 Okay. Good afternoon, everyone. And this is our first Provost Lecture Series in 2023. And welcome. And in fact, this is our fourth lecture, which we started in November 2022. And this slide, again, just as a reminder for those of you who are here the first time. So we created the Provost Lecture Series mainly to focus on the OIS faculty members. And we want to celebrate the faculty members when they either get promoted to tenure or promotion, and also for those who are going to retire very soon. So we're very lucky we're able to celebrate Professor Ichiro Maruyama three days before his departure from OIST and followed by Professor Mahesh Bendi, who was promoted to full professor. And also Professor Tom Bouguignan was promoted to associate professor with tenure. And so today it's my great honor to present, also to introduce Paola Laureno, who will be introduced properly by our colleague Professor Simone Pigalotti. And I just want to, again, to thank everyone from the admin side and from the provost office who helped us to make this process, to make the Provost Lecture Series possible. And in particular, people from CPR, Brett, Scotty, and Michael and others. And Patrick Kennedy, the section leader from the engineering support section. So he has been able to do 3D printing, DIY for special picture frames with lower budget. That's always what we're aiming for. And of course everyone from the provost office. Indeed, just for fun. So I'm sharing this scheduling. As you can see, everything has been planned in advance, at least one month who is doing what? Microphone, podium, food, and everything. So I would like to thank you again to everybody from the provost office. And so I will hand this over to Simone. But before I do that, I also want to alert you our next Provost Lecture Series will be given by Professor Oaf Skoglund, who has been the full professor at OIST and the Dean of Graduate School. He's going to leave very soon, going back to Sweden. So we welcome, so mark your calendar. It's going to be February 16th. And after the Provost Lecture today, so we, again, so we will serve some snacks and coffee outside. And again, everyone please be mindful. So we were doing our best to prevent COVID-19. Thank you. So now Simone. So welcome everyone. It's a great pleasure to introduce Paola's Provost Lecture today, called from so simple beginning. So there's an old saying in science that if you look at research institutes around the world, they all have two things in common. They all have a lot of coffee machines and a lot of Italians. So when I first visited OIST, I was surprised because I saw lots of coffee machines around, maybe not so many Italians then. A little I knew how quickly things would have changed in this place. And so, yes, so Paola actually arrived shortly after I visited here for the first time. And so quickly we all got a chance to know her and we figured out how lucky we are to have her here at OIST with us. So Paola comes from a previous research experience in some of the best research institutes around the world. She has a PhD at ETH Zurich in 2011. Later, she moved for a postdoc at the Weismann Institute in Israel. And then she came here for her faculty position as assistant professor. And today we are here to celebrate her promotion to a tenured associate professor. So Paola's research has to do with proteins. And she studies evolution of protein function from two different sides. One is the natural evolution, but also from the engineering perspective. So how we can artificially evolve protein to perform functions that we want. And so there is these two sides of natural and synthetic biology. And over the course of these years, Paola has, Paola's research has branched in many different directions, really making best use of what OIST has to offer. Reaching fields such as synthetic biology, biophysics and up to a recent work on liquid, liquid phase separation that she will tell us about today. So again, Paola will tell us much more about this. What I want to say is that she has been a very effective PI. And to be a very good PI, you don't only need good research topic and good research skills, but social skills are also very important. And in fact, what Paola managed to do here is to build up a great research group, the Laureano Lab, that not only has been very productive in these years, but has also a great atmosphere. And I know that she really invests a lot of energy in nurturing her group. And if you know her, it's not a surprise that last year she received the Faculty Excellence in Mentoring Award from OIST. Paola is a very accomplished experimentalist, but she's not only at ease in the lab. She has other interests outside the lab. She likes the great outdoors and I have to thank Take for sharing the pictures. She loves exploring the great natural spaces. And she's even more adventurous than that, as witnessed by these pictures. If you know her, you know that she's a very energetic and very determined person. And these pictures only confirm that. And also show that she has everything it takes to be a successful scientist. So Paola, congratulations again on your achievement. And I'm happy to leave the floor to you. Yeah, I just want to forget. That traverse. That is the most scary stuff I did in my life. Thanks