 Gauru, can she send you her slides? Yes, I will ask her. I told Steve to do that, but I will call her. OK, thanks. She's not online here, so I can't write to her. I don't have her phone number. I will call her and let her know that she has to actually send the slides. So can I go on? Yes. How do you see my slides? Yes. OK, I'm sorry. I was in the other meeting and I was there as early as 125. So I want to talk briefly on complexity of biological macromolecules. And I'll be using lipid transfer and on constant risk constants as the title I will just look at. So I'm Jonathan Babalala from the University of Ibadan in Nigeria. I'm basically a physical chemist. So I'm basically a physical chemist. I work on biophysics. I work on biophysical chemistry. So I move around within that range. So this is a typical lipid balea, which is very important for all membranes. And you can see the structure here is really complex. You have oligosaccharides. You have alpha helix proteins. You have lipids in between. You have globular protein. You have phospholipid. So you have this complex. And this must be this way for the membrane to function well. And then the three major type of lipids you actually have will be the phospholipid, the failure site, the glycolipid, and then cholesterol. So I decided to pick on cholesterol because of the kind of idea we have about cholesterol. The problem with cholesterol is that it is synthesized. It can be transferred, but it is not degraded. So most other lipids, when they are synthesized, they can be broken down. They can be transported. So it's easier to regulate where and where they go, how and how they move, and how they bow accumulate within the system. The major problem is that the moment you cannot make a balance of the synthesis, the transfer, and the degradation, it becomes a disease. So in this case, to say cholesterol cannot be degraded, then it makes it a special repeat that has to be watched. So what we actually do, since it cannot be broken down, what we think we should do is actually to find a way of how to transport it from places where we discover that it's already accumulating. Some people have tried to do this before, but the kind of experiments they perform are looked from up. It's like when you want to monitor cholesterol, the molecular mass is known, and you bring a fluorescence, if a fluorescence label, and you are 33, the fluorescence label itself is much heavier than cholesterol. So when you are looking at the movement of cholesterol, inferior to what you are seeing will not be the exact movement that you have within the membrane. So what we decide to do in this case, so I find that error, is to actually get a cholesterol and liberate it. So there's little difference between a cholesterol that has been liberal, carbon-14, and carbon-12. So that gives us relatively what we actually need. And when you have cholesterol accumulation, you have three types of diseases that can naturally occur. You can have the Neimanpik type III disease. You have type I and type II. You can have the hypercholesterolemia, and then you can have the Wordman's syndrome. The Neimanpik type III is what we actually want to use. There's a protein called MPC2. That is one of the lipids that are associated with this disease. So I just give these are the facials that will publish the same error during the presentation. So when you look at a typical endocytosis in the suma membrane digestion, you discover that cholesterol is more at the membrane ends and it reduces as you go into the inner cell. Whereas BMP, one of the phospholipids, you don't have much in and then it increases as you go inside. And then ceramide is lower at the top and then at the center, you have more, then you have lower at the other end. So what we have done here is to actually prepare our liposomes. Liposomes will replicate what you actually have within the body system. So we make sure that we have composition that looks like what you actually have within the body system. Of course, you cannot replicate it exactly just to give us an idea of what to do. So we did two things. One is our cholesterol label here. This is our cholesterol 14. We had a BMP. We had a PC. PC is just to make up for it. Then we had Boutin PE. This Boutin PE is to help us to be able to remove our magnetic bits that we are going to add later to separate our cholesterol from the rest of the lipid. So we have a donor that is carrying cholesterol. And then we have an acetyl here that has just BMP, PC, and has MBDP. MBDP is the fluorescence liver. Not on our lipids, but we want to use it to monitor whether we still have our membranes intact. So if it is liver and we're able to separate it, then we know that whatever we have is intact. So this is the process that we use for the experiment. I want to go into details of this. So we had so many controls. You know, in biomedical systems, when you are doing the experiment, you want to be sure that one thing has not contaminated the other. So we have a donor alone that we can check at the end. We have the acetyl, we have the donor, an acetyl together. We have the donor and we have our bar mark that's to separate Boutin from it. And then we have the acetyl and the bar mark. Then we have all this kind. And the acetyl scone, the 8th ear, is actually to help us to destroy that what we have is within control. Because this will be a protein that is not known to transport cholesterol. So I would just go on. So this is the kind of experiment that we have in mind that if you have this ear, your MPC2, as you move between one membrane to the other, you have a donor here, that this will be picking from ear and of course it will be just going around and then you transfer cholesterol from