So welcome everyone to the second of two Steambock lectures presented by Ron Vale. And I will just say that Ron has been a, it's been really wonderful to have him visit here and I hope that everyone has enjoyed having a chance to talk with him. He's about to head out of town right after his talk. So I'm just going to say thank you, Ron, for visiting us and I look forward to your presentation. Great, well thank you, I've had a really fun time here. I wasn't as cold this morning as I hoped for, but...  So today I'm going to talk about a very different subject that we're working on in the lab, although I will try to maybe illustrate a few similarities. So the general topic of this talk is efforts to reconstitute elements of the T-cell signaling system and I'll explain to you what I mean by that. But maybe I'll just start off with, there's just a word of what reconstitution is. So reconstitution has had a very important role, I would say, in kind of the history of biochemistry, biological research in the 20th century and the paradigm goes something like this, that we should always start with cells because that's what we're trying to understand and it starts off by examining cells and defining some phenomena that may occur in a cell and then the goal of reconstitution is to recapitulate that phenomena using a defined set of components, often in a in vitro setting. And of course if you go through the textbook you realize that a lot of the textbook or understanding that's in textbooks has been derived from reconstitution experiments. So DNA replication, the enzyme DNA polymerase was discovered through in vitro reactions by Arthur Kornberg, transcription, vesicle trafficking, this past year's Nobel Prize was awarded essentially for a combination of genetics and reconstitution, ubiquitination. And I guess the one thing that is similar, which has drove our interest in doing this in T-cells was of course that it's also been very powerful for motility, the fact that you can take this phenomena of biological motility and recapitulate in a test tube with a defined motor protein, a defined substrate and a plastic bead was really essential for being able to dissect the mechanism of motion. So the question is can one use the same strategy for something that is as complicated as T-cell signaling, which certainly has a very cellular component of not just a phenomena occurring in one cell but actually involves interaction between two cells. So this just shows an image of what's going on in your lymph node when T-cells which are here in red are interacting with these antigen-presenting cells and these antigen-presenting cells are collecting various peptides and in case of if you're infected by bacteria or virus or etc., some of these foreign antigens get presented on the antigen-presenting cell and if the T-cell interrogates the cell and it has a T-cell receptor and remember the T-cell receptor has an enormous genetic diversity so there are many different sequences for the T-cell receptors encoded in each T-cell but if there's the right complementarity between the T-cell receptor and the ligand on the antigen-presenting cell which is the peptide MHC complex then that T-cell will become activated and proliferate and so there's also I should just say a great deal of interest in understanding the system of T-cell so they can be manipulated in other ways that might be productive for medical applications. In addition just to fighting foreign antigens in your body there's been a great deal of interest recently in getting T-cells to kill cancer cells and I won't go into this in great detail but this is currently the hottest thing in cancer right now and there are some antibodies that have already been commercialized that effectively allow T-cells to more productively kill cancer cells and there's been a great clinical success in this and many companies are now starting to move into this arena but even here if we truly understand how these T-cells work and how the signaling system works we probably could help design better strategies for getting T-cells to kill cancer cells so it is important to really understand the fundamental mechanism of the T-cell signaling mechanism even for pragmatic outcomes such as I show you here so Anouda is going to take you through some really basic players of the T-cell signaling pathway and again like any signaling pathway it's a sea of names but I'm going to try to make this as simple as possible and just highlight the main players so one is the receptor which I already alluded to which is the T-cell receptor it actually consists of six polypeptide chains and this interacts with molecule the ligand which is the MHC on the antigen presenting cell which presents a peptide in its groove and this could be a peptide again from for example a virus or bacteria or something like that and if there's the right complementarity and a tight binding interaction between the two then a signaling system gets set into motion where a tyrosine kinase that's a member of the Sark family called LCK phosphorylates the specific tyrosine residues on what are called ITAM domains on the intracellular side of the TCR and a prominent chain I'll describe later is called the Zeta chain of the TCR complex and once these ITAMs are phosphorylated they become a new binding site for a second kinase which is called ZAP70 and ZAP70 binds to these phosphotyrosine residues which allows it to become activated from an inhibited state and it then signals to it can then phosphorylate downstream targets a major downstream target that I'll describe later in my talk is an adapter protein called LAT which has many tyrosine residues on it that can be phosphorylated by ZAP70 and this adapter molecule then recruits many other proteins that I'll describe but at this point the signaling system just starts branching off in many different directions map kinase is activated, calcium is mobilized PI3 kinase is activated, the actin cytoskeletal is activated so the signaling system starts branching in many different directions so the first part of the talk I just want to tackle this first question which is the initial event of T cell receptor phosphorylation and this is called also T cell receptor triggering but it's the key event that gets this whole cascade moving in the right direction and the interesting question here is how does this extracellular binding interaction lead to this cytoplasmic event of phosphorylation of the T cell receptor on the other side of the membrane so that's the question we'll tackle in the first part of the talk and then in the second or very end of the talk I'll come down to this question which is how do you move beyond this initial phosphorylation reaction to then start triggering these downstream signaling events so well used by analogy with dining well you understand kinesin so you should understand dining well we understand lots of receptor tyrosine kinases so certainly you understand the T cell receptor well there's a very fundamental difference between the T cell receptor which is tyrosine phosphorylated and many of the other receptors that you know about as receptor tyrosine kinases and the fundamental difference is that these receptor tyrosine kinases such as the EGF receptor or insulin receptor have the kinase built in to the polypeptide chain so there's an extracellular binding domain that interacts with the hormone and on the cytoplasmic side there's a built in kinase and there's a great deal of information known about how hormone binding on the outside leads to receptor dimerization and leads to structural changes of this kinase that cause its activation now the interesting thing about the TCR is that it has no kinase of its own it has these ITAM motifs which are the phosphotyrosine acceptors so these are where the tyrosines are but there's no built in kinase so it's completely slave to another kinase that's diffusing around the membrane to phosphorylate these tyrosines so there's something intrinsically different from the other receptor kinases the other intrinsic difference is that the other signaling systems EGF and insulin involve a ligand that is secreted by one cell the ligand or hormone is soluble and then the soluble ligand interacts with the receptor on the receiving cell but in the case of the TCR this is a case where both the receptor and the ligand are both bound to plasma membranes and the two cells have to come in contact for the ligand to be presented to the receptor so I'll come back to this because I think this is actually a key also point in the mechanism and a difference from these other receptors now the last thing I want to just say by way of introduction is in addition just to a ligand receptor interaction there is a lot of spatial reorganization of components that happens during T cell receptor signaling and this has been observed for years by various labs this is just a paper from our lab but here we're visualizing on the surface of a T cell the T cell receptor that's interacting with a ligand this case on a planar lipid bilayer but you can see that the T cell receptor gets organized into little clusters which contain dozens or even hundreds of T cell receptors and then also they move as you can see to the central domain here by actin flow so this is something in the end we need to understand why is there this spatial organization why do things come together in these big clusters and again I'll come back to that at the end of the talk now in terms of ideas of how the T cell receptor works the T cell receptor has been around for decades since Mark Davis described it but even you know I would say even up to now there's great debate about how the T cell receptor works so this is actually taken from a review just a couple years ago which in fact presented in the review eight models for T cell receptor signaling which I'm going to condense it down to four but just to give you an idea of the flavor of ideas out here and don't worry about all these little details here I'll just conceptualize it one is that there's a conformational change just like what happens with the EGF receptors that somehow ligand binding on the outside of the cell changes the conformation of the T cell receptor for example such that the the ITAM domains now become exposed and accessible to a kinase so there's some transmembrane conformational change the other idea and don't worry again about the details you can just listen to me is that when the ligand interacts with the receptor there's another protein called a co-receptor that essentially chaperones the kinase that LCK to the receptor ligand complex and that co-receptor essentially delivers the kinase to phosphorylate the T cell receptor another idea is that this interaction results in the formation of lipid rafts which are lipid domains in the membrane and that the TCR and the kinase co-habit these lipid rafts and lead to phosphorylation and another idea that's been proposed is what's called the kinetic segregation model actually by Vendemur and colleagues which actually I'm going to favor in the end this is a prelude and that is that there's kinases putting on phosphate there are phosphatases taking it off and during signaling the phosphatase and the kinase become spatially separated and the kinase wins in the reaction now so these models were all at play and it's been difficult to really sort out one versus the other in part because during T cell signaling many things are happening simultaneously and it's really hard to know what comes first what comes second, what's essential what is maybe secondary so because of all this it's really hard to understand what is fundamental and critical for receptor signaling so John, a very brave postdoc took on what I think was an extremely interesting but extremely challenging project from the onset which was to reconstitute T cell receptor signaling in a nonimmune cell and apply the same ideas of reconstitution that if we can identify the key components that are needed to get ligand mediated TCR phosphorylation then we can understand the minimal components that are needed for their reaction and begin to manipulate them to really understand the mechanism so he undertook this not in vitro but in taking a nonimmune cell which is a garden variety kind of human kidney cell that people use normally just for protein expression and convert that if you'd like into an artificial T cell not