TI Flashcards

Question

What happens in LTBMC?

Answer 1

We use flasks in which the stromal cells are at the bottom. The stem cells bury into the stromal cells, which stimulates them to divide. The stem cells emerge from the stromal cells as committed cells (stem cells remain in the stromal cells) and move into the supernatant. There is a direct relationship between early stem cells and later progenitors.

Answer 2

1. Research - basic haematopoeisis and carcinogenesis 2. Testing toxicity of chemotherapeutic agents and carcinogens 3. Generate cells for stem cell transplantation/manipulation

Answer 3

1. Density centrifugation 2. Fluorescence activated cell sorting (FACS) 3. Immune-precipitation

Answer 4

Mechanical and enzymatic disruption using collagenase, dispose and trypsin

Answer 5

Unmodified

Answer 6

- aberrant expression of some genes - variable contamination - poor growth characteristics (50-100 divisions) - inter-patient variability - phenotype instability - molecular manipulation is difficult

Answer 7

- Good growth characteristics - Phenotype stability - Defined population - Molecular manipulation readily achieved

Answer 8

1. Spontaneous - from tumours or prolonged culture, multiple ill-defined mutations, transformed phenotype 2. Genetic manipulation

Answer 9

Processes that regulate growth and ageing... - p53 - retinoblastoma gene (Rb) - telomerase

Answer 10

- telomere binding protein protects the telomere ends (TTAGGG) - when the telomere gets too short, the binding protein is lost - p53 binds to the unprotected chromosomes - activated p53 triggers growth arrest or cell death

Answer 11

A viral protein which interacts with normal p53 and Rb. This can cause increased growth without loss of function.

Answer 12

A viral protein - E6 targets p53 for degradation, E7 binds to Rb

Answer 13

- SV40; T and t antigen - HPV; E6/E7 ...p53 and Rb bind to different viral proteins

Answer 14

Telomerase prevents the erosion of telomeres. Some cells need telomerase and silencing of Rb for 'immortalisation'. E6/E7 and telomerase are believed to maintain a differentiated phenotype.

Answer 15

Plasmid...Gene for selection...Growth promoting gene Transfection Selection pressure added Colonies selected

Answer 16

- getting the DNA into the cells | - getting the cells to stably incorporate the DNA once inside

Answer 17

``` CaPO4 co-precipitation Lipofection Electroporation Viral transfection Nucleofection ```

Answer 18

1. positively charged lipoplexes interact with the negatively charged cell membrane 2. taken up by endocytosis 3. release from endosome 4. transport to the nucleus 5. entry to the nucleus is inefficient and may need mitosis

Answer 19

An electrical field is applied to the cells in order to increase permeability of the cell membrane, allowing chemicals, drugs or DNA to be introduced to the cell. The pores reseal when they have been created by the electrical current, and the rate of this resealing is dependent on temperature.

Answer 20

- Exploits the normal mechanism of viral infection - Usually has high transfection efficiency - There are three gene-targeting vectors commonly used based on three types of viruses; retrovirus, adenovirus, and adeno-associated virus.

Answer 21

- combination of electroporation and lipofection - increased efficiency particularly of non-dividing cells - technology is protected under patent - different solution and protocols are used for each cell type

Answer 22

- RAPIDLY DIVIDING CELLS OFTEN LOSE DIFFERENTIATED FUNCTION - therefore in making cell lines, growth and function are at odds - the ideal solution would be a cell line that divides when you want more and stops when a study of function is requited

Answer 23

- conditional mutant eg T-antigen of SV40 | - changing culture conditions such as the ECM, co-culture or 3D culture

Answer 24

An artificially created environment in which biological cells are permitted to grow and interact with their surroundings in all three dimensions

Answer 25

- genes expressed as in vivo - cell-cell communication is re-established - cells are orientated in the same was as in tissues

Answer 26

- histology - phase contrast - time lapse

Answer 27

Due to energy loss, the emitted light is shifted to longer wavelength relative to the excitation light. It is the difference between the two peaks on an excitation-emission graph.

Answer 28

Bleaching of fluorochromes - due to high intensity illumination the flurophores might permanently lose their ability to emit light

Answer 29

- Work with reduced excitation light intensities or use grey filters - Use shorter exposure times/higher gain settings and longer intervals during time lapse studies - Use anti-bleach in your mounting media

Answer 30

These proteins are naturally found in light-producing cells of cnidarians. Fluorescent proteins can be fused with other proteins and introduced in cells via transfection. This allows live study of fluorescent tags in living cells/organisms.

Answer 31

Higher z-resolution and reduced out-of-focus-blue make confocal images crisper and clearer

Answer 32

Only a small volume can be visualised by confocal microscopes at once. Bigger volume need time consuming sampling and image reassembling.

Answer 33

- can self-renew | - can differentiate into a range go more specialised cells

Answer 34

Their ability to differentiate

Answer 35

can only form one type of cell - i.e. HSCs can only form blood cells, but various types of blood cell

Answer 36

Can produce all of the differentiated cells in an organism. Can give rise to the embryo and the tissues that support its development, such as the placenta. These supporting tissues are known as the trophoblast.

Answer 37

Can give rise to all the cell types that make up the body. This is similar to totipotency but don't make the supporting tissue, only the embryo.

Answer 38

(aka Adult stem cells) function as a repair system for the body, replenishing old or damaged tissues

Answer 39

produces one stem cell and one differentiated cell. QUIESCENCE

Answer 40

Produces 2 stem cells. ACTIVATION - leads to expansion of the stem cell pool and occurs due to injury/disease

Answer 41

This is the area in which stem cells reside. It is the microenvironment where you find the stem cells, and it interacts with the stem cells to determine their fate. It also regulates stem cell activity. The niche helps to maintain the balance between symmetric and asymmetric stem cell division.

Answer 42

- key component of the stem cell niche - two way interaction; stem cells also remodel the ECM - critical regulator of stem cell function - mechanical features can determine cell fate ...brain stiffness induces neuron formation... ...muscle stiffness induces muscle formation... ...bone stiffness induces osteoblast formation...

Answer 43

- can undergo more cell divisions than somatic cells - length of telomeres determines how many divisions can occur - have high expression of telomerase which rebuilds the telomere

Answer 44

- have clinical applications - can be autologous - won't be rejected - fewer safety concerns - no need to immunosuppressants, not linked to tumour genesis - easier to get sufficient numbers

Answer 45

``` Primarily found in bone marrow but also in... placenta umbilical cord blood adipose tissue adult muscle corneal stroma dental pulp ```

Answer 46

- homing capacity - tend to home to damaged tissue sites - high differentiation potential - production of trophic factors - immunomodulation

Answer 47

Also known as 'brittle bone disease', this is a genetic disorder in which osteoblasts produce defective type I collagenase. Symptoms include osteopenia, multiple fractures, severe bony deformities and shortened stature.

Answer 48

Corneal renew and repair are mediated by stem cells of the limbus.

Answer 49

The process by which an adult cell can change from its initial differentiated state to another. This is largely an in vitro phenomenon.

Answer 50

The growth of tumours is fuelled by limited numbers of dedicated stem cells that are capable of self-renewal. Many studies have suggested that CSCs are resistant to cancer therapy which could explain relapse as the CSCs can relocate and form a tumour in the new location. Therefore, if we target therapeutics specifically to CSCs we may be able to cure the cancer permanently.

Answer 51

- surface markers - signal cascades - ABC cassette - the microenvironment

Answer 52

Meaningless Random Differences

Answer 53

difference is worth considering

Answer 54

statistically unreliable differences

Answer 55

fairly convincing differences

Answer 56

Immediate visual summary of effect size and confidence

Answer 57

- does not give a precise measure of confidence | - comparing one confidence interval to another cannot be translated into a numerical unit

Answer 58

gives a single number that summarises whether your data looks random

Answer 59

- very little information - doesn't tell you effect size - doesn't tell you confidence levels

Answer 60

When we get p

Answer 61

When we get p > 0.05 even though there is a real effect. This often occurs when you haven't repeated the experiment enough, or your sample size is too small.

Answer 62

- mathematical transformation e.g. take a log of the data - non-parametric test - Mann Whitney U test instead of T-test - Wilcoxon signed ranks test instead of paired T-test

Answer 63

- not valid to do a series of t-tests for each pair - multiple testing increases the frequency of the type 1 errors - an alternative is to use 1-way analysis of variance (ANOVA)

Answer 64

Looks at data from all groups and asks how likely or unlikely it is that the data as a whole is random. ANOVA compares the variance within each group to the variance between groups. The variance within each group gives you a measure of random variation.

Answer 65

This suggests that the different between groups could be explained by random variation.

Answer 66

This suggests that it is unlikely this can be explained by random variation.

Answer 67

- unlikely the differences between groups is random - valid to ask which differences are responsible for this - can do individual tests between each pair of the groups - t-tests or other 'post hoc' tests

Answer 68

the ability of a cell to give rise to every tissue in the body

Answer 69

embryonic stem cells (ES) and induced pluripotent stem cells (iPS)

Answer 70

- cleavage stage embryo - cultured blastocyst - isolated inner cell mass (grown on layer of irradiated mouse fibroblast feeder cells) - cells dissociated and replated to avoid overgrowth (and new feeder cells) - established ES cell cultures

Answer 71

only 10% of embryos produce these stem cells

Answer 72

- migration - cell division - differentiation into different types (tissues) - apoptosis

Answer 73

proteins secreted by cells called growth factors

Answer 74

- formation of the GUT TUBE - formation of the dorsal and ventral BUDS - BRANCHING - endocrine progenitors MIGRATE in mesenchyme, where they differentiate and form ISLETS

Answer 75

the insulin-secreting beta cells

Answer 76

- biodegradable polymers for nerve regeneration - hydrogels for musculoskeletal repair - electrospun fibres to allow generation of artificial tendons - windpipe made of glass

Answer 77

- heart disease - type I diabetes - CNS trauma - neurodegenerative disease

Answer 78

- ethically contentious - transplantation would involve immunosuppression - immune matching involves generating >1000 variable ES cell lines in the UK - far more difficult to derive than first anticipated - currently 300+ human ES cell lines world wide and very few clinical trials

Answer 79

a type of pluripotent stem cell that can be generated directly from adult cells - formed when you force the expression of 4 genes in fibroblasts (skin cells)

Answer 80

When first investigating iPS cells, scientists found there were 3 genes that caused complete loss of colonies when they weren't included: - factor 14 (oct 4) - factor 15 (sox 2) - factor 20 (klf 4) They also noticed that removal of cymc (factor 22) allowed colonies to form but they didn't look right. They went on to show that these 4 factors alone could cause the formation of ~160 colonies, whereas the most a 3 factor combination could form was ~50 These 4 factors are known as the YAMANAKA FACTORS and are still used today.

