And we will get started. So as humans, we tend to think about life from the perspective of individuals. My great grandmother had 14 kids and raised them in Wyoming. My grandmother was very playful and a great comfort to have around when the Easter egg dying goes wrong. My mother is both an engineer and the artist that drew this fractal. So clearly, we should keep thinking about life from the perspective of individuals and the relationships that we share. But I'm here today to tell a different story about life from the perspective that I learned during my PhD research. And that is that we're all bound together by germline stem cell reproduction. So female humans with two X chromosomes are born with all of their eggs. The egg cell that eventually became me once shared the same space with my mom and my grandma. And egg cells and sperm cells are the germ cells and germline stem cells are responsible for creating and maintaining egg and sperm cell production. So in one sense, all that love we share between individuals is a result or even like a byproduct of germ cells perpetuating themselves over eternity. And broadly in the Kimba lab, we study the molecular mechanisms behind one piece of this endless cycle. We don't study humans in the lab though. We study fundamental biology using a tiny nematode or non-segmented roundworm called C. elegans. We also don't specifically ask about this cycle as a whole or what individual worms think of their moms and grandmas. But the piece that we study is the regulation of the germline stem cells in the adult tissue. But I wanna spend time on the cycle as a whole because I want to emphasize the importance of germline stem cells. Life couldn't exist without them and their importance is reflected in the fact that we can see germline stem cell regulation at every step of the cycle. So we're all fairly familiar with the part where sperm meets egg to create a fertilized embryo but germline stem cells need to be regulated properly so that the animals have enough healthy germ cells when they need them. Even single cell embryos are already starting to organize the next generation's germline stem cells. And then as you'll see later, this process can also break going from the juvenile to the adult because these four germline cells that the animal starts with need to be able to grow and develop into the full adult tissue. So here we are again at the Kimballab's main time point of interest, the adult germline stem cells. Blowing up this tissue, there's a pool of stem cells at the very tip of the tissue. And I've mentioned that germline stem cells are responsible for creating and maintaining germ cells and that happens as a result of cell division. So when stem cells divide, they make daughter cells, make more daughter stem cells in a process known as self-renewal or they can make daughter cells that will develop into germ cells. So if stem cells only or mostly self-renew, you'll have a tumorous tissue. If on the other hand, the dividing stem cells only divide to make daughter cells that will become germ cells, the result is a puny tissue that is also dysfunctional because it can't produce enough healthy germ cells when the animal needs them. So how does the animal pull this off? The short answer is niche signaling, but I'm going to step back in time for a minute to give you some context of what signaling is. So the most famous embryology experiment was done in 1924 by Hilda Mangold and Hansh Feynman. The picture I'm showing is the same experiment repeated in 2004 and using embryos from a different amphibian than the original experiment. But back in 1924, embryologists had microscopes and they could watch embryos divide and got very clever about the ways that they cut them up and observed the results. So in the Spayman Mangold experiment, a specific clump of cells was taken from the top of one embryo and grafted onto the bottom of a second embryo. And that results in this twin head being formed with all the right organs inside. And this experiment won the 1935 Nobel Prize for establishing that cells induce fates like head versus tail and cells tell each other what to do and what to become. And over the next several decades, the question was how? How are cells talking to each other? And the common theme that arose from those publications was that there was some cell to cell signaling. So signaling means that there are two cells, two cells involved in inducing a yellow fate. One cell releases a call and the other responds to the call. So now we're in the 1970s and signaling is emerging as a developmental driving force for all kinds of fates and vertebrates. But it wasn't clear that this was happening in our invertebrate nematode C. elegans. And something that makes worms very powerful to study is called an invariant lineage. So if we start from the single cell fertilized embryo, it will divide into two and then four and eight. This is the invariant lineage map because every time the single cell embryo will divide to make one cell on the white side, one cell on the yellow side, and you can trace the lineage from cell to its daughter and so on. And eventually you'll reach a terminus, which is a specific cell in the adult animal. And this is a very famous diagram because it's both awesome and useful to know that from the single cell, worms will invariantly have 959 somatic cells that follow these lineages. And my mentor, Judith Kimball, mapped out this chunk of the lineage as her PhD project. Then she went to do a postdoc in a place fondly known as Worm Mecca. And there in the coffee room, she put together her work with Rob Horvitz and John Silston's work, and it was the person that actually drew out this diagram. So some of the thinkers in Worm Mecca, also known as MRC Cambridge, thought that maybe worms were not like vertebrates. Maybe the neighboring cells were not important for directing the cell fates, and cell fate was instead determined by the cells' ancestors. And Judith wanted an answer to that question, and she was working with a scientist who developed a laser microbeam that can obliterate specific cells. So in Judith's own words, the