 para usar o atome do microscópio para extractar as propietas físicas de vírus e aqui você temo como esta máquina está funcionando, você temo o cantelebre, no cantelebre do Iberiano, você temo um típico mágico. Of course, isto não é para a escala, vemos unha escala na escala later on e con este sistema podemos manipular, podemos provar, podemos manejar para tomar imagens de estas vírusas individuas e também podemos extractar unas propietas mecánicas. Então o típico hoje, nesta escala eu vou fazer a introducción brief, então eu vou... Eu vou dizer un pouco dos experimentos que nós performamos no nosso lab sobre as mecánicas de vírus humanas, nós tentaremos explicar como as mecánicas de toda a escala e o coro de vírus humanas nos da uns perspectivos, as intenciones sobre a interplanera biofísica para que se perfilie o vírus biológico de estas vírusas. Então nós conectamos a o ponto 2, vamos explicar como nós monitoramos o rellénio de genome, como nós veamos o vírus humana do Andrés, ok? No ponto no nº 4 nós vamos cambiar o típico un pouco e tentaremos explicar explíxan que son o rolo mecánico de proteínas de sementín na sessas sistemas. E finalmente, eu tentarei coxer. Então, o vírus, o típico dos títulos que usem x-ray e eletro-microscopia e con estas duas técnicas é possível coxer bonitos estruxas A loa de then, con muy detención, con muy particulares detencias sobre as proteínas, e actualmente, o náxido de estas técnicas é que, of course, nós entendemos a loa de estruxas e podemos estabilizar relaciones entre o estruxas e a facción. However, beyond the struxas and the facción, nós temos also properties. E, en este caso, nós temos unas propietas físicas, como, por instancia, thermal stability, mecánicas, estratix, vibración, etc. E todos estruxas nós podemos clasificar them as physical virology, que son as propietas físicas de viroces. E, por mesurar estas propietas, podemos tentar acomplir e completar este esquín between the stretcher, the function and also the properties. Por instancia, podemos mesurar, podemos palpar a single virus para extratar a presión, a presurización que nós tenemos dentro, e também podemos mesurar as estratix properties de viroces. E, atomiformeroscop is a single molecule technique que allows mesurar the properties of viroces and to get images of viroces in physiological conditions, so we can see functional protein cells and in the second term also it's possible to make manipulation and real time experiments. In order to make these experiments in liquid, we need to submerge both the tip of the cantilever and the surface in liquid. So, when we put our viroces on top of the surface, we just seek each one of them in order to perform the experiments. In this image you can see a real comparison between one of these AFN cantilevers. This is the shape of one cantilevers, which is about 50 microns of width and about 200 microns of length. And each of these small dots you see here are fluorescence-label viroces. So here you can see in a glance how is the comparison of size between the cantilever, the AFM and all individual viroces. Actually, atomic for microscopy can be understood as a blind person with a white cane, which is probing the environment and is trying to get a virus and to see if there's a chair, a virus or whatever. So, if you get the picture of this guy here, you get the idea about atomic force microscopy. The typical approach we use for imaging viruses is jumping mode. In jumping mode we are trying to see this guy here, this virus, by going back and forth with the AFN cantilever in such a way that when we are down, we are in contact with the surface or with the virus, we measure how much force and we control the force we apply on the virus and then we release the surface and once we are out of contact, with the surface we make the lateral displacement of the cantilever. In this way we avoid dragging forces acting on the viruses that can destroy them. Here you have an example of one human aviravarous particle where it is resting on top of a triangular facet. Here you can see a pentameric stretcher, just the penton, and also you can see the exon. Actually we apply a filter, we can distinguish, we can resolve much better the details. Here you see the penton here, surrounding 1, 1, 2, 1, 2, 3, 4, 5 capsomers. Actually you can see the individual proteins which are on the capsomers too. On the other hand also if you make a zoom here, also you can see the trimmers of the exons, so here you can see each trimer and each trimer is conforming in this case one capsomer of the human aviravarous particle. So the main tool that we are using in order to extract the mechanical properties of viruses, we are using the atomic force microscopy cantilever as a hammer, well, it's just a rough comparison, but somehow we are poking on top of the virus with the cantilever, with the tip, in such a way that first you deform the caps a little bit and after a while you break it, you use surpass the breaking force. Actually here in this cartoon you see a better explanation what I said before. Here you see the stage number one where the tip and virus are out of contact which corresponds to this deformation which actually is zero, there is no force applied to the virus. Then in point number two the cantilever starts touching the virus at this point and you see actually it's bent a little bit and this bending is detected in this sort of linear deformation that is provoked by the deformation of the virus. We see the combined spring constant between the cantilever and the virus. The third part of the experiment once you go above the breaking force you