Thank you very much for the invitation or for the opportunity. I'm a human geneticist and I got into comparative genomics because we reasoned that it might be a good idea to look at differences between humans and non-human apes and other primates in order to understand what makes us human. We failed. We don't know what makes us human. We find a lot of interesting things along the road and it's been quite a journey. As I said, what really motivated us at the beginning is to try and understand the basis for adaptation of complex phenotypes in humans and those include adaptations that ultimately make us more susceptible to diseases. But we didn't know how to do that and we were committed to try to do that by studying humans and our close, extant evolutionary relatives, not go to mice or other model systems and that of course makes everything challenging in some respect because of the ethical and practical considerations of accessing humans and non-human apes. Back in 2003, we started simply comparing gene expression levels between species with the hope of understanding combined with the comparisons at the sequence level, understanding of some perspective on how gene expression changed, regulatory change and perhaps ultimately connected to some form of adaptation of complex phenotypes and we failed. Around 2007, when we couldn't really push it through to the complex phenotypes, we decided to take a step back and since we have now such great maps of differences in gene expression, transcript expression between species and we understand something about the evolutionary forces that shape those transitive expression even though everything we say about complex phenotypes is just at that point storytelling, we can go back and study regulatory mechanisms and that has been work that is still ongoing and work that continue to evolve as new technology evolves and new protocols evolve and the ability to visualize and characterize new regulatory mechanisms evolve and it's work that opened up a whole I think window into studies of regulatory variation within population between humans, the entire EQTL field and regulatory QTL field and I think that for me for us the journey was very satisfying and that interplay between comparative genomics with large effect size where causalities out the window to within species where effects are smaller but you can make more inference about causality using some form of mediation analysis was really gratifying but really the first time we were finally able to take one step towards some measure of complex phenotypes was when we studied changes in protein expression, still everything that had to do with complex phenotypes itself remains just storytelling and then in 2011 where finally in 2013 we established it we started working on adaptation in cell culture now that's a far cry from adaptation in vivo or anything you can do with actual model species and in this conference we're clearly at a disadvantage where a lot of you are working on actual organisms and animals in a multiple species and can perform experiments we are committed to working on humans and chimpanzees and species that we don't want to really touch and so everything is still in vitro but it's using a system that we couldn't use before which is the IPSEs so this is a legacy slide which for sentimental reasons and some bet I'm never changing but the numbers are outdated we now have close to 20 validated chimpanzees IPSEs and actually close to 200 human IPSEs and a lot of them are done exactly in the same technique using the same technique and they're really highly validated and of high quality we validate them using a large number of assays including of course making sure that they're fully pluripotent making sure that the clones that we choose represent the particular individuals from which the clone was derived routinely when we wake them up we check their carotide and and those are fluid and so a lot of time we have to just terminate some clones and go back to clones that are healthy and have healthy carotide at the beginning of this journey we used to do teratoma assays and thankfully the field moved away from those as a critical checkpoint of course we do eb's for every cell and actually we continue to do these eb's routinely to just make sure that the lines continue we have a lot of early stage line but even the late stage lines are continued to be fully pluripotent as you may have heard I worry a lot about batch effects and so we have studied at great length and at great detail the potential batch effects that might impact work with stem cells what I'm showing you here specifically is the relative impact of starting the IPSC generation from individuals that are from different populations so different population of species of chimpanzee what what what some or maybe most cold subspecies different populations in humans and we showed that whether you study gene expression gene emethylation and I didn't put other slides here on histone modification accessibility and so on the obviously the species effect is much more pronounced than the effect of the different population but you still have the effect of different population visualized which is a good thing because it means that the stem cell reprogramming maintains the genetic identity of the donor and whatever variation you see actually between individuals an interesting observation that we confirmed and and now using a large number of tissues is that there's less regular variation in IPSCs than in practically any other tissue that was studied terminal cell types here specifically you see the difference between fibroblast LCLs and IPSCs and the bifurcation the trees on the two sides are the different species and and in the IPSCs you can see you can see in the IPSCs there are there's just much less variation than in any other cell type and that is interesting of course because everything that intuitively we immediately associate with early developmental stages canalization and I'll kind of get back to it when I demonstrate two utilities of these IPSCs we spent a lot of time