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may i first of all acknowledge the traditionalcustodians of the land upon which we are gathered today, the kombumerri people? may i also acknowledgethe chancellor, leneen forde ao; mr terry robertson, doctor at the university, chairof the gold coast advisory council and deputy chair of the institute's board of advice;miss susanne gallagher, representing the gallagher family today; board of advice members; otherdistinguished guests; ladies and gentlemen. may i welcome you all to an event, what iconsider very special to the institute. that's he peter gallagher memorial glycomics lecture.it's a very special event because peter gallagher was very special. he was a long-term supporterof the institute and before that he was a legendary rugby league football player, asyou can see in this wonderful diagram, an

australian representative and indeed a captainof our national team, the kangaroos. peter was also an adored member of the gold coastcommunity and did an enormous amount of work in the gold coast community, particularlyin his roles within the racehorse industry. he was very much a can-do person. that iscertainly what he brought to our board of advice that many years ago before he passedaway. so we celebrate peter's life every year for his contributions that he's made not justto the institute but also to the gold coast and to australia in the broadest context byhosting this special public lecture in honour of his life. so moving to the lecture todayand by way of introduction our 2014 lecture is indeed a distinguished glycol-scientistresearcher from imperial college, london.

it's my pleasure to welcome professor annedell who is by the way, even though based in imperial college, and she knows i was goingto say this, is an expat western australian. in fact, she graduated from the universityof western australia in chemistry at that point in time and then left australia in theearly seventies to advance her research opportunities, taking on a higher degree at the universityof cambridge, supported by an 1851 exhibition research scholarship. for those who don't know that's a very prestigiousscholarship award that enabled particularly australians to head to the uk to undertakehigher degree research. after completion of her phd anne then moved to imperial collegeto commence her career where she quickly rose

through the ranks to professor in 1991. icould go on and on and on about anne's career trajectory but i won't. just to summariseit, anne, at least i think in our community and in the scientific community, is one ofthe world's outstanding research leaders in the area of carbohydrate chemical biologyand has made enormous contributions to this important research field. importantly, anne is indeed a marvellous iconfor all students and for those students particularly who aspire to great things, particularly femalestudents. anne achieved all of her outstanding contributions with time for a family and ithink that's such a wonderful thing that we see that you don't have to choose one or theother. you can actually do both and anne's

such a wonderful icon that i think all ofour students can look up to. in recognition of her research outcomes and achievementsshe has been bestowed numerous awards including appointment as a commander of the order ofthe british empire, importantly elected to the fellowship of the royal society and awardeda wellcome trust senior investigator fellowship. all of these are signals of a person thathas done amazing things in their scientific career, so we're delighted to have her. ihave no doubt that you are going to enjoy her lecture entitled communication with sugars.anne, the floor is yours. anne dell thank you, mark. it's wonderful to be here.thank you very much for the invitation.

thank you very much to the audience for coming,i think, some distance, some of you in traffic, to hear all about communicating withsugars. so having been told that we were celebrating peter gallagher, i thought great,i can say i know all about rugby because i actually live a couple of miles away fromtwickenham rugby stadium and the supermarket i normally go to is here. that giant big car park is usually used asan overflow car park when the twickenham car parks, which are always full, are filled up,which means i can't go to the supermarket. so when there's a big match on i complain.however, having prepared this slide nicely one of my colleagues at work said to me, "ispeter gallagher a rugby union player?" and

i said, "what do you mean, rugby union player?rugby is rugby, isn't it?" so a couple of hours later i was by then an absolute experton everything to do with the difference between rugby union and rugby league but i thoughti would continue to use my slide anyway. i am now sufficiently expert that i know thatthe grand final or whatever it's called here, of rugby league was played only a couple ofdays ago, weekend or whatever, with the rabbitohs winning. i also know that the reason thatthe rabbitohs won is because they had three english players playing for them, three brotherswith one of them winning the man of the match, yes? communicating with sugars, now, i do appreciatethe audience is quite diverse and many of

you will feel you don't know anything aboutsugars but actually all of you do. i don't think anyone in the audience would be unawareof glucose, particularly with all the publicity given to type 2 diabetes and the need to havethe right levels of blood glucose. so glucose is a sugar. it is, if you like, the most famoussugar ever. we wouldn't be here on the earth if glucose wasn't around. it is, as far asmolecules are concerned, molecules containing carbon, which are organic molecules, it isthe most abundant organic molecule on the planet and it is largely that abundant becauseof cellulose in trees and other plants. cellulose is a polymer of glucose. the glucoseas well as it being part of all the plant world, it is the most important energy sourcefor everything including ourselves but i am

not planning actually to talk very much aboutglucose today. i want to talk about relatives of glucose. glucose is a relatively smallmolecule with six carbons and there are a lot of other related sugars that actuallylook very similar and i don't expect you to remember or even think about the structureson here. most of the time, for the rest of the talk, i'll be using these little symbols.so all you have to remember are a few colours and a few shapes. what this slide is indicating is that theother sugars that i'm talking about can be derived, and usually are, from glucose andare actually very, very similar to glucose. for example, the sugars which are being shownas circles of different colours, if you're

eyesight is good enough and without my glassesmine isn't, the colours are indicating what we call stereochemistry or, in other words,shape. the difference, for example, between the blue colour here, which is glucose, andthe yellow colour, which for some reason keeps shimmering, the lactose. there is an oxygenwhich on this sugar is pointing up and on this sugar is pointing sideways. that's theonly difference. it's just whether it's up or whether it's sideways. that makes it adifferent sugar. it's called galactose. similarly mannose over here is � in thiscase you can spot the difference. it's actually this oxygen is pointing in the other direction.i'm going to tell you a lot about mass spectrometry today. you might think mass spectrometry issomething either you've never heard of or,

if you have, it's not very interesting. whati'm hoping to tell you is that it is interesting. what a mass spectrometer does, as we'll seein a minute, is to measure mass. and with respect to these colours the only thing thatyou have to remember, you don't even have to remember that but it will help you to understandsome of slides, is to remember that anything which is the same shape has the same mass. the colours indicate also a relationship butyou don't need to worry too much about that. but if they have a different shape they areactually a different sugar and you'll notice this one has a nitrogen here instead of anoxygen but that is all you need to understand about this slide. i promise you that afterthis hopefully things will get a little more

