 to allow for bacteria to take that DMA up. So to illustrate this process, we'll zoom in on this conjugation exchange of DNA between bacteria. So here are those bacteria in a little bit of a different way. And so this donor cell is providing this DNA here with arrows to a recipient cell. And the recipient cell will take this DNA in and it will insert it into its chromosome. And so now the recipient bacteria has picked up some new genetic material, but it wants to mitigate those risks, right? So it needs to make sure that it wants to express this DNA before, yeah, before it starts to want to keep it. And so to uptake this DNA, the bacteria have this protein HMS, which combined to this newly acquired DNA and through protein-protein interactions can cope all of this DNA to form filaments. And HMS can work with these other proteins that I mentioned, STPA and HHA to form these filaments. And this HMS filament will silence the gene by preventing at least two steps in transcription. It can block promoters, so preventing initiation, and it also can affect the long-living RNA primates that I encounter in the filament. So when I started in the lab, we didn't know very much about these different kinds of filaments and the details of how they're affecting transcription. So really there was a big question of what is the mechanism of silencing by HMS? So to answer this big question I focused my thesis research down on a two-part question. I asked what is the structure of HMS and mixed HMS filaments and how do they regulate transcription So today I will tell you a little bit about what we knew about how HMS can form filaments and how they affect elongating RNA primates, which was known when I started in the lab. And then I'll tell you a few more details about investigations I did into the structure of these mixed filaments where HMS and HHA are interacting with each other. And then I will finally end with what the cell gets from these diverse filaments. HMS is a small 15-pill dot in protein and it has two domains an N-terminal oligomerization domain and a C-terminal DNA binding domain. And these are connected by a flexible linker. So there's no full-length structure of this protein, but we do have structures of the individual domains. So I'll first show you the oligomerization domain which I'm showing you a few of the HMS monomers here in the different colors. So the red one here is one monomer and the tan one is the second. And these oligomerization domains won't have a DNA binding domain coming off here. I'm showing you the circles. But what the structure told us about the oligomerization domain is that it can facilitate different protein-protein interactions between HMS monomers. It can form diners at this site in the middle and the second site facilitates longer oligomerizations of HMS. So there's some restriction of the HMS DNA binding domain which I'm showing you here interacting with the short DNA duplex. And this DNA binding domain binds into the minor group of DNA and it binds to most of the AT-rich sequences. And while I combine AT-rich sequences more generally it has been documented to have a high affinity for a 10-base pair sequence that will appear where the key feature are these T's and A's in the middle. So together between these DNA binding domain structures and the oligomerization domain we know that HMS can bind to DNA and they form filaments but we really are lacking a full-length structure of these proteins. So I want to tell you a little bit more about these filaments and what they look like. So we know that HMS can form filaments throughout the polygenome. I'm showing you here some chip signal from HMS binding throughout the genome which is shown on this circle here. And so each of these peaks are HMS binding sites. And these regions vary in length but they're averaging about 2 kilobases long. And so HMS can bind over various regions in the DNA or in the genome. And we know from in vitro studies that HMS can adopt two different conformations. They can adopt a formal linear filament which is where HMS binds to just one segment of DNA. And this is in contrast to the second confirmation of the bridge filament where HMS can bind to two segments of DNA. And I'm showing you proteins for these filaments here because we really have a lot of questions about what these filaments look like. We don't have a structure of the whole protein and so it's difficult to imagine how all of these monomers can arrange themselves within either the linear or the bridge filaments. And so I'll also mention here that HMS is not acting alone. Instead it is modulated by HHA and other factors. So I want to tell you right now about what we know about HHA. So HHA is an even smaller protein. It is all about 8 kilodomans and it lacks a DNA binding domain. But rather its single domain is similar to the HMS oligomerization domain. And when I joined the lab, the structure came out showing how HMS can interact with HHA. And so this is the structure here. And there are two portions of HMS monomers interacting with the HHA to join in blue. And so if we were to take this interaction between HHA and HMS and assume that they're interacting together to form what I'm calling a mixed filament so a mixture of HMS and HHA, they might look something like this where the blue circles are HHA that I've added into the bridge HMS filament. And