloride, they see this as a stressor and so swim more rapidly
or
Take some skin from another fish and mash it up and drop it into the water. They think that this means that one of their peers has been eaten by a bigger fish and so they swim away
Have devices that measure the movement of the fish
So, in the normal stress response fish would do this
In Disc1 fish, they do not respond to either sodium chloride or the ‘alarm’, find that they do not respond and also they do not show elevated levels of cortisol

Question 209

what was found when the behavioural and physiological studies of the stress pathway in Disc1 fish was done?

Answer 209

that they are unable to mount a proper stress response

Question 210

Which drug is the most harmful? why?

Answer 210

alcohol
In 2013, 8416 alcohol related deaths in the UK
Has an enormous social, economic and healthcare burden

Question 211

what are the receptors of some drugs in humans?

Answer 211

• Different drugs have different primary effects
• Opiates – miew opiate receptors
• Nictone – acetylcholine receptors
• Cananbs – cannabinoid receptors
• Alcohol – several; major being GABA (gamma-aminobutyric) and MDA (N methyl d asparate) receptors
  (Doesn’t act on specifically 1 type)

Question 212

what system do most drugs work secondarily through?

Answer 212

Most if not all drugs of abuse act secondarily through dopamine and the mesolimbic ‘reward’ system

Question 213

What are the Contributing factors to drug addiction?

Answer 213

Contributing factors to drug addiction:
Genetics: sensitivity and development of tolerance
Social environment (past and present)
Personality and personal history (for example trauma)

Question 214

Are flies good models for drug addiction?

Answer 214

Flies are small insects that go through metamorphosis
Many offspring generations can be grown at any time and so in this way they are very deal and cost-efficient model organisms
grown quick

Question 215

at 18 and 25 degrees, how long is a drosophila cycle?

Answer 215

cycles take 10 days if the drosophila are grown at 25 degrees and 25 days if they are grown at 18 degrees

Question 216

what human diseases can be analysed well in flies?

Answer 216

• Neurological disorders
• Cancer
• Developmental disorders
• Metabolic disorders
• Cardiovascular diseases

Question 217

what makes flies not ideal as an animal model for alcohol abuse? what are some questions that must be asked?

Answer 217

•	not all genes implicated in actions of drugs are conserved
o	GABA, NMDA and dopamine receptors
o	(no opioid/cannabinoid receptors)
•	do flies respond to ethanol?
o	And is this response similar to ours?

Question 218

what is one experimental approach that can be used to test flies’ response to alcohol? does it show that the flies respond to ethanol?

Answer 218

Experimental approach 1 the (‘booz-o-mat’)
There is a chamber where the flies move around in and has air in it
There is another chamber with ethanol and a scientist can control the air that comes in through as being ‘clean’ air or air that has had ethanol diffused into it
See how flies respond to different types f air
Video made showing their behaviour in response to the different signals
Do flies respond to ethanol?
Compared to clean air, the flies exposed to ethanol air moved around very fast and a lot. Then, after a while, some of them stop responding as they could be ’drunk’
This can be quantified by locomoter velocity profile
Startle response then an increased locomotion then sedation. This is a similar effect at the same EtOH concentration as in people

Question 219

describe the 2nd experimental approach used to test flies’ response to ethanol

Answer 219

Experimental approach 2: the ‘inebriometer’
Similar to previous
Tube where flies are moving and at some point, ethanol tube is fed through to this tube and see that the flies are awake, move around and stay in the chamber and if they’re drunk oxidative, they cant move and just fall down and out
Can measure the elution time – the amount of time taken for the flies to get so drink that they fall out of the chamber
Can count at each time point how many flies are falling out
See some variation with a peak of ~20 minutes
See that sensitive flies drop out quicker than WT ad resistant ones fall out last
Want to understand the genetic differences between the flues that are dropping out earlier/later and if these genes are what cause them to be more sensitive/resistant to alcohol

Question 220

what are the 2 independent genetic factors that contribute to drug addiction?

Answer 220

1. Sensitivity

2. Development of tolerance

Question 221

What, if anything, does ethanol sensitivity have to do with alcoholism?