interesting. mark has already said that i'mat imperial college. i thought it might be nice, if you haven't had the chance to goto london and therefore you will not have had the opportunity to visit this part oflondon, which is hyde park, kensington gardens and the most important bit which is imperialcollege. imperial college sits in the middle of thisrectangle. up here is the royal albert hall and down here are a variety of museums, naturalhistory, science museum and over here the victoria and albert museum. mark has alreadygiven quite a nice introduction to what i plan to talk about at this point because hementioned the 1851 award which i went to the uk on. and 1851 is the year and it dates backto the first great exhibition, as it was called,

but the first of the science world fairs.so the uk held the great exhibition in 1851 in hyde park. the amazing thing about thisexhibition was that it was housed in this so-called crystal palace, which was a largelyplate glass building of cast iron and plate glass. the interesting thing about it is the abilityto make plate glass had only actually been worked out just a few years before 1851. sothe modern technology was being exploited. the other thing, particularly for the youngerpeople in the audience, as to how far you can go very rapidly, is that the design ofthis building came from a person called joseph paxton who had been a gardener and was actuallya head gardener for the duke of devonshire.

joseph paxton was the younger son of a farmer.at 15 years old he was apprenticed as a gardener and went to work for the royal horticulturalsociety in the uk. so apart from farming training he had virtuallyno training but he was a bright lad and he did well and the duke of devonshire used tovisit the rhs gardens in london and used to chat to this young gardener. when he was about20, i think, the duke of devonshire mentioned that because he knew so much about gardeningperhaps he'd like to be head gardener for his at chatsworth house. no one really knowshistorically whether this was a serious invitation or not but within days joseph paxton had packedhis belongings and headed to chatsworth. the duke wasn't there at that point. he'd gonetravelling in europe.

he had got to know the head housekeeper andchatted with her and got to know his future wife, who was one of the servants, and essentiallystarted to rebuild the gardens of the duke of devonshire. although he was not a trainedarchitect he drew a sketch for the commissioners for the 1851 of what he thought would be anice house, as it were, home, for the great exhibition. and it was accepted and withinnine months it was built. so this is a remarkable achievement for engineering. it also is aremarkable achievement for public outreach because the exhibition was open from may of1851 to october and in that time a third of the population of britain attended. bearing in mind that the railways had onlypretty much just started at that point and

people had to get to london. so somethinglike six million people went to the great exhibition in that time and the great exhibitionmade a profit, a very handsome profit. and from the money of the great exhibition allof the land here, all of that land and in fact land all the way out here, which wassubsequently sold, was purchased by the royal commission and in addition to that, as markhas already mentioned, they used profits to set up an education trust to enable studentsfrom the colonies, as they were then, to study in the uk. i was very fortunate to be going to one ofthese. i thought especially for the younger people in the audience you might like to seewhat was the email of the 1970's. this was

the notification that i had one. i was veryfortunate because i was able to go to cambridge to study for my phd but i was even more fortunatebecause although i did not go to do my phd with howard morris i for a variety of reasonsfinished up being his very first phd student in his very first academic appointment incambridge and became a phd student studying mass spectrometry. i just want to point out here this was sortof the pot of gold at the end of the rainbow. it was the opportunity to be a student atkings college in cambridge which, as you may know, cambridge and oxford students join colleges.i chose kings college because i thought it was a very traditional college and thoughtthat would be nice, to go and stay at a traditional

college, again, complete mistake but it turnedout to be a really good mistake because it suited me very well. kings college was actuallyincredibly left wing. keynes, the famous economist, had been the bursar of kings college and hadactually built it up very financially successfully but also very much created the culture. emforster was a fellow and lived in residence there. it was the kind of college which attracteda great variety of students from countries such as the soviet union. the first year iwas there, i remember in the summertime we had visitors from countries like the sovietunion staying at the college. for me as a parochial west australian meeting these peoplefor the first time was a tremendously stimulating

environment. so mass spectrometry, a littlebit about what it is, so you'll have some idea of what i'm talking about. essentiallyit is a fancy weighing machine. it allows you to measure the mass or the weight, inthis case molecules, very accurately. it's very, very widely used. in fact, i'm suremany of you will be familiar with some of its application. for example, the testing for performance enhancingdrugs is almost entirely done by mass spectrometry these days. fuel testing for the formula oneraces, if you watch a formula one race you will see just before the race starts a guyrunning from one car to the other. what they are doing is sampling the fuel and that within24 hours is analysed by mass spec to make

sure that the right fuel is being used. forensicanalysis, analysis of chemicals, but from the point of view of today's talk the analysisof biopolymers. and associated with that the modern biologic drugs, again, some of youwill be familiar with the biologic drugs. although the names might be different here,herceptin, for example, the anti-breast cancer drug, which is widely used in the uk and therest of the world, is a biologic drug. what that means is it's a biopolymer. so i wasvery lucky because i started my career in mass spec at a time when mass spectrometryfor the very first time was being applied to biological molecules. the reason it couldbe applied to biologic molecules was largely because of ideas of my mentor, howard morris,who had come up with ways at least in his

head and then industry responded by buildingthose things, of being able to analyse very large molecules. and a biopolymer is simplya polymerised pieces of polymers, as they've called, or individual sugars, for example,polymerised together to make a bigger molecule. for mass spectrometry to work on these largermolecules you needed instrumentation which at that time was essentially very, very powerfulmagnets. and these actually had not been built on til then, so theoretically they could bemade. practically they had not been built and the very first ones, the first one isthis one here, the second is this one here. these were built and delivered to us at imperialcollege and at that time nobody else had them. so that was brilliant.