so like with the HMS oligomans we had a lot of questions about what this overall filament might look like. So before I get into the structural work that I did to investigate these filaments I want to tell you another reason why we care about these different filaments. And that's how they affect transcription. So I want to go back to my operand and tell you a little bit more about what we know about how HMS can affect transcription. And that affects all the stages of transcription. So in the first step of initiation HMS can form filaments over the promoter that could be bridged or they could be linear. And I'm just showing you the two confirmations here with different colors so it's easy to see that in the bridged HMS is interacting with two DNAs for the linear HMS only buys to one DNA. So HMS can block or informers bind into the promoter but it can also affect the second step of transcription elongation where an elongated RNA polymerase might encounter an HMS filament in either a linear or bridged confirmation. And a previous postdoc in the lab of Matt Kulayich he investigated the interaction between RNA polymerase and these two confirmations of HMS filaments and he found that only this bridged filament can simulate causing an RNA polymerase. And this pausing is a normal part of elongation where RNA polymerase will dwell as certain sequences throughout the DNA and this pausing can be enhanced by a variety of factors including the bridged HMS filament. And so this pausing the stimulation of pausing has an RNA polymerase to dwell at that sequence even longer and delays RNA polymerase from getting to the very end of the DNA. And in addition to slowing down elongation overall this paused RNA polymerase is a substrate for the last step of transcription elongation where this bridged HMS filament can increase the number of termination events that happen in a gene. So together these three effects of HMS on RNA polymerase result in overall gene silencing. And so my question coming into the lab one of them was to investigate how HHA might affect HMS activity. And so like I mentioned Matt, a previous postdoc, had investigated the effects of these filaments on elongation and so I wanted to use the same assay to ask what this HHA HMS mixed filament might be doing to RNA polymerase. So to do these experiments we wanted an example operon that HMS might be regulating and the big operon was a really good option for us. This operon typically is used to make enzyme so the cell can utilize adiablucosides as a nutrient source. And here I showed you a portion of what that operon looks like. And the key features I want to point out are that there are two regions in these gray boxes that HMS can bind to and this results in formation of an HMS filament over the promoter for the bagel operon. And this results in silencing of the bagel operon. The second thing I want to point out is that there is a promoter downstream of the filament that I labeled P on and Jason Peters who was a grad student in the lab identified this promoter and if RNA polymerase were to initiate at this promoter transcription would be headed into the HMS filament. And so this gene, this operon, provided us a way to ask two questions about HMS and these mixed filaments. The first is what is the effect of HMS on an elongated RNA polymerase? And the second is to ask more structural questions about what these filaments look like. So I want to talk first about the effect of HMS on elongated RNA polymerase which focuses in on this situation where RNA polymerase would be transcribing into HMS filament. And so we developed a neutral assay to look at this. So we could poise RNA polymerase downstream of the filament, either the bridge or the linear or any combination we could come up with. And we could then, before elongation through the filament by adding NTPs, RNA polymerase is substrate and RNA polymerase will then transcribe down the template. It would pause at very useful pages before it reaches the end of the template creating the full length RNA product. And so we could look at RNA polymerase's progression through these different filaments or in the absence of filaments to see how the filaments affect transcription. So I want to show you first the effect of these two filaments on RNA polymerase. So the bridge to HMS in the red and the linear HMS filament in the purple. I'm showing you here the RNA's made in the presence of these different filaments at one time point. And so the RNA length is on the x-axis here and the abundance of those RNA's is on the y-axis. And so at this time point the RNA process may be down the template. It can make a longer RNA which I'm showing you here in the black trace which is RNA polymerase transcribing through Bayer DNA. And when RNA polymerase is in the presence of this or transcribing through this bridge HMS filament in the red we notice that there were shorter RNA's in higher abundance than without any filament present. But this effect went away in the presence of the linear HMS filament the purple trace here which looks a lot like transcription of Bayer DNA. So from this experiment we concluded that the bridge HMS filament was preventing productive movement of RNA polymerase down the template by stipulating pausing RNA polymerase at these sites. But this was not occurring in the linear filament. I wanted to add