one to the other. This is just a typical thing. You have a goal here, you have a donor and here you have an receptor. So what do you want to do? Well, this is the goal. You want the two to be in this way. And then the next thing is you have two options. The first option is that you get into the water here, you swim here and get to this place. Or the other option is that you take a parachute and then get to the other side. So MPC2 here is like a parachute. The reality is that when you add donor and receptor without any protein, they will still be transferred. So that would be like, sure, but it's not going to be as much as when you have MPC2 to help with the transfer. So this really looks like what the membrane, the leprosome looks like. So you have the donor that has 100% reductivity, no fluorescence at all. It doesn't have NBDP. And the receptor that has no reductivity at all, but it has fluorescence. So when you mix these two together and later you add your biomarker activity and then you separate. You ask this in the supernatant and then you ask this so you can separate the two from one another. And then now you see you have fluorescence in one and then you ask your cholesterol will have probably gone to the other side. So the kind of research we got from this kind of transfer is so complex. Now the problem is you are transferring cholesterol. You are using MPC2. What we discover later from previous experiment with it is that even some of the other lipids are also being transferred. It's just that we monitor only cholesterol. So we cannot say from the complexity that the complex situation we have, you cannot really say that that has only transferred cholesterol alone. But looking at the amount of cholesterol we have transferred you discover that at the various temperatures you have this transfer. No at a zero degree, 25 degree, 37 degree. So that means the higher the temperature, the better the transfer that we have. And if you look at this, this is without MPC2. The maximum you have, which is the spontaneous transfer is around 40. Now with MPC2 you can see what we have is close to 10 times. So that gives us the impression that using MPC2 you can always have a better transfer of cholesterol. If you do it with a function of pH, we have some, we have a provider that lives this way, but of course with pH we have to change the type of buffers. That's why you can have a smooth curve because of the buffer capacity. This is when you do it with concentration, concentration of MPC2. And I need to say that the MPC2 we used here, we extracted it from both amic, that's calmic. It took close to six months to do that. And then, this is also time dependent and temperature dependence if we look at them. And when we added, we changed ionic strength. You also have quite a number of things. So you can see all these things affect whatsoever we have. Because of time I think I will skip some of these. So here we change the ratio of the donor to acceptor. The one that we found that would probably be best would be to use a ratio of one donor to five acceptors. And so we have to figure out the ratio of donor to acceptor and see where we have the best option. And then this is what actually happens in the body, the physiology. So this is what we have when we figure out the ratio of donor to acceptor. And then here we look at various donor lipersome when we find the concentration as well. And just like I said, if you look at this graph here, you'd see that there are so many other proteins that can actually transfer cholesterol. And all of them are present within the body system. So that is very, very complex. Because we are not talking of one factor and not two factors, not three factors, about four or five factors working together. So you can see here that the glycosylated MPC actually has the ISM level of transfer. Now we had a problem. The problem we had was that after the experiment, we discovered that our fluorescence also appeared in the sample that we would do from the transfer that we got. So that means that some of the donor actually has some fields with the acceptor. So if you look at this, so instead of just getting and going, it has formed another liperome. Now this has become a big problem. And then so that is what we think we have here. It's not purely the acceptor any longer, but a mist of donor and acceptor. So we have to use fluorescence to begin to measure and see what quantity of our acceptor has actually gotten into the donor and then phase of it. So we look at what to do. So initially we are thinking of how to do this theoretically, but we discover it's very difficult. So eventually what we did was to find a lipis-panning membrane to resolve that issue. So I will just jump all these and then go to the next one, which looks very simple, but very interesting to us. One five minutes left, please. OK, yeah, I'll be very fast. This is a simple structure of hemoglobin. It has four polypeptide chains. And the expectation is that when you have a monocle reacting, this is a typical equation for it. And this is the formula for calculating the equation. Normally we react this with DTMB because we use sulfide and we use sulfide agents to react to the sulfide group that we have in hemoglobin. And essentially we look at beta-93, system beta-93. So we have all these complex profiles. So let me just go to this. Now, the same hemoglobin. You react at the same, you react with the same quantity of reagent DTMB. And then when you react at 5 micromolar, you add 5. When you react with 10 micromolar of hemoglobin, you have this profile. When you react with 15 micromolar of hemoglobin, you have this profile. The expectation is that normally when you feel it concentration, your rate