so i was a post doc by then at imperial collegeand people came from all over the world to us, asking us if we could help them with generallytheir biological problems. what i'm hoping now to do is to give you an idea of the kindsof things that we do and how that information that we get from our research can be usedto understand biology better but also after that to translate it into new medicines andgenerally better healthy lifestyles for people. so what is mass spectrometry? as i said, it's a fancy weighing machine.what we do in mass spectrometry is we take a sample of any kind so that �as you imaginethis is a biopolymer with the individual units joined together. it's a very happy one whichis why it's got a smiley face, its tentacles,

as it were, antennae, are kind of droopy.what happens is we ionise it and that simply means that we take that molecule and we giveit a charge. it has to have a charge because otherwise you can't measure its mass. andthere are many ways of giving a molecule a charge and back when i started one way ofdoing it was to take an atom or ion gun and effectively fire accelerated atoms or ionsat the molecule. this again was a new technology. it was developedin the uk in manchester by micky barber and colleagues at the university of manchesterinstitute of science and technology. and all of this happened pretty much at the same time.the developer of those very big high-field, as they were called, magnets and this reallynice technology for ionising our samples,

allowing us to get mass spectra, as we callthem. what are mass spectra? they are simply its data or information that tells us herethe mass of the molecule and these other components here. notice that i've changed the colour as i convertedit to a charge. and also as you can see its face now is no longer happy and smiling. itis actually looking, "ouch, i am very hot." and the heat, if you like, that's put intothe molecule in doing that is enough to make it vibrate and fall apart into pieces. sowe get what's called the mass spectrum, which is just simply an access along here with ionsof different mass value, which tell us about the mass of the overall molecule and the massesof bits. what you'll notice here is that we

can differentiate between the ends of themolecule because this has got the now rather scared face on it and this, for example, isthe tail. so we can piece our molecule together. soit's a bit like thinking about a jigsaw puzzle that we've dropped the puzzle of the floor.it's all in bits but not entirely completely smashed up. then we try to reassemble it andwork out what it was in the first place. so that's mass spec. why are we interested insugars? there are many reasons why we are interested in sugars but one of the main reasonswe're interested in sugars can be exemplified by just thinking about biology and what itis in biology that essentially codes for our biological system.

now the first thing that most people �even,i would imagine, many of you who are not scientists will think about is the dna because genesin dna is what people often know about. but about 15 or so years ago when � slightlyless than that when the human genome project, the outcome of that, was announced by thepresident of the united states and the prime minister of britain, that's how high profileit was, it was realised to a lot of people's dismay that the number of genes in humans,despite earlier predictions that it ought to be at least 10 or even more times greaterthan this, was very little different from the number of genes in this, which is a very,very tiny worm which is about a millimetre long, this, which is the fruit fly and thiswhich is a weed.

it's a little cress weed which at least inengland you get in car parks growing and it's used as a model organism in the lab. so it'srather pathetic. it has tiny little flowers but that's about all. it doesn't do anythingspecial. but notice it has more genes than we have. the other thing that is also discovered,but again perhaps not quite so surprising, was that the chimpanzee and the human actuallynot only have exactly the same number of genes but actually the sequences are 99.9 percentidentical. so biological complexity is not linearly related to the number of genes. andthere are many reasons. i'm sure there are many in the audience who will say what about,what about, what about for the other reasons why we think we are more complex than, forexample, a one millimetre worm.

but one of the most important reasons, andfor this audience the most important reason, is because the products of the genes, so thegenes encode information that leads to proteins. so dna goes to rna goes to proteins, thisparadigm of life. but the proteins do not stay as proteins. they become modified andthe greatest diversity for modification is putting strings of sugars on them. and whatwe get from that are what we call glyco-proteins or proteins that have sugars on. so the functionaldiversity of the gene products is vastly amplified by having sugars on them. whereas we can sequencethe dna and know exactly what the dna sequences are � and of course nowadays that has becomemuch faster and much cheaper � knowing what these glycans are is still a very difficultthing to do.

so today i want to give you a glimpse intothe research that's been going on now for 30 years or more but also i hope by the endof my talk to give you an idea of actually what still needs to be done. in other words,we do know quite a bit but there is so much we don't yet know. what we do know is thefollowing. every single cell in every organism that's looked at is coated with sugars. now,this is a picture that's called the electron micrograph. essentially, what is is, it'sa slice through a cell and then an electron microscope, because the cells are tiny, isused to visualise that slice. and on the surface of the cell here there is dye which has beenused. it's called ruthenium red, which has been used. it's shown in black here becauseit's an electron microscope picture.

the black fuzzy bit around the edge of thecell here, this is a portion of the cell, this is the nucleus, a portion of the nucleus.and this is what's called the cytoplasm or cyto-cell. you will notice that the wholeof the cell surface is covered in this black coat. the dye is recognising sugar. so whatthe dye is lighting up, as it were, even if it is lighting it up in black, is the sugarcoat. and every single cell in our body is coated with sugar. we are sugar coated people.these sugars are linked on, so this is the membrane. don't worry about the details ofthis. this is the membrane or the outer surfaceof the cell. and the sugars which are shown by the symbols that i've shown you alreadyare linked on as these sugar polymers onto

proteins which are shown in these differentcolours here, blues and brown. and they are also on lipids but we're not going to talkabout lipids today. so as i've said they are on our proteins and we have very, very goodevidence that's been built up now over two or three decades that these glycans are involvedin recognition processes. so for example these are cartoons of cells cut out just so youcan tell it's the cell, showing there is all sorts of organelles inside. but what we're showing here is that when wehave a cell from, for example a mammal, perhaps a human, then when a pathogen, an infectiveagent like an infective bacteria or a virus, and of course influenza is something thatobviously this institute is very famous for,

when the virus is infecting the cell it willengage with a sugar on the surface of the cell. and what it has is a very specific proteincalled a lectin on its surface that recognises that sugar. similarly if you have, for example,this is an e. coli pathogen which is a urinary tract pathogen, is recognising sugars. when a mammalian cell meets another mammaliancell exactly the same recognition is taking place, families of lectins, and families ofsugars, which they recognise. for example, these are white blood cells, so this is apicture taken when a camera has been put up a capillary. this is a white blood cell andthe reddish colour that you can see here is the layer of cells that lines the inside ofyour capillaries. and the white blood cell

engaging with the endothelial cell, as it'scalled, that recognition involves sugars and lectins. and what our mass spectrometry cando is it can tell us what kind of sugars are on these cells because we're trying to understandthese recognition processes and to do that we need to know what the sugars are, we needto know what the recognising lectins are. one of my very early achievements, which issomething which at the time we had no idea of the importance but actually it's takena decade or more and even now we don't fully understand the importance, was the discoveryof � because of a collaboration we were carrying out with a scientist who had cometo us from southern california from a cancer institute in southern california. and he wasinterested in how leukaemia cells differed