break the particle and sometimes the particle is going away, sometimes the tip is penetrating inside the particle, well, this depends on each experiment, but the important thing is that you see the third part as there is a sudden decrease in the deformation force which is going on. If you keep pushing at the very end, eventually you can find the slope one, the infinity slope, sorry, which means that you are now deforming the cantilever on top of the surface. Here you have an example, in this case this is a p22 bacteriophage particle, this is the particle before and this is the particle after. So here we perform one of these single indentation assays and then after you break the particle you see you can see a little hole right there. You can control how big is this hole by controlling the maximum force you applied to the experiment. Let me make here a small digression about these experiments in order to focus in these cracks. When you make a crack in one of these viruses usually this crack remains forever. Well, at least forever means that you stay hours and hours scanning there and the crack stays there. However, there are certain protein cells such as for instance the ball particles which are protein cells that don't have a viral origin and this particle which is also an automatic size is possible to make one of these holes as you can see here and then after waiting a few minutes you keep scanning the surface and at some point the hole disappear. This property of healing that some of the protein cells do have has been so far little investigated and I think that this would be a nice place to go on with this kind of research just to see the determinants of how these little particles can recover the cells. Let me go back a little bit to the other slide because from this curve we can extract just let you know we can extract two main mechanical parameters such as the spring constant of the virus which is given by fitting a linear regression to this data here and also the breaking force which is telling us about how much force do you need to break the particle. So now let's go to the second topic which is about the mechanics of human adenovirus particles. First as a brief introduction to human adenovirus this virus has a size of about 100 nanometers and one of the particularities of this virus is that it packs double standard DNA together with histone like proteins. So it's not the same as the virus we saw this afternoon ok, where we have a third of a cheese packing a spool double standard DNA in a spool without the help of any protein here these virus need to have proteins inside and actually 50% of the molecular weight of the core are the histone like proteins and the other 50% of the mass of the core is given by the double standard DNA. So it's absolutely condensed by these proteins. In the process of these virus first is recognizing the wall of the cell there is an endocytosis of this particle and this endocytosis there is a stepwise uncoating of the genome. So it means that in this case the particle is losing some parts such as for instance pentons, fibres, exons etc etc. So after a while the kinesins are transporting the viruses to the nuclear pore. Once in the nuclear pore what you have there is a semi-disrupted particle. So it's a particle which is not intact anymore is the genome is already can go outside. One is reaching this place the nuclear pore there is a diffusion process of the double standard DNA through the nuclear pore to the nucleus. Once the DNA is there of course then we have the replication, transcription, assembly and packing. Another important issue about these viruses is that of course here we have an immature particle. So there is a protease that is cleaning sand of the proteins inside this core in order to make a mature particle. So we are going to concentrate actually in these two parts. We are going to concentrate in this assembly of the particle and how this assembly is affected by the fact of having an immature particle or having an immature particle. So this is the maturación process just a cartoon. Here we have the proteins is cleaning sand of the proteins here and at some point this is affecting to the physics to the physical stage of the physical state of the double standard DNA that we have inside. So we will see then two consequences. The first question we are asking here is if the DNA is modulating the mechanical properties of the viral particle and the second question is what is the interplay between these physical properties and the various functions. I will try to give you some insights about this. How can we understand this implication? So we are going to focus in this assembly and in the DNA diffusion. So in order to start experiments with AFM it's always very important to control the symmetry or the absorption symmetry of particles. So here you have a two-fold symmetry axis particle actually you see here two triangles so the particle is resting on the reach between two triangular facets. Here you have a five-fold symmetry axis particle but you can see actually the pentone right there. And finally here you have the three-fold symmetry particle where you see a triangular facet. We are in order to make the mechanical experiments here we are going to focus only in this particle because it's more stable than the other two particles. This particle is more stable because it's resting on top of the whole facet. So it's better to make mechanical experiments. So the first thing we can do is for Easter to make to try to see how much is the spring constant of what is the mechanical evolution not only along maturation but also