Good morning and welcome to CSIS for those of you who haven't experienced our beautiful new building. I hope you enjoy it I'm John Alterman. I'm the director of the Middle East program and it's my great pleasure to introduce to you Walter Douglas Walter was with us as a visiting fellow a couple of years ago and worked on a very interesting project on engaging audiences in Muslim majority countries and and the point that Walter was trying to work through was this problem That he'd been out in the field and Felt that that Washington was somehow disconnected from the field and there had to be a way To not only go from Washington out, but also to the field in especially when you're trying to Pursuade audiences Walter and I actually met out in the field. We met in Riyadh several years ago when he was Spokesman for the embassy there Walter for those of you who know him has had a large string of jobs Talking to Muslim audiences on behalf of the United States not only in Saudi Arabia and Pakistan where he was just before he came To CSIS now. He's in India which has one of the largest Muslim populations in the world, although it's not a Muslim majority country I went and saw Walter in India about a year ago and Walter brought the same Combination of intellectual acuity and enthusiasm Which so much of us have come to appreciate through his career Walter start off in advertising something I found a little bit surprising because Walter has been focused from the beginning and How do you actually speak to audiences? Not only telling the audience what you want the audience to know But how do you tell the audience in the way the audience wants to hear and Walter? Did something that I think very few people in the State Department are able to do not only did he sit down and Put his thoughts into a document a document, which I hope you'll read But then he did what a lot of State Department officers do is then he went through the clearance process That explains a little bit of why Now that he's been in India for a year. He's back with us. I am delighted to welcome him here I'm delighted to hear some of the ideas he conveys in his presentation And then I'll look forward to helping moderate a discussion with all of you on his findings So ladies and gentlemen, Walter Douglas John thank you very much for the introduction as John said we met in Riyadh in 2006 when I was the PA over there and actually I see Bob Keith who was in Riyadh with me also at that time but anyway, we hit it off and I've always found him to be one of the sharpest observers of what's going on in the Middle East and I find myself still quoting him on What's going on? Over the years, so it's it's wonderful to be back here with John and when I was at CSIS I sat up with the Middle East program of which John's the head and so was able to benefit from his input and specifically he read this report twice First time taking a very rough piece and shaping it and then second time refining it So it would be nothing it'd be a shadow of itself without John's input. So thank you very much I'd like to just thank a few other people who are here today Before I get into it one is I should thank the State Department for giving me the detail to come over to CSIS and it was Really an eye-opener for me and a lot of the strategic thinking I learned when I came here I've been able to give back to the State Department in many of the things that we're doing in India today I'd also like to Jeannie Neal is sitting up here in the front You'll I will might refer to we sometimes and she was with me She was the intern on the project and knew nothing about public diplomacy I think what she started and left as an expert But she went through more reports and that sort of thing like ever imagined was a fantastic research assistant So I just want to thank her and I see her up there and you'll see her name is on the report as well Terrence Smith is here somewhere Terrence was at CSIS with me And then he went to Fletcher and came out as an intern in India this summer And he actually helped do some logistics to finish up the report And then of course nothing is final without thanking my wife and my family who were over there And so they put up with me going to places like Riyadh in Islamabad without them And probably drove the family a bit crazy that way, but I must say the result was I got to do this report and And see a lot more of the world that was very interesting. So thank you John was hinting at you know why this report, you know What was the origin of it and I hope some of you have had a chance to read it it came up it went up on the CSIS website last week and I'm not going to go through it blow by blow because I think you can read it But I just want to point out some of the things of how this came about and where it takes us But I mean the as John said it was just fascinating as we looked at 26 reports since 9-11 dealing with the Middle East and The most amazing thing is how few of them had spoken to the officers in the field Yes, there are bits and pieces here and there, but nobody had really said how do you actually do this stuff? Overseas and the sense so we had we had a lot of advice coming out from here But the best that they could come up with isn't the overriding recommendation is just give public diplomacy more money That's easy to do, but money doesn't solve all your problems It's how you actually use it and how you employ it when you're overseas And so that was one of the incentives for doing this what actually goes on over there How does it work? And so over the years, I've been associated with public diplomacy for quite a while And it's been the past ten years really working in that region the era of Middle East Pakistan Afghanistan and so it's a look at what our officers are doing in that region to come up with a number of suggestions the challenges what faces us out there I've stayed away from policy because that's really the White House's prerogative But really as implementers, what do we do overseas when we get there? I would like to say I have taken in a lot from what I've seen and heard but everything I put in this report is mine It is not the State Department's report. I came over on a detail, which means I could do some independent thinking So I just like to make that because that was important through that clearance process That I make sure I speak in my voice and nobody else's One of the Something else that that really I think was very interesting coming out of this is sort of defining what public diplomacy is There's been a lot of talk about that since 9-11 But basically and there's been a lot of reporting out I don't think public diplomacy officers were active to ask to actually define what it is and one thing that was really Striking I'm just gonna one thing that came out in the report by find a very interesting part of it is we get it We hear a lot about messaging Washington, are you messaging or you're getting this message right? But public diplomacy really is something larger than that Messaging is certainly something we do in the press we get out there, but public diplomacy is a full panoply of programs and Platforms that we use to Engage the audiences out there to make them more receptive towards American foreign policy Well messaging's one what we really spend most of our money on actually these public diplomacy programs that go on overseas Adam Aroly who Was as always pointed out he repeats it so often I can actually say something it really is the sum of these two these two things Adam's no longer with the State Department, so I guess I can mention that and it's certainly true We probably spend three-quarters of our money working on programs such as Fulbright international visitors and really targeting those people that We think can make a big difference to the opinion landscape that's out there and that way in sense You have the rifled effect of targeting certain people who we think are very important Then the shotgun effect when we use the media to reach broader audiences And I think that's something that back in Washington was not fully understood and that's something I really wanted to put in the report That it's not just messaging how you get your word out It's not just messaging how you engage foreign audiences and it's not just messaging how you sort of make them see what we're doing and Show them we've got a point of view. That's very respectable I Think it's important if you look at the cover of the report if you see who's on there It's not a spokesman who's on there that is actually an English teaching officer He's someone who was in Pakistan with me And I thought that he really stood for that that other side of public diplomacy that isn't often reported But the fact is English teaching is something we use very effectively out there We target to certain audiences that we think perhaps are more at risk And but it's it's a way that we get out our word To a different audience and give them a broader sense of what's going on in the world And maybe they'll be able to inform other people about what's going on as well as get ahead in their own lives Because English is such an advantage to anyone out there But I thought it was really important to have that on the cover and not a spokesman at a podium Finally I'd like to say just for a wrap up why the report I think there's something else that motivates a lot of us public diplomacy officers in the field are very patriotic and So many of us just feel really strongly about what we're doing But nobody had really looked at who we were and what we were doing in a sense. This is a report I wanted to come out and say here's how we see what's going on overseas Here's how how we think it should be implemented This is how we will take something that comes from the White House and then shape it for the audiences out there But in a sense the heroes of my report is that public diplomacy Function in the State Department and they really are the ones who are charged with leading the public diplomacy efforts worldwide But I wanted to sort of tell their story and get out what goes on And so that's why I took what I've got here as I spoke to public diplomacy officers and non-public diplomacy officers And over the years I've been engaging on this This is a summation of this but I really wanted to get that story from what goes on over there Because I think the real heroes here are all these my colleagues in the State Department That are overseas working in very difficult environments trying to get that word out and engaging audiences and making them more receptive towards our policy initiatives If you look at the report itself, and I don't want to go through it all but basically I think as I said earlier I haven't engaged in the policy structure because that really is something the White House does But I very much done is try to sort of give a how-to outline of how we do it overseas some of the challenges and opportunities We have and just to highlight a couple of them More than a couple, but one thing I think is very interesting is when we approach societies I divided it in this for the report's sake in telling America's story versus engaging behaviors and attitudes within a country And I think a lot of people are sort of unclear exactly which one do we put emphasis on and what I've said is I've said throughout this report is Every country is going to be different and that's why the guys in the field are so important because they can help us work through What goes on out there, but to everyone should weigh up what are we trying to do here? Are we trying to engage to tell them about what we're doing about our policies? Or do we want to actually take people overseas who might turn into turn violent and sort of make it so okay? They don't take that route not that they know they're going to love us But simply that they