what happens to transcription when we add HHA to these different filaments. So I'm keeping my bridge in linear HMS results at the top. And now we compare this to the HHA HMS filament. We see some different effects. So again I'm showing you the RNA length on the x-axis and the RNA abundance on the y-axis comparing transcription of Bayer DNA and the black linear HMS filament in the purple which again has little to no effect on RNA polymerase. But this HHA HMS filament in the blue only allows RNA polymerase to make really short RNA's. So these are less than 64 nucleotides long whereas these were about 10 types that length. And so the presence of these really short RNA's suggested to us that the HHA HMS filament is also blocking RNA polymerase progression by stimulating pausing. And so it might be a bridge HMS filament since we knew, or bridge filaments since we knew that bridge HMS filaments can stimulate pausing. So it was really this transcription result seeing differences between bridge and linear filaments that drove the session that I had spent most of my thesis work addressing was what is the structure of these filaments. So this is a really big question to investigate the structure. So I'm just going to tell you about two aspects that I investigated the structure of these proteins. So first I want to tell you about the fact HHA has on HMS filaments and then tell you a bit more about the arrangement of the monomers within those filaments. And I'll just continue to show you this image to remind you that these two different combinations of HMS filaments can have different effects on RNA polymerase during transcription. Okay, so I want to first tell you how I investigated the structure of these HHA HMS filaments. So I used a technique called atomic force microscopy and this allowed me to form individual filaments and visualize them on a surface. And so I can mix them together with HMS to form something like this bridge filament or maybe a linear filament and I can take these molecules and I can apply them to a surface and so this is one of my real life samples. And the DNA protein complexes are on the top of this small square kind of sticking out on the face. You can't see them here with your eyes. But if you put them in the AFM, which looks like this, this instrument will allow us to see any changes in the height of molecules on a surface. So I take this surface here and I put it in the AFM and so now the surface is this funny green block, so my molecules. And there's a tip shown in the blue which will tap along this surface. And so the tip is going to be tapping along at the same rate if the surface is flat. But if there's any changes in the height, that tip will pick up those changes and then they are detected by this laser and then the computer can turn this changes the height and ability for the tip to tap on the surface and I can get an image that looks like this. And in this image all of the dark colors, these black or red are the surface and any molecules that are on the surface would appear as this white or light orange color meaning that they were high off of the surface. And so in this image I'm showing you two DNA molecules which are pointed out by these white arrows. So this is one squiggle of the DNA molecule and this is a separate molecule. And so this DNA is when they're just by themselves on the surface and adopt this confirmation which is kind of like a squiggle because of the properties of DNA. And we can use this to compare what HMS or HHGNHMS how they're interacting with the DNA. And so if we compare this DNA alone image to the same where I've added a low concentration of HMS, I can compare formation of the bridged HMS filaments. And I'm showing you in this image four of those bridged filaments and I want to point out a few key features. And so the bridged filaments the number one thing was that there were two DNA molecules and I could see those by looking at the very ends of these molecules. They look kind of like a V meaning that there's two DNA molecules there. And these DNAs are bound by protein and I've observed with these wider spots meaning they're higher off of the surface and the two DNAs seem to be very close together so I can't resolve them. And so we concluded that that was where HMS was finding these DNAs allowing them to be brought together. But there were some areas of those filaments that were separate these kind of loops where HMS either wasn't bound or fell apart when we made the samples. But in these conditions we could observe these bridged HMS filaments where two DNA molecules were being brought together by HMS. So I also could form filaments at a higher HMS concentration which we determine formed linear filaments which look like this. And in this image there are two linear filaments with the blue I'm pointing with the blue arrows here. So this is one molecule and this is the second. And these linear filaments could be identified because they were straighter than the DNA alone so if you compare how straight this molecule is to these two over here. The HMS is stiffening the DNA in the linear filament and I also didn't see any of the these at the end of the DNA that I saw in the bridge filament suggesting that there was indeed only one DNA molecule bound by HMS. So we could form these two different HMS filaments by varying the HMS concentration and I wanted