Answer 81

- compare cell morphology - look for expression of pluripotency associated genes using PCR - check for expression of markers by fluorescent staining - look for proteins that should be expressed in the nucleus (oct 4, Nanog and sox 2) and on the cell surface (Tra160 and SSEA4) - functionally assess their pluripotency - functionally to form chimeric mice using green fluorescent protein, the GFP gene

Answer 82

- using range of viruses and plasmids to deliver the genes | - safety is a consideration because many of the genes put in to make iPS genes are oncogenes

Answer 83

- initiation stage - maturation stage - stabilisation stage

Answer 84

A key feature of initiation stage is MET. The starting cell population has a mesenchymal phenotype and when reprogramming is initiated they start to become more rounded and closer together. This is completed by day 6. By day 21 they have formed colonies of pluripotent stem cells which have a very high nuclear-to-cytoplasmic ratio, and are tightly packed epithelial-like cells. As well as MET they cells increase proliferation as a result of the cymc, lin 20 and shp 53 expression. They also resist signals from other cells causing them to apoptose or senesce.

Answer 85

- share most of the attributes of ES cells i.e. self renewal and pluripotency - can be generated directly from patients own cells so they are autologous and won't be rejected - no ethical issues - present us with the opportunity of taking patients blood or skin cells and can reprogram then to iPSCs to study disease in test tubes

Answer 86

- current production methods involve inserting genes that could cause cancer - these current methods are too inefficient to guarantee production of iP cells for each patient - iPS cells may not differentiate as reliably to all types of cells - great potential but need to address these challenges

Answer 87

Antibodies produced are a heterogenous mixture of Igs to different epitopes. They have variable regions and constant regions, light chains and heavy chains. The variable regions vary amongst different Igs and is the specific region that 'sticks'.

Answer 88

The normal immune response is polyclonal - involving several monoclonal lymphocytes recognising different epitopes and their progeny

Answer 89

part of an antigen on its surface

Answer 90

- promotes a range of diverse specific antibody binding sites - not just a SINGLE antibody recognising a SINGLE antigen, will be a single antibody recognising a single epitope. - different antibodies recognise different parts of the same antigen

Answer 91

- high specificity - possible avidity (how well they tick together) for virtually any antigen including novel synthetic molecules - better than alternative - peptides need conjugation with large 'carrier' molecules to be immunogenic

Answer 92

- cost - amount of antigen available - volume of sera required - phylogenic relationship between Ag origin and host

Answer 93

1 - immunise antigen to activate B cells (add adjacent and then boost several times a few weeks apart) 2 - 4-8 weeks collect blood, clot, aliquot serum (test for activity) 3 - store (freeze) at -20*C or 4*C

Answer 94

- ethical issues with using animals - every animal is different so may not be reliable for a specific test - need to make sure the antigen being given to the animal is the same each time which is difficult if the virus is rare e.g. HIV or ebola

Answer 95

making monoclonal antibodies

Answer 96

they were first seen when looking at cancer cells. the cells are immortal so constantly produce the same antibody (as they come from the same cell). Overproduction of the same 'monoclonal' antibody can be dangerous, useful in diagnosis of some B lymphocyte tumours.

Answer 97

- high specificity - low possibility of cross reactivity with unrelated Ag - inexhaustible supply of consistent homogenous reagent - commercial applications

Answer 98

1 - immunise animal with antigen of choice (test bleed reactivity for 3 weeks - antibody should be going up, then boost every day for 3 weeks) 2 - one day post booth harvest B cells 3 - take a myeloma cell (=neoplastic B cells) from a tumour culture 4 - fuse in a gel for 2/3 days then treat with a drug [unfused B cells die - not immortal] [unfused tumour cells die - from drug] [fused cells are both B cells and tumour cells so they LIVE as they are immortal and resistant to the drug] 5 - plate out individual fused hybridomas and grow 6 - test each hybridoma medium supernatant for specific antibody by testing binding to antigen (ELISA)

Answer 99

- can re-engineer DNa encoding heaving and light V region which can be fused to activate molecules to target activity e.g. a toxin that kills tumour cells = therapeutic usage - can combine antigen binding site engineered in mouse with Ig constant regions form humans

Answer 100

separates and detects specific proteins from a protein micture

Answer 101

1 - denature cell line by treating with denaturing reagent e.g. SDS 2 - fractionate by length of polypeptides using SDS-PAGE 3 - transfer to a robust membrane eg nitrocellulose or nylon sheet 4 - hybridise to specific labelled antibodies to see what was specific (hybridise with a specific primary antibody, was, hybridise with a secondary antibody specific for primary antibody conjugated to fluorescent label, chemo-luminescent label or dye label)

Answer 102

- to determine the size (MW) of antigen - to see how a disease is progressing - to screen samples for antibody presence

Answer 103

- to screen samples for antigen presence - use antibody to capture beta-HCG antigen from urine (pregnancy test) - rapid ELISA for fluA antigen - to screen samples for multiple antigen presence

Answer 104

we can use this when we have a sample full of other things that we don't want to denature (as would happen in western blotting when they were put through SDS system) - sample labelled - add and bind specific antibody - add Fe binding protein A and precipitate - wash to purify: add SDS to elute Ag from Ab

Answer 105

- to detect proteins of interest in a sample - to purify native proteins of interest - to characterise MW of proteins - to study structure of proteins or antibody/antigen complexes - to detect specific antibodies in a sample

Answer 106

- purifies a soluble molecule from a mixture | - requires specific and reversible binding

Answer 107

STAGE 1: couple of ligand (antigen antibody) to a solid matrix in a chromatography column STAGE 2: pass the sample through column allowing binding and discard the rest of the mixture STAGE 3: elute off bound molecules. Low pH buffers disrupt non-covalent bonding

Answer 108

- reusable | - can purify late quantities (manufacture)

Answer 109

The same idea as affinity chromatography except instead of beads being stuck in the column, they are magnetic, and you use a magnet to pull them to one side.

Answer 110

``` 1 - mix sample and beads and bind 2 - apply magnet 3 - pour or wash supernatant 4 - resuspend purified beads 5 - elute off beads if necessary ```

Answer 111

- mostly pulling pathogens out of sample that are very complex - pathogen specific Ab's allow purification and concentration from complex sample e.g. toxigenic E.Coli from faeces

Answer 112

They have tight junctions between the cells that restrict the passive diffusion of ions/substances across the epithelial cell layer.

Answer 113

luminal, apical, mucosal membrane: faces a hollow or fluid filled chamber basolateral, serial membrane: normally close to a network of blood vessels

Answer 114

- tight junctions - polarised epithelium - vectorial transport of ions (ion channels, pumps, transporters) - transcellular and paracellular pathways for water movement

Answer 115

the driving force is the different between the electrical and diffusional forces acting on an ion

Answer 116

1 - across the apical membrane 2 - across the basolateral membrane 3 - via the paracellular pathway

Answer 117

the membrane voltage (Vm) at which the driving force for net ion movement across a membrane is zero and is given by the Nernst Equation

Answer 118

in vivo/in vitro | water/solute transport

Answer 119

``` ussing chambers short circuit current (ISC) ion flux ratios membrane permeabilisation micro electrodes ```

Answer 120

vesicles patch clamps artificial bilayers

Answer 121

1 - clearance methods i.e. absorption of glucose 2 - gravimetric measurement of net fluid absorption/secretion (Iv) 3 - marker infusion

Answer 122

1 - Short circuit current (ISC) | 2 - Radiotracers

Answer 123

- the resistance of epithelia to current flow by paracellular pathways is high - creation of a transepithelial potential (Vt) - values for Rt and Vt can be obtained by passing a current across the epithelium and applying Ohm's law - the current flow required to maintain Vt at 0mV is the short circuit current (ISC) Sum of ALL active electric ion fluxes across the epithelium --> ion replacement, pharmacological blockade, permeabilisation studies

Answer 124

the measurement of unidirectional transepithelial radioisotope fluxes can be used to identify transport of specific ions irrespective of the transport mechanism net fluxes across an epithelium are calculated from eh differences in oppositely directed unidirectional fluxes ussing predicted that the unidirectional fluxes of an ion moving via simple diffusion should be of equal magnitude (Ussing flux equation) fluxes that do not obey this equation are active transport processes

Answer 125

- identical solutions either raid elf the membrane (Ca=Cb) - no PD i.e. short current conditions (Vt=0) Jab/Jba = 1. exp (0)=1 --- diffusion Jab/Jba > 1 --- active absorption Jab/Jba

Answer 126

Active transport correlated with ISC If PD=0, ISC=sum of all net ion fluxes (I ionic)

Answer 127

a technique which simultaneously measures several physical characteristics belonging to a single cell in suspension. This is done by light scatter and fluorescence

Answer 128

flow cytometry = measuring properties of cells in flow flow sorting = sorting (separating) cells based on properties measured in flow. Also called fluorescence-activated cell sorting (FACS)

Answer 129

- fluorescence microscopy | - flow cytometry

Answer 130

Cells in suspension flow in single files through an illuminated volume where they scatter light and emit fluorescence that is collected, filtered and converted to digital values that are stored on a computer.

Answer 131

Need to have cells in suspension flow in single file. this is accomplished by injecting the sample into a sheath fluid as it passes through a small orifice. Same fluid flows in a central core that does not mix with the sheath fluid - laminar flow. Introduction of a large volume into a small volume - hydrodynamic focussing.

Answer 132

Lasers have a single wavelength of light (or more rarely a mixture of wavelengths). They can provide from milliwatts to watts of light. They can be inexpensive, air-cooled units, or expensive, water-cooled units. Lasers provide coherent light (single frequency).

Answer 133

When light hits a cell it is scattered in two directions. Forwards scatter is forwards and proportional to the size of the cell. At 90 degrees is side scatter, which is proportional to the granularity of the cell.

Answer 134

Because there are filters and one detector detects only light from one fluorochrome. Emission is overlapping - PMT1 will only measure a very narrow wavelength of light, as will 2,3,4 etc

Answer 135

- processing of signals from detectors - analogue-digital conversion - emitted light is converted into an electronic signal by the detectors. We can capture our data and analyse it on the computer.

Answer 136

the energy difference between the lowest energy peak of absorbance and the highest energy of emission

Answer 137

FLUOROCHROMES AND DYES - fluorescein isothiocyanate (FITC) - GREEN - phycoerythrin (PE) - ORANGE - peridinin chlorophyll protein (PerCP) - RED dozens of flourochromes on the market so lots of options about what laser and what antibodies you want to use.

Answer 138

- peripheral blood - bone marrow - fine needle aspirate - fresh tissue - CSF and other fluids

Answer 139

DIRECT: monoclonal antibodies (MoAbs) are pre conjugated to fluorochromes. This is a one-step-process. INDIRECT: unconjugated monoclonal antibodies. The primary antibody doesn't have the fluorochrome, but we put a secondary antibody on the antibody which does have the fluorochrome, and it binds to the primary antibody. This is a two step process. This may allow a stronger signal (more fluorochromes). This method may have to be used if you can't buy or make an antibody with the fluorochrome on it.

Answer 140

the cells that have the particular fluorescent integrity of whatever parameter is being measured

Answer 141

A more sophisticated method. We can draw regions around the cells we know because of the side scatter on a dot plot and can make the machine only display cells in that gate on the basis of the two fluorochromes, so will be able to see the populations what whichever cell type we decide to look at.

Answer 142

8 with 2 we can only see 4... but if we used 4 we would see more, etc etc

Answer 143

in the simplest method, cellular DNA is detected using a fluorescent dye that binds preferentially to DNA. Propidium iodide is most commonly used. It undergoes a dramatic increase in fluorescence upon binding DNA. It requires permeabilisation of the plasma membrane. There are no antibodies or antigens - we are just adding something that binds to DNA. We have to permeabilise the cells to get the propidium iodide in - basically we have to punch holes in the membrane to allow it to enter.