in comparison with empty particles. So here we have an empty particle, no DNA inside. Here we have a immature particle and here we have a mature particle. And as you see roughly the spring constant we could say that is going up. So why is this one? We can apply a simple model by considering that the shell is contributing with the spring constant and that the genome or the DNA is contributing with the spring constant. So in this case both the spring constant are adding. This is a very simple model but if this model is correct the results I just showed you in the other slide imply that the spring constant of the stiffness of the core of the mature particle is bigger than the spring constant of the core of the immature particle. So this will be the implication. But is this implication true or not? Can we try to dig more a little bit in this question? Well, the problem is that we have the combination and we are assuming something about the core which is inside but we don't see the core. We don't know what is going on inside. So something we can do is we can manipulate the protein shell to open the shell, crack it and access to the core inside. So in this way we can also take a look to the mechanical properties of the core itself. So in order to do that we make these experiments we start the sequential indentation of this particle so in total this particle has been indented ten times. So we pocketed ten times. So here are all the curves. So you see that at the beginning when you don't break it you have sort of like a linear deformation but after a while in curve number three you break it and actually you see there is a hole already there. And through this hole it's possible to explore the mechanics to put the tip through the hole and explore the mechanics of the curve. And actually you see that the behavior of the curves is quite different at the beginning because here you see that it's completely... it's earthy and it's non-linear. Also happens something similar with the immature particle. You start making sequential indentation and at some point you open a crack and again you get this kind of curves which compare very well with that. So what is the difference between both? Well we can try to adjust the deformation curves of the core by using the dimitriadis model. The dimitriadis model is taking the hertz model, the hertz model of the formation and it's adapting to the fact that usually when you do the biological sample it has a finite... just one thickness. I mean it's not infinite. So by using this it's possible to get information about the job models of the course. I will tell you don't pay too much attention to the absolute numbers. The numbers are not important here. The important thing here is the comparison between both numbers because both numbers have been obtained using the same approach. So the important thing here is the comparison. Here you see that the job models of the mature particle is very low, it's reaching about 0.3 megapascals and in the case of the mature particle they are reaching about 1.2 megapascals. Actually you can see already by eye that the curves are quite different, the green and the blue. So the blue are from the immature particle and the blue are from the mature particle. And this then is in contradiction with the thing that we saw before because before we saw that the spring constant of the inert particle in the case of what time was bigger than the spring constant of the inert particle of the mature particle. So this is a guess thing that probably we have some persuasion inside. In order to accomplish for these two things. You see that the curve behaves completely opposite to the behavior of the spring constant of the inert particle. In order to see if we have persuasion or not we can try to play with counter ions in this case with spermidine. By using spermidine our goal is to condense the genome and try to make it harder after condensation. So this is what we do here. Here we make a new experiment. Here now we have the red curves. The red curves are provided by the deformation of cores that have been condensed with spermidine. An actually you see a very interesting behavior that consists that the value of the jump modulus is going from 0.3 megapascals about 0.8, 0.7 megapascals. But still it's not reaching the condensation state of the immature particle. So this is telling us that we are able with the spermidine to condense the genome of the mature particle but still the condensation is not good enough to reach the values of the immature particle. Well, in order to estimate the pressure we have inside there are two approaches we use. The first approach is just to use this analytical formula that you can find in this nice publication. It's a formula that is giving you how much is the persuasion of a spherical shell as you measure the spring constant with and without pressure. If you make the numbers you get about 3 megapascals. The other approach is consider the virus, I mean the DNA because it's condensed with histone proteins. So it's not the same thing that bacteriophages. So here you can consider as an unbranch polymer and without going into details you also can obtain the value of about 3 megapascals when you adjust the numbers to the expected values of the persistent length of one genome which is condensed with histones that you can find in the literature. OK, so what is the model we propose? What is going on here? In principle, here we have these condensing proteins. For instance you have protein 7 which is this sphere where the genome is wrapping around. But also you have protein mu which is this green one