a truly artificial T cell that behaves like a T cell but one that can recapitulate the early events of T cell receptor signaling and also for the students or postdocs in the audience this was a project that required a great deal of patience because it involved like step by step introducing components, understanding how they work one by one and building up a system over a period of like two or two and a half years where you weren't getting any results but you were just setting up the system however at the end of the day there was great reward because suddenly all these experiments became possible so you go from this time when you're not getting any quote-unquote results, nothing exciting or publishable and then suddenly being a kid in a candy store where you could do all these experiments in very rapid timescale so this is what John set out to do and these are all the molecules he had to introduce into this artificial T cell and artificial antigen presenting cell so I'll just go through conceptually that there is a set of adhesion molecules between the T cell and the antigen presenting cell that bring the two cells together so he introduced those molecules he introduced the ligand in the receptor and even this was quite challenging because the T cell receptor is a complicated protein complex made up of six different chains so that was non-trivial I should say a lot of these were made by combination of stable cell lines and transient transfection using lentivirus for those of you that are interested and the other set of molecules are a series of kinases which are involved in positive regulation and CD45 and a second kinase are involved in negative regulation i.e. keeping the system quiescent so basically I'm not going to show you all the results that led up to this but just to the final experiment so this is one of those things go bypass two years of John's life but let me show you the key readout that he used for phosphorylation we could look at phosphorylation with radioactivity but in more powerful ways if you can get a visual readout and see the phosphorylation in real time so as I mentioned when the TCR gets phosphorylated the second kinase zaps 70 binds to these phospho-item domains and it translocates from the cytoplasm to the plasma membrane and that is a reaction you can follow by microscopy this is just a simple slide to illustrate that of transfecting this artificial T-cell with the LCK kinase and the TCR and the ZAP 70 the ZAP 70 is labeled with GAP or a cell without this kinase and you can see here without the kinase it's ZAP 70 is all over with the kinase it's recruited to the rim so this is going to be our readout and here is the final experiment that John showed to demonstrate you could get ligand specific signaling so this experiment here is a control where the artificial T-cell is interacting with the antigen presenting cell here is the ligand the peptide MHC it's all over this antigen presenting cell and ZAP 70 is all throughout the cytoplasm and that's because the wrong peptide in this case was loaded into the MHC molecule so this peptide MHC complex is not recognized by this particular TCR that was John introduced into this artificial T-cell now we can introduce the proper peptide that it is known to be recognized by that TCR and you can see that here now the ligand interacts with the TCR right where the two cells come in contact and gratifyingly the ZAP 70 gets recruited to these phosphorylated TCR right at the interface so John was able to recapitulate ligand specific TCR triggering in this system and just to say these are what the two cells look like interacting with one another the T-cell actually kind of wraps around the antigen presenting cell it actually looks a lot like images that are taken of a real T-cell interacting with the antigen presenting cell and the reaction also happens very fast so this is a movie that John caught when the two cells just come in contact with one another and the ZAP 70 is normally cytosolic so here's an image of yeah, here's the T-cell here's the antigen presenting cell and when the two cells come in contact you're going to see the ZAP 70 which will appear as a cyan image due to the color overlay right when the two cells come in contact so here they are, they're the two cells and that's the ZAP 70 being recruited to the membrane so the signaling mechanism happening very very fast so the exciting thing is the experiment worked that's all really good but then the next question is why how is it, what is critical for the signaling reaction to happen and this is the beauty of the system because now you can take it apart and dissect what is happening and the mechanism by which it's happening okay, so first thing that was very dramatic is when John looked at the various components of the system and visualized where they were there was a very dramatic result that when the two cells came in contact this is where the phosphorylation reaction is occurring this is the localization of the phosphatase it's basically everywhere else except where the TCR is being phosphorylated the nice thing is John can also show that this exclusion of the phosphatase is absolutely critical because an experiment won't go through in great detail but he could re-engineer the phosphatase has an extracellular domain and an intracellular domain he could swap out the extracellular domain and put another extracellular domain that would bind to another protein target on the antigen presenting cell and that would drive the phosphatase back into this interface if he drove it back into the interface this phosphorylation reaction didn't happen so this phosphorylation absolutely requires this spatial exclusion of the phosphatase and that's very consistent with this kinetic segregation model that I described to you earlier but now I can take the question one step farther okay we need CD45 exclusion but what is driving the spatial segregation of the CD45? how does that work? and so that's the question and now having built up this complicated system now John could slowly take it apart he can deconstruct it and ask what are the minimal set of components that are needed to drive the spatial segregation of CD45 and the answer to that was gratifyingly simple and that is you don't need any of the kinases or other signaling components don't need signaling at all the peptide MHC-TCR interaction itself without any as I said any associated kinases is sufficient to drive the spatial segregation of CD45 so what you're seeing here is now these two cells interacting with CD45 here and just the TCR and peptide MHC and you can see here we're not looking at signaling we're not looking at recruitment of ZAP70 we're just looking that the TCR and peptide MHC interact at the interface and the CD45 is excluded and that is something specific to the peptide MHC-TCR if you look at if the cells interact just with these adhesion molecules the cells come together but the CD45 is not excluded so what's going on with this well I'll give you some hints I can't say we know the complete answer to this but one thing that a very nice experiment that John did is to look at how the two cell membranes interact when there's TCR-peptide MHC interacting across the membranes and you could use that by putting an M-cherry on the cytoplasmic domain of the peptide MHC a GFP on the cytoplasmic end of the TCR so you can now image this by microscopy and if you do that you get a green image, a red image and if you overlay them they look like they overlap but for those of you that know about super resolution you can take a line scan across the red and the green image and you can get a very detailed profile of the fluorescence intensity and in fact you can average that across the whole line and you'll see that these two peaks if you look at the precise peak of the Gaussian distribution are slightly shifted from one another by about 30 nanometers which corresponds to like about a third if you include where the GFP and the M-cherry are about a 15 nanometer separation between the two plasma membranes now if you look at how far the cells are apart when there's a control peptide in there so where the peptide MHC and TCR interacting the cells are just interacting with the adhesion molecules the two membranes are far apart when you have the TCR and peptide MHC the membranes are now coming very close together so let me tell you a provisional model of what we think is happening and that is we think that the two cell membranes are initially coming together first by interacting with these adhesion molecules but these membranes are probably not like flat boards they're probably fluctuating they're moving by Brownian motion maybe also through the actin cytoskeleton and occasionally some of these fluctuations allow the two membranes to come closer and if there's a peptide MHC and TCR that interact the two membranes get trapped just like fly paper so normally the membranes would fluctuate and come back but now they've got this sticky fly paper that's sticking these two membranes together now this is unfavorable because it's creating bending energy in the membrane here which is illustrated by this red normally this would like to flatten out again and this is also a lot of unfavorable energy because we've now created these four points of membrane bending so a lower energy state would be to bring these two points of contact together and that can happen because these things are probably moving laterally in the membrane to create this state here where all of these interacting molecules are now clustered in a point and now we've gone from multiple points of membrane bending to just two points of membrane bending and we think this is what is driving the TCR, peptide MHC and potentially other things that are interacting across the membrane to a cluster the phosphatase on the other hand has this big extracellular domain which doesn't have a binding partner on the other side so we think it's basically being squeegeed out of this region where the peptide MHC TCR are interacting the LCK on the other hand doesn't have any extracellular domain it's just attached by a lipid anchor and we know that it's not excluded by this set of interactions so this is our working model of how the binding energy of peptide MHC leads to spatial segregation of this kinase and phosphatase so this suggests a kind of a simple ingredient for this signaling system now I'm not going to say that the signaling in a T-cell is as simple as this this might have been the ancestral version on which a lot of bells and whistles have been added so I don't want to imply this is all that's going on but that essentially what the system may be using is this binding energy between the receptor and the ligand I personally don't think there's a conformational change that's going on or that is necessary for this but what this binding energy is being used to do is to cause spatial segregation of proteins in the membrane and typically that of a phosphatase that's a negative factor largely it's taking away phosphates that's excluded from this region and a positive kinase that doesn't feel these exclusion forces and that gives rise to T-cell signaling now one thing if this idea was right you can then say okay well what's so special about the TCR and peptide MHC I mean if what's needed is just to create binding energy across the membranes maybe that can be replaced by some other form of a ligand receptor pair that if you attach the cytoplasmic signaling domains to something else that's creating binding energy across the membrane maybe you can also get a signaling system so we've tried this in several cases and it does work and I'll show you the most recent incarnation which I think is pretty exciting and that is to replace the TCR with DNA so in this case this is worked by Marcus Taylor another really wonderful postdoc who's actually a joint postdoc between my lab and G2 Marin he's got a lot of help from Zev Gardner at UCSF the strategy here is to on the T-cell take the normal cytoplasmic domains that get phosphorylated but on the extracellular side just put a domain to which you can covalently attach a strand of DNA and replace the antigen presenting cell with a lipid bilayer and anchor via another protein tag with another strand of DNA onto the bilayer now of course we want to bring so this is the mimic of the peptide MHC this is the mimic of the TCR we now want to