don't go the violent route and I think in this part of the world after 9-11 It's a question we have to ask and every country is different out there every one of them Will have a different percentage assigned to one side or the other, but it's one thing I wanted to highlight Very much, and I think not everybody understands that we sort of have these dual functions out there. I Also felt and I mentioned this in the report that sometimes we even need a third office And we created one in Pakistan really to engage on that changing attitudes and behaviors a little different than traditional press work or cultural Affairs work and exchanges and that was really important to put out there that there are a number of different ways You can approach this question even outside the existing structure in the embassies in places like Pakistan and Afghanistan We have so much money that's given to us to do public diplomacy We have the luxury of being will create anything we want and that's what I was trying to capture here But I think even offices without all those resources can do a lot to change things and to relook at the structure of what they do Another point I brought across all the time is the diversity of audiences One thing is you do hear people say the Muslims or something like that And I really want to say there are many different audience and many different types of Muslims believing a lot of different things out there What's important is I think people in the field become very attuned to that right away and understand certain communities Believe one thing other communities believe another Sometimes when you're back in Washington people don't understand those those differences so well And I thought it was really interesting to sort of look at how the field looks at when they get into a country Trying to size up who's what who is more susceptible to a message Who do we have to work harder with who's important who's not that sort of thing? But the diversity out there is incredible It also ties in with The need therefore to speak to these people in the languages that they speak and I say this in India It's very funny. I constantly bring this up. You ever see two Indians speak in English to each other Basically unless they don't have an Indian language in common In which case they will use English, but even the best English speakers guys without an accent our embassy will speak Hindi to each other You get the same thing out in the Middle East You can have people who can communicate wonderfully in English, but the moment they engage each other they're using their own language Therefore as public diplomacy practitioners, we need to be in those languages. It's where they say what's most important to them It's where you basically have to listen to them to what they say not rely on English It's kind of interesting those English language newspapers you see out there I remember actually in Pakistan. I was speaking to a An editor I said could you tell me what what are these about? He said these are for you the foreigners in other words this is not something that Pakistan issues to communicate with each other and And I thought that was a really important insight and it's one thing is we have to make sure we don't rely on the English Language out there to try to interpret what's going on because that's not where the real action is It's in the vernacular and it's very important to be there. I Wanted to talk a bit about security which I did in here and I said I made us you know Obviously in Afghanistan Iraq Pakistan and Libya and Yemen could go into it today Security is a huge concern for us But in the other places while we do have these embassies that are much more secure than anywhere else Real public diplomacy work takes place outside of embassies In fact, I even argue that the people we want to reach generally don't come into our facilities That's where the real value is going into their institutions and meeting them out there And I think that's something I quoted Thomas Friedman who came to I remember a turkey after the The bombings that in Turkey he looked at our new iman consulate and remarked how how this was a bad message It can be or it's a difficult message out there It can be but it's public diplomacy offers the fact that officers you get out of that environment And you want to go to the people who who really don't care what our what our embassies and consulates look like and that That's absolutely vital and that's why that's not such a key stumbling block for public diplomacy officers Except in those very high-risk security posts that I just mentioned But it's very important to see these other places you get out and that that's what public diplomacy officers do Finally or not finally sorry For next steps in the report, and I hope you do all get a chance to read it Set out a series of recommendations. I think one thing that was very important for them is and I go back to those 26 reports written since 9-11 The one recommendation as I said for everybody was to spend more money on public diplomacy that's not going to happen and Basically what I tried to come up with or I said genie with genie's help Was to we came up with we're low-cost or no-cost solutions because the State Department's not looking to dump tons of money into something New and in this budget environment, it's going to be a while before they're able to do that And so I think what we came up with a lot of things we can do by certainly shifting emphasis Looking at the field a little bit differently real things that the State Department could do actually I believe that some of them are underway to a certain extent And I'm looking forward to this afternoon going and speaking to a lot of people to find out just where we are that way in in in the State Department Finally, let me wrap up and say The limitations of this report or it's a just report the limitation is obvious It just dealt with one part of the world, but there is a whole world out there people said why didn't you cover Indonesia? That's that's Muslim or something Muslim majority. I think be great if somebody does I'm certainly in India now I'm finding a whole series of public diplomacy Ideas that are different than I had my report because I'm dealing in a totally different environment But we're running a country where we're working with a country with say 60 to 70 percent approval ratings Very different than what we had in the region that's covered in this report So what what I'm what I'm saying is there were there were limitations Just because of time money effort all that sort of thing But I'm hoping this will spark more reports to actually look at what we're doing overseas I think this is an absolutely vital area that we understand public diplomacy. I'm delighted I'm going over the State Department actually to speak to a number of people who do that and I'm going to give them a Mini report on my report and talk about the importance of what goes on in the field as something very different than what they've seen here I'm gonna stop there what I'd really like to do. I hope I've given you a bit of an overview here I hope you've got a copy of the reporter if you read it or looking forward to reading it Because I think there's a lot here that really will define something. It hasn't been seen before I was really glad that some of the academics that I brought into this project said, you know We have not had a report from the field and they've been eagerly awaiting this and as John mentioned It was a little delayed in coming out the research it for this basically ended the summer of 2012 but But now they have something in the public diplomacy courses where they can actually look at What we're doing in the field and try to make some judgments that might be a little different than what we'd hear if they were Aligned solely on on on material coming out of here. So thank you very much and I'll look for some questions and answers Thank you, Walter as you suggest there's a lot to chew on there One thing that you didn't talk about at all in your report really was the issue of metrics and One of the things I see in the State Department Is a fascination With metrics for social media because social media tends to produce very precise metrics From your position in the field What is your sense of the role of metrics? How do you use them and how are people misusing metrics? In ways that we have to Metrics is a question that is really a difficult one for public diplomacy You mentioned I was in advertising before and you always had does the product sell or not? It was a very interesting metric and if it didn't sell well, they fire the ad agency So you would pay that way not that it was always the ad agency's fault, but they usually tended to blame the ad agency's first Metrics are something that is very difficult We know that public diplomacy and USIA contributed to the decline in the fall of the Soviet Union But nobody's been able to measure exactly how that happened We've talked about it, but it did play a role in undercutting the intellectual livelihood of the Soviet Union When we get to today trying to measure metrics, it's very difficult because you can measure inputs But you can't measure outputs There is some work done and the private sector mostly looking at when someone takes an action to understand that they have Absorbed that idea so example. I mentioned it's not just the tweet you do It's the retweets that you have to count that you've actually scored something with this That's one way we do it But nobody's really come up with an idea that says they have changed their way of looking at the United States Based on what we have given them many have tried to do that very difficult to do But I did come up with the recommendation and this is in in over the years speaking to hill people Hill staffers is they have said one thing we need our narratives about what you're doing How does this stuff play out writ large and it's something I think we can all improve our In what we're doing is putting together all these public diplomacy programs around the world in certain areas say what are they achieving? What are we what message are we getting out pointing to some success stories not having the exact metrics saying we move the needle from from You know 50 to 60, but simply saying we are putting out these ideas there It is a concerted effort it makes sense and if your narrative makes sense people can probably trust that you're on to something I think that's one of the recommendations I have there is get up to get out to the hill get back to the State Department for the field more with these narratives about what's going on And would that suggest that that the number of people engaged the number of people Who are in our audience is Less important than creating a dynamic and we we need to be more tuned to the dynamics We create rather than the size of the audience I mean, it's it sounds to me like you're recommending a sort of looking at waves and and creating ripples rather than Rather than trying to measure the force. Uh-huh. Yeah, I think that's right If you go on broadcast television, you get huge numbers How many of those people are important to the influencing policy? That's a that's an open question We have those exchange programs and other things we do on a narrow basis where we said that's someone who's important to the debate We want to expose them or maybe get the person on an exchange program with the United States where they can see what we do So you're making judgments all the time about the value of the audience. It's out there some more valuable than the others When you put it together, you try to then draw the conclusion of what you're getting out there What words going out there? How much you're you're you're causing a debate but to actually measure the impact is difficult But you've got to