that actually the stretch is acting like a staple which is grabbing two DNA strands together OK, and are holding together two DNA strands. However, when you have the maturación process the protein is cleaving this protein and then after cleavage what happens is that this protein is not holding the DNA strands together any more. So the DNA strands are free to fill the reparsion the restorative reparsion between them and this is exerting certain preservation against the world. So what is the biological implication of this preservation in maturación? Well, we know already from a few of these references for instance by Birklaq and also by in our lab that the weakest points on a shell are the pentons, OK, because they are under stress. So in this process of uncoating what's happening is that there is changing several conditions of the environment surrounding in this case the adenovirus. For instance the pH or other ions. So what we propose is that this change is weakening even more the pentons and this precipitation is helping to pop off the pentons from the particle and helping for disruption that has to take place in order to have the final infection, OK. Well, this was with the uncoating about what is going on with the diffusion of DNA. Is helping that for that or not? So this is the next question we are going to try to address. For this experiment we are using a different mechanical approach. We are going to use fatigue. Fatigue means that instead of making a single indentation and say where we completely break the particle we are going to gently touch and manipulate and massage the particle in order to make it lose some of the parts. So we can do that with the jumping mode. Actually with jumping mode you can touch the particle thousands of times for getting images. This is what you have to do. So at the same time that you apply a low force in this case we are applying 100 piconewton that I tell you is very low compared with the 3 nanonewton for nanonewton you have to apply to break the particle. So this force is really low compared with the breaking force. So when you do that you can obtain, you can monitor how is this assembly process induced by mechanical stress and this is actually what you see in these movies. Here you have the assembly process of a mature particle. If you keep an eye on that you can see that the first parts that are lost are first the pentons and after a while the particle collapses completely. The same thing happens when you are monitoring the mature particle. Again you see that there are some cracks, some voids that are opening right at the pentons and then after a while also collapse. So now we want to analyze the data that we can obtain from these movies. So in these curves we are monitoring the size of the particle as a function of the time. So the time is telling us how many images are we taking or one of these particles. And here we are seeing the height of the size of the particle. Each curve is a single virus. So here we have 1, 2, 3, 4, 5, 6, 7 viruses. Here we have something similar in this case with the mature particles. Each curve is a single virus and again we have something about 7 viruses. Well, so what is going on in the mature particle? In the mature particle you see that first the particle is keeping the size quite well. But at some point after a few images the particle collapses and actually the tip is getting very low heights inside the virus. You see are actually very low heights and all of them are random, okay? However, in the mature particle the things change. You see that first the particle is keeping the size but after a while the size is going down a fixed value for each virus. You see it's about 25 nanometers. For each virus the same. So here is a pattern. Where is coming from this pattern? We think that this pattern is coming from the fact that you are removing only the shell of the virus but you are not able of digging inside the core. So this is like taking an orange. You take an orange and you peel it, okay? You can remove the wall of the orange and the orange remains intact inside. So here is something similar. You remove the wall but here because the genome is condensed, okay? Then you cannot manipulate the core and then the size you see here is the size of the core plus the size of the wall there. And actually what we propose is that the mature particle the DNA escapes as soon as you break the cage. However, in the immature particle you break the cage but the genome remains condensed. However, again the question arise here is can you really visualize the genome on coating? It's possible to see the DNA. Well, with AFM you can see already some gas. For instance, this is the final state of one mature particle where you see that the core has been divided into pieces, okay? And actually we put a yellow color like that one to the height of two nanometers. And actually two nanometers is the size of the diameter of the DNA. So we could assume and we could think that the yellow color here is telling us where is the DNA. Also you see these black pieces and the black pieces are the capsules that have been removed during the experiment. Here in the case of the mature particle it happens something similar. However, now you don't see so much yellow color you don't see so much DNA, you must say. And also the core remains intact in one single piece not in two pieces right here, okay? And again you see the black pieces that are the capsules coming out from the experiment. So still the question remains, can we see the genome? In order to address this question we are going to use fluorescence microscopy combined