bring these two strands of DNA together which Marcus can do by adding a trigger strand that basically forms complementary base pair Watson-Crick base pair strand interactions between the DNA on the T-cell side and the DNA on the bilayer side to bring these two membranes together in this kind of interaction now he's also introduced this not into an artificial kidney cell but he's introduced this into well maybe somewhat like a more T-cell an immortalized T-cell that people standardly use in culture which is called a jerkat T-cell and the experiment is if we introduce this DNA system here so instead of using the normal T-cell system can we replace this just by DNA basically interacting across these membranes and will that signal downstream in the T-cell to the normal signaling system so two of the outputs that we know are involved in T-cell receptor after T-cell signaling are actin polymerization and also map kinase activation so just in terms of actin activation let me just show you this is what a T-cell receptor looks like after it's been activated the actin becomes the cell spreads on the T-cell receptor spreads on the surface because the actin network becomes very active for polymerization so the question is do we get this kind of behavior so I'll show you the experiment now this is a T-cell receptor we're actually looking at the DNA receptor tagged with GFP and now we're adding the trigger strand just now to bring these two membranes together and you can see the cell now triggering the actin cytoskeleton beautifully spreading out after these two strands of DNA across the membrane are brought together by the trigger strand of DNA and you can also see just like the real T-cell receptor this artificial DNA receptor if you like is also becoming organized into clusters very much like the way the normal T-cell receptor does during the signaling process as well and it also activates the map kinase pathway so we can follow that just by measuring a phospho-erc with an antibody and we can measure the number of phospho-erc responsive cells as a function of ligand density so that's the DNA ligand on the artificial bilayer so we can vary the ligand density and see if these cells now become phospho-erc positive so you can see you get this activation curve with the more ligand you have the more cells become activated but the beauty of this system and the reason why we're going to DNA is that although this is still early work in progress is you can manipulate DNA in ways that is much harder to manipulate protein so we can control the length of the DNA the thermodynamics of the DNA-DNA interaction, the off-rate of the DNA just by controlling the oligos that we use so we can vary and explore the entire space of what is needed in terms of binding energy across the two cells to trigger the signaling system and you can even see this if we go from a 16-mer or Marcus goes from a 16-mer or 13-mer there's good signaling drops the base pairing down by two nucleotides and signaling is off so what we're trying to do is to really understand and to connect to the signaling system and activate the downstream signaling pathway and this has been a big debate in the field about what a T cell receptor sees in the peptide MHC to cause it to activate so we're hoping to understand this in a much more controllable way using DNA where we really can control these parameters much better so I'm now going to switch so I kind of gave you a flavor of trying to reconstitute elements of T cell receptor signaling by making an artificial cell if you'd like and even that we can make artificial receptors that can connect even to the normal T cell receptor system this is going to be a different approach now of making pure protein so there's no cell here at all to reconstitute elements of the T cell receptor signaling system using a completely defined system in vitro and there are various things that I think are interesting to reconstitute one is this initial model can you bring membranes together and lead to protein exclusion can we really understand this network of kinases and phosphatases to understand how they behave and can we reconstitute elements of T cell receptor signaling so I'll give you a progress report where we are on all of these this is the least advanced but just to show you that it's possible that we can take a completely artificial system of a supported lipid bilayer these are lipids bought from Sigma and a vesicle and put the extracellar domain of CD45 on the GUV so this you can think of I guess as the T cell and a ligand receptor pair that you can control with a small molecule to bring these two membranes together and the short answer is if you don't have rapamycin they're all even but if you bring this pair together with rapamycin you can bring the receptor and the ligand and the exclusion of the CD45 here in a completely biophysically accessible system so I think this is going to be interesting in terms of the kinase phosphatase reaction this is maybe somewhat infuyu probably a product of your great graduate program so thank you very much infuyu is really just a super fantastic scientist now doing a postdoc in my lab he was from Ed Chapman's lab and he has taken on I think a very important project of understanding the entire network of kinases and phosphatases that are leading to the phosphorylation of T cell signaling so the this is the cytoplasmic network it's all impinging upon whether phosphate goes on or taken off of the TCR and in fact it's quite complicated because there's LCK which is putting phosphate on but it has a number of complicated regulatory mechanisms for turning that kinase on or off or changing its activity there's a negative regulatory kinase that is controlling the activity state of LCK there's CD45 which is taking phosphates off of here but it's also removing an inhibitory kinase from LCK so the bottom line is this network is really easy to draw as a model diagram on your Macintosh but very hard to understand on first principles about how this network really works when it's all combined so towards the end of the day of really understanding this whole system in silico you really need to get good quality data of how these enzymes are behaving and also how they're behaving when they're put in combination which again one cannot drive a priori so his approach was to purify all these enzymes and put them on the surface of a liposome and then study, develop assays to study the in vitro phosphorylation of the TCR and he did that by developing a FRED assay where here is the TCR cytoplasmic chain and when it gets phosphorylated it recruits a domain of that other kinase I told you about ZAP70 so he takes the interacting domain of ZAP70 puts a fluorophore on it and when this fluorophore is recruited to the TCR there's a FRED interaction between this tagged reporter and a rotamine that's present on the liposome membrane the net result is that your reactions look like this that here is that signal that's going on when this reporter comes in contact with the membrane and here we're activating the kinase by the addition of ATP so he can both measure the rate of phosphorylation as well as the extent of phosphorylation in the reaction this is just to show that this FRED assay is doing just as good a job as a really laborious blotting assay so this allows him to get excellent kinetic readouts and I'll give you just a flavor of a few results that he's gotten from the system although he's gotten an enormous amount of information this just stresses a lot of people in the field study these kinases in the following way and this is true in the SARC field as well they purify SARC with a solution with a single time point IP often with an artificial substrate like enolase or a peptide or something like that these enzymes are meant to be interacting on a membrane and with a real substrate that has you know physical features that are different from a peptide but this shows the difference in rates in which if you're studying something in solution on a membrane for example here is the rate of phosphorylation for LCK and the TCR if both components are in solution effectively hardly any over this time scale hardly any reaction occurring if you increase the amount of kinase by tenfold you can begin to see something if you put these components on a liposome membrane each other on a 2D surface the rate we estimate is about 700 fold faster than in solution so I think really for understanding a lot of the enzymology of membrane bound kinase phosphatase reactions you really have to study these things on a membrane surface the other thing another thing that ENFU try to deconvolute is that LCK which is this kinase is actually a really complicated machine it has it's phosphorylated at C terminus and this creates like an auto inhibited state which after this phosphate is removed goes to some kind of open state and it can be further enhanced by auto phosphorylation at the active site this is known for SARC but again a lot of the first of all SARC need not necessarily be the same as LCK and also a lot of this study if you really go through the literature is more qualitative than quantitative in terms of the real understanding of what these states are in terms of real catalytic effects on LCK so ENFU basically was able to dissect this by first of all creating biochemically pure states of LCK where he could knock out one phosphorylation residue by knocking out the tyrosine and then phosphorylating the other residue essentially to completion so he can create basically the four major different states of LCK and measure the reaction rates get the KMs get the KK and all of that so now he actually has a really detailed map of the catalytic activity of every single state of LCK which again I think is going to be critical for mathematical models in the future but there are also some this is just a summary on the log scale of the differences in the catalytic rates of these four different regulatory states of LCK but there are even some really interesting things about this that the order of phosphorylation if you start off with like naked LCK it really matters which phosphate gets phosphorylated first because LCK autophosphorylates itself so if this negative regulatory phosphorylation residue gets hit first the kinase activity goes dramatically down well at least five fold now this relatively inactive LCK has a hard time autophosphorylation itself on this active residue on the other hand if you proceed through the cycle in this way you phosphorylate the active site first the second phosphorylation of the inhibitory tyrosine doesn't really do that much to inhibit the activity so there is probably an important kinetic effect which I can't say we totally understand about which tyrosine actually temporarily gets phosphorylated first in these reactions the last thing I'll tell you about what ENFU did was a really detailed understanding of this whole network where he can actually use 96 well plates and by this FRED assay this is the FRED assay curves that you see all over here but systematically vary the concentrations of LCK and CD45 or in some experiments adding additional components in the reaction and measure the reaction rates as a function of concentration of these opposing kinase and phosphatase in the reaction to look at the net effect of phosphorylation and from this he's been able to essentially make phase diagrams of the enzymology of what this system looks like as a function of different concentrations and there's a lot of information here but first of all I should say this is blue is low reaction rate so little phosphorylation red increasing amounts of red so increased phosphorylation of the TCR so that's what you're seeing here this is kind of thought to be the physiological range of LCK and CD45 you can see it's largely in a quiescent state so you either have to go down in this direction of decreasing the phosphatase to move into a phosphorylation reaction or increase for example in this direction the level of the LCK which I'll come to in the next slide but this phase diagram basically tells you when the system is going to be in an on state or when the system is going to be in an off state and you can measure this looking at the change of this phase diagram with for example if you knock out one of the regulatory mechanisms of LCK the negative regulation the phase diagram shifts over basically becomes