Answer 144

- excited at 488nm | - emits in the red

Answer 145

- the main peak is G0-G1 - the following smear is S phase - there is another peak when they're in G2M there is sometimes a peak at the beginning showing apoptotic cells (or can be argued to just be fragments of DNA) we can draw regions around these cells and see what proportion of the cells are in these phases

Answer 146

- PI can not normally cross the cell membrane, so living cells will look negative on flow cytometry - if the PI penetrates the cell membrane, it is assumed to be damaged - cells that are brightly fluorescent with PI are damaged or dead, they will look positive

Answer 147

- by staining with PI (cells fixed) - phosphatidyl serine, can be detected by incubating the cells with fluorescein-labelled Annexin V, and PI - i.e. using two stains together (cells not fixed) - by staining with 7-aminoacinomycin D (cells not fixed)

Answer 148

Annexin V goes to the phosphatidyl serine. In a live cell it appears totally negative because the PI can't get in and the annexin V can't bind as the phosphatidyl serine is all on the inside of the cell. In early apoptosis the PS is flipped onto the outside of the membrane so annexin V can bind. However, the membrane is still in tact so PI can't get in, so the cells will appears green but not red. Later on in apoptosis/necrosis we lose the integrity of the membrane. The annexin can bind (green) and the PI can also get into the cell (red) so these cells will be positive for both green and red.

Answer 149

- Excited at ~488nm - Emits at ~660nm - DNA specific - intercalates in G-C regions - long emission wavelength, with FITC and PE labelled Ab for simultaneous evaluation of DNA content and 2 colour immunofluorescence using only 488nm ex. We can label any of these things with fluorochromes and can look at different caspases at the same time. We can measure where the cell is in the apoptotic pathway.

Answer 150

- immunophenotyping of leukaemia and lymphomas - detection of MRD - stem cell enumeration - CD4/CD8 in HIV - measurement of intracellular cytokines - study of cell cycle, viability and apoptosis - measurement of cell proliferation - assessment of transfection efficiency

Answer 151

When it recognises one of the droplets that fits into the category that we want the machine charges the stream so that the droplet containing the cell that we want breaks off the nozzle from the vibrations, and it is pulled into the plates and separated from the rest of the population.

Answer 152

LIPITOR: the biggest drug blockbuster in history with sales of $11 billion (before patient expiry)

Answer 153

TORCETRAPIB: raised blood cholesterol, strong scientific basis and rationale, but failed clinical testing in 2006 after Pfizer spent $1 billion. Increased mortality rates as compared with placebo.

Answer 154

1. ) DRUG DISCOVERY: candidate molecules chosen on basis of pharmacological properties 2. ) PRECLINICAL DEVELOPMENT: non-human studies. Toxicity testing, pharmacokinetic analysis and formulation 3. ) CLINICAL DEVELOPMENT: volunteers and patients. Efficacy testing, side-effects and potential dangers.

Answer 155

- functional proteins - receptors - enzymes - transport proteins ...not all are actually 'draggable' however...

Answer 156

cost and complexity of drug discovery and development (regulation)

Answer 157

- cloning of target protein - assay to measure functional activity - automated systems to allow for speed and economy - high-throughput screening of large compound libraries - natural products, fungal, plants, bacteria Lead optimisation, complex chemistry to increase potency, selectivity and stability

Answer 158

- pharmacological testing for hazardous acute effects - preliminary toxicology testing - pharmacokinetic testing for absorption, metabolism, distribution and elimination - chemical and pharmaceutical development to assess feasibility of large-scale synthesis and purification as well as stability

Answer 159

volunteers and patients, efficacy testing, side-effects and potential dangers... PHASE I: safety and tolerability (MTD) PHASE II: small scale efficacy/placebo controlled PHASE III: large scale efficacy/randomised and blinded PHASE IV: post marketing surveillance

Answer 160

- first specific anti-angiogenesis drug - in 2013 was the second biggest selling oncology product - forecast to be the top selling cancer drug for 2018 - approved for colorectal, lung, kidney and ovarian cancers and eye disease

Answer 161

the formation of new blood vessels form pre-existing vessels

Answer 162

- blood vessels supply oxygen, nutrients, remove waste and allow immune surveillance - physiological angiogenesis is essential for organ growth in the embryo and repair of wounded tissue in the adult - abnormal genesis leads to disease --> inefficient vessel growth: stroke, MI, ulcerative disorders and neurodegeneration; excessive vessel growth: cancer, inflammatory disorders, pulmonary hypertension, blindness

Answer 163

- developmental angiogenesis/vasculogenesis - normal angiogenesis - pathological angiogenesis

Answer 164

organ growth

Answer 165

wound repair, placenta during pregnancy, cycling ovary

Answer 166

tumour angiogenesis (gives the tumour nutrients from the blood and allows metastatic spread via the blood), ocular and inflammatory disorders

Answer 167

vasculogenesis is defined as the differentiation of precursor cells (angioblasts) into endothelial cells and the de novo formation of primitive vascular network, whereas angiogenesis is defined as the growth of new capillaries from pre-existing blood vessels

Answer 168

hypoxia is a strong stimulus for tumour angiogenesis. Hypoxia is low oxygen tension (

Answer 169

- monoclonal antibody - binds to VEGF - prevents VEGF binding to VEGF receptors on endothelial cells

Answer 170

- T1 relaxation - T2 relaxation - Water diffusion (micro) - Flow (macro) - perfusion

Answer 171

- anatomical - functional - response to treatment

Answer 172

- abnormal tissue or vascular structures - presence of lesions - structural changes (learning, ageing)

Answer 173

- blood flow: perfusion, brain activation - motive taste or experiencing pain - metabolic (biochemical changes)

Answer 174

- lesion change: disappearance, shrinkage, change in appearance - functional changes: normal blood flow, activation changes

Answer 175

There is a very strong uniform magnetic field along the bottom of the magnet (whole thing) 30,000x stronger than Earth's magnetic field. MR imaging is formed with magnetic field gradients to code the image signal in space. Linear variations of magnetic field coding the signals so you know where they're coming from so hat you can see slices instead. You can then build up the image. MRI scans are good because you can rotate things if the patient is not lying straight etc

Answer 176

it comes from the protons in water and the fat in tissue water within hydrogen atoms gives most of the magnetisation. the nucleus is a proton that spins on its own axis - it has a single positive charge. Nuclear Magnetic Resonance can interact with this magnetisation.

Answer 177

- Oblique: any slice orientation is possible - Multislice: acquisitions on many different slices within the TR - 3D: acquisitions from a full 3D volume of tissue - Gated: RF pulses triggered to ECG for cardiac imaging

Answer 178

T2 is very dependent on how mobile the water is in the tissue and increases with: - oedema, an increase in the water content - demyelination, a loss of brain tissue structure

Answer 179

T2 is reduced by the presence of paramagnetic ions - Fe from blood breakdown products - Gd from contrast agents

Answer 180

T1 is very dependent on the presence of paramagnetic ions that reduce T2 T1 is also dependent on how mobile the water is in the tissue, and T1 increases slightly with oedema

Answer 181

lower in white matter because of myelinated neutrons

Answer 182

- water in the vicinity of the contract agent experiences strong fluctuating magnetic fields, hence T1 and T2 are reduced. - bind strongly to the Gd - don't get free Gd ions in the patient. Wherever the Gd is, the T1 and T2 are produced.

Answer 183

- gradient echo images (GRE) are very sensitive to the rpesense od non-uniformity of the magnetic field in tissue (T2* relaxation --> T2* decreases as the uniformity decreases) - deoxygenated blood contains deoxyhaemoglobin (dHb), which is paramagnetic, and so distorts the magnetic field - if blood becomes more deoxygenated, then the dHb levels increase and the T2* decreases - In a GRE image, this means that the image gets larger

Answer 184

vasodilation to increase blood flow to areas of the brain require for function (moving, reading, speaking)

Answer 185

A 1H spectrum of biochemicals can be obtained from a localised region of the brain using three slice selective pulses

Answer 186

A stronger shielded nucleus has a lower resonant frequency

Answer 187

No ferromagnetic objects in the examination room; scissors, stethoscopes, wheel chairs, gas cylinders - hearing aids, watches, spectacles (dentures - image quality)

Answer 188

- pacemakers - first trimeter pregnancy aneurysm clips - metallic foreign bodies (orbit x-ray, shrapnel)

Answer 189

- mechanical radiation, propagating by vibration of elements (molecules) of a medium - manifests as longitudinal pressure (compression) wave and transverse (shear) wave - sound production requires a vibrating source Sound is a physical wave that requires a medium to propagate through.

Answer 190

Longitudinal: waves same way as direction Transverse: waves perpendicular to direction

Answer 191

because we need very short wavelengths to be able to resolve structures in the body that are very close together

Answer 192

density and bulk modulus (stiffness) the density of the material and how rigid it is determines the speed. If a material is squishy it slows down the speed, and conversely dense materials will increase the speed of sound. the waves travel a bit slower in fat, very much slower in air, and much faster in bone

Answer 193

- ABSORPTION: ultrasound is converted into heat by friction - REFLECTION: some energy reflected from a boundary - INTERFERENCE - REFRACTION: some or all of the energy is diverted from its original path when the beam hits the boundary away form the normal, this can produce artefacts in the image - DIFFRACTION: divergence of beam from sound source when it goes through a narrow gap. this can give us less focus or no information at all - SCATTER: some of the energy is dispersed in all directions. This causes the grainy texture of the images produced, this is also an artefact

Answer 194

the tissue properties, frequency and the distance travelled. The higher the frequency, the more you are vibrating the molecules so you will lose more energy. For deeper structures we have to alter features to get enough echo strength coming back to be able to see something. We use lower frequencies to see deep structures for this reason, but can use higher frequencies when looking more superficially.

Answer 195

At tissue boundaries where there is a change in acoustic impedance (Z)/ a tissue interface the amplitude (or intensity) of the reflected wave depends on the difference in the acoustic impedances (reflection coefficient) - the greater the difference, the stronger it will get reflected back

Answer 196

Returns echoes to the transducer only when the sound beam is perpendicular to the interface. If you hit the surface at 90 degrees you get a crisp white bright line in the image. When you hit it away from the normal, it will bounce back at an angle and you will lose some of this clarity

Answer 197

she of the little wavefronts coming off the scatter can summate together and line up which will increase their intensity - this is constructive interference if they don't line up you will get destructive interference as they cancel one another out. These effects together give the grainy appearance - it is totally meaningless and is nothing to do with the texture.

Answer 198

the loss of power or amplitude the ultrasound signal as it passes through tissue ATTENUATION = ABSORPTION + SCATTER + REFLECTION usually attenuation is nearly proportional to frequency - attenuation increases with frequency: absorption factor.

Answer 199

- TRANSMITTER - TRANSDUCER - RECEIVER, ADC AND PROCESSOR - IMAGE DISPLAY

Answer 200

to energise the transducer (PRF). Issues pulses all the time, like a clock, everything is related back to the transmitter for timings of echoes coming back etc

Answer 201

piezoelectric ceramics convert the electrical energy into mechanical vibrations. The ultrasound probe, the piezoelectric material which makes the ultrasound and directs the echoes coming back. Made of ~250 elements of varying materials - often silicon or a composite material. They are all individually wired up to individual electrodes.