really a fair amount of time in our lab validating differences or studying differences between differentiated cells from these IPSCs and primary tissues and I'm gonna take a minute to explain how important that is and to acknowledge that this well not perfect is something that we think about and and does show that this model is useful so the the issue here is that we are doing in vitro work and we are doing in vitro work because we are unable to do in vivo work and when you're a human geneticist the two competing models that you have are always in vitro cell lines or go to mice or other model system that are acceptable depending on the phenotype that you're studying and mice are not humans and in vitro is not in vivo and you never can never get out of those types of criticisms they're all valid but the fact is that you're not going to experiment on humans and you're not going to experiment on chimps and so we are choosing the in vitro system we are choosing to work though not on immortalized cell lines that are more standard but at all those differentiated cells from IPSCs and we study at great detail the similarities and differences between the differentiated cells and the primary tissues that they're supposed to model to understand to what extent those systems in vitro systems are faithful to what extent they're useful and we optimize different protocols in order to create cells that are closer to the primary tissue so in this particular example we going for cardiomyocytes and we're using different protocols to generate cardiomyocytes and we're testing the gene expression the methylation other phenotypes in those cardiomyocytes comparing them to the same data collected from primary hearts sometimes from primary hearts from the same animals from which we actually created the IPSCs because this is this has been a long process as samples that I and my lab have been collected for actually almost 20 years and some of these chimps passed away and then we were able to collect their hearts not anymore the NIH has a moratorium on collection but we have a lot of those tissues in our freezers so I'm not going to ask you to read all this this is this is a large slide that shows that essentially cardiomyocytes are most similar to hearts but there's also a take home message is which is that you'll never confuse cardiomyocytes with hearts cardiomyocytes form their own clusters they don't look like anything else other than hearts but they also don't exactly look like hearts but when you actually look closely to figure out what are those are going to be differences between cardiomyocytes and hearts you discovered that the vast majority 70 75 percent of those genes can be explained by the culture in conditions there are not necessarily artifacts associated with the fact that this is an in vitro differentiated cardiomyocytes but for example differences that are associated with the fact that you don't have lipids in your culture in media and so a lot of the genes that respond to lipid metabolism are shut down and those the vitro cardiomyocytes you can perform experiment with primary cardiomyocytes culture the same way and you can get a little bit closer profile so the take home message here is that these systems are useful they're far from perfect but you can characterize the extent to which they're useful and the extent to which they might not give you faithful information so without in mind I want to I want to just tell you two quick projects without getting into too much details just provide examples of the utility of this system in comparative genomics and of course I chose two studies that you couldn't perform using frozen tissue samples or the standard immortalized cell lines the first study is a study of comparative of a developmental trajectory that of course is impossible to do other than in vitro in humans and chimp and the second study is really the reason we started this back in 2011 which is to study some phenotypes that are a little bit more complex than just study the molecular profile of a sample and I call this project the in vitro heart attack we're changing the oxygen level in the cardiomyocytes so here's the first part is a comparative study of endoderm differentiation we started with this honestly for no other reason that it was just the first differentiation protocol that worked in the lab for both species it's a three-day protocol it's easy of course since we its comparative genomics and we can't have batch effects we require that the cells will differentiate to the same endpoint using the exact same protocol and it's surprisingly difficult actually to have one protocol working with similar efficiency in both species and so as we develop more protocols that and today we have quite a few for hepatocytes for cardiomyocytes for chondrocytes for other cell types but the first one was was the definitive endoderm so it's a three-day protocol we did it in four IPSCs from chimps in four IPSCs from humans plus a couple of replicates and skipping nine months of QC I immediately will jump to the fact that most regulatory trajectories here are highly conserved in human and chimp and that was completely expected that definitive endoderm is such a fundamental basic tissue shared in all mammals and obviously highly conserved in human and chimp and so we expected that and that was actually in many ways good to see because that means that our IPSCs in human and chimps and the differentiation protocol performs equally what was surprising to us but I think also very rewarding is to observe this reduced within species variation in gene expression in primitive streak which is the samples in day one in observation that we didn't have in any other day so what you see here is the the p-values rejecting the nipals is that the f-test that that assumes that there is no differences in variation and these plots are a little more complicated to explain these rely on the pi zero estimate from one species given that you observe reduction in other species but the take-home message is that this pattern is highly shared so it's not that