laser beam engineer went on sabbatical and left her to play with the lasers. And as I mentioned, this chunk of the lineage, she knew like the back of her hand, so she went for this cell. And when she killed that cell with the laser microbeam, the stem cells were lost. The stem cells, by the way, are not included in this diagram, but they come from these two up here. So she proved that neighboring cells are important in the worm, and she also established that this cell, this red one at the tip of the gonad, forms a niche that keeps stem cells self-renewing. This niche cell produces a call, and the germline stem cells respond to that call. So stem cell niches were only theoretical until Judith's work demonstrated that stem cells rely on niches to support their function. And the specific signaling that comes from the niche is called notch signaling, which is one of the 11 major signaling pathways that shows up all over animal development, invertebrates and vertebrates alike. The call is a notch ligand, these little red dots, which is received by the notch receptor. Part of this notch receptor goes into the nucleus and is involved in the response, which is turning on the notch target genes. So the last piece to know is turn on which genes. There are just two direct notch target genes that support germline stem cell self-renewal called SIGL-1 and LST-1. SIGL-1 and LST-1 are functionally redundant, meaning that they both do the same job of supporting the stem cell self-renewal, and they both need to be removed to lose the stem cells in the same way that you lose stem cells when you laser beam the niche cell. I studied just SIGL-1, so I'm going to go through the next slide, only mentioning SIGL-1, but the same is true for LST-1. So back to this diagram illustrating the consequences of too much or too little stem cell self-renewal. Now we know that notch signaling from the niche is important for keeping this balanced. Notch is the call. Our lab and others have seen that too much notch gives us this result and no notch gives us this result. The response is turning on the SIGL-1 gene, and turning on SIGL-1 too much gives us this result. Having no SIGL-1 at all gives us this result. So SIGL-1 is a potent stem cell regulator, mystery solved, right? But if SIGL-1 is the response to notch signaling, how is the response controlled so that SIGL-1 is at the right abundance? The gap that my project fills is just shifting from identifying the molecules to understanding what combinations and quantities will support biological function. So in the early 20th century, the groundbreaking research was observing that cells tell each other what to do and what to become, and over the next 100 years, we've come a long way in uncovering what molecules are driving these processes. But it's important to remember that different biologies and different traits of different animals are not so much due to having more genes. Worms and humans have approximately the same number of protein coding genes. It's about the different gene regulation. So biology happens because of the complexities of not just what molecules are expressed, but with what timing and where and with what other molecules. And so understanding all of these complexities of the gene regulation will really make a difference as the research community is trying to understand health versus disease and trying to optimize therapies. So my project dives into gene regulation at multiple levels. Often, as is the case with SIGL-1, the protein is the molecule that actually carries out the cellular function. Proteins are made from RNA and RNA is transcribed from DNA. And now you'll notice that this DNA helix is here both at the beginning of gene regulation and involved in turning on SIGL-1. So my project investigates the DNA that regulates SIGL-1. DNA is the molecule that contains their genetic information. And these rungs on the helix represent different faces, each represented by a different letter. So an arrangement of these eight faces forms a binding site, which is a landing pad for proteins to bind to the DNA. These SIGL-1 binding sites are lag-1 binding sites specifically because they bind the lag-1 protein that helps turn on the SIGL-1 gene. I'll be calling them LBS for lag-1 binding sites. And the wild type version of the SIGL-1 gene found in nature, like what you'd find if you went to your compound, your compost bin has six total LBS, two clusters of three, one cluster for each chromosome. I used CRISPR-Cas9 gene editing to create mutant LBS based on several studies from test tubes that evaluated which bases disrupted lag-1 binding most. So for the rest of the talk, I'll be zooming out from the DNA helix and indicating the DNA as a line, the LBS as arrows that indicate which way the binding site is facing. And I'll be representing these mutant LBS with Xs. I then created every combination of wild type and mutant LBSs and tested them. And I'm gonna recap that all quickly using this inverted pyramid, which is a science communication tool I learned during my time. It's inverted because the broadest questions we need to drill down and ask more specific questions to actually learn something about. We started here, the germline stem cell regulation isn't just crucial for health or disease of individuals, but on a species level, this chain of events has to remain unbroken for eternity or the species will die out. A proper balance of the germline stem cell pool is crucial for any type of stem cell and also impacts individuals' health versus disease. The C. elegans germline stem cell pool is balanced by niche signaling. The notch pathway turns on the SIGL-1 gene. SIGL-1 is a potent stem cell regulator, so its abundance is one very important factor for balancing stem cell pool size. And one way that SIGL-1 abundance is regulated is by the DNA that helps turn on SIGL-1 transcription. The SIGL-1 regulatory DNA I'm showing are these clusters of lagoon binding sites for LBSs. And then I can test several different things about the LBSs that control SIGL-1 production. I decided to go for number and I made these mutants that have four or two or zero