Answer 221

Low sensitivity to modest doses of ethanol are associated with a significant increase in the risk of future alcoholism, perhaps by increasing the chances a person will drink more heavily and more often

Question 222

define sensitivity and the development of tolerance

Answer 222

• Sensitivity: inability to tolerate the adverse effects of a drug: start point. How the fly responds o alcohol the first time its exposed
• Development of tolerance: (independent) 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 223

How can you measure sensitivity to alcohol in flies?

Answer 223

• Irradiate flies to induce mutations
• Screen mutants (pick flies that elute first and last)
• Identify the gene mutated

Question 224

describe 1 mutant that has been used to study sensitivity to ethanol in flies

Answer 224

Increased sensitivity to ethanol
They get drunk and fall out easier
Have a 20min mean time in WT but much shorter
Independent on concentration
Cheapdate mutant are as a whole more sensitive and elucidate much quicker

Question 225

Which gene is mutated in the cheapdate mutant?

Answer 225

Cheapdate is an allele of amnesiac, a neuropeptide that activates the cAMP pathway
cAMP pathway is involved in several important pathways
cAMP can activate PKA which can move into the nucleus from the cytoplasm and activates CREB
in the activated from, CREB protein binds to CBP (CREB-binding protein) and find on the DNA, CREB binding elements and so can activate target genes and induce transcription of several target genes
then lead to change in subset of gene is in the nucleus
in the mutant, this pathway doesn’t work

Question 226

what was done to testwhat aspect of the cAMP pathway was causing the change in it?

Answer 226

tested ethanol reposne in rutabaga (adenyl cyclase), DCO (PKA catalytic subunit) and dunce (cAMP phosphodiesterase – inactivates cAMP mutations.

• Found that mutation in dunce doesn’t affect the sensitivity but the others do
• Shows that it’s due to the entire cAMP pathway as dunce is what inactivates it
• Activity of cAMP pathway decreases alcohol sensitivity

Question 227

how can you measure the development of tolerance in flies? what is found?

Answer 227

Repeating the same experiments on the same flies
Then screen mutants to see if they become tolerant over time
Found that flies are less sensitive to ethanol on their second exposure

Question 228

what is the name of the fly mutant that have reduced tolerance?

Answer 228

Hangover mutants have reduced tolerance – the flies don’t develop tolerance and so Every time they’re exposed, show same response

Question 229

what do hangover mutants show on a graph of sensitivity to alcohol?

Answer 229

The increase is much smaller than in WT upon second exposure
They do increase but not nearly as much as in the WT
Shows that there is a genetic component that allows tolerance to develop

Question 230

what does the hangover encode for?

Answer 230

Found that hangover encodes a Zn-finger protein
Has multiple domains which work in nucleotide binding
Found that its expressed in the brain
The signal is lost in the brains of the hangover mutants

Question 231

what genes/pathways are affected by hangover?

Answer 231

Turning off cAMP pathway is required for tolerance

In dunce mutant, there is no change in sensitivity, so don’t develop tolerance (similar to that of hangover)

Hangover binds RNA
Immunoprecipitated RNA from the cells and tested if hangover proteins immunoprecipitate with RNA. They found that it does
Dunce – cAMP phosphodiesterase (inactivates cAMP) function is required for developing tolerance
The dunce and hangover counteract each other

Question 232

How does hangover regulate tolerance?

Answer 232

Hangover encodes a RNA binding protein that binds dunce RNA (cAMP phosphodiesterase that inactivates cAMP) and increases amount of transcript (stabilises the RNA) so increases the amount of RNA to increase amount of protein in the cAMP pathway but then increase dunce to inactivate this pathway which inhibits hangover
Balance between hangover and induce expression determines degree of tolerance
A reduction in cAMP level is required for tolerance

Question 233

what are the 2 interacting systems that are thought to be working together to control if you’re asleep or awake?

Answer 233

1 = sleep homeostatic drive and 2 = the circadian clock

Question 234

what are circadian rhythms? what are they though to be influenced by?

Answer 234

Circadian rhythms are 24hour rhythms in physiology and behaviour generated by molecular clocks, which serve to coordinate internal time and the external world
It works independently n the 24 hours in a day (not connected) as this is an internal clock
The circadian is a biological intrinsic clock
it is thought that through evolution, our intrinsic clock has become aligned with the extrinsic ‘light’

Question 235

what is the link between the circadian rhythm and melatonin?

