Cafe Scientific, Southampton, UK, past talks

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Some summaries etc of past talks held at the venue, St Denys Community Centre
Some hosts are not alowing remote linking now , so to view a "forbidden" picture you have to right click on the mouse and select "view". Not verbatim, and there will be homonyms, transcription, transliteration, typing and spelling errors and misattributions in the following write-ups, and also untranscribed potential litigious stuff that sometimes emerges. Q&A , grouped under one "Q" or ? terminator tend to be dialogue with / multiple questions from one enquirer. ? for unheard / masked words , ??? for phrases.

Tuesday 15 May 2018 Bob Stansbridge , Soton Uni: Micro-Electro-Mechanical Systems (MEMS); what they are, what they do and why they are jolly useful. 1.5 hours I lecture in instrumentation to mechanical engineers, aero and ships engineers. When I started at Soton Uni decades ago, I was in the highest tower the Faraday building. I was involved with 2 people working at the top of the tower, manufacturing integrated circuits, sub-micron lithography, geometries on silicon less than 1 micron wide. A big advance at the time , but the tower was too wobbly and they had to move the plant out. Every time the lift moved, they could tell . They were working on making shapes out of Si , using the same IC manufacturing techniques , etching and depositing materials, removing or building. One was making a cantilever, like a bracket hanging off a wall. So they cut out a shape and then eroded underneath. You could see the little structure on an electron microscpe, but said its a pity there is no use for it. But there was a use for it, accelerometers. A visual aid of a happer on a string, the hammer is the proof mass and the bit of string is the meter. Move it slowly horizontally and the string stays vertical, but move it quickly , then because of inertia the mass stays back, and then catches up. Soif i could measure the angle , then a measure of the acceleration. The bigger the angle , the more acceleration. Put a mass on a cantilever and if you've a way to measure the bend , then a measure of accelertion. Not much use for them , then. A major use was crash-test dummies. Newton's second law F=m*a. So a dummy of known mass, given a known deceleration and measure the forces. The old style acceleraometers were a mass set between 2 piezo-electric materials, in a can 12 x 12 mm or so. One side is squeezed by the mass , when accelerated. Squeezing out a few elecrtons a few microcolombs. They cost about 250 pounds then and involved a special charge amplifier , that cost about 350. Very few were made , as only specialized use, so very exp[ensicve. They were highly specified, each with its own calibration #curve , frequency and temp response etc, all traceable back to the NPL. So 600 pound to measure acceleration on 1 axis. The were exploring a cheaper way of doing this. Another use was to take one of those cans and stick on on the side of an engine or particularly a gearbox. When running it would shake and you can measure the vibration of the casing, condition monitoring. A frequency spectrum wiht picks corresponding to cogs rotating at different rates with different gear ratios. Something big likand expensive e a turbine can have its condition continually monitored likke that. A chiped gear , or a lack of oil will show up in the vibration, knowing the construction of the gearbox. They can then do preventative maintainence. The first use of the MEMS version was in air-bags. They got yhe price down from 600 pound per channel down to 30 pound. Also mounted in top end cars, not requiring high spec, just detetecting hitting a wall say. With this extra use, the price droppped to about 8GBP. Now they were that cheap , they could go in games consoles, the wii. They are very small, and now in gaming machines , the price dropped again. They went in mobile phones and the price dropped again. So now you all cary one around. From the silicon, a central mass remains after etching around and under it, leaving 4 corner supports, hanging in space. There is air inside, that acts to damp. A visual aid , large MEM accelerometer. A shallow wood box , a central plate supported by 4 springs to the corners of the tray. A pointer fixed to the central plate and a scale on the left and right. Move quickly and the mass and pointer moves relatively to the tray. So how much force involved with moving those various amounts. Turn it throuhg right angle and gravity is acting as a force and the pointer moves to +1, turn upside down and it moves to -1 , a change of 2g. From that I can calibrate the rest of the scales, quite a simple calibration procedure. So how to measure the tiny movement electrically. Less than 3pF capacitance, and the change is about 1 part in 1000. No chance using a capacitanc emeter. Instead of 1 finger, produce a comb of fingers , 100 times the capacitance. Dimensions of the electrode combs about 30 microns. We cannot measure the movement externally , it must be done internally. The combs are about 100 micron long, 3micron deep and 1micron between fingers. The central mass weighs 1 microgram, so a millionth of a spoonful of sugar. They put a simple oscillator in there, gives pulses, 2 sets of pulses , one upside down relative to the other, anti-phase. Because the fingers are so close, the effect of trhe signal can jump across. The fingers move closer to the A electrodes, so picks up more of the A signal, amplified gives +5,+5.... +5. If its in the middle , its about the same for both sources, roughly midle when amplified. Go the other way and B signal is transfered mainly, giving 0V when amplified, just for the few moments when accelerating. This amp and demodulation circuitry and some comms circuit is all inside, nothing outside except some power source. Why didn't they measure the resistance of those very fine corner supports? The advantage this way is its differential and very linear. Q: what is the frequency response? Its very high as they are so small and light. With the old piezo type they would not do low f at all, a flat response except for an enormous resonant peak , they had to stay away from. Something like 100 or 200KHz, mainly limited due to the speed of demodulation. As they are silcon , then the same temperature limitations as transistors etc of about 150 deg C. For any app hotter than that then piezo or strain gauges, or capacitance. You can add extra combs around the sides, but for a long time were stuck wiht just measuring x and y, eventually they could introduce z. But as they are so small they could just place 3 orthogonal together. That is just the beginning. Moving from a to b , say just a metre, you have distance and time. If it took 1 second then the velocity is 1m/s. So keep on measuring tme and distances you can work out velocities. If you know the vel at point c was 10m/s and at d was 12m/s, then you know it accelerated 2m/s per s. You can go the other way, knowing acceleration , you know hte time those readings were made, you can determine the velocity, then with velocity you can work out how far its moved. So now with an accelerometer you can tell what forces there were, the accelerations in diffefrent directions, the velocities in different directions and how far you moved. So a navigation system. So electronic op-amp integrators , doubled up, integrate a to v and then v to distance. These days that integration process is done purely mathematically in a microprocessor. Such as this is called an IMU, inertial measurement unit, to measure x,y and z plus other things. Add a memory card to it . A video of someone with one fixed to a shoe. He walks along straight, climbs a spiral staircase and walks 10m on the upper floor. Showing the vectors of each footstep. Q: so a small stone in his shoe, changing his gait , would have shown up on that plot? Yes Another app, a version placed inside a ball , for measuring turbulent flow in water. It stores all the movements on its memory card. Its called a smart ball, footballers use it. Another app, a gyroscope. Also twisting movements , measuring the rate of change of angles. A barometer using much the same principal, or an altimeter because of change of pressure with altitude. In 2007 Albert Ferrer and Peter Groomberg awarded the Nobel prize for dicovering giant magneto-resistance. Current flows more easily in a layer aligned with the direction of magnetism With a very thin layer of metal , not a piece of wire as the wrong scale. They could do that inside a MEMS device, so they can tell which way is north. If it turns over , they can still say which way is north. With 3 of them,x,y&z, they can tell which way is north in 3D, a 3D compass. It always knows which way is north and always knows which way vis down regardless of which sense itself lies. Its called an IMU. So its a navigation unit, so on planes, space vehicles and satellites. The one with me is 3 axis accelerometer, 3 gyroscopes , 2 magnetic sensors, pressure sensor, 2 GPS receivers. Even sophisticated GPS receiver has to know which way its up. Initally these cost 800 GBP for 1 accelerometer measurement. Now you could buy a MEMS accelerometer, 2mm long, 2mm wide, .5mm thick, with 10 degrees of freedom, x,y,z , pitch,roll and yaw, 3 axix magnetic sensor and temperature, so 10 columns of numbers come out and it cost 95 pence, if you bought 1000. It had an incredibly long and detailed data sheet. It could self-calibrate. Its all microstructure , no coils . For the magnetism its like a barber's pole, the conductor in a helix. Dependent on its orientation ,makes the magnetic field spin. With those things you can involve feedback. A single gyroscope with a servo system, so turn the unit and a follower moves in sympathy elesewhere, with no observable latency. A quadcopter demo of stabilisation using an IMU, with the numbers coming off the IMU with and without stabilising. Without, a human finds it impossible to control. Another example , balancing an inverted pendulum on the top of it while flying and also a glass of water. The numbers showing angular velocities and angles in degrees, pitch roll and yaw. You see drones all over these days, because motors have been around for ages, but batteries are lighter these days, and of course the introduction of IMUs. You can use such systems under water or underground, where there is no GPS or even in a warehouse without GPS inside. There are problems with it , but a basic navigation system on board. They are ordinary motors but pulse-width modulation powering. You can use them on UAVs. These aren't precision scientific type units. The microcontrollers can be built in there also, and so cheap. About 50GBP but cloned versions are about 3GBP. Another curious use, reforesting. Send a drone up with a pack like a dart with soil and fertiliser in around each sapling. Drops it like a dart penetrating the ground. Can reforest a big area done remotely. Yesterday I heard that in Leeds doing an experiment . Over a million GBP in Leeds mending potholes. The potholes only got repaired after damage to car suspensions. They will use drones to scan the road surface for defects. On finding a defect, the drone will do a sort of 3D-printed patch over it. So they fix the pothole before its become a pothole proper, so a lot less expensive. Doing it at night avoids holding up the daytime traffic. Now the fit-bit, not just for the fit. Old people's home with a new alarm system that monitors all human movements carrying a sensor unit. If someone doesn't move then someone will investigate. Q&A Worst case of these sensors in a mobile phone, if you drop it on concrete , the screen survives , would the MEMS cantilever component survive that sort of high g direct impact? They are very robust, they can take something like 100g. The cantilever is no problem , its about 1 microgram. Its like you could drop a mouse off the Empire State building it might survive, as very little kinetic energy there. Thats not MEMS failure mode . That jet-pack man or Iron Man, I keep saaing video of, I bet he has a few MEMS in the control of that, considering 3x 200 horse-power thrusters on each arm? If he puts his right arm out , then he should turn over, but there must be stabilisation on there to bring him back up again. ? ? calibration ? You don't have to tell it where north is, it will find it itself. There is metal around and also lots of stray magnetic fields from motors etc. So they would tell it where it is , before it takes off. There are hard magnetic fields and soft magnetic fields. Hard would be from a magnetic material, and like a compass needle , would affect the MEM. With variable fields from the motors, they would calibrate it at the start. With the gyro and the magnetic field sensor , if the gyros say a test turn has turned and the compass says its turned, it has turned. If the compass says its turned but the gyros say its not turned , then its not turned. Al lthe fixed magnetic fields will still be fixed, it will see north fluctuating as it turns, and they can work out which field is the Earth magnetic component, and what the othe rfields are associated with the structure its mounted on. It is a problem and its not fool proof. Bu tthere are techniques to imrove the situation. Could they put it in a mu-metal box? They don't really have to . The sensing element is a mechanical structure, ? ?. How do you get tiny GPS receivers, on a tiny chip, surely some sort of largish antena is required? Yes there would be an external connector to an antena, beyond the chip. How GPS works , is satellites with high precision atomic clocks on board. They transmit the time and with 3 satellites minimum in view, you can get a 3D fix of position. They all transmit a code containing the time . The receiver picks them up at different times , whether near or far, then the speed of light , works out how long the radio wave took to travel. The intersection of the circles each radius being the transit time from each satellite. Nothing is perfect and with 4 or more can get a better fix. The use of MEMS with otherwise mainly GPS improves things in the areas where there are complications for GPS, in urban environments etc. Threy can correct each other. S 17

Tuesday 19 June 2018 , 20:00 to 21:30 St Denys Community Centre, Main Hall, SO17 2JZ David Johnston (Light Microscopy Facility Manager, Biomedical Imaging Unit, University of Southampton and University Hospital Southampton NHS Foundation Trust) Small is beautiful. Microscopes, sample preparation and imaging technologies have all developed rapidly in recent years, allowing us to look at biology in ways that we previously never imagined possible. Show-casing the different sorts of microscope technology available for biomedical research (using light, electrons and X-rays) and the many types of multidimensional image data that we can generate. 1.5 hours Some of us in the unit are NHS employees and some the university. We all mix and match supporting both sides. So most people have tried school type microscopes (M), and could barely see anything. They don't focus properly, smeery images. You can pick up reasonable Chinese made instruments these days for about 100 GBP and they're not bad. The ones I will be talking about tonight cost between 50,000 and 250,000 GBP. Orders of magnitude better and in terms of what they can do for us. Size and scale. Take a mm and expand it up , microns or a millionth of 1m. Simmilarly diividing a micron , to 1000 nanometres a billionth of 1m. Scale - an Airfix kit 1:72 scale, buy 72 of them , stick them end to end, be the same size as the real thing. If we represent 1 micron by 1m in the real world , starting from here we'd get to Paris. Go to a nm, and go from 1m in the real world, around the equator 25 times. We can magnify anything by as much as we want, but we get no reward going higher, we see no more detail, we same the same detail but just bigger and more blurry, we've reached the limit of resolution. So resolution relates to how small things are and how close they are and we can srtill see 2 separate objects. Different biological effects occur at different scales. The very best eye can only see down to 1/250 inch , for most people its 1/120 inch. So we go to Ms to enlarge and get better resolutuion. There is a limiit on mag of optical and to go to smaller scale , we have to go to electron Ms. The unaided eye can see down to about 250 pixels per inch. Apple retinal diplays are 326 pixels/in , you can't see it. The new Sony Xperia S5 is 806 pixels/inch, completely pointless , its a marketing tool. What limits our engineering ability to make good lenses- its the laws of physics. The human eye can see light from deep blue to far red 390 to 700nm. The limitation is the wavelength at which we observe thigs. The resolution is about half the wavelength of the red light. So things closer than 200nm , 1/5 micron, we cannot tell apart by the very best light M. To go further we have to use things with smaller wavelength. Its not our abilities to build good M that limit us. We can do some clever things with light though. 2 basic problems of observing biology with light. Most biology is composed mostly of water , we are about 67% water, about 80% for muscle and 30% for bone. So mainly transparent , so most biology is transparent. So how do we see tsomething thats transparent. The next problem is engineering, we cannot make the perfect lens. Every lens has a point at which it focuses, but it still captures info from both in front and behind that point , but out of focus. So your focused bit is always superimposed on out of focus stuff either side of it. So you can never see everything fully clearly, whether a lens in a camera, telescope or M. The human eye is a bit different has it can continously adapt focus, for different distances. To get around this, we can make our samples very thin, so the thickness is what the lens can hold in focus at any one time. Usually our samples are embedded in a wax block and then cutting very thin sections from it, that roughly correspond to the focus range ability of the lens. A piece of human tonsil about 5/1000 mm thick , originally a raw red colour but cut it down to that thin , 80% water and its effectively transparent, w ecan't see it. Counteract that by usually by staining with a cromatic dye , giving it colour to be seen by. Thousands of dyes available but all are pretty limited and crude. Purple on the nuclei , pink is the cytoplasm , we get the gross structure but not any real detail. Even the best dyestuffs, a combination of 5 stains, w ehave more colours there, but its still relatively crude as to what its labelling by the colours. Q: The dye is just saturating the tissue? All the dyes have slightly diferent specificity , one pair could be haemotoxin and diosin, H&D is the most common one, The H one tends to go into the nuclei of the cell and th eD to the connective tissue , cytoplasm . With the pentachrome, 5 dyes, one would go for elastin and collagen . They are crudely selective like that, they've been around for centuries, very good what they do but very limited. For a piece of lung tissue we've had to fix it, kill it , dehydrate it , embed in wax, cut thin sections, stick to a slide, get rid of the wax, stain it, dry off again, pedastal? on top , and then ready to look at. Can't do that to observe living material as it killed and preserved. To view living cells grown in a nutrient soup , we come back to the problem that biology is transparent. No contrast in the cells, we see nothing. Phase contrast microscopy gets around that, producing contrast in transparent material by changing differences in refractive index, how different bits of cells bend light. Using a trick in physics to do that. One of our phase-contrast M. Its built upside down ,, for technical reasons, an incubator housing around it, so we can grow cells in there and observe them ove rlong periods of time. A video of how cells in a body move around. A sheet of epithylial cells, lining cells, from the airway , grown into a confluent sheet, then we put a scratch in it. Cells don't like that space, they grow into it and when they touch from both sides they stop dividing and stop growing. A sort of model of wound-healing in a body. The trick we use is the back lens of the objective on the M , that does the magnification has an etched silver ring that blocks light. On the condenser bit of the M, that focuses the light onto the sample, there is a blocking plate with a circular slit in it. Matched and aligned with the lens etching. Nothing gets through until you put something between them , that bends the light. So quite simple in some ways , but it works very well. Another trick is to use polarised light, same as in polarised sun glasses. A polarising filter , it restricts light to light vibrating in a single plane. Place 2 such together , that are crossed, cross-polars , no light passes as what passes one is blocked by the other. Unless you put something between them that has a highly organised structure of crystal. If you use crystals, crystalised out on a slide, you get