enormous honor to obviously be awarded the Phelps foundation medal and I hope to do it justice in terms of delivering on on the future promise in my career so I wanted to tell you today about some of the work that we’ve been doing not actually to do with sleep or Parkinson’s disease or anything like that it’s actually very very basic research that we hope in the future will translate into medical care and I’ll come back to that right at the end of my talk so it’s probably not escaped you’ll notice that we live on a planet which has an intrinsic period because of the rotation of the earth and that rotation has basically programmed everything on the planet to have a reciprocal rhythm embedded within it and we know that organisms all the way from bacteria to complex organisms like us have these cicada in clocks and in us there are a variety of different rhythms that you’ll recognize or maybe even take for granted that occur on a 24-hour basis so every day even before you wake up your body is basically getting you ready for the day by upregulating your hormones such as cortisol your blood pressure getting you ready for the day ahead and then at nighttime it’s putting you to bed in the same manner by changing your temperature secreting various hormones to coordinate this and this is done proactively and not reactively so the reasons we have a biological clock is that we can preempt what’s coming because after all the daily cycle is eminently predictable and occurs every single day so why not have a system that actually is able to predict what’s coming if it’s going to come every day and that’s in fact why the clock exists to anticipate stuff that’s going to happen before it does happen so that we’re actually able to better utilize our environment and that’s proven by an experiment I’ll come to in a second but before I come to that I’ll just tell you a little bit about about how clocks are distributed through the body if I was talking to you twenty years ago I would have very much been talking to you from a neuroscience perspective because we believe that the basically resided in the brain in a tiny little nucleus called the super charismatic nucleus consisting me about 50,000 neurons in US but it turns out actually that that’s not the case and in fact every tissue within the body and in fact every single cell even skin cells like fibroblasts have the capacity to exhibit these circadian oscillations these daily oscillations of about 24 hours so how do we know that they’re important well this is an example from a mammalian system but people have done similar experiments in plants and bacteria and even in fruit flies and this is from an experiment done in chipmunks and what they did in this experiment which was done in the early 2000s actually the real experiment was actually done the 80s this is from a review the suprachiasmatic nucleus which I told you about just now if you knock it out by doing electrolytic lesion you can basically convert an animal with a very robust rhythm here the black stuff indicates activity the white stuff indicates rest you can basically turn that very rhythmic animal into an arrhythmic animal so its overall activity is not that much change it’s a bit lower but it’s basically distributed all throughout the day so it has no idea what’s going on now if you put animals with lesions back into the environment with sham controls that have had surgery but not actually knocked out there Sen you can see that they are out competed very quickly and they die off in the environment and as I say people have done this with various different organisms and showing the same thing so it has a survival advantage and that’s probably why we’ve kept circadian rhythms going in complex organisms even like us the thing is we it’s very difficult to see the effects of these things because we as humans have become so good at controlling our environment that we no longer a hostage to the environment like our kind of cousins were in the distant past so clocks are also now obviously implicated in many diseases because we have these clocks absolutely everywhere in our bodies not least in our brains where they’re associated with niraj gentle conditions in the heart and the immune system and particularly in the metabolic tissues such as the liver pancreas muscle and fat so clocks exist everywhere and normally they’re coordinated in order to make us better physiological machines and if that goes awry because people do shift work or get jet lags then you can upset the normal patterning and we know that that’s really important for predisposing people