Answer 202

to detect and amplify backscattered energy, i.e. detect what is noise and can be discounted

Answer 203

a flat screen device which present the ultrasound image

Answer 204

it is there to allow as much of the sound through into the body as possible. If it was not there and we put the transducer straight onto the skin then there would be a big difference and we would get a lot of reflection; the matching layer is a pathways between the elements and the body

Answer 205

it is a damping element to stop echoes coming out of the back of the probe and bouncing around which would cause confusion. Sound coming back will hit it and cause it to vibrate gently, and produce avery small electric current - a piezoelectric element.

Answer 206

Continuous

Answer 207

B-mode is 'brightness mode' - this gives us little dots on the display instead of a line. the brightness shows the strength of the echo and further down the image means it took longer to get back - i.e. placement on the image shows the time it took to get back. These dots gradually build up over time

Answer 208

- large number of pulse echo lines - each echo is displayed along a line as a dot - the brightness of each dot is determined by the strength of the echo - the distance down the display relates to its depth below the transducer

Answer 209

This means that the operator has control. When the elements fire, its not just one but normally a group of them that operate. If we adjust the timing, the outer ones fire a bit earlier, then the inner one. The wave fronts interfere with each other and focus in a tissue - this allows you to focus in the area of interest. Varying delay times enables focussing at different depths.

Answer 210

echoes coming from deeper structures are weaker and must be amplified to produce uniform tissue echo appearance. this is manually controlled and has a profound effect on the quality of the ultrasound image presented for interpretation. they go through standard linear amplification systems the echoes coming back from further in the body will be much weaker and so the machine has to compensate for this or the images will get darker and darker until it is black. the echoes that take longer to come back are amplified a bit more to compensate for that loss - "time gain amplification system". The operator can alter that using the slides on the machine and this will change how much amplification occurs.

Answer 211

we can just alter the front or the back of the image, basically to make the image look more aesthetically pleasing

Answer 212

we can look at tissue movement by bouncing the US off RBCs and detect their forward movement - we do this by looking at the Doppler Shift Effect. If we have a target that isn't moving and hit it with ultrasound waves, then its reflection will have the same frequency. If we have a target that is moving towards us, then we will get a compression of the wavefront and the frequency is higher in the reflection. If it is moving away then the wavefront will get stretched out and the frequency will be lower in the reflection. US machines use this to analyse blood flow as the sound bounces off RBCs. We can also get a good estimation of its speed using this technique and a complicated equation.

Answer 213

RED: anything flowing towards the probe face BLUE: anything moving away from the probe face there is a scale at the side with the hue of the colours giving us an idea of the speed of the sound.

Answer 214

a measurement of speed, gained by using a spectral gate, which you place over the blood vessel, which you flick into spectral doppler mode and get detailed analysis of it flowing through the gap. this will give measurements of peak velocity, systolic velocity and average velocity. this method is a lot less invasive as we don't have to inject anything but we still get a lot of information

Answer 215

- information on speed and direction of blood flow - image noise results in random flashes of colour - angle dependent (no flow shown at 90 degrees)

Answer 216

- only detects blood flow, but more sensitive to low volume flow - angle independent - better boundary detection - very sensitive to soft tissue motion