LBSs. So thanks to the pioneering work of the worm community over the decades, I started my project knowing quite a bit about the relevant molecular players in this biology. And the next challenge after we identify the components is to understand how biological function emerges from their collective when, where, and who of how they're being expressed. So how did I actually investigate this? This is a picture of the worm under the microscope. The gonad is outlined and we can also dissect the gonad out of the worm. And I did that and treated it to get snapshots of several different stages of the regulation of gene expression. This is just a preview of what the images from different treatments look like. So I used one assay that Judith mentioned, the SM fish to look at nascent transcripts that SIGL-1 RNA coming off of the DNA. In the same assay, I could look at SIGL-1 mRNA and I used a different assay to look at SIGL-1 protein. One major advantage of my project is that I can investigate these molecules in their native developmental context with spatial resolution. The second advantage is that I'm not limited to looking at the molecular response to my manipulation of SIGL-1 DNA. I could also assess how well the manipulated animals were carrying out their biological function. So I'll take you first through the molecular part and then the biological part. I previewed these different stages of gene expression and now I'll show you results starting with this group of mutants that have one LBS mutated per cluster. I studied the molecular response in animals that still have LST-1, the redundant partner of SIGL-1. So I can look at the quantity of SIGL-1 and know that I'll still have a functioning germline tissue to look at. I'm showing you three different mutants here and only one picture. This is because there are so many more instances of active transcription in the wild type. There are an average 24 spots per gonad up here and only six for the mutant.  So this is the first clue that we're hampering an intermittent process is that we see so many fewer spots. And because we have so many fewer, we pooled the three different mutants together to have a bigger data set to analyze. In an average gonad, we caught one of those nascent transcript spots in a little bit more than half the total nuclei that are very close to the tip of that tissue compared to only about 15% in the mutants. And those green nascent transcription sites were on average a little bit dimmer than the wild type ones, meaning that less RNA is being transcribed. So we concluded that mutating these LBSs weakens the notch dependent turning on of the SIGA-1 gene, which is what we expected, especially thanks to research done by Chung-Hwan who studied SIGA-1 transcription in live mutants. And so also caught this process on video. And in that same SM Fish assay, I could look at the SIGA-1 mRNA out in the cell. In these images, you can see still those bright, oh, that's not the laser. You can see these bright active transcription sites still, but you also see this population of dimmer purplish mRNA. So we can see individual molecules out here. There's a lot more mRNA data than there is nascent transcript data. But even after the individual mutant analyses, we saw that each of these three mutants were all fairly similar. So I'm again only going to show you one picture and one graph. And the takeaway from our mRNA quantitation is that all of these mutants have slightly less than half the mRNA abundance at the tip compared to wild type. So my SM Fish assay, let me count the mRNA molecules. And for this talk, I just set, like this is not one mRNA, I just set the wild type to an arbitrary one so that we could quickly see that it's half. We also see that it extends a little bit less far into the tissue. And finally, we looked at protein, a reminder that SIGA-1 protein is the molecule helping carry out the job of stem cell self-renewal. You can appreciate from the image that the wild type is brighter by eye. And when we quantitate the pixel intensity of those images, we can see that the protein is a little bit less than half as bright as wild type. And again, it doesn't extend as far into the tissue. So to summarize, when one site per cluster was mutated, leaving four of the original six, the remaining clusters are a little bit less than half as efficient at turning on SIGA-1 gene expression. But it's slightly less than half efficient, good enough to carry out the biology. I mentioned that we left in SIGA-1 to look at the molecular response, but LST-1 also masks any effect that SIGA-1 has on stem cell self-renewal since they do the same job. So we removed LST-1 and I asked if the SIGA-1 response could keep the stem cells self-renewing into adulthood. I used three different assays to estimate the size of the stem cell pool and they all agreed with one another. I'll show you data from just one of the assays. In our field, we have an established proxy for estimating size of the stem cell pool. And each of these three mutants, I've again made this one and each of these three mutants have about half the size of the stem cell pool. So first group was about half second group. What about them? I won't take you through the whole gene expression path again, but this group as a whole had about zero or near zero molecular response. But I again removed LST-1 and asked, can a near zero molecular response maintain germ-like stem cell self-renewal? No. And I thought that would be the end of the story, but it turns out that this group of mutants had more to teach us. It turned out that because they were all so close to zero, they could tell us about marginal differences in between individual binding sites. So consistently across multiple assays, this one mutant in the middle expressed no mRNA or protein and the other two produced detectable mRNA and protein. And when I say detectable, I mean just detectable. I'm showing you SIGI-1 protein and this is a detectable mutant. This one's undetectable. The detectability is the difference between that dashed line and the orange line. So how do we know if this is real? We looked into biological