easier to phosphorylate the receptor and by measuring for example the rate of the reaction keeping one of the components constant he can measure essentially a concentration dependent reaction rate of how this reaction changes as a function of changing one of the components and that's what's shown in this system so out of this CD45 is how constant we're looking at the variation in LCK or here LCK is kept constant and we're looking at the variation of increasing CD45 so here we're activating it here it's active at low phosphatase and it's being turned off but the bottom line is and what we can measure here is the hill coefficient so this is how cooperative the system is and interestingly in both cases we see some cooperativity in the system as a function of changing the enzyme concentration but the hill coefficient is for a reason we don't completely understand honestly is steeper if you're varying the CD45 concentration so if you keep LCK constant and you end up decreasing CD45 you get a steeper switch like behavior to TCR phosphorylation this is interesting because we think this is how remember what I told you before we think what's happening is reaction is we're removing CD45 from the reaction zone where the phosphorylation is occurring which means we're decreasing the CD45 concentration and so we think this is probably important leading to kind of when you start segregating out the CD45 to a steep transition point where the system is going from an off-state to an on-state so it's at least consistent with what we're thinking about okay in the last five minutes I want to talk about this clustering because these are some cool experiments here I'll have to rush through this a tiny bit but remember I said there's a lot of clustering going on in the system it turns out many years ago actually this is our first paper in the T-cells Adam Douglas found that this protein called LAT seems to be important for these clusters this is another marker for these clusters it's a co-receptor called CD2 but in a LAT dependent cell he transfected in LAT normal LAT you could get these clusters in the mutant cell line but if you transfected in LAT that didn't have any tyrosines the interesting thing about LAT is it has lots of these phosphorylation groups which bind to SH2, SH3 adapters and what might be possible is like LAT is an octopus it has lots of arms, lots of adapter proteins these adapter proteins have multiple arms and you start to get this multivalent system that can lead to clustering so to test these ideas you know ideas one could go to purified proteins so again I'm rushing through this a little bit but as part of a collaborative grant that we all got together we brought a number of people PIs to the marine biological laboratory in Woods Hole and one of the goals for that summer, this was the past summer was to reconstitute some of these protein protein interaction systems that are going on in T-cell signaling this was like a tremendous amount of fun I think this is how we should do a lot of our science these days none of the work I'm going to tell you about would have been possible in any of our own labs everyone here had different expertise they brought students or postdocs that had different expertise in different proteins we all worked cooperatively to bring these proteins together in Woods Hole so we prepared almost for a period of seven, eight months with ideas and reagents and just when worked around the clock at Woods Hole to do experiments and it was just like you could pull stuff out of the freezer and interact, you know, come up with an idea at noon and have it realized that, you know, midnight so it was really fun and actually one of the few things we actually got more work done than we anticipated which it's usually usually the opposite. Anyway these are some of the key people again in the project I'll tell you about John Ditlav is in Mike Rosen's lab Xiaolei Su is in my lab and Enfu also helped with many of these experiments other folks helped too but the first thing I said LAT might be an octopus and create clustering so in this experiment we took LAT and some of these adapter proteins phospholat and this is experiment okay, the phospholat here is on a bilayer I thought I had another movie here but anyway it's completely diffuse but then we add these adapter proteins and then at the end of the experiment we're going to add phosphatase that's going to take the phospho groups off of LAT and the phospho groups are needed to interact with the adapter here's what happens boom, you add these adapters they form these clusters very similar to what you see in a T cell you add the phosphatase they go away so we really do think that the ability to form these multivalent interactions is creating actually a higher order complex of proteins and the question is are these involved in signaling so at the very end of the summer we took on the ambitious goal of actually trying to reconstitute the entire T cell signaling system all the way from the TCR which ENFU was an expert at to this LAT system which Chalet was an expert at with John and we had other people involved that knew a lot about actin polymerization and these are all the proteins that had to come together in their reaction very impressive number of proteins here's the reaction to this movie Ryan but we had tags on that's the actin coming so all of this is on a membrane bilayer there's no cell involved the TCR is on the membrane bilayer the kinase is on the bilayer other proteins that should be in the cytoplasm are just floating around on the glass cover slip where you're seeing in green is a flash of the ZAP 70 recruited to the planar lipid bilayer which then phosphorylates LAT which then forms these clusters which then recruits activators of the actin polymerization system which involves a string of events that lead to activation of R2-3 and the polymerization of actin filaments which emerge from these LAT clusters yeah so these are just the components that's the flash of ZAP 70 recruitment after that we get the LAT clusters forming and after that with a longer time lag the actin being formed out of these little clusters so you know we think we can use this system really understand a lot of the biochemistry and so these are the folks involved in the work John again all these folks are amazing John now is his own lab Marcus, DNA work, Gele and John a lot of the LAT clustering infu thank you Madison for sending him to my lab a lot of really nice enzymology and thank you for your attention I probably could take a couple questions and then I have to run to a bus to get to the airport because my plane was cancelled ok yes that's a good question yeah so is it just that it's really big it has this really big extracellular domain and it's just being pushed out sterically the answer is that's not the total answer if we chop down the extracellular domain of CD45 so it's exact same size as the TCR it's still excluded it gets some added benefit by being bigger you get more exclusion but you still get a very effective exclusion it's the same size the reason why we think that's true is that part of it is being big pushed out of the way but also if you don't have a binding partner on the other cell you end up losing a seat at the table that's what we think so if you have this zone where these proteins are being clustered and you have a partner on the other cell you're kept in that zone if you don't have a binding partner you occasionally step out just by diffusion and someone that has a binding partner takes your seat at the table so we think that proteins that don't have binding partners that aren't contributing binding energy across the membrane eventually lose their seat at the table and get excluded from these protein-rich zones that are forming binding interactions between the cells do any proteins get excluded? yes and he's tested that by putting in different again he could do that and we hope to look at that the liposome system too in various test cases and look at what gets excluded I mean we're still trying to understand exactly what that means but that's at least an idea yeah exactly well that's what we're I mean there are obviously a lot of people are looking at this question I mean people have looked at antagonist peptides and measure their binding affinity with surface plasma on resonance so that's been an approach people are also pulling on T-cells and angine presenting cells to look at force dependent binding interactions are some people think that on-rate is important some people think off-rate is important I don't know the answer to that it's a really key question so our approach is try to use DNA I haven't told you anything yet but that's like where Marcus is going now now that he has a system working he's hoping to vary because we can vary and actually measure all of these parameters for DNA DNA oligo interactions and you know the very well understood thermodynamically that this may be a good system to be able to understand those questions so but we don't have the answers to that yeah no, I mean again there will be a reason to go to a GUV system because there are all kinds of experiments you can vary lipids you can vary membrane tension osmotically things like that which you really can't do in cells I mean there are a lot of people that take cholesterol away or add cholesterol but there are so many complicated things that go on in those experiments I think again we'll try to get at that but I think at least at the first approach looking at that in a completely defined GUV system may be a better way to go well there are all these classes of proteins, bar domain proteins that actually stabilize membrane curvatures and you know of course in a real cell too in addition just to lipids and membranes there's actin so actin is like constantly acting on the cell membrane and we also know that I didn't provide any role for actin in anything although in reality we know actin is actually very important for T-cell signaling if you had latrunculin there's no T-cell signaling going on so yeah so I think it's obviously more complicated when I just presented but and yeah so I wouldn't be surprised if actin is also like inputting the energy into the system in addition just to the physical properties of the lipids


Where are we stopping? Darned upset stomach. We'll have to turn back. When upset stomach strikes, tender walls of both the stomach and the lower digestive tract become irritated and you have that well-known stomach ache. I thought you had an upset stomach. Hey, hi, what's going on gang? Welcome back to my channel.  