everybody for coming here. First of all, I would like to thank Emi for this initiative of the Provost Lecture. And I would like to thank the Provost Office to organize this and to put together the poster. I really like the poster they made. And Simone for the kind introduction. This talk will be divided in three parts. In the first part, I will tell you a bit about my personal and scientific journey is going to be very short. And then I will introduce you what we do and we have done in general in our laboratory in these last five years. And then I will just dig in one of the projects, the droplets project, which I'm very proud of as well as all the other projects in my lab. I was trained as a chemist, actually as a medicinal chemistry as a medicinal chemist. I got my degree master master degree from Milan University in Italy. Master degree is a big, a big thing. So and actually this is the day of my degree. This is two of my close friends. We are currently still friends of mine. And yeah, we put this lauren crown like in the ancient Roman time for the emperor when we get master degree. After this, I moved to Leiden University again with a small fellowship from the European Union. I spent there one year and again I was synthesizing a small molecule, medicinal chemistry. I was in the medicinal chemistry department. This is a group picture. They used to have a lot of event and the perspective is right. I'm very sure to compare to Dutch people, which are very well known to be the tallest in Europe or maybe around the world. After this, I decided to switch. And I went to do carbohydrate chemistry to synthesize carbohydrates. Carbohydrates are polysaccharide, commonly called sugar. And for this, for the chemistry is very different from medicinal chemistry, where we do small molecule synthesis. For me, this was a stepping into unknown, a completely different kind of chemistry. I moved to Ithiazure to do my PhD in organic chemistry, synthesis of polysaccharide. This is a few lab members, maybe the closest friends. Simone mentioned we are making a dumpling in this case. And my PhD advisor moved in the middle of my PhD to Max Planck Institute in Berlin, Professor Sperger. And so I had the opportunity to work in two of the most prestigious institutes in Europe. After this, I had the end of my PhD synthesizing polysaccharide, sugar. And polysaccharides actually bind to protein. At that time, my knowledge of protein was something like this. Maybe I was imagining some cloud, which bind protein, not very well defined. And again, in my career, I decided to step in something unknown. And I wanted to see what is this binding protein, and maybe engineering and modify. I was lucky at that time because someone support this completely change in field. And this was my postdoctoral advisor, the late Dante Alphic. I moved to Weizmann Institute of Science. This is a picture which was taken just before I left Weizmann, is half of the group. We used to prepare a happy hour, again, food, departmental happy hour. And it was our turn to prepare this. So basically, I end up, I was trained as a chemist. And actually, I became a protein evolutionist. Why I'm saying this? Because I had the hard core training in Dante Alphic Club on the origin of function of enzyme. And what I've spent most of my time in Weizmann, not only to understand and characterize the protein, but also to define a minimal and ancient protein geometry to distinguish between convergent and divergent evolution. And here we were looking for something very ancient, something that the origin of life, some geometry which is retained from 3.5 billion years. Then I came to Weizmann in September 2017. I couldn't find our real picture. This is a picture of my group probably 2018. And my group in these five years expand. And this is a picture we took last November. Actually, it was celebration for my tenure. What I would like to tell you is what I learned from these five years, not sorry, from my personal and scientific journey, from my master until now. The most important things, I think, is support of people. And this can be family, can be friends, can be older person, so advisor, but also student. What is very important is enthusiasm for science. And I think for me, the most important things was to be able to step in the unknown, a different stage of my career. I really like to keep my learning curve very steep. I titled my talk from so simple a beginning. This is a quote from Charles Darwin, from the Origin of Species book. And the full quote says, from so simple a beginning, endless forms, most beautiful and most wonderful have been and are being evolved. I take this because this is very inspirational book for any kind, any form of evolution, from protein to, of course, species. In my laboratory, we are working with protein. Now, when we think about protein, many people might think about food, actually. I'm not good in Japanese, but actually someone tells me that here it's written protein in Katakana and I think most of you know. Instead, when I think about protein, I'm thinking about something like this. A protein is a long chain of amino acid which actually folds in a three-dimensional structure. This is a structure, it's a real structure of a protein that we're working on in the lab, methionine