output. So this is a newly hatched larval worm. It has four cells in its germline tissue and as it grows, these cells will divide and develop the adult tissue. We know that in larvae lacking both SIGI-1 and its redundant partner, LST-1, these yellow cells can't self-renew to build a stem cell pool. So this is work done with a former post-bacmante caza. Caza looked in older larvae with the idea that if the LBSs are driving production of even a puny quantity of SIGI-1, the puny quantity of the SIGI-1 in adults, if those LBSs really do have some activity, then maybe their two yellow cells can divide just a few more times and the end result will be more total germ cells in the mutants that do have some detectable molecular response. Caza found that in older larvae of the mutants with detectable SIGI-1, they made it five times as many germ cells as this mutant with no detectable SIGI-1 and that mutant made the same number of germ cells as our control that doesn't have the SIGI-1 gene at all. And I am careful not to say that the number of molecules we observe in adults is what's driving X number of divisions. We need to measure the molecular response in larvae to make those kinds of conclusions, but the important part here is the distinction. Consistently, this arrangement and only this arrangement of LBSs is like having no SIGI-1 at all. So now at the end, I've cut off a lot of worm heads to extrude a lot of gonads. I've spent what feels like years of my life taking images on the microscope and then quantifying the image data to understand the molecular response. I could also link my DNA manipulation and the resultant molecular response directly to the biological outcome, which was the ability of stem cells at the tip of the tissue to sell for new. So to summarize, one big conclusion is that the number of LBSs is a major factor for tuning the SIGI-1 response to the notch signaling call. Wild type and this group with one LBS mutated, they both produce a healthy functioning germline. The mutant LBS clusters drive only about half the molecular response and half the stem cell pool. The group with two LBSs mutated per cluster produce next to nothing, but we also went and verified that a single LBS on one chromosome could produce. So we still wanted to see if we could get a quantity in between half and practically nothing. And this was the project of my former undergraduate mentee, Min Yu. Min Yu did a lot of work to look at this kind of mutant. Like the two LBSs per cluster mutant, it has two total intact LBSs, but they're both on the same chromosome, only nine base pairs apart, whereas these two are separated onto different chromosomes. Min Yu measured SIGI-1 protein. She saw that four LBSs produced about two thirds of the SIGI-1 abundance of animals with six LBSs. Animals with two LBSs produced about one third of animals with six LBSs. So this is really fun data because on one hand, the SIGI-1 is produced really straightforward, forwardly and additively, two LBSs produce half as much as four, and this mutant with two thirds the LBS number produced about two thirds of SIGI-1. However, this experiment also shows us the LBS regulation of SIGI-1 is not only based on number. As I mentioned, this mutant with the two LBSs together, only nine base pairs apart, they give us a decent quantity of SIGI-1 protein that can maintain a functioning germline in adults. But two LBSs on the separate chromosomes, especially if they're these two LBSs in particular, are like having no SIGI-1 at all. So from this, we conclude it's not just LBS number, there's also a greater ability to turn on the SIGI-1 gene if these binding sites are close together. And a big general question in science is how the numbers and sequences and arrangements of binding sites in the DNA is contributing to getting the right quantity of the right molecules in the right place at the right time to drive healthy biology. Scientists have learned a lot of the effects of different DNA sequence from test tubes or cells in dishes or putting trans genes into animals. But there's real strength in testing these kinds of questions in the most authentic context possible. So I mutated the endogenous DNA in its natural context and observed the results all the way from nascent transcription to protein and the biological output. We learned that these LBSs regulate SIGI-1 using a combination of number and proximity to each other. And we also have a better idea of what minimum quantity of SIGI-1 might be required to carry out its function. We can think about our findings not just for transcription of SIGI-1 in worms, but for not directed transcription more broadly. Because the group of single mutants were all approximately the same as each other, despite some differences in the distance between the sites or their orientations relative to one another, those differences in spacing and polarity make it the most small changes in the ability to turn on the SIGI-1 gene. However, that group of double mutants also showed us that our assays are sensitive enough that we can use this kind of exploration to make a distinction between some and none and learn about some of the things that do make a difference for small changes in gene expression. So in my work, I investigated LBS number, further explorations could investigate spacing or polarity or different mutant sequences. We also saw that DNA manipulation changes the level of SIGI-1 protein. And we can think about how different SIGI-1 protein abundances interact with other critical stem cell regulators first and foremost LST-1, but also with the other critical regulators that we already know some about in this well-established system. So we can use these mutants that I made to help us learn more about molecular germline stem cell regulation in C. elegans. And finally, I mentioned that my group of LBS single mutants maintains a half size stem cell pool. That half pint pool was functional both for self renewing stem cells all the way from juveniles to adulthood and for successfully producing offspring. However, I regret to report that despite the length of my PhD, I was unable to test whether