random genes have reduced variation within the species across individuals as the differentiation moves from day zero to day one these are genes that consistently have reduced variation in both humans and chimpanzees and so this idea that you really canalize this differentiation to a particular developmental state a particular network state that you need to hit roughly at the same point at the same time with hundreds of genes is something that we seen in both species and that that's not something that we've ever seen before and in fact it it really changes a little bit the way that we now think about analyzing these types of comparative data because we're no longer just looking at these changes in gene expression thinking well in one species it went up by this much in another species it went up by only this much so that's different we're now thinking of hitting a particular state a particular growthary state and if you're already there maybe you don't need to change much that doesn't mean that there's a difference between the species if you hit the same state that might mean that you were already where you're supposed to be and that state might be conserved and it's a little bit different way of of analyzing the data the second project I want to tell you about is really where I think most of this lead and we have three projects like that now in the lab but I chose to speak about this one because this one is the most mature in fact already published and so what we do here is we take kadiomyocytes and we grow them in neuromoxic oxygen conditions and so for those of you who don't do much cell culture or did cell culture only you know briefly during your training you're probably cultured cells in just atmospheric oxygen which is 21 percent that's not neuromoxic conditions that's high oxidative stress for your cells they're actually a which is a topic for another whole different conference or there are a large number of papers that got their results a little bit wrong because they measure everything in oxidative stress conditions so these cells start from neuromoxia by the way most of my lab obviously does it at oxygen levels that is atmospheric so i'm sure that we get that problem too but in this particular case since we have to give them heart attack we can't start with oxidative stress conditions so we start with eight to ten percent oxygen that's condition a we move them to one percent that's condition b we keep them there for six hours or and move them back to neuromoxic condition and we have two recuperation two conditions where they can stay in that high oxygen level eight percent before we measure the RNA as well again either six hours at sea or 24 hours at the end let me tell you immediately because you might wonder by 24 hours namely 24 hours after reperfusion the cells are nearly indistinguishable than what they had the starting point at a which means that this entire process was generating back the the cells we start with and you no longer or almost no longer have any indication of stress so uh reperfusion give them high oxidative stress before it goes back b of course is is lack of oxygen and that's kind of the you know fondly i refer to this as the heart attack now much of the of the of the response is conserved but you see in these plots are dust the dot plots of gene expression differences in chimpanzee on the y's and the humans on the x from a to b from b to c and from b to d and everything is or almost everything is near the diagonal you see something a little bit of a subset of genes in color that represents the differences between species in that response but most of it is conserved here are examples of conservation these are genes that are important because we know something about their function and they respond very similarly i gave this example mainly because of this gene where you can see that the actual level of expression sorry i thought that you see the the gene in the middle you can see that the actual level of expression between the species is quite different but the response response pattern across conditions is actually almost exactly the same now i'm not going to take you through this slide in great detail but tell you the take home message that that these genes with interspecies conserved response are actually unusual and they're important for human disease in many ways they're enriched among cvd g was there enriched among particular genes that are seen to participate in different disease process that affect the heart and they're depleted for ecu tails that are identified in g-tech so so those ecu tails that are basically high effect sizes shared among a large number of tissues those are not the conserved response genes for heart attack and reperfusion so they have a lot of signatures of of importance and and they create these they generate this list of genes that provide additional promising targets that for follow-up and this is not you know for the first time in my career this is not this hand waving uh you know these seem to be under selection let's follow them up we never will because i don't believe the selection in the first place these are actually gene that respond to particular medically relevant intervention in a conserved way between two species they're highly enriched in process that we care about these are real uh worthy targets of follow-up and and some of them we do uh there are obviously also differences and those are extremely interesting differences in response genes that respond particularly in chimp genes that respond particularly in human as you may know uh there is uh quite a lot of work about the differences between human and chimp in the way that the the two species experience cardiovascular disease and some of these genes are just you know the storytelling about them that what we know about them may fit right in to try to explain those phenotypic differences and so i think for the first time we have through this in vitro system some access towards these processes that we couldn't address using frozen tissues standard cell lines and clearly not anyway in vivo