to metabolic disorders now and also in particular to cancer so there’s very good evidence that the other stuff there’s less good evidence for at the moment but it’s emerging with time so what my lab is particularly interested in is how these clocks actually work now the fact that they work at a single cell level is actually pretty useful for us because it means we can really confine the problem to looking at individual cells so this shows some cells in a dish completely separate from the world they’re in a dark incubator at constant temperature so nothing is telling them what time of days is but these guys have been transfected with a reporter that basically glows during the daytime and is dim during the night time to them so their subjective time and they’re able to tick and tock like that without any intervention for days on end and in fact tissues can be looked at in the same way and they can stay like this for up to a year in continuous culture ticking and tocking away so how does this work so the model that’s been proposed over the last 20 years built on observations in drosophila is basically revolves around something called a negative feedback oscillator which is probably how all oscillators work whether physical or biological and in this model you have a positive regulator which switches on clock genes these clock genes then get made into their mRNAs and eventually into their proteins and then the proteins then negatively feedback to switch off the positive drive and then gradually as the clock genes aren’t stimulated to be produced anymore they switch off the negative regulation eventually goes and then the whole process resets itself the clock genes turn back on and another cycle starts so this whole thing takes about 24 hours to work which defines the circadian clock work now in mammals the players in this in this circuit are shown here so positive drivers are these transcription factors call clock and B mouth and the negative elements of these genes called genes or proteins called pyridine cryptochrome now a similar thing happens when you look at the fruit fly which is where most of the model was developed and you have similar positive drivers and negative negative suppressors on the other side but you’ll notice there are differences creeping in as we step back in evolution now if you look at plants it’s a completely different ballgame so all of these components now are completely different there is no homology between these components and the ones in fruit fly or as the genetic level or the protein sequence level and if you go back to the furthest cousin of ours if you can call them a cousin the sign of bacteria again there is no homology between these proteins and genes and plants or us so that it doesn’t seem to be any overlap in these basic pathways so if you kind of summarize that there’s a lack of conservation evolutionary conservation in the transcriptional oscillators that we know exist in these model organisms and these are basically the five principal modalities that we study clocks in different researchers around the world study them but you’ll see that the clock genes that we’ve kind of been looking at over the past few years aren’t the same so this begs the question as to is this transcriptional Drive actually important for sustaining circadian rhythms there another there are lots of other observations that have got time to go into why this transcriptional oscillation might not be the whole truth but an experiment that we did to try and look into this in further detail was to actually use red blood cells and we thought what’s the best way to actually look for non transcriptional oscillations will pick the right model and then try and show or try and prove or disprove what we think might be the case and red blood cells obviously are known for their ability to transport oxygen around the body and a pack full of hemoglobin for that purpose but also there are actually a great model because they’re basically a bag of cytoplasm and they don’t have any organelles and they don’t have a nucleus and they don’t have the ability therefore to perform transcription so we can use this as a model to inform us about whether trans non transcription oscillations can occur the problem is as soon as you try to ask this question you run into a problem because all those proteins that I’ve been telling you about clock b mao period proteins they just don’t exist within the red cell so even if we wanted to simply just look at the same stuff that participates in this transcriptional loop we can’t do it because the stuff isn’t there in red cells so we ran into a problem but thankfully we also had a solution to this problem by looking back at some of our previous work and that work was done in 2006 while I was a postdoc in mackay stings lab and back then we were trying to look at proteins that oscillated within the liver and it turned out in this study we found that ten percent of all proteins in the liver oscillate and that’s actually known now for multiple tissues in the body so every day ten percent of your proteins in any

individual tissue will undergo a biological oscillation and we found that obviously interesting from from a global point of view but what was really interesting was one of the proteins that we found during this experiment looked even more interesting because it exhibited a post-transcriptional or in this case a post translational modification that occurred on a circadian basis over 24 hours and this is a gel don’t need to worry about the details but it basically has two spots on it and basically those two spots are different because one of them the one in blue is basically oxidized and the one in red is not oxidized so there’s basically the same protein but they’re moving between two different forms and you can see that on the graph here the blue one does that and the red one does that they’re in anti phasic oscillation what’s happening is they’re basically shuttling between the two the actual level of the protein isn’t actually changing and that’s important because if that was the case we could also potentially use that same marker in a red cell which can’t make new protein and look at oscillations and it turns out that that’s well-trodden paths in a different field and antibodies exist to actually probe for this oxidized form of protein so just a little background on these proteins these proxy reduction proteins they have cysteine residues which dies by hydrogen peroxide and then they can be further oxidized into what’s called a hyper oxidized form and that can be recycled back to some initial state by an enzyme called sulphur dachshund and we as I said could probe this bit using an antibody and probe this bit using a different antibody and therefore work out cycles in red blood cells so we did a simple experiment we took blood from people purified it and then basically incubated it in the dark at the same temperature so had at no idea what was going on in the external world and then map their oscillations over time and when we did that we found a triplet of oscillations within the red blood cell we observed oscillations in the peroxy reductions that oxidation reduction reaction that I was telling you about occurred within these red blood cells which obviously was incredibly surprising we didn’t expect to find that and what was even more impressive is that when we thought about what these where this hydrogen peroxide might be coming from within the red cell we figured it was probably emerging from oxidation of hemoglobin that occurs within the red cell so one of the reasons why we were really interested in proxy dachshunds is that because they’re highly expressed in red blood cells and they exist there to mop up hydrogen peroxide that’s actually being created by this process called Auto oxidation which happens in red blood cells and one of the things that you see in in peroxy redox in mouse knockouts is that the red cells become unstable and that you actually get hemolytic anemia the red cells actually break down and they’re actually there to protect the red cell from this oxidation process this hydrogen peroxide flying around so we figured you know is there as something going on with the hemoglobin and we looked with a different essay based on fluorescence and we found that again you could see these oscillations in these isolated cells with no idea of what’s going on in the outside world and we figured the reduction back to these these forms that can be used carry oxygen would also be going on and that’s dependent on reduction factors within the cell these archetypal reduction factors nadh and nadph and indeed they exhibit circadian oscillations so by three different methods we were able to show that in these red cells you had pretty robust oscillations that couldn’t be explained by the current model of how we think the clock works now proxy reductions aren’t just in us there in practically everything on the planet they’re incredibly conserved proteins all the way from bacteria even in archaea the third kingdom of biology and also in eukaryotes like us and what we were really lucky with is that the antibody that we use to probe those oxidation-reduction reactions was actually because because the proxy reduction is so highly conserved around the the business end the active site that actually takes part in the hydrogen peroxide metabolism we’re able to use that same antibody potentially in multiple different species and that’s what we did clubbing together with basically all of our friends who we could get organisms off around around the world we basically map these oscillations in a variety of different organisms including prokaryotes and eukaryotes so bacteria even archaea we managed to get hold of flies fungus plants whatever we got hold of we found that we could see these circadian oscillations in peroxy reduction oxidation reduction rhythms and even an algae that we managed to get hold of through Andrew Miller in Edinburgh so in virtually all species that we were able to well all the species that we were