these are sufficient to keep going for eternity. But another thing to mention is that I'm studying worms in the lab environment where they're literally swimming through a pile of food and worms in the wild live in boom and bust population cycles between sources of food. So another direction to take these mutants and help think about those questions would be to stress test them with age or food source or over many generations. And like with all worm research, we don't just learn about worms. Worms are a model organism in which we can learn about development more broadly. And I wanna bring up again, worms aren't apparently less complex than humans because they have fewer genes. We have about the same number of protein coding genes. And it's all about, oh, and I also wanna mention some of those genes are similar or have similar function. So just like Judith showed with her lasers, all animals share a lot of fundamental biology even if they don't have a backbone or eyeballs or brain. So I hope I've convinced you that taxpayer dollars are well spent on worm gonads. But I'll leave you with this puzzle activity that I made. Model organism puzzles are more tractable to put together like because of their color pattern. So my rainbow stripes were things like the fact that I can see directly into the stem cell compartment. I can manipulate the DNA really easily and we have a really well-defined stem cell system. Then once we piece the puzzle together, we get clues about how to assemble this more complicated puzzle. And the clue here is that the pieces in each row have the same shape and these puzzles are cut identically. So once we get this thing, we can put together this puzzle more easily. I thought you'd like that Camille. So while at times, I've definitely felt the shock or dismay of this scientist thinking that I've devoted so many years of my life to worm gonads. I'll always think that worms are small but mighty. And this part too was drawn by a former UW graduate student who's now a professor in California. I saw him speak and he said that he was a student and he said that this cartoon is indeed based on the Kimball Lab. And you can tell because of the red buildings out the window over the way our benches look. But I will say that we label our pipette tip jars with a P and also unfortunately we can't have plants in the lab because we like to minimize the risk that worm eating mites will wreak havoc on our experiments. So one reason that I'm not yet bored of worm gonads is that I came to a place where I'm surrounded by people passionate about science. I chose Wisconsin because people do rigorous cutting edge research and care a lot about graduate student training and collegiality. So thank you to my committee and especially Judith for your high standards, but also making it clear that you're building me up. Thank you to group members both past and present for your mentorship and your expertise and your friendship. Thank you especially to the people who contributed to the work I showed today, my co-authors on my manuscript. And so without Chung Won, my MATLAB analysis would not be possible at all both while we were Bay Bates and once he moved to Albany. I gave Kazza that larval germ cell counting assay that I had never done and she took it, got it working just fine. It was also delightful to have an edgy engineer in our lab and she's now doing awesome stem cell engineering work at Medical College of Wisconsin. And then Minyu very bravely and competently took on a huge project. I showed you just a tiny amount of the work that she did and she's now off being brave and competent at Imperial College London in a biotechnology masters. I also want to acknowledge support from the broader iPad community. I mentioned collegiality and thanks to the work of many, I'm standing here today and literally standing in this room because Katie Henson Wildman moved to her lab meeting so I could be here when there was a problem with the other auditorium. Rick Amesino was on standby to ensure that I had enough committee members to actually defend today. And I also would have been gone a long time ago without the administrative and media and IT and building support. And I'm not just thanking you people because you've done your jobs well but it makes a difference that everyone is so competent and compassionate and really a pleasure to get to know. And speaking of people who are instrumental to me being here today Brian and Afon spent hours with me on Tuesday working out the kinks and helping me prepare and thank you Brian for running the zoom right now and I can't look anyone in the eye. And I think grad students in general share a really special kind of camaraderie because we are there for each other for every bump and milestone of which there are many. In fact, we're so in sync we tend to match outfits a lot. But it's not just the graduate students that make this place so supportive. One of the things that I want to thank Judith for the most is assembling such a great team of people to work with and also keeping us socially connected even during the pandemic. Judith cares a lot about workplace culture and that's really important. So thank you for the mentoring subgroups that we did where we worked through entering mentoring case studies as a team. Thank you for the lab meeting where we read strong inference and talked about science more broadly. Our Kimble Lab logo is to find your niche. And yeah, this is the niche self that Judith ablated with her lasers. And that's what it looks like it wraps up the cells and it keeps them supported and functioning. And the Kimble Lab has been such a fantastic niche and I'm really glad that I get to stay for just a few months longer. But the Kimble Lab wasn't my first niche. This analogy starts to break down a little bit because I'd be a weird stem cell but I lived in so many niches. But I'm not sure I would have gone for a PhD if my undergraduate lab wasn't such a family to me. Thanks especially to Gina for your mentorship. Thank you for teaching me how to pay attention to detail while doing bench work to figure out the why of the protocol to stand up