to know if either of these filament confirmations were present when I added HHA to these HMS filaments. So here are my HMS filaments again and so if I added HHA to either of these filaments I observed a variety of different molecules including this one I'm showing as an example and this molecule is quite large and very high off of the surface. And so if you just compare these scalar bars which are all the same length then the HHA is much larger than these filaments over here and in addition to my fortune a little bridge filament landed over here next to my HHA and HMS filament so you can even see in one image how much bigger this HHA HMS filament is. Okay so I spent a long time staring at these images you know one this example otherwise what is going on? What is this filament? And what we determined at the end was that HHA was facilitating lots of bridging events so a multi-bridge filament which I'm showing with this cartoon down here where the DNA in the black can be interacting with different polygomers of these HMS HHA complexes. So all these different interactions can facilitate a formation of a bridge complex with more than two DNAs in them. For this experiment I concluded that HHA is indeed stimulating bridging by HMS so it's either increasing the amount of bridged interactions that are already present in a bridge filament or it's switching this linear filament to a more bridged combination. So I want to bring this back to the transcription result I showed you at the beginning. So what would be the effects of these filaments on transcription? So I'm showing you those results here again these are the same ones I showed earlier showing that these bridged HMS filament in the red and the HHA HMS filament can stimulate pausing but the linear filament here in purple does not. And so these transcription results where some filaments stimulate pausing but others do not is consistent with the results we observed with AFM showing that bridged HMS or the bridged HHA HMS filaments can stimulate pausing but linear filaments cannot. So this provides some structural information about what filaments are able to affect the transcription machinery during elongation. So to summarize this first part I'll use the top of course microscopy to visualize these different filaments the linear HMS, the bridged HMS, and the HHA HMS filament and I found that HHA can modulate the conformation of HMS filaments. So I've added this HHA arrow here to show that HHA can convert a linear filament into a bridged filament or continue to stabilize that bridged filament. And I'll just remind you again that it's this bridged filament that can inhibit or affect the elongated RNA filaments but the linear filament only seems to affect energy RNA filaments. Okay, so that was the first part of my structural investigations into these filaments. And now I want to talk about my second question, kind of zooming in on these filaments and looking a little more carefully at how are these molecules arranged. So we're going to zoom in on the DNA binding interactions between HMS and the DNA which despite the fact that HMS is a DNA binding protein, aren't really well characterized. So I get to this investigation I had a distinct hypothesis. I hypothesized that the arrangement of the DNA binding domains might be different in a bridged and a linear filament. So there was a little bit of data that went into drawing these cartoons, but at the end of the day we thought that maybe in a bridged filament the spacing between these DNA binding domains would be larger, so I'm just showing you that difference with those arrows. So we thought if we could see this difference in the DNA binding domain then it would give us more of a clue how the molecules of HMS are arranged within these filaments. So to look at the DNA binding domain location, I added a probe to the DNA binding domain that looks like this. So here we're going back to that structure of the DNA binding domain, but I'm showing it in purple now. And I attached this iron EDTA moiety to the HMS DNA binding domain. And the reason we picked this is because iron can perform a number of reactions including defective reaction by reacting with hydrogen peroxide and it generates this very reactive hydroxyl radical species. And this hydroxyl radical can interact with the DNA and it can result in DNA cleavage. And because this iron is attached to the HMS DNA binding domain in my system, this DNA cleavage is limited to a small area right around where the iron is. And so in this experiment, if I'm observing DNA cleavage, it is because free radicals were generated by the iron which is attached to the DNA binding domain meaning that HMS was bound to the DNA. So said another way, I can only observe DNA cleavage if HMS is bound to the DNA because that's where the iron is. So I wanted to look again at our filaments that I can form on the bagel opera like I showed you with the AMF. And so those were large filaments and so I'm going to zoom in on one area of this filament to look at about a 100 base pair region of this filament and look at where HMS is bound to the DNA. So I can form my filaments with my labeled HMS which is now purple in this case also a linear filament. And I can add the hydrogen peroxide which will generate those free radicals and result in DNA cleavage within the filament again nearby the DNA