stunning pics. Some parts of biology have a suitable structure, like collagen, the protein strands line up in a precise controlled way, a sort of pseudo crystal and bends light that is polarised. A pair of images of lung tissue comparing to the polarised view which shows where the collagen sits. With high mag , we can only seea small section at a time. We can do "google-earth " with M slides. We have 2 types, one that will do up to 4 slides at a time and another that will do 100. A robot loader that picks slides out of trays, loads them in, the M has a very precisely movable stage that can take 1000s of individual pics , covering the whole area of the slide. Then use auto-stitching when asked to look at a certain part. A piece of spinal cord, comprising 2500 individual pics , then with a web-browser type set up , we can zoom in where required, jumping around. Low bandwidth required as only the bits of image interest are manipulated when required. One uses a very high quality digital camera with lens with no spherical aberration. There are line scanner ones that are much faster, that traverse the whole slide in a scan. So we make a slide from some piece of patient anatomy, with a problem that we don't know what it is. Someone on the other side of the world with expert knowledge can help you zero-in on a diagnosis of the problem, without the use of large bandwidth pipes. This is what is useful in our context. We still use the same sort of staining and slide preparation. It allows us to produce digital archives, relating to rare metabolic diseases. We can make them available asa teaching resource. Much more freely available than a physical slide in a lab somewhere. Instead of using chromatic dyes we often use flourescent dyes now. Chromatic dyes are very non-specific in their staining , but we can make FD almost any colour we want, that are highly specific. Often they are involving antibodies. You go to your doctor , for a flu jab , you will be injected with a deactivated flu virus. Your body can then produce an immune response to an assault. Antibodies, very specific proteins, have a lock and key relating to that protein and that protein only. So essentially we purify the protein we are interested in , inject into a mous eor a rabbit, it is recognised as foreign , produces an immune response to it , take a blood sample and find antibodies in there. Put them on some tissue that has that protein and the ABs will bind to it with a flourophore?, exite the flourop[hore and can see the sites. So very specific localisations. You can use multiple fluourophores , for multiple diferent targets, and a good range of colours. So image of heart muscle , with bands of contractile protein in it. Another pic is a piece of spleen where one group of immune cells have been labelled up and nothing else. Everything so far is 2D, but biology is in 3D. Q: ? Different dyes are excited by different colours, but always give off a red-shifted colour. We might excite with UV and get blue light off, or excite with blue and get green light out, or green in and red out. It depends on the dye and we can pick and choose what we want, deppendent on the requirement. To go to 3D we have to remove the out of focus part that is super-imposed on the in-focus. No trick of p[hysics required. So for focussing on the nucleus of a cell, we also get light from above it and below. But a form of m, called confocal-M , sticks a plate with a tiny hole in the light path. Then only light from one level gets focussed at one viewing level. So we can produce an op[tical section , then refocus slightly , and repeat and producea 3D dataset. Ech slice of the datasetr is just the in-focus bits. So for the nucleus image now, we so nothing of the organelles above or below it, or we can move to the organelles levels. It works with laser beams to do the excitation , different colours, bent into the M light path and 2 mirrors in the head that oscillate in a very precise and controlled way, to doa tiny scan across and down. Any flourescence wil lcome off and some will go back into the M, past the mirrors as differnt frequency of lightr, dichrome splitter, past the pinhole and on to the detector. The detection is just a light intensity meter rather than pixel array. But it knows where the surveyed spot was at any one time. So builds the pic, lightness values 1 pixel at a time, but very fast. So any ordinary M viewing parts of a worm , we can see there is different stuff , but we never see it clearly , because of the superimposition of out-of-focus stuff. Looking at the same 1mm thick sample by confocal-M , layer by layer, out of focus stuff is rejected and nice sharp images. In response to the flourescence light coming off at different colours , glows from different probes in there, the light is split by prism intio a rainbow, focused by lens systems into parallel light paths and one of the light sensitive meters sits in front of each path. Before them are 2 mirrors and the gap between them , can be opened and closed, up and down . So the gap is moved across parts of the colour range , corresponding to each flourescence colour of each dye . Anything that does not go thru the gap bounces away. Other optical set-ups in the other detector paths. so simultaneously we can detect 5 different colours, with infinite tuneability and nm precision in terms of the wavelength of those colours. It is really easy to use, just manipulating slider bars on a pc screen and the actual manipulation is done behind the scene. It allows us to look at lots of different things all at the same time, very specifically. Some new stem cells grown in culture, stained with 4 different flourescent dyes against 4 differenr cellular components. Blue is DNA stain of nuclei , a stain that is active antibody to a protein that only occurs in the nucleus of some cells, the white, then 2 antibody stains that recognise different proteins of the cyto? structural scaffolding. The filaments, one labelled green and the other red, and different cells express different amoints according to what they are doing. Some green , some red and some between of orangey yellow, that are expressing both. So by repeated digital slicing we can get multi-section 3D datasets. The head of a tiny shrimp, going through it , section by sectuion just by moving the M focus on the con-focal. We can take all those images , kind of squash into 1, and can see everything in one go. You can see individual muscle block for individual joints, in all about 2mm long. Another job, with the NOC, looking at mudstar fish, living in the deep oceans. They burrow into the mud and eat fallout from upper levels. They are interested in how they reproduce. As they are in the deep dark ocean, do they give off environmental signals. They reproduce all year round as no seasonality or do they have to have seasonal reproduction. We are looking at the ovaries of these and counting , via confocal M, the number of eggs and what stage of developement they are. Because its a 3D dataset , we're not just restriceted to looking up and down , can look in x&y, x&z etc. We can manipulate the data set to look at any angle and that is very powerful. Vey early mouse embryo cells, about 1/10mm diameter, but they've started to develop. Stained with 3 flurescent dyes, 1 for the nuclei as blue, and some as green a marker for one particular cell fate? , and red for another cell fate. 3-D veiwing glassess , red over left eye and relax eyes and can see the 3D effect of the video clip. If we have a 3D biology dataset . This is blood vessels ina brain, injected with tiny flourescent beads. Some are closer to you and some farther away. Part of a study looking at Alzeimers and neuro-degeneration and changes in brain vasculature. The tiny capilliaries of a human placenta. Another type of M is light sheet M, where excitation lighting is shot from the side, very thin but wide band of lazer light to optically flouresce. We make our samples clear , sitting in a bath of organic solvent that makes it transparent, light from th rside . It only excites F at a particular level in the sample , capture with camera at the top and move the sample . This is much more macro in scale , up to 1cm x 1cm. The whole head of a mouse, starting from scalp top . The F for this was a specific probe , for neural pathway of the brain, we can isolate just that signal which is green here. Again as 3D dataset can be manipulated any whichway. We can also do cool stuff with electrons. We are still limited by the rules of physics and how close we can get and see things apart. So for snaller dimensions than the wavelength of light , we move to electrons, that behave as waves but their wavelength can be up to 1 million times smaller. 2 forms of electron-M , scanning e-M a source of electrons at the top , a variety of electronic lenses in the vertical colum that focus the beam and scans across the sample surface. The sample is in a chamber at high vacuum. Sample fabrication is difficult, must be fixed , chemically killed , preserve it, dehydrate it in a precise way so it doesn't just collapse. Any water in the vacuum would just boil off so must be totally dry. The surface must then be coated with a very thin layer of gold or gold/palladium mix . The electron beam , as it scans across, interacts with the coating, bouncing off and be detected or hit the coat and generate more electrons, secondary e that are also detected. So we get surface views, not internal structure. The great +, is it has incrredible depth of focus , close and distant parts are held in focus at the same time. A x400 pic of a dandelion seed, and a great depth of field, that is achievable by light-M. Go closer x1200 can see individual pollen grains , about the limit of light-M. So moving to e-M, individual bacteria at x7000, and at x14000 the surfaces of individual cells, the processes are micro-vili, and long thin processes are cilia. Little motile structures . On our e-Ms we can go to about x60,000 but only of surfaces and because electron beams, always monochrome. The images you see in the media are false-coloured, to make different things stand out. A small airway in the lung, red blood cells, and cillia on the cell lining the airway, the mechanism that keeps the lungs clear of grime. Q:How is the false-colouring done? Basically photoshop, masking out different areas , so entirely manipulated . The other type is the transmission e-M, pushing e-beams thru a sample. To do this with e, we have to make the sample very thin. Start with tissue sample, chemical fix it to kill and preservr, embed in block of resin so about 1mm cubed . Trim that black down , then with often a glass knife . Take a long strip of glass , cut into squares across the diagonal, and get incredibly sharp but short lived knives. A plastic boat around that , sealed with wax and fill with water. Then a microtome, a sophistacated very thin slice "bacon-slicer" advancing minisculy onto the knife, we get very thin slices, of the order 50nm. Essentially about as this as an oil film, very delicate and very skilled handling required. That needs to be picked up on a tiny copper grid , stain wiht heavy metals , to give contrast in the e-beam. That sample goes on a rod that is entered into the e-M through an airlock, into the column. We shoot the e-beam thru the sample , with mags up to x200,000 . Goijng up ins scale, start with clls of the intestine, the absorbtiv e layer, one cell ith its nucleus , a goblet? cell producing mucus, includes gland filled with mucous. A big surface area for absorbtion, mag x2000. Up-scale a cross-section thru one cillium , much less than 1micron across and can see discrete structure, x50,000. Individual virus particles , x200,000, a sort of cross between a hypodermic syringe and a moon lander. A head with the DNA in it , then a needls and a contractile outer sheath with legs. They land on a bacteria cell , with the legs stabilising it, the contractile bit contracts to force the needle thru the bacteria wall into the cell, to pass the DNA thru. If I took a peanut and magnified it 100,000 times it would cover the centre of Southampton. Back to cillia , very complicated internal structure, individual linked tubes , hair like wafting in a controlled way. An efector stroke and a recovery stroke , lining the whole of our airway. Every time we breath in , we breathe in dust , bacteria etc. If left to accumulate in the lungs , would clog them up and cause infections. Many of our cells produce mucus, so dirt is trapped in the mucus and the cilia , wafting in syncronised fashion move this dirt al lthe way up the airway, 24/7/365 to the top of the windpipe where its swallowed and then neutralised in the stomach, for the entirety of our lives. Its a complicated structure with hundreds of different proteins involved in it. Any mutation in any one of those proteins , can effect how those cillia function and the lungs don't get cleared well. A spectrum of diseases that come under Primary Cilia Diskynesia? where scilia don't beat as they should do . Another M technique is high speed video. Scilia on the edges of a small group of cells, healthy cells from a normal healyh person. A tiny mascarra brush is pushed 6 inches up into the nose. Scrape out some cells , place in nutrient so we can view at 500 frames per second camera and high mag. The scillia are all wafting in a co-ordinated way, over the patch of cells. Q : Do the cillia rotate as well? Not the ordinary funcioning cillia . A longer cillium is called a flagellum , like the tail of a sperm . Bacteria have a flagellum that have a different struture and some of those do rotate and twist. But in higher organism its a beat and recovery stroke . An abnormal scilliun can do that or be still or be hyper-active, but normal ones are effector then recovery stroke, like a rowing action. I don't have pics of rotating ones, but an abnormal set from a patient with PCD, they are not beating efficiently, out of sync with each other , not a nice regular stroke. This is part of the dignosis of this disease. We are 1 of 3 centres in the country to do this , looking at high speed video. We go beyond that to look at e-M , as often bits of the internal structure is missing, depending on the overall genetic cause is underpinning . Outer tubule doublets in inner pairs and linking arms , which are like the motors , link tothe next and cause the bending. Some patients with a particular genetic mutation of their genome, bits of that structure are missing or disorganised . It requires very high power e-M to do such diagnosis . But sometimes these differences look perfectly normal by ordianary e-M. This is where we do e-M tomography. We take a thicker than normal section , image it in the e-beam and then tilt it , so we get views , of hundreds of cillia fro ma patient, average them all together , make a 3D model based o nthat data. An outer doublet with 2 dynime ? arms, 2 motor arms on it, fro ma normal patient , from yomography. The outer dynime arms of one is solid . In a patient where we know the cillia ar enot beating normally , look normal by normal e-M , do tomography on that patient sample . As we tilt the view we can see a hole, that is the mutation that exists below the resolution of normal x150,000 e-M. But with this extra trick , we can see what is going wrong. TEM is complicated , 3D biology , a 50nm sample of tissue, but what is going on above and below, we cannot tell. Whenwe remove that string of microtome sections , we cannot recover each slice intact, process it, register it, all in sequence is virtually impossible to do. We can get a 3D image of e-M, combining scanning and transmission e-M, called serial block face scanning M. A very high quality scanning e-M , with special sample chamber that includes a mirotome , slicing machine, with a diamonnd knife, that lasts for years when looked after. Take a sample and prepare as for normal transmission e-M , this one the sample goes black because we use osmium , combines with lipids and turns everything black. That 1mm cube , mounted on a pin near the diamond knife . The cut sample is scanned by e-M, that slice is chucked away , image again , throw away and repeat this for thousands of sections. Because we don't need to recover the section , that is fine. So allows us to do 3D biology with TeM. It allows us to look at stuff we could not look at before. We can let it run and do thousands of sections. Bone marrow cells in gel, visualiesed by serial block face. Going down cell by cell with perfect registration with all the detail, phenominal bit of kit. Q:The gel is transparent? Its completely black but transparent to electrons. Its stained with heavy metals that interact with the electrons, to give the signal. They were embedded in gel before being embeded in the resin to hold it all together. They came out of the marrow as single-cell suspension , then packed together, surrounded by gel to hold together and then fixed and processed to be embedded in the resin block, to keep it all tight. . Phenominally com[plicated datasets and no compute rcan analyse that . AI and patern recognition is not yet clever enough to look at something like that and be commanded to find a nucleus and segregate the nucleus of each of those cells, out from all else. It requires a trained biologist who knows what he's looking at to do that. It is laborious requiring step by step , each slice, drawing on the screen the things you want to segment. The initial dataset can take a week t o generate and then a year to analyse. So a whole PhD can be done on 1 or 2 datasets. Now a single cell from within bone, an osteocyte, segmented manually at multiple different levels. The nucleus, the mitachondria the energy factoies, the cytoplasm of the cell then the exciting bits are all the processes. Tiny holes in the bone, never been seen before. Not just visualising , you can do constructive maths on this the important bit of the analysis. X-rays, in terms of wavelenghth , fall between light and e-M. The resolution isn't better but allows us to do clever stuff. A regular medical Xray image, Xray source and a detector behind the point of interest, film or these days electronic sensor. 