for myself and my ideas. I spent an awesome summer doing research and goofing around with my American friends in Dresden. Thank you, Nicole, for opening up such a cool project to me for finding me a place to live in a place where I only know words like blubbery and squirrel. And thank you for hosting me at your home in Leipzig. And I also found a niche at the UW Writing Center. So I've been meeting up with these four women every week to set goals and make writing progress for nine months now. And I will see you all knock this up coming Monday but the next one. And not pictured here other writing mentors, Angie and Chrissy and Nicole and Ending and Vanessa Union and all the rest in our Slack group. And now we've arrived at the people and activities that kept me sane all these years. Thanks for the memories and the lasting friendships. Joining the discursators of the Frisbee team was something I did on a whim because I thought Frisbee was an approachably easy sport. It's chill, but I quickly learned that it is a regular difficult sport. Instead of quitting, I immersed myself because I really liked the people and how playing Frisbee is an essential part of my life. So thank you, Tots. Tots for making me feel welcome even when I couldn't play and even though I still run the wrong way, at least once a season. Not even joking. Thanks to my friends for spending quality time with me on holidays, out dancing or going to shows, cooking food, riding bikes, creating art, watching TV shows. And thanks to my original niche, my family. Thanks to my parents, which includes both my biological mom and dad and also my second parents, Mike and Camilla. Thanks for unquestioningly making it happen to be here today. And thanks to my brother, Glenn, and my sister-in-law, Jessica. I'm sorry the schedules couldn't work out, but thank you for offering to drive through the night until I told you that was crazy. Don't do it. I see you now. And thanks for visiting me through the years.  I couldn't be here without his logistical support. I couldn't be here without his encouragement to take time for myself.  And one last story. I mentioned my great-grandmother had 14 kids. This is only some of us. And we make t-shirts. So I want to zoom in on the t-shirt design that my parents designed when we hosted the reunion in Estes Park. I have lots of lineage diagrams in my life. And worm scientists love this diagram.  So thanks for the nerd gene family. It's really helped me fit in with the friendly and collaborative group of nerds that is the C. elegans community. And that is all. Thank you. Thank you. I have a question. Anyone who doesn't know this, no graduate students have a personal responsibility for their work. I'm curious. Is every LVF specific to how well you serve tomorrow? Yes. All three of the ones that I mutated are the same sequence. I'm not sure. I'm just going to say that they are very canonical. Some of the bases tend to be a little more degenerate than others. But we know the canonical sequence, which is why I knew to go mutate those is because they're very well conserved and they were computationally probably there. And Erin mutated them in a reporter. Yeah. Yeah, let's see, I need to start this again. And then this guy. Yeah, there is. It's using truncated peptides of the proteins, but there is a crystal structure several. Yes, there's actually a whole story that we can talk about later. Yes, so NICD. So one of the things about notch signaling in general is that this complex gets degraded fairly quickly. And this domain at the end of the notch receptor is intercooled to that degradation. And just all throughout the notch receptor, many different mutations cause disease. I'm not so sure about specific disease causing ones on the other two, but probably. I have questions from Lauren. Awesome. Scott says good job, Tina. Thanks, Scott. What do we know about LBS member conservation across metazo and notch signaling? Always greater than one, always three. So I don't think there's an always. It's often in clusters. I looked across all of the C-interab diodes, and there's always at least two in the SIGA1 gene. But there are, there's so many different ways that notch is expressed that like what we're thinking about, sometimes they're in clusters to help it respond faster. And so I think sometimes there's just a couple or they're really integrated with other transcription factor binding sites. So I think there, yeah, there's no one general rule for the LBSs. What do you think is special about the one LBS that's capable of increasing a small amount of SIGA1 transcript relative to the other two? Yeah, I'll go back to that diagram because I think about that. And the most the sequence is the same. So the number one thing that pops out about what might be different is the polarity. It's the only, this one is the one that's not producing anything. And it's the only one facing forward. So like maybe because we know something about the crystal structures and the proteins in the complex, and that one lag three has a really long terminus that interacts with other transcription initiating factors. So maybe there's something about, even if there's no other sites to interact with, it's interacting with something that's helping turn on transcription. I'm not sure. Yeah, as I thought, and what I've known is that some genes overlap and I'm talking to people about that maybe happening now. TBA, yeah, yeah. Or like we don't even know about the sequence next to it. Well, that's the same. And that like that wouldn't change double mutant to double mutant. But or I'm trying to think if it there's anything that matters that SIGA1's in an operon or no. I think you have to exclude that possibility. Yeah, unlike the genes that's really well mapped on the coming genomes. Right, exactly. And in worms at least, the elements of your gene closer to your genes than humans, they can be down in those where the genome is still out of effect. But in worms, they can be closer. But we never know. We never know. We can't exclude it. The other questions from Roger and Anonymous were David Watson's question as well. Thanks, Al.