put all these factors together on that one idea You want to get across then maybe there's another idea and how those blend together, but you've got to use all these tools They're probably at any one post you could easy drop a PD toolkit that would have 25 things that are up that we basically use And it's all from using our local staff in the vernacular language to using the ambassador as a speaker to using an econ Officer as a speaker using exchange programs to bringing out speakers Into these countries all these different things in our toolkit to get the message out hitting different audiences that we think are important But how you measure that impact where it moves the needle. That's very good as a manager How do you think about the problem as you allocate your resources across the entire toolkit? at the beginning of Going anywhere. I think you have to sort of sit back and say what our objectives here and try try And this is something I learned at CSIS and going through a number of exercises with you and others around here Was trying to say what the goals are up front and don't worry about the implementation right away But simply set up the strategic goals I think there's a tendency often to mix implementation and strategic goals And you've got to really see them as something separate and then go reach into that toolkit of what works best in that environment And that becomes a judgment call. It's probably more an art than a science But for example, if you're in a very open country like India, you can do a lot of things I guess I'm getting out of my my Where the report is you might have difficulties in other places where it's more closed and in the Middle East you find a tremendous variety of Country some more open than others But some can surprise you certainly when I was in Riyadh I was amazed at how how open they were to have to engaging on so many topics that people probably outside of Saudi Arabia Don't realize and when you were over there I think you've been able to engage quite a few audiences and you sort of know that receptivity to what's going on Anyway, you try to judge. What's the best way to engage the audiences? I want to reach and usually there's always a blend of those two things the broad media with a more narrow Targeted approach So let me just pursue one other line before we open it up. You Have mostly spent your career overseas But you had a lovely year in Washington when you were presumably part of a target For other embassies outreach programs for other embassies efforts at public diplomacy here in Washington Who do you think was especially good what kinds of things did you see as a target where you said, you know what I Recognize what they're doing and they're being effective And we should we should try to take a page from that and do it when we go overseas That's an interesting question. I guess when it struck me is how rarely I would think people did engage me You know often that the officers were working on specific countries that one embassy will engage them But engage you more broadly you don't come across it as much in the State Department That said you do end up with some events You do end up with certain things. You're at CSIS. I mean, we're Right when I was here. Yeah. Yeah, we engage with a lot of Audiences I have to say and it was it was very interesting. I don't think anybody targeted me the same way I would have expected And I would think they could probably do more of that. I think that the think tanks CSIS being my favorite obviously Have a lot of these ideas and a lot of these people they want to engage more with But I I don't see it as much as I would have expected and certainly when I looked at audiences I was here I went to other think tanks as well CSIS and I've looked at the audiences and I was sort of struck by There's some embassy people, but I would have expected more and I think probably the Maybe it's because I mentioned this the public diplomacy function the State Department is kind of unique other countries Don't seem to quite have it the same way And so I found that State Department people were Much more around the city getting around and I see that overseas engaging with think tanks overseas I know some other countries may be doing a bit less than we do and perhaps because they don't have a dedicated function They'll have a spokesman, but not a guy who sees that broader picture of how am I going to engage that foreign audience? How am I going to influence it about what we're doing? I see that less and less I As I look in the audience, I see a lot of people who know a lot more about public diplomacy than I do So let me turn it over to you. Yes, sir. Do you could do me a favor three things wait for a microphone? identify yourself and Ask only one question to have a chance to go around thank you so much Douglas a good presentation My name is Akbar Hwaja. I'm a World Bank retiree originally from Pakistan You recommended approach in Muslim country is the same yardstick for Kingdom like Saudi Arabia or you know military generals like you know some of the Middle Eastern country and Pakistan like a democratic country. Thank you. Yeah Well, one thing that really comes out here is is the variety that's out there and that we have to shift gears And I at one point I talk about like a good arabist can take 15 years of training kind of guy You can understand, you know Differences in the regional cultures the local cultures the different Arabic that's spoken out there So I think though what's important is I think we do recognize the diversity and everybody who works overseas sees that And what works in Saudi Arabia? Isn't necessarily something that's going to work in in Jordan They have different levels of openness for example in Saudi Arabia We had a American archaeology team out there and that was something kind of new and different but in Jordan They've had them there for 100 years even longer Or even going back into the Ottoman days. It's nothing special there or it's something special in Saudi Arabia It's different every country you have to sort of look at it as as a different activity And that's one reason why so much I believe and you have to have the guy in the field The man or the woman trying to understand and interpret these societies and then getting back and saying what works best here because as you said Some are open some have a democratic political process others don't other have a monarchy all these Systems really demand a different parts of the toolkit to go into action. How do you think about? Working a monarchy. I mean you're now in the world's largest democracy, but you've served in One of the world's most important monarchies How do you think about? the targets for public diplomacy in a system where you're not Trying to target elected officials. Yeah Some people say there's an audience of one when you have a monarchy, but I think it is much broader There are a lot of influences there the king goes out and regularly goes on on trips to meet his I guess his subjects and so there are a lot of people giving advice And so I think the important thing is who are those people who are opinion leaders there and you want to engage them as much as possible I think All over that region you deal with very bearing degrees of democracy and Lack of democracy, but you still always have people who influence the debate You're always gonna have a guy who will go on TV and talk about something Talk shows are huge business out in this part of the world those guys even if they support the government We'll have different ways of portraying it And then there are those who don't support the government so much. You'll have a different way So I think the key thing is to find out who are those people for example a systematic watching of television something kids are really good at is Can often you'll find who are the people who speak most about the subjects you want and within three weeks of say watching You can come up with oh, I'm noticing these certain people keep coming up and they are influencing the debate in some degree You probably want to engage those guys They're gonna be they're gonna be important to you And so I think it's listen look at look at who's out there and and come up with who are those people who are important It's not just the one guy at the top Hi Bruce Gregory George Washington University You make a strong case for looking at public diplomacy in the field and for the most part looked at the State Department's role in the field But you've also served in large platform embassies where there are a variety of departments and agencies of the US government and in India You have an annual strategic dialogue that involves many many different agencies Could you reflect on who does public diplomacy in the field in addition to the State Department? And what's the State Department's role in leveraging those actors? Yeah Well, certainly when I could think of in a lot of places USA ID and in Pakistan. It was a huge USA ID operation there In India, it's much smaller in other countries. It varies in Egypt. It's huge and it varies ever you go They are certainly influencing the public debate out there They do have a public role. It's a bit different than say what the State Department is They generally coordinate with us and everything they do and and so you hope there's a unified message going out But there are certainly key communicators when you're overseas, but there are some other agencies like that Health and human services can be a big one Generally doing CDC. There are all these other people are out there doing things the key thing is you want to capture a lot of the good works that they're doing and Explain why it's important what they do and show say that partnership between the United States and these other Branches of government. It's not just strict policy. There are a lot of these other other things that are going on It's important that people see that you're right though in my report I've really just focused on the State Department because as I mentioned we have the lead role But there are a lot of others who support the efforts and give a lot do a lot to to support public diplomacy It's coming Good morning. I'm Penn Agnew from the Bureau of Middle East North African Affairs at the State Department Walter and I worked together in the office of pressing public diplomacy Walter I'd love to hear your comments on how you approached issues of religious faith and tolerance both in Saudi Arabia And in Pakistan and some of the creative programs that I know you did in in Saudi Arabia I think would be of interest, but I'm what I'm really curious about is how you avoided an inflammatory Exchange or engagement but rather kept it more than a level of mutual understanding and of interest in The issue on both sides. Yeah Religion is a fascinating one. I speak about it. My report is that I think we have to realize Establishment clause isn't to avoid religion. It's a void to a specific religion and that it's a very effective way to engage people I keep thinking of someone who came out to Saudi Arabia and I was there Who is Jewish and his father had recently died and in the Jewish faith you read every day? Certain passages from the Torah elsewhere to honor your father and when he mentioned that to Saudi audiences said that's great Honoring your father through your religion. They just love that So here was someone where he was quite open about what he was doing religiously and they respected that I think we have to be open about that. It's something. It's very important for them There's probably there's much more religion in there in there in their daily debate Then you might find here and I think we can't shy away from that but rather embrace it as something that's that's very worthwhile and explaining Well, I guess I guess one thing that's explaining how the