Answer 217

``` TRANSVERSE SECTION (TS): right side of patient on left side of image LONGITUDINGAL SECTION (LS): cranial aspect of the patient on the left of the image ``` this is the reverse for cardiologists/echocardiography

Answer 218

- real time guidance - multiplayer - visualisation of blood flow - speed - portable - cost - non-ionising radiation

Answer 219

- sound penetration (bone and gas) - operator dependence - only one person doing the images and they give the definitive opinion which is often a diagnosis - patient size and very gassy patients - worst imaging, 100% deflection from air, fat slows down waves - artefacts - have to be careful with early pregnancy as the warming effect can be measurable in a foetus

Answer 220

When a region has a lower attenuation coefficient than adjacent tissues the machine overcompensates. This provides a clue to tissue composition. Echoes coming back are stronger and look brighter, and this can be a useful artefact in the cases of liver cysts for example.

Answer 221

- ABDOMINAL IMAGING - PELVIS (GYNAECOLOGY AND PROSTATE) - SMALL PARTS - OBSTETRICS - VASCULAR - PAEDIATRICS - CHEST - BRAIN - CARDIOLOGY - ENDOSCOPIC/INTRAVASCULAR - INTRAOPERATIVE/LAPROSCOPIC

Answer 222

- real time and portable - offers advantage over the image guidance - biopsies, FNA, drainage procedures, vascular access - guidance for ablation procedures (e.g. RFA) - sterile probe covers required

Answer 223

- Diameter 1-7 micrometers (able to cross pulmonary circulation) - gas filled - cross capillary beds - vascular markers - duration: vascular phase 5-10 minutes - resonant frequency 2-5 MHz - very safe

Answer 224

Ultrasound transducer is used to compress tissue. The compression is simultaneously imaged and the images are further processed to generate stiffness images - "elastograms"

Answer 225

- BREAST: improve differentiation between benign and malignant disease, assessment of appropriate therapeutic treatment - THYROID: provides additional features of malignancy, targeting biopsy of complex lesion - PROSTATE: to improve visualisation of prostate cancers, targeting biopsy - PANCREAS: to differentiate between benign and malignant tumours - LIVER: (WIP) to observe progressive degree of liver fibrosis

Answer 226

- volume measurement - viewing the orthological plane - assessment of surface and complex shapes - separation of B-mode data from colour

Answer 227

- acquisition - movement artefacts - artefacts in general

Answer 228

produces images in the 3 orthogonal planes. Can be rotated or scrolled through. Power doppler information can be combined and on 4D systems this can be viewed in real time.

Answer 229

- increased demand for imaging - variable want for ultrasound service and out of hours access - affordable, high quality, portable machines - move towards point-of-card diagnostics - improved patient pathways/service improvement - guidelines advocating the use of ultrasound in procedures

Answer 230

- performed in A+E for detection of haemoperitoneum - scan for free fluid - sensitivity 94% and specificity 98% - in the low risk patient a negative ultrasound has a very negative predictive value - recognises increased demand for ultrasound - training to be adequately funded - training to consist of a combination of practical and theoretical - standard of training to be equal to that of a radiographer - foster relationships between radiological and non-radiological practitioners

Answer 231

- evidence of scanning protocols - minimum scan members (20 per year at level 1) - minimum equipment standard - regular equipment maintenance - policy on infection control - policy on image recording - reporting of images BFCR (06) 1 - incident reporting

Answer 232

generations of action potentials, synaptic communication

Answer 233

Na+/K+ ATPase produces an unequal distribution of Na+ and K+ ions to reduce osmotic pressure and prevent cell lysis. This leads to creation of electrochemical gradients for Na+ and K+.

Answer 234

Ohm's Law: Voltage (V) = Current (I) x Resistance (R) Another way of describing resistance is as a reciprocal of conductance (Siemens,G1) R=1/G The less resistance to the flow of current between the pd of two points, the more conductance there is... (V=IR, rearrange to V=1/G) = Ohm's law = I = GV

Answer 235

- extracellular recording - intracellular recording - voltage-clamp recording all measure activity of many/different types of ion channels - macroscopic recordings

Answer 236

two micro electrodes are places outside cells. Records cell-induced membrane potential changes between two electrodes e.g. APs. Single cells: single-unit recording. Group of cells: multi-unit recording

Answer 237

Record electrical activity in anaesthetised/conscious animals, e.g. Hubel and Wiesel showed specific neurones in visual cortex responded to different visual stimuli

Answer 238

One micro electrode is places inside, the other ground electrode is placed outside the cell. Records membrane potential across the membrane of single cell - allow manipulation of external solutions (e.g. control electrolytes, apply drugs). This does not alter intracellular composition of cells.

Answer 239

- Record electrical responses to external stimuli e.g. neurotransmitters - measure changes in conductance - opening/closing of ion channels

Answer 240

- one microelectrode measures voltage - which is 'clamped' - one microelectrode applies current to and from the cell - uses a 'clever' feedback amplifier - measure V and I at the same time The power of the voltage-clamp is to measure membrane current produced by opening of ion channels at a specific membrane potential

Answer 241

- reduced background noise - can record very small currents (

Answer 242

- cell-attached patch - inside out patch - whole-cell recording - outside-out patch

Answer 243

USES: - agonists applied to pipette solution - eg ligand gated (eg nicotinic receptors) - voltage steps applied through the pipette - eg voltage gated (Na+ channels) - agonists applied to extracellular solution (non-pipette solution) - eg GPCRs (muscarinic receptors) PROBLEMS: - resting membrane potential cannot be controlled - can not control the composition of the intracellular medium

Answer 244

USES: - agonists can be applied directly to the cytoplasmic surface of the cell membrane - eg activation mechanisms of 2nd messenger systems Ca2+, DAG, IP3, protein kinases (PKC, PKA) - control composition of both extracellular and intracellular media PROBLEMS: - very reductionist approach - will the channels behave normally?

Answer 245

USES: - agonists applied to pipette solution - eg 2nd messenger gated - agonists applied to extracellular solution - eg ligand-gated/GPCR - voltage steps applied through the pipette - eg voltage-gated channels (Ca2+ channels) - control composition of both extracellular/intracellular media PROBLEMS: - measures only macroscopic current - not individual channel openings - important intracellular components may dialysis out of the cell

Answer 246

USES: - unlike whole cell recording can measure single channel openings - agonists can be applied to pipette solution - eg ligand gated/GPCR - voltage steps applied through the pipette - eg voltage-gated channels (Ca2+ channels) - control composition of both extracellular/intracellular media PROBLEMS: - important intracellular components may be dialysed out of the cell

Answer 247

BP = CO x TPR

Answer 248

blood flow = pressure gradient/vascular resistance

Answer 249

Vascular resistance relies on vascular tone. It regulates diameter and resistance.

Answer 250

- endothelium - smooth muscle - these are the muscles that contract, giving the vascular tone - perivascular nerves - elastic and fibrous tissues

Answer 251

If the fibres contract they will narrow the vessel and vice versa. They lie perpendicular to the blood flow. This is what constitutes vasoconstriction and vasodilation.

Answer 252

resistance is INVERSELY proportional to the 4th power of the radius (r) of a vessel, and proportional to viscosity of blood (curly n) and the length of the vessel (L) POISEULLE'S LAW

Answer 253

measure tension generated by the vascular wall while the diameter of the vessel remains constant can be done in large organ baths or wire myograph widely used in vitro techniques to investigate the functional properties of isolated vessel segments

Answer 254

- for large vessels (internal diameter > ~1.5mm) - segments are mounted as ring preparations on two fixed steel hooks - kept in a chamber with physiological salt solution at 37*C with oxygen - they are viable for >6 hours

Answer 255

- for small vessels (internal diameter ~100-400 micrometers) - segments are mounted as ring preparations on two fixed steel wires - kept in a chamber with physiological salt solution at 37*C with O2 - viable >12 hours - smaller arteries play a bigger role in vascular resistance --> DISSECTION --> MOUNTING -->

Answer 256

the concentration at 50% of the maximum response

Answer 257

- examine functional properties of small or large vessels - freshly prepared vessels are viable for many hours - detailed investigation into the mechanisms of action - compare vascular function in control vs diseased states - can be combine swift other experimental techniques

Answer 258

- dissection and mounting of vessels requires skills and suitable apparatus - isolation of vessels may remove influences from surrounding tissues - difficult to study longer term changes in vascular function using the same vessels - cannot measure myogenic contractions - cannot measure flow induced relaxations

Answer 259

- for small vessels including arterioles (>50 micrometers) - segments mounted on 2 glass microcannulae and pressurised to an appropriate transmural pressure - kept in a chamber with physiological salt solution at 37*C with oxygen

Answer 260

- an intrinsic property of vessels (auto regulation) - stretch of smooth muscle causes vasoconstriction - stabilises blood flow despite increase in BP (between 40-100mmHg) - contributes to basal vascular tone Shear stress = frictional force on the endothelium

Answer 261

- shear stress on endothelial calls leads to vasodilation | - impaired in cardiovascular diseases including atherosclerosis and ageing

Answer 262

- examine functional properties of small vessels, including myogenic constriction of flow-induced dilation - freshly prepared vessels are viable for many hours - detailed investigation into the mechanisms of action - compare vascular function in control vs diseased states - can be combined with other experimental techniques

Answer 263

- dissection and mounting of vessels requires skills and suitable apparatus - isolation of vessels may remove influences from surrounding tissues - difficult to study longer-term changes in vascular function using the same vessels

Answer 264

- cannulate supply artery to a vascular bed - e.g. measure perfusion pressure under constant flow rate - flow = perfusion pressure/vascular resistance

Answer 265

- cannulate the ascending aorta (retrograde perfusion) - measure coronary perfusion pressure under constant flow rate - viable for a few hours - can also examine left ventricular contractions and heart rate (if eft beating naturally)

Answer 266

IN VITRO - isolated small vessels - isolated vascular beds IN VIVO - ultimate preservation of surrounding tissues and regulation through hormones and innervation?

Answer 267

- examine changes in blood flow in vivo (natural surroundings are largely preserved) - compare vascular function in control vs disease states - can be adopted for non-invasive measurements - allow long-term measurement of blood flow in the same vessels

Answer 268

- relative measurements of blood flow - quality of signal (signal:noise ratio) depends on tissues and machines - invasive procedures and anaesthesia may be necessary - expertise requires for experimentation, data analysis and interpretation

Answer 269

- schizophrenia - schizoaffective disorder = schizophrenia and bipolar disorder - delusional disorder - some depressive and manic illnesses

Answer 270

- early onset - prevalent - disabling and chronic

Answer 271

- affects up to 1% of the population - no significant influence of culture, ethnicity, background, socioeconomic groups - increased in urban areas - no different between men and women (men 1-25 years and poorer response to therapy, whereas women 20-30 years)

Answer 272

Before the disease is recognised there is often a phase in late teenage years associated with social isolation, interest in fringe cults and social withdrawal

Answer 273

For one to be schizophrenic, 2+ of the following must be present for a significant period of time: 1. delusions 2. hallucinations 3. disorganised speech, emotional blunting and meaningless behaviour 4. grossly disorganised or catatonic behaviour (e.g. sitting still for ages/abnormality of posture) 5. negative symptoms (eg affective flattening)

Answer 274

- must have positive and negative symptoms - gradual or sustained deterioration of work, interpersonal relationships, communication and self care - duration of symptoms has to be at least 6 months of disturbances - exclude substance abuse/general medical conditions - relationship to pervasive development disorder

Answer 275

no but there is some familial correlation e.g. twin studies and parents etc

Answer 276

- born in winter months increased risk - some form of damage to the child, e.g. damage to corticolimbic system - viral epidemics - development in vitro - obstetric complications (oxygen deprivation)

Answer 277

genetic makeup deemed to be necessary for the development and manifestation of schizophrenia, along with extreme stresses within the surroundings

Answer 278

an early symptom indicating the onset of a disease or illness

Answer 279

1. abnormal ideas 2. abnormal perception 3. motor, volitional and behavioural disorders 4. formal though disorders 5. emotional disorders 6. psychophysiological changes

Answer 280

peculiar forms of motility, stupor, mutism, stereotypy, mannerism, negativism, spontaneous automatism, impulsivity - stereotypes: purposeless, repetitive acts - bizarre postures; strange mannerisms - altered expression - grimacing states of catatonia or bouts of extreme hyperactivity impulsive behaviour e.g. violent acts and reasonless murder

Answer 281

- disturbances in thinking - intellectual speech - derailment of speech - loosening of associations; failure to follow train of thought to its conclusion - poverty of speech - manifests as distorted or illogical speech

Answer 282

- loss of natural reaction to social cues (affective flattening) - reduced facial expression - apparent indifference to emotive topics - cold and detached - cannot establish a rapport in social dialogue - inappropriate affect - shallowness of emotions (e.g. laugh at death)

Answer 283

EVENT-RELATED POTENTIAL (ERP) ABNORMALITIES - deficit in processing of sensory information associated with medial temporal lobe dysfunction - a reproducible finding in schizophrenics EYE TRACKING CHANGES OBSERVED IN 50% OF SCHIZOPHRENICS - dysfunction in connections between frontal eye fields and the temporal and parietal cortices - a robust phenotype marker of familial risk of schizophrenia