able to get hold of at least in terms of model organisms we were able to show robust Acadian oscillations of these proxy redox and proteins so although you don’t have conservation of the transcriptional elements the clock genes we showed that you could have conservation of these peroxy reduction rhythms which really makes you think that the proxy redox inside of things or at least redox reactions or more fundamental in terms of the oscillations that are occurring then the clock genes that we previously focused on and that’s really important because it means that we might be able to get further insights into how these mechanisms actually work which is what my labs basically focused on at the moment so we have to kind of rethink how the clock looks so everything I’ve told you in the first part of the talk was about this transcriptional translational feedback loop but in fact that’s important we know that through some of our experiments as well so that we can’t just chuck this bit in the bin but also we have to factor in this bit this new bit that we never even knew about before and that’s because in the complete absence of the nucleus in the red cell we could still see these robust oscillations of reduction and oxidation within those cells so why is that important well transcription factors are actually pretty tricky to target in terms of drugs for example so targeting those transcription factors such as clock and B miles is pretty tricky and they look very similar to other transcription factors in that same family there’s about 160 within that super family of transcription factors so specifically targeting the clock ones is actually pretty difficult but peroxy dachshunds are enzymes which are potentially more druggable so this offers a kind of way into the clock so one of the things that we’ve been trying to do recently is to actually target proxy redox in molecules using a virtual drug screening approach this is a structural representation of proxy reduction for one of the homologs and painted red is the active site region this is actually five of them joined together then actually form a donut in it and in a kind of polymer of 10 normally and and this is one of the stable forms that you can actually crystallize so what we did was to use our super computing cluster in Cambridge to actually doc drug molecules into that red pocket virtually so we used about eight million compounds and docked them in virtually to see which ones would fit best and these are the top 100 that you can see docking into the the active site and it took about two hundred computers three days to do what you’re what you’re seeing here with eight million molecules this is showing the top 100 but these are the best fit ones so we’ve isolated about 50 of those and started doing high-throughput screening or relatively high throughput screening for us to try and see if these actually have any biological effect it’s all well and good having virtual screening but you want to actually prove biological function and we’ve done that with some assays that are quite commonplace in the circadian arena we use these 384 well plates each one of these wells is basically a collection of 5000 cells that we’re looking at from above and each one of them is expressing one of these circadian reporters that I showed you oscillating within these individual cells and these are actually two plays plates with exactly the same conditions on them so some of them have drugs in some of them don’t and actually they’re expressing reporters that are actually anti phasic to each other so if you look carefully when I play the video you can see they’re actually kind of oscillating in anti-phase so we can what we can do is we can take pictures of this over time and then quantify these individual dots and see what happens when we throw on these drugs and that’s our kind of a safe or whether they can have effects on the on the biological clock work and if you graph that out this is the raw data on the left this is what we call the detrended data so we can see patterns more easily most of the drugs do this they didn’t do anything over wide concentration ranges we do usually do ten concentration ranges but they don’t mostly do anything but some of them do do something in this case prolong the period you can see the black line here is that is the control and this one here is treated with a drug and likewise in a kind of dose-dependent manner down here a completely different drug which is completely novel can affect the biological clock works so we’re hoping that by using this type of approach we can actually develop molecules that can adjust the clock either prolong it dampen it or shorten it to do what we want it to do and as I say this is far more promising than any previous approaches that we’ve had available to us because you can actually target these molecules in a structural structural way which is pretty important for finding possible therapies so I just want to end my talk there and acknowledge the people who did the work I did some of it but not all of it people in my lab Rachel Edgerton Vela Cringer alexandra stan Galen ian

robinson gamma ray Lisa Wu Nick and William who are PhD students and rotation students at the moment respectively and then a variety of different labs across the world and across the country that made this work possible and obviously immense amounts of funding from the Wellcome Trust in the ELC who bankroll my lab at the moment thank you very much

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