United States is religious That's always a surprise for audiences when they come here I quote a journalist who said when he was studying in Boston He was on my ten churches on his block or within a one block radius of where he was and he never realized Americans were so religious and Al Jazeera did a program on Islam in America and found out to their surprise that it was Pretty good and that the religious situation was good here I think that's something that is a barrier and and and breeds a lack of trust in who we are because religion is so important to them And sometimes what they see coming out of America is a television show in a movie or something like that It doesn't portray that as much as that is part of our lives I think in the State Department we have to embrace that not proselytize obviously I'm not saying that but be very open about that. We are religious and believe that too because it opens a lot of doors for you What when when when you speak about that the other piece of course is when American religious figures do things that are polarizing in the Middle East or seem to be disrespectful is there Is there anything you can do other than explain, you know Nobody speaks for the government and we have a big messy democracy We do that a lot And there are a couple of very well publicized cases recently in which we we need to consistently engage on that and try to explain the First Amendment The First Amendment is a tough one to because most countries don't have it I mean we're it in a sense and there are a lot more restrictions even in Western Europe on what you can say than the United States It's a hard concept to get across Because those people can use that and they generally can get a lot of press even though they have very small following in United States So while you can talk about the First Amendment You also have to explain that these are not mainstream people that what they're saying is not something that a lot of Americans are picking up or a lot of Americans feel Yes, certain ones do But the best the best offense is to be open as you can about it Don't try to ignore it. Don't try to hide it try to explain it and And in some ways let these let the message fall flat in the United States and say okay What effect has this had on what we do and often that the proof is it has very little effect on us so but we're constantly dealing with those those guys and It's it's troublesome It's but we we have to explain this is our society and how we do it but look at the results Do they throw back at us the same the same thing of all the people who are endorsing terrorism? You throw us all in the same. Oh, yeah in the same basket. Yeah. Yeah, that's right we hear that constantly how varied you know most of us are and When you're in the field you really get a good sense of that and appreciate it you realize most of them are like you and me you know, they just Want a little a middle-class life get their kids educated all that a lot of them recognize the problem. They've got When I was in Pakistan one of these guys a very senior government official I became friends with used to say, you know America don't abandon us. You're our best hope It's not a message.   You don't hear back here as much But on a daily basis when you're out there you hear that a lot and these are people who are trying to distinguish themselves From the stereotypes of what Muslims are like and they're just speaking from their heart and you can't help But be affected by it and understand those those those differences in a society for the troublemakers out there Sure, there are but most of those societies know it as well, and they don't like it any more than we do Thank you question all the way in the back Thank you My name is Lynn while and like Penn and a number of others in this room I've overlapped with Walter professionally First while I was a Capitol Hill staffer and led a staff delegation to the region He was very gracious to help us with that and then later during his last days with the Undersecretary for Public Diplomacy and Public Affairs For whom I then worked for a year and a half now I'm at the Broadcasting Board of Governors where our governing board has an opportunity to change the Emphasies and focuses of the agency over time and in the wake of 9-11 for example They created the Middle East Broadcasting Network Alhura TV and Radio Sawa and I wondered well Walter if you'd like to address how the personalities and changing Emphasies of the Undersecretaries over time may have affected how the Muslim majority countries are approached either through the way the United States speaks to them through public diplomacy or programs that have been launched Or relaunched over time. Thank you That can be a minefield I could speak most about the one I worked for Judith McHale and Her approach was kind of different and one that I think still lives on today in a way I very much like and that is that when USIA merged with the State Department in 1999 It wasn't a complete merger and a lot of strategic planning function didn't follow from USIA And she'd been ahead of discovery and realized the importance of bringing that in and I thought she did a very good job of Giving us a tool to do strategic planning and to get us really focusing on that. This was 10 years after the merger and It's invaluable I think more work needs to be done on that But I certainly think this planning we've got limited resources and the more you can plan what's important and not spend your money And your resources on personnel and all that on what's what's unimportant that that makes a huge difference. I think one of the Most difficult thing about things about that position is how much it's been vacant and Matt Armstrong Has written about it in his blog a mountain runner about I think the position has been vacant about a third of its existence And the turnover has been pretty fast there that hurts public diplomacy We like to have people in there and and emphasizing what we can do and putting resources and giving some direction to what we do Why do you think it's been empty so so much? It's an interesting question. I would think there are a lot of people who want to do it and usually when I spoke to Spoken with them. They enjoyed it. Someone like James Glassman who only got six months at the job said he really had a great time and and wished he could do more and my sense is that It's harder. They have generally looked out At they don't use career people there. So to find the right person who's out there who they think can do the job They I mean seven four the State Department takes a little more time, perhaps than something else, but Probably every case is separate individual why one takes a long time to fill I mean Judith McHale came in quite quickly when the Obama administration came in so clearly, you know They moved very quickly to bring her in as fast as the confirmation process would allow I'm not sure why on an individual basis. The others took so long And I wish it it weren't like that Hi Walter Mike Schneider from the Maxwell school I wonder if you could describe for us some of the ways in which your program in Saudi Arabia tried to foster civil society Okay Well, I Guess I was there at a time 2006 2007 We just had a number of hot spectacular terrorist attacks earlier the Saudis were doing a lot on their own and have done a lot On their own for it, but I guess what we were just trying to present is a View of how the United States works is it maybe there's some things there they could see that would be worth borrowing and part of it was just exposing people to the United States in a way they never had before and It was everything from using Muslim Americans in Mecca to talk about things like how we deal with health problems To a photo exhibit that we worked with National Geographic with Tom Abercrombie their great Middle East guy really introduced the Middle East the United States and With that photo exhibit we could go to places that Americans traditionally didn't go we partnered with the so Saudi Ministry of Culture to do that and Hoping that when people saw that some of those those The extremists there would say well, maybe America is not all that bad and that therefore maybe dislike of America is not Something these extremists would say it's going to get us the mileage that there's another side that make them hesitate and say maybe you know Well, not all Americans are bad. I look right in front of you at Safiya Khilji who was with me in Saudi Arabia who was very innovative and going out and working with a lot of these communities and places where American embassy people have not been before and She was a wonderful asset to have with us But I think just the exposure to an American like that could talk to them somewhat about about who they were There was a another project out there that I greatly admired and that was It was a breast cancer project tying up Susan B. Coman Foundation with MD Anderson out in Texas and Breast cancer was a terrible problem there and Saudi Arabia was not equipped to handle it And so tying the awareness of Susan B. Coman with the treatment of MD Anderson was a wonderful project out there that Women came together but fully supported by men. They all have wives daughters and that sort of thing And that I thought was just a brilliant program to sort of show How you can tackle this problem in what you might say civil society in a very very fine way Using us as the example because we do that we tie the awareness together with the treatment stuff like that I think Had a lot of impact and judging from the Saudis you'd speak to and the media attention it got and the attention from the king Tells you that we're on to something there. Can you say a little more about gender? I think that there's sort of a presumption that farm policy is is a man's game But I know from from what I know of your programming that that like with Susan B. Coman I mean you were you were reaching out to all kinds of audiences as the United States thinks about Engaging broadly and We know that women play roles in families determining who participates in boycotts and those sorts of things how should we think about Engaging women as distinct complimentary to male audiences and To what extent should we just say we're gonna talk about foreign policy and whoever's interested will come. Yeah. Yeah Well, I think the thing is you have to Engage women on both levels as Professionals as foreign policy is they they fit those opinion leader positions because why there might be fewer of them There are women out there in all these societies who have an impact To watch Lebanese TV is to see all those Announcers and reporters who are women and realize that NBC is broadcast quite broadly in the Middle East And that they are certainly you're gonna engage them because so many of the presenters are so attractive Whatever it takes You know, the fact is that they are presenting news in points of view and there you want to work with with them on on those Very serious hardcore political issues, but I think then in looking at how you engage a larger Grouping you have to find what's important to them and make sure the things we're talking about are important to them so for example, if you Sit there talking about glass ceilings and corporations or something and things like that. That's important for a very small group It's important, but it's not a broad broad based effort I think what you have to do is look at something that perhaps does have a larger impact on women And that can be education. It could be gender-based violence It can be all these other things that really hit a broader society and maybe share some of her ideas and really share It's not about lecturing or doing anything like that You have to show what we do and see where they can borrow something that might fit with their