from normal white blood cells. just generallyat that time just wanting to know the fundamental biology and obviously subsequently the potentialexploitation of that for diagnostics and possibly anti-cancer drugs. so he asked us whether we might be able totell him what kind of sugars were on the surface of white blood cells and we said we'd giveit a go. and one of the sugar clusters we discovered was this little cluster here whichagain i've put the symbols on so that you can see the sugars we were looking at earlier.and this is a very, very famous sugar cluster now in glycobiology. generally it is calledsialyl lewis x. this arrangement of having one of these diamine sugars, it's a very importantsugar � this is the sialic acid. in fact

that's what the flu virus recognises. thisis galactose, so called glutanac, and fucose. so this little cluster we're going to seein a number of later slides. now, what we identified at that time, which subsequentlybecame very important, we observed that in leukaemia cells they had a lot more of thissialyl lewis x structure. about five years later the partner, the lectin partner, ofsialyl lewis x was discovered. it turns out to be a family, so there's a family of threemembers of the family that will engage with sialyl lewis x and recognise it. and theselectins are actually called the selectins. what was worked out, again over a number ofyears with quite a lot of research labs around the world being involved, is that the waythat a white blood cell like, for example,

a neutrophil � a neutrophil is one of ourwhite blood cells whose job it is to kill nasty things if we get infected. so normally it's circulating in the blood.if we get an infection in our tissues, an inflammation occurs, then the neutrophilshave to know it's there. they have to recognise that they need to go out of the � so thisside, this is the endothelial layer which in that proper photo was pink-red, now codedgreen, just to confuse you. so we have this layer of endothelial cells, as they are called.this side of it is the capillary. this side is the tissue. so what the white blood cellshave to do is they have to get from that side to this side. and of course they're moving.if you think about it they're being pumped

by the heart. it's flowing along here. what they have to do is they have to cometo a stop and then they have to squeeze between the cells that are lining the capillary andget to the other side. medical people call this extravasation, getting to the other side.so this is a cartoon showing what's happening that we have our sialyl lewis x on our whiteblood cell. it is engaging with the selectin, which is being expressed on the endothelialcells, and it's a bit like rolling over velcro. if you think about when you have a ball andyou attach it to velcro and then you try to move it along. it will obviously very rapidlycome to a stop. then it flattens and squeezes out.

this engagement of white blood cells withthe endothelial layer absolutely underpins everything to do with white blood cell traffickingin the human body. so for example, as i'm sure you all know, you have two circulatorysystems. you've got the blood and you've got the lymphatics. and the lymph nodes are thereally important connections where things from the blood can go into the lymphatic system.and some of the white blood cells have to do that. for example, there are white bloodcells called lymphocytes that need to go into the lymphatic system in order to circulateand do their job as immune cells. some of them, like the neutrophils, do not. they haveto stay in the blood system until there is some kind of infection out in the tissue.

so again there has to be regulation at thelymph nodes defining when these white blood cells, and which of them, are allowed to leavethe endothelial layer. that's why the discovery that sialyl lewis x on cancer cells is muchmore highly expressed was a very important observation that was rapidly proved by a wholevariety of other methods in the 1980's where it was shown that pretty much all solid tumours,especially those that metastasise, migrate, to other parts of the body were expressinghigher levels of sialyl lewis x and almost certainly use this trafficking pathway toget from the site of the primary tumour. so what i'd like to do now is to tell youa little more about how we can use � so that was the 1980's but as you will see ina minute we are still looking at those kinds

of structures. but i want to give you a betteridea of how we can use our data in a wider spectrum of applications. and this is justto point out what we are doing overall. what we are trying to address is what kind of glycansare we making in particular in the human, because we're trying to understand human biology.we very often use the mouse as a model system for the human because obviously it can essentiallyreproduce us faster, for a start, and it is clearly ethically more appropriate to be doingexperiments on a mouse than it is on a human. we also will look at cells and in fact wecan look at a whole variety of biological substances but the aim is to get pools ofglycans that are representative of the material we had in the first place to address the questionof how might these glycans be evolved in recognition

processes that lead to biology. and this isone of our students in london with one of our modern mass spec systems. now, i'm goingto have to give you a little bit of biochemistry knowledge. otherwise you are not going tounderstand my cartoons. don't worry, it's very simple. at least, i think it will besimple. so basically what you need to know is that inside a cell we have a productionline for making glycoproteins. so this is the nucleus of a cell. again, it'sonly a partial cell. so this is that plasma membrane with the sugar coat. it's not beingshown on this cartoon but inside the cell we have what is called an endoplasmic reticulum.don't worry about it. everyone calls it an er because they find it a bit hard to sayendoplasmic reticulum. and this is where the

protein is first made. so this is where theinformation from the dna in the nucleus is translated. essentially, the dna is convertedto rna and then that information is translated to a protein sequence, which occurs here. then a glycan, a sugar polymer, is put onthere. now, what is absolutely amazing about that � and this is our glycan polymer withthe cartoon, as i showed you earlier. but what's absolutely amazing is it doesn't matterwhether it's a human or a yeast or a plant or any other animal which is what's calleda eukaryote, in other words, an animal which has a nucleus. this glycan is absolutely conserved.this has been conserved since the beginning of evolution, as it were, which i think andmany people in glycobiology think is just

an absolutely amazing conservation but itreflects almost certainly the importance of this particular modification, so absolutelyconserved. what then happens is that in the er and thensubsequently in another apparatus within the cell called the golgi, there is a productionline where this is trimmed down and then built back up again. i'm just going to show youwhat happens. it gets trimmed and trimmed and trimmed further but then it starts toget built back up again. and then it finishes up on the surface. and the marvellous thingabout this is that all of the glycans have a common, what we call, core as they are attachedonto the protein because it never trims right down to take the core. so we know what's inthe core but the other thing that's important

is that we get what are called antennae andthose antennae are decorated and you already know one decoration. that's here, sialyl lewisx. arrange it with exactly the same set of individualunits but in a different arrangement and we get a related sugar cluster called sialyllewis a. take off the sialic acid and we get lewis x. lewis x is on all of your white bloodcells. sialyl lewis x is on most of your white blood cells. you will have heard of the aboblood group system. those are sugars. so basically again you will see they are all sort of relatedto each other. so they are just little patterns of sugars. and these sugars are there to,as it were, identify the surface of the cells. so what do we use our mass spec for? well,we use our mass spec, as i said earlier, to

