It's truly a pleasure to be here again, at Lindau, and to have such wonderful interactions with all of the young scientists! So, what are telomeres, and more importantly, why would you care about it? So, I thought in the next half hour I'll take you through the journey that I had and which many others are continuing into, which has been a journey all the way from pond scum, rather unlikely journey to take us to something in humans, that we can call, the mind. And the general message and what comes out of this, quite surprisingly, from this journey, was implications of telomere maintenance for human health and disease. So, I'm going to tell you how I started on this journey, what took us into it, and then all of the things that have grown out and there are questions, of course, that'll be still exciting to answer. So, if we looked inside a cell that was about to divide, and took a picture of the chromosomes, which you can see in blue, you would see that the cell is about to divide, so all the chromosomes have replicated, so the DNA which is all compacted and looking blue, you can see is actually discernibly double. And at the end of the chromosomes, each end of each of those replicated chromosomes, you can see that there's a little blob. And that's actually lighting up the telomere. And what the telomere does, was for long time sort of known just by, a blob, but it was very functionally important It was known that it was actually protecting the end of the chromosome in ways that protected the genetic material, and without something, whatever that was, a protective structure, at the end of the chromosome, that chromosome could become very unstable, lose information, sometimes even stick by its ends to other chromosomes. Not good news! So, the telomere was a kind of a cap, capping off the end of the chromosomes. So, there was a mystery though. What was this blob? How could we answer that question? It required being able to get your hands on the molecules. And so, this is where the pond scum came in, because this little creature, called Tetrahymena thermophila, is a beautiful single-celled organism, it's lit up green, but it's not actually green. It lives in pond scum, but the point is, it has lots of very tiny and linear chromosomes. So, lots of ends per rest of the DNA. So, that enabled me to very directly analyse it molecularly and found out, sort of surprising, there was this very simple DNA sequence. It wasn't coding for proteins or anything, and the simple sequence was just repeated, over and over again. I'll show you an example in a moment. So, this was very curious and then we found that this was more general, this wasn't just confined to this wonderful creature, it was in yeast too, and that was work that we did together with Jack Szostak. And I collaborated with Jack and we found that, oh, it extended to yeast. And then, more and more people looked and found that actually, for eukaryotes, which have linear chromosomes, and, by the way, you're going to wonder about bacteria, they're just so much smarter, they have circular chromosomes, they don't worry about this stuff. So, we're talking about the eukaryotes, all of us, all the way down from us to pond scum. So, this is at every end, and this is the little repeat motif that you see at our ends. Now, in fact, there's much much more than I've shown, there's thousands and thousands of DNA building blocks, but the point is they make a scaffold, and that scaffold is a protective sheath of special protective proteins. And the point is, it has to be a big enough scaffold for that accretion of protective proteins, which are very well studied. I'll give you some names soon, that it has to be big enough to make a true protection. Now, that it has to be big enough. Okay, there was another mystery, and this was a real zinger, because people knew how DNA was replicated, they knew the machinery by the 1970s, and there was a real problem here, because the beautiful machinery that replicates all the rest of the chromosomal DNA is stupid enough that it can't replicate the very end. It's just the way it's built! So, if you just took this to its logical end, excuse the pun, it would be the, every time you replicated the DNA, you'd lose something from the end! And as the cells divided and replicated each time telomeres would get shorter and shorter and shorter. This would not be a great idea! And, in fact, it was predicted, perhaps this would lead to some kind of finishing off senescence, if you will, of the cells. But nobody knew what might be going on. So, at least we knew by then that the telomeres had repeated DNA at the ends, but also another observation was that they would, the numbers of repeats were actually more fluid, they were going up and down and changing at the ends of different chromosomes. Hmm, this was much more dynamic! Now, the shrinkage we could understand, the growing, that was weird! So, anyway, bottom line was that I decided that we should hunt for an enzymatic activity that maybe was adding DNA to the ends. So, very simply, here's what Carol Greider and I succeeded in