society You can't impose this on them And they will work in there at their own speed and at their own time to do it But probably exposing them to some of our better practices can't hurt You've also got to explain the things we do that aren't so right and one of the things you get is Especially they might look at certain aspects of our society Some of our TV shows show quite a lot of licentiousness in America And you have to put those in context with what they are One, they are television shows and not reality And you have to deal frankly with these issues because I found that sometimes these make the greater barriers to understanding the United States Than anything else is they see that portrait of America whether it's accurate or not or generally It's not accurate and say I don't like that and you say well There's more to America than what you see on those shows reality TV isn't reality Jerry Springer presents a view of America And they certainly watch it and love it But I also like to think that Oprah I think is the most popular show out there And probably put some more positive image out but also speaking about real women's issues to a broader audience So you've got to make sure they see maybe a little bit more open a little less Jerry Springer Hi, I'm Steven Canada retired from the US Council for International Business, and I'm looking at this paragraph called the private sector And you say a free conversation with private sector executives might be an inexpensive alternative So if I'm the head of the IBM office in Mumbai And my job is to make IBM global a seamless operation What can I do? What should I do? Do I want to get engaged with this and it's sort of like walking on eggshells? So to what extent can US global companies? play a positive role without Cracking the eggshells Sure. Well, it's interesting because we do work with those if you take something like Microsoft They have every interest in intellectual property rights is that we do and they go are very vocal about it They have they bring people together. They send them off Including on training programs in the United States. So they do a lot of things like that So a lot of companies that are augmenting what we do or complimenting what we do for their own reasons But they happen to Hit a lot of our ideas one thing I was mentioning though about that what I thought was so interesting I really saw this in Pakistan we put out with AID a contract to do to add agencies and There was I was part of the committee to choose one and I found that and I'd been in advertising so I could I could read through some of the mumbo jumbo they put there and What was interesting is when you have an ad agency talk about what works you're not just getting the guy thinking about your one thing There's some guy with 20 25 years experience selling products there and has a good sense how this stuff works So one thing that came through in a lot of these contracts for example was it don't use SMS that's spam And at the time in Washington we got use SMS. So who you gonna believe you know kind of thing Well, I think these ad agencies were really trying to move product and all that have a good sense of what the tools are What can work and what can't I? I think we can also learn from some of the failures I mentioned a McDonald's campaign in here that was covered at the New York Times about how they thought they were doing one thing But social media backfired on them and it came it turned out to be something very different I think we can learn from what these companies do But my final conclusion and it's funny because when I go back to India I have a guy right now hooking up a session with an ad agency I say I want them to tell me for an hour bunch of us go over there What would be surprised to know about how you? Communicate in India. I think there's a lot of experience that we can tap into there We tend not to be so good marrying with the private sector on that, but they've got a lot of experience and can tell us an awful lot We have time for one more question right there, sir Thank you very much Richard Lee Smith from the British Embassy I just I feel I need to ask a question just to show that we are here and we are listening I Wanted to ask about the balance between effort from Washington and effort in country Obviously, you're blessed here with a huge number of foreign journalists To what extent can can you use them to influence the message that you receiving in country and And what what sort of have been the best experiences you've heard of that of that happening What's that last part then what you're sort of good examples of engagement of the forum media here to to influence okay, we regularly engage the foreign journalists that are there and We do it one with especially with the Americans out of courtesy because they are fellow Americans who we were there to help out but also one thing that's very interesting is how what they write can boomerang right back into the society and The fact is say when I was in Pakistan anything that appeared in the Washington Post the New York Times New Wall Street Journal was covered and entered into the debate there So we found them very valuable to public diplomacy simply to make sure they have our perspective and what's going on We also know that they engage with an awful lot of people in whatever host country you're in and so Just by the questions they ask you learn a lot about a country When we have chats with them The one of the difficult things though is there they have difficult to getting access to a lot of these places Some countries let them in some don't some throw them out some have minders So it's not something you can't use them everywhere all the time that way or they're just not around The usually you always will have a wire reporter or something like that. He's often a local But to get a New York Times or Washington Post guy in there is something different in Pakistan We're very spoiled because almost every major media organization had a had a representative there In India I can say although it's outside the scope of this report New Delhi is a huge hub for international journalists and and every major medium is there So wherever we are we like them we use them We think it's important to communicate to them if they ask us questions But they also are not our target, you know, who they're not the guys we're out there to reach It's sort of what we do in addition to reaching out to whatever that the host country is And I think that's always something to be aware of for us a real success is when we see a story that appears say in in the In the Middle East press or in Al Jazeera something we've worked with them on much more than if something would appear on BBC And once again, I also get back to that vernacular trust and all that that certain Organizations will will burrow deeper and have a broader audience than others So we want to be sure we're clear who we're really trying to you know work with the most here Walter, I want to thank you for presenting not only a fascinating report, but also a really Broad-ranging discussion that demonstrates just how rich a field public diplomacy is I want to thank you all for coming We look forward to seeing you again soon. Thank you. Thank you. Thank John. Yeah


Bakhmut, Avdivka and Morinka at epicenter of combat actions. Russian forces spotted the arrival of fresh Ukrainian air defence forces and capabilities to the Kiev-controlled settlement of Chasov-Yar in the Donetsk. Russian retired Lieutenant Colonel Andrey Marachko told this. In the area of the settlement of Chasov-Yar, the arrival of fresh enemy air defence forces and capabilities were observed. They arrived with surface-to-air mounts. The air defence systems were the highest man-portable air defence systems and drone jamming stations, he said. Andrey Marachko earlier told that Ukrainian troops were redeploying reserve equipment and personnel from Bakhmut to the Krasnyi-Lyman area. Chasov-Yar is located in the north of Donetsk about 11km west of Bakhmut and 25km south of Kramatorsk. As the Republic reported, placing Chasov-Yar under the control of Russian forces is a major tactical objective that will make it possible to protect the southern flanks of advancing units in the Bakhmut area. The general staff of the armed forces of Ukraine has reported that the towns of Bakhmut, Avdivka and Morinka remain at the epicenter of hostilities. It is said that the enemy continues to focus their main efforts on conducting offensive actions on the Lyman, Bakhmut, Avdivka and Morinka fronts. The hottest spots are still in the areas of Bakhmut, Avdivka and Morinka. General staff announced.


So, good morning everybody, my name is Theo Javel, I'm working in Basel in the laboratory of X-Fan Invasion and today I will talk to you about my PhD work that consisted in studying the response of E. coli to carbon starvation at the senior cell level. So what happens when we put bacteria in the flask of fresh nutrients, well it's been well established and we have seen a couple of examples of that last week, that bacteria will start growing exponentially consuming the nutrients and once those nutrients are exhausted they will reach what is called the stationary phase where the number is stagnating and if we leave them long enough in this phase they will start dying. And as we've seen last week most of what we know quantitatively about bacterial gene expression has actually been established on exponentially growing cells and arguably one of the reasons for that is that it's very easy in exponential growth to put the cells in a reproducible steady state. But if you think of it and we have had a glimpse of that in the talk right now, in most environments nutrients are limited so we actually expect bacteria to spend at least a substantial part of their lifetime in a state of starvation for some nutrients. We also expect that the selective pressure associated with starvation have therefore shaped the evolutionary story of bacteria and it's fundamental for understanding it to understand what's happening during starvation. Stationary phase is in a lab proxy for this state of starvation because the cells stop growing due to the exhaustion of the nutrients as I was explaining. Given the fact that the bacteria seems not to be active and seems to not change their numbers we can ask okay can bacteria do anything at all in starvation or is it simply dormant and waiting for new nutrients to come in order to regrow. In 2014 it's been shown by Geffen and colleagues that bacteria are actually able to sustain gene expression during starvation. To do so they use genetically modified E. coli carrying a transcriptional reporter where a promoter of interest is driving the expression of a fluorescent protein. By following the fluorescent signal emitted by the cell they were able to follow the ability of the cells to express genes. So they put those bacteria in a flask of fresh nutrients as I explained before let them grow exponentially until they reach stationary phase. They ask what is the production rate of the fluorescent protein a long time and they could see that as expected as bacteria enters stationary phase this production rate is dropping massively due to the exhaustion of energy. But the surprising result is that instead of dropping to zero this production rate was dropping to about 10% of this exponential phase value and remain relatively stable for an extended period of time. The authors call this constant activity in stationary phase and this really signals the fact that bacteria are not dormant in stationary phase but are able to keep on expressing genes even though they are not growing anymore. Now it's been reported many times that the concentration of various gene products in star cells is fundamentally different from exponentially growing ones and this