2D slicing of stuff that is high contrast, we can see the bone but not the muscles other than diffuse blob. Bone has lots of Ca that blocks Xrays. We can use a CT scanner to view patients in 3D, just a glorified moving Xray machine. Xray source and a patient and a detector and the whole lot spins round as the patient moves thru the Xray beam. By taking loads of images at different angles , we use a computer to generate the 3D appearance, Computed Tomography. In the lab we can use micro-CT, instead of movin ghte detector and the source, we rotate the sample thru 360 degrees. Even sub degree, for a dataset , then analyse to wha tthe whole lot looks like. This image I'm not allowed to show the inside of it as its the only one in hte country, made by Nycom? I think in the UK. The source and detector are designed to work with much softer samples, much lower contrast. There are micro-CT in the main campus where a whole jet engine can be scanned, very hard Xrays, to penetrate inches of steel or aluminium. Our tissue samples have very litle contast to Xrays. Its still a prototype and we're still working it up . A slice of human lung tissue , embedded in wax visualised and then #analysed to see where the blood vessels and lymphatic vessels are and segmented them out from the CT data. Then you can do maths on this segmented dataset, the important stuff. Then she modelled fluid flow of the blood vessels to work out the functioning in the lung. From the images, a mesh that can go in different computational resources and work out where the fluid flow goes in that bit of tissue. Q: some sort of doppler system to determine the flow? No its computational modellling, not measuring real flow , plugging previous sampled flow data into that dataset. It allows real measurement and do predictions as well, not just producing beautiful and complex images. We have all that range of tools in our unit , we have some of the best toys in the country, its a great job. Q: With the e-M, to get the electrons to move requires a charge, a high voltage, from the anode, what sort of voltage? Depends on the magnification, for low mag 12 to 20,000V, then # up to 200,000 volts, required toforce it through the tissue. Q: the anode is on hte other side of the sample ? There are electrons above and below . Q: The reflection M, the anode is on the sample itself? No it sits ou tto the side, but as the electrons scan across it samples the intensity of the signal doming off, millions of times, to produce the image from secondary emission a pixel at ta time. So 2 modes of operation. Often we coat the sample and get the bounced off electrons. But use a carbon coated sample , requiring earthing again as it charges up, but generating secondary electrons within it , also Xrays generated and different elements wihhin generate a different spectrum , for an elemental composition map across the sample. Q: do they do M with gamma-rays these days? Anything that has a wavelength you can functionally use, will be used by someone somewhere? Q: The Diamond light machine is something completely different to any of your machines? That uses a synchrotron, we can do a similar Xray procedure but ther's is massively hard energy and much highr resolution. Q: So you can see better with e-M, than Xray? In terms of resolution , yes unless you were doing things like difraction crystallography. Say a purified protein in crystal form , then Xray source and the scatter pattern, then deduce dimensions from that, a completely different process ot us. We can do that sort of thing on our eM. Big processing power? Our 2 main workstations for these datasets are 30core with 200G of ram, serious computer power. Often we have to go to a mainframe though. That is a limitation, the cost of RAM as its being bought up for crypto-currency mining. Q:The pressures in the chamber, of the order?, and you talked about gold coating , but the stability of a sample at such low pressures must also be key or you would get breakdown? Its pretty stable, once they've been dried in the appropriate manner and coated in gold , they will last for years. What you do find , if a complicated surface topology , like spikes, you often get discharges due to them and a flash bar on the image. Q: But generally not deep in the samples, instability at such low pressure? No because it must be porous enough, that as you take the presure down it can accomodate that, but has to be completely dry or it would boil off. The scanning e-M can operate in low vacuum mode and also in environmental mode as well , but only for samples that will not be destroyed by that, so usually high vacuum mode. For serial block face imaging , because of charge build up on the resin block , the resin is not conductive ,so impinging electrons have no natural earth path . The top slice is thrown away but charging can be a problem. So using a low vacuum mode allows the charge to dissipate through the residual air. A year or so back someone came up with a mod , which is a extremely tiny jet of nitrogen on a sling-arm, so when the knife moves back out the way , a tiny carbon fibre needle goes close to the sample and a tiny amopunt of gas to neutralise. That has revolutionised the quality of images we can get. The contrast under high vacuum is many times better. Q:With your 5-way optical splitting process , if in conjunction with an incubation chamber , presumably means cycling through temperature regimes, do you loose calibration ? Normally we would not be deliberately cycling through , we would turn the incubator on the day before, so everything is temperature stable. But yes, small changes in just room temp would change the M stand very slightly and can have an effect. Its not so bad with confocal , because of lots of ?, lots of slices at each time point and that will largely ??. With ordinary phase-contrast M, it can be mor eof a problem, room temp changes can move the sample by say 1 micron which is enough to go out of focus. So we heve ? that will rack up and down, record the images , looking at the changes in contrast and find the sweet-spot and use that one. So all kinds of add-ons. Some systems use a reference laser beam onto the glass at the bottom of the culture vessel and back down to capture the sweet spot instead. Q:For the movies of the beating scillia , what sort of M for those? Thats standard phase contrast M at x1000 , 50fps on very fast cameras. You can have high quality cameras or you can have fast ones. With such high mag , very little light is going thru, then you need an ultrasensitive camera, that is fast, so its not a cheap camera, about 10,000 GBP. You can tell all the different beat patterns from such images , fast Fourier analysis on the moving patterns on the sample . Q: what is the maximum mag you can get with light? The max useable mag is roughly x1000, x100 on the objective and x10 on the eyepiece. You can always mag more but you don't see more, jus tthe same stuff bigger and mor eblurry. Q: On such limits of viewing, in the early days of astronomy, when the optics got good enough to observe features on Mars, Hershel or whoever saw canals on Mars. Apparently that was due to night vision, the limit of resolution of the eyes and total internal reftraction in the eye or something, his brain interpreted the veins in his eyes as canals on Mars. Do you get the same sort of business with M at the limits of eye resolution , the brain seeing things that aren't actually there? Not that I've ever noticed , but you've got me worried now.;-). Normally that would not apply. Lookinvdown a M you sometimes see floaters , old red blood-cells floating across your retina a sa shadow and that is more marked down a M. Multi lens elements of an eyepiece are all coated to avoid such internal reflections. In the early days it would have been a problem,yes. Q: You said looking at the cillia and some patients had particular diseases, stopping them beating correctly . Do patients actually gret their cells checked , via your unit , to find what the disease is or by others? No thats what we do, we're one of 3 centres in England that does this . A patient would be referred to us if they had chronic lung infections , not clearing their lungs properly , what is breather in gets trapped in . They will come with that clinical history of problems and investigated in a variety of wayd. Noone knows why but the concentration of nitric oxide in the exhaled breathe of someone with PCB is slightly higher than a normal person. So a NO test, also a nose scrape that obtains cells for a high speed video. Its the same cillia there as in the lungs? Yes. Its a genetic mutation and will be the same in all such cells. What is the big pattern, even if it looks normal there might be something behind that disease. If showing abnormal then go on to TeM. Unlike something like CF which is a single gene , single base mutation, one amino acid in one protein affected. With PCB can be hundreds of mutations in hundreds of genes, contributing to that ciliary structure , any one of which could go wrong. It might not be a protein in the scilia , but a protein error in the assembly process. This multiplicity is why its called a syndrome rather than a disease. The incidence is about 1 in 20,000 of the population . CF is a killer in terms of short life span PCD is a cronic debilitator and allied problems like the female reproductive system involves scillia, interation with sperm. In males the sperm cannot swim because the flagella ... . When you are an early embryo there is 1 scillia , the primary scillia that beats and sets up a fluid circulation , in a hollow spherical housing, and that single cell beating is enough to cause the body to twist to give the asymetry of our bodies. With scillia failure there, the body will go 50% that way or the other. Half the people with PCD have sinus ?, where half they're body is the other way round, just by randomness. Thats not life changing, but from the communities we have most of our referals from, is first cousin marriages and this becomes more common. The bad genes tend to stick around and get amplified, and the incidence in such communities can be more like 1 in 200, similar with island communities. Q: What would be the cost of the nose sampling procedure? We don't cost it like that. Because of all the rare diseases , the NHS has a specific budget , that funds certain units. So its not patient by patient funding. TeM is not a fast process . At the clinics are consulatants , specialist nurse to do the brushing. High speed vdeo takes a couple of hours to set up and do, the TeM can take several days or weeks , but not continuous as stepped. But presumably several thousand pounds. Other than physiotherapy to clear the lungs there is not much that can be done. One day there wil lbe better treatments, but I doubt it will be gene therapy, as its an inconvenience rather than a killer so not a priority. Q: In the video of scillia , there seemed to be a local group synchronised in their beating in a wave motion, what is the mechanism that causes the synchronising? We don't understand it. The entire lung system is syncronised , because if one scillia wanted to push something up and the next one down , it would get nowhere. The entire lot is syncronised together . Is it related to heart-muscle in that an isolated heart muscle will beat .? The lung scilia twitch with a mexican wave, effector stroke and a recovery. There must be some sort of mechanical connectio nas the cells are glued together. In the heart you have the pacemaker cells a group of neurons that work together and send a pulse out on a regular basis. A manmade external pacemaker is just taking over that natural function. For scillia there must be some sort of mechanical feedback between cells. If you held these cells horizontal does that function still work? They are tiny, perhaps 2 microns, cannot be related to gravity as when you lay on your side , they still work, pushing outward of the airway now horizontally rather than vertically. Q: ??? The first thing we do with a patient is make sure they blow their nose really well as the last thing you want with a sample is the cells to stick together with muccus. Another process with lung conditions is a bronchial lavage? a fluid into the lung and wash the lung out and lok at that. Bacterial cultures and counts on that etc , looking for anything unusual ,if nothing odd found, then they come to us. 28/80/59 B 40 95min W86 pint W87-94 air Q W96 july 17 W97 basingstoke S17-19 bob

[ An expanded version of her talk , that won through 8 doctoral research challengers per faculty and 8 faculties of Southampton University, so 64 challengers all together, in their 2018 Three Minute Thesis competition. ] Tuesday 03 July 2018, Rachel Owen, Soton Uni: Using the cellular origins of two contagious cancers to identify vaccine targets. The Tasmanian Devil, an endangered carnivorous marsupial which exists in the wild only on the island of Tasmania, is under threat by the emergence of two independent cancers, Devil Facial Tumour (DFT) 1 and 2. Both of these cancers possess the remarkable ability to transmit between animals, and have quickly spread across most of the island. By using the cellular origins of these cancers as a guide, I am aiming to identify cellular targets in both cancers which could be used to develop vaccines against them, stopping the spread of the disease and protecting an already vulnerable species from extinction. Cancer is a group of diseases caused by uncontrolled cell division . A healthy cell aquires a mutation and it become3s cancerous. This cance cell continues to divide uncontrollably , potentially forever, and thats how a tumour forms. For a tumour to form and survive it must overcome a lot of cellular defences. Like the immune system , constantly fighting the growth of tumours, mechanisms in place to control the cell-cycle, mechanisms t stop the growth of cells and mechanisms that replace cell death. Hence the hallmarks of cancer, a set of things that cancers need to be able to do , to form,grow and survive. Mainly avoiding the immune system, sustan ing qualitive? signalling , avoid growth suppression , avoid cell death, bu tthe one I'll be focussing on is that they have to evade the immune system. So contagious cancers, usually people have not heard of. So how does a C become contagious . A few things need to be in place initially. The cells have to have a physical route to be able to transmit between 2 different individuals. The cells that do transmit have to be able to invade tissue , to divide, and grow like aC would. They have to evade the immune system and specifically the anti-tumour response of the IS. They have to evade the anti-graft response, which most Cs don't come up against. At least 8 instances in nature where Cs have gained the ability to contagious. One is canine venereal transmission tumour , a sexually transmitted tumour that circulates in dogs ,wolves and foxes , hyenas. Its widespread, across most of the world. THe Uk is one of the few places that doesn't have this C. Mainly associated with coutries with lots of stray dogs or where they let pet dogs run around free. Generally this tumour is not fatal. It spreads and grows fast but reaches a point where the tumour stops growing and often it disappears, the dog is fine and rarely needs treatment. First identified late 1800s , but thought to be about 10,000 years old. Probably emerged in a wolf or an ancient dog breed. The are 5 of these Cs circulating in marine bi-valves. Muscles, clams and cockles, described over hte last 3 years and realising they are contagious. We know these Cs have been around a lot longer, only recently is start to understand them. One of them has crosed the barrier between species. Formed in one species but now only exists in a different species, so has managed to avoid the IS. As we have 5 types in clams , it suggests these sorts of tumour may be quite common in the marine environment. There are no truely transmissible Cs in humans, we don't tend to have the routes of transmission they need and our IS is pretty good. But there are rare instances where Cs to transmit between peole. 1 is during organ transplant , donor to recipient, rare and usually because the recipient is on immuno-supressants. Another is mother to foetus during pregnancy, even rarer. The foetus has a dgree of immune priviledge to stop the mother's body rejecting it as foreign. So only when the IS is dampened in some way. The tape-worm that turns into a tumour. A man in Colombia with late stage AIDS, soa bad IS at that point. He had lots of stange nodules in his lungs , not lnowing what they were. When he died , he had a massive tapeworm in his gut, that had developed a C and the nodules in his lungs were Cs from the tapeworm. Leaving the ones I'll talk about Tasmanuan Devil Facial Tumours , 2 different T that circulate i nthe TD, TDFTa and 2 , both causing necrotic rotten Ts around the face and mouth that kill the TDs most of the time, high mortality rate. The only mammal we know about that harbours 2 different contagious Cs. TDs are the worlds largest carniverous marsupial. Endemic to the Oz island of Tasmania. They went extent on the mainland about 400 years ago, after humans settled and killed them off. Like all marsupials they have a pouch, give birth to very undeveloped joeys, so first few months it lives in the pouch. When fully developed they become independent quite quickly. They are generally solitary and nocturnal. So how did such a cute looking animal get the name TD. A video explaining the reason, unearthly yelps and growls and snuffling , wrestling between a pair over a carcas. The first setllers only ever heard them as nocturnal , not saw them , and hearing that sort of noise out of the darkness of night , you'd think demonic. The feeding frenzies when they come across a carcas involve a lot of fighting , snapping at each other, biting each other. They have one of the strongest bites in the animal kingdom relative to size. They damage each others faces a lot during these frenzies. They were historically persecuted by farmers , who thought they were hunting their livestock. Their populatin was decimated a few times . In reality they don't hunt often , going for roadkill , primarily scavangers. Farmers now realise they are beneficial as they clear up a lot of dead animals, stopping infection spreading to their animals. There has been lots of TDs on Tansmania for a long time, sometimes they were killed but their numbers came back. Since 1996 the populations have been in steep decline. Oerall 60% drop in TDs, in some parts of the island its > 90%. In 2008 they were reclassified to endangered. This later decline is very much due to the emergence of the TDFTs and particular the FT1. Looking a tthe number of sightings in a 10km area and by years, plot showing when FT1 first emerged and numbers dropping steadily. We're not sure how these FTs emerged. No evidence of any external causes, not caused by UV or external pollutants etc. The fact they've been historically persecuted and recovered a few times has reduced the genetic diversity in the species, reducing species health and being prone to diseases , is a risk factor. They bite each other so often is also a risk factor for transmissible Cs like this, but we don't know a specific reason for FT appearance. FT1 was first identified in NE Tasmania i 1996 but unsure how nuch earlier it arose. Nowadys it exists over most of the island. they recntly found a population in the SW that is currently disease free and they will do some genetics on them to see if there is a reason for that, or its just not reached then yet. FT1 emerged in the Schwarm ? cells, in the peripheral nervous system, not in the brain , but in other nerves of the body. They wrap around the nerve axons , forming the myalin sheath, a fatty layer that speeds up nerve conduction. Only 2 types of cell produce this myalin sheath , 1 is the Schwarm cell and the other is the equivalent in the brain the eli?cyte. A specific protein periaxin ? in Schwarm cells is a marker for TDF1, which was the first evidence of where the FT1 came from. The FT2 emerged in 2014 in south Tasmania and cannot find evidence of it existing any earlier than that. It may have emerged only 4 or 5 years ago, and is currently yet to leave that region. Only been officually documented in 11 TDs but about 10% disease prevalence in that region. And we dn't know where the FT2 came from at the moment. Despite the Ts looking the same, they are different. They evolved independently. Slices of T tissue and darker purple dots are the T cells. The FT1 the T cells are in nests or bundles ,same in all FT1s but FT2 doesn't , a completely different architecture. Periactin? the marker for DFT1 , the DF2 is completely negative for that. The brown stain is Periactin positive but none for the DF2 tumour. The 2 brown stains at the bootom are Schwarm cells where there is periactin and so staining there, The T itself is negative. The chromosomes we see in a normal male TD, FT1 and Ft2 , FT1 has 4 marker chromosomes which we see in all DFT1 Ts and we never see in healthy tissue. That is caused by the C. DFT2 has a Y chromosome , so its emerged in a male TD. We cant find any evidence of a Y chromosome in DFT1 so it looks as though its emerged in a female TD. 2 different origins, emerged separately, they are separate Ts, despite looking the same and spread the same way. C is not normally a contagious disease. Our IS can recognise and remove foreign cells. So 2 mice that are different, try a skin graft from one to the other , there is rapid graft rejection, because of the IS of the recipient mouse, recognises it and kills it. This is called MHC class 1? , which is a sort of cellular barcode, consisting of 3 lelements a heavy chain which is very variable within a species , a small accessory protein Beta2M ?, and a peptide. A peptide is just a short fragment of protein , 8 to 12 amino acids long, and is generated as proteins in a cell ar ebroken down and reformed. Normal protein turnover, some of them get put on the cell surface. This complex forms together, sits on the cell surface and acts as a buffer to the IS , giving a snapshot of how healthy the cell is. The IS can scan those molecules , look at the heavy chain and the peptides and if either is foreign , it will destroy the cell. The situation where we have 2 genetically different mice , whats most likely to be fdifferent is the heavy chain , the IS rapidly recognises trhat and get rapid graft rejection. The IS can also recognose the [eptide , so2 mice genetically diiferent but both with the same MHC class1 heavy-chain , but hte peptie is different ,the IS will still recognise this and reject the graft. But doing it much slower. Surviable grafts , without immune suppression is if they are genetically identical. So MHC class1 is the same and peptide bound to it is the same. So why don't TDs reject FT1. They have a functional IS and from experiments we know thry will reject skin grafts quickly. These contagious Cs are essentially cellular grafts and should be rejected, but we don't know why. Itts been found recently that FT1 doesn't express MHC class1 on its cell surface, so to hide from the IS, its just got rid of the barcode, nothing for the IS to scan and find as foreign. TF2 does express it , so we don't yet know how it evades immunity detection. The fact that FT1 is removing it and that TDs can reject skin grafts , makes us want to see if there is a way to exploit this system , that TDs can recognise foreign cells to see if it can start recognising TDFTs . Our lab is working on how to reduce FT spread and working on something called a peptide vaccine. We are in Southampton, not Tasmania , so how do we get samples. We predominatelty work in cell-lines. Get a T and cells growing in it , smash up the T and isolate some of the cells. Plsce in a media that is just sugar and nutrients and things the cells need, in a flask, the cells will stick to the bottom and grow. We get them sent once and then we can grow as many as we want. We grow them up, freeze them down, grow them up again. We can get thousands or billions of them and can do what we like with them, essentially a limitless source of biological material. There ar elimitations, this is not how Ts grow in the wild, so there is debate on how well these cells reflect what the wild C looks like and acts like. So on top of this we also use fixed T samples. So from a biopsy from a wild TD , fix it so everything stays in the same place , embed it in wax, cut and slice or smash it up and look at the genes expressed. No one keeps TDs in a lab , they're an endangered species. So derived from wild TDs, so a need to catch them first. They are nocturnal and shy so difficult and we often can't catch the same TD twice, so statistical significance when working on wild TDs is a real problem. Hence we tend to stick to dell lines. We have collaborators in Tasmania and perform regular trapping trips. Taking white bucket like things out to sites where TDs are known to be , return in the morning and see if any TDs in there. So visual assesment for any signs of a T around the face , if they do then take a biopsy. If the T is really bad then the T is euthanised if its quality of life wil lbe affected. Most of them are released . So we have 2 contagious Cs emerging in 1 species in just over 20 years. That suggests a risk of maybe developing mor eof these. The fact they have such a reduced genetic diversity means the species health is not too good. So if another C emerges then it will cause more damage to the TDs that are left. It is urgent we find ways to help them. So work on breeding and re-release, on one of the small islands skirting Tasmania has been populated with healthy TDs with genetically diverse breeding. The Morura? Island genetic diversity project. Also zoos breeding them in captivity and re-releasing. And biologists are trying to work to some sort of vaccine. So what is MHC class1 doing. FT1 does express MHCclss1 , flow-cytometry and cell count on the Y axis and expression on the x-axis , dotted line negative H2M and pink line is stained H2M , negatve not expressing MHC class1. FT2 is expressing on the surface , shown in our lab by another PhD student last year. Treat them with an inflammatory cytokine , interferon gamma , inflates the cell going into an immune response, we see an upregulation of MHC class1. So inflame the cell and they will express MHCclass1. Even though FT2 already expresses MHC class1 , I've recently shown it also upregulates MHC class1 if treated with an inflammatory cytokine. 