in these species so the eyepieces are really cool and you should have some we are sharing these eyepieces we have been sharing them for many years we can't share them overseas because of cites especially with the new rules it's it's practically impossible it's just impossible we tried but we are sharing them domestically we shared them with 26 labs already in the united states there are in fact more papers using panels of chimpanzee eyepieces that we created from other labs than from my own lab and we're pretty proud of that we're sharing them with no restriction limitations we don't need to be a collaborators we don't ask you what you do with them we don't care if you compete with us it's just the resources and that's how we treat it here's a you know quick and dirty summary that i'm kind of proud of of the type of topics that people use our eyepieces in their papers that's not papers from my group and and all it takes is an email and a couple of forms with your keys and we give you the cells i'll end by acknowledging your keys and southwest foundation and dri because i wouldn't have a career in comparative genomics if not for these primate centers hard work and continued commitment and and and collaborations with our scientists that rely on these samples and of course energy ri as well as energy message bi for for funding this type of work for so many years thank you


Hi, my name is Starr Cochran and I'm an accounting officer here at Cal OES. I work in the travel unit within the accounting department as an accounting officer. To support the agency's responsibility of mitigating the effects of disasters and emergencies, employees must travel. To respond, direct and coordinate state and travel resources across the regions. As an accounting officer, I'm responsible for auditing travel claims for the administrative units. This includes Cal OES's director and chief WD directors as well as legal, finance, crisis communications and IT units. I am also responsible for processing and issuing all travel advances. I love being a part of an organization whose mission is to help and support others as well as being a part of a unit who desires that as well. I've been here about two and a half years. I'm proud of my position here, although my unit's not on the front lines providing support to those that need it. We absolutely admire and appreciate those that do. And if we can help by reimbursing employees accordingly for their efforts or provide advanced funds to assist, we're proud to be a part of that. One of the best parts about working here is also working with my unit. The camaraderie and just the unity between us is good. When disaster hits, things are connected, so we step up and help each other. My name is Star Cochran and I am Cal OES.


Welcome back to the 21 convention. I hope everybody had a great lunch. Our next speaker has been supporting the convention since 2008. He is the world's number one pickup artist. Welcome, Adam Lyons. Awesome. Hey guys, it's good to be back. I like it in Orlando. I know the 21 convention sort of like traveled around and being sort of everywhere. Orlando feels like home. It's like this is where it started. Got a lot of new faces. There's so many people here that I sort of haven't seen before, which is really cool. I want to talk a little bit before I get going. I've got a pretty fun talk for you guys. We're going to be covering something. It's one of those topics that's like a staple in the pickup community. It's like everyone knows it and yet no one actually knows how to use it or how it works. So I kind of want to just bring that up a bit and sort of explain to you a number of different ways to use this really powerful technique. But before I get into that, I kind of want to explain about getting an idea of kind of like the kind of people that we've got around us and the kind of people that you guys are. So very quickly before I start, how many of you guys are like a very would consider yourself sort of like amateurs? You're like new to this. You want to get good? Just a show of hands. Two, three, four, five, six, a few of you. Okay. And now how many of you would say you're sort of quite proficient at this? You've been doing this for a while and you're pretty good. Raise your hands. So, okay, so that's that's much less. One guy's like, I'm not sure. I'm in between. I'm kind of, I'm kind of, you know, depends who you compare me with.  You can pay me somebody who's really good. I'm bad. Okay, that's that's where he is. Okay, cool. First of all, I like to think about where did we come from? What, you know, why, why do we suck? Why, why were we not very good? And I've got a lot of theories about this and we're going to be looking into that a little bit. One of the big ones is I think I can pinpoint the exact moment my game started dropping. I came from a really interesting background. So I'm going to share this with you. I've never shared this before. It's a funny thing. In my local layer, it became like a funny thing to talk about like Adam's background. They'd be like, when it first came out, everyone's like, oh my God, really? They're your parents? I'll be like, yeah. And they're like, man, it's crazy. My, my mom was a stripper and my dad was a, like a croupier for Playboy Casino. And so they were, they were my parents and everyone's like, oh my God, you came from that background. You needed to learn games like you're the one, you know, you're bringing foundation, pulling it all together and showing everybody how it should be done. You learned the game and you went back to where it was. And I'm pretty sure I would have been a natural, pretty sure a long time ago, I think I would have been a natural, but I know when it happened. I can exactly pinpoint what happened. There I am going out at school, hanging out with all of my classmates at school, sort of like little Adam. And as I'm hanging out there, I see this girl and we start floating.  It's not real floating.   