adenosine transferases. And this is a ribbon representation, so you see this represented as a cartoon. And the proteins are actually not rigid, but they are very flexible and we like to say they breathe. And this is actually a 3D structure that we generate here at OIST. You can pass around if you like. Protein are very important. One example to give you about their importance, if we look at the illustration by Goodsell of a section of a bacterial cell, this is the inside of a bacterial cell. This is the cell membrane and this is the flagella. Each of these elements are actually protein. Proteins are highly packed and constitute most of the living material. In particular, in my laboratory, we are interested in enzyme. Not all the people in my lab, but most of the people work with enzyme. Enzymes are protein, but they can catalyze reaction. So basically they take substrate and they transform in a product. It's like a fuel that goes into the enzyme. Enzyme catalyzes reaction and makes this reaction much faster than how they would occur without a catalyst. This is another enzyme we are working in the lab. It's called DNA methyl transferases. This string that you see here is actually DNA. This is an enzyme and this enzyme, the substrate is DNA, but it has also a small molecular substrate and it can transfer group to this DNA, which is the substrate. Why enzymes are so important? Because metabolic reactions actually require catalysis and require a fast catalysis. Metabolic reactions must occur very fast to sustain life in an order of magnitude between 10 to the minus 5 to 100 seconds. But actually most of the reaction that occurs are very slow. They are very slow when they are not catalyzed by enzymes. As you can see here, there are some examples of some reaction that occurs basically every second in every cell of your body. For example, amino acid decimization will just take 1000 years to occur without an enzyme. Again, to stress out this point, if we look at the inside the cell, these are all the schematic representations of all the metabolic pathway. Each of these metabolic pathways is actually catalyzed by this enzyme. In my laboratory, we study enzymes and proteins and now we study this. We look at the history of this enzyme or protein. So we have this long chain of amino acid, which falls in a three-dimensional structure and over a billion or a million of years they accumulate mutations to perform optimally their function or to acquire a new function when they have to adapt to a stress or a challenge. So we try to understand how these mutations are accumulated and we try to define general rules behind that. Then once we understand this, what do we do? We do something that humankind has been trying to do, I mean, has done since centuries. I'm Italian, so I pick up another example from Italian history, Leonardo da Vinci. This is just an example. He studied for a long time how board can fly and then he applied this rule to try to make a prototype of our plane. Basically, what we try to do is define these general rules that nature apply to evolve new functions, which between function and engineer enzyme for new function. Why this is important? Because by engineering and creating new enzyme, you can, for example, manipulate cell or solve other problems, for example, bioremediation. So what we are trying to do in our laboratory is to define these general rules. We don't study one single enzyme. We study different enzymes. We are particularly interested in some problematic or some problems of the evolution of enzyme and the emergence of function. But yes, we don't focus on one single enzyme, but rather to define the general rules. Now I will give you just an overview of a few projects which we succeeded in the last five years and then I will focus on one single one. Something that we have been interested is how, in the essence, expand enzyme functionality. As I mentioned to you, protein is a long chain of amino acid and this long chain of amino acid can acquire mutation. This mutation can be substitution. So one amino acid exchange with another amino acid or you can have shortened or elongation of the chain by addition or deletion or one or more amino acid. We call this addition or deletion indels. Indels are much more difficult to study because they change the structure of the protein. So this traditional structure often comes is disturbed. And we were particularly focused on indels. This one example from my laboratory, we look at the Rosman-Fold enzyme, which is the three-dimensional structure that is going around and is blue and white. And this enzyme Fold is very important because it's 20% of our proteomes. And this enzyme Fold is catalytically very diverse. It can catalyze more than 300 reaction, while most of the enzyme Fold can catalyze only one reaction. We were particularly interested in functional switch between reductase and methyl transferases. These are completely different reactions. And basically we find out that the deletion of free amino acid in the catalytic center of this Rosman-Fold can promote the switching functionality. And this is not only for one enzyme, but is actually for a class of enzyme and is highly concerned in nature. Something else also related to indels that we have been studying the last few years