constant ought to be constant. So we describe that our rate constant is not constant. Now you have a problem. We just think, what do we use this problem to solve? So what we eventually did was actually now to take very concentration of hemoglobin and we calculated the second other operative constant for each of them. And we have profiles that look like this. Now, our final conclusion is that when you have hemoglobin in lower concentration, you have it more in dimer form. So that means the hemoglobin, we are all actually not in tetramer form. So you have tetramer now going to dimer. So what we did was just use a simple kinetic thing that you have OK, when you have all tetramer, you have one. And then the amount that is substituted is alpha. And of course, a simple dissociation for that would be the equilibrium constant, the k that we have minus kT and this. And of course, our k42, which is now our dissociation constant, is going to be this equation. So this we did. And what we have the k apparent, we have the alpha and we have the failures pk42, which this actually should be constant. But of course, we have to cut out here. We cut off and then we don't take the rest value. So as you can see from the total concentration we've taken is just at 10. This is in tetramer. So this is just at 10 micro in. So these values that you have here has given us a value that we eventually now plotted our pk42 against pH. And this has given us an important biochemical value, which you call the acid boron effect. And from here, we could calculate our boron effect, which ordinarily we have to do all that kind of experiment that we may not be able to perform in our environment to determine. So we did that for oxymoglobin, we did that for carbon monosimoglobin, and we did that also for methymoglobin. So now we had a problem and that problem we have turned into another instrument to be able to come up with biochemical values. In this case, the acid boron effect and the alkaline boron effect of our hemoglobin. And the transitions for oxymoglobin and carbon monosimoglobin are given here. So I think I will just stay here and thank everybody for listening. Thank you. Thank you very much, your talk was really nice. And actually it's open to questions. Ali, I guess you. I wait for some students to ask and then I'll ask. Okay. I don't see any hand raised. No question in the forum. Does anyone has written in the forum and that I cannot see? I can't see anything. So Ali. Hi Jonathan, thanks for your talk. Thank you. Of course it was, as you know, our audience here is very diverse. Yeah. Okay, so just to try to make some connections with other people in the audience from different fields. You mentioned very flirtingly that you would try to do some theoretical modeling or something. Can you describe, for example, what type of problem that you're interested in experimentally would need or would benefit from some modeling? Or what types of questions are you interested in asking or helping getting interpretations on? Yeah, let me go back to this. We have our carbon-14 cholesterol in the donor and we have our fluorescent label MPCBE in the acceptor. The, what we expect is that by the time we have the two and MPC2 as carried, all the cholesterol from the donor to the acceptor, whatever quantity it will take, that at the end, we should have, whatever we separate, without fluorescence. Now that means we are not using any fluorescence from our acceptor. That means our acceptor should maintain its 100% fluorescence. But for all the experiment we discovered at the end that they lost fluorescence. Now what happened, we discovered that what happened was that the acceptor, some of them match with the donor and form something that looks like, so there was a fusion. I see, I see. We should be able to separate what quantity has fuse, what quantity is the acceptor and what quantity is in the donor. I understand, okay. You know, we got so many values. We now felt, okay, is it possible for us to be able to simulate theoretically and see this should be the value that you have at a particular pH, at a particular emission since we cannot differentiate between the fuse one from the ones that are not fused. You know? Okay. So when you even check from electron microscope, you will be able to see so much of the difference. Are you, have you thought about using techniques like FRET, so fluorescence resonance energy transfer? Yeah, we did. We did, it wasn't making so much, so much difference. I actually wanted to use FRET to monitor the transfer, but the energy level generated by this was not strong enough. I see, okay. So that was the basic thing we wanted to use initially. Okay. Ali, there are two other people. Yeah, perfect, perfect, perfect. Online, Estelle, very quickly. Well, for me, it was like, thank you first about the talk. So I just wanted to know if you try as a technique which Ali had already asked, but my question second about, they have different shape. So maybe if you try to see with a technique, for example, to see the structure, like maybe FM or other technique, which is allowed to do the biological sample under, I mean, in liquid. So you are in a safe place. Did you try to do that? Or you are not interested to know which, what are you having as a structure? Well, what we did was to prepare our liposomes ourselves. And the liposome we prepared are just 100, the size is 100. Now, after whatever, we pass through the pressure that we have liposomes. Now, the set of liposomes we have for a septal is the same type we have for dinner. Okay. It's not likely to make a difference between the two. Okay, so how are you sure that this is what you are getting? It's not about fluorescence. Okay, is the fluorescence or