results in very different phenotypes of the exponentially growing cells compared to the star cells. Star cells are notoriously associated with for example relevant traits such as increased tolerance to a broad range of stresses going from osmotic stresses to antibiotic passing by oxidative stresses and phages and is therefore fundamental to try to understand what sets the concentration of the gene product to understand this phenotype inside the star cells. One fundamental difference between those cells and their exponentially growing counterpart is the fact that the turnover of protein is now greatly limited by the fact that the dilution stopped entirely because the cells are gross arrested. So we expect that star cells are not able to change the protein very efficiently. So the question we want to answer is actually what determines quantitatively the protein concentration inside the cells during starvation. Starvation is notoriously associated with heterogeneity and so we want to answer this question at the single cell level in order to understand how this variability can be characterized. To do that we use a microfluid device called the mother machine. We've seen a couple of examples of that last week where individual bacteria that you can see as those black rods are growing into dead end gross channels that are those white lines and we can feed them whatever nutrient we want through the main channel. In our experiment we used about like in my work I was studying about 20 promoters of E. Coli and this was made possible by the fact that our microfluid chip is multiplexed so we can study up to eight strains in parallel and all of those strains are carrying a different transcriptional reporter for a different promoter. Now we use time-lapse fluorescence microscopy to obtain a long time, a time-time resolution images about the phase contrast that will inform us about the morphology of the cell, their length typically and images about the fluorescent that is an information about their gene expression. I don't have the time to enter into much details but we use a sophisticated pre-analysis pipeline that allows us to recover quantitative measurements out of these images. By that I mean that we recover information about lengths and fluorescence inside individual cells where the measurement noise has been removed so that we have a very precise estimate of what is the actual length of the cell and the fluorescent content. On the top plot here you can see an example of individual cell traces in terms of fluorescence and we also get a reliable estimate of instantaneous rate. Instantaneous rates are the instantaneous gross rate at the time of the measurement and fluorescent production rate so the volumic production rate that is the amount of protein that is produced per unit time per unit volume and this last rate is a proxy for the activity of the promoter we are interested in and you have an example of these individual cell traces of this rate on the lower plot here. In the stationary phase the population is constant in that mono curve. Now that could be because every cell is not growing or because cells are growing but also dying. Yes. The numbers, right? So your traces show cells to be growing. So no. What about death? So this is not in stationary phase. This is in exponential phase. Sorry. Oh this is in exponential phase. Yeah, because basically typically what we do is we switch the cells from exponential gross to stationary phase and I will come to that. Sorry. Yeah, this is just an example of the way we treat our data but this is exponentially growing cells. Yes. But so my first question is that in the stationary phase is it that cells are growing and also dying? So no. That's why the number is constant? Yes. Or is it that cells are not growing? So cells are entering gross arrest. This is one of the results. Within our time frame we observe no gross and no substantial death of the cells. Just wanted to add one thing. If you call it generally once glucose runs out, for example, it takes one or two days before you start seeing viability going down. So when you, it takes like a long time before cells start actually dying. So within this time scales, yeah, I think it's just like cells not growing. All right. So as I said, we want to study starvation using this device and what we typically do is we connect to the microfluidic chip, a media containing glucose, M9 plus 0, 2% glucose, and a media containing no carbon sources that is simply M9 not supplemented with any carbon source. Using a pressure controller and a feature of our microfluidic device, we are able to switch very rapidly from M9 glucose where the cells are growing exponentially to M9 0 where the cells will be starved for carbon. And what we observe is that the cells enter very quickly a gross arrest. So on this plot you can see the gross rate and the average gross rate across the population is the black line and it shows that the cells in general at time zero where it's the moment of the switch are entering gross arrest. And you can see that individual cell traces that are showed as these thin blue lines and the ribbon corresponding to the standard deviation across the mean shows that individual cells are also entering gross arrest. So there is an homogeneous gross arrest that is very abrupt at the moment of the switch. So now that I've showed you that we are able to follow individual cell and their gene expression a long time as we switch them from exponential gross to carbon starvation, I can come back to the question that we want to answer and show you what determines the present concentration inside the cell during starvation. So we primarily follow the volumic production rate that is a proxy for the activity of the promoter we're interested in. And out of the 20 promoters that I've studied today I will show you only three of them that are covering the spectrum of behavior we observe for all the promoters we studied in our work. On this plot you can see the first example for RPLN that is ribosomal promoter and on those plots the thick colorful line corresponds to the average across the population while once again the thin colorful line corresponds to individual cell traces. Here I show the volumetric production rate normalized by its value in exponential phase and I'm showing you 20 hours of starvation out of the typically 60 hours that we perform simply because most of the dynamic is happening at the entry into starvation. In the case of promoters such as RPLN the volumic production rate is sharply decreasing at the time zero and then remain relatively stable around zero for the rest of the stationary phase. On the other end of the spectrum we have promoters such as BOLA that is controlled by RPOS. RPOS is an alternative sigma factor that is implicated in the response to starvation and stress. And as expected for those promoters we see an increased activity but remarkably this peak of activity at time zero is then followed by an exponential decrease of the production rate, so the promoter activity, towards zero. Finally we have promoters such as HSLV that is a heat shock promoter in this case where we don't see an up regulation or down regulation at the entry into starvation but we simply see that the production rate is bettering out. Now since most of the dynamic seems to happen at the entry into starvation we were wondering if we consider only simplified dynamic at the entry into starvation are we able to predict what would be the concentration resulting from this production activity. And concentration is the interesting parameter here because that's what will drive the phenotype of the cell ultimately. So we are wondering how those difference of behavior are translating into differences of concentration. So here I will show you a very simple model where we consider the interplay between production the simplified production dynamic, dilution and degradation that are like coming together to lead to a certain concentration. In our experiments the dilution is dropping to zero instantaneously so we model it like that. The degradation we can measure experimentally for fluorescent protein. I don't have the time to explain it here but it's not going to be different for different promoters so we really expect that actually the difference will be led by the difference in terms of production activity. In the simplest case that corresponds to our ribosomal promoter case the production rate drops to zero at the same time as the dilution so the promoter simply stops being active and in this case we expect the relative concentration to actually go down under the effect of the degradation. Now simply introducing a gradual decrease similar to what we've seen for the promoters at Petor's app this heat rock promoter is enough to produce an expected increase into the relative concentration and this is due to the fact that even though the dilution has stopped the production keeps lingering on for a while and therefore the concentration is expected to increase. Now if we add a peak of production at the entry into starvation such as we've seen for about an hour deep we've modulated further the expected concentration. And so here it's really fundamental to realize that it's the delay between the arrest of growth and of the production that shapes the concentration in the cells. And so of course yes dilution stops so that means the turnover of protein will be diminished but the fact that the dilution stop also offers the cell the opportunity to change dramatically the concentration of certain gene product even though there is no upregulation of the promoter. Sorry just to be sure so but in the previous plot so I should expect that for longer time scales of course the concentration should drop. So given that the degradation rate in our that we measure is exponentially decreasing we actually expect that it sort of stabilize and also like yes in this model since the production drops to zero you would still expect that it sort of decreases but like at very long time scale. Because the degradation goes to zero. Yeah I like sort of yes it exponentially decreases. So we basically use inducible promoter so we keep the production of the fluorescent protein then we let the cell enter starvation and stop the induction of the protein so they start with a certain pool and then we measure very at very long time dynamic the fluorescence and see it decreasing. So that we neglect the photo bleaching effect because we measure very rarely and we are able to measure the level of the protein. Now when we look at our to come back to our experimental data when we integrate the production along time along with the concentration that we measure the degradation that we measure exponentially we indeed observe that the concentration of the for the three different promoter is behaving as we expected for a more very simplistic model. So that really means that the dynamic at the entry into starvation is really important in setting this concentration and what we were surprised by is the fact that it seems that individual cells as can be seen by the standard deviation like the ribbon around the mean seems to behave quite similarly and more similarly than to in the other cases of nutrient switches or something like that that we observe in the lab. So we wanted to quantify how homogeneous the cell are responding to starvation and to do that we can look at a metric that quantifies the amount of variance that is explained by the fluctuation of the average behavior. So I will explain that with this cartoon on the top of the cartoon you can see that we have individual cell traces that are those seen lines and all cells are considered acting independently. In this case looking at the variance across all the observations or across cell and time we can see that it's much bigger than the variance of the