3 different DFT2 cell lines, DF2 regulates up to MHC class 1 significantly more than DFT1 does . So can we vaccinate against DFTs . A small number of wild TDs, trapped and tested that show an immune response to FT1 , this is correlated with ?ion of the Ts and some TDs survive for 3 years with no evidence of the disease reurning. This is good news, these TDs in their blood antibodies against MHC class1 + DFT1, so the MHC class 1, on the TDF1 cells is eliciting an imune response and if you take those MHCclass1+ cells , you can vacinate against the DFT1. Some success in doing this , but currently a bit limited and the responses a bit limited. So we want to refine this or thier peptides to vacinate against TDFts. MHCclass1 is definirely involved i nthe immune repsonse to TDFT1 and its presence on TDFT1 means the TDs immune system can clear out the cells a lot better. The fact it upregulates TDF2, makes it relevant to FT2 n as well. A peptide vacine would be feasible. It has been extensively explored for human C but is very complicated. The basics of it is; you have a healthy cell , its expressing a normal peptide on the cell surface , the IS is not bothered it doesn't flag up anything. When it becomes a C, ther's a mutation which messes up a lot of the proteins in the cell . If just one of these proteins to be expressing a peptide on MHCclass1 suddenly we have a new peptide on the cell suface, that is potentially foreign to the IS. If we can use that as a vaccine target , maybe we can ellicit an immune repsonse that only affects the cancer cell. In theory this is T specific and there should be no adverse side-effects, hence its use in C treatment. So an attractive option biut they're difficult. A study compared a healthy mouse genes with a cat-mouse C gene and they found despite many thousands of mutations in the DNA, very few become T specific, the antigens. Very few are on MHC class 1 and even fewer have any response from the IS. Even if you get to that point, you can have similar peptides on other cells that will cause a response . Hence target effects that can be fatal in some trials, so a difficult area to work in. So we start , we really need understand what the peptides see on these Cs. So how to find these mutated peptides. In single organism C its relatively easy, the organism forms the C, so go back to the organism that formed it, isolate the cell type the C mutated from , its healthy #cell-type of i=origin and make a direct comparison . Comparing all the peptides and can see which one is defective. Its a bit fifferent with TDs, so 2 TDs which have both undergone mutagenic events to form DFT1 and DFT2 , both TDs are #dead. Either died because of the disease or because old, they don't live long . We cant make direct comparisons, w ehave to do instead is look at the current healthy TD popultion , isolate the healthy cell-type of origin , pool all that together and then compare to the Cs. Trying to identify specific mutations. We can do this for TDFT1 because we know it came from a Schwarm cell , but we don't know where TDFT2 came from yet. This is a huge part of my project, what cell type did the FT2 originate. The top 20 proteins that ar eover-expressed in FT2 compared to fibroblasts that are a healthy control and TF1. Every protein in red is a protein with a direct function in the nervous system . Evry protein in blue is a protein we see over-expressed i nthe nervous system . The TF2 is enriched compared to the fibroblasts yet it over-expresses loss of proteins from the nervous system, so it looks like a nervous system protein . Now looking at the gene expression , RTDSTRs ? a way of looking at genes expressed in cells. The control should not change between cell lines. All these are myalin associated genes which we should be seeing in only 2 cell-types and we are seening them in all the DFT ? , at alower level in FT1 but they are there. Which means DF2 has come from a cell-line that produces myalin. When you stain the FT2 sections for Schwarm cell markers NB and S100 and ciatic nerve as a positive control which is highly enriched for Schwarm cells, can see if there is brown staining on FT1 and FT2. So DFT2 Ts are positive to schwarm cell markers. My conclusion is that TF2 has emerged in a similar cellline as as TF1 , it produces myalin from a myalin producing cell-type which brings it down to either of 2 cells ,Schwarm cell or Eligodentacyte? We think it originated in a more immature cell version of this, than DTF1 did. It suggests TD myalinating cells are predisposed to contagious Cs , so it highlights how important how are vaccine strategies are. We have 2 independent Cs , both emerged from similar sell-types, we know if we treat these Cs with interferon-gamma we can upregulate the amount of MHC class1 on the cell-surface. This means we are putting more peptides on the cell surface , which the IS could notice are foreign. Most of these peptides would be differnet beteen the 2 Cs. But what if we can find 2 peptides which are both mutated in the same way , so a peptide on both Cs which we don't find anywhere else in the body. If we can find that peptide , then a single vaccine is possible to help attack both Cs. This iswhat our lab is going for. So currently I'm looking at what peptides of MHC class 1 are actually binding in Ft1 and Ft2 , are any of these peptides mutated in eithe rFt1 or Ft2 and are any of these peptides , that are mutated, are actually present on both Cs. If we can find those, then thats a foundation for a vaccine, that could help Tds survive both these unusual and agressive Cs. Thanks to our lab coleagues, our collaborators at Monash and Melborne . Q: When you've got the IS to recognize a foreign peptide and it attacks those cells , how successful is it in attacking enough cells to make the recipient healthy or not healthy. We don't really know yet. There are a lot of factors involved. Some peptides just don't ellicit much immune reponse, and it depends on a lot of things. Again a factor full of complicated things in the generation of vaccines. Sometimes you find peptides that are unique, nowhere else in the body , and for some reason the IS will not recognise them. Or it will and the reponse is muted. Others have a huge response to a peptide, so completely varied . If we find a peptide that is really immunogenic then theoretically you wouldn't need that nmuch to get the IS reallg going. But more likely not going to be strongly immunogenic, which is probably how they're spreading. It would be a case of using adjuvants ? and things that enhance the immune response ot the vaccine in the first place. Q: So you don't have enough knowledge as to why some peptides don't bring abou tthe response and others do. Sometimes it seems a bit random. Certain amino acids, the ones with big side chains and rings in them essentiallly they tend to have a bigger immune reponse, why i'm not sure and noone else either. Bigger bulkier peptides tend to activate the IS more than small peptides. But no guarantee of finding the big ones, as presumably those wil lbe the first targets for the Cs to get rid of. Q: ? T-cells ? if you could isolate them from immune cells , then screen those against the peptides ? Q: You don't use radio-tracking to zero-in on repeat samplings of the same animal in the wild? They've just started doing that. There is a problem with the shape of their necks , squat necks and easy for the trackers to fall off. They don't really have a neck. Q: Any TDs at Marwell or any UK zoological sites ? I don't think so. Thats not many outside of Oz. San Diego and Denmark has some. Q: How did these problems emerge? I can understand the Island Effect on genetics . Is there othe rhuman involvement beyond trying to eradicate them, I'm thinking pollution ? A lab we work with in Cambridge has looke dfor it , for specific signatures in the genome for something common from UV damage etc. The most common signature for these Cs is its age-related. It appears in quite young TDs. Its seems to be appearing from a natural source rathr than something external. Q: It all seems such a strange situation, I'd have thought it was fundamental to nail down the origins. Could be viral possibility. It certainly doesn't ? spread ? but that doesn't mean original transformation was not due to a virus. The original transmission could have been by a virus and traces of those viruses are now lost. Especially now showing that the Cs are similar cell-types , then that makes more sense ??? Q: Being marsupials , does that mean they could be exposed to more exotic illnesses. No other marsupials have anything like this . Al lvery strange. It probably comes down to TDs are unique and has a very easy method of transmission , they fight so regularly as part of their behaviour. Maybe these sorts of Cs are forming oin lots of species but because they don't interact in the same way, we don't see it spread. Form and then die in that one animal. Its got to the face because they bite one another. Q: so there can be a feedback mechanism to assist how these Cs can evade IS processes. If a C forms in a body, that body dies, with no transfer to another host , then its purely down to random generation. With a feedback process it could accelerate an otherwise random event? In the human C process a C can metasticise to anothe rorgan , the C starts in one place and then move . Those cells then have to overcome new immune challenges in the new environment. That in a way is similar to these Cs , instead of going to a new organ they go to a new individual. But can that speed up the evolution of these sorts of C. Q: For the marine animals with these sorts of Cs , that is just co-evolution , just isolted random events with no cross-over between species? Other than the one that now exists i na different species, I think they all emerged separtely. They seem to have different mechanisms as to how they have become transmissable and formed in different ways. It looks as though the emergence of contagious Cs , at least in the sea might be quite common. The bivalves off the USA were dying off , but no one realised it was due to Cs. Q: Were they geographically isolated or around the world? When fisrst noticed off N America , they thought it was geographically isolated , now its found in the Med off Spain . Q: the proposed TD vaccine only protects individual animals , doesn't protect any offspring and so a massive vaccination procedure required? Yes. It would e a combination of capturing what you could. Plus the captive ones on the outer islands , were all vaccinated on relaease, it would take a while, but eventually there would be herd immunity, the point where the Cs can't spread any more. If we gat a vaccine, then yes it will be a massive undertaking, a lot of money to get that running. The government of Tasmania is fully interested, some releases in northern Tasmania , been quite wiolling to trial of vaccinating the whole cells, the problem is its really expensive ot do a vaccine that way. Not viable perhaps fo rhte whole population and no way of monitoring the anomals after vacination . We can't do a proper scientific study. Q: THe trick where it doesn't have the code, ??? would that work for humans ? That is common with human cancers , like how they put up a mutated peptide , Cs are aware they do that, that comes in with the immune pressure and happens a lot with human Cs. It correlates with more agressive Cs, they tend to get rid of MHC class1 on their cell surface, they just stop expressing it, then they re really hard to treat. There is a lot of effort for humn Cs , honing up that ?? , in the past when people have tried making the inflammatory cytokines that we are working with , it can cause terrible immune reactions, called a cytokine storm in patients, the IS goes into overdrive and the patient dies. So its not seen as a viable treatment for humans unfortunately. Finding other ways of manipulating C cells so they upregulate MHC class1 , is something people are looking in to. Unkowing the geography of Tasmania , is there mountain ranges and rivers that can isolate communities , so you can re-release knowing it is very unlikely to be cross-connect with other communities, as I assume they are otherwise free to roam? The main one we know of is in central Tasmania . The is a mountain range thru the middle . It was thought at one time that as the disease spread east to west, that that range would stop FT1 but it keeps spreading. Birds or something could be a vector for transfer? No reason it couldn't be , I think they assume the TDs have simply got up there. We don't even know how long the cells can live outside of the TDs and active cuts or sores. Presumably they could live a bit like a virus ???. I was thinking of concerns over here of culls to supress badgers , because of bovine TB , but a theory goes, kill them off in one area and then others from outside move in with TB and then back to square1 ? Human manipul;tion does not always work. How do they ensure non-disturbance of existing populations, via re=releases? I don't think thye worry too much , they just want more TDs back on the island. That badger type UK research I don't think has been done for the TD situation. How many in the wild in Tasmania at the moment? About 20 to 30,000 , it was 120-150,000 . The last surviving one on mainland Oz was when? There is aa bit of debate on when the extinction but consensus is about 400 years ago, shortly after European settlers. Have they ever become pets? I don't think so . Apparently they are very nice natured until about age 2 and sexual maturity and they become a nightmare. A young TD you could go up to and pet, nice and placid. With animal rescue centres that deal with TDs, the people handle the babies , ut stay clear of the 2year olds or older as the only way they interact is agressively. They also smell terrible as well. If they do attack a human they can do a l;ot of damage. For Oz generally although very large, it seems that if anything can go wrong with human interactions with fauna, it does go wrong. I'm thinking of the mice, the rabits , the cane-toads, it just does not seem to happen elsewhere? Is that situation even worse in smaller Tasmania? THere always is a problem with island populations becuae they generate very specific eco-systems , that becomes very finely tuned , a delicate balance . When there is nothing that can come in and balannce it out, limited to what is already there on the island. The second you bring something in it can all go out of kilter, no way for the eco-syetem to fight back. For example compare to Europe, it doesn;t happen on such an intense scale , there is natural stuff that can come back in and refil gaps. If you accidentally wipe out sonmething on Tasmania , there is nothing to fill in. Its isolted from everywhere . Oz gets it becuase it sa very ig island. Things get wiped here quickly ,by small changes i nthe environment . Even in the UK the red squirrel can only survive on outlier islands of the UK, in the south of the country. So that is a precaripus situation if anything gets on to Brownsea Island, just one grey squirrel and that population of reds would be gone. So climate and genetics can affect the island effect? In more ways than you may intially expect, on an island, over a long time the genetics effect and then nothing else that come in and replace. One thing goes and then the entire island has to change. Or one thing comes in and all change. All highly responsive to outside interference. hat made you want to do TD for your PhD? I was always interested in C as a biologist/chemist at uni, but I wanted to be avet when a lot younger, but decided I didn't like the people side of that. I put the ides of animals out of my mind and went down the C route, then this topic turned up as research , involving both endangered species and C at the same time. So a perfect role for me. You apply at PhD level to a specific project and this was the perfect fit for me. It does seem a bit odd, yourself and a lab on the other side o fthe world heavily involved in this research? And another lab in Cambridge in the UK and a lab in the USA. Is that fairly common, not just TDs , that structure of worlwide spread? I think it comes from people who have an underlying interest in a biological process and they end up studying a sole gene or disease that allows them to study a molecule . The Canbridge group emerged from tumour evolution , using the TD ones as a model. We have an objective of something very practical from our research , but along the way explore the underlying mechanisms ???. In the USA they're modelling the spread and the factors that influence it. Its a combination of people seeing a real issue of the moment, a problem they can address , and a scientific interest in something a bit more broad. I suppose 20 or 30 years ago you could not have done this trans-national research , withput the principle of cell-line culture, requiring people to physically going to the relevant country? Yes, the cell-line stuff is so integral. For these cell-lines, if you came back 50 years time , it would still be the same cell-line? If you keep freezing them , when you're not aactually growing them , then they will keep reasonably stable. But they do change. The most famous cell-line is the Heeler-cell ? a cervical C cell line from an Afro-Caribbean woman in the USA. But look at those cells these days , there is an obscene ampunt of chromosomes that have appeared from nowhere. They don't have half the organelles that you expect to see in a cell. Its come to the point where some people are questioning if these really are cells any more. There are limitations . You bring them up from frozen, grow them on a plate , but when they fil lthe plate you have to subdivide onto more plates for growing more. ut there is only a certain number of times you can do that , and you start to see the cells changing. All cell-lines not jus tthe Healer-cells. eg DF2 which normally spread out and look like stars , lots of sticking out projections but the longer you grow them, those projections get shorter , start growing strangely , stick to each other , all sorts of weird things when they are farther down the propogation line. So even without looking at the genes or the proteins or whatever you can simply see the cells start to look different after too many divisions. Thats why we have multiple cell-lines , isolated from different TDs and different tumours at different times. The idea is that if they are stil lreflecting each other , they can stil lreflect the tumour samples , and so still a good enough modelfor our use. ut you have to be careful as a lot of limitations with cell-line work.