doing: We made a synthetic little piece of telomere DNA, the little building blocks you can see here, from this Tetrahymena, the one that had lots and lots of linear chromosomes, so I thought maybe it has lots of something that makes those linear chromosome ends, and you could make the right sorts of extracts from these cells, and do it by chemical reaction, and just put in two building blocks, notice that the telomere DNA is just of these two in this species, and you could make that synthetic DNA grow. So, when we examined this a whole lot, we found it was a fascinating thing. It was putting on these nucleotides, doing something that normal cells were not thought to do. It was copying a bit of an RNA sequence information into complementary nucleotides added on to the DNA end, thereby elongating the DNA. So, this RNA is much bigger than this short sequence portion of it, but this RNA is built-in to this enzyme, which is partly protein and partly RNA. And the RNA helps the reaction, but it also provides this short portion of it as a template. Okay, so we're in the course of studying all of this mechanism, when serendipity happens. Okay, so, for various reasons we were making very precise mutations in the telomerase RNA, and we happen to make one at a particular place, which had actually an effect that was really useful for us. It kind of came back at the enzyme and made it stop. So, what that enabled us to do was ask a question, "How do cells feel if their telomerase isn't working well?" We had been doing things in the test tube and, lo and behold, we could go to Tetrahymena, which, I didn't tell you, but it's a lovely organism because it grows immortally, it just divides and divides and divides, so long as you feed it right, talk to it nicely, it'll keep dividing. Okay, and it had, as I showed you, relatively plenty of telomerase. So, the telomeres got a little bit shorter and longer, but there they were. So, what would happen if, we know inactivated it, using this very precise, surgical stiletto at the heart of the enzyme? It made it stop. And what happened is that the telomeres slowly shortened, took about 20 cell divisions, and then the cells simply stopped dividing. So, now our immortal cell, if you will, we'd turned it into a mortal cell. We had in fact, dealt it a mortal blow right at its heart. It now no longer was immortal, we just done this very simple, we knew what we were doing, we mutated just the telomerase, and it became mortal. So, the conclusion was then, the cell needs plenty of telomerase, it has it and it balances the shortening. Now, the shortening still happens, but the balances that you get, elongation when you need it, so these cells can keep going. Okay, so, now we'll go forward to lots and lots more work for many many different groups and synthesize what we now know is a much more complicated situation. Although the essence of what I told you, hang on to that, because that's exactly what's going to hold through all of what I'll tell you as we now move to thinking about this, essentially always from now and in humans. So, we have the RNA components, it's got a name, hTERC. We have a protein, which carries out this Reverse transcriptase, the core protein, hTERT, TERT, and that's the basic part of it! Now, what's known in humans is that this is extraordinarily complicatedly controlled. You don't have to read all of this, this is just to tell the aficionados that every kind of molecular control mechanism you can think of for the action of this enzyme, to make it more or less active, you can think of it'll be doing it. Telomere proteins themselves protect the telomere from too much telomerase action. They actually ration it a little bit. Telomerase levels, how much telomerase is made of the telomerase genes? How much the RNA products of the telomere transcription, telomere gene transcriptions are actually put into different spliced forms? How much they're assembled into the enzyme? Etcetera, etcetera. Okay, you get the idea, it's really complex how on earth we're going to work out which of these ones is actually the important ones, in humans. They're all doing something, somewhere. So, what you have to do, is you actually have to look in actual humans. And this is where it's really important to now think about scale of time, because humans, you might remember, we have a life expectancy, in many developed countries around the world, to something like, approaching 80 years. So, we can look at our experimental models, like a mouse, but it only lives for two years. And you look at a fruit fly, it's only about six weeks, and a worm, and it's only about three weeks. So, while we all have the same molecular and cellular building blocks in our cells, how that plays out in the whole organism is going to be on this very, very different time frame. And you don't know that what might be the critical rate limiting aspects of it, are going to be the same from organism to organism. And in fact, I'll tell you, each of those model organisms, the mouse, the worm, and so on, they don't die when they die of old age, they don't