Answer 235

melatonin increases on circadian cycle (so maximum melatonin at night time)

Question 236

what are some examples of hormones being synced with the circadian rhythm?

Answer 236

behavioural outputs are appropriate/optimised to the appropriate time window each day
cortisol levels – (lets you anticipate stressors) – get blip at dawn, co-ordinates with higher coordination, faster reaction times etc.
peak of melatonin secretion at night time

Question 237

what controls the synchrony of the circadian rhythm?

Answer 237

Most cells in the body possess a molecular clock and are maintained in synchrony by a master clock located in the suprachiasmatic nuceli (SCN) of the hypothalamus
The superchiasmiatic clock entrains all other clocks

Question 238

what do the clock controlled genes orchestrate in terms of the genes/genomes?

Answer 238

Thousands of clock controlled genes orchestrate the oscillation of tissue-specific metabolic and physiological functions: between 2 and 30% of each tissue’s transcriptome displays a circadian rhythm
mRNA is regulated dynamically by the clocks
higher levels at 1 point of the day and low amounts at other times

Question 239

what things are controlled by clock genes? how does it relate to growing as children?

Answer 239

Core biological/physiological/metabolic processes are under control of clock genes (body temperature, cortisol hormone, blood pressure, melatonin, growth hormone, testosterone and prolactin)
this is why you only grown at night – due to release of growth hormones from hypothalamic neurones
wrapped around tanocytes that only let out the growth hormones at night time

Question 240

How do we know about the circadian molecular clock?

Answer 240

All initial studies on circadian rhythms etc, were found in drosophila
It was in the drosophila that the first genes involved in this pathway were isolated

Question 241

what is the name of the system that entails the ways that circadian rhythms work

Answer 241

cell autonomous transcription-translation feedback loops

Question 242

describe the way that the cel autonomous transcription translation feedback loops work in circadian rhythms (including CLOCK and BMAL)

Answer 242

Transcription factors (e.g. CLOCK, BMAL1) come together I the nucleus and activate/drive expression of other genes (e.g. period (per1/2) and cytochrome (Cry1/2)), whose protein products in turn feed-back in the nucleus to inhibit CLOCK and BMAL1. This inhibition means that these genes aren’t activated anymore so the protein product breaks down and this expression is alleviated then BMAL and CLOCK can express again
This takes 24 hours (the feedback involving transcription, translation, making the protein stable breaking it down etc.) – this is what is taking the 24 hours
There is strong entrainment of this internal clock by external sources

Question 243

how can sunlight affect the clock genes?

Answer 243

Sunlight directly activates the protein product of cytochrome cry gene (CRY) and promote its binding to (TIM) and so affects the working of the clock
(light is thought to be stabilising CRY)

Question 244

how did a study on the fibroblasts of schizophrenia vs wt patients show a difference in clock genes? can this be applied to other disorders (e.g. bipolar disorder and depression)

Answer 244

• taking fibroblasts from someone with a robust circadian cycle, culture the fibroblasts and would be able to see the clock genes going up and down with normal rhythmicity over 24hours
• Can see tis using qRT-PCR or antibodies or make GFP construct for one of the genes that are regulated on a 24-hour cycle
• Take fibroblasts from mice, shine light on the cultured cells and will be able to see that for 12hours, its green and 2=12 hours they’re not
• This would be reflecting that this protein is expressed in 12 hours out of 24
• See that cells have the capacity of intrinsic clock activity which persists in a dish for 4-5 days but will break down without the entrainment using external stimuli like the superchiasmic nucleus
• Fibroblasts isolated from schizophrenic patients show a loss of rhythmicity in CRY and PER1 expression, and their peripheral blood leukocytes have decrease and/or disrupted diurinal expression of CLOCK, PER1/2/3, CRY1 and a functional CLOCK homologue NPAS2 in comparison to healthy controls
• GWAS an SNP analyses show the genes encoding the core components of the molecular clock have been associated (albeit weakly) with schizophrenia, bipolar disorder and depression
• Interestingly, they also show dysregulation of the core molecular clock

Question 245

how is the hypothalamus associated with the circadian rhythm? is this a cause or consequence of mental illness?