get a mass or complete weight and then thepieces. so this is modern mass spectrometry where i had guns earlier on. unfortunately we don't use guns anymore. it'snot quite as exciting. what we use instead are lasers like a very high-power laser pointer.that's what the l in this acronym stands for � don't worry about the rest of the letters� or we spray the sample into the mass spec which is what that other acronym is. so it'sjust a different way of making the ions. it happens to be more sensitive than the wayswe used 20 or 30 years ago but we get our molecular ions and then, as i said, we getour fragment ions. the difference between now and 30 odd years ago is we do these intwo experiments as it were. we do it in two

stages in the mass spectrometer. so we can do this stage just by itself, justgetting the masses, or we can choose to do this as well. so what does that mean? becauseyou now are absolute experts in the kind of glycans that can be made by a synthetic pathwayyou will hopefully not find it too difficult to understand that if we have a mass whichwe can fit to a composition, and don't worry about the fact that this is not the wordsi've been using � hexose just means that we can't tell the difference between a greencircle and a blue circle and a yellow circle because we said they were the same mass. sowe can get the composition in terms of these what we call isomers but the important thingis we can immediately map it onto potential

structures. so we use that knowledge of thebiosynthesis to do that. if we then want to know what structures theyare we do [0:41:18.3] where it falls apart and gives us beautiful ions that tell us exactlywhere our decorating sugars are. so what that means is that we can take a range of whiteblood cells. and these are examples. i've already mentioned the neutrophil. there'sanother cell called a mast cell. there's another cell called [0:41:35.0]. these are cells thatare really important in our immune system for helping to keep us healthy. and all thati want you to look at is not any of the detail. it's not intended to show you the detail butjust to show you that we can take the whole cell and we can get from the mass spectrumthese populations of glycans and we can attribute

structures because we know these biosyntheticpathways. so how can we use that information in a reallife context? i want to talk about two, briefly one and then a little bit more time on thesecond example. so i want to firstly tell you about the fact that there are � andthis has been known for some time � a number of diseases which are called congenital disordersof glycosylation. and all that means is that congenital of course means born with, so there'sa problem already at birth. disorders of glycosylation means something went wrong with those glycosylationpathways and many of these actually unfortunately really devastating diseases because, as isaid, these recognition events are so important. they are important in early development thatif something goes wrong the chances are that

that's going to cause a lot of problems. so many of the really severe congenital disordersof glycosylation have really been known for some time because usually children do notsurvive very long if they have them. there are actually screening programs. now thatwe understand the glycosylation there are screening programs around the world for identifyingchildren who are born with these diseases and finding out very rapidly what they are.however, it's become very clear in recent years that there are less severe congenitaldiseases of glycosylation which people have not understood. and understanding them betterobviously can lead to better therapy and hopefully a better quality of life for the childreninvolved.

so i'm going to give you one example of thatand then i'm going to spend a little bit of time to tell you about recognition in humanreproduction because recognition and the correct recognition is incredibly important from manyaspects of human reproduction. so let's start first of all with the cdg. i'm afraid there'sa few medical and slightly complicated acronyms but i will explain them and hopefully it won'tbe too complicated and you'll understand the theme of what i want to say. the first thing,however, i want to say is that all of modern science is done with teams of people ratherthan single individuals. one of the great things about not even so much modern science,even when i first started, was that science is very international and very often involvesexpertise from a range of places.

so in this particular case we were carryingout the work in collaboration with christof klein in munich and the patients that he wasworking with were children in germany. and in london, university college london, linkedto great ormond street hospital, which is the main children's hospital in london, professortony segal and a phd student, actually a trained doctor that decided to go and get a phd afterhaving had medical training, abu hussein, was involved in the project with tony segaland in our own group aristotle [0:45:12.5] a post doc, did all of the lovely glycomicswork that i'll show you in a minute. now, what christof klein discovered aroundabout six years ago, 2008, 2009, is that a type of dysfunction which is not uncommonin children, which is called severe congenital

neutropenia. what this means again, congenital,you know what that means, severe, neutropenia simply means there's not enough neutrophilsbeing made. so there are a lot of instances of so-called scns and very often having anscn is not a major issue clinically because it often just simply manifests itself as problems,for example, with infections. so these children and then young adults, they do have neutrophils.they just don't have as many as a normal healthy person has. so they are prone to infection.obviously they can be treated for that. there are some scns where there are otherphenotypes or other clinical symptoms. and the one that was being looked at in particularwere families where the children with severe congenital neutropenia had other physicaldefects such as short stature. they also had

heart problems and importantly as well notonly did they not have as many neutrophils as they should, those neutrophils didn't actuallywork very well. now, remember the neutrophils are the white blood cells that go out intothe tissues to kill things. the way they kill things is by spewing out chemicals. they doother things as well but the main thing is to spew out chemicals and actually what theydo is they make hydrogen peroxide and related high energy oxygen intermediates. so these children were actually � theirneutrophils were not producing the hydrogen peroxide they should. this is shown on thisslide which i apologise. i have had to put a structure on for you. what the stuff kleinhad discovered was that the gene that was

mutated in these children was the gene thatcoded for an enzyme which is called g6pc3. it's written out up there but all you needto know is it's important in glucose metabolism. what it does actually is to take a phosphateoff what is called glucose 6 phosphate. so glucose metabolism is being affected by theproblems in this gene. this little bit of data here, i haven't put much data on theslides but the little bit of data here is just showing the neutrophil dysfunction. so what we have here are three children calleda, b and c from three families. child a, a boy� actually, i shouldn't call him a childbecause he's now about 18 years old � is the brother and actually twin brother of whatis called health controlled here. so the health

control, these are not identical twins. thehealthy control in terms of age matching and obviously environment is a perfect controlfor this child. and the other children are shown here. and the height of this bar graphis just showing how much superoxide is being produced by their neutrophils, by each millionneutrophils. we have lots of neutrophils. so a million neutrophils is producing thatamount of hydrogen peroxide in the case of a healthy control. as you can see, the otherchildren, particularly child a, has a very low level of hydrogen peroxide showing thisneutrophil dysfunction. so what we were asked by tony segal, who at university college,had identified families in london whose children had this same defect, so there were familiesin germany and families in london and also