Before looking at specific teaching ideas for the present simple tense let's first look at some considerations for teaching ideas in general. We have to remember that these activities will be used in the activate stage of your lesson. Here the focus will be on building fluency and using the language in a bit more of a free-flowing way yet still focusing on that class's language point. We also have to consider the appropriacy for a number of factors, timings, whether the activity will be too short or too long. We want to look at ages making sure that the activity is appropriate for young learners or for adult learners. We also have to take into consideration our language levels. Will the activity be appropriate for our starters or pre-intermediates or our intermediates? Will the activity be appropriate for a small class or a large class? And last but not least certainly we have to take into consideration our students culture and their interests making sure that the activity is appropriate for either one. And finally we need to make sure that we're maximizing our student talk time. Are the students using the language as much as they possibly can while they're still in the class? Remember that the more appropriate the activity will be the more student talk time we'll get. Our first example of a teaching idea for the present simple tense will be a classic activity called find someone who. Here we'll be trying to find someone who has a general truth such as the ability to play a musical instrument or the fact that they have a brother and a sister. This results in students going around the room asking questions in the present simple tense such as do you play a musical instrument? Are you a teacher? Do you have a brother and a sister? The answers resulting will also be in the present simple tense. They will be generally yes I do or no I don't. The students will go around trying to find somebody who fits the general truth and when they do they will write their name in the appropriate space. The students will mill around the room for generally five to eight minutes depending on your timing of your class. When this teacher feels it's appropriate he'll have the students sit down and begin feedback. Feedback will also be in the present simple tense resulting in questions such as who goes to bed before 11 o'clock? Students will raise their hand and also report in the same tense saying the name of a student in the class such as Jim goes to bed before 11 o'clock. During the feedback the teacher can also expand upon these general truths. When the teacher hears that Jim goes to bed before 11 o'clock the teacher can ask why do you go to bed before 11 o'clock? Do you always go to bed before 11 o'clock? The same can be done for musical instruments. What musical instrument do you play?


Hello, in this lecture we're going to talk about the objectives of the closing process. The closing process will happen after the financial statements have been created, so we will have done the journal entries, we will have compiled those journal entries into a trial balance, and then we will have made the financial statements. And then as of the end of the period, in this case we're going to say as of December, when we move into the next time period, January, what we need to do is close out some of the temporary accounts, those accounts including the income statement and the draws account, so that we can start the new period from start in a similar way as if we were trying to see how many miles we could drive, say in a month. If we wanted to then end December and then see how many miles we're going to drive in January of next year, then we would have to reset the odometer. That's what we're doing here, we're basically resetting the odometer, the odometer in the case of the financial statements being the income statement accounts, the draws accounts, those temporary accounts. So if we look at the adjusted trial balance as of the end of the time period, end of the time period is December 31st, in this case, we see that the accounts up top, the assets, the liabilities, and the capital accounts, those are accounts that are going to be permanent accounts. And then everything from draws, the revenue, and the expenses, those are going to be the temporary accounts. These accounts down here from draws, revenue, and expenses, those are the ones that we need to make zero. So as of January, we need to make those zero, we need to reset the odometer on those accounts. We can also think of that in terms of the balance sheet. Remember, the balance sheet is as of a point in time, meaning those are the permanent accounts. So we can see all the assets from the trial balance are listed here on the balance sheet, as well as the liabilities. Then in the equity section, we only have this one account called owner's capital. It looks like this owner's capital over here, but it's not the same number. Why? Because this owner's capital represents the amount as of a point in time. This owner's capital represents actually only the beginning balance of the capital account, not including the timing stuff, which would include draws and revenue and expenses. Therefore, in essence, what we're saying is all these blue accounts, that's what that number is that 742 800 is including all these numbers. That's where we're at as of the point in time, combining all those numbers together. And everything that is from draws revenue expenses down our temporary that need to be closed out for the temporary accounts and are already part of the ending balance sheet account. We can also see that in terms of the financial statements. So we can see that the net income on the trial balance is the credit of the revenue minus the expenses, the 88 980, that's represented on the income statement. This income statement is a temporary account. That's like our odometer type account that we're going to reset to zero. The entire income statement as of January 1st, the first day of next month will be zero. There won't be anything there because we haven't started just like when we start a new race and we're trying to see how many miles we're going to go. It's starting at zero as of the next time period. That 88 980 is also on the statement of equity. The statement of equity is in essence a temporary account as well, meaning we have the beginning capital. That's the permanent part of it that was from last time period, plus what has happened during this time period, meaning net income. And then we've got that draws the two temporary items. And of course, net income clumping together all the income statement accounts revenue and expenses. That's how we're going to get to our ending capital number. Once again, as of the point in time, that ending capital number is what will be on the balance sheet. Everything else is the temporary section. That's what we need to close out. Remember that 742 800 same number that we