is the chitinase enzyme. Chitinase enzyme degradate chitin. Chitin is a long chain of polysaccharide and is the most abundant one after cellulose. Chitin is very important because it constitutes the cell membrane. So basically you can degrade the cell membrane by the use of this enzyme and others. Chitinase, we are particularly interested in the chitinase from plant and we see in nature two of them. This chitinase, which you can see doesn't have this ornament, while this other has is a longer chain and it has much more ornament. This ornament we call them loop. And what happens in nature is that the loopful, so the enzyme with all this loop, acquire a new function, an antifungal activity, while the one without loop doesn't have this antifungal activity. So basically the addition of this loop far away from the catalytic center allows a new function, a new biological function. What we find out by structural, biochemical, biophysical characterization is that one of these loops actually promotes the access of the fungi cell wall. And this makes completely sense that this enzyme acquired this function because fungi is the most dangerous most dangerous microorganism for plant. So we study the origin and the mechanism of the acquisition of this loop. Something else we have been interested is always related to enzyme functionality, but in this case is specificity. I mentioned to you that the enzyme take a substrate and generate a product. But actually enzymes often are not specific for this substrate. We focus on one case, the mat enzyme. Mat enzyme is the enzyme, the first structure I show you that it was moving during the presentation. And this enzyme has the identical catalytic pocket between the bacterial enzyme and the human enzyme as identical catalytic pocket, but completely different specificity. While the bacterial enzyme is very specific for the substrate, the human one is not specific at all. We study the mechanism behind this specificity and we also demonstrate that in cancer cell line, this promiscuity is actually relevant and it goes to basically generate other product in cancer cell line. This can be of course, this can be considered biomarker for cancer cell line. We have been interested in mistranslational error. This is something that we started just recently and we will work more in the next few years. So the mutation that we have been discussed so far, they are all encoded in the DNA that encode for the protein, but actually we are interested at error at the protein sequence which gets encoded at the translational level. And finally, we develop a system that mimics cytosolic condition and why we generate this system to study enzyme activity. Today, I will focus all my lecture on this system. Why I decided to focus on this? Because this is a very interdisciplinary project which goes from biology to physics to biochemistry. It's something that I, it was something like stepping in unknown again for me when I started my tenure and it wouldn't have been possible without the support of OIST. We usually study enzyme in buffer condition. What do I mean? We study enzyme in water with salt. These conditions are diluted and are very controlled conditions. At the beginning of this project, we were wondering what is happening if we try to mimic or reconstitute something highly packed like the interior of a cell. The concentration of this protein is about five millimolar, so very high. If we could generate this kind of system, we could probably study how the protein crowding, the macromolecular crowding, can have a positive or negative effect on enzyme activity. Because this molecular crowding could, for example, affect the stability or the activity of the enzyme, the diffusion of substrate, or the other properties, physical properties of the cytosol. Basically, we were aiming to have a simple and controlled cytosolic-like system and we wanted to basically match the same macromolecular crowding than the cytosol in the cell, the inner of the cell. We wanted to have a steady state activity, so a control activity inside this cytosolic system and we, ideally, we wanted to have avoid external perturbation. So we didn't want to add mechanically the substrate, but let it freely diffuse into the solution. If we could have achieved something like this, this system will have allowed us to study enzyme kinetics, dynamic structure in almost a real condition or at least mimicking some of the real conditions. To do so, we decided to mimic what is inside the cell. This phenomenon, liquid-liquid phase separation, was discovered a few years ago. And basically, liquid-liquid phase separation is when a solution condenses into two phases, one more dense and one less dense. And these two phases are not separated by any membrane, but they can coexist in one solution. Now, if you think this is like these granules, liquid-liquid phase separation, if we can reassemble something like this, and you think this is not in small size, but in micron size, and we can mimic something like this, some cellular condition, we can basically generate some protocells, right? Because we have the inner of the cytosol, and this active as well as maybe we can reach the same activity than microorganism. To do so, what we explore is basically we generate some droplets, membranes