average behavior across the population. On the contrary if all cells are behaving exactly the same we expect that the average behavior across the population recapitulates the average the behavior of individual cells and the fraction of the variance of the total observation that will be explained by the variance of the population average will be big. Now when we look at individual cell traces of concentration for different promoters in the case of RPLN we see that before times zero it looks like indeed the cells are fluctuating a lot crossing each other and after we switch them to starvation it looks more coherent. This is even more true for HSLV and BOLA where we look at individual cell traces it really looks like after we switch them to starvation they respond very homogeneously both in terms of timing and in terms of amplitude to starvation. And it seems that most of the variability or most of the dynamic is most of the action is happening early on in starvation so we want to ask how homogenous is the response of the cells or how homogenous is the behavior of the cell at different time points in our experiment. So we can divide the total span of our experiment into three different time windows what's happening during the exponential growth, what's happening during the early starvation that we fix at 10 hours because this is an arbitrary threshold but that is based on the fact that most of the changes we observe are happening early on and what's happening during late starvation that are the remaining 50 hours of starvation. And when we look at this fraction of explained variance we see that in all case the blue bar the corresponding to the transition is substantially higher than what's happening during growth and during late starvation and this is particularly the case for HSLV and BOLA. So this really says that the cells individual cells are acting much more homogeneously during this transition than they would during growth or during late starvation in terms of this promoter activity. So this really sort of like shows that the response to this abrupt growth arrest is maybe a programmed response. Now I've shown you that like most of the dynamic is happening early on in starvation but if you remember one of the point I started with is that in the literature there was some report of constant activity during starvation. So this means that we have to decipher okay what fraction of the phenotype of the cell is dominated by sort of basically what is the importance of this early production dynamic. And can we decipher whether this plays a big role in the fact in the phenotype of the cell or whether the residual promoter activity is actually dominating because over 50 hours of residual activity maybe it doesn't really matter that the cells have been very active at the entry into starvation. So once again what we can do is divide our time window into three different time periods what's happening during growth what's happening during early starvation and what's happening during late starvation in terms of production. And at every time point we can ask out of the pool the total pool of protein present in the cell for reporter during starvation what fraction come from which production phase. So we expect that at time zero the 100% of the protein will come from what has been coming from growth because we didn't start considering production that production that is happening in starvation. Until time 10 hours we don't expect any green to be visible because we consider early starvation to span from zero to 10 hours and after 10 hours we will start considering the protein produced in the late starvation so the remaining 50 hours. So in the case of LPLN you can see that across time the red sector is largely dominating and this is not surprising because the production drops almost instantaneously to zero. So the state of the cell at any time point in starvation is dominated by what the cells have been inheriting from their exponential growth phase. Now if we go from to our promoter that is petering out you can see that the blue sector is taking over very quickly at the entry into starvation. So this means that the phenotype of the cell like that they had in exponential growth is quickly forgotten and quickly replaced by a new phenotype that is set by the early production events. And after 60 hours of starvation you see that the blue sector still dominates the fraction of the protein so it means that after 60 hours of starvation the phenotype is still dominated by the early production events. And this is even more true when we look at promoters such as Bollard at Displayopik where here the blue sector at the end of the starvation is largely dominating the pool of protein. So this really tells us that the phenotypes of the cells are set early during starvation and are kept for a very long time. And so a way to interpret that is when cells are running out of food they have limited resources and limited time to act and change their phenotype. And they will do so very quickly and after that they will be set in the state they managed to reach for a very long time. But of course the question that we can ask is okay does that matter at all for anything? And so like if what we think is correct if the phenotypes are indeed really set early and that relevant traits are set early we could ask, okay what is the consequence of perturbing this gene expression on some relevant traits? And one of the relevant traits that I was mentioning in the introduction is the fact that star cells are globally more tolerant to a various range of stresses. So we decided to expose the cell to an oxidative stress, yes? Hey Drew, is this the plot that you just showed? How much production contributes to the protein pools? Can you say something about how much do you think an active remodeling would contribute to the protein pools, you know active degradation and then making proteins out of? So what we would expect is for example if the cell were able to regrow now if I would switch them back to exponential phase and ask the same question, I would expect that due to dilution the turnover would be very quick and that like the phenotype will be reset very quickly, right? But the thing is protein degradation has no chance to reach this amount of turnover because it consumes energy. Most of the proteases in E. coli are ATP dependent and in the absence of carbon source the cells are just unable to do it. So in the literature there are some reports of active degradation at first and we also observe that actually on our fluorescent protein. The degradation is faster at the entry into starvation and then starts decreasing. So it would increase the turnover and we would expect to have a faster replacement but practically we don't think it's really happening somehow. So in your experiment in order to induce the starvation you just switch the media from glucose to nothing basically to minimal medium. So what's your intuition about where do the cells take the resources in order to express the other protein? I mean through degrading the protein that are already there through some medium that maybe it's still around or? So we don't expect that there is medium still around because the switch is very, very quick and actually the fact that the cells and our growth are so quickly is showing us that we replace the media very quickly. So we would rather bet on something like a bit of recycling of internal components although this is limited by the protein degradation. And also potentially some reserves in the literature there are some reports of glycerol stocks that are very short lasting and stuff like that. So I would expect the internal pool of the cell to play this role but we actually don't know. So in your experiments you shift very quickly right to the carbon free medium? Yes. So how much of this very quick response determines this initial condition dependency in some sense? So if you were to shift it more slowly I guess you would see less of this effect. So we also conducted these experiments by using batch cultures where we flow batch cultures through the chip and the cells are sort of then growing in this limited nutrients and enter progressively stationary phase. And so the cell in the chip are experiencing the same sort of progressive change of media and we see a remarkably similar pattern in terms of gene expression. So it's actually I think it would be interesting to push it in even slower growth and so on. But for a standard like starvation stationary phase entry we actually observe the same thing. Okay thank you. What is the mechanism of very rapid activation of RPOS? Is there an anti-sigma factor for this sigma factor? So how can it you know again I know that the sigma factors are usually sequestered with anti-sigma factors and the purpose of this is to do everything much faster than you would do if you just relied on transcriptional regulation. So what is what is going on here? The main up regulation is linked to the fact that RPOS is during exponential phase constantly degraded and this degradation stops when the ATP gets depleted. So the pool of RPOS is actually piling up. On top of that you have a tiny bit of transcriptional up regulation and you have the activity of a protein called CRL that will actually sort of help RPOS to win the competition for the RNA polymerase, RNA polymerase versus sigma 70. So you have like a couple of different mechanisms that are actually pushing RPOS activity to shoot up very quickly. But the main one is really like that you stop degrading it and so suddenly sort of the constant expression is getting bigger. Okay and a related question again or maybe not so related. So here maybe not but I kind of got confused so here in the left panel you see virtually no degradation so everything stays roughly constant. And in the previous few slides you were showing that you measured rapid degradation. So what what is the difference between? So what I've seen what I've shown in the previous slide is sort of basically is that like the production activity is dropping right? And so that's why here you don't replace the pool of protein whereas here you sort of like your protein production keeps on lingering on for a while and that's why you start reshuffling the while you start replacing basically adding up some protein and sort of diluting the other ones out basically.  