just talked about on the balance sheet. How are we going to do that? Well, on the left hand side, this is our adjusted trial bounce that we used to make the financial statements we need to make it look like on the right hand side like it does here on the right hand side. So this is the adjusted trial bounce has a 1231. This is what we have as of January 1 of next year. We can also think of it as the post closing trial bounce. So right after we closed everything out, we'd have a trial bounce that would look like this. Notice everything that's the same. All the assets are the same permanent accounts, all the liabilities are the same permanent accounts. And then the equities, the capital is a lot larger here on after we've closed it. And then it is over here. That's because of course we took all these numbers, we crunched them together and we put them over here. We can see that two different ways. We can calculate that two different ways. We can say how did we get to this 742 800? We can say, well, we're going to take the credit. I'm going to do it in plus and minus rather than debits and credits.  That will give us the amount of the 742 800. That's what we need in the capital count represented in the equity section after we close out everything else. In essence, we closed out all the temporary accounts and plugged all that numbers, all those numbers into the one number of owners capital. We can also calculate that this way. We can say, well, if that's the only number in the equity section and assets minus liabilities equals equity, we can say, hey, let's add up all the assets adding up all the assets here cash on down subtracting out the counter assets of the accumulated depreciation. Got to make sure to do that. And then we're going to subtract out all the liabilities. And that will then give us the same number that 742 800. So again, we could think of those in two different ways. And of course, that 742 800 we have seen before that's the number that was on our balance sheet as of the end of 1231. And of course, we also saw that number on the statement of equity, the ending number, being the point in time, the activity in the equity section being temporary accounts. The question then is, how are we going to get here? In terms of just logistics, the journal entries, what are the journal entries going to look like? How do we get from something that looks like this to something that looks like that? Well, we're going to do a four step process traditionally, we're going to say first, we're going to step one, close out income to the income summary, we'll have a journal entry to do that. Then step two, we're going to close out the expenses to an income summary, which is a new clearing account we'll talk about. Step three is going to be to close out the income summary to the capital account. And step four will be to close out draws to the capital account. In essence, what we're doing is making a journal entry, we could make one large journal entry basically closing out this trial balance to the capital account. However, it's traditional to do in this kind of four step process and we'll discuss why next time.


All right, so pop quiz Hank, do you know what this is? Good morning Hank, it's Tuesday. So I'm at the Indiana Medical History Museum in like the room where they would do the autopsies where all the students could watch because we're filming a new kind of crash course where we take a deep dive into one particular topic and you'll never guess what my one particular topic is unless you guess it's the oldest deadliest and in my opinion, least understood infectious disease in human history tuberculosis. Hank, you pointed out in your video announcing that you had cancer that lymphoma was originally believed to be a type of tuberculosis, which makes sense because they have some similar symptoms, right? Night sweats, fevers, sometimes you get coughing with both. Not as young as I used to be. This is an iron lung for infants who got polio. That's the little mattress. This is a very helpful thing for me to look at because it reminds me that progress is possible. Laboratories have come a long way in the last hundred years. They sure have. Oh, I didn't know that you would be here. Yeah, no, I'm always here. Right, I guess that is kind of the issue. Wherever I go, I bring myself with me. The other symptom that TB and lymphoma have in common, of course, is swollen lymph nodes. In the case of TB, when it invades the lymphatic system, it's called scrophula and it can actually cause the lymph nodes to become so swollen that they rupture through the skin.  I know. All right, let's go get a check of the weather. Hank, I know you're a microscopy enthusiast, so I just wanted to make sure you got to see this fella. People are always like, oh, we're the worst, and sure, but look how hard we've worked to get better. I'll let you in on a little secret. The 30-foot microphone cord never stops being funny to me. It's the funniest thing in the world. Is it gonna be enough? It's close, but I made it. By the way, I'm not gonna explain all these costume changes.  That's a nice day. Let's talk about swollen lymph nodes. So yours turned out to be lymphoma, which, of course, is not ideal. Almost all the time, when you have swollen lymph nodes, it's no big deal. You may not even feel sick. It's just your body kind of dealing with an infection, but in your particular case, not great, but people who swollen lymph nodes turn out to be TB also have a significant problem, of course, especially if it ruptures. And it wasn't that long ago, Hank, that at least in Europe, it was widely believed that the number one most reliable cure for scruffula was, wait for it, being touched by the king. It was called the king's touch. The king would like touch your head or else your wound, and then you would be magically cured because, of course, the king got healing powers through his direct apostolic line that could trace all the way back to Jesus. If you're ever in Indianapolis, you have to go to the Indiana Medical History Museum. It's just awesome. Being here reminds me that, yes, history is a nightmare from which I am trying to awake, as Stephen Detta was famously put it, but it's also much else. History can also be an encouragement. We no longer treat scruffula through the king's touch, which I would argue is good news on a few levels, both because we no longer think kings are that powerful and because we now have much better treatments. So, yeah, Hank, I'm not gonna lie, I'm super pissed off that you got cancer, but I am really grateful to the people who accumulated and shared and built upon each other's knowledge so that we could share a better and healthier world. 300 years from now, I hope we look back on a lot of things the way today we look back on the king's touch. Hank, I'll see you on Friday. Hopefully, but if you're not feeling good, that's fine, no worries.