binding domain. And I can then observe these cleavage events by running these DNAs which have been labeled on a gel. So here is my one and only gel for my top to show you these different cleavage events. And so at the top I'm showing the DNA by itself with no HMS because I have one DNA product because it's big black blood at the top. And if I add in my iron labeled HMS what I have sort of is all these different shorter DNA products. And it seems to be a nearly every base on the DNA. But I did notice that some of these bases were cut more than others. Those bands were much brighter. But I couldn't really tell from looking at this gel what was going on. And so I qualified this to investigate how much each base is being cut on the DNA. And during that one location I can get a graph that looks like this where the location of the base is on the x-axis and the y-axis is showing me bases that have less cleavage to more cleavage. And what I observed was there were distinct bases that would be cut more than others. And so I've indicated those with these red arrows here. So this probably doesn't look anything like HMS binding to DNA to many people including myself for a while. So we wanted a way to investigate before turn this cleavage pattern into an arrangement of HMS on the DNA right to address our original question. So to determine where HMS is binding to the DNA I had to do a few other things. So I took the cleavage pattern that I just showed you before and combined it with the cleavage pattern on the other strand of the double strand of DNA. And then I also worked here on Daniel Roxton, who was a postdoc in LA for just a little bit of time. And he helped me do some molecular dynamic simulations to basically predict where this iron EDTA bound HMS might cut the DNA. And I wish I had time to tell you about all the great work he did, but I'm just going to tell you he did it and it's great. And because he did that great work, I could then build a model of HMS binding to DNA, which looks like this where the DNA is in gray and the DNA binding domains are in this purple surface representation. And I'm showing you the iron again just so you know that's how I put this together. And so these DNA binding domains are in a very regular arrangement on the DNA, meaning that HMS is one of the sites in all of the filaments I've worked in my solution. So I told you my first experiment was looking at a linear HMS filament. And my hypothesis was that the linear embrace might be different. So my next question was, is the pattern aligning the same in a bridge filament? Is it the same as what I've shown here? So here's my cleavage pattern again that I got from a linear HMS filament. And with the help of my processor, Christine Hussmeyer was going to be amazing at HMS for the next help to me do this new experiment where we could add HHA to stimulate bridging, which I already showed you the AFM, which was pretty convincing in showing that HHA first formed a bridge filament. So by adding HHA, we could form this bridge filament and look at the cleavage pattern in this filament. And here in the green is that cleavage pattern, which looks pretty much the same as the linear filament. To my surprise, I was so surprised and also very happy. And so from this result, I concluded that the DNA binding domain must be in the same arrangement in both the bridge and a linear filament to generate the same cleavage pattern. So I'll show you my model again and say that this model represents where we think the DNA binding domain is found in both the bridge and the linear filament, which was different than my original hypothesis. And so we needed some other explanation. So we made an estimation for how HMS is switching between this bridge and linear confirmation. So I looked to the literature and it turns out there's a number of other groups who study HMS of course. It's an interesting protein. And there was one paper from some colleagues of ours Raymond Stame's group. They published in 2017, which suggested that HMS can adopt different confirmations. So I just want to illustrate those with a cartoon. So the first confirmation answer was called an open confirmation. And so if you were to look at one HMS dimer here, so the red and the yellow are numbers forming a dimer. In this open confirmation, both of the DNA binding domains, and in particular the residues of HMS that can bind into my group, which I'm showing in the gray, are open and available for binding DNA. So that confirmation HMS could bind to two separate DNA molecules. The second confirmation Raymond Stame's group observed was the closed confirmation where one of the DNA binding domains moves and now it's interacting with the internal domain of the protein and is in essence sequestered and unable to bind to DNA. So in this closed confirmation, only one of the DNA binding domains would be available for binding DNA. And so these two confirmations fit into our idea of the bridge and the linear filaments, respectively. And so to put these two confirmations in our filaments, we can add the closed confirmation HMS into our linear filament because only one DNA is bound by HMS. And so here are closed confirmation HMSs, regularly arranged in our linear filament. And this open confirmation could explain how HMS binds to two DNAs in a bridge filament here where two DNA binding domains are able