But in a couple of slides ago you had something which I interpreted the overall proton white degradation but maybe I was wrong. Yeah something like well there was some experimental data you said that you measured it. What we measure is the degradation of the of our fluorescent protein and this is what drives RPLN concentration here to go down on the left. And this is an effect that also touches on HSLV and BOLA but because those are producing more than the degrade you see the concentration increasing. But in the case of RPLN because the production drops very sharply the degradation is actually contributing to reduce its concentration. Yes so does that matter at all and what we can do is test that by submitting the cells to an oxidative stress. And asking okay now if we start perturbing the gene expression in various ways how does this affect the relevant trait that we're interested in. Here tolerance to oxidative stress. So the first condition we use is not perturbing anything at all. We simply switch the cells from M9 glucose where they grow exponentially to starvation just as I did up till now. And then submit the cells after 20 hours of starvation to 5 hours of oxidative stress. And switch them back to fresh media in order to ask what fraction of the cell is able to regrow after those 5 hours of oxidative stress. And this is a measure of viability how many cells are able to regrow within 20 hours in M9 glucose. When we do that and we follow the BOLA reporter as previously shown we observe the concentration is ramping up very quickly and is relatively stable until the point where we hit them with the stress. And when we ask okay what fraction of the cell are able to regrow after the oxidative stress we see the fraction of surviving is almost 100% in our cases. So like we use a rather mild oxidative stress when the cells are let to express gene as normally. Now the second condition we can use now is okay if we kill our POS we expect that actually the starvation and stress response will be killed as well. Because this is the main sigma factor implicated in this so it's kind of like the extreme where we prevent the cells from having this stress and starvation response. And we can submit them to the same treatment. When we follow the concentration of our reporter we see that as expected it remains extremely low during the entirety of the starvation. And now if we ask what fraction of the cell is able to regrow we see that almost none is able to survive this oxidative stress. So this really highlights the fact that our POS as was reported before is fundamental in this process and we believe that part of this is due to the fact that it allows cells to kick up some genes during starvation. But if you pay attention you'll see that during exponential growth there is already a much lower level of the concentration or like a lower level of the concentration of the reporter. So this means that the RPS knocked out strains are not able to upregulate upon the entry into starvation but they also start with a lower level. So probably this response that we observed is a combination of not being able to increase the level but also starting with a lower level. And what we're really interested in is what would happen if we simply prevent the cells from expressing during starvation. So we use chlorophenical in order to inhibit gene expression only during starvation. So we let the cells express normally during their exponential growth and as we switch them to starvation we also put them into chlorophenical that is expected to reduce their ability to express genes. Following our fluorescent reporter here we see that we don't get a complete inhibition but we still decrease substantially the level of expression of these reporters. So this shows that the chlorophenical condition is not killing gene expression entirely but is dramatically reducing it. Now if we ask what fraction of the cells are able to regrow we see that we get about 20% so it means that compared to the situation without perturbation preventing the cells from expressing their gene normally during starvation is decreasing 80% their ability to survive an oxidative stress that is concurrent with starvation. So this shows that like expressing gene is important during starvation for a relevant train. But my main point was to say that actually what matters really to set the phenotype is the early production events. So now we can ask okay now if we let the cells express normally during the first five hours of starvation would this change anything we would expect from what I explained earlier that it would actually allow the cells to recover quite a bit of their viability because they would be able to set their phenotype early on and then expressing or not expressing wouldn't make such a big difference. What we see is that the concentration of our fluorescent reporter is indistinguishable from the concentration without perturbation which was expected because we've seen that most of the dynamic in terms of gene expression was happening early on. And now looking at the fraction of the cell that is able to survive we see that we recover an extra 50% of viability compared to inhibiting gene expression during the entirety of starvation. So this really shows that having early gene expression being allowed to express only during five hours is really allowing to recover like 70% compared to the RPS knockout. So this really tells us that this early gene expression as we were hypothesizing before is fundamental to set the state of the cell and then the cells will keep this state for a very long time during starvation. And so it's fundamentally different from the exponentially growing case where we can look at the cell, check how much it expressed or what is the concentration of the protein at the time t and because of the fast turnover we get an information about the gene expression status and like it's not really required to know the full history of the cell instead of exponential growth to understand its current state. In stationary phase we think that it's really fundamental and everything is played very early on, all the action is happening early on and then the cells are frozen in this state and they have to deal with whatever comes with what they were able to do early on. So I will summarize with this. I showed that the delay between production arrest and growth arrest is resulting in a rapid reorganization of the protein across a single E. coli cell. I've then showed that the phenotype is then set early on in starvation and is kept for a very long time and that this early expression is critical for relevant phenotypes such as the survival to concurrent stress. With this I would like to thank the Sanimvegan group and especially the people that have been directly implicated on this project and I would also like to thank you for your attention and I'm happy to take any questions. Thanks a lot. We have time for a couple of questions. So maybe I missed it, but so you measure degradation rates. Do you observe any specific time course of degradation from exponential phase to the shift until long term observation? So we are not able to measure it with our method in exponential phase. But it also matters much less because the dilution rate will set the rate of the turnover of the protein. But because we are relying on the fact of being able to sort of shine light once and then 10 hours later on the cell and measure the level of protein, we wouldn't be able to do that on exponentially growing cells because obviously this is not the same cell that we can observe across time. So in the way I'm doing it, I'm just assuming that the starting level in starvation is the level of degradation in exponential phase, but it's anyway a negligible effect compared to the dilution rate. Sure, sure. So you wouldn't be able to tell whether cells increase their degradation rates from exponential phase to whether they activated. No, that we are not able to tell. Yes. So the transition into starvation phase, the processes that are happening to the protein, do you think it's a program that once it's triggered it will just run until the end? Or do you think it would be stopped and adapted when in that very moment nutrients become available? So I think that the RPOS activity is a program response, but I think part of it is also coming from the dilution rate that stops, right? And if you would put them back into fresh nutrients, I expect like in the middle of this transition phase, I actually expect that it would start to regrow quickly and that all the sort of processes that are responsible of like... Well, basically you start returning over your protein very quickly and your phenotype is able to recover very fast. On the other hand, if we let the cell long enough in starvation, then you see a very long lag phase before they start regrowing and the duration of the lag phase has been reported to be a function of the duration of the starvation. But if you let the cell in one hour during carbon starvation and you switch them back to glucose, they are able to regrow without a lag basically. So maybe there could be some kind of feed forward loop trigger, like making this asymmetric process, like that switching on and off are different, yeah? Yes, yeah. So my question is exactly about this lag which you mentioned in answer in the previous question. So do you have any intuition of what sets the duration of it? Because here's where I'm confused. So the main message of your talk is that first five hours or maybe 10 hours are critical and then nothing changes after this. And that means that there should be no duration or no influence on duration on the lag if you stay 20 hours or 30 hours or whatever. Yet you just said that the duration of the stationary phase is very variable. So just tell me what is how do you understand this lag on reentry to the exponential growth? So it's a fair point. What I'm mostly focusing on here is sort of the phenotype that is relevant for starvation. But of course, as you mentioned, the longer we let them installation, the more they will struggle to get the right phenotype when they have to regrow. I think this could be due to several things. Like you have this ribosome, a hibernation system that starts coming in. And also, we also know that if we let the cells long enough in starvation, they will eventually start dying. They cannot sustain this state for a long time. So they accumulate some damages and that they have an accumulation of misfolded protein and components that are toxic for the cell. And then they get sort of, they have to get rid of that and put things on their feet again before being able to regrow. So this is sort of, I would say, this is sort of an aimed phenotype in the sense like it feels like the cells start kicking in. So lots of responses, osmotic stress and so on, that they might not primarily need in starvation, but where they wouldn't have the time to adapt in case these kind of stresses would come later on. So some kind of preventive response, if you wish. And another aspect is the thing that are going to degrade anyway because in the absence of energy, the cells are not able to sustain for a very long time. Okay, so we have one last question. So I was wondering, you showed for three different promoters kind of the dynamics. And now then in the end, if I understood correctly, I mean you showed the knockout, the behavior for the knockout of RPOS, which I guess is not surprising. And then the inhibition of most, how would you say, of most processes. So I was wondering, did you take any more, do you can comment anything on take home messages from the different promoters that you studied? So it looks like if we see which promoters are upregulated and which are downregulated, typically our ribosomal promoters are following the trend that I showed for the ribosomal promoters. They sharply go down and sort of this is also looked like an active process, an active sort of downregulation. This is in general true for the promoters that we would associate with sort of exponential growth, whatever it means, but like fast growth, such as ribosomes and so on. It feels like this is an active process of saying we don't want that. Also what I find interesting in the case of the ribosomal promoter is that they usually consume a lot of resources. It's like very costly to express ribosome. And so downregulating those very quickly is actually also allowing the sales to use the pool of resources that remains to express a lot of other things. And because the dilution stops, with this limited pool of resources, they're still able to do a lot somehow. That is my vision of the way why these promoters are getting downregulated. Then I think the very important promoters such as Bola and the one that our RPS regulated, they get kicked up very high so that they get a very high expression. And you also have some promoters like this HSLV that is sort of simply pattering out but even this decreasing activity is enough to increase the concentration and to understand really what sets exactly sort of the level of the peak and sort of the time dynamic of this decrease of production activity is not clear yet. But yeah, that would be definitely an interesting question to answer. Okay, let's thank the speaker again. Okay, so we move upstairs for coffee.