Answer 284

- delusions of perception and loss of control of own thoughts and actions - bodily sensations due to outside forces - alienated or disorientated thought - sudden fully formed delusion in the wake of normal perception - thoughts make no sense to normal people - hallucinations - voices talking about the person - bizarre behaviour

Answer 285

- loss of empathy with/in others - inappropriate or blunted mood; repetitive activity - incongruity of emotions (blunted emotions) - social withdrawal - inability to experience pleasure - poverty/paucity of speech - attention deficit - difficulty planning - impaired judgement NEGATIVE SYMPTOMS ARE REFRACTORY TO DRUG TREATMENT

Answer 286

- deficits of neuronal matter: smaller brains and longer ventricles particularly in medial temporal lobe and left hemisphere - alterations in thalamus and basal ganglia - functional analysis of brain: electroencephalogram abnormality reported in 80% of schizophrenics, but the changes are non-specific

Answer 287

decreased blood flow to the prefrontal cortex

Answer 288

memory, emotion and behaviour

Answer 289

- limbic system - prefrontal cortex - prefrontal cortex MRI scans show dramatic loss of grey matter (parietal, temporal, frontal cortices) during the evolution of schizophrenia

Answer 290

subcortical forebrain structures (hypothalamus, hippocampus and amygdala) - influences emotion and motivation

Answer 291

- altered personality - impatient - impulsive - unpredictable - undecided - tendency to indulge in profanity (at odds with previous behaviour) - decreased ability to alter behaviour on basis of experience - attention is prone to distraction prefrontal cortex is important in cognitive function and emotional expression in survival. damage or infection of temporal love structures can cause psychotic symptoms.

Answer 292

drug-free: stopped for a while so its not in their system any more drug-naive: never taken the drug before

Answer 293

DOPAMINE: a key mediator CHLORPROMAZINE: dopamine receptor agonist, provided first significant improvement in schizophrenics - symptomatic relief LYSERGIC ACID DIETHYLAMIDE (LSD): acts on several 5-HT receptor subtypes Hyperactivity in the mesolimbic dopaminergic system and hyperactivity in mesocortiyal dopaminergic projections.

Answer 294

- infers that hyperdopinergic activity leads to psychosis - dopamine receptor agonists(eg amphetamines) can induce psychoses - especially in those who are already predisposed - there is a correlation between D2 receptor affinity (Ki) and clinical antipsychotic efficacy - structural deficits in the brain suggest a loss of control of dopaminergic function - multiple studies have shown an increased dopamine release in striatum (SCZ > controls) after amphetamine treatment consistent with increased phasic dopaminergic activity - consistent evidence of abnormal D2 receptor number or affinity is lacking

Answer 295

5-HT, GABA and glutamate may also play a role in the disease process. BUT most of the neuroleptics interfere with dopaminergic transmission. Dopamine receptors: D1-5

Answer 296

There is a link from LSD to psychosis. Phenylcyclidine (PCP/Angel Dust), antagonist at NMD (N-methyl D-aspartate) glutamate receptor type - use is associated with psychotic state - paranoia - agitation, hallucinations - social withdrawal - reduced emotional expression PSP (and ketamine) can precipitate psychotic episodes in susceptible patients. PSP use suggests that deficits in glutaminergic transmission may be associated with schizophrenia. Postmortem on brains of schizophrenics show loss of neurones in mediodorsal thalamus.

Answer 297

1. loss of thalamic axon terminals 2. loss of dopaminergic innervations of prefrontal cortex - impairment of GABAergic cortical transmission - reports of significant alterations in subpopulation of cortical GABAergic cells in schizophrenics involves many NTs: dopamine, 5-HT, glutamate, GABA, CCK, somatostatin

Answer 298

- inhibit most florid and subjective behavioural disturbances of psychosis - restore patient to normal life in society - some agents diminish the negative symptoms of schizophrenia (amotivation, flattened effect and social withdrawal) Amotivation:incapacity to perceive one's behaviour and its outcome

Answer 299

There are two classes: typical and atypical. Atypical neuroleptics cause less motor disturbances. There is a strong correlation between the potency of the drugs and their affinity for D2 receptors in the CNS. The agents bring about improvement in acute positive symptoms of the disorder. Reduce disturbances of delusional thinking, hallucinations, inappropriate saviour and anxiety --> the drugs take time (days or weeks) to work

Answer 300

- you get secondary effects which may be important for therapeutic effects - a range of pharmacological effects are associated with the agents

Answer 301

PHENOTHIAZINES eg chlopromazepine, thioridazine, fluphenazine D2, alpha1, M receptor agonists, H1 blockage - causes sedation BUTYROPHENONES eg haloperidol D2, alpha1 receptor antagonist THIOXANTHINES eg flupentixol less sedating; EPS ``` The atypical (novel) neuroleptics differ from each other and typical neuroleptics in their pattern of receptor binding eg respiradone, aripiprazole ```

Answer 302

weight gain, aesthetic jaundice, etc needs to be administered in a hospital setting as it causes sedation is also interferes with H1 receptors to some extent

Answer 303

idiosyncratic reactions, anticholinergic reactions, antihistaminergic reactions, antiagrenergic, increased prolactin secretion - impotence etc, miscellaneous reactions (rashes, liver and bone marrow toxicity) mainly due to the mode of action of the drugs, but can be unrelated --> dopaminergic pathway, blockade of muscarinic, alpha-adrenergic and histamine receptors some drugs have individual side effects. Some work on a variety of receptors - they are 'promiscuous'

Answer 304

parkinson's like symptoms... | rigidity, tremor, restlessness, slowing of voluntary movements, postural instability, dystonia

Answer 305

- uncontrollable involuntary movements of face and limbs | - benzodiazepines and beta-blockers may be helpful

Answer 306

1. ) mesolimbic (SCZ - increased DA causes positive symptoms) 2. ) mesocortiyal (SCZ - DA hypoactivity: negative and cognitive and affective symptoms 3. ) nigrostriatal (drugs - EPS and TD drug side effects) 4. ) tuberohypophyseal (Drugs - hyperprolactihemia side effects, D2 antagonists reduce activity in this pathway therefore prolactin levels rise)

Answer 307

A subcortical part of the forebrain that helps coordinate body movements associated with behaviour and motivation. Loss of dopaminergic innervation to the striatum is involved in Parkinson's disease.

Answer 308

normal secretion acts to block prolactin to a certain extent. typical neuroleptics interfere with the pathway, producing elevated prolactin levels as side effects

Answer 309

R.M.I.V.U.X.G

Answer 310

- radiation that causes ionisation when it interacts with matter - types used for medical imaging are gamma rays and x-rays - we use ionising radiation because it is PENETRATING

Answer 311

This is when the radiation is incident upon the DNA and can cause damage at that level. Direct effects occur once a threshold level of damage has been reached.

Answer 312

Because most of the body is water, the indirect action is most predominant. The radiation is ionising so it splits the water into ions. They can also combine to form H2O2 which is highly reactive and can go on to interact with other molecules in the body and cause damage

Answer 313

The cell could mutate to such a level that the cell dies, could lose control of its growth function (cancer) or could be affected at the DNA level and cause genetic changes.

Answer 314

- only at higher radiation dose - threshold effect - e.g. Erythema and hair loss

Answer 315

- risk of cancer induction - risk of genetic change in subsequent population - effect proportional to radiation dose - no threshold - ALL RADIATION HAS RISK

Answer 316

alpha particles: 2 neutrons, 2 protons (positive) beta particles: electrons (negative) emitted following the radioactive decay of an unstable nucleus

Answer 317

positrons: the electrons interact with matter to create gamma rays emitted following the radioactive decay of an unstable nucleus

Answer 318

gamma rays: penetrating radiation emitted following the radioactive decay of an unstable nucleus

Answer 319

x-rays: spectrum of electromagnetic radiation artificially produced in an x-ray tube

Answer 320

X-rays are essentially an attenuation map - the image appears darker in areas where more x-rays penetrate the film. Attenuation increases with... - higher atomic number - higher density bone has a high atomic number and high density so the beam will be highly attenuated, meaning it stops some of the beam transmitting through it so we get quite allow x-ray signal. muscle is less dense with a lower atomic number, and the lung is just air with a bit of tissue so very low attenuation.

Answer 321

- radiation directed through the patient - transmission map collected is essentially an attenuation map - good at showing structure, especially between tissues of different densities and atomic number

Answer 322

- the radiation is administered to a patient in the form of a tracer - emitted radiation is detected outside the patient patient is injected with a radio-pharmaceutical, putting the radiation inside the patient, and then detect the radiation/gamma rays which are emitted FROM the patient and then looking at the distribution of that emission.

Answer 323

X-ray tube is within a vacuum. We have a positively charged target. The target and the filament are separated by a potential difference. Heating the circuit controls the voltage. We have control over the amount of x-rays produced and their energy. the current controls the amount of electrons, and so, therefore, the x-rays produced. Control of the energy of the x-rays produced is done by varying the voltage across - it accelerates the electrons so that when they interact with the positive charged target, the interaction produces x-rays.

Answer 324

FILM HARDCOPY: film processor with tanks of chemicals, high resolution COMPUTED RADIOLOGY COMPUTER SCANNER: phosphor plate, special laser scanner of CR reader that reads and digitises the image, digital enhancement and archiving DIGITAL RADIOLOGY: flat panel detector, fully digitised system

Answer 325

Used to reduce the breast thickness, improves resolution and lowers radiation dose.

Answer 326

- real time imaging - a catheter is fed inside an artery and radio opaque dye is injected - show blood flow inside vessels and can be used to assist with interventions - interventional - want to see if the surgeon is guiding it through the right place

Answer 327

- real time imaging using an image intensifier called fluoroscopy - a cardiac catheter is fed inside the aorta - radio-opaque contrast agent used to identify areas of occlusion - treatment may be either balloon angioplasty or insertion of a stent

Answer 328

- cannot distinguish between overlying tissues - tissues other than those being observed reduce the contrast in the image - historically partially solved by moving the film cassette and x-ray relative to the patient to blur out overlying tissues, called 'tomography' - superseded by Compound Axial Tomography, now abbreviated to CT.

Answer 329

the patient is on a bed and is moved into the scanner. The scanner has an x-ray tube and a detector which rotates round the patient the bed moves out.

Answer 330

- AKA spiral scanning - introduced in the late 1980s - continuous rotation - continuous table feed - the patient moves through as the scanner rotates

Answer 331

- multislice - faster scan - more coverage each rotation - modern variations operate in helical MSCT - multiple slice CT - can cover more of a patient per rotation of the CT tube so much faster scanning technique

Answer 332

- measurement of the size of the left inguinal lymph nose shows progression of disease - imaging is used for monitoring response to therapy

Answer 333

- external beam radiotherapy irradiates normal tissue as well as tumour - multiple beams are used to spare normal tissue - CT is used to define the area to be treated and the direction of the radiotherapy beams that are used

Answer 334

- indirect radioactive tracer, patient is emitting the gamma rays - time amounts of tracer (nmol) (x-ray is 10's of g!) - image depends on the metabolism of the tracer: FUNCTIONAL IMAGING We are making an image of the distribution of a radioactive tracer. Nuclear medicine only shows function. It may reflect anatomy but without metabolism, the tracer will not be taken up. Nuclear medicine is a functional modality.

Answer 335

- uses single photon emitting radio nucleus - can operate in 2D (planar) and 3D (SPECT) - SPECT: single programme emission computer tomogram Gamma cameras have imaging 'heads'. They re used for radionuclides that decay with direct emission of gamma rays - the most common radionuclide is Tc-99m (half life is 6 hours)

Answer 336

- positron emission tomography - uses positron emitting radio nucleus - always 3D - a ring of scintillation detectors supported in a fixed gantry - operated in 'coincidence mode' - only photons emitted from an annihilation event are recorded

Answer 337

half life is the time taken for the radioactivity to reduce to 50%

Answer 338

- cameras positioned above the patient - Te99m DTPA injected IV - camera positioned able the patient - gamma camera records gamma rays and collects image over time - functional time-activity curves are obtained

Answer 339

DaTSCAN --- I-123 FP-CIT --- Ioflupane - Binds to the dopamine transporters (DAT), on the neutrons - Parkinson's disease has reduced uptake in the Putamen differentiates from Essential Tremor

Answer 340

- both positron and electron are annihilated | - 2 gamma rays are created at 180 degrees to one another

Answer 341

FDG is a glucose analogue which enters cells in the same way as glucose. There is good reflection of the distribution of glucose uptake and phosphorylation by cells in the body Glucose --(phosphorylation)--> glucose-6P --> glycolysis FDG --(phosphorylation)--> FDG-6P --X--> metabolically trapped

Answer 342

- FDG reflects metabolic activity - alzheimer's disease (hypo metabolism, mostly in temporal and parietal regions) - Pick's disease (fronto-temporal hypometabolism)

Answer 343

- PET-CT - SPECT-CT (used to localise uptake) - PET-MR

Answer 344

- relates velocity to [substrate] - Km = substrate concentration at which 50% maximal velocity is reached We can measure the velocity of an enzyme and plot it against the concentration of the substrate. Vmax is when the enzyme is working at full rate, achieved when you have an infinite amount of substrate. A very high Km means the enzyme doesn't bind the substrate very strongly. We can use this information to set up our assay, we want to be close to the Vmax and so we can approximate how much substrate is needed. We would possibly use around 10xKm because we can't put in an infinite amount. Measuring the production of the product is another way of measuring the velocity of the reaction. Take the gradient of this to get the velocity of the enzyme reaction. If you have a very valuable substrate and can't put in 10xKm then it might be depleted over time and you may get product inhibition. If there are some impurities in the reaction then it takes a long time to get ready and then goes into a linear phase before the reaction tails off.