Tuesday 17 July 2018 Jonathan Ridley, Head of Engineering ,Maritime Science and Engineering, Solent University : Hydrofoil design and use (second talk, previous talk on yacht design) An example of one of our one-time students Jason Kerr graduated 1994. The pinnacle of yacht design is the Admirals Cup and the last one had 3 teams with some of our graduates working for them. By the third year their doing 3D CAD work designing vessels from scratch , structural design and theory , power, systems, aerodynamics, everything. A hydrofoil we built in 2014 called Solent Whisper and this is where our interest in small craft hydrofoils (H) took off. A history of hydroils. Our hero is Sir George Hayley. A scientist who made astounding contributions to sci and eng, but we've almost forgotten about him. He re-invented the wheel, the tension spoke-wheel, with little knowledge of ship-stability he invented self-righting lifeboats, he invented tracked vehicles calling them universal railway. Before internal combustion engines came along he invented an internal combustion engine that ran on gunpowder, invented automatic railway crossings, also the seabelt. He started to lok at aerodynamics . Late 1700s /early 1800s anyone who wanted to fly simulated birds and flapping wings, fundamentally flawed for humans. George Hayley observed birds and started to derrive a theory of fligth. 1809/1810 he published his scientific paper on aerial navigation. His gunpowder engine would have worked better with a gaseous fuel. It was the same with powered flight , but no engine light enough and powerful enough, but if we did, then this is what we could do. He was the first to look at an aerofoil shape, at lift ,drag , thrust and weight and would have to be balanced together to make it work . He didn't have a wind tunnel he created a long rotating arm, a foil on one end and a counterbalance on the other end and a motor to rotate it. Measuring the rotating force, and the lift, simple experiments but he learnt a lot. He learnt about the control of foils moving thru the air and how to control lift. He came up with heavier than air principles and understood and derived the centr of lift and the centre of pressure, how forces move and where they move. He looked at camber effects of different shapes and different lift andhow to control it and in 1848 he had something we'd recognise today as an aircraft, with a set of wings , tailplane and rudder for control , the world's first gliderr. He got a local boy to fly it , as fairly light , and launched him off a hill. 1853 he has the second successful glider , using his coachman this time to fly it. One longish flight and landed and the coachman would not repeat it. The functional knowledge of foils is essential for aircraft and essential for Hs. All goes quite but Henrico Fullerini ? starts to look at Hs. In 1911 he achieved 42.5mph on water with just 60hp of engine, He did a lot of background work and published a lot about H, from an experimental point of view. 1889 to 1901 John Thornycroft on the Thames ???. 1905 William Beecham did deep scientific analysis of Hs. In 1919 Alexander graham Bell had a go, the HD4 hydrofoil with 700hp he achieved 71mph on water. That would be pretty impressive even today. The fastes object on the planet in 1919 was the Sopwith Dragon aircraft that would do 150mph. Developent on from then comes from the aircraft industry. They have sea planes that need to take off from water, water is sticky stuff and difficult to go fast enough, put some hydrofoils under such planes and get some lift then can get more air speed. Not particularly successful as going faster creates more drag , your aircraft becomes less efficient. In 1954 the next big step in hydrofoils. Frank and Stella Hemingley , Frank is a naval officer who survived WW2 and he wanted to set the world water-speed record. While in USA he marries his wife Stella , a pair keen on breaking the speed record. They built a hydrofoil they called the White Hawk . All Hemmingly had was a drawing of what they thought their hydrofoil should look like. They needed someone with more technical knowlege and enquired of Imperial College as to whether they had a student who could do stress calculations for us and design the vessel. They lent them a Ken Norris , went to their house in Chelsea , and with no proper plans , had to start from scratch. It looks a bit like Bluebird , because Ken Norris went on to design Bluebird. His brother was also an experienced engineer , Lou Norris who worked for Sir John Chobham, trying to get the world water speed record in similar time. Ken Norris designed the fatal for Cambell K7 Bluebird , over 300mph . Ken Norris also designed the car the CM7 , stands for Cambell Morris. He then went on to work on Thrust2 and then ???. Somehow with his connections ot the Navy , Frank gets hold of a Whitley turbo-jet, 1943. They go as fast as they can down Lake Windermere, a few runs , getting faster and faster, without any timed runs, just to see how well the vessel works. They get up to a certain speed , then a bit faster and it nose-dives and sinks. A jet engine running at full speed, suddenly immersed in water , is a no-no. They managed to get Rolls Royce to lend them 2 Derwent? jet engines and the technicians to run them. They return ot the Lake District, see how fast they can go and the same thing happens, at a certain speed, it nose-dives. The 1950s its a tandem cockpit and both are in there. Frank did the running and testing but it was alwys his intention, when they broke the world speed record that Stella would be driving. As not a formal record attempt , it was not recorded. A pitot-static tube , measuring their speed didn't work. Frank reckoned they were doing over 100mph , but from the physics and difficulty in judging speed while on water, that speed was unlikely. They spend winter in Windermere , working on the boat, waiting for a break in the weather. The break never comes and the Americans get interested in rreally fast Hs. They go over to the states and the US Navy gets interested, and the technical points are discussed. On the technical front Frank and Stella were happy, the US Navy was happy but contracts had to be arranged. After several years of lawyering Stella does a few demo runs and the US navy decides it was outdated and no longer interested. As a ps to this vessel , Frank brought the vesel back to the UK, bringing it through Southampton Docks. HM Revenue and Customs turned up , saying Frank owed them money, and thought it was an import and impounded it. Nobody knows where that boat ended up , it did not leave Southampton. Perhaps in the corner of an old warehouse somewhere in the docks , it may still lie. They had 4,000 pounds force of engine , delivering 17.8KN of thrust . Go back to Alexander bell 700HP and got 71mph. This time with all that thrust they got 70.6mph. Al lthat developement and time and just short of the previous record. Then a few commercial Hs , the Boeing Jet-Foil , they still operate in Hong Kong harbour etc. Fairly successful, roughly the same passenger payload asa 737 and much the same cost. Still operated today . There was a lot of military investigation into Hs as to performance and speed . They did lots of trials but did not get very far . One problem for the military is the generatio nof huge amounts of spray behind them, the spray is very cold , so not stealthy along with the noise. Also hit any debris in th e water and Hs are relitively delicate. They run between Rhodes and Greece and Tukey , on the larger rivers of East Europe and russia . So why have Hs not become bigger and faster over time. Datapoints of commercial and successful Hs , horizontally the power to weight ratio versus the top speed. If not a lot of power or a lot of power, and you can get to 70mph , and you basically hit a brick wall. With Hs there is a top-speed that cannot be exceeded. 1950s and 1960s , the Hs themselves were made of aluminium , ? with steel, moving very quickly thru water . Hit something submerged and they are easily damaged , then back to the shipyard for a very expensive refit. The quote is Hs are very nice until you slice a dolphin in half. It all went quiet in the early 1970s . Then the sailing world got interested in the International Moths?, the closest sailing gets to a blood-sport. They are lethal, the rules for Moths were they have to be within a certain set of dimensions and since 1970s carbon-fibre just about comes into the cost-range of people into these sorts of craft. We start to see there developement, going faster and faster . Moth sailors realised quickly that if you wanted to really hurt yourself , then get above the water and you can fall off at greater speed than ever. From 1974 a lot of individual trials and experimenting, empiricalwork but not university reasearch. Going round Portland harbour as fast as possible. Even today , the Moth class is a pinnacle of sailing in terms of small craft performance weight and speed and technology. 1980s/1990s,to 2000 the Americas Cup , by 2003 was using the IOCC 72 ? a very graceful monohull , but from an audience perspective , 2 of them racing far off, pretty boring. They developed new rules and looked at multi-hulls, catamarans, trimarans. The team to beat recently has been Team New Zealand, the world leaders in this sort of tech. For the 2013 America's Cup, everyomne agreed on big cats. The Kiwis launched their boat and the spies were out for the trials. It was sailing but extremely slow. Second day out and everyone else was really pleased at how slow it was. Daggerboards. As you are sailing the wind is trying to push you sideways as well as forwards. So to stop that you have some vertical board underwater , the daggerboard. the Kiwis found a little loophole in the rules, it did not say what you could do or not do with daggerboards, just that they were allowed. They bent the tips of the dagerboards in , and on day 3 of the trials in Aukland harbour and the boat shot off into the distance. Mass panic and a huge amount of plagurism everyone tried to quickly ament their daggerboards. This was the start of really big boats going faster. Far better hydrodynamics and far better materials allowing to build such large structures. Unfortunately they were inherently dangerous. In one of the races a UK sailor was killed on one of these vessels. The organisers decided for the next cup to throttle things back a bit. The next race, still cats going as fast as possible but a bit slower. The speed record was just over 40mph ,fast enough. For the next cup coming up AC36 in 2021 , a whole new type of H , the foils cant or rotate in or out of the water as required and the vessel can sail along balanced on 2 foils. Mathematically its possible, it will be interesting to see in practise. A designer involved with this say they look as though they should crawl up a beach, lay an egg and return to the sea. They all work by lifting the vessel bodily out of thw e water. Another area is where we use Hs not to lift the vessel but to control the flow of water around a vesel. In 2016 the Vendee Globe race , non-stop single-handed around the world and 60 day circumnavigation was possible. So a H on each side, they extend out once its at sea. They produce a lifting force to roll the vessel upright allowing to sail much faster. Video of Schetana ? designed by one of our graduates who graduated in 1993 an Open-60? class. Horrible weather conditions, but the H keeping the vessel upright . How fast its going, the Hs in extended mode . Doing about 25 kn, in those sort of waves, quite fast enough. When it was racing in the southern ocean just south of NZ it hit some debris in hte water , severely damaged hull and limped back into NZ, all on board safe and well bu tthat was the end of the round-the-world race. We are starting to see Hs fitted to very low speed vessels. You don't necessarily want to lift the whole vessel out of the water. But a fairly small foil just lifting the stern slightly can present a better underwater shape and can reduce drag. Not a huge saving perhaps 1 to 3% but for a vessel in almost continous operation , that saving can be significant. I'm involved with a PhD study of Hs on pilot boats for the Port of Southampton, burning 1 million litres of diesel a year. Another project, our towing tank , a higher speed vessel with a bolted on 3D printed H , testing the effect. Assume our interest is to lift a vessel out of the water and to go as fast as possible. An America's Cup boat , sailing along with sails providing the driving force forwards, to go faster a nd faster. Working against that is resistance , water against the hull. Along as our driving force is bigger than the resistance, we accelerate. As we get bigger, the resistance gets bigger , and balanced forces then a steady speed. To go quick we must reduce the resistance as much as possible. The components of resistance, simplified. Air resistance pushing against the vessel, complex as its tied in with the sails , ignore that for the moment. Viscous resitance - water is sticky stuff . Consider a stationary vessel , water flowing past it like a stream . Looking at individual molecules of water , on the surface of the object there is friction with the surface and the molecules slow down. The next layer of molecules above that , a bit of friction with the molecules below , but less , getting less with each layer away from the object. Farther away the molecules can move faster and faster. This is the boundary layer , Newton tells us that force = rate of change of momentum and if we are slowing down these molecules , a change in momentum , must create a force, of drag. Drag force can be significant. Viscous resistance depends on water density, the vessel velocity, the friction coefficient which itself depends on density , the vessel length and the velocity , the dynamic velocity in water , the shape of the object or form-factor but most important is its directly proportional to wetted surface area, the amount of the vessel under water. If I can halve the wetted surface area , I instantly halve the frictional resistance. For an Americas Cup vessel , the wetted surface area . At the maximum draught 40cm of hull underwater and 30 sq m of wetted surface area. If I halve the draught that wetted area drops to 15 sq m. Then wave making resistance. As the vessel moves thru the water , it creates waves, it creates pressure distribution around the vessel under water. But also creates a disturbance at the surface, the Kelvin wave pattern. Waves from the bow , diagonal waves coming off and waves coming off the stern . These wave patterns interact with each other and create drag. Wave making drag depends on water density , vessel speed , wetted surface area , bit also on something called the wave making coefficient which is really complicated. Its difficult to calculate, thats why to solve this problem , we stil lbuild model boats and tow them down a tank of water. Its more accurate and more fun than trying to do the maths for it. WMR depends on speed and the shape of the underwater volume and wetted area. We have some equations to allow us to calculate WMR , the Reynoldsaverage Navier Stokes Equations, non-linear, partial f=differential equations, to be solved simultaneously. Imagine something like 25 million separate variables to get this to work well . If we ganged together the worlds supercomputers and asked them to solve it for us, we would die of old age before the solution. A graph of an AC catamaran, with no foils on. As it goes faster and faste rthe WMR goes up. If I can lift the vessel out of the water , the amount pushing the water and waves out the way reduces , the WM coefficeient goes down , WMD goes down and in theory the vessel can accelerate. Ignoring air resistance. VR is relatively small, WMR is bigger and if we add them up we get the total drag. There is a dip in this curve, caused by waves interacting ewith each other in the WMR at different speeds. We accelerate our vessel, go faster and faster , until so much resistance we reach a fixed maximum spped. For the H version of the AC yacht, stating to generate lift. At about 15mph , 7m/s the lift htey generate, is sufficient to unstick the vessel and start to lift. The problem with Hs is they increase the wetted surface area, counter to what we're trying to avoid. At slower speed we get an increase in the VD but at about 7m/s the lift starts to overcome that, less hull in thr water. As we accelerate furthe rthe VD does not increase too much. The same with the WMD ,not so much an issue as not so dependent on wetted surface area, with the stasrt of lift, reduce the underwater shape and we start to control the drag , and add the 2 plots together. When the H craft is up to about 15m/s or 30knots we're generating about half the drag as the vessel without Hs. Hence instead of limited to 20 knots, we can do 40 knots. A bit of H theory. A theory often banded about is the intelligent fluid theory. H or aerofoil, does not matter, fluid hits the front of it and the fluid splits , some around the top, some around the bottom . We apply a bit of logic to Bernoilli , if we have a shorter path the flow must be going slower and if you slow a fluid down , Bernouilli says the pressure goes up . Along the top, a longer path , so the fluid must go faster , then via carburetor or Bennoilli , the presure must drop. so high pressure under and lower pressure under, must push my foil up. This is the classic theory , taught as an explanation of how a foil works. The reality is different. What goes further must travel faster , but there is nothing to say it must travel faster. Everyone says the top molecule must go faster than the bottom one, so they can meet up again at the same point behind the foil. much research but no one has conclusively proven that water molecules mate for life. With a big foil in a small wind tunnel , because of blockage effects, this van happen. In reality it doesn't work. So start with a flat plate and at slow speed , flat plates make remarkably good aerofoils , the paper aircraft scenario. Take the flow in from the left, hit our flat plate , inclined at some angle of attack , so it will try and create some lift. We will assume there is no viscosity with our fluid , the molecules flow perfectly over one another, an ideal fluid. Plotted here are streamlines , like isobars on a weather chart. they tell us the direction of flow and hte pressure and speed. The closer our streamlines are together , the faster the fluid flows, like isobars and wind on a weather chart. For out foil , the top fluid flows around the top , bottom fluid around the bottom and inbeteeen ther eis a point where our molecules hit the foil and have to decide whether to go up or down. They sit at that point, the stagnation point. At the trailling edge , again a stagnation point . There is a symmetry here in the shape of the streamlines , mirrored on the centre-plane . The symmetry tells us that if we do the maths of this and try to calculate the lift and drag , that in terms of a vertical force , the force lifting the foil up and the force pushing the foil down are identical , cancelling out and no lift and no drag. Referred to as the DeLambert ? paradox. now put some viscosity into our fluid, a real fluid and look at the trailling edge . The water comes down around the trailling edge and tries to go around the corner and go back to meet the stagnation streamline. If no viscosity then that would be fine. The viscosity tells us we have a different scenario . Viscous fluid does not like going round corners. It tries to run around the bottom of the corner , but it runs out of energy due to the viscosity . So instead it rolls around , and disappears up itself and rolls downstream , the starting? vortex. As soon as we move a foil from static, a starting vortex appears at the trailing edge. That starting vortex disappears downstream. For a good demo of this, when you have abath , put some talcum powder on the water . Get a credit card , carefully place i nthe watr , just 5 or 10 degrees angle of attack and move very slowly. You will see the starting vortex. Same with canoe paddles. Vortices are incredibly powerful , such as tornados. This vortex is very small but very powerful. It acts like a gear wheel. It starts to pull the rest of the fluid , round in the opposite rotation , behind it, circulation. Our starting vortex is going round very quickly , very small diameter , is creating a much bigger circulation of flow around the back of the foil. Again take your credit card and move it thru the water and you carefully lift it out, you will see a little vortex of the starting vortex , going downstream and you'll also see the talcum powder rotating round showing the circulation pattern. A simple experiment you can do at home. Our vortex runs off down stream , the circulation stays that hits the back of the foil and gives it a bit of impetus to change and it pulls the stagnation streamline right back to the ?. When it does that , we've lost the Delamber paradox, lost the symmetry , we're starting to get accelerated flow along the top and higher pressure along the bottom and starting to get the lift force. We can now put a bit of camber or curvature into this foil to control the lift, we can put some thickness in the foil for strenght so it doesn;t snap off. There is a calculation for a flat plate with a small angle of attack , streamlines coloured by velocity . But it gets a bit more complicated. This is a 2D foil with no end to it, infinitely long. With 3D foil we get problems . Look at such a foil from behind and down on top. At the bottom of the foil is high presure, top of the foil is low pressure , the high pressure rolls around the tips of the foil in to the low pressure and rotates dowwn stream as a pair of tip vortices. The plan form of that foil really contols the tip vortices , and they themselves create extra drag, induced drag. When R J Mitchell was designing the Spitfire there was a piece of ironically German theory called the Bettz ? Minimum Energy Hypothesis, that said if you want to minimise tip drag , then you need a lift distributiuon across the foil that is elliptical. So you need a foil with an eliptical shape , hence the Spitfire wing , tryin gto control the tip vortices. A H similar to what we used on the Solent Whisper. If I want to control the