droplets, by mixing BSA, bovim serum alumine, which probably most of the biologists here use as not only biologists, and PEG, which is a long polymer chain. By mixing these two phases, these two phases separate according to their molecular weight into droplets, BSA-rich droplets, and a continuous phase constituted by PEG. Here you can see the bright field, these are the droplets, the BSAs inside the droplets, the PEG phase is outside the droplets, and I mean mainly outside the droplets. Then we decide to characterize these droplets. So we basically play around the concentration with the buffer condition, and what we manage to achieve is high macromolecular crowding of BSA inside the droplets. Here you see bright field image, and then what we did, we flourished label BSA, and we took an image at the microscope to show that most of the BSA is actually inside the droplets. We also have a quantitative data, because we separated the two phases, and we could measure the concentration of the BSA. The concentration of BSA is around 5-8 millimolar, and the E. coli cytosol is actually a protein content around 5 millimolar. It's interesting, because this BSA concentration is so dense that we almost cannot pipe it, and it's 2,000 times more dense than water. Then, as I mentioned to you, we were interested to have these droplets actually active, because if you think this is a protocell, something that mimics, something which contains not living materials, but mimics living cells, we want to have enzyme inside, and still we want to have a pretty simple system. So, we have BSA, we want to add only one enzyme. We took, I will show you which enzyme, we took one of our enzyme, we labeled with a different fluorophore, and again we went to the microscope, and we can see that the enzyme is partitioned inside the droplets. And this is schematic representation. Basically we can still give the substrate to our droplets, which is now a microorganism or a microreactor, depend how you want to see this, and generate product. What is interesting is that the substrate can freely diffuse inside the droplets, and the product can also freely diffuse. And what is also interesting is that it's kind of infinitive reservoir. So, few droplets in a very big volume, and then you will understand why this is very important. We had also quantification of the catalytic activity of the continuous phase and the droplets phase. So, we know also by numbers that all the enzyme is in the droplets phase and is not in the continuous phase. So, basically what we achieve here, as we wanted, is high macromolecular crowding. The droplets in certain conditions in low concentration of enzyme are uniformly active, and they are membranless. The fourth enzyme we decide to study is lactate dehydrogenase. Lactate dehydrogenase take pyruvate and transform it to lactate, reduce it by the use of NADH. And this very important enzyme belongs to the glycolysis, but it's also very well studied, and that is the reason because we start to use this. First of all, we again check the partition that all the enzyme is inside the droplets, and then since we are normally, but we are biochemistry, we retrieve mycal mentis parameters. Here you can see the mycal mentis curve, which is given by different concentration of substrate plotted against the initial velocity of the enzyme. So, when the enzyme is still in a linear phase, this is the buffer, the mycal mentis for the buffer, and this is for the droplets. Now, this is not standard mycal mentis, as you can see it goes down, usually the mycal mentis goes to plateau, but this is because the lactate dehydrogenase suffers of substrate inhibition. So, after this point, the substrate basically gets in the catalytic pocket, but in unproductive conformation. What was interesting to notice here, and we actually have tested this for many other enzymes, is that we had increased in catalytic efficiency. So, how we define the catalytic efficiency as k-cut over km, so the turnover rate, and we could see two-fold increase in catalytic affinity. Now we are getting this, not only for this enzyme, but also for other enzymes, because we see the other enzyme behaving the same way, at least with enzyme with active catalytic loop. And what we have found out by MD simulation in crowded environment is that the closed state, so the active state of the enzyme is favorited in this condition compared to the open state. This was done in collaboration. The MD simulation has been run by UGA, Sugita, in Riken, in the supercomputing facility in Kobe. Again, we wanted to have high and sustained enzymatic activity. Now here we plot this in a different way. This is the rate of consumption of chemicals, but basically this curve is exactly the same curve than the Michael Mantis curve that I showed you before. It's just that here we use different concentration of enzyme. And if we plot this and we look at more or less what is the estimated activity of a microorganism, we can see that we have same metabolic density of the angriest microorganism. Another very important characteristic of these droplets is the fact that if we run, this is the same reaction in solution in buffer. So this is time and this is the product formation. The reaction is basically over in five seconds in buffer condition. But