[Music] good afternoon everyone thank you very much parently for that introduction and for giving me this great opportunity today first to see some of my colleague and my friends some of them I didn't see them for more than 20 years now and to talk about heat stress and got integrity in the the birds and its ecosystem when Pierre Andre invited me I was a hesitant because I didn't know if I fit in the scope of ecosystem but when we look for the definition of ecology what's the first time was coined in 1866 by the German zoologist Ernest hiccup it's the it came from the Greek word home or place to live however the word ecosystem was used for the first time in the ecology German in one publication by the bridge botanist Arthur tensley who draw the attention to the importance of transfers of materials between organisms and their environment and here you see the environment today the definition of ecosystem and I took an example from Wikipedia is the community of living organisms in conjunction with non-living components of their environment interacting as a system and I I found here this nice picture from Smith group from Netherland who published last year in frontiers in microbiology illustrating the chicken ecosystem my lab has been interested for a long time in the effect of heat stress and the regulation of energy homeostasis and thermo regulation at the central level of the hypothalamic level this year in 2019 there was an elegant paper in PNAS journal showing that heat stress has a direct effect on the gut integrity but that paper was mainly interested in the and filtration of macrophage like cells in the genome of dairy cow yet the molecular molecular mechanisms by which hit stress after that gut integrity are not well defined in that regards my lab has become recently interested in the effect of heat stress on the gut which is considered at the second brain I am NOT gut anatomist or physiologist by any means but I am mainly interested in the defining the molecular mechanism by which heat stress after the gut epithelium and as dr. Gong mentioned early the epithelium integrity is under the control of protein complexes and here there is is a somatic representation of the enter epithelial junction which is composed of tight Junction at the injection this Muslim and gap Junction the tie junction are mediated by addition protein including occluding Clawdeen's and john form from junction adhesion molecules the others appearance ejection are composed of mainly cuddling and alpha and beta catenin and the gap Junction are mainly the connecting hexamers all this protein are tightly regulated by different signaling pathways and their signaling pathways varied between species and also depend on the physiological context of the animal and the stress and the stress the duration of the stress the type of stress and also the severity of stress there are many signaling pathways are involved in the regulation of this protein and i hesitate whether i present another review of all those pathways which i don't think that you need that you don't need me to do another review you can find by yourself in PubMed or I present some of our results and that's what I prefer I would like to show with you some of our work because I need your valuable input I hope during the discussion we can talk about that among this molecular pathway there are heat shock protein and their related transcription factor heat shock factor TLR dr. gonna talk about the other four nitric oxide synthase and p53 map kinase and phosphatase the autophagy machinery the interlocking one pathway and TNF pi3 kinase and protein kinase B microbiota and their metabolites and also non-coding RNA including the micro RNA and long non-coding RNA start for signal current user and activator of transcription and win a wind signaling for the limited time I would like today to talk about only and Hitchock protein and enter looking one data pathway and show with you a share with you some of the results that we we had for this we we have conducted an experiment the physiological environment - research lab at the University of Arkansas we use the complete randomized design we have 12 under each chamber contains two pens 24 tens and we use 25 birds per pen 300 birds per group and each environment air-conditioned the reading conditioner standard the temperature was gradually gradually decreased from 32 degrees Celsius to 24 by day 21 and we increased that up at day 28 to 35 degree within 10 minutes we have 35 degree and the heat stress was cyclic 12 hours per day from 8 a.m. to 8 p.m. and we sampled that tissue after two weeks of heat exposure we took several metabolically important tissue from the brain the hypothalamus to the liver to the gut segment and and muscle we also recorded the growth performance feed intake and water conception on the daily basis body weight on a weekly basis and also we measure the core body temperature by using a thermo logger here as you see on the Left the environmental chamber works as we designed is a beautiful cyclic heat stress the relative humidity was between 20 on the right side between 20 and 40 percent as we expect it's stress increased the core body temperature in our experimental condition there is an increase between 1.5 to 2.5 degrees Celsius and it is also cyclic one of the most prominent effect of heat stress is depression in feed intake and as you see half heat stress significantly reduce individual and cumulative feed intake which in turn results in significant reduction in body weight and body weight gain increase of FCR by eight point and syndicate reduction of feed efficiency and we didn't see Cynthia difference in term of mortality three versus five percent in term of carcass quality and parameter all the parameters that we measure except the fat content who were significantly reduced by heat stress as you see the carcass the breast yield the tender the wings the leg and the rack however fat content there was no significant there's a slight decrease but is not significant different and this data is quite different from the one that PR andriy published in 1986 where he or 96 were he showed that heat stress increase fat abdominal fat content and thus this discrepancy might be related to different factors the birth of 1986 and the produce world today are different the experimental condition I think are different the diet may be different and also the duration of heat stress different than I am not surprised that we see this difference indistinctly we found that heat stress reduced the incidence of woody breast the moderate woody breast here from 57 to 24% and the severe woody breast from six point five to two point three percent and this is not surprising because the bird eat less and they have lower body weight we also found that heat stress significantly increased the individual and cumulative water conception in terms of blood parameter we used the I start a lenity system that allow us to measure 13 parameter including pH glucose electrolyte and hematology and here I presented only the pH the co2 and B carbonate and as you see all of them were significant were increased the pH significantly increased but other one was who they were increased which indicate we are in state of alkalosis and this is also known from the effect of heat stress and we did also some behavioral measure measurement including time spent eating and time spent drinking the frequency of eating and activity and also panting and all of all our data indicate that the birth heat stressed now the next question what is the effect of heat stress on the gut and degree to do that we used fitzy the extreme technique which has been optimized by Billy Hargis and mimmo Chile's group and we found that his stress increased the serum fitzy compared to the thermal natural counterpart and we have one paper and the revision in poetry science where we evaluate