die of short telomeres. But they're on a really different time frame from us, so we have to look in us. Luckily, we can measure telomeres, we can take blood samples, we can take other samples. Commonly blood, sometimes just spit, our saliva, filled with useful cells, we can get a window inside our body, We can measure the telomeres and there's nice methodologies for doing that. I won't go into it, but I'm just tell you, you can measure in various ways and get good, reliable numbers out. So, bottom line is, what do we find? Well, we start off with about 10,000 nucleotides worth of these repeats. Really nice, long telomeres when we're born but by the time we get really old it's down to about half of that. Now, that half of that might sound like plenty to you, but what it's doing is it actually is now putting the cells in grave danger, because that protective sheath is no longer big enough. And what happens then, in humans, is really interesting. And there was no real reason to think this would be the case, because the telomeres gradually shorten as you look at most cell types, as we go through our decades and decades and decades, and the cells we gestate, called Senescence, which is a signal that those overly short telomeres cell send to the cells, and the say, "Stop!" And they say to do some other things too, that I'm going to tell you, but, first of all, they stop even multiplying, so if it's a replenishing cell type, it's not going to anymore. So, of course here was this gradual process, it's taking place over years. Is it like a life candle burning down, causing, eventually, is it related to our death? That's the question! Here was the cellular part of it, on this side and what was going on down here, what was going on in our whole lives here. So, and remember you've got that highly highly regulated telomerase, and now you could've imagined, well it could be saving things, but it isn't, it isn't doing a great job! It is good in certain cells, like certain cells that have to keep replenishing tissues over life, it's not bad, but not perfect. Luckily, it's really good in the cell lineages that give rise to our germ lines, otherwise we wouldn't be here. So, it does get maintained throughout our germ line, the sperm and egg-producing cells, that produce each of us and all our forebears and all our descendants. So, that's good, but it's pretty low in our body cells, in the many, many cell types. Now, there's also another variation, remember, I said this is complex. Turns out that human cancers, which of course, what are they, they're cells that just keep going and shouldn't keep going. They're not necessarily immortal, but they certainly are doing way too much replicating. They love their telomerase, and they actually rev it up really high. But they do a lot of other very bad things, too, as you know about cancers. Okay, so there we've got the situation in humans. How is all this going to play out? Now, we know a lot about the consequences of what happens if you look inside a cell and ask about the telomere. So, here we've got a nice human cell, looking at it under the microscope, so coiled up inside the nucleus, is all the chromosomes in purple, and out, filling the cytoplasm volume, lots of it is the mitochondria, which of course are the energy powerhouses of the cells. So, now we have 92 telomeres, 46 chromosomes, 92 telomeres, and so, here's one. Now, what is going to happen over life is that telomere in cells as they replicate, and even just as they undergo damages, you'll see, they will get shorter and shorter and now it will become too short and become uncapped. Now, that telomere is not silent, it talks to the cells, and one thing it talks to is mitochondria, and actually pushes them down, through a set of signalling molecules. Makes them less good. When mitochondria are not doing all their wonderful energy stuff really well they can start malfunctioning and producing reactive oxygen species. Really bad news, and particularly bad news for telomeres, and it actually sets up this vicious cycle, where telomeres can get even shorter. So, I've just shown one, but there are 92 and variously, they can get worse and worse. So, this is bad enough for the cell itself. But, these things are like these cells now, with these Uncapped Telomeres sending these signals, they're like a rotten apple in a barrel. They can now start affecting other things, because what happens is, another of a kind of signals that this Uncapped Telomere, which is a very worried telomere, is sending, is, for whatever reason, it is sending alarm signals which include secreting outside the cell. Factors that include those that are increasing inflammation. Now, through a whole series of things that go on in the wonderfully complex immune system, that also can up the body's reactive oxygen species, too. So, you've got even worse situation. Okay, so you can see a senescence cell with telomere damage is not a really good cell to have! So, this kind of cellular pathology is quite well studied, in cells and also in certain mouse models and so on. And there's every