tip vortices, control the drag, to go fast and lift the foil and vessel out of the water, I need to look at the plan form. How long the foil is compared to fore/aft dimension. Span and chord , dividing and that is the aspect ratio. A low aspect ratio is short and fat, a high aspect ratio is like a glider wing long and thin. More equations but basically our lift coefficient , measures the lift of a foil depends on the inverse of the inverse of the aspect ratio. The bigger the aspect ratio , the longer and thinner the foil, the more lift you can get. The induced drag coefficient is invesely proportional to the aspect ratio so the bigger the aspect ratio, th esmaller the drag. Al lgood news, if we want lots of lift , not alot of drag a long/thin foil . So a fast jet at one end of the graph and glider at the other end. With Hs there is no single neat mathematical solution. With a short/fat foil , then the ends of the foil don't bend up much , don't deform , no stress within the foil and easy to build . We have low ? of lift , quite a lot of drag and effectively small pressure changes around the foil. But fo r a glider type aspect ratio , then very high tip deflection . If I double the length of the foil , keep the same ;oading , the same shape, the long foil tips will move up and down 16 times more than smaller foil. That creates structural damage. But we also get large pressure changes , that gives us lots of lift. This is where going back to WhiteHawk , get to a certain speed , about 70mph , we hit abrick wall. As we go faster and faster , we start to affect the water around us. A substance having phases, a solid,liquid and a gas phase. For wate r, not in the arctic is liquid. If we play around with the temperature or pressur ewe can convert it to gas. Boling a kettle increases the temp and we get water vapour. If we combine that with pressure then some different effects. Boil a kettle on Everest it will boil at around 70 deg. Hs creating lift as they move along, on the top surface, the pressure drops low enough , that we can turn seawater into wate rvapour. A simple lab experiment of a flask of water with a partial vacuum above it, not dissimilar to Donald Trump. Create a complete vacuum and the water boils at room temp. In slow motion , we can see the inception point is not from heat at the bottom , but nucleating about dust in the fluid body. We effectively build a bubble around our H , if too fast. A water H works far better in water that in a bubble of gas. As we get to the magic 70mph , we start to get cavitation. We can control that to some extent by the shape of the foil. A typical shape for aircraft wing and a typical shape for a H. For the wing , plotting the air pressure on the top of the wing , we'd find the peak of the low pressure , when flying straight and level is close t the nose of the foil . A big peak , then drops off, the pressure recovery, going back to the foil trailing edge. Hs , this one an ekla-H ? ar eparticularly designed so ther eis not the big pressure peak at the front , a nice gentle increase and then amuch flatter line dropping off towards the back. So we have similar areas of lift but the peak pressure is lower, the point where cavitation starts , is much lower. Its referred to in hydrodynamic as a rooftop section. Then we get another problem. Underwater video from our towing tank. A yacht stationary and then accelerating, a keel at an angle designed to create lift a sa partial H, and a certain point it just taps the surface of the water. There is low pressure on the top of our foil , not going fast enough for cavitation , but that low pressure on the foil is attractiv eto the low pressure of the open air above it. The low pressure sucks in air from above , pressure drawdown, ventillation . The carbon fibre strut is bouncing around due to the amount of forces. If our foil is too close to the surface , we get ventillation , we end up in a big bubble and we loose lift. Solent Whisper being tested , you can see a tip vortex appear when a bit too close to the surface, water vapour starting to form , the white line is the water vapour under water. The foil gets too close to the surface , ventillate and the whole foil become covered in a white cloud , loss of lift and a nose-dive. Hobby-horsding along the surface, nose-dive recover , nose-dive ,recover. This is what happened to White Hawk, going so fast cavitation starts, it lifts too close to the surface , looses lift and drops down and with a turbine beehind you while doing 70mph , pretty hairy. So getting H design right to control lif and control cavitsation is really tricky. We need to control the foil. We don't want it too close to the surface , sufficiently underwater so it won't ventillate , so some sort ofcontrol required. Gravity trying to pull our vessel down . In the wate rnormally and not foiling , displacement mode, we have bouyancy force Archimedes. As it accelerates and lifts out of the water our bouyancy force disappears , gravity is still there , a little bouyancy from the foils and w ehave lift. As we go faster and faster , creating more and more lift and the vessel wants to lift out of the water and we'd get to the point of the foils ventilate or cavitate. So for our foils we need lots of foil to lift us and then suppress it so we can just balance with equilibrium of our vertical forces and sail at constant height over the water surface. 2 ways of doing this. 1 is the ladder foil , its what Alexander graham Bell used. Ken Norris described them as Christmas trees under water. At slow speeds and low in the water, all the foils are submerged and creating lift. As you accelerate, the top foils come out of the water and air is 1/1000th the density of water so the lift of the top foil drops by a factor of 1000, ie no lift. So down to 3 foils , then 2 foils and you try and tune the foils to match the weight o fthe vessel. Tricky for Mr Bell as , going along and burning fuel , he was getting lighter, so easier to do with sail as the power. This is the simplest and mos t straightfrward but carrying a lot of dead weight fo rthis and low speed drag. So , for commercial vessels , go for a V form foils. Therre are some additional benefits to this, but mainly as the foil creates lift , more of it comes out of the water and less lift from the parts of the foil left in the watwer . Problem there is oscillation , from ventillation near the surface being drawn down and we need a bigger foil to offset the loss from ventillation. A vicious circle. They work to a certain extent but not hugely efficient. The modern , as used by AC vessels is to use L foils or T foils. On these we can change the angle of attack. The AC yachts will cant the entire foil , forward, level or aft for positive , neutral or negative attack angle, so they can trim for best balance. It is a person doing this, computers are not allowed, a human trimmer has to fly the vesel , playing the angle of attack and get the vessel to run at at a particular height above the water. This is difficult and the early runs required the knowledge and skill of pilots to teach the relevant skill to these human trimmers. For Whisper , a simple mechanical solution , like an aircraft, we build a flap on the trailing edge of our foil . Its difficult to do in terms of structure becuae the flaps ar esmall out of carbon-fibre and the hinges out of kevlar. Behind the foil , called a wand, which has a float on the end and bounces on the surface of the water. We can tune the wand to the foil, so if our boat is too low in the water , the wand is pushed up from the water surface. That works a mechanism thru the board , drops the flap down that gives lift . At the perfect height we make sure our mechanism is such that the flap is level and no aditional lift . If we go too high out of the water , the wand drops down , th eflap ? a tthe back , we dump some lift and the vessel drops back to the original height. A simple mechanical solution but works well. In Whisper we could change the gearing in the system to tune for different ride height ,from 20cm to 47cm above the water. Unfortunately though simple mechanically, it is expenside to manufacture. In terms of materials for building Whisper , the cost is about 20,000 GBP but of that , the one foil is about 5,000 GBP. If you hit the bottom while out sailing then quite an insurance claim . At the tip , the end of the foil is bent down. This is a sharklet? an attempt to try ad control the tip vortices , by changing the lift distribution just a t the very end. Go too thin and it will just break off. One main foil that supports the vessel but if you have only one foil in the middle , you will tip forward or backward so need t support it at more than one point , sovthe rudders have another foil and we can change the angle of attack on those to tune. The design and materials of Hs is such that they must be strong despite a thin foil and not to flex is tricky. A real challenge in designing Hs and getting them to work properly. Thanks for choosing me rather than going to Tim Peake's astronaut talk ,also on this evening, in Winchester Q&A ? ? multiple foils , problem with tuning.? Its difficult to get it to balance up. Requires a certain amount of lift from the front foil , certain amount from the back foil and need to balance the two. The control we used was like a motorbike, a twist grip for the rudder and change the rear foil and angle of attack to balance up. The fastest that boat got to was over 30 knots. With the tendency towards climate change, there is big urge to reduce the power consumed by large ships, is there a large ship application for Hs? This is another area where Hs have an upper limit. an upper limit of about 600 tons. As you scale your vessel up , the weight of the vessel goes up with your scale cubed. Double your weigth and 2x2x2 . The lift generated from the Hs as you scale them up , scales by the surface area, so squaring. So scale up and not enough lift is possible. The power for a motorised H is quite large and in terms of passenger carrying capability , dependent on the deck space , you can get far more seats on a wide catamaran than a relatively narrow H vessel. The efficency commercial driver for passenger transit is to go for catamaran rather than H. For smaller vessels, use of Hs to reduce power, in controlling th e flow around the vessel . For small commercial vessels, we still need to get thedrag down more, then that would be possible. Last week was the announcement of the world's first diesel-electric hybris pilot vessel , the next developement to that will include Hs for efficiency purposes. If and when you hit something , firstly do they have shear-pins and secondly I went to IoW yesterday on a Red Jet. It was spring tides and low tide. I'm aware that spring tides lifts all sorts of nasty stuff that otherwise sits beached on the land. I have seen a big bit of ex-quayside baulk of timber weighted down by large ironwork so neutrally boyant and only just piercing the surface. Yesterday the vessel turned at Town Quay and started to pick up speed and there was a great clunk , you could feel through the hull and seat. He slowed down to a full stop and he did not go backwards and nothing came over the public address. I was trying to imagine what was going on. Perhaps he hit something on the bottom , as low tide, would he have had underwater cameras to see if he'd snagged a chain or a something and could see if it dropped off if he reversed? Undoubtedly what happened was something got sucked into the water intake , the depth there at low tide is 12m so plenty of water under them. The prime candidate would be a plastic bag , sucked into the intake, that would shake the vessel . This would have been cavitation , changing the flow into the water-jet unit , causing the impeller to cavitate , which is very violent , and the whole vessel shakes. Cavitate for too long and its perfectly possible to eat holes in the blades of the impellor. A fairly common event, due to the huge amount of debris in the water , natural and otherwise. Neutrally bouyant debris , typically carrier bags are the prime candidate. The shear-pin business, designed in for worst case ? You would design a fail-safe scenario . For racing vessels and high speed sailing vessels , you can make the risk as low as reasonably practical but can't negate the risk. The fisrst Whisper prototype , was sold to people who accidently hit Sweden with it. That removed the foils and did a fair amount of damage . You need it to fail at a certain point, but not rip the hull to bits as well. Rip off the foil and that disappear cleanly away rather than risk hull integrity. Red Funnel Shearwater Hs when they wer running pure Hs did loose a front foil once , the hull nose-dived and came up again . Scared everyone but no one injured. Rather than the catamarans which had foils to help the drive control , Red Funnel about 20 years ago had full Hs that would lift the vessel completely out of the water. They were built in Italy, still actually operating in Ireland. Similar to hitting debris, once you are up on the foils and moving quickly , if there is a lot of other traffic around you , navigation becomes difficult. The current red-jets if they want to stop quickly , they can just drop the buckets at the back of the water jets and it will settle fairly level , fairly quickly in a few boat lengths. With full Hs you have more distance to carry and in crowded waters that is risky. I was thinking there may be some sort of gyroscope action and that a H craft could not turn on a sixpense.? It depends how much you want to scare the passengers , a relatively good turning circle but it will bank very steeply. That asymmetric system for the 2021 series. I would have thought the forces on a simple blade keel were bad enough , but an off-centre H arrangement , looks like pretty horrendous forces involved? The whole thing just does not look right . For that assymetric structure I can't imagine what the internal bracing must be like? Its probaly milled titanium with a carbon skin around for the hull. They will be interesting , there is a move afoot I believe, that says lets work together on some of these really complicated components and then we'll see who is the fastest when it comes to driving them. Have you pics of those Americas Cup 21 series sailing? No generic name for them , not assymetric foil or anything other than AC 21. Some next generation H sailing boats. They look terrific fun but I'd not go anwhere near them personally. Flat out in the Southern Ocean they'd be doing 30 knots. Is there a sweet spot for the depth below the surface for avoidance of bumping or whatever? Debris is at all heights in the water column , but if your H is too deep under water , there is a lot of supporting strut to hold it , additional wetted surface area and extrra drag. If too close then ventillate or cavitate and so extra drag. So a lot of time spent trying to find the sweet-spot , 1m under water, 2m under water . All quite difficult to test at model scales, it can become an expensive process. For Whisper it was cheaper to build the full-scale boat and suck it and see. There are no servo-systems built into that control system? The loads going thru the foil are so great , to create an electo-mechanical system to try and move the H up and down is probably something possible in theory only. The weight of the kit would probably negate any benefit. W96

Tuesday 21 August 2018 Prof Mark Cragg, Soton Uni - Antibody immunotherapy: overcoming cancer by engaging the immune system Antibody immunotherapy (AB I) and how we use it for anticancer treatments. We're based at the SGH, just moved to the new site of cancer I. Some general introduction to ABs, monoclonal (MC) ABs which is what we use in the clinic, ABs that are now treating patients successfully, fovusing on 1 AB I've spent 20years working on Rituxamab (R) and how it works. A paradigm on how lots of different types of ABs work. The target of that AB is a molecule called C20, discussing other C20 MC ABs and some of the other ABs now being used in the clinic. An AB is a Y shaped molecule , it is dimeric 2 identical domains to the sides with a line of symmetry. The bits , the variable domains , the bits that do binding. ABs are essentially recognotion molecules, the bit at the bottom is the Fc domain which does the interaction with the I system. A ctrystal structure of what an AB looks like in 3D at the atomic level. Then ribbon colouring to show where different parts of that AB are. The FAB domains are identical on eithe rside, the Fc domain at the bottom . The bits inj the middle are sugrs, carbohydrate part of that molecule that helps keep this in the right orientation and structure. The important parts of the molecule , as d=far as AB function is to do with recognition. The loops ar ehypervariable loops and they are different between different ABs. One area is particularly hypervariable , each having a unique sequence particularly in that one region . Within your body , the diversity of ABs is enormous. So w ecan recogniose billions of different types of molecules , because the sequences in thwese regions are different. When I say they combine to specific targets, they try to recognise specific things. Its all part of what the IS does, differentiate between self and non -self. We are trying to recognise the difference between cancerous cells an d normal cells. ABs have the problem that many molecules llook similar or are quite different and we need to be able to distinguish between them. ABs will bind with one very specific molecule , and ignore all other molecules. This is important when distinguishing between self and non-self, pathogens ,bacteria,viruses all those , compared to normal human body cells. One of the primary things ABs are involved in is fighting infection. Al lthe time lots of circulating ABs in your blood. They are recognising those different molecules, particularly molecules on viruses and bacteria. The way they are generated in the body -we have 2 phases. A primary response , when first exposed t ta pathogen , we generate ABs. It sees the pathogen snd removes it from the body. The beauty of the IS is that it has the capacity of memory. If you encounter the same pathogen agsain, you are immunised, having had memory of encountering thst same pathogen . when encountered the second time you get a much bigger response. And a much more rapid response. The magnitude of response the second time is 100 times bigger. So we can fight off infection. Instead of feeling ill before the system gets going. This way the repsonse gets going before we can feel ill. Wha tpeople have been trying to do for the last 100 years is to understand their utility possibility for treating different types of diseases. Particularly for anti-C treatments. Paul Erlick had the idea of magic bullets , postulating that ABs existed before anyone had any physical evidence for that. Way before we had structures , before we could clone things or any of the moder=n biochemical tricks we have these days. He postulated the body could recognise things . Then nothing for 60 years. Millstein and Khola in the 1970s found a way for ABs to be generated in the lab. They demonstrated ABs and could isolste them and grow them in the lab. In the lab they made MC Abs. The different ABs are recognising different parts of a virus, hundreds of things on the cell surface differnt from humans , generate aBs to each of those different biits . We can generate different ABs to diferent parts of a specific molecule . So lots of ABs involved , of different specificity, a polyclonal response. Lots of different ABs mounting against one particular pathogen. Khola and Millstein generated MC ABs by taking a single immune cell, a B cell or a plasma cell, a normal cell for making ABs and they physically fused it with a chemical PEG so 2 cells fusing and the cell they fused it to was an immortal cell , a myoloma cell. A cell capable of producing lot of protein. So a single B cell which can only make a single specificity of AB with something that was immortal and would contine to produce that particular AB. They won the Nobel Prize for this. 30 years ago, now we use them for a lot of detection kits, pregnancy detecting stem cells, cancer cells in the blood , and for diseases the early detection of cardio-vascular disease, deep-vein thrombosis. This is the specificity of ABs , of great use for detecting, binding to something uniquely. A massive repertoire of their bility to recognise something. But these days we are starting to use them to treat diseases. To treat neurological disorders, auto-immune diseases , allergies and treating C. We are trying to recognise something different about a C cell. So an aberrant protein , something expressed differently on a tumour cell , not expressed on the normal cell. Then get an AB to recognise it and then in some way interct with the IS to destroy it. So why do we need new therapies, whats wrong with things like chemotherapy. A plot representing evolution of chemotherapy over a few decades, for treating lymphoma, B-cell cancers, blood cell cancers. Each line is a ore intense/more agressive chemotherpy , so capacity to kill cells with increasing intensity . But this survival curve shows it makes no difference, chemo can only take you so far. For the patients surviving out to 5 years , chemo has done a good job , but all the others have not survived. Getting more intense chemo will not work. So the use of MC ABs to use the IS to fight cancer or disease. So the AB using the variable domains will find something specific on a tumour cell, and engage the IS. The IS has multiple ways it can delete a cell thats been tagged by an AB. A protein component in the blood C1q , a member of the complement system , of a protlytic cascade and can also interact with receptors on immune cells. The reptors that find that in the Fc are called Fc receptors. Fc means it was fragment crystaliseable , one of the first parts they could crystalise and so getting the atomic structure , very early on. It was particularly straightforward to do that. So we'll bind an AB to a C cell and get the IS to delete it. Since 1997 this route has worked , for effective therapies. A curve since the first years of getting ABs thru into the clinic, approved therapies. So in 1997 there was 6 . The original ABs were mouse ABs , mous eB cells , the mice had been immunised with a human protein , the mice made an AB to that human protein, then the cells were immortalised to make the MC AB. The first ones to go into humans were also mouse ABs. Putting a mouse AB in a human will probably generate an immune response and get rid of the AB. This happened , so subsequent generations of ABs became firstly chimeric the Fc part of the AB that was mouse was then changed by genetic engineering to become human. So much less of a problem as less of the AB was derrived from mouse. More recently as we've got more sophisticated with microbiology tech, we've either humanised or generated fully human ABs. Humanised is where we took the mouse sequence , and look at the human sequence noting which bits ar edifferent between mouse and human and convert them with molecular biology. For human we generate them originally form human B cells using cloning techniques or we do a phase display library , where we take all the possible B regions and do some selection in-vitro , not in an organism. The original ABs were generated by immunisation via mice , now we can take human B cells , isolate all the different AB molecules and then a panning technology to identify things that bind to the things we are interseted in. So fully human ABs. Since 1997 essentially an exponential increase in the amount of aBs that gain approval. I looked at the table yesterday and we are at 70 or 80 ABs approved. Plus 100s in phase 1 trials , on the route to come through, phase 2 and phase 3 . This will continuie for at least the next decade, with all the ones just starting thru at the moment. Canonical means just normal ABs in terms of their structure and function, just the same as the wild type ABs , normal ABs . Non-canonical where we're a bit cleverer and identified a particuar function of an AB is either useful or not useful and then augmented or removed it. C1q is one way the ABs can target a cell, the C1q binds , then a cascade that enables various things that happen with a complement cascade. One is we get immune activations , we get release of anaphalatoxins where you get redness and swelling inflammation. C3a, 4a/5a they bring in the immune cells to the point where these things are released. You can get coating of part of this cascade C3b which targets the cells where this activity is stimulated. This gets coated on the cell surface and in there ar evarious cells that have receptor components C3b which again allows a recognition and destruction of a target cell. Then a membrane attack complex , a multi-protein complex forming which physically punches holes in a plasma membrane of a target cell. The Complement Cascade A second thing we can take advantage of , all these receptors especially when talking of targetting C cells, are there for a reason. There not there so we can conveniently tag them with ABs , they are doingg something generally. So if a tumour cell has upregulated a protein , its probably there for a reason. THat means when we then bind it with ann AB , you potentially perturb the signalling that comes from that particula molcule. Then lastly the ineraction between the Fc and the Fc receptors on various immune cells and particularly importantly are cell types such as macrophages and natural killer cells. Anyone studying biochemistry has to learn about complement cascades, 20 protein complexes with lots of things leading to lots of other things. It starts with the C1q protein , that does the recognition , it recognizes when Fc parts of the Abs are close together, so you need a certain orientation of these Fcs to C1q , to bind. That starts the cascade, conformational change happens when they interact and they start the protolytic cascade of cleaving , C4, C2 comes in and cleaves to form a C3convertase, essentially a whole process of proylytic cleavage , releasing the next fragment that goes on to start the next cascade. It ends up with the MAC which has polymeric amounts of the last component called C9 , one molecule of C8 , C5b the coating on the cell surface. Its essentially punching holes in the plasma membrane, which then allows those cells to be destroyed. In terms of signalling , ABs can do various things , they can physically transmit a signal thru the receptor that causes growth inhibition or death of that cell. It seems counter-intuitive as to what a tumour cell would want to do, by upregulating on its cell surface, but unless there is any selective pressure, for that receptor to be detrimental , then why would it down regulate it. If it just happens to be a particular cell that evolved and has a particular receptor , which can target for destruction , we can take advantage of that with our AB. Thts certainly the case with anti-ideotype AB. Also ABs can block a positive signal coming from a receptor cell surface. If a tumour has upregulated a protein , to help it grow/proliferate/survive , then using an AB , we can block that signal. Either blocks the receptor interacting with other receptors on the tumour cell surface or it can block interaction between say a growth factor and a growth factor receptor. This is what happens wiht hte drug Herceptin , which is an aB that binds to a receptor called Her2nu, which is over-expressed on a proportion of breast-C patients. One of the successful treatments for secondary breast-C after initial treatment. ABs can also block host / tumour-cell interactions, a particular thing of interest when we consider mestastasis. When a C metastasies to a different site , it then has to generate its own vasculature. It has to get blood vessels, get nutrients into it and it upregulates the protein VegF and we can then generate ABs that block VegF , so we can stop that process, or happening less efficiently. This is hte action of the drug Avastin . These are AB drugs , that you may not have known. THe bit I'm interested in , fo rthe last 10 years. Fc receptors tht engage cells of the IS not just the cascade complement. An interesting but complicated family of receptors , parallel systems activating in mouse and in humans. Humans have to be more complicated than mice , but the principle is straightforward. We have Fc receptors that are either activating or inhibitory. Activating ones stimulate cells of the IS and inhibitory ones inhibit cells in the IS. They do that with different signalling molecules on the inside of the cells. The only real difference between the mous eand the human system is tha t we have more members in the human family . This happened during evolution , duplication of the whole genome locus and we got twice as many. They are highly conserved when you look at their sequence and structures. For understanding how these work, I'll go thru some gene-KO studies we've done in mice. One is the gamma-chain Knock Out. The gamma chain is associated with all the mouse activatory receptors, meaning that if we have a gamma-chainKO, none of these receptors are expressed or signalled. We can they say , are these gamma-chain receptors important for the function that we want to study. We still have the inhibitory receptor left , because that doesn't need the gamma-chain for signalling or expression. What happens when you engage an Fc receptor. On natural killer cells, they only express an activatory Fc receptor called 3A or CD16? , just one of them. When it finds an AB thats been tagged with its Fc domain you get a big signalling cascade and activation of that natural killer cell. The NKC can bind to a target cell and kills it, by releasing cytolytic granules ,in close proximity to the target cell. That process is called AB dependent cellular cytotoxicity, one way that Fc receptors activate a NKC to kill a target. B-cells are the opposite of NK cells , only expressing an inhibitory receptor , no expression of activatory receptors. The purpose of that in normal biology is it prevents excessive B-cell proliferation. When B cells recognise something and go on to make ABs , if we've already generated enough ABs , we need a receptor to stop that process . THis process is regulated by the inhibitory Fc receptor. Then a load of other cells in the IS that express both activatory and inhibitory. Cells called dendritic cells , monocytes, macrophages , different cells with discrete functions in the IS. Expressing both activatory and inhibitory, like a rheostat model. The more activatory you've got, the more likely you will activate that cell type or more inhibitory that cell expresses, then the more likely to inhibit that particular immune cell. We can change the expression of the activatory and the inhibitory Fc on these different cell typpes by giving them different types of stimulation. Complement factors C3A, C5, the anaphalotoxins that are released when complements get activated, can also activate cells so they have more activatory Fc receptors. Cytokines, ?receptors things about inflammation , recognition of foreign pathogens , all upregulate these activatory Fc receptors. A correlation with genetics , certain polymorphisms , different mutations seen in different individuals which correlate with more or less function , more or less expression in Fc receptors. Perhaps 50% of the audience have one polymorphism for the CD16, high affinity allele so you bind ABs better than the other half of the audience. One of you here probably has a particular polymorphism for one of the inhibitory receptor functions. We have 2 alleles of each of each of those Fc receptors. Given we have 6 Fc receptors , and multiple polymorphisms, the genetics can get quickly complicated. But it certainly has an impact. The last thing is AB isotype. ABs are formed of immunoglobulins , 4 different classes of aB. When we generate the initial immune response to a pathogen , we don't always generate the same type of AB. Could be IGB1,2, 3 or 4 and they're there for different reasons. If we are trying to recognise a carbohydrate mo;ecule, then we tend to generate more of an IGB3 type response. Trying to generate a very potent response , a good way of deleting things , we geneate IGB1 . This is generally the isotype we use to treat C cells. We try to use the most effective isotype of AB to interact with Fc receptors. For tumouur destruction we want more activatory Fc signalling and less inhibitory Fc signalling . So the ABs in the clinic. Now 31 as of checking yesterday approved ABs for C. 28 approved, 3 are pending . Ritaxomab, Aflatumomab , all with mab at the end as monoclonal ABs. Ibutamib ? , tocitumamab ? are all directed to the same target . HER2 , EGFR, VegF the Avastin targetting molecule . All the trailblazing was around the target CD20 . 4 ABs initially approved . CD is for a Cluster of Differentiation giving it a designation so they could be compared with other people's ABs around the world. To know that one AB bound to the same target. So clustering all the ABs together if they bound to the same thing, then named that thing CD20. CD20 is the 20th one that got a name. In 1980 we were working on murine ABs, purely generated mice. 1F5 was about the first AB that ever went into a human. Subsequently another AB abrutonib, tioxitan? followed by ritotomab which is a chimeric AB having a human Fc domain rather than mouse Fc. It was approved in 1997 and since then a whole host of other ABs. As they progressed into the clinic, so has the technology . So from mouse to chimera to humanised , to human , the non-canonical AB. THey have modified Fc functions, doing things differenty to normal ABs . R was the first monoclonal approved for the treatment of C. It has had the singlemost action releating to patient responses , in the 30 years since licensed for lymphoma. R binds CD20 which is expressed on lymphoma cells , its expressed on leukeamia cells , white blood cells. Its a bit like the chemotherapy slide , that doesn't always work. Chemo1, more advanced chemo2 and chemo3, the mortality from lymphoma, going up over time. Thats no tto say these drugs are not effective, just that the incidence of lymphoma is goiing up and we're not curing people with these chemotherapies. THen R licensed in 1997 and the mortality starts to come down . Even though the incidence is still rising. This is using R and chemo, R only works in a transient fashion on its own , dependent on the type of lymphoma. But they are used entirely in combination. THese days, treating not just lymphoma but also a number of auto-immune disorders, like rheumatoid arthritis , SLE , MS. It deletes normal , auto-immune and malignant B-cells, the whitr blood cells that generate ABs in the fisrst place. We're using ABs to delete them because they cause different types of disease. In the lab we tried to understand which of these maechanisms was really important for R to work. If we understand that , we could decide which mechanisms , to enhance, to make it even better. To do that we took advantage of some mouse models . So a mouse that expresses the human CD20 gene , so its transgenic expressing human CD20 meaning we can use anti-human CD20 ABs . We do adoptive transfer experiments. Taking cells from such a mouse that expresses the human trans-gene, label them with flourescent dye , we mix 1:1 ratio with normal B-cells from a mouse , that don't express the trans-gene . We put into a recipient mouse , then we allow those cells to traffic the normal organs of the body , the spleen predominately . We give a therapeutic AB one day later and read out how well the therapuetic aB deleted the cells we put in. A complicated assay but straightforward to read. Using flow-cytometry , labelling up your cells to identify B-cells. Identifying the cells with low levels of the dye or high levels of the dye. A log-scale plot of CD transgenic B-cells, control cells. Expressed as a histogram there is the 1:1 ratio. What we look for is whether an AB can then delete transgenic B-cells, which it does. So we showed the AB deletes the target cells. The assay is semi-quantative , ratioing of peaks and we can see what happens putting in a control AB , we don't get any deletion . But put in R AB , we get 80% deletion. We can change the recipient mouse and assess which effector function is important. C1q the important protein from the complement cascade, has the potential for doing Membrane Attack Complex ? cells . But if we place in cells ofa mouse that lacks this protein, or the next one along the cascade , it makes no difference. The ABs are perfectly able to delete those target cells. Complement is not the really important mechanism. The ability of an AB to transmit a signal that might cause a cell to die. Protein VCL2 ? is an antiapoplotic protein , it blocks death, it stops cells dying thru the signalling process. Again the ABs can delete them . If we did the same experiment and gave those cells chemotherapy , these cells would be protected from chemo, because thats how chemo works , it causes apotosis , the death of the target cells. But ABs and chemo work in completely different ways. Chemo would be made resistant by this particular gene , but the AB doesn't care, its able to delete it , as if it wasn't resistant. The take-home message is, if we go back to the gamma-chain knock-out mouse, which doesn't have any activatory Fc receptors , we get no deletion. This is exactly the same as if we didn't treat the mouse. If we give the mice chlozinated liposomes? which are taken up by macrophages, the phages are phagocytes, they eat cells and eat things clearing up the body of debris. If we get rid of the macrophages , with chlozonate, again we get no deletion . If we chop off the Fc part of our AB and make a FAB2 fragment , done enzymatically, again no deletion. The same is true with complement killing . B-cells express the inhibitory Fc receptor , the only Fc receptor they express. THe more they express, the more quickly they internalise the AB, if you have lots of the inhibitory receptor at the cell surface. The FAB domain doing the binding to CD20 , the Fc sticking at the back, and the Fc can then bind the Fc receptor on the same cell surface , and that causes an internalisation process. It gets dragged into an organelle caled a lisosome , which degrades it, like a recycling bin. Images via confocal microscopy, labelling each component with a different colour. The R labelled green , inhibitory Fc in blue, and they are coincident. Looking at another cell , the AB, the Fc receptor, and the organelle the lissosome , all trafficked there together. In the lisosome it gets chopped up and degraded. The AB is binding at the cell surface , Fc binding to the Fc receptor , internalised and degraded. We postulated that for lymphoma , that could be a possible resistance mechanism, the internalisation and removeal from the surface. To check for that, we go to clinical data. 2 different clinical trials, 2 lymphomas that are treated with R. We get the diagnostic blocks from those patients , stain for the inhibitory receptor, we quickly see a difference between different individuals and lymphoma. Either completely negative or incredibly strongly stained and then if you stratify your patients according to that , the patientrs who don't have lots of inhibitory receptor on the surface, do much better in survival , than those who express high levels. Mandelson Lymphoma in the context of chemo with AB . The other ? lymphoma , is given as monotherapy , the only incidence where we give R on its own without chemo. Where we don't have the inhibitory receptor, we do better than where we express medium or high level . THe AB binds to the CD20 molecule and if the cell did not express the inhibitory receptor , the Fc gets bound by the macrtophagre, gets deleted and all is happy, getting rid of the C cell. If the cel l expresses inhibitory receptor anfd allow the internalisation process to happen , then it gets trafficked into the lisosome, degraded and then the possibility of resistance. The next question is how do we overcome resistance. Some early lab work , we recognised that though we can generate ABs to the same target, they might have different activities , definitely the case. We classified as type1 or type2. The type1 do clustering in the membrane, R causes all of the AB molecules to cap together on the cell surface . Type2 stay all around the periphery of the cell. There are othe rdifferences but the key point is when you go back and do the internalisation stage , the AB gets internalised. Toxitumolab which is a type2 doesn't do that anywhere near as much. The second AB doesn't internalise as much and our hypothesis was such that the internalisation was the reason for resistance. We tested whether the type2 ABs might be better. the first assay we did was to see whether we could delete normal B-cells better in the CD20-transgenic mouse . Deletion on 1 axis and repopulation , red type1 ABs, blue type2 ABs . One deletes better and for longer the type2. A tumour model so a transgenic human CD20 cross to a tumour model and looking at the data the type2 aB can delete better and for longer. This is what we do in the lab but ultimately someone has to test this in the clinic. So the human model Opatuzamab a typ2 Ab , given a particular chemo versus R and the same chemo. A difference in the survival curves. Improvements each time, learning and understanding what the different mechanisms are, to improve that. The idea of pimping your ABs, based on the effector function that you think is important. Macrophages are important, the Fc receptors are important. So we would improve the Fc so it binds to Fc receptoras better. Some people are using ABs with other types of drugs, small molecule drugs, targetted drugs rather than chemo. BTkyanase ? inhibitors is one and the data is pretty convincing. R on its own , doesn't cure these patients, but give a PI3kynase ? inhibitor its much better. But you have to undersatantd the mechanism and which disease to apply them in. The new kids on the block are immuno-modulatory ABs. ABs that don't work by targetting the tumour cells directly, they engage with cells of the IS. They bind to an immune cell , stimulate it and then the immune cell goes off and kills the tumour cell. THey can be agonistic , meaning they stimulate receptors or they csan be blockers , meaning they block an inhibitory receptor thats already been upregulated. Both are trying to do the same thing, trying to boost an existing anti-C immune response. By predominately increasing T-cell responses . THere are 2 arms of the adaptive immune system, one is the humeron ? system that is ABs and B-cells and the other arm is T-cells. T-cells have a receptor on their surface , the T-cell receptor, and they work like ABs. Similar sort of diversity, can recognise lots of different molecules and if correctly activated , go out and seek out a tumour cell and destroy it, they don;t need another AB molecule to do that. Fc receptor system have 6 , the T-cell system with an interacting partner called an antigen-presenting cell. On one side of the diagram the positive side to stimulate the T-cell to go of and kill something. On the other side are the negative regulators, the things that shut T-cells down . The tumour cell upregulates all the negative regul;ators and down-regulates all the positive ones. Because its rtrying to hide from the IS, so we try and reverse that. We try to restimulate the receptors and not the others. Being AB people , we use ABs . So a whole host of them for upcoming trials, for the activators and another host of them for the inhibitory molecules. The rewarding thing, is the kind of responses you can generate. A response in Melanoma , a patient with a big lesion , given the AB interlumamab an immune-modulatory AB , by week 16 obviously downsizing, and the patient remained tumour free. This is the hope and the dream of ABs. The reality is, most patients aren't sucessfully treated with that particular AB intralumamab but some of those patients who are successfully treated can have long-term remissions , to the point of them being cured which is not what you see with chemo. Where you have people coming back with regressions. If you are in the cohort treated with infralumamab and you are tumour free by 2 years, you are likely to go out to 10 years. This is partly due to the IS having memory. We can generate T-cells to generate memory, then they are there and ready to fight off C ,if it comes back. No othe rtreatment like that, chemo is given then its out of the