now known to be families elsewhere, we wereasked could we explain how a defect in glucose metabolism was influencing how the neutrophilswere functioning. and one of the questions we decided to ask was was the neutrophil ableto make its normal glycan population. what we're showing up here � so this isthe healthy twin and this is a mass spectrum of the neutrophils taken from that healthytwin. again, all you need to look at is just simply an idea of the patterns showing, asyou can see here, that we have lots of different glycans. they have those patterns of antennathat i showed you earlier. and what was really dramatic in his twin brother was the completeabsence of all of these multi-antennary glycans. so the conclusion from that, if you just simplylook at this picture here, was that this is

what we expect on normal neutrophils, lotsof antennae, lots of these decorating sugars, those diamines and red triangles which arefucose and sialic acid for anyone who wants to know the sugars. but this is what was happening essentiallyin this � not only in the affected twin but also in the children in the other families.so it was immediately obvious to us what the problem was and i think it's probably obviousto you. the yellows are not going on because if you don't get the yellow going on to makethe first bit of this it can't put anything else on. and the yellow is galactose and this,for anyone who knows about biosynthetic pathways, is just simply showing the complicated interrelationshipsof molecules inside the cell. the important

thing to note is that glucose here is feedinginto what is called here udp-gal which is essentially galactose in an activated stateready to go onto a glycan on a protein. so this is a very important observation becauseit immediately suggests that it might be possible to improve the clinical symptoms of thesechildren. and in particular it might even be � although we don't yet know. we arehoping that it might even be possible to do that just by giving galactose supplementsin the diet and this is currently under investigation in germany. the child being treated in germanyafter six months does appear to be better but it's early days. i don't mean better asin able to be perfectly normal. i mean that the symptoms are not as severe in terms ofinfections and so on. but it is very early

days but this is the way that we can takebasic science through to translation. so the second example i'd like to tell youabout in a little bit more detail but not too long because i notice time is gettingon, is to talk about human reproduction and in particular to address the question of howit is in human reproduction and in the reproduction in fact in all mammals that essentially havethe developing embryo in an environment where the embryo is foreign, as we'll see, to themother or the female animal that is carrying that embryo. this issue of self and non-selfbecause the whole of the immune system's function depends on it being able to recognise whatit knows as self and what it recognises as non-self. and this is programmed to attacknon-self and to not attack self.

and of course, as i'm sure many of you know,if that programming goes wrong in some way, in for example autoimmune diseases, then theimmune system will be starting to attack self when it shouldn't. now, a lot of what i wantto tell you about today has been done with our collaborator, gary clarke, who is nowat the university of missouri. we started working with gary in the early 1990's at atime when i knew nothing about any of this. i was trained as a chemist. i've learnt somebiology along the way. it's sort of paper thin at times. one of the things that i hadn't really thoughtabout at that time were some fundamental questions that had actually been posed a long time ago,like this question here that had been posed

by a very famous british immunologist, petermedawar. he asked way back in the early 1950's this question. how does the pregnant mothernourish within itself for many weeks or months a foetus that is antigenically a foreign body?leaving aside the fact that he calls the mother and "it" this is a very important questionbecause the key thing to remember is that as soon as the embryo starts to go from thesingle cell stage when it was, if you like, the mother's egg, it is expressing foreignthings because half of its dna comes from the father. now, there are other questions which are relatedwith are, for example � and again this is slightly complicated but don't worry aboutthe cartoon, is that the way that the immune

system recognises that our self-cells andthe immune system shouldn't attack those cells, is because on most of our cells we have theseproteins. they are actually glycoproteins that are called human leukocyte antigens orhla. and they're essentially the molecules that say, "please do not kill me. do not killthe cell. i belong to you." now, the thing about both of the gametes, the egg and thesperm, in the human is that they actually do not have these hla antigens. in addition to that when the embryo implantsinto the womb, and this is the placenta here. this square here is now blown up to show here.what the embryo does as the placenta, which of course comes from the embryo and thereforehas father's genes in it, these cells start

to infiltrate in order to connect with themother's blood system. and so we have a whole variety of cells with all fancy names. eveni can't pronounce them. don't worry about it. we have all of these cells that are goingto engage. some of them will engage with the mother's cells. they also do not have these hla antigens.so again in principle they're foreign. and in particular there is a class of white bloodcells that i haven't mentioned up to now. there are lots of different kinds of whiteblood cells which have a really interesting name. they are called natural killer cells.their job is to kill cells that lack hla antigens. so they go around looking for cells that don'thave hla antigens and they kill them. so the

really important question which i have tosay i'm not going to give you the answer because this is research on the go. we're a little way to understanding it, ishow do women, for example, protect the developing embryo? how do they essentially protect spermwhen they enter women because they don't go and kill off all the sperm? when natural killercells are in effect trained to kill these cells that lack these antigens but they don't.and the important thing is you might say, well, maybe there's no natural killer cellsin the human reproductive tract. in fact, about 70 percent � [0:57:14.1] just simplymeans immune cells that are active in the human uterus. about 70 percent of them arenk cells. so we don�t actually know the

answer to this as yet. there are all sortsof experiments and it's not going to be a simple answer either. there are going to be a lot of different factorsthat are involved but it almost certainly is that a very key factor involved in recognitionand glycans will be involved in that. and so gary clarke, the person i showed you earlier,back in the mid to late 1990's, when he was thinking about this, he suggested � andhe called this again a fancy term, fetoembryonic defence system, or human feds. he suggestedthat essentially if we have hla negative molecules they have to have something else on them thatsay to the natural killer cells, "please don't kill me. i'm actually friendly. i am not foreign."and he suggested that there were sugar sequences.

and another very important thing that he suggestedat that time was that the sugar sequences that are on the human egg may be importantfor two roles. because remember the human egg then becomes the embryo. they will beinvolved in recognition of the sperm with the egg because in the process of fertilisationthe sperm has to recognise the egg. you don't want the sperm recognising a neutrophil ora t-cell or any other random cell that it happens to encounter. and he was proposingthat the sugars that are on the coat � it's called the zona pellucida of the human egg� may perform a dual role of being involved in sperm binding but also to help to protectthe gametes from immune attack. and it took some time to get evidence forthat and i'm very pleased to say that once

we were able to get evidence of what is onthe human egg � and there are teams of people involved here from a number of countries � wefound a little bit to our surprise because we didn't think it was going to be quite thateasy that the major glycan that is on the surface of the human egg is this epitope thatwe have already identified earlier on in the talk as being very important with respectto the functioning of the immune system, sialyl lewis x. and what was very surprising to us,and i thought i'd put a bit of data just so that you can see some data, was that whenwe did our profiling that sialyl lewis x was pretty much on everything. and we were also very pleased that this kindof work was considered important enough because

as scientists what we like is to have a paperspublished and secondly published in the very top journals and thirdly noticed by the editorsso that they make comments about it. and this we were pleased happened in this case, especiallybecause they thought it would be a nice story, so it was called "the sweet meeting of spermand egg" and it started off that first your parents had to meet but another � etcetera,etcetera. and of course that was the paper where we were reporting that the egg is coveredwith these sugars. however, if i am allowed another five minutesor so, i don't want to leave you with the fact that you might be thinking at this pointthat science is all very straightforward and research gives you the answer you wanted inthe first place and you have your hypothesis,

it's given you the right answer and thereforeyou know everything. because it turns out that it's not quite as simple as that andwe know that we don't know everything because one of the things that we had known for adecade before the discovery of sialyl-lewis x on the egg is that surrounding the babyas it's developing the liquid, the so-called amniotic fluid, that bathes the embryo duringdevelopment has during the time of the development of the embryo a glycol-protein called glycodelin-aor gda, which is a very high abundance in that fluid and is known to be really, reallyimportant for helping to protect the developing embryo. and what was also known about glycodelin-a,using the technology that's used in in vitro

fertilisation is that glycodelin-a was ableto block sperm egg binding. so in other words somehow it is able to mimic one or other ofthe molecules that are involved in that binding. and so we had done the characterisation ofglycodelin-a and regrettably it has absolutely no sialyl-lewis x on it. so in other wordsit is possible to take a protein which has a range of sugars on it and block that interaction,even though those sugars are not sialyl-lewis x. so we don't yet know the whole story; howeverwhat we do know is that glycodelin-a is � there's a lot of words on here. don't worry about the detail of it. but glycodelin-a,as i've mentioned, is very important in protecting that foetus during development. what we alsoknow, and that's why i've shown you a complicated

slide slightly faded out so you can't seeit, is that it has lots and lots of different glycans on it and they are summarised downhere. we know that glycodelin is important for helping with respect to the environmentof the baby of ensuring that it is in a good environment and in particular that it is inan immunosuppressive environment. so the immune system is not in there attacking the developingbaby. we don't know � so this is a "don't know"as opposed to a "know". we don't know which of these glycans are involved; however naturehas helped us a little to understand a little more of this system because a significantnumber of women develop diabetes during pregnancy. this is called gestational diabetes. it'sa bit like type 2 diabetes but it goes away

once the baby is born. so it is associatedwith pregnancy. when we characterised glycodelin-a from these diabetic people �this is normaland this is the diabetic. what we noticed is that the diamines, which are the sialicacids, are missing from the diabetic patients. so we know that sialic acid is important forimmunosuppression because we also know that the diabetic gda is no longer as immunosuppressiveas the normal glycodelin. so now at least we have a little bit of insightinto how this glycodelin is functioning as an immunosuppressive molecule. but we're nomeans well informed; however we have another little bit of help and that's from the guysin the audience because you folks make glycodelin as well. so it's okay, glycodelin is a wonderfulmolecule. you make it as well. you make it

in your reproductive tract, so it finishesup in the seminal fluid surrounding the sperm. early on before we have characterised theglycosylation a lot of people were very worried about this because, as i said, glycodelin-a,i didn't say it quite like that but what in effect i said is it's a potent contraceptive. and the idea that a potentially potent contraceptivewas bathing the sperm was not being received very well by my male collaborators. but isaid, "don't worry, guys. i haven't done the sugar structures yet but i'm absolutely surethey're going to be different." and, as you can see from these pictures here, this isthe girl, this is the boy. they are very different. what was particularly interesting in the maleglycodelin was the presence of these sugars

here which have two of these triangles. thesetriangles are called fucose, on the antenna. that is called lewis y. it's another clusterof sugars with the name lewis y. now, for many years lewis y was thought to be a cancerantigen only. it had only ever been found on cancers. itis rarely ever found in women and if it is found it's mostly associated with cancers.in men, on the other hand, it is the most abundant cluster of sugars in your reproductivetract. so if we look at sperm, this is just simply human sperm, saying what kind of sugarsare there, they are absolutely coated and these sugars are known to be recognised bylectins of the immune system, for example, [1:06:06.0]. we also know, as i've said, thatlewis y is a major [1:06:10.7] cancer. you

may not know what these pictures mean buti think you can tell that that looks very different from that. what this is showing is the expression oflewis y on this extra tissue within the pancreatic cancer cells here. so what does this all mean?i don't want to sum up saying that i've got a complete story. what i want to sum up sayingis that what in effect we are looking at there is the issue of self and non-self and thisdilemma that in the reproductive tract there has to be a situation where the immune systemis effectively being told do not kill non-self. and in essence this is associated obviouslywith reproduction which is placentally associated reproduction, having the development of theembryo linked via the placenta to the mother's

blood stream. if you like, as this is the slide i borrowedfrom gary clarke, is an achilles heel of the whole system. the hypothesis here is essentiallythat the price we pay for having that long development of the baby is that we will havean immune system process of suppression which can be hijacked by parasites, for example,and also in a sense hijacked by aberrant cells in our own body by cancer. so that is, ifyou like, the price we can pay but on the other hand i don't want to end on what mightsound a bit depressing because i think it does offer, if we understand it, quite excitingopportunities. so in principle if we can fully understandthese processes we should be able to intervene

and prevent, for example, the cancer developing,let's say, by switching back on again the ability of the immune system to kill the cells.but we really need to fully understand the processes. so in summary what i've told youabout today is that sugar recognition is incredibly important in cell-cell engagement. and whatwe know is that when cells come together you can see the sugars meet the lectins and thewhole cells light up. in other words, they send signals into the cell to tell those cellsto behave in particular ways. now, we know that's the overall, if you like,summary process, the headline process, but a lot of the detail is missing. until we understandthat detail we won't be able to take this information as far forward as we would like.in summary, i'd like to thank many of the

people who have been involved in this work.these are people from the group currently and many people over the years. i want toflag up again the importance of howard morris who has been my mentor all my academic lifeto other senior members of staff in our group, maria panico and stuart haslam, and a wholerange of students and post docs here. and without all our collaborators worldwidewe wouldn't have been able to do any of what i've told you about today and any of the otherthings we've done. i do want you to note what this map is. it's take off the web not thatlong ago. and it is the national happiness rankings of the world. with the darkest bluebeing the happiest and the red the least happy, you'll be pleased to know that australia iscompletely dark blue. sadly, although ireland

is dark blue the rest of the united kingdomis not quite making it to those happiness rankings. thank you for your attention. [applause] mark von itzstein thank you, anne, for a very inspiring lecture.it certainly delivered a lot of messages to me personally about your career trajectory,actually. i'm pretty sure that that young lady in that picture was you. is that right? yes, it was, actually. i thought it might well have been. i thinkfor me as a scientist that has engaged mass

spectrometry the technique of accurately weighingmolecules has been fascinating. and the development, as anne has described, she's been there rightfrom the beginning. so you can understand why her career trajectory has been as it is.she's developed so much and is recognised as being such an important player in massspectrometry generally but particularly one of the leading proponents of this techniquein the world. it's great detective work. that's the way i really look at it in termsof how you can understand these big biopolymers of sugars and drill down to the nth degreeabout saying, well, how they are connected together. more importantly, linking that thento disease, i think it's absolutely fascinating. so congratulations, anne, for that. i havea scientific question but i want to ask the

audience first. we'll just have a moment forquestions, if that's okay, before we retire to celebrate this wonderful lecture. so i'llopen it up first of all for questions from the audience. catherine tindal thanks for that. i really enjoyed the massspec part of that. so i suppose following on from mark a little bit, do you take thosemass spectra and sit down and investigate manually or is there software these days? perhaps before you answer the question i mightjust repeat it so we capture the question. the question simply was when you collect allof this wonderful data, all those wonderful

spectra that you saw during the talk, do youhave software that can automatically analyse that or do you have to go through this bitby bit yourself? it's a mixture, actually, is the answer tothat. so for 25 years we looked at it manually. in fact, in the very early days, for the firsteight or so years of my academic career, the way the data was recorded was on photographicchart paper. it was exactly the same kind of photographic paper that you could buy froma photography shop. rolls of kodak, actually, we used to get, paper. and we just simplyrolled it out and we counted from 28 which is the mass of nitrogen, which there is alwaysa bit of nitrogen gas that ionises, and we counted it one mass unit at a time up thespectrum.

then computers came along that were actuallyable to do the counting, if you like, and then the interpretation for certainly 10 or15, 20 years was entirely manual until we did get a bit sort of bored with the factthat we were getting by then too much data to do that really realistically. so thereis software. we had a very talented bioinformatics post doc who wrote some really nice softwarewhich is open access on the web. and what that software does effectively is to createthose cartoons using the information. it is not completely automated and that is deliberatebecause we don't want people to be misinterpreting their results. there is a variety of other software thatis available on the web, so it is in between

being manual and automated. but i would alsosay that the most exciting bit is getting that data and then being able to work outthese ions. the zona pellucida egg data was just absolutely amazing. there was no waywe used software to interpret that. we did that manually. any other general questions that people wouldlike to ask and then i'll turn to my final question? if not i'll ask one final questionthen. so we know that men are becoming irrelevant. the y chromosome is shrinking, we know that.it's going to be completely gone, eventually. i have no doubt about that. well, accordingto my colleagues in the academy of science here that's what they tell me. the questioni have is going back to your sperm glycan

carbohydrate story and the egg engagement.we know that actually there's an enzyme in the sperm, in fact, in the cap, in the topof the sperm, that apparently is secreted that actually chops [1:14:48.9] acids away,the wonderful sugar that you spoke about. how does that relate in terms of the storyabout engagement of the sperm? okay, so what i would not perhaps have shownyou as well as i should have done to answer this question is that the egg is a very, verytiny thing. it's about the size of a full stop, if you think about an ordinary sizedpen or pencil as opposed to making any size on the computer. but surrounding it is thissugar coat, the zona pellucida. so when the sperm arrives at the egg it engages initiallywith this sugar coat. after that there is

a reaction which is called the acrosome reaction,which is what mark is referring to. what that essentially means is that insidethe sperm, the sperm is a pretty boring cell, actually. those nice cells i showed you incartoon which had all of these different compartments inside to show they were interesting cells,the sperm doesn't have all of that. it just has a little package of dna which is sortof important. then it has a bag of enzymes which are called the acrosome. yes, the firstthing that happens upon that engagement, again, not fully understood how it happens but isfor, if you like, the front of the sperm to blow apart and for these enzymes to spew out. their role is to do all sorts of things, includingmaking a way for it through that sugar layer.

although we don't understand whether or not� one could argue that the sialidase is needed to make sure other sperm don't engagebecause that's the critical thing. remember that these millions of sperm start off. theyfinish up two days later, maybe hundreds, but there's still hundreds. only one is makingit. so it is very important that you don't get the egg being fertilised by more thanone sperm because that can create problems. so that is almost certainly the reason forboth enzymes spewing out, a combination of it then eating its way into the egg but alsoto make sure that other sperm are not going to fertilise. sialic acid is really, reallyimportant. yes, absolutely. i completely agree with you.so before i invite susan gallagher up to award

you this wonderful plaque in recognition ofyour outstanding lecture i'd like all of us to join in thanking anne for what i thoughtwas a very articulate lecture. we have this tradition, anne, of actuallygiving a plaque to our peter gallagher memorial glycomics lecturer and this is it. i'm sorry,i've got fingerprints all over it. how rude of me. susan, do you want to come on thisside and award anne with this wonderful plaque. susan gallagher on behalf of my family and the peter gallaghermemorial fund at the university here, i'd like to thank you for a most informative lecture.first of all, dad would have been chuffed that you spent so much time researching hisbeloved rugby, despite whichever code it was

you were looking at. also i've used his nameto help the university to raise money to buy one of those mass spectrometers, so i feelparticularly attached to this. thirdly, you used lots of words i understood this time,so i don't think i'm getting better. i think you guys are getting better and making surei understand, so thank you very, very much. [applause]end of recording]