This channel contains videos that can help heal more than 90% of any health, wellness and body image challenge. So please do share this channel. Please subscribe for me down below. Subscribing is where the support does come from. And being that it's April, it is IBS Awareness Month. I'm pretty sure a lot of us are aware of IBS and what it is. IBS is irritable bowel syndrome. This is one of those things that I've been very successfully helping people overcome way more easily than I thought they might for so many years. I absolutely can't not share this info. IBS is seen as one of those normal things. It sure isn't normal that we sometimes get. Just one of those things people have. You either have it or you don't. And if you do, you just kind of live with it. You just help ease your symptoms. Nope. Stop it. Get some help. IBS is not a normal thing that any of us should be having. A lot of people have IBS that really don't need to be having it and can get rid of it so much more easily than otherwise was thought. When you go to the doctor to discuss IBS, the allopathic, regular family, general practitioner doctor, what they're going to do is zero in on those symptoms, help you deal with the symptoms without searching for the root cause of where IBS is coming from for you as an individual because all of our bodies are completely different. Nowadays a lot of people will take medicine if they have the acid reflux, just anything to kind of put it off. The whole conventional medical system is based on treating the symptoms. That's why people are on medications for years to come and the problem is never gets resolved because we are treating symptoms. Having a West America training myself, I can tell you we are treating the symptoms. We are not getting to the root problem. That's how the system has been set up. That's how the education is being done. The whole system is based on chasing the symptoms. Technically, nobody when you treat the symptoms gets cured. So the cure rate while you treat the symptoms is zero and getting to the root problem means the patient problem can be corrected long term. The root causes of IBS though, pretty much the same across the board from what I've seen in my own practice. The number one cause of IBS are food allergies or intolerances and food sensitivities that you may not realize you even have. Oftentimes a lot of people do have sensitivities or intolerances that they're not quite aware of and these things will irritate the bowel and cause all those yucky IBS symptoms such as... Among some of the more annoying symptoms of IBS is the sensation that you suddenly have to go to the bathroom. Many people who experience this sensation also report the feeling of not being finished when they do get a chance to break away from their responsibilities for a moment. Intensification during menstruation. For women, IBS can be a very difficult experience because a large percentage of women report that the symptoms of IBS intensified during times of menstruation. One of the main issues that causes food intolerances, sensitivities, and allergies is inflammation in the gut. The leading cause of inflammation in the gut is stress. So it really does help to keep that stress in check to help your digestive issues along. Another thing that causes IBS is unbalanced gut bacteria also known as intestinal dysbiosis. This usually means an overgrowth of the gut bacteria Candida. We all have levels of gut bacteria that can be healthy for us. However, these can tend to grow out of control leading to things like S-I-B-O which means small intestine bacterial overgrowth. This is so unbelievably common. There is a test called the spit test that can be done at home. This test is not medically recognized. However, what you would do is at night before you go to bed get a small cup of water.   It should stay at the top of the water in a cohesive blob. If you see strings of saliva reaching down from the top cloudy specks in the water or cloudy saliva that sinks to the bottom of the cup overnight you then should be looking into having Candida protocol done on your gut. If you want any help with that or have any questions please comment below. I would be absolutely delighted to help anyone there. I am out here for you. Help me. Candida overgrowth is caused by an imbalance of sugars whether that be alcohol it could be eating too many sugary foods to non-sugary foods and especially antibiotics. Think about the word antibiotic. This is why we need probiotics to help re-colonate our gut with healthy bacteria. Even one round of antibiotics as a child can wreak havoc on the gut and cause Candida later on in life. So that's another area of IBS that I see a lot in my clients and general inflammation issues in the gut. The five most inflammatory substances are alcohol, gluten, which is anything made from wheat, barley, or rye, pasteurized dairy, refined sugar, corn, and soy. So if you do have IBS, if anybody comes to me with IBS the first thing I tell them is to absolutely completely avoid any processed food with refined sugar, alcohol, gluten, wheat, barley, rye, corn, soy, pasteurized dairy for a minimum of three weeks and you will notice likely huge changes in the way your poor little bowels are treating you. So it really can be as easy as that. Like I say, it's not necessarily the exact same reason for everyone but those are the absolute root causes of any case that I have personally seen of IBS, irritable bowel syndrome. If you want any more information on that or would like any help, please do comment below. Again, please subscribe to this channel and if you would like notifications of future videos as they're posted, please hit that little bell notification icon down below and you will be sent a notification each time I do post a new video.  And until next time, see you then. Bye.


And as you probably noticed on your agenda, this is our first session right after Tai Chi of the entire program this year. So welcome, you're all early birds. As we sit here today, over 800 million people across the globe still lack access to electricity, while human demand for energy is set to rise by 30% over the next three decades. Yet even now, energy production unused with electricity, industry, construction, transport all included is the largest source of global greenhouse gas emissions. And if we compare the numbers in 2022, the global demand for coal increased by 1.2% and first time in history is surpassed 8 billion tons. Meanwhile, IEA predicted the renewable energy will replace coal as the worst largest source of electricity by the beginning of 2025. Why are we talking about the numbers here? Because what we see here is a mixed picture and a fast changing one. And the choices we'll make today on energy transition will shape our collective future. And how do we achieve that? We'll have a distinguished panel, highly capable of guiding us and navigate this transition map. Let me quickly introduce our panelists today and remind for everyone we'll leave 15 minutes for Q&A, so take your time and think about your questions. First is a lady on my left-hand side, Dr. Sekai, Irene, and Zenza, Minister of Industry and Commerce from Zimbabwe. Zimbabwe has committed to reduce greenhouse gas emissions by 40% by 2030, if I'm not mistaken. And the gentleman in the middle is Mr. Xinbao, an executive chairman of Stay Great and Stay Great, and for those not based in China, is not only China's major supplier of electricity, but also the world's largest utility company. And Mr. Simon, sitting right across from me, Simon O'Connen, CEO of SMV Netherland Development Organizations. It's a nonprofit international development organization working on food security, water sanitation, and I'm sure you'll let us know more details in a minute. And most importantly, the access to affordable energy in low-income countries. And last but not least, Mr. Franklin Serven-Schreiber, CEO of Transmutex, the Swiss startup dedicated using technology to reduce the stockpile of existing nuclear waste. So we'll get started. Let me start with Dr. Nzenza. The transition to grain is incredibly important and also is set up as one of the priorities for the Zimbabwe government. But there's still significant room of improvement in your energy transition speed. How to address that and what are the key hurdles? Please. Thank you very much, Lin. Lee, let me begin by saying good morning to everyone and also to bring you greetings from Zimbabwe. And I've noted that there are people here who have been to Zimbabwe or lived in Zimbabwe. So I'll bring you greetings from the President, his Excellents, Dr. Mnangagwa. In Zimbabwe, we are going through a transformation. We have recently, recently as in the last year or so, launched the National Development Strategy 1. And the focus is mainly to transform three key areas, transformation in agriculture, moving on to agro-processing, transformation in the area of manufacturing, where we are focusing more specifically on value change. Having realized Zimbabwe's natural resources, our major challenge, Lee, was to do with exporting raw materials. So the major transformation in Zimbabwe now is to process the natural resources locally. And we're also looking at mining. And within the mining sector, we're focusing on value addition. So in all these areas that I've mentioned, from mining to agriculture, as well as to manufacturing, what's absolutely key is energy and energy efficiency. And we've noted that there is a deficit here. So if you have deficit in energy, you cannot produce, you cannot trade. So what are we doing about it? I would like to just pause here for a while so that for the next question, I can then discuss areas in which we can focus on increasing our energy resources. Thank you. We'll come back to you shortly. And let me move on to Mr. Zimbabwe. Stay great. As China and the world's largest utility companies, stay great in industry and people's lives should speed up the transformation to clean and affordable energy. How do you do that? Thank you, moderator. Ladies and gentlemen, good morning. It gives me great pleasure to participate in today's session. As we all know, effectively step out to the challenge of climate change. The transformation of energy is the key. Low carbon, clean and effective development is critical part. As the largest company in China, we are conducting our obligation in energy transition. And we're building new type of electricity system in China. We've come up with action plans for the reduction of carbon emission. And we've enhanced our investment into key projects as well as scientific research. And we've established new type electricity system innovation alliance. And we are improving our capacity to absorb renewable energies. And we've produced 33 ultra-high pressure grid and more than 4.4 million stations, transmission stations for electricity. Fourthly, we're developing energy storage station, which can support new types of energy storing capacity. And we're facilitating the electrification of end users. As we all know, electricity is the secondary energy, which is clean and effective. So we should raise the mix of energy in the end user consumption. That is the key for energy transition. So energy to replace oil, energy to replace natural gas are the key works we're doing. Now in China, electricity and consumption percentage has reached 37%. In the future, that presentation will climb up. As we all know, there is cost to energy transition. On the one hand, we must promote low-carbon transition. Secondly, it must be sustainable and affordable. So our key is to guarantee the production and to make it steady and stable. Now the average tariff of energy in China has been remaining at a relatively low level. Residential and agriculture use remain at the lowest level. The safety operation of grid has been improved drastically. Our business environment in the world ranks top. So the satisfaction degree and happiness of the end users have increased. Thank you. Helping us lay out the outline of China's energy transition, some of the challenges, but also achievements. These are massive scale what China is doing. China has been working on things not just massive, but micro-scale as well. Like Minister mentioned, you lived in Zimbabwe. So in your work through SMV, how do you see energy equity among the developing countries? Thank you and good morning, ladies and gentlemen. First off, if I may, I'd just like to say what a great pleasure it is to be back here in Chang'an in China. I think we all know, we all recognize that the energy transition, the transition to cleaner energy, the climate crisis, these are global issues that need, yes, contextually tailored solutions, but global solutions. And to have that, you need collaboration, you need relationships, and of course you need trust, which is why it's so, so important and why I'm so delighted to be here in China. You know, to your good question, it's not actually that micro. The issues that we're grappling with, you rightly said in your introduction, there are 800 million people that still lack access to any form of electricity. I could add there are 2.4 billion people worldwide, 2.4 billion people who lack access to clean cooking. These are, you know, big, large scale macro issues as well. And to come up with the right kind of solutions, we need the right kind of partnerships with, yes, development actors, NGOs, civil society organizations, but vitally important as well, the private sector and governments at all sorts of different institutional levels. I think there are three things that we, at SNV, so yes, we're a global development partner, three things that we really encourage a focus on. One is the importance of context and contextual understanding. I'll just give you a couple of examples. I worked for several years in South Sudan. I'd worked in Sudan previously, then went down into South Sudan in 2005 when the Comprehensive Peace Agreement was signed. The country was then formed as a fully-fledged independent country in 2011, fast-forward from 2011 to 2023, so 12-plus years, and only 7% of that country have access to electricity. In Mozambique, where my organization is working, 34% of the country