the, I mean, theoretical technique and tell you what is going on and so on. But what I am interested to know is there is a possibility to know that what you are having, it is real. Is this correct as you are having here as a model? No, can I get the question again, please? I mean, how you characterize whatever you are showing here? Well, actually, what we have done, we used, we messaged the activity. From the activity, we are able to know what amount of cholesterol has been transferred. We messaged the fluorescence to know what we are and where our septals are. So our assumption initially is that all the septals, we have full fluorescence, which we discover was nuts. And then our donor that will lose some legality to the acceptor that will tell us the amount that we have. All this we were able to precisely do. But the problem, the problem we had was that we discovered that we... King Internet is back again. Christian, unfortunately, maybe we won't have the time. Lost fluorescence. And therefore, that means that you are... So... I think we lost him completely. Yeah, yeah, I was even surprised that... Yeah. Yeah. Okay, okay, go ahead. So it will have been difficult to say this is what you have and this is what you have because they are the same size. Okay, Prof, I have to take another question from Estelle. Yeah. I just have a very nice thought, by the way. But I have a very naive question from somebody who is not in the field. And so what will be the impact of your research, let's say, in the world? Can it help somebody to reduce maybe cholesterol in his body or something? Yeah, it's not... Well, actually, people believe that the factor you had, the more cholesterol you have, that is really not true. And the location of the quantity of cholesterol you have, where it's... The location where you have it is really much important. And I said initially that when you have it more in a place you ought not to have it, then you have disease. And there are three types of diseases I talked about. The Neiman Peak type C disease that I talked about is a rare degenerative disease. You probably have about one in... About one million. I think that kind of disease, and it's hereditary. So the experiment is actually to investigate that disease and be able to follow the pattern of the movement. If we have to follow the pattern of the movement, please avise the other lipids that are present within the membrane. Then we can do a lot of other things, see what kind of energy level we need to be able to locate them appropriately. Easy, thanks. Thank you. So any other question in the audience? I have a question still. Actually, Jonathan, you had a slide. You don't need to put it up now. Where you showed as a function of pH, there was some strong sensitivity to something. I don't know what that something is. That is hemoglobin, hemoglobin. But what happened was, after we discovered that our... The rate constant was not constant for hemoglobin reaction. And of course, we simply discover was because the Tretama was associated to dimers. Dimers are faster, the Tretamas are slower. So to know the ratio of Tretama, the dimer that has distributed, we did the calculation we had there. So what we now have is pK42. That is the solution constant, vis-à-vis the pH. And when... So what is the molecular reason why pH affects things so much? Actually, you know, within the body system, the pH should be around 7.4. And once you have anything lower, the hemoglobin molecule will just hit more. But if you use an alkaline pH, you split off the Tretama completely, but it will use you on wines. Okay, so it creates like... It does, it unfolds. It unfolds completely. If you use very alkaline pH. So this one is still within stages where they are not folded. They are just separated into Tretama and Tretama. And that actually helps us to calculate the amount of hydrogen release and hydrogen uptake by hemoglobin, which is very essential for body functioning. I see, okay, thanks. Okay, professor, thank you for your very nice talk and nice explanations. It was a really good honor to have you for this session. Thank you. Thank you. I'm very grateful. It's not easy to handle because of problems, internet and then speakers who confuses links. I had so many emails. So the moment that for my CTP, I thought it was, I registered, I entered, I tested my microphone by around 1.30, they are about, and nobody told me that was not in place. When it was time for me to be called, I thought I wasn't called. So I'm sorry for all the people in the audience for the small delay that we had at the beginning. So it was due to not the fault of the technician, but all these reasons that I just mentioned. So thank you very much. Now we'll have a break for 25 minutes. And then I would really advise you or invite you. Thank you. And the only talk that we have in 25 minutes, that will be given by Amanda Weltman. You know what the name means. And then particularly to young students who are interested in cosmology, I think there are many in the room. So let's see in 25 minutes. Yeah, I mean, I would just say stick around in the meeting room. And this is a good opportunity to socialize maybe. Go get yourself a coffee and have a virtual coffee as you chat with people. I just want to remind you tomorrow morning, we have the poster session between 10 and 12. So there are 30 posters that are gonna be presented by the participants who submitted posters. In the first hour, everyone will have, every poster participant has two minutes to quickly present their poster. And then in the next hour from 11 to 12, everyone will go into their own rooms where they can present their poster over a longer period of time to different people who come into the Zoom room.