to bind to two separate DNAs. And again, the spacing between the DNA binding domains is the same between these two filaments. So our hypothesis that the spacing of the DNA binding domains between these two proteins was not working out from my experience, but rather it suggested that since the DNA binding domains are in the same arrangement, this other confirmational change in the protein could facilitate one or two DNAs bound by HMS filaments. And again, the switch between the bridge and the linear can be facilitated by proteins such as HHA. So my work investigating the structure of these filaments showed two things. One, HHA can stimulate bridging by HMS. And also that the DNA binding domains are arranged the same between bridge and linear filaments so just being a confirmational change happening in HMS to facilitate binding one or two DNAs. And again these filaments have different effects on gene expression where the bridge filament seems to be responsible for affecting elongating RNA from races whereas the linear filament can only affect this initiating RNA from race. And so these different effects of HMS filaments on transcription have put together a model for us. The model is that modulating the confirmation of HMS, so whether or not one or two DNA binding domains are available, allows the self-tune how it regulates gene expression. It can facilitate either inhibiting initiation or affecting e-oblution, all by modulating the HMS confirmation. So I told you about one way in which HMS can be modulated and that's by protein HHA which stimulates bridging. It turns out there's a bunch of other factors that can modulate the activity of HMS, including set of protein STPA, the parallel of HMS, which also stimulates bridging by HMS. And there are at least, at least three other factors that can modulate the confirmation of HMS. And work from many others in the field have recently identified that nearly all of these factors can change the confirmation of HMS in a similar way to what I showed you before. And it's this change in HMS confirmation which changes how HMS is regulating gene expression. And so together my work helps put together this picture of modulating the confirmation of HMS and modulating the effect that HMS has on gene expression. So I want to bring this back to a horizontal gene transfer situation. So I told you this process is important for bacteria to pick up new genetic material. And the material that it's picking up is very diverse. Either my encounter of bacteria has never encountered before and therefore new genetic material. So all of this DNA, this transfer gene here, could need to be regulated by a variety of different ways. And what these bacteria possess is a host of different kinds of filaments and modulators that can sign these different kinds of genes. And so by having these diverse filaments, the bacteria can silence a diverse pool of acquired genes. I just want to end with kind of a bigger picture question. What does it take to understand bacterial content? I think if I mentioned this at the beginning I was really excited and starting to ask a little like, I can figure all this stuff out. It's going to be great. But it turns out that these questions are really hard to answer. Really hard. And I have to look a little bit. But we still have so many. I wrote a little chapter on it. It's a lot. But what I did find was that these different filament confirmations can silence genes in different ways. And in order to figure that out, I had to use a bunch of different techniques. And I only told you about two in detail, the AFM and my feverish assay, in addition to our vitro transcription assay. But it had a bunch of other stuff that I didn't really get to talk about today. And all of those experiments together have taken us steps towards understanding what these filaments look like and what they're doing to interact with our nuclearis and silence genes. But what I've done is only one small portion of all these spectrochromatin proteins. I would have said HMS, which silence genes. But there are a bunch of other chromatin proteins, like at least 13 of them, maybe more. And we still have a lot of questions about how they interact with DNA and how they can regulate gene expression. And so what I've learned from my thesis work is that it really takes a lot of different strategies to get at these complex questions of understanding how gene regulation occurs inside of a bacterial cell. So with that, I have a ton of people I have to thank. So, silence does not happen, like on your own. You know how they say it takes a village to raise a kid? Like it takes a village to get the thesis. So, I need to first thank everybody in the Lambda Lab who have been amazing people to come in to work with every single day. There are many people in the lab now, many people who have been in the lab. Since I've been here, they've all been wonderful people to work with. I need to thank my committee who have met with me once a year and provided valuable suggestions for directions to go with my project. I just spent generally supportive. Every time I met with them, I felt like I can keep doing this. This is great. I have a few specific people I need to thank for project related things. Kaylee, one of my classmates has been in their reagents so I can do AFM. So, glad to have organic chemist friends. Kevin, who