now have access to electricity, and at SNV we're working already on a large-scale initiative called BRILLO with the British and Swedish government and a range of private sector partners to create access to solar household systems and solar mini-grids for 1.3 million people. So in the grand scheme of the world, perhaps that doesn't sound that large, but in the context in Mozambique it is enormously significant. But the point is all contexts are different from South Sudan with 7% access to electricity, 34% in Mozambique to many, many other geographies. So we have to understand context. Secondly and relatedly, then we need to design solutions that are most appropriate to those contexts. In Mozambique it wouldn't be appropriate for any organization to start setting up solar mini-grids unless, and I believe you just rightly highlighted, you've got your tariff structures in place if you've got a good understanding of your tariffs. In Ethiopia, we're working with the Global Energy Alliance for People and Planets so a large-scale energy initiative to set up 9 solar-powered mini-grids for irrigated agriculture. We can't possibly do that unless the tariffing structure around those mini-grids is set up. And then the third point is relatedly, again, the right financing forms. To have access to an improved cook stove in Mozambique, you need to spend about between 7 to 10 US dollars. There are many, many people in Mozambique that do not have 7 to 10 dollars in their pockets. It may seem extraordinary to some of us in this room. Don't have 7 to 10 dollars in their pockets to buy and improve cook stove in one go. So you need your pay-as-you-go models, you need your lending models, you need your financing structures at those micro levels to enable people, the extreme poor in a country such as Mozambique, to start climbing that energy ladder. And ladies and gentlemen, it's a ladder that we're talking about here. We're not going to go from the 7% access to electricity in South Sudan to 100% coverage on a full grid anytime soon. So we need to be thinking about that energy ladder and the steps we can take accelerated to scale that access to electricity. Thank you. Thank you. We'll come back to many of the points you mentioned here, but thanks for giving us a picture of how comprehensive this challenge is. It's not just energy, there's also finance, there's also governance, everything coming together. Let me move on to Franklin. Well, your company is only four years old as you just told me as a startup, but you are in a very specific and interesting space, the nuclear space. Tell us what's the difference of the role of nuclear energy 10 years ago compared to today or if we fast forward 10 years from now. Thank you, Lee, and thank you, everyone. Good morning. It's a pleasure to be here talking about energy. We definitely need the World Forum because we are together, and CO2 has no frontier, so it's really key. Why are we here is because we are a startup, but we are a spin-off from CERN, and we are based on solid scientific references. And the key thing to understand is that when we talk about electricity, electricity is just a transmission medium of primary energy. What is primary energy? It's coal burning, it's oil burning or gas burning, it's solar panel wind, but it's also nuclear, and nuclear is the fundamental energy of the universe. It is the most powerful primary energy we can find in the universe, so if we could master that, it would be key, and that is our mission. We are now in the uranium fuel cycle, which has amazing advantages, but also one key disadvantage for us, which is the nuclear waste, the long-lived nuclear waste, which is something that is difficult to imagine on a human scale because it's 300,000 years, but there are other ways to do nuclear. Fusion is one, and what we're working on, which is thorium fuel. It's very hard to make a change as fundamental as going from uranium to thorium because of the military need for plutonium for their bombs. We have developed the uranium fuel cycle, the costs are sunk, and therefore we can do commercial operations. We have to start from scratch with thorium, and it's always much more difficult to motivate states to do something new when there is no war. So the war against CO2 is not clearly as motivating as a real war, unfortunately. But that's where we are. As an environmentalist who was fighting plastic pollution of the ocean before, I saw the destruction of the ocean firsthand. That's why I got into energy because we were warming the ocean, we were acidification of the ocean because of CO2. Therefore I got passionate about this. The only thing I saw that really enabled us to have massive clean energy was nuclear. In Ontario, Canada, they went away from coal in 10 years because of nuclear and wind. In the United Arab Emirates, where they have all the sun and all the space in the world that they want for solar, they are already producing twice as much clean energy from nuclear as from solar. So I believe nuclear is going to be indispensable to have clean energy but also to give energy availability. We need, in all countries that are not nuclear yet, a way of making nuclear available which is non-proliferant. That means it has no use in military, which will not make long-lived waste. And which will be safe and cheap because the fuel cycle for thorium is everywhere and there's plenty of it. So this is our focus, to make energy available everywhere and to make the energy transition possible at scale with nuclear. Thanks for sharing that, very exciting. And as promised, I'll come back to Minister and just like Simon mentioned, solutions must be appropriate to the country context and reality. You said you wanted to share with us what actions in but we are taking to accelerating energy transition. Let me just begin by an anecdote. I was born in a very small village and there was no electricity then and at that time I know my mother was using paraffin lamps. I don't know who still uses paraffin these days but there are many places in Africa where we still use paraffin. And we were also using candles. And I went to school studying using candles. And for cooking it was just firewood. So that means cutting down trees and also making fire every single time you make a meal. But what has changed in my little village, in my home state? We've got solar. That was a life transforming, especially in my own home village but we haven't got enough of that yet. There's so many schools that are off the grid. So many clinics still off the grid. So what we're looking at is a transformation, giving access to solar energy at household level, at village level, at institutions and also at schools. But when we move now to business, what we're looking at is bringing in that technology that I just talked about, the solar energy. But our vision is to reduce carbon emissions by 30% by vision 2030. Now that requires a much larger goal, larger strategy. So from a regional perspective, we are looking at partnership and collaboration within the region, the Sadiq region with commessa and more importantly, now looking at the role of the Africa continental free trade area and how best we can as Zimbabwe be part of that regional integration. And also not just the regional integration but looking at the role of the private sector in ensuring that there is access to reduction of carbon emissions. I must also mention that Zimbabwe has had this long-term relationship with a number of Chinese companies and also especially the Chinese government. And we are focusing on looking at new investments that bring in new solutions to our energy crisis. And already we are seeing some very positive developments. At the moment, we do have one of the largest iron and steel company in Zimbabwe and they've only just started introducing new methods and new ways of bringing in solar energy. And going forward part of our strategy is that any new investments must also look at how best to introduce new solutions to the use of reducing carbon emissions. I also just want to mention that we are in the stage of transition and this transitional stage can only continue to improve through collaboration and partnership and by focusing on the private sector. Thanks. Thank you. You mentioned collaboration quite a few times. Collaboration with the regional player with the international stakeholder and also collaboration between the public and private. I want to bring that to Mr. Shing as well. How is China from your angle collaborating with key stakeholders internationally? In terms of lower climate change and to seek international cooperation, in your perspective, how China seeks regional collaboration with the stakeholders. And thank you very much for your questions. Just now you mentioned the key issues in the energy transition that is to ensure the power security and the security in supply and the reliable production. It is true power-centered, electricity-centered. The transition is very, very challenging. China, 1.4 billion. We have realized household-based and the village-based power accessible. And in this development, we have formed much experience through our experience, through our practice. We want to seek collaboration and to enjoy the result. On one hand, we develop green energy and also we want to utilize more renewable energy to advocate green consumption. And also to promote carbon neutrality, we try to provide regional solution. In Brazil, invested and built and plus-minus 100 kilo-voltage ultra-high voltage transmission. And also, we can have the clean power to the hundreds kilometers away to the rail and some polo centers injecting the green power to the regional development. Secondly, we have the connectivity of the power infrastructures, enhancing the connectivity in power with other countries and to emphasize on the policy coordination to promote energy production, energy consumption and internalization of the process. At present, together with 10 countries such as Mongolia and other countries, we have over 10 cross-border transmission lines. The other thing I want to mention, we have collaboration on the low-carbon green technology with the digitalization and energy technologies in depth integration. New tax and new facilities equipment keeps emerging together with the universities, institutions, international organizations. We want to enhance the fundamental research and fundamental technologies collaboration with international partners engaged in the international standards formulation. The other one, international cooperation in the transition. For example, the summer doubles and winter doubles international cooperation and dialogue to reach consensus to work together. And we want to leverage international network of energy in a promote the energy transition and the low-carbon development. We try to contribute our wisdom from our state grid cooperation. Thank you very much. A picture of the international collaborations. I want to bring that to Simon, you next. Throughout your work at the developing countries, how to balance at the same time the need for development and as soon as possible and also the necessity of energy transition and like the previous two speakers mentioned, what kind of role the international collaboration plays in that? Great question. I think I just come back first of all to Minister Nzenza what you said about, you know, your story of paraffin lamps.  The transformation of just having that basic access to lighting to enable reading in the evening to enable some productive use of energy activity. So that transformation is so extraordinary from darkness to light.  Currently worldwide about 3.2 million people, mostly women, are dying prematurely from respiratory illnesses from smoke inhalation. 