with atomic force microscopy. Both at the same time, both simultaneously. And we are going to use a fluorophore which is called Jojo-1. Jojo-1 is an intercalator of DNA that is absorbing in the blue color and is emitting in green color. But it is only emitting in green color as soon as it binds to the DNA. If it does not bind, there is no emission in green, okay? But if there is emission in green means that it is intercalated right there, okay? In this place. So what is the experiment we propose? We propose to break the particle in the presence of Jojo-1 molecules. So these green balls here are the Jojo-1 which are expecting, which they want to access. They want to access to the DNA but the DNA is in the cage. So we don't have any light. What do we have to see light? What we have to do, we have to break the cage, okay? If we break the cage then the Jojo is going to be able of intercalated to the DNA and then we expect to see some light coming out. So basically the experiment we propose this is an artistic rendition just to show the experiment we want to poke on the virus. We want to see the light coming from the DNA. And actually this is what we see here. This is a bright field image. This is the cantilever and the tip is somewhere there. So I ask you to pay attention to the center of the circle, of the orange circle where we are going to locate the virus and we are going to poke it, we are going to destroy it. Now we change to total internal reflection, fluorescence and we change the cantilever disappears and we see only what is going to happen at the surface with total internal reflection. So we start the experiment at the beginning as you can see there is no light coming there. Now we go there, we poke the virus, we break it and you see there is a spot which is going up there that is arising. So this light there is coming from the DNA that was packet in one of these particles. So here I can show you the whole experiment in the better way this is the inter particle with atomic form microscopy. This is the background noise of the fluorescence, no light at all. Then this is the particle after you break it and this is the light that you have after you break it. So you see that when you break it you have light, you don't break it, don't light. So the judge is telling us the DNA is there, is being released. Actually here you see the combined curve of the orange one is the force curve, the indentation curve which is telling you that the particle is breaking at this point. And the green curve is telling you the behavior of the light. So first you have background noise and as soon as you break it the light is going up and up because you are releasing in this case the genome. So if you make some statistics you can compare how it released the genome from the mature particle which is the green curve and the mature particle which is the blue curve. And actually this axis is the time of the experiment so once at the beginning you see that there is no light because we don't break it. After you break it the light is going up very fast for both kind of particles but at some point you see that the mature particle is growing faster, is growing faster than the light coming from the mature particle. So this is telling you again that the genome released by the mature particle releases better or let's say the other way. The yoyo one is for the yoyo one is more easy to find the released genome in the case of the mature particle and in the case of the mature particle. And we think that this has to do something with the condensation of the DNA. Also the other parameter you can measure is the width of one of these spots. If you measure the width you can see that again the width of the wild type particle is larger than the width of the mature particle. Again it's telling you that the yoyo access much more easy to the genome of the mature particle and the genome of the mature particle. Well, in this experiment we are playing something similar but in this case we are making fatigue. We are just breaking the particle step by step and you see that as soon as you break the particle there is some light already coming out from the virus. So it means that the yoyo is interacting somehow with the DNA. We don't know still, we don't know yet if the genome, if the yoyo one is going inside through the pentons, through the cracks we are inducing or if the DNA is going outside and then is finding the yoyo. We are still trying to make experiments to figure out this issue. So, well, the conclusion here is very easy. First the mature particle spreads more the genome and in this case the mature particle emits less photos. So it's very general response that drops very well with the mechanics that we saw in the other part of the talk. Now let's go to the last part of the talk which is about the mechanical role of cementing proteins and the cementing other decorative proteins are an alternative strategy to strengthen the various capsids during maturación. I mean some particles when they mature they change the interaction between the capsomers, they change the contact area between the bonds in the capsomers but some particles they have the evolution, they provide with different mechanics and the different mechanics is to strengthen the particle by using cementing proteins. So these proteins are covering the external part of the virus. So we already were doing some work quite a few years ago on landafage and the landafage here you see in blue color is the co-protein and in orange color you see the cementing protein. And in this case we figure out two things. The first thing is that there was a change