is here, was helpful in analyzing some chip-sync data that I didn't show you today. And then the voice lab next door have been invaluable resources for things that I didn't know how to do, like run a nice gel to resolve face-pair glue pitch vents on DNA. And so, Wilma and Albert were incredibly helpful for that. And I probably will share my experience with them. So, thank you. Okay. I also need to thank Kate and all of the biopian people who make sure that I check things on my list when I'm thinking about science and forgive to do any sort of paperwork. So, they're wonderful. And Kate, who came in with me the last one, a Class of 2012 one, we're here. We're here. Kate and I started at the same time. Okay. So, I wanted to share about Bob because you shared one story or more about me. This is one of my favorite pictures of Bob. And it's the shirt that Bob's wearing. And I feel like this embodies Bob's attitude about science which I have come to appreciate. Bob loves doing science. It's great. It's repassionate and I love that about you. And I remember in this conference we went to an inscription meeting in 2017 and we all like flew in and drove up to the meeting in the middle of nowhere and talked together. And then the first event is this cocktail hour. And so we're all there, like ready, socializing with people. And Bob shows up with this shirt on which I've never seen before. And I mean, I've seen Bob's shirts. I think I know most of his shirts. I have not seen this one before. I said, what is the shirt Bob is wearing? Have you seen this before? No, no. What is it? So I was like, walk over to Bob. Bob, what is this shirt? It's my party shirt. Because we were at a conference and it's party time. It's like a time to like, interact with our colleagues and socialize and just have fun and learn about what other people are doing in science. And I, no, he wore this all week. All week. That's a good question. Yeah, but then as soon as the conference was over, the party shirt came off because it was time to get back to work. And I think I will always remember that. All the conversations we've had about getting science done and doing it the right way have been invaluable. And I don't think that I would be the scientist I am today without you. So thank you very much Bob. Thank you. And thank you for living in the lab and doing all this stuff and trying things out and failing this reliever in over years of things. Okay, so Bob has been great. I also have to thank the HMS support group as I call them. So just like HMS modern words have to interact together to silence genius. There's many people that have been involved in getting this project to work. So I have to thank the alumni of the project, Matt Halage, who I don't think I would have a PhD without him because he put together this Ambitur transcription assay which allowed me to work on stuff. And he taught me how to do things, how to work with HMS, how to do these complicated Ambitur assays. Daniel was an undergrad in the lab who started like one month before I did and he purified STPA, which took him like six months. So I felt bad for him but he did it and then I have to do experiments with him and stuff too. And then finally Eric who was the in vivo side of the HMS group and his cat Linus who was just as supportive to him as he was to me. So these people have been invaluable for getting my project to double in the lab. I also have to thank our group at Ida University. Ravis invited me over to do experiments with Ramon for our student in the lab. I took this screenshot from the video we took about doing some experiments because they were complicated and we thought it would be fun to take a video. But Ramon took two weeks out of his basic life to show me how to do some experiments also I didn't have time to show you those. But it was really great and I appreciated the guidance that Ramon has provided. Ramon has worked with HMS for years and I think without him I would not have been able to purify my HMS units for my experiments I showed you. And then Andrew who is a grad student in the lab now also helped do some experiments for my paper. Okay I have to thank some recent additions to the HMS support group Christine who is going to take over and she's going to do great. And then Daniel who did the simulations for me. He became an honorary member of the HMS group. And then I also had a bunch of people did in for bioinformatics help because my project morphed into a bioinformatics project like in the last year. And I was like what's going on? I was like Jess and Kaylee and Kevin you guys have all been so helpful and patient with me as I'm like how do I take this command in two minutes? So it's great and help me think about the right options to be asking. I also would not have made it without you guys. Thank you. Okay so those were the HMS people but the lab has been amazing. I pulled out this picture. This is from 2012. This is my third day in the lab. And he was taking the photo for the holiday car. And also in row he started like you know two days after I did it or before me or something. But these people have been invaluable support. They've listened to me. Talked about things that didn't work. They cheered with me. When my people Jess they finally worked and they have just been the best people and become some of my dearest friends and I am grateful