We were before the stage of handing each other notes in class and probably just before the stage of telling our friends to tell the other we liked each other, but we'd gone past the point where we hated each other and we're hitting each other. You know, it was like that kind of stage.  And there's like a little old me, maybe I was 9, 10, whatever, but I was only a little.  And all her friends are with her and they look at me and they go, no. She wouldn't want to go and dance. Why would she want to dance with you? She's much better than you are. And that was the start of the end of my ability to observe what was going on. Because what happens is I learnt that when a girl is attracted to me, when a girl is showing me a sign of interest, actually it means she doesn't want me. And that takes shape in the rest of my life. So you can now fast forward 15 years from that point and now I'm sort of like, you know, 23 and I see a girl in the bar and she looks at me and she smiles and she looks away.  She's going to make me look bad. She's going to make a fool of me. I better not talk to her. That's what I learnt. That was like my process of growing up. And then it took coming to game to realize that actually that isn't necessarily true. What happened in the past was she had bad friends. There were social groups going on. Her friends were jealous or whatever the reason was. But that literally was one of about a thousand things that stopped me from potentially being the natural. Like I may have been able to be. And my argument about game, I don't think everyone's like natural is not natural. I don't think there's such a thing as a natural. I think we're all naturals.  Just get an idea. Who here agrees with that? Who here thinks actually inside them, raise your hands high so I can see. That's like the whole room, right? Every single one of us does believe that. And like, this is a really big point.   Just some of us have different problems in our past. We have issues and we don't see things necessarily the way they should be. Perception is a very important part of reality. It's a very important part of game. Today, I'm going to be teaching you a little bit of voodoo mumbo jumbo stuff.  So I want you guys to pay attention to what's going on. Not just what I'm saying, but the way I'm saying it. Because we're going to be revisiting some of what I'm talking about and showing you that actually what's going on up here might be a little bit more than meets the eye. And there will be people that don't see it. There will be people that are like, no, there's nothing going on there. But they're just not really focusing on actually what's going on behind the words in the subtle subcommunication. If you think about it, the majority of communication isn't done by the actual words. It's done by the subtle, nonverbal communication that we use. And I'm going to start getting you guys to maybe wake up to some of the verbal subcommunication we use. Not the nonverbal stuff, but the verbal subcommunication, which is also going on. So we're going to be revisiting that. It's going to get a bit complicated, but try and bear with me and hope you guys understand. Before I go on, just out of interest, obviously we've spoken about guys that are shy and sort of like a little bit unconfident, which is definitely where I was. Standing up on stage was something that was always going to be scary for me. I always liked the idea of being up on stage. I remember as a kid, I wanted to sort of like do public speaking, but I was always scared of doing it. And I forced myself to do public speaking classes as a kid. And so I'd get up there on stage. I remember being really scared, and it was only the more I did it, the better I got at it. And games, the same thing. When I first came up with the idea of doing game, it was very scary. The idea of talking to someone I've never met before, talking to a girl, that's a very scary process. And yet, the more I did it, the easier it got. So again, just a question. How many of you here are scared of approaching still? How many of you is that a problem for? Raise your hand. That's a very small number, right? There's a few of you guys. Some of you are still scared. Okay. The more you do it, that lessens, but it never really goes away. As a complete set of everything, how many of you would be okay coming up on stage? Sorry, how many of you would be scared about the concept of coming up on stage and giving a public speaking talk? Raise your hand if the idea of standing up on stage scares you. Just... No one. That's awesome. Now everyone's hands are going up. One, two... Anyone else? Raise your hands high, guys, so we can see one, two, three. Yeah, right. All right, cool. Let's just get an idea. Cool. So what's really fascinating is a lot of you guys would be a lot more comfortable coming up on stage than you would actually going out and meeting girls and talking to girls, which is ironic, because one of the biggest fears that people tend to have is actually public speaking and going up on stage. So all of these things you can get rid of by practice. The more you do something, the better you get. But being armed with the correct tools for practice helps. You guys see what I'm saying there? Having the correct tool when you go out in practice ensures that you've got a much higher success rate than if you went out there alone. Imagine going to fight a war and someone said, all right, Son, are you ready? Here's your little pea shooter here, your catapult. You're going to go with that. Everyone else has got AK-47s. But you've got good spirit, good heart, I believe in you. No, they're setting you up for failure. If you've got the right tools before you go out there, you've got a good chance of success. Now, having said that, the situations that you're going to be meeting, they don't require actual physical tools. They require verbal tools. And so during this talk, we're going to be talking about the power of qualification and how qualification can actually empower you in a number of different situations to bypass situations and problems that you may be having and also how to use it to refine the work that you're doing. Now, let's start off with...