body and if the tumour comes back , its not there any more to do anything. A T-cell has memory and can come back and fight the same tumour. Its considered that infralumamab is cutting the brakes. There ar elots of signalling pathways going between antigen-presenting cell and a T-cell, so making it activated. Tumours stop this T-cell becoming activated. It upregulates the receptor called CTLA4. In the normal part of immune regulation , you want to sometimes stop T-cells becoming too active as you get auto-immunity. So we need to be careful about stimulating T-cellls too much. Tumours co-opt this system and they artificially cause the upregulation of the CTLA4 molecule. We come along and block that interaction, so releasing the T-cells fro mthat suppression. Movie of the T-cells coming round to find the tumour cells, they are dynamic in their search. Like an NK cell , they punch cytolytic granules into them, and the tumour cells die. T-cells are serial killers, they keep on searching and destroying tumour cells. These drugs have only been licensed the last 3 or 4 years . T-cells are regulated by the tumour , we sort of knew anecdotally but we now know in a lot more detail . If w ehave lots of T-cells in a person's tumour, that is a good indicator that it will work. Its showing the T-cells can recognise something but they're being suppressed by the tumour. If we can intervene at that point the T-cells can then kill the tumour. There ar elots of different things they can recognise on the tumour . One thing they recognose is immutonome , bits of the genome , expressed as proteins that are different between the normal host cells and the tumour. C is essentially a disease of mutation , so differences between normal host cells and the C cells and the T-cells can recognise those differences. We are at the beginning of this, trying to understand how all those receptors interact, hoew tumours do things differently and trying to work our way thru , to find the best treatments for different types of C. A cautionary tale. Switching on T-cells is good , we want to kill tumour cells. But switching on T-cells , across the body, can be dangerous. If we just switch on T-cells , without anty regulation , they can attack cells of the host. With different types of immuno-modulatory ABs , a certain proportion of people have adverse side-effects. If we use anothe rone , with a different type of blocking molecule and then combine them. We can get an incremental increase in response , but we also see an incremental increase in toxicity. So its a balance of understanding when and where to use the different interventions. This is the path where we are at the moment. ABs can interact in this complicated system , the potential to reverse the exhaustion? and they have great potential but starting to combine one AB with another, increases the complexity exponentially . In haematology particularly its complicated and using against solid Cs , lung, breast C , it is tricky. Bu t the only way to understand even better , is understanding the tumour-host cell interaction in much more detsail. This is what we spend a lot of time doing. Combining direct-targretting ABs with immuno-modulatory ABs is what we are excited about and is one of the trials we've just got running in Southampton. A big team here, just in our AB and vaccine group there are 50 people , not including the patients who give us the samples, the nurses and the clinicians who provide us with primary material, which is critical to what we do. Q&A The tumour often metastacises and there wil lbe further mutations and then the later mutations can be resistant to a certain extent , is that the case? Very much the case with chemo , a type of treatment to induce apoxtosis, to kill cells. Lots of ways a cell can modify itself so its resistant to that kind of thing. So for chemo to work, needs the cell tobe in cycle and proliferating. So firstly you select the cells that are rapidly proliferating , which lots of cancers are doing. What it doesn't then do is kill the ones that are dormant, because its not part of that process. There are multi[le mutations that people have documented , that are the reason why some chemo doesn't waork. D53? , the Guardian of the Genome , a massive regulator , it recognises DNA damage which a lot of chemo induces. Most Cs have some form of dis-regulated P53 , by mutation or by another mutation. If a tumour has escaped the IS, from a reasonably large pool of tumour cells in the fisst place. We've learnt in the last decade, that every tumour cell is diferent, in any individual, either genetic or epigenetic , a minor mutation in one particular clone which is 0.0001% of a tumour . If that one does not get killed , then it gradually progresses, taking on further mutations and it re=appears. The clever thing about the IS , is that the IS can evolve along with a clone, typically what happens. In terms of the IS process , we look at it in 3 phases , mostly a host cell will mutate , start on the pathway to a tumour, the IS recognises it and gets rid of it. But sometimes, for whatever reason the IS doesn't recognise it , it expands, takes on furthe rmutations and then there is a dynamic balance between control by the IS and the tumour trying to expand. That equilibrium is not a problem for the host until it goes to the next phase where it escapes, largely escaping the IS. When the IS cannot ewgulat eit, it can expand and mestacicise to another site. All that is disctated by genetics, mutation and epigenetics. What proportion of tumours are knocked out by the IS and we never know about them? It must be really high. You look at every time a cell divides there is a chance of mutation. Do such calculation and we would all have died by C , by the time we got to a million cells or so. We have billions of cells and we change them all the time. P53 ? is one of those helping in the process, it recognises a mutation has happened and makes the decision either to kill that cell or stop it proliferating. That why there is massive pressure for tumour cells to escape from P53. So the control by the IS and by P53 are probably the 2 most important things to tumour developement in the first place. I'm assuming , for a clinician, there's a large computer system to for all different variables and permutations , its getting beyond any human? I'm not a clinician, they are clever, but not that clever. The way we analyse these data nowadays . We've gone from a system of biopsy, pathologist examining that, do a section, incredible insightfaul ability to know whether grade1, 2,3 , recognising the type of tumour just from what it looks like . Even really sophisticated machine learning algorithms and supercomputers, can't do what patholofgists can do, yet. But they will be able to , as somewhere along the line there is a pattern. We are good at pattern recognition but humans have innate bias so a reason why you would like computers to do some of this. You can teach computers some of this and it does it quite well. Thats just at the level of looking at a bit of tissue, then factor in every tumour cell is mutated relative to all the others, in an individual, its incredibly complicated. So if you can let the IS do your thinking for you , because it can evolve with the tumour as it changes. We need to understand how the tumour and IS interact on an individual level. Each individual has differnt mutations , and each of those has a consequnce as to how it interacts with the IS, how much it proliferates , its ability to metasticize . Only by careful study and understanding of tumours can people start to learn that. At some point you have to abandon one gene , one understanding type approach, you have to have pattern recognitin software looking for trends. We will guide them , as clinicians, in the concept of personalised medicine, looking at their whole mutonome. How its changed, that is the dream of 5 years ago, the mutation burden of any individual. Now we have to do it on single cells, to understand how every single tumour cell is different. Some of those will be resistant to treatment, the ones we need to know about. At some point I think you get commonality of process . Although sinlge mutations will be different they are all impacting at certain nodes and certain processes. So, this breast C patient is deregulated in pathwayA and pathwayB, then we should rtreat him with this. If its patwayC&D then with something else. So a bit empirical , I think that will be quite a successful approach. We don't yet know which we should try together, there are all those receptors on that pic and lots of combinations to try. The issue we have at the minute isthe more info, the more detail we know about how individual patients are different the more difficult it becomes to then design trials. We think a number of those things will be important, we are reducing the number of people with a coherent set of mutations , together , to test. What used to be the case, taking breast c, we treat it with thais and see how we do. Then a meta-analysis , seeing some survived and try to understand why that is. Once you've done that , you can design your next tier of treatment, based on the 20% that did much better. For the 80% why did they fail , what will we try with them next. I've 2 members of my family who had R treatment and survive. For me , someone interested in data, that no one has really followed-up on their survival. No checking what their lifestyle might be etc. I ask this of many of your colleagues who have presented this sort of research. When might we see the impact of patient data. THe patients are very keen , completely involved in the treatment. We have the internet and daily data could be collected from them on their lifestyle and whatever else. Without it , you have quite a crude test of mortality rate. Did they die, if they die in England, then you get the data. If they move to Oz, then you never hear if they die. Why is it not possible to enter into an agreement with a patient , not all of them may agree. Thatt he reciprocal nature of what you're doing to save their life, they need to participate in providing regular data. So salt intake , or holidays , where they live or whatever can be fed back to you. In the past this sort of data could not be collected , but it could now be collected to the nth degree. ? It all comes down to money. The NHS is under pressure, just for giving the drugs. R took 20 years to develop. Say NICE put this drug outside your limit, however Mr Roberts, if you're prepared to daily , via the internet, and answer 100 questions, then you can participate in the trial, but we're not collecting? One is trial and the other is treatment. Within a trial, the data is reasonably well collected, money provided to take samples. In other diseases, done much more holistically. So the MRC funded 100,000 people just to be monitored, blood pressure, lots of routine blood tests and then a population study to see what of those people get a disease . Very expensive . But if the people generate the data themselves, via questionaire? In terms of resources, I don't know who would push to get that data collected and who would pay for it. NICE is already making decisions not to fund drugs that will extend people's lives , because of cost. That is something that could potentially extend someone's life. We have discussions with drug companies about what is the benchmark , the lowest level they can afford to sell and similar discussions. I don't disagree with you , the power of data is enormous. Some of that data gathering is exactly what we do , looking for patterns, in things we don't necessarily think to look for. But its a masive thing to do, just to collect that data. The 100,000 genome project is starting to do that for multiple Cs. 10,000 lymphomas in terms of mutation, but I agree they're not collecting patient data. Technically the NHS should already have that data, but even accesssing that , as a scientist who wants to access data , is a problem. Isn't Google there already, R and what it does? Yes but not at an individual level. If I asked our clinicians for all the data on 20,000 people treated with R , the answer is no because of data protection. I agree with you, if at the time of treatment people signed things to say they were happy for us to use their data , that would be fantastic. Currently we have ethics committees worrying about samples and access to them. It is a real problem , and something wider society struggles with. What are we goiung to do with this data , when we start sequencing people's genomes. We are not looking at individuals but we have people in ethics boards that make it very difficult to access such data on the understanding that the general public find it a problem, but I doubt the general public consider it a problem. When being treated , they're very willing to engage and would like to help in subsequent research. There were leukaemia clusters , is that still true? There always was hotspots and I don't know if thats been resolved. Radon was a suspect one time, or living near power cables etc, stil lnot resolved as far as I know. Is the legal and ethical constraints exclusive to this country, or also USA , France and Germany etc? Not so sure about the USA , at a higher risk of litigation. There is a different dynamic in the US , where you're paying a clinician in a much more direct way than the UK. You can buy a particualr treatment, and you as a patient are more involved in that selection. Here you have standard treatment, if that fails , you can enter a clinical trial, if you wish ot do so. Have you heard of Avalon/Babylon? Health, an AI engine run by IBM. It is intended to diagnose 100,000 ailments , collecting data from people who are suffering or asking questions and synthesises likely diagnoses, and it will happen? Those kind of providers are the sorts of people who could set these up. For them its demonstrating the poweer of their supercomputers, so a vested interest there. Whether the NHS or us should be funding that , or C charities, I'm not sure. The general public is concerned about their data and the likes of Facebook, and such concerns are feeding into9 the workings of ethics committees. There have been cohort studies. Since the 40s or 50s there have been children , followed thru, looking at their lifestyles over lengthy periods. We're in such a good position now to get more data, as we now feed it straigh in, not via paper . There is still so much biomedical research rather than social research which then may suport diagnostic work. A friend of mine , his wife had something , eventually he got her clinical records ,foot high, scanned them in and put the whole lot on the internet for access by anyone who might read and learn from them. Presumably these treatments ar einjected? and how often? Yes but some ABs are given subcutaneously now. R has been reformulated and can be given oncw weekly. They looked at the dose . A body of research suggested that rather than internalisation, there is a second process called tromacytosis? , where when the IS macrophages get full to capacity , they stop engulfing the tumour cell and they start chopping of fthe receptors, which would then leave the tumour cell alone. A second school of thought was that that would be a problem, a way around was to give less AB initially so the system never got over-saturated. That trial stopped early as it became clear that giving less AB was not a good thing. A lot of clinical practise was based on what was successful before . When you start into patient studies , you start to get into millions of poundes. Doing what was done before , although the AB is different , but its already a procedure , everyone is signed off on it and it seems to make sense. But its not necessarily optimised for that particual treatment. If a trial works , thats it. Some people measure the half-life of ABs , they are good in hanging around for a long time . The average half-life of an AB is 21 days , so its not like small molecules. Small molecules get turned over quickly , in hours, so you need to take a tablet every day or whatever. With aBs weekly or monthly injections are possible. Particularly lymphoma, treatment R only and no chemo , that is given initially weekly , then maintainence shots every 2 months. Now people are doing more modelling studies , to get some analysis of the aB half-life , then mathematical modelling to say when the optimal retreatment time is and its dosage , for that particular AB. We're developing technologies to extend the half-life even longer . Talking of canonical/n0n-canonical Fc parts, you can change that Fc , so it goes out to 60 days rather than 28. Then those technology changes have to be tested in the clinic. Immunotherapy effective against solid tumors or just ? ? That relates largely to how mutated the tumour is. Things like melanoma have lots of mutations, so that gets treated quite successfully with some of the immuno-modulted ABs. The lymphomas we work on less ? mutations , so there we use things like R as immuno-modulatory don't work so well there. Its based on mutation burden , that why for some things like lung C , actually having smoked increases the chance that you will be successfully treated because you have certain types of DNA damage from smoking, that are recognisable by the IS. It seems unfair , but that sthe way it works, the correlation exists. You mentions MS as well , in the media recently, getting approval? I think its now at phase3 after a couple of phase2 trials. For other diseases, lots of immunologies polarise to certain individuals , thinking their particular cell type is mor e important and the drive rof all diseases. Such goes through phases. One moment everyone decides dendrytic cells are the most important, then T-cells the most important. I work on B-cells and I think Bcells are the most important. For a long time people thought B-cells were a bit stupid and all they did was make ABs, that they were'nt involved in likes of regulation. Its only when people started deleting B-cells with R , they began to understand they were much more involved in the regulation of the IS. It should not be surprising as all these cells talk to each other, in feedback loops etc. I find it surprising with MS though. Auto-immunity made a bit of sense , ABs might be involved somewhere in the disease, you're getting rid of a cell type that is part of generating that auto-immune process. With MS it was not so obvious a jump. I don't know how the first person gets treated with that, I'm not sure there was a huge basis but it all looks interesting. As people look into it in more detail there are extopic organelles and lymph nodes in some of the disease sites, which may be affected by deleting B-cells. Or it might be something secreted by b-cells , that snow been removed and so less of an effect. A b-cell effect? Thats what you're doing deleting B-cells , that not necessarely negates that your doing something to T-cells ??. Its not part of something that seems to have been around for the last 20 years or so , repurposing of drugs like Thalidomide suddenly found a completely different use. I'm assuming because of big computer systems that can pick up such unconnected things? One of the other AB drugs Alantuzomab? used originally in chronic lymphocitic leukaemia and works for a certain proportion of patients , that have particualr mutation status , that has now been repurposed for MS at a much lower dose. This again comes back to the questin of why that was done, it was a decision made on population size . They were treating a very small proportion of patients quite succesfully , not making much money. They've repurposed the same drug and taken the anti_C drug , no longer available on licence, but they are using it to treat MS which is a much larger pool of people. Again that is outsdide of our control, its a pharmaceutical company decision. Then CRUK might come in and treat what is a rarer di=sease and licence a drug for that particular purpose. Is proprietary strictures a problem in this research area? For R , the patent is now off licence, so people are making it in India or wherever bio-similars. It was patented before it was licenced, less than 20 years . Those ABs exist, and a big area of research involving bio-similars, how ABs are made and produced. They are not like simple small drugs, not compounds with a couple of rings, 150 Kilo? of proteins , a carbohydrate that is not identical in every molecule , a lot of range. Normal human ABs and look at the carbohydrate, there is a huge range in how big they are, what they're composed of, it changes during pregnancy , a lot of modulation. When you doi a monoclonal and make it in the lab , its still an issue that 1 cell will go thru the carbohydrate bio-synthetic route slightly different from another , depending on what cell cycle its in , what nutrition it has , all sorts of things. A whole area of QC and validation of what makes that drug, what are its parameters of use. Aggregation of ABs is one thing they control for , carbohydrates have to be within a certain limit and a field of work around that. But then if you want to start treating patients with a bio-similar then technically, but the laws ar echanging and evolving, you have to do new full-randomized clinical trials. Any drug company has the next drug in waiting for the next 20 years. As long as they can demonstrate superiority , lots of governments and health initiatives buy from the original source. At the minute lots of ABs are coming on-line at the same time, lots of big pharma and small bio-techs , see that its a huge market with potential of having a block-buster drug. They're all crowding into the market. So 10 anti-PD1 ABs which probably all do the same thing but only 1 will get licenced for a particual thing. Then someone else wil lget their licence for a slightly different thing. But probably working in the same way as the simplest type of AB are those that block something, because you don't have to engage the IS, or involve lots of complex biology , just have to stop one thing binding to something else. If that is the action , then almsot any AB wil do that. As to one being better than another, no one will ever do the multi-million clinical trial to show A is 0.1% better than B and get it licenced.

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