of the mechanical properties induced by the decoration of the cementing protein so here you see the breaking force and the spring constant and this is for the undercorreted particle, you see about this value. However now you put the correct proteins and what happens is that both the spring constant is going up to this value and also the breaking force is going up. So the particle is getting stiffer and the particle is getting also less fragile because you can apply more force without breaking the particle. This is the case with landafage. Now also something similar happens with fatigue. When you play experiments with fatigue we saw that when you have the particles that are decorated with the cementing protein they need more loading cycles in order to be destroyed that the case of the particles that were undercorreted. So now the question is can we use this philosophy to recover for instance weekend protein shells? Let's assume that you have a weekend protein shell with some defects and you want to recover it because you know that it's very weak. Can we use the approach of putting cementing proteins like a general rule? Like in this case you are putting these bolts in order to fix these panels to the wall. So the wall here is very weak and can fall down. But here once you put the bolts you don't have this problem. So we are going to use to in order to address this question the P22 bacteriophage capsid. With these capsid it's very nice because you have a large variety of structures. For instance here you have the expanded particle but by heating the expanded particle at 65 degrees during 20 minutes you can remove the pentons. So you can actually remove the pentons and you can create a new structure which is called with a wall. Which is the same structure now before but in this case without pentons. Now, another interesting thing you can do here is that you can use the cementing proteins of the L bacteriophage also in this bacteriophage in P22 bacteriophage you can use them to cover not only to interact not only with the expanded particle but also with the with a wall. So as you can see here we have four kinds of particles. We have the expanded particle without cementing proteins we have the expanded particle with cementing proteins you have the with a wall without cementing proteins and you have the with a wall with cementing proteins. So we expect that with all this variety of particles we have here we will be able of addressing how is the mechanics or how can we manage to manipulate the mechanics of the particles with the cementing protein. So the first glance that you can see here is is the partial collapse of the particle on the surface. Actually I was discussing we bought an about the launch about this issue. So here you see for images this is a 5-fold symmetry axis oriented expanded particle this is a decorated expanded particle this is a with a wall and actually the with a wall you can see the three these three pentons are missing because it's a with a wall and here again even if it is decorated but the three pentons are also missing. So what happens when you put one vira particle on top of the surface? If this is the vira particle you put on the surface and there is a partial collapse of the particle on the surface. So if you pay attention to the size of the particles when they are absorbed on the surface you can already learn something about the resistance or the mechanics of the particles. So let's see that. So here in this graph I am showing you the size of the average size of the height of expanded particle and expanded particle plus the collision protein, okay. So I finish. So here you see there is an increment in the size so it means that the quality particle collapse less than the under quality particle. Something similar is happening with the with a wall. The with a wall is collapsing a lot. You see it's reaching collapses of about 80%. So it's getting on only the 80% of the original size. However, as soon as you put the cementing protein you are recovering almost 88% of the original size. So this is telling you already how is going to be the mechanical behavior. Also you can play, you can check the statistics the chemical resistance against for instance SDS. SDS is a protein in the natural, okay. So you put SDS for expanded and with a wall particles you see that the expanded particles are resisting much better than the with a wall. However when you put the cementing proteins and you compare the with a wall with cementing proteins here you see that the black curve is remaining almost constant. So the cementing proteins are making the particles more resistant. Well I just changed the last slide here. Here we calculate how much energy you have to apply to break one of these particles. You calculate this energy as actually this area below the curve and you see here you can compare the energy in KBTs. You can compare that the with a wall you have to apply very low energy about 700 KBTs. But as soon as you put the correction proteins you have to apply an energy of about 1500 KBTs in order to break the particle. And something similar is happening also with expanded but this more is exaggerated in the case of with a wall. So with this I finish my talk and I want to thank to mainly to my students which are taking part of my work here. And actually I tell you that for instance Manuel and Paco they have two posters that they will be very happy to show in you. And also I want to thank to my collaborators Melitú Carlos Catalano, Carmen San Martín, David Reguera, Iván Escab, Núria Verdaguer, Antre Bordaglas. Thank you very much for the attention.