3.2 million people, mostly women, annually. You look at your time savings. Imagine, ladies and gentlemen, an improved cook stove, the 50% time saving you can gain from an improved cook stove. So again, these may seem like micro things, micro initiatives, but they can be extraordinarily transformational. What needs to happen, I hugely embrace the terminology, the language from the Forum Centre for Energy and Materials, you know, really highlighting this transition has to be sustainable, secure and just. And for it to be just, in fact for it to be all of those things, it has to be inclusive. It has to be inclusive. So how do we ensure this inclusive transition? One, we need to take a systems approach and was just said in the examples of collaborations with Mongolia and many, many other countries. You know, energy is rarely just a national issue. It cuts across borders and as Franklin, you rightly said, it's of course a global issue as well. So we have to get the framing right around a systems perspective and it would be wrong for me working for a global development partner to think squarely within that global development framework or box. We have to build those collaborations with the private sector. Second, and governments of course. Secondly, and as I said, tailor solutions to context, but particularly think about the most economically viable solutions. I think there are currently about 420 million people worldwide accessing solar mini-grids and the IEA would say that's the most viable economic model for those 420 million of the 800 million people still waiting to have access to electricity. Solar mini-grids offer affordable viable economic solution. Thirdly, in building those collaborations and particularly engaging with the private sector let's not forget that smaller medium enterprise segment which is by far and away the largest segment within the private sector that we need to be talking about how we can stimulate the right types of financing. So at my organisation we're working on things called results-based financing where we're incentivising those smaller medium enterprises to go those extra miles to go to those more peripheral geographies in the countries where we're working and providing access to the products that are so needed in those geographies through creating additional payment mechanisms for them. And then lastly, I think it's so important for us to think about the optimisation element, the energy efficiency side of things or what my good friend Roberto Boca might talk about in terms of energy intensity, I'll just give you one little example because I guess part of my role here is to try and contextualise some of the conversations we're having. In Western Uganda, SNV, we're working with tea farmers, fairly large scale, great, very productive tea farming country but it's hugely energy efficient because you're needing to cut down a lot of wood to fuel the drying of that tea which then goes from Western Uganda over the border into Kenya down into Mombasa where it gets sorted and then sent off around the world. Who knows, perhaps we're drinking tea here in Changin from Western Uganda this morning. But the challenge is there to produce a more clean, energy efficient tea so you don't have to cut down all those trees to dry that tea. You need to connect those tea plantations, those tea producers to the grid to cleaner forms of energy and the truth is ladies and gentlemen to do that, ultimately you need to pay a bit more money for your tea that you drink in places like The Hague where I'm based and to pay more money or to be prepared to pay more money you need to have your tracing sorted out so that you can trace your tea back to those more cleaner productions in countries and contexts such as Western Uganda and that's one little example but you could say the same for the textile or garment industry in Bangladesh where I was a week before last or in many, many more countries so you need your tracing and then you need your preparedness for us in those more affluent societies to pay more for the goods and services that we are consuming so that you enable that more rapid transition to cleaner energy in contexts such as Western Uganda. You've mentioned that it's financing, it's tech change it's also the mindset change and now consumer behaviour change these are all coming together and finally you can have the energy transition that we are talking about Moving on to Franklin your nuclear energy project is quite ambitious although it's young and since we are talking about collaboration how do you see we can leverage international collaboration in that specific area and partnerships to accelerate the deployment of advanced nuclear energy in the safe way you just described Thank you Collaboration is essential, first of all energy is government the private sector works very much within the framework that the governments give it for energy and nuclear is the summum of this we have security, safety, all kinds of issues and nothing is going to happen without government and collaboration between government because it's expensive and ambitious What is the startup doing there? A lot of people ask me what is the startup doing there? You have big collaboration, governmental collaborations such as ITER for example for fusion and other big collaborations and they work but very slowly What we bring as a startup is speed and dynamism I don't need to ask anybody for approval in order to go and meet somebody to make a collaboration agreement with the research lab here or there So we have this concept which is very much like CERN where we have a core team of super talented people and then we dispatch the work across the best labs and the best private companies in the world So we do bring the world together we think everybody's talent this is such an extraordinary moment an extraordinary need for a new energy that we need to bring together the best talent in the world and that's where the collaboration works We have America, a lot of European countries together and now we're reaching out to Asia to bring the world together The problem is nuclear, is also weapons and therefore there is a special sensibility but I feel the urgency is such that we will find a way to collaborate even if there is this nuclear sensitive aspect to it So that's why a startup is here to bring the world together in a much more nimble and productive way Thank you And we do save time as promised for questions coming from the floor and as people thinking about your own questions all through one until the stage first We talk about the developing needs for many countries How to avoid power crisis that's in the mind of a lot of governments while you do the energy transition especially to the affordability and sustainability Who wants to take this first? Prime Minister? How to avoid power crisis Zimbabwe is now the second largest producer of lithium second to DRC So we are looking at how best to utilize the lithium into electric batteries And let me just say we are actually looking for investments in that regard Secondly, we have almost 365 days of sunlight in Zimbabwe So we are best placed to utilize solar So in that regard we are looking at investments in solar packs And I would be very interested to talk to any investors who might be here interested in establishing solar packs in Zimbabwe That would be one of the key solutions One solution coming from investment For energy transition we believe the first key is to stick to the direction of clean and low carbon with the prerequisite to guarantee the delivery of energy to assure that there is stable production and supply of energy Secondly, we should enhance the safety and security of global value chain and industry chain as well as the smoothness of these chains Thirdly, we should step up policy support to energy transition to stimulate entire society to participate in energy transition Thank you With that, we'll take questions from the floor Please raise your hand and quickly identify yourself The lady first and the gentleman Please I think my question is for Xinbao and Xinzong So I use Chinese In your presentation you said to guarantee the delivery of energies to prerequisite and you've mentioned state grid's achievement in that In China, when we are improving our electrification how do you interpret the situation in China when we are faced with the consumption increase of electricity? I'm from TMA News, I'm a journalist Thank you for your question As a matter of fact, to guarantee the delivery of energy is a task although there is increase of power consumption looking at the current status we could guarantee the delivery of power in China but I want to touch upon the point that is about climate change and fast economic development there is drastic increase of power consumption in China So in 2023, overall, the delivery of power is controllable and could be satisfied but in certain regions during peak hours there might be some small gaps However, we've optimized the deployment of power on our grid We believe we have capacity to ensure power delivery even during peak seasons and peak hours So please rush it short Thank you, good morning I am Juan Emilio Posada from a company called ISA Electricity Transmission in Latin America and we operate in most countries in Latin America What we have seen connected to some of the subjects in this panel is that there are citizens who do not have access to electricity and they do get to see the power lines and that is not a fair and just transition So we invite anyone of you to join us in a campaign to innovate and research on how to sustainably provide electricity to anybody under our direct influence of transmission lines in Latin America And add to that data connectivity since we usually have fibre optics on top of the transmission lines So we are very much open to that type of collaboration globally Thank you, thank you for the comments and do you have anything to add? Yeah, I think there are of course many many contexts where that is a lived reality for many people It goes to my point about the importance of understanding context We need to understand why indeed that is the case Why are power lines going straight across certain geographies What are the barriers to connecting the people living in those geographies to the grid, is it? Is it the obvious one of finance or is it something different? And then when we better understood what those barriers are we can come up with contextually relevant solutions to overcoming those barriers I would say as well that we continue to be somewhat fragmented as a community working to address some of these issues I think even within the sort of non-profit development actor side of things we have work to do to better share information, relationships and technical know-how and in doing so we can go a long way to underwriting the risks or de-risking that development finance institute money that is so desperately needed We're still not quite connecting enough the different parts of the ecosystem as it were so that we de-risk that financing which is which is needed Thank you And the lady behind? And I'm from the Rockefeller Foundation We were part of setting up the global energy alliance of people in planet that Simon just referenced I guess my question is that you know for most of the developing countries the challenge is really how do you balance economic growth with decarbonization Energy transition is something which needs a lot of capacity building in the developing world because you know we're just assuming that the way energy access happen energy transition will happen the same way certainly not you know even basic stuff around how do you write regulatory policies for energy transition etc there's a lot that needs to be done on the capacity side on the other side we're seeing there's a huge deficit when it comes to commitments around climate finance so my question to the panel would be you know we've been going into cop after cop you know if you had your kind of vision what would be the two things that you really would want cop to deliver on this time because this is a real crisis I mean climate change is not knocking on a door it's right here and issues around energy transition need to be addressed right away but with the capacity gaps financing gaps how can the global south really be able to be on a path of economic growth which is absolutely critical Thank you, thank you for the question we have a session on climate finance tomorrow afternoon in collaboration with Caixiang as well so welcome to that session and who would want to comment on that probably start with the minister I think Simon wanted to go first No you go first, just need to write down if I can Happy to go first, I'll be lovely to be in a room with you Deepali and thanks for the question I think for me one massive point is the climate financing money that famous 100 billion that we've been hearing about it must be additional to the odor monies it cannot be a substitution it has to be new money we're seeing huge pressures on official development