Answer 345

Temperature: Q10: relative reaction rates at 2 temperatures differing by 10*C Optimum pH: can vary from 1.5-10 Precise substrate concentration and ionic strength of buffers. We have to optimise the temp as if its too low the reaction will be too slow, and it it is too high then it may denature the enzymes (~40*C) and it will become labile. If you increase the temp by a factor of 10 you get approximately doubling of the rate - this is Q10. Ionic strength of suffers is also important - some require a certain amount of salt in the buffer for them to work.

Answer 346

INTERNATIONAL UNIT (IU): one unit catalyses conversion of 1micromol of substrate in 1 min (temp not defined) KATAL: one katal converts 1 mol substrate/second (1kat = 60mol/min = 6x10^7 IU. 1 IU = 1micromol/min - 16.67 nkat) - can standardise them in some places and constitutions - under defined conditions - katal is used in industry and is on a much larger scale - normally just use the international unit (IU)

Answer 347

- enzyme purity and nature - in a lab we want a very pure enzyme - enzyme stability - under assay conditions - quality of substrates, solvents and buffers - high quality, uncontaminated - assay mixture - make up everything together, only thing to do is add the enzyme to start the reaction - mixing to start the reaction ASSAY: measure the activity of that enzyme under particular conditions

Answer 348

ABSORBANCE: one component may absorb UV lots, and this may decrease as it changes to product FLUORESCENCE: of the substrate or the product OPTICAL ROTATION: when we convert the substrate to the product with a different chiral form, it will change the rotation of polarised light CONDUCTIVITY: release of proton/consumed proton VISCOSITY: polymer into monomer - change viscosity RADIOCHEMICAL: when impossible to use any of the above, measure formation of product by measuring the radioactivity. Need separation to separate the substrate and the product to do this.

Answer 349

- if substrate or product absorbs light, reaction can be followed by the change in absorbance --> A - log10 I0/I - I0 is intensity of incident light and I is that of the transmitted light - Absorbance is related to concentration of light absorbing substance (Beer-Lambert law: A = eco) Relationship between [substrate] and absorbance/ Proportional to path length that the light has to go through. If you double path length, absorbance will decrease by 2x. It is dependent on the intrinsic absorbance of the molecule you are following. We can plot for an enzyme reaction the change in absorbance and can work back from this to the concentration of the substrate.

Answer 350

- standardisation of assays - quality control (must know the expected variation of the control samples - coefficient of variation (CV)) standard deviation (SD) of control values/mean of the control values x 100 (%)

Answer 351

there is protocol for this. to enable yo to standardise the assay you need to put tubes in with known amount of enzyme activity. every so often these come up and throughout the day you will measure the activity of the enzyme in these controls. you will know if the machine is working properly throughout the day because of this. Ways of knowing if it is to isn't by looking at the standard deviation of the control values and monitoring that throughout the day.

Answer 352

Takes lactate and NADH and converts the lactate to NAD. NADH is highly absorbent and we can follow the reaction by watching the absorbance of NADH fall. NADH is more stable than NAD - we can follow this continuously by looking at the decree in absorbance of NADH. The forward reaction from pyruvate is favoured because: a. equilibrium to the right b. maximal velocity higher to right c. NADH more stable than NAD Lactate dehydrogenase is released by blood cells in anaemia.

Answer 353

Alanine is produced largely in the liver. In liver dysfunction the level of ALT increases massively se we get a large increase in activity. ALT switches over the amino and kept groups in L-alanine and 2-oxoglutarate. We can use the previous reaction in an assay. There is another assay as liver enzymes using aspartate which makes oxaloacetate which is a substrate for malatedehydrogenase.

Answer 354

4-nitrophenol phosphate (colourless) --> 4-nitrophenol (yellow) + phosphate (colourless) We can set up a reaction to measure the formation of the product directly without enzymes first. We can remove the phosphate from the substrate, which is done at very high pH (10.3). We can set up there reaction to use a particular phosphate which is colourless (4-nitrophenol phosphate), this is a synthetic molecule which is not found in the body. When alkaline phosphatase works on it we get 4-nitrophenol + phosphate. 4-nitrophenol is yellow and by monitoring how much of this we have, we can work out how much enzyme we have.

Answer 355

Liver dysfunction, especially bile duct problems, cause increased levels of alkaline phosphate. There is also a bone form which is released in bad conditions. Bone and liver alkaline phosphatase are slightly different in structure as they have different sugars on them and they are different isoforms. they can be detected immunologically. We can run a gel and see which is increased in its presence in the blood, which is a very useful assay.

Answer 356

We want to measure glucose in diabetics for example. One of the things we can do is use the reaction of hexokinase with ATP that will make G6P and ADP. We can use absorbance to measure any of these. G6P is the substrate for G6PHD, which converts NAD to NADH. This can be used to measure the amount of starting glucose and the final NADH level. This reaction is completely specific for G6P, meaning it doesn't measure anything else in the blood so this is an advantage. Glucoseoxidase is specific to glucose.

Answer 357

Amylase*: acute pancreatitis Alkaline phosphatase*: liver, bone Alanine aminotransferase: liver disease Aspartate aminotransferase: liver infarction Gamma glutarmyltranspeptidase: hepatobiliary disease Lactate dehydrogenase*: haemolysis, lymphoma Creatine kinase*: skeletal disorders * = isoforms - separating of bone and liver ALP useful

Answer 358

- correlation - regression - chi squared test

Answer 359

Measures whether 2 variables that you have measured vary together i.e. does X tend to go up as Y goes up? ``` R = 0 --> no correlation R = 1 --> perfect correlation R = -1 --> perfect negative correlation, one variable goes up as the other goes down ``` (some statisticians use R^2 instead of R) You also need a p value to say if the correlation is statistically significant. The p value is a measure of whether yo results look like they could be purely random data.

Answer 360

- the two variables are independent of each other - both variables are normally distributed; but the test is robust, in practice shape of distribution is not crucial, but others can cause an artifactual positive correlation - the relationship between the variables is linear

Answer 361

Correlation is used when both variables are measurements, which may have random errors or random variation. These usually result from observational studies. Regression is used for experimental studies where we have set one variable in advance e.g. time or dose of drug

Answer 362

Linear regression estimates the best straight line through the data. It returns a value for the intercept (a) and slope (b) for the line y = a + bx If you know biologically that y must be 0 when x=0, then you can ask for the best line that that goes through the origin (intercept = 0). While regression estimates the best straight line through the data, it is just an estimate as our data is subject to random errors.

Answer 363

sometimes the data from a study are counts for the number of individual events falling into a particular category. There may appear to be an association between two factors but this could just be random variation. If the data can be presented as a 2xn table, then you can test for differences using the chi squared test.

Answer 364

Split brain patients have undergone surgery to cut the corpus callous, the main brain bundle of neuronal fibres connecting the two sides of the brain. Input from the left visual field is processed by the right hemisphere and vice versa. A word is flashed briefly to the right field of view and patient asked what they saw. Left hemisphere is dominant for verbal processing so patients answer matches the word. When a word is flashed to the left field of view and the patient is asked what he saw, as the right hemisphere can't communicate with the left, the patient is unable to say what he saw, but he can draw it. The corpus callous can be cut to reduce the spread of epilepsy. These patients helped prove hemispheric lateralisation of function.

Answer 365

EEG is the recording of the brains electrical activity through electrodes on the scalp. P300 wave related to cognitive function.

Answer 366

Electromagnetic induction induces weak electric currents - can cause activity in specific or general parts of the brain - repetitive transcranial magnetic stimulation (rTMS), is being tested as a treatment for various disorders including migraines, strokes, Parkinson's disease and strokes.

Answer 367

the model has a sound theoretical rationale (neurobiological or psychological mechanisms, aetiology), the way humans get the disease is similar to how animals do.

Answer 368

phenomenological similarity between the model and the disorders being modelled (symptoms)

Answer 369

manipulations known to influence the pathological state should have similar effects in the model (drugs work the same in both)

Answer 370

- global ischaemia: 2 or 4 vessel occlusion - focal ischaemia: middle cerebral artery (MCA) occlusion - haemorrhagic: inject collagenase into rat brain - causes bleeding

Answer 371

Morris water maze - test of memory where rodent uses visual cues to try and find the underwater platform

Answer 372

Loss of dopamine neutrons in the substantia nigra

Answer 373

6-OHDA: toxin kills dopamine (and NA) neurones but no extrastriatal loss and no Lewy bodies. MPTP: toxin kills dopamine cells (see MPP+) Rotenone: agrochemical but neurotoxic MPP+ from MPTP: inhibits mitochondria LPS: lipopolysaccharide which induces inflammation

Answer 374

5+ of these symptoms need to be present in the same 2 week period (nearly every day/all day) and represent a change from previous functioning... 1. depressed mood 2. markedly diminished interest or pleasure in (almost) all activities 3. significant weight loss when not dieting or weight gain or decrease in appetite 4. insomnia or hypersomnia nearly every day 5. psychomotor agitation or retardation 6. fatigue or loss of energy 7. feelings of worthlessness or excessive guilt (inappropriate) 8. diminished ability to think or concentrate, or indecisiveness 9. recurrent thoughts of death, suicidal ideation without a specific plan, or a suicide attempt or a specific plan for committing suicide

Answer 375

- chronic mild stress (different small stressors most days for ~6 weeks) - learned helplessness - social defeat - maternal separation - olfactory bulbectomy - disruption of pair bonds

Answer 376

Rat learns that it cannot escape form an electric shock and gives up trying. Also demonstrated in dogs - in the end they just lay down and whine.

Answer 377

smaller rat bullied by a bigger rat chronic stress that occurs in the presence of persistent loss in aggressive social encounters especially in the absence of social support

Answer 378

Take young pups away from mother very early. We get an increase in ultrasonic vocalisation (USV) of some frequencies and on return of pups to mothers we get an increase in USV at 50Hz (happy sound). There is some debate about what each USV frequency might mean.

Answer 379

destroy the olfactory bulb. Whiskers and smell are very important to rats - a lot of brain is used in processing the information from its snout and whiskers. A rat that has has its whiskers or olfactory bulb removed might be 'depressed'

Answer 380

separate male and female gerbil partners oxytocin? mongolian gerbils mate for life so if you separate them, they become 'depressed'

Answer 381

- sucrose choice test (good model) - forced swim test - tail suspension test

Answer 382

'Depressed' animals may seem to choose water over sucrose when offered the choice. Chronic mild stress reduces sucrose intake, wuline (chinese herbal medicine) appears to be antidepressant as it normalises sucrose intake. Most clinically used antidepressants would normalise sucrose intake.

Answer 383

Measure swimming and escape behaviour (climbing). Depressed rats are less active in both respects. antidepressants (fluoxetine and desipramine) caused increased swimming and escape behaviour

Answer 384

Mouse/rat struggles to escape and eventually gives up - measure latency to giving up. Antidepressants increase this latency.

Answer 385

- elevated plus maze - social interaction test - black/white box - object burying

Answer 386

rodents prefer close arms to open arms. placed in centre and number of entries and amount of time spend in the open and closed arms is recorded. Anxiolytic drugs increase time in open arms.

Answer 387

used to examine social interactions between 2 rats or mice in a novel environment. 2 rodents that don't know each other will sped some time interacting, anxiolytic drugs increase this time.

Answer 388

rodents prefer dark to light spaces - a rat or mouse is places in the dark part of the box. Measure: 1. latency to leave the dark box 2. time spent in light box 3. number of crossings between the boxes anxiolytic drugs may increase time spent in the light box

Answer 389

introducing a new object of aversion to a familiar cage causes anxiety in rats and mice, stimulating them to engage in burying behaviour. Anxiolytic drugs reduce this behaviour.

Answer 390

- use a conditioning paradigm - associated bell with food - if we use the bell many times beforehand, it takes longer to condition the animal/person - not the case with schizophrenics - they learn the conditioning (e.g. the bell) very quickly

Answer 391

1. recurrent use resulting in a failure to fulfil major obligations at work, school or home 2. legal problems resulting form recurrent use 3. continued use despite significant social or interpersonal problems caused by the substance use 4. recurrent use in situations which are physically hazardous (e.g. driving while intoxicated)

Answer 392

1. substance abuse, PLUS... 2. increase in tolerance 3. withdrawal symptoms 4. compulsion to use the drug

Answer 393

if you give a rat cocaine always in the white compartment and placebo (saline) in the black compartment, the rat will always choose to spend relatively more time in the white compartment as it associates this with feeling good