Answer 245

Hypothalamus co-ordinating optimal internal environment with external environment
this would work best if they are perfectly aligned

So, it is known that lack of sleep/poor sleep pattern is associated with poor mental health
Now, disruptions to the intrinsic circadian clock are thought to be causal to poor mental health

Question 246

how has us being able to understand the mechanisms ofthe ‘clock’ helped us to understand mental health diseases and develop treatments?

Answer 246

1. As the mechanisms of the clock have become more-clear, it gives us a handle to understand the link between the clock and key physiological processes – e.g. metabolism
   Many metabolic tfs and output regulators are under circadian control. We are beginning to understand how this works
   Also, how the clock genes affect the immune system (I.e. children getting fevers at the beginning and endings of the day)
2. By understanding molecular clock mechanisms, we begin to get a handle on factors that might help in co-ordinated therapies
   e.g. melatonin is a hormone that is produced in the pineal gland with a circadian rhythm
   - low level during daytime, high levels during night
   pineal regulation of melatonin Is regulated by the master clock in the SCN1.
3. An understanding of core clock mechanisms is helping to best-harness chemotherapies in cancer
   Time of day may be critical to the optimal response to the chemotherapy

Question 247

what is melatonin? and what is its primary function?

Answer 247

Melatonin ubiquitous in nature (one of the most phylogenetically ancient of all biological signalling mechanisms
Primary function - a potent antioxidant
Used to get rid of free radicals that are thought to build up during the daytime

Question 248

what physiological functions is melatonin involved in?

Answer 248

• Circadian rhythm regulation
• Sleep propensity
• Blood pressure regulation
• Immune function
• Detoxification of free radicals
• Regulation of bicarbonate secretion in GI tract

Question 249

why is melatonin referred to as being better than other antioxidants? what is it better at?

Answer 249

More efficient than other antioxidants; devoid of pro-oxidant side-effects

a. Scavenging of free radicals
b. Up-regulation of antioxidant enzymes
c. Direct inhibition of free radical formation

Question 250

why is it being suggested that our understanding of clock genes can be involved in improving chemotherapy?

Answer 250

Clock evolved to help organisms protect against UV-induced DNA damage. Particular 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 the body is least-able to function optimally in repairing this damage
So, this understanding of core clock mechanism is helping to best harness chemotherapies in cancer

Question 251

what is ageing? and its occurrence in UK (maximum lifespan)

Answer 251

A progressive, irreversible decline in organismal performance
Cause of death after 65 years old in UK: 2013
• Maximum life span is about 125 years. The oldest known human = 122 years
• It’s possible to increase average if we know factors that influences ageing

Question 252

what are the factors influencing ageing?

Answer 252

• Genetics
o Genotypes at birth and accumulation of mutations with age
• Environment
o Diet, lifestyle, exposure to various external factors

Question 253

what model organisms are usually chosen to study lifespan and ageing in? why are they chosen?

Answer 253

• Lifespan is studied mostly in c. elegans, drosophila and mice
• This is due to their relatively short lifespan
o C.elegans – weeks, drosophila = months and mice = years
• Small, easy to culture
• Short lifespan
• Genetically tractable: homogenous/many tools available
• Controllable environment (food intake/temperature etc.)

Question 254

• How can we measure ageing?

Answer 254

o No direct method of measuring ageing
o We really measure age at death
o Demographics: large cohorts of identical animals
• Do a survival curve with proportion alive (% age) on y axis and time (age in days) on x axis. Median lifespan is measured from this

Question 255

how do genetics affect ageing?

Answer 255

• Genetics
o Genotype at birth and accumulation of mutations with age
o Genetic screens and short life-span makes drosophila a powerful tool to find genes regulating life-span

Question 256

what is the metabolic syndrome?

Answer 256

The metabolic syndrome – when cells become resistant to insulin so have high likelihood of developing T2D, visceral obesity, atherosclerosis etc

Question 257

what is insulin?

