Modelling Human Disease Flashcards

Question

what does the NHS 100,000 genome project entail?

Answer 1

• 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

Answer 2

in situ hybridisation and immunohistochemsitry | - can make an antisense mRNA to a variant gene, do ISH and see where its been expressed

Answer 3

• Conditional deletion of a gene | If you find a variant gene, can KO this gene in the area

Answer 4

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)

Answer 5

* 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

Answer 6

transgenic reporter line

Answer 7

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

Answer 8

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

Answer 9

* 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?)

Answer 10

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

Answer 11

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

Answer 12

- 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

Answer 13

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)

Answer 14

- 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

Answer 15

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

Answer 16

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

Answer 17

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

Answer 18

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

Answer 19

Nearly 90% of patients with DM have Type 2 diabetes

Answer 20

* 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.

Answer 21

- 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….)

Answer 22

- 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)

Answer 23

* Impaired insulin secretion * Insulin resistance * Increased glucose production

Answer 24

- 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

Answer 25

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

Answer 26

neurones in the brain regulate glucose homeostasis

Answer 27

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

Answer 28

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

Answer 29

adipocytes

Answer 30

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

Answer 31

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

Answer 32

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

Answer 33

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

Answer 34

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

Answer 35

a selective deletion

Answer 36

they found that leptin neurones causes obesity in neuorones | mice with leptin receptor KO in the liver were normal

Answer 37

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)

Answer 38

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

Answer 39

``` 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 ```

Answer 40

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

Answer 41

NPY - make a neurotransmitter | POMC - makes a long protein

Answer 42

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

Answer 43

* NPY neurones stimulate food intake | * Pomc neurones reduce food intake

Answer 44

- 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

Answer 45

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

Answer 46

- progenitor cells | - stem cells

Answer 47

in the arcuate nucelus of the hyporhalamus

Answer 48

NPY (neuropeptide y)

Answer 49

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

Answer 50

o Leptin stimulates pomc neurones

Answer 51

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

Answer 52

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

Answer 53

lie very close to the 3rd ventricle

Answer 54

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

Answer 55

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

Answer 56

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

Answer 57

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

Answer 58

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

Answer 59

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

Answer 60

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

Answer 61

--- 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

Answer 62

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)

Answer 63

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

Answer 64

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

Answer 65

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

Answer 66

* 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

Answer 67

• 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

Answer 68

* 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

Answer 69

neurones that are involved in energy formation

Answer 70

no | eg in a caged animal, • not making new neurons because its in a homeostatic norm (optimum)

Answer 71

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

Answer 72

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

Answer 73

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

Answer 74

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

Answer 75

¬ 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

Answer 76

¬ 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

Answer 77

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

Answer 78

♣ The single injection reduces blood glucose levels to norm

Answer 79

¬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

Answer 80

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

Answer 81

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.

Answer 82

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

Answer 83

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

Answer 84

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

Answer 85

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.

Answer 86

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

Answer 87

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

Answer 88

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

Answer 89

• 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

Answer 90

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

Answer 91

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

Answer 92

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

Answer 93

¬ 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).

Answer 94

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

Answer 95

* 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

Answer 96

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

Answer 97

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)

Answer 98

it i sthe cells that resnd to wnt signallling

Answer 99

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.

Answer 100

mammillary tumour

Answer 101

¬ 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

Answer 102

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

Answer 103

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

Answer 104

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)

Answer 105

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

Answer 106

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

Answer 107

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

Answer 108

- 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

Answer 109

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

Answer 110

* 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

Answer 111

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

Answer 112

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

Answer 113

- 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?

Answer 114

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

Answer 115

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

Answer 116

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

Answer 117

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

Answer 118

- 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

Answer 119

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

Answer 120

* 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

Answer 121

• 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

Answer 122

* 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

Answer 123

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

Answer 124

• 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

Answer 125

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.

Answer 126

• 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

Answer 127

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

Answer 128

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

Answer 129

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

Answer 130

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

Answer 131

- 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)

Answer 132

- 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.

Answer 133

• 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

Answer 134

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

Answer 135

- 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

Answer 136

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)

Answer 137

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

Answer 138

1% of people have heart defects | 10% have small holes in the heart that could predispose you to get various heart conditions

Answer 139

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

Answer 140

• 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

Answer 141

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.

Answer 142

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.

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

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- 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

Answer 145

Early embryonic cells are pluripotent

Answer 146

- 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

Answer 147

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.

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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

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

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• 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.

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

Answer 152

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

Answer 153

No expression of genes indicative of differentiation | And ES cells form teratocarcinomas when transplanted in permissive environments

Answer 154

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.

Answer 155

``` 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. ```

Answer 156

the 3D approach and the 2D or adherent approach

Answer 157

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

Answer 158

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

Answer 159

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

Answer 160

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

Answer 161

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

Answer 162

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

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* 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)

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

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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

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

Answer 167

• 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

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* 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

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- 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

Answer 170

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

Answer 171

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

Answer 172

``` • 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 ```

Answer 173

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

Answer 174

* 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

Answer 175

* 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

Answer 176

• 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

Answer 177

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

Answer 178

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

Answer 179

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

Answer 180

that they are unable to mount a proper stress response

Answer 181

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

Answer 182

- 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)

Answer 183

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

Answer 184

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

Answer 185

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

Answer 186

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

Answer 187

* Neurological disorders * Cancer * Developmental disorders * Metabolic disorders * Cardiovascular diseases

Answer 188

``` • 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? ```

Answer 189

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

Answer 190

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

Answer 191

1. Sensitivity | 2. Development of tolerance

Answer 192

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

Answer 193

- 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)

Answer 194

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

Answer 195

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

Answer 196

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

Answer 197

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

Answer 198

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

Answer 199

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

Answer 200

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

Answer 201

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

Answer 202

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

Answer 203

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

Answer 204

1 = sleep homeostatic drive and 2 = the circadian clock

Answer 205

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’

Answer 206

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

Answer 207

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

Answer 208

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

Answer 209

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

Answer 210

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

Answer 211

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

Answer 212

cell autonomous transcription-translation feedback loops

Answer 213

``` 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 ```

Answer 214

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)

Answer 215

* 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

Answer 216

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

Answer 217

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

Answer 218

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

Answer 219

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

Answer 220

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

Answer 221

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

Answer 222

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

Answer 223

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

Answer 224

• 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.)

Answer 225

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

Answer 226

• 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

Answer 227

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

Answer 228

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

Answer 229

* 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

Answer 230

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

Answer 231

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)

Answer 232

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

Answer 233

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)

Answer 234

• Environment – dietary restriction | Reduced nutrient intake extends lifespan

Answer 235

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

Answer 236

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

Answer 237

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

Answer 238

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