assistance budgets in all sorts of countries so we have to ensure we protect, preserve and in fact even grow some of that official development assistance financing and then the climate money comes in comes in on top secondly we have to explore those opportunities for leapfrogging the transition needs to be secure, sustainable and just slash inclusive to do so I'll give you an example a country like Laos 90 plus percent of Laos is on grid now and the grid in Laos is relatively clean it's largely hydro so there can be opportunities to leapfrog in the clean cooking space to go rapidly towards making more available your electric cooking, your e-cooking technologies so constantly being aware of and in fact encouraging or pushing towards those leapfrogging technologies has to be somewhere a part of the the solution to this Minister and Mr Shinwon's to ask you to I'd like to look at this at two levels firstly just pick up on Simon's point in terms of taillight to two contexts so from a household village level firstly is the the awareness in Zimbabwe for example when we started talking about climate change we struggled to find the right language because in our language the language is right language because in our language we didn't have climate change but we found a way of packaging it in such a way that people then understand what climate change is so at that household village level we're looking at inclusiveness which you just mentioned but also looking at affordability accessibility and affordability and then that brings in the technology and then the second point is to do with just to pick up on financing that you mentioned that is indeed a challenge Zimbabwe has been under sanctions for just over 20 years and we are now focusing on ensuring that we utilize the natural resources that we have looking for investments in many other areas within the global context so financing is really absolutely important and that again coupled with our partnership and collaboration fantastic and as she mentioned what would be the two items you wish to happen on the COP28 that's part of the question what would be the two and part of my answer was ensure that at COP28 the climate financing that is required is truly forthcoming on top of the official development assistance allocations that are also necessary and minister I'll say also access to the carbon credits and we actually haven't spoken about that yet but I think there's a forum for that but again within the African context we're looking at reducing carbon emissions through carbon credits are the gentlemen over there for question and then on this side a mic please I was speaking Chinese I'm from Jingke Energy and we are a major global solar panel and also PV solution providers just now we talked about economics behind the solar energy and 90% lower price in China's energy supply I mean the power supply in solar economic performance is a very important consideration has been addressed by lower price in the solar energy and the other thing like countries like Zimbabwe and the other countries and the relevant regions we haven't mentioned stability of these countries even economically stable but without the stability of the country and the solution may not be sustainable so we have some comments solutions to that would you just unpack that a little bit for me so basically he says the scale can bring down the cost and solar is one casing point that the solar cost is brought down by 90% already because of the production and now for companies like theirs when they come to the new market the worry is more about the stability especially political stability whether they can invest and work over there to address the stability a security of that we've come out of a difficult time in Zimbabwe but I would like to say quite clearly and categorically that we are in a good place and that there is political stability in Zimbabwe we are going towards elections and we expect them to be free and fair and that going forward investment and the open for business mantra that the president continues to speak about will continue and also just to enter that we have increasingly noted the number of investors coming into Zimbabwe and not just from China but also from Europe thank you so we have time for take the last round of questions I want to collect all the questions and then go around the title so the gentleman over here please bring a mic Philippe Patron from Orano I just wanted to comment on the nuclear energy I think that we have to emphasize that the nuclear energy allow recycling of energy and the spent fuel and it was to be said because it allows to enter in kind of circular economies and I think this is the only this is the only energy that we can do that just to give an example in France in France we do recycling since now 40 years 10% of the electricity that we consume in France is coming from recycled fuel, spent fuel that we call the MOC fuel actually China as well has adopted this policy for on the nuclear side to recycle in the future the spent fuel I think it was to be said thank you and the gentleman behind let me collect all the questions from the audience yes just a second so I work for an environmental NGO in China and I have a question to the gentleman of the state grid of China so China's emissions are still a very large share of the global emissions and also still slightly rising so my question to you is there's also been quite a few approvals of new coal-fired power plants in China so what do you think would be the best way to accelerate the energy transition in China which is so critical for the world my understanding is that the state grid plays a very important role in that thank you and the gentleman writing front thank you thank you very much and the manager in China we have been China for 40-50 years my question goes to all the panelists here if possible would you please to share with us and by 2050 by 2060 what will your perspective of the energy mix win the solar or nuclear in the coming years down the way and the 2050-2060 what will be the energy mix thank you hi my name is Baruch Halperdan from Electric Global hydrogen storage company in power part of our dealings we see all the time questions about microgrid and how the energy transition could be utilized to live for like you mentioned Simon the old ways of electricity transmissions lined so do you see a path forward by microgrids utilizing solar and wind and expediting I would say electricity all around in the third world like it was done in telecommunication in the 90s thank you so we got five questions already and five minutes left but before we answer those questions let me introduce the first speaker of us Roberto Boca is leading the center for energy and materials at the forum he can make a quick comment and we can start answering the questions as well thank you very much so a couple of comments actually you asked me a question on how we avoid power crisis I think it's important to look at the element that Simon mentioned the energy intensity the waste of energy that is happening is a big element so if we can reduce the waste so address the energy intensity that is the best form of energy the one that is not consumed that will allow us being on the cooking stove or in the big industrial system I just wanted to mention one piece of work that we are doing that will be issued tomorrow a report on the energy transition taking the energy triangle as we call it so the energy system has to support the three dimension of sustainability security and equity has been discussed today in this panel tomorrow there will be the ranking 120 countries out there doing with a view of this transition so I won't tell you much about the content because I would like you to look at it tomorrow but it's very interesting to see how the two countries that are represented here both China and Zimbabwe are really doing a lot of good things so thank you for giving the space thank you and let's come back to the question Neil Cleary and then on the investment to China's energy transition that is about the emissions in China in the energy transition how could we accelerate the energy transition especially the utilization of the coal is rising and then I want everybody to have a quick comment on the energy mix you are seeing in 2050 so Star Wars probably in nuclear thank you we've seen many cops coming and going and we've broken record every year of CO2 emissions in the world so we have to question if the world is organizing a way to truly combat CO2 production and to do the energy transition as best as we should I definitely think nuclear is necessary and I think for the 2050 energy mix the director of the international energy agency states that we need to invent 50% of our energy is going to come from technologies that have yet to be deployed and invented so this is also where we should focus we shouldn't just say it's going to be the way it is today just projected we need to reinvent something completely new that's where we are reinvent something Simon thank you for the question I think mini grids absolutely recognises and as I stated earlier have to be a part of the solution going from your solar household systems to your solar mini grids you get an increased productive use of energy you therefore start to generate an economic dividend as well which enables that climbing of the ladder and we're seeing of course countries like Mozambique there's now the regulatory environment starting so that you get your tariff structures and then you're enabling of indeed that overall mini grid structure the global energy alliance for people and planet have been an important part of that I do also think there was a question earlier about the operating in more insecure or volatile environments still extremely fragmented you look at the sort of supply chains and the battery side in particular there's work to do to think about how we aggregate some of those initiatives form deeper collaborations and in doing so de-risk so that we can get more of those technologies into those more fragile places so mini grids yes indeed a big part of the solution thank you and just now talked about the coal based power coal based power utilisation in China the energy demand is growing however the GDP the power consumption per GDP is going down dramatically and which has been stated in the government working report at the same time the coal based power development in China we advocate a clean and efficient development of it we have certain restrictions imposed on the development of coal, fire power which is to provide room for renewable energy development and to provide absorption and support function to renewables we know that there's still intermittency and instability and renewable energy so we need fundamental energy to support the development of renewables that is why we are developing clean coal to support the development of renewables thank you Mr Xin energy mix 2050 in 2050 I would like to see a household that no longer uses firehood and also to see a mix possibly of biogas and using the latest technologies that would have been available by then through innovation and research and the use of technology and secondly I would like to see a situation where at policy level you have the private sector working very closely with government to find sustainable solutions to energy usage thank you well we have to wrap up but before we go I want to insert a sense of urgency like Franklin mentioned wearing a war combating CO2 emissions and combating global warming so if you identify one most urgent action we need to take on energy transition what would that be start with Franklin I think we need to find alternative to nuclear the way it is I think complimentary everything has to be on the table we need renewable and we need new energies also that we're going to develop so investing in research and doing fundamental things everything on the table investing in research mine would be to accelerate and scale locally led solutions by providing the right forms of financing local solutions right financing I believe firstly we should develop renewables secondly to build renewable internet energy internet energy that's accessible and affordable to the majority of people and also at industry level to have a stronger focus on use of solar I see solar affordability gentlemen thank you extraordinary panelists for the discussion this morning