if we use these in droplets because of the continuous diffuse of substrate, the reaction can actually, the reaction lifetime increase a thousand-fold. Finally, we were interested to see if this enzymatic activity can generate some macroscopic effect in the crowded environment. To do this, we use a different enzyme urease enzyme. Why we use a different enzyme? Because this enzyme is much more catalytically efficient compared to other enzymes. Urease decomposes urea in ammonia, which is a strong base and CO2. As we did before, we compartmentalized the urease inside the droplets, as you can see here. And then we look at the activity at the microscope. Now I will show you, we put into the droplets some polystyrene nanoparticles and you can see nothing is happening. And this is actually the picture we used to get also from the lactate and the hydrogenase. But once we had the substrate, we see a directional flow. And this was very, very surprising for us because you can see this even without nanoparticles, just looking at the droplets. This is the comparison. Now the urease generate a strong base. We were in buffer condition, but not so strong buffer condition. We will start to wondering if it's actually something new to the base that we are forming in these droplets. What we have done is to take the BSA and label with a pH dye, which is called SNARF. This pH dye basically change color according to the pH environment where it's sitting. And as you can see, while the reaction goes on, the pH change from pH 6 to pH 9. We had quantitative measurements of this where the pH increase over time. And also we reach around the pH 9, which is not so surprising because the pKa for the ammonia is around 9. And then, of course, we did the characterization of these droplets. We can see that keeping the same concentration of urease, but with different concentration of substrate, the change in pH is different. And by increasing, if we look at the droplets size and the change of pH, we can see that the change in pH is bigger for bigger droplets. And this is hinting on something about the diffusion of the substrate and diffusion of the product that we are currently investigating for. So basically what we managed to do is generate, we self-generate a pH gradient inside the droplets. And this drives a directional internal flow. This project and especially this part, which was the characterization of this flow, the directionality, and the physical reason behind this directional flow has been studied in collaboration with a group of physicists in ETH Zurich. The head of the group is Eric Tufresne and these PhD students spend almost one year in our laboratory here studying this phenomena. Not only this, we push this further. And now when we were doing these experiments, we have a glass and these droplets basically pin on the glass. So all the video that I show you, we look at from the microscope from the bottom and it's a kind of semi-sphere, right? And we look at from the bottom. But then something that the post-doctor was working on this project did is basically change the focus from the glass a bit higher and see what was happening. And what you find out here is that these droplets, when they are in solution and we generate this flow, they're actually merging and moving around. So basically what we managed to do, I mean, and now we want to study further, is to see how these droplets can be influenced by nearby droplets. And this can maybe mimic or recall some other cellular behavior. For example, motility of merging, not merging, but motility of microorganism. And something that we're very proud of and just came out last week from one of my PhD students. We managed to make these droplets not pin down anymore on the glass, but actually now they're able to swim around. And this means that we can maybe direct them. I think for this protocell project we are only we see only the tip of the iceberg and there is much more that can be discovered and used in the future. So I would like to conclude here. I hope not everybody fall asleep. I first of all need to thank my PhD advisor, Peter Seberger for the initial support and enthusiasm that it has made to me for science. I need to thank Dan Taufik because the reason because I could do this step and move to protein is thanks to him and I miss him a lot. I would like to thank all the past and current collaborators and then a special thank to the research support division, the imaging section which helped us with all the video and the discussion and in particularly we work very closely with Paolo Barzaghi. I would like to thank the OIST instrumentalist section as a whole because we do a lot of measurements there and in the project on the enzyme specificity with particularly working with Alex. The OIST sequencing section for the next generation sequencing and the OIST scientific computing and data analysis for the support during these five years. I would like also to thank the CPR for the help and then my family, my husband and my kids which are the most important things and finally all of this has been possible just because of my group and of course for this thanks to the support to especially OIST but also the private foundation Iwatani and Takeda and the support from Kakenki and JSPS and thanks to all of you.