Answer 257

Insulin: hormone that promotes the uptake of glucose from blood for storage

Question 258

describe the basics of the Insulin/IDF-1 signalling pathway

Answer 258

• Insulin binds to insulin receptor and intracellular component chico
• If the receptor and chico are stimulated it triggers the pi3 kinase sig pathway that has effects on 2 tfs: foxo which governs cell number ands6k which governs cell size

Question 259

what was found to be the effect of muattions in chico in drosophila males and females? how were these things found?

Answer 259

the reason the insulin signalling pathway was found to be so important is because when people did these large-scale screen
took lots of drosophila and di large sale mutagenesis
looked for effects on lifespan
identified that mutations in chico leads to drosophila having a longer median lifespan
female median lifespan = 40 days, male = 50 days. When there are mutations in chico, female median lifespan reaches days 60 days, males reached 62 days from 50 days. So, increases a lot in the condition

Question 260

describe the chico pathway and how it can lead to an extension in median lifespan

Answer 260

Turns out that the insulin chico pathway is an ancient conserved pathway
In mammals, c. elegans and drosophila, there are insulin like peptides that act on a conserved receptor (InR in mammals, daf02 in c.elegans and dinR in drosophila)
This ineracts the cytoplasmic component of the insulin receptor which binds to the protein chico (has a mammalian counterpart
When this all binds, it triggers the pik3 pathway which represses foxo or homologues n others
Foxo stimulates a repressor of growth and lifespan
If there is an overexpression of any inhibitors or mutate he components of the pathway, you will get an increase in lifespan (repressing a repressor)

Question 261

How does insulin pathway control life span?

Answer 261

If insulin goes down, foxo increases
This means that there is an increase in autophagy, DNA repair and decreases oxidative stress which all in turn increase health and longevity

Question 262

what is teh name of the mutant which was thought to have an ncreased lifespan? what was the issue with it?

Answer 262

Methuselah mutant
Methuselah – encodes the receptor which triggers insulin release in response to nutrients
Methuselah mutants are long lived
A peptide antagonist of methuselah extends life span
However, the functional abilities are below control level at old age (flight duration is lower than control at old age from ~50-70 days old)
(so you have increased lifespan but not healthy ageing)

Question 263

how is the environment thought to affect ageing?

Answer 263

• Environment – dietary restriction

Reduced nutrient intake extends lifespan

Question 264

what is DR in ageing?

Answer 264

Dietary restriction (DR)
• Oldest known proven method for lifespan extension
• Modest DR extends lifespan across species
• Fully fed drosophila had a significantly increased median lifespan
• Can make a survival curve become the probability of death curve

Question 265

Is diet required throughout life?

Answer 265

As time goes by, a higher probability of death in the flies
Another graph shows that flies were fully fed until they were DR at day 14, this massively decreased their probability of death. Another group with flies that were fully fed until day 22 had a similar probability of death as in the previous condition. Shows that don’t need to have DR all lie, just more in the later stages is enough to significantly lower your probability of death

• DR reduces the mortality risk at any time

Question 266

what is one disease that uses flies to understand its occurrence and how to study it

Answer 266

Alzheimers disease:
Accumulation of B-amyloid peptide
Overexpression of b-amyloid peptide in drosophila is neurotoxic
This makes it possible to screen for genes that reduce neurotoxicity

Question 267

what was found about the overexpression of UAS and how this affects insulin and lifespan includingthe various genes/pathways involved? describe it fully

Answer 267

Overexpression: UAS/Gal4 system
Make 2 fly strains
Promoter of a gene expressed in a particular tissue
Gene – b amyloid peptide

So, cross these and get a fly that expresses the Gal4 in every neurone and expresses b amyloid peptide
So gal4 – made and works to express many UAS genes
So, have overexpression of the UAS

Found that through these flies, the neurones would normally die so when you cross this, they do a genetic screen and mutagenesis screen which could restore lifespan
Or, can look at effects on particular neuronal tissue i.e. the omnatidia (pattern that can be detected/measured very easily)
In the diseased fly eye the omnatidia are visually worse off
So, see effects on longevity and also see genes that improve the eye
Found a gene called insulin degrading enzyme (IDE) which is an evolutionary conserved metalloendopeptidase which was found to be mutated

IDE increases lifespan, decreases neurotoxicity that is due to the over expression in b amyloid so shows its involvement in the lifespan ad the heathy life span/healthy ageing