reason to think that it would go on if you get such cells in humans, as well. So, now let's say, well let's look at some consequences of what happens to telomere length. So, now, we had the good fortune to join a very large cohort project and we could measure telomere length in 100,000 people, so we can really start to get some good ideas about what happens in humanity, and first of all in a part of California. So, the first thing we did, was we looked at telomere length in 100,000 people just in spit samples. So you've got a nice sampling of cell types in the body. You look, just looked at average, over time, as people got older and older it was just one time point for each person, and what you saw was what I told you! There was this gradual decline. But then we saw something really interesting! Now, let me point out most mortality in this population is happening around here. So, you got this decline, most of the mortality, overall is around here. But there are people who you can see are surviving longer and longer. Now, it's just one time point, but what you see is the longer a person has survived, the more likely they are to have an average telomere length that's longer. So, this is very, very interesting, because it never been seen before! We replicated that males and females are different. But we did show that the difference, which is a bit less before about age 50, it sort of really splits off, between females and males. And then after this critical time, when most of the mortality, just population mortality happens to both of them show these sort of rises. So, really interesting. But now we can say what will happen if you actually ask if this has any consequence or any predictive value. And so, the DNA was taken in the year 2009, so the clock tick, and by 2012 you could go to the records and say who was still alive. And who had died in that time period. And then compared that, as a function of what were the telomeres like at the beginning. And, lo and behold, if you looked at the people in the bottom quartile and set their likelihood of dying to one, just as a reference, then you found that anybody whose telomeres where in the other quartiles, that is people whose telomeres three years earlier, or within that three year period you looked at deaths, you just found that, oh, longer telomeres actually had a decrease chance that you would be in that group of people who had died, within three years. We saw that age and sex clearly have an effect, so the blue line's actually, the blue bars show that that was already corrected for. But we also know that a lot of other things affect mortality. A lot of other things also affect telomere length. And we know what they are, and we can get the major ones out, and here's the big list. We did the age and gender, age and sex, race-ethnicity, education, cigarette packyears history, physical activity habits, alcohol intake, body mass index and so on, and, doesn't budge. What that's telling you is this is independent. This is a very diverse group of people, but California's, but you know Californians, meh, you know (laughs). Right, so, let's look at some serious, sober Danes, and see if the same thing happens. This is the Copenhagen study, Rode et al. published this beautiful work where they looked at 64,000 people's blood cell telomeres, just average telomere length, baseline and then time goes by and they looked at a bigger range but the average was about seven years, so longer than what we'd looked at. And they said, of those many people who did die, how many, in which telomere length category to begin with, how many had died? So, in other words, the same question, does the chance of you dying within that period of averaging about seven years, does the chance of you dying have any relationship to the telomere length, at the beginning of the baseline analysis? And, lo and behold, again, after correcting for all of these multiple possible variables that's called the Multivariate Adjusted, this is what this is, referring to telomere length shortness, as you see trends as worse and worse chance that you will have been in the group of people who died, the shorter the telomeres are, and it's a real kind of a trend. So, we get the message now, we got big studies, I think it's pretty clear. Reduced mortality, and I've shown you all causes is related to the observation, just observing the telomere length in people. Didn't show you the data from Rode at al.'s study and from others, that actually, when you start to divide it down into some of the major killers of the elderly, cardiovascular, all-cancers lumped together for the moment, all of those are related to reduced mortality from those causes, as well, are all related to observed having longer telomeres. Okay, and though the news is good too about longer telomeres in another parameter we care about. Well, lifespan, that's one thing, we want to know how long, how many years of healthy life we'll have, how long is our, quote, healthspan. And, in fact, again you see a relationship, here's a bit of numbers, but the point is they're related. Now, you