Answer 394

decreased social interaction 2 rats initially spend ~70s of 5 mins interacting. if one rat is given cocaine (1mg/kg) then it spends more time interacting (~120s on day 7 and 140s on day 14). if the rats cocaine is stopped at day 14, on the following day it will only spend ~10s interacting - this is a withdrawal symptom. Some drugs (e.g. ondansetron) can reduce these withdrawal effects

Answer 395

on 1st occasion a rat is injected with cocaine is moves around a little. if it is given cocaine every day for a week then given the same dose it initially got, it moves around more. after about 2 weeks of cocaine it moves around even more - this is locomotor sensitisation

Answer 396

the rat has an IV line in its jugular vein. it can press a bar for cocaine through the IV line. at its preferred dose it will self administer cocaine all day and eventually overdose if allowed - rats like cocaine and will work hard for it

Answer 397

schizophrenics have problems with this test. the subject has to determine a rule (e.g. must attend to colour/shape/number of symbols on the card). a related test has been developed for rats, not involving cards. this is a test for cognitive function in humans.

Answer 398

digging task for cognitive function: attentional set-shifting the rat must learn rules e.g. food is in the cumin disk - only allowed to dig in one pot for food in some cases PCP (phenylcycxlindine) leads to long term cognitive dysfunction and PSP treated rats can take longer to learn the rule changes i.e. they have cognitive deficits

Answer 399

1. anaesthetise animal 2. place animals head in stereotaxic frame 3. drill hole into skull 4. insert guide cannula (sometimes) 5. insert probe 6. do experiment then kill (overdose) or allow animal to recover

Answer 400

ADVANTAGES: - animal feels no pain, less stress? - able to better control the experiment DISADVANTAGES: - some anaesthetics influence transmitter release - cannot correlate transmitters with behaviour

Answer 401

increased dopamine levels found during sexual encounters

Answer 402

with 5-HT rather than glucose there is an increase in exercise from 90 minutes to 270 minutes (in male rats)

Answer 403

- brain slices - organotypic brain slices - prisms - cell cultures - synaptosomes

Answer 404

- kills rodent humanely (anaesthetised or cervical dislocation) - open skull - take out brain - cut out region of interest - slice section of brain of interest with 'vibrate' - typically 300-400 micrometers thick - vibrate moves blade forward slowing with sawing action

Answer 405

A typical brain slice chamber can be kept alive for hours with heated (e.g. 32*C) oxygenated artificial cerebrospinal fluid (ACSF, contains salts and glucose). In this case a water bath heats the ACSF

Answer 406

Same as for brain slices except... - take thinner slices (e.g. 200 micrometers) - take tissue from very young rodents (e.g. PND7) - use antiseptic technique (sterilise) - culture slice in 'incubator' (long-term experiment) - use an air-liquid interface - use -culture medium' (e.g. horse serum and antibiotic) not ACSF (just salts and glucose) - can use 'co-cultures' e.g. a piece of cortex cultured with a piece of accumbent - will they make appropriate connections? - NC3Rs - reduce animal use in research --> reduction, refinement, replacement of vertebrates Recent data suggests that one can use older rodents (e.g. 40 days old). Normally we see a loss of cells and structure after culture. Here, and antisense, which blocks gap junction formation, promotes slice health after 14 days in vitro.

Answer 407

A rush block of brain, not used much anymore - record from thin site where tissue is viable - it gets more oxygen and salts than deeper tissue and so it is healthier.

Answer 408

- neuronal cell lines (from frozen) - dissociated tissue from neonate (e.g. PNd7) - other cell lines with neuronal characteristics and use nerve growth factors to induce a neuronal phenotype (e.g. PC12 cells) We can record 'quantal' dopamine release from adrenal chromatin cells (quantal release: release from a single vesicle)

Answer 409

neuronal phenotype in PC12 cells - increased dopamine transporters and 'dendrite growth'

Answer 410

the functional expression of dopamine receptors in cells (Missale et al)

Answer 411

the brain sample is homogenised then spun in a centrifuge at different forces (g) until the sample has a band of ''synaptosomes'' which are basically just synapses but contain many receptors, uptake sites and enzymes which are involved in transmitter synthesis, release and metabolism.

Answer 412

- the different afferent and efferent connections are intact - can drugs cross the BBB? - subjected to metabolism in the gut and liver

Answer 413

- no information on systemic metabolism but this cn be useful if one only wants to determine the exact pharmacological mechanisms of a drug - can measure NTs with better spatial accuracy - one can see the brain nuclei down the microscope, in vivo one must use a stereotaxic frame - only have some afferent and efferent connections

Answer 414

- no afferent or efferent connections - limited information on drug metabolism - but a much simpler system to get simple mechanistic answers e.g. on QUANTAL release from single or few transmitter vesicles

Answer 415

1. radio-labeling 2. HPLC 3. push-pull cannula 4. microdialysis 5. voltammetry 6. aperometry

Answer 416

- incubate brain slice in tritiated dopamine [3H]DA - dopamine neurones take up radioactive dopamine through DAT - when dopamine is released one can measure the amount of dopamine which is proportional to the radioactivity - not used so much anymore

Answer 417

sample is injected into HPLC column, which separates the transmitters and metabolites based on their size and charge. in this example, 2 dopamine metabolites DOPAC and HVA come out of the column first, then dopamine and finally a metabolite of 5-HT - 5HIAA Typically, areas under the curve are measured and compared with standards i.e. known quantities of transmitter

Answer 418

not used much any more. buffer is pushed down the cannula, which is in the brain, then drawn back up - test to see what transmitters are picked up. micro dialysis has superseded this technique.

Answer 419

The micro dialysis probe is implanted in the brain under general anaesthetic some days prior to experimentation (can be used in humans). The tip is the only part exposed to the brain and transmitters cross the semi-permeable membrane by diffusion down their concentration gradient. Note you can also add drugs to the brain by having them in your buffer.

Answer 420

- uses small carbon fibre electrodes inserted directly into the brain - electrode oxidises the transmitter and the transfer of electrons is measured as a small current - oxidation varies with the potential of the electrode

Answer 421

ADVANTAGES: - near to real time (e.g. 10 samples/second) - great spacial resolution (8x30 micrometer tips) DISADVANTAGES: - currently limited to only a few compounds: dopamine, noradrenaline and serotonin and some of their metabolites, NO etc - they must oxidise at low voltages

Answer 422

a glass electrode filled with a single CF is pulled (heated and pulled). The carbon tip is trimmed to about 20 micrometers. Apply a triangular voltage waveform (-1 to +1.4 V) to the CFE and record the current flowing through it. At about 600mV dopamine oxidises (more current) and at about -200mM dopamine reduces (less current). V = IR, current doesnt follow voltage because of capacitive properties of carbon. A sample takes 20ms

Answer 423

- in brain slice - in brain slices can measure anoxia dopamine release in caudate - in anaesthetised rats

Answer 424

Voltammetry is fast enough to correlate transmitter release with behaviour on a sub-second time scale - can look at many different behaviours, and behaviours that only last a second, not just sleep, locomotive etc

Answer 425

This is similar to voltammeter except that the voltage is not changed, i.e. a constant voltage is applied to the carbon electrode (for dopamine = 600mV). Thus, we do not get any information about oxidation or reduction peaks. The main advantage is that sampling is essentially continuous, i.e. 'real-time'.

Answer 426

- test in simple system (e.g. synaptosomes, cell culture) - go on to more complex system (e.g. brain slices) - test in whole animals: toxicity data, effect in animal models - test in humans

Answer 427

- shrunken brain - ventricles are swollen - thickened and sunken sulci - thinner grey matter ribbon

Answer 428

We now know that the most common gene mutation is on chromosome 21 and it is APP. This protein can have a little bit cut off of 40 aa. This fragment can form Abeta which forms the plaques.

Answer 429

Presenilin 1 on chr 14 and presenilin 2 on chr 2 Presenilins are mutations associated with familial, early onset AD. It is quite rare and occurs before the age of 30.

Answer 430

There is a swedish/london/netherands family and all have familial AD. They each have a slightly different mutation and as it is rare they are referred to as for example the Swedish family. Swedish family mutation: double mutation at aa670-671 in APP. These families are sometimes mentioned within the same breath as Down's syndrome. The Down's trisomy is an extra version of chromosome 21 and they have an extra copy of APP. It is possible that Down's Syndrome is a kind of mirror of AD.

Answer 431

- in recent years deaths from many other disease have decreased - however deaths from Ad have increased - this may be because people are living longer so more people are ending up getting it - causing a massive drain on healthcare budgets and time and is a sad way to live

Answer 432

as we get older pathology accumulates in our brains and can take us to a recognised state of dementia. We can't be diagnosed as having dementia until a symptomatic threshold is reached. This may have been preceded by a preclinical phase we don't know about... the brain may have absorbed some of this pathology - loses some neurones and can still cope.

Answer 433

If we could delay AD onset by even two years this would be very good. Therapy and a 'cure' may not be about stopping it, but slowing it and reducing risk. Once you have full dementia there is nothing that can be done - there is no reversal and lost neurones can't be replaced.

Answer 434

Cognitive scales (MMSE, CAMCOG, MOCA) allow you to assess the patient. This is useful as even if a patient is very good to begin with and is then average after time, this still shows a decline.

Answer 435

The patient is given a list of words to remember and after two minutes they are tested to see how many they remember. This is a good method but is hard to replicate reliably over different countries because the words have to be different in different languages so this makes it less fair.

Answer 436

extracellular deposits of Abeta fibrils also in downs syndrome teenagers

Answer 437

Beta is cleaved from APPby secretase enzymes: alpha, beta and gamma. - beta secretase = BACE - gamma secretase - presenilins are essential cofactors and produce Abeta42 and Abeta40 The three secretase enzymes have an intracellular C terminus and an N terminus that sticks out.

Answer 438

In healthy patients alpha-secretase cleaves APP forming fragments and no harm is done. If it gets excessively chopped by beta secretase and then gamma secretase, we end up with tiny fragments which form plaques from the Abeta - they can be 40 or 42 aa in length.

Answer 439

- used wth Abeta in diagnosis - small molecules have been developed and are given to the patient - they pass through the BBB and bind selectively to amyloid plaques - Abeta is everywhere in AD patients and there is loads of binding - this is a way of determining amyloid load in live patients - would be great if we had a way of getting rid of the amyloid but we don't know how at the minute

Answer 440

- Tau is part of the neuronal cytoskeleton - essential and is a type of scaffolding for neurones - it is bad if it is HYPERPHOSPHORYLATED (some phosphorylation is normal) - this makes the filamentous tau protein crinkle into a tangle - fragments inside neurones and cerebral cortex Hyperphosphorylated tau looks like little balls/clumped, when it should look like neat parallel railway tracks.

Answer 441

- filaments are in nerve cell process - wants to keep it intact and going where it is meant to via connections - if filaments become defected and tangle, the axon becomes pathological and may die - neutron may live but the circuit won't work SOME PHOSPHORYLATION IS NORMAL - BUT TOO MUCH IS NOT

Answer 442

We don't really know how plaques and tangles come and how they contribute. There is debate as to whether tau or amyloid is more important... - nerve cells grown in petri dish from mice - grown in culture and treated with Abeta - 4 days later many of the axons are gone and those remaining are regressing and fragmented - this was done again wth mice genetically altered to have the tau protein gene removed - the same process is done with its nerve cells - they are resistant to Abeta and are unaffected The logical conclusion of this is that tau is needed for Abeta to have an effect. Abeta comes first, causing the defective tau and tagging and therefore the neutron dies

Answer 443

Amyloid not only found in plaques - can be found around blood vessels also. Abeta40 is found clustered around the vessel and Abeta42 forms a messy corona around that - they are very distinct. In someone who has this, they tend to have both 40 and 42. 40 is a feature of the vascular type and 42 is labelled a a plaque. Can have vascular amyloid as well as plaque based. Vascular is also damaging but less so than plaques.

Answer 444

CHOLINESTERASE INHIBITORS: - Donezepil - Rivastigmine - Galantamine Memantine - NMDA receptor antagonist ...and Immunotherapy

Answer 445

The cholinergic system is involved in memory and Ach is a major contributor in these circuits. Many people with mild cognitive impairment (MCI) see a benefit from the cholinesterase inhibitors but not all.

Answer 446

We know that Abeta is a protein and that it is what causes the problem. Therefore, if we can make an antibody then it will find the Abeta in the plaques and stop them from forming and could even get rid of them.

Answer 447

- vaccine AN-1792 - active immunotherapy (n=372) - full length Abeta 1-42 peptide and adjuvant - 15 AD patients: aseptic meningoencephalitis - terminated in jan 2002 after negative results and a death As the remaining 14 patients died over time their brains were examined. It was seen that in 3 of the patients all the plaques had completely gone. However, these patients still had a score of 0 on MMSE (highest 31 and is healthy) and so this showed that Abeta does its damage very early on and so we think that it is too late in severe dementia to do any help. If we looked for tau in these brains it would be abundant and neuronal cell death would be prevalent. Basically, the vaccine worked - but it didn't. It is too late when the AD has become symptomatic.

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