to have been around so many wonderful people. I also need to thank my classmates who have been through this whole process with me from our awkward like we finally don't really know each other at the zoo in our week one to be really close friends and people I felt like I could call anytime if I had questions about anything related to science or life in general. So they have been wonderful people and I'm grateful to know them. I also have to thank a bunch of my friends. Yeah. These friends have been wonderful people. I would not have been able to make it through the many years I've been out just like moral support and like you can do it. And some of those people have been and some of them have been outside of the lab but they helped me have fun when I am stressed and also been there to celebrate with me. And then a lot of weddings. There were a lot of weddings. I also have to thank my family, my parents. You guys were like I don't know what you're doing but have fun. So thank you for that and thank you for like bringing me to science stuff so at age 10 I can be like I want to be a scientist. Here I am. I also have to thank my new family, Mike and Love. Hi guys. They're watching them. They have been just as supportive of me even though they also don't know what I'm doing but I appreciate all of their support all the way from Florida. I'm my sister who understands grad school. She got a master's and has been cheering me on through all these things. Okay. I finally have to thank my husband who has listened to me the other side who has listened to me be excited and I probably would not have made it to grad school without you. So thank you. Okay. With that I'd like to take any questions that anybody has. So I assume from what it looked like in your diagram that that was the region of the anti-sense transcription from over within the bagel opera? Yes it was. Yeah. So that begs the question did you ever do it in the presence of a plume race and did it walk completely? I did not try that experiment. No but I think that would be a really interesting one. So other people have done some footprint take experiments on other plumes that are regulated by HMS and it's really not clear but there's maybe some changes in HMS binding to DNA or any plume races there but it also might not be any changes in HMS binding. So I think that's definitely a big question and maybe that's other people to actually give better way to get that back. I think we have a positive side of that. Which requires more. Yeah, absolutely. This is all... but the... can you do a work shift or not? Yeah. Can you do a work shift and determine whether or not you do some patterns? Most of great questions Kevin and I wish I had answers to them. So there's one. So about the HHA chip. One group looked at HHA chip where HHA was expressed from a plasmid. So it was over expressed and it seemed to divide in any HMS filament but in actuality there's not enough HHA for it to find in all of the HMS filaments. So it's still unclear how HHA interacts with HMS and Vivo. And as to the bridge versus linear that is like the golden question and I think that it's hard to tell from just the chip experiments if it is a bridged or linear filament. I think some other kind of like chip plus something else or like the high C types experiments, people have thought maybe those could enlighten whether they're bridged or linear but I think it's... nobody's done that yet and it's I think a source of much debate as what filament matters like there's a linear camp and a bridged camp and everybody's like fighting about it and if we can identify like this bridged and linear filaments in the cell, things maybe could be resolved. I think the question is a little bit more conceptual about the horizontal gene transfer and so you said that all of this HMS binding is sort of to science the genes that's picking up in case they're dangerous. So what does this cell do? Do you want to decide to keep something or not keep something and like how does it get rid of the HMS? Is it just a matter of time or... Yeah, so there's a variety of ways that cells have evolved to base a deep across HMS silence genes and I think some of those have just like evolved over time where okay this gene doesn't seem to be detrimental from a basal level expression but there's a bunch of factors called counter silencers that can displace these HMS filaments or some elongation factors can aid, we think, transfer came through the filaments. So I think there's a few ways to evolve based on whatever the gene is. So this one is more when you were looking at the big locker it seemed like there were two HMS binding sites. The gene both formed one filament or the other, did you try eliminating one seeing if you had a preferential formation of certain filaments? So I did not do the HMS directly looking at that but Karen Schnitz, her group looked at these two binding sites to determine which one was basically required for gene silencing and I think both of them are required to form some filament. And me though, we don't know which one it is but I think it was, if both of them were there it's like more gene silencing is more repressed than if you just had one or the other. So yeah, we don't know what filament is actually forming on them. So if there are no more questions let's take Beth again for a very nice seminar event.