can see another thing. You can say, now let's go the other way, what about bad news? What about diseases and shorter telomeres? Do we see a relationship? And the relationship goes in that direction, shorter telomeres correlate with, not only just finding an association, but actually also predicting, like I saw, I showed you predicting mortality, predicting, getting some disease, and there's a whole group of these diseases, and you can see they're very common kinds of impairments and diseases that happen with aging. Now, the observant of you have been noticing something, all of these arrows have double heads. I haven't said anything about causality yet. That's what we'd like to know! So, genetics! Genetics is wonderful because it gives us, if we can see a gene we can attribute a gene, especially one whose function we know, we can say causality. And wonderful beginning began in 2001, Vulliamy et al. found that if people inherit in families a mutation, fortunately rare, which is in the a Telomerase RNA Gene, remember that hTERC that I told you about, that RNA component of telomerase. If you have a mutation that knocks your telomerase down to half of its normal level, there's a very clear causality, we know what hTERC does, it's part of telomere maintenance, and people get really really short telomeres. Now the point is that really matters, there's a clear disease impact, related to how much the telomeres shorten. And the first things they've found were a bunch of very interesting diseases, they already showed certain overlapse with some of the diseases, which just occur in the population with aging. Here, we've got real causality, now, it's a bit extreme, these people are very much more likely to die much earlier, and as the telomeres get shorter and shorter, going down through the generations, because the short telomeres get passed down from person to person, they get worse and worse and die, sadly, earlier and earlier. So, the causality is extremely clear, but it is kind of extreme. Now, the genetics has just grown and grown since 2001, so more genetics has told us the same story, but really filled it out! So, again, inherit a rare mutation, half the telomerase level or other ways of reducing telomere maintenance, not only reducing telomerase levels, but also other ways that you can reduce it, which are to do with directly binding telomere proteins. So, what you see though is exactly what I showed you, but now, because there's more and more cases round the world there are various family pedigrees, people really, they find these sorts of things and the list now has got longer! So, now it's starting to even broaden out to neuropsychiatric diseases, very interestingly. This is looking like the big set of things that can happen, it doesn't all happen in one person. It varies on how much shorter the telomeres have got with succeeding generations. The genes though, and this is just for the specialists who'd like to look and say, "Yes, what genes are they?" Well, there's 11 of these now, six of them are related to telomerase itself or the biogenesis directly of telomerase, and there are five now, which are known telomere binding proteins. So, we know exactly what these things do, they are directly doing telomere maintenance. So, we got real causality here. So now let's look at the rest of us, who are fortunate enough not to have been afflicted with these rare, although extremely informative mutations. Now you can say, well, what about common alleles that actually just impair our telomere maintenance a bit? We had a wide range of telomere lengths, by the way, those means that I showed you with age, they're much much tighter than the actual range of telomere lengths at all ages. But you can do statistics on large numbers and in this beautiful study, looking at just what genes are associated with having shorter telomeres in the population at large. So, causality, because genes cause things. What was amazing was, the five top hits were our old friends, the known telomere telomerase genes. So, this was very clear, we know just what these do! And then they said, well, if you look at these alleles that actually are related to the shorter telomeres, and these are common ones, that can occur in all sorts of combinations in any of us, is there a disease impact? And there was, in cardiovascular in a particular form, coronary artery disease, and so, in fact, if you added up the short version alleles for each of these genes plus a couple more that were less related but showed up in the quantitative analyses, but these were the top five ones, we know what they do. Just out of the bat, you would have a 21% higher chance of getting coronary artery disease at some point in your life than the population at large. Now, to have all seven, right, there five plus two, seven genes, to have all the bad alleles of all, combinatorily it goes down and down, so it's probably only one in a few hundred or at most people. But, you can get that. Most of us have a mixture of these, because they're just all sorts of different genes. But this is really major thing, because again, it is saying that at least this short telomere thing, because we know what these genes do, must be able to contribute, contribute this particular form of cardiovascular disease. If you believe in the logic that genes have causality, and I think that's a rational thing to do, then, that's what we can say for this case. So, it's really useful! Now, telomeres is not all good news, because in the context of cells that are normal. You remember Dr Jekyll and Mr Hyde, it was one person in the daytime, he was this nice, good citizen, well-behaved, but at night-time, same person, was a horrible criminal, violent, right? And telomerase, in the setting of the night-time setting, if you will, cancer-prone cells that had changes. This can be really dangerous! Genetics, again, has told us. Now remember, I told you, if you have only half as much telomerase as you ought to have, one of the things that is caused is cancers. And it's actually a specific subset of cancers. But now, more recent genetics has come in, and we're finding another very interesting thing, that is just about cancers. Now, when I say, we, I mean the world at large. And so, just a little bit too much, even just as, less than twofold, too much expression of telomerase itself, actually causes some other cancers. You can see actual contributions here. And there are completely different set of cancers, and they're not the most frequent of the cancers in the world. But now, you can see where we are. Cancers first of all, vary. We are really living in a trade-off world here, right? We're precariously on a knife-edge here, because this is what has been learned by looking at actual humans and actual diseases and genetics and understanding the molecular basis of what's going on. You're going to say, "Well, telomeres, wait a minute, they are not cause to all diseases, diabetes, it's caused by other things. It's caused by insulin not working properly, there are other things that go on, it can't be all telomeres!" And I couldn't agree more! But I want to pose the idea that, in fact show you the data! Actually, it'll come from mouse models, because it's so clear that telomeres interact. So, now we've talked about this idea, the more the telomeres shorten, the more cells get engaged in harmful kinds of consequences, and the more severe the pathologies. And in a mouse, you actually have to delete telomerase completely, but then eventually if you breed the mice you can start to see these sort of graded effects. So now, let's look at mouse models, where we're looking at just single-gene mutations, which cause disease in humans. And so, here's three. One is Progeria, very premature, fast telomere shortening, sorry, premature, fast aging, actually, it has telomere shortening, too, in humans. Another one, completely different, a muscle wasting disease. Duchenne Muscular dystrophy, known mutation, another completely different one again, diabetes, not enough insulin in this mouse, single-gene mutation. So, completely different things. Now, each of these models, you put the same mutations that you find in humans, you put them into the mouse and it's actually a little bit disappointing. You don't get all the phenotypes, the Warner's doesn't have the full phenotype, the Duschenne Muscular dystrophy shows the skeletal muscles, but actual people with this disease die of cardiac disease. So, it's not really mimicking it perfectly! So, now let's go through with time and now let's superimpose in the mouse model a Telomerase deletion. What happens is really interesting, because well before the telomerase deletion is really showing any effect by itself, the telomeres only partly done some of the cells, you get an interaction, and what gets worse is the Warner's syndrome phenotypes. And you'd starts to look now like the human one. The right cells start to be showing pathologies. The same thing happened with the Duchenne muscular dystrophy in that setting, as the telomeres got shorter, now they started to get heart problems. And now, the same, the diabetes, part insulin deficiency, it became much worse and it was the diabetes symptoms that got worse. Okay, so bottom line is we've turned mice into furry little humans! Right? By removing telomerase and setting that as a background. So, interactions between telomeres and disease, we can see that in these mouse models. Now, I just want to tell you that it's not all genes, and in fact, people have long known that something you might think is totally different but, coming from the mind is something that does have an impact on telomeres. On disease, good example is heart disease, in fact. And so, we and others started studying this, and we started and found some effects on telomere maintenance. And now, the list is huge, I'm just going to show you this huge list of all these sorts of things, terrible things that happen in people's lives, which really argue, since it's the length of exposure that is often very much a quantitative predictor, or the duration, duration severity, length of abuse in these situation... It quantitatively predicts how short telomeres are. So, we think there's real causality here. And we know that Chronic psychological stress has impact on disease, but one of the things it does do is it reduces telomere maintenance. So, the big question is going to be, disease impact itself, and what can you do about it?" And why would you care? This is all very statistical. And now, I want to finish with something really remarkable. Which is: People who have bladder cancer, and you look at an interaction between something that you probably would never have dreamed of looking at if you're asking how much a bladder cancer patient will survive. An interaction of the short telomeres in the blood cells, the normal blood cells, with depression. Now, depression is fairly common. So, they just did a simple thing in this beautiful study, which was they divided people up at the time of their diagnosis, of course the bladder cancer had been developing all those years. Got to the point of being clinically diagnosed and they said that the person had depression, but not short telomeres or short telomeres and not depression, or neither, short telomeres or depression, or did they have both. Now this just says, well, they did the study right, this group at MD Anderson in Houston, Texas, I'm going to show it to you visually. Here are lots and lots of people! They had over 400 people. They're all just sorts of different people, and they've got bladder cancer. And at the diagnosis, they fell into one of those categories. One or either of depression and shorter blood cell telomeres, or neither, or both. After two and a half years, if you only had depression, or you only had short telomeres, or you had neither. it actually statistically didn't make much difference. So, this many people are no longer with us after two and a half years, they died within that two and a half years. But if you had both, it was that! So, this is a big difference, over half! And now, five years, is a very critical time for cancer. And now, if you only had one, well of course, more and more people did die, either depression or short telomeres, or neither, it's about the same. But if you had both, everybody had died. So, that starts to look clinically significant and I think the game that you've been seeing is, it's all about interactions. And so interactions between all sorts of factors. We've observed in lots of stress-like and other situations, things that will make your telomeres shorter. It has been observed things that will make them longer. The real thing is going to be finding out which of these things, quantitatively, will actually, in true, proper studies, that are not just looking, but really testing in trial-like arrangements, which of these kinds of things, and they'll be more, could actually improve telomere maintenance. And it's got to be this right balance, because remember, if you push it too far, cancer risks go up, of certain kinds of cancer. So, it's got to be really, really tuned right, and probably physiologically is important. Okay, so what I've done is I've said there are all sorts of inputs into the telomere shortening and the how it much it shortens is really a complex set of things. But you can measure all of that, and see what the telomere shortening is, and you can see these quantitative relationships, associations. And as I've shown you, the sum aspect of it, which is presenting causality, and of course there's plenty of other causality, all interacting as well, but we think that we have this sort of underlying situation, where we can say, here's something that's changeable in life, you can see it's influenced by a lot of things besides genes and it partly, at least, contributes to the very common diseases of aging, which account for so much morbidity in people. And that just is the long words way of saying what I just told you in a diagram. So, maintenance of telomeres is important, and it's something else that we think now is actually contributing to these diseases of aging. So, finally, I just want to finish with, the journey began, with curiosity, you have to be always willing to play with ideas, you had to had background knowledge to make all these kinds of discoveries, not only in the pond organism, but in humans. You had to have wonderful collaborators, so that meant working with people, working in a research environment that made this possible. So, I'm immensely grateful that I'd been able to have this journey, and the science community people, so important. I'm in the Bay Area, in San Francisco, but I have colleagues everywhere. This journey began in looking underwater, and I just thought I'd finish with this most beautiful graphic, it's a photograph taken just outside where I was born, in Tasmania, Australia. It's the shoreline at night, and beautiful, single-celled organisms, living under the sea, living underwater, just like Tetrahymena does. These beautiful organisms are shown lit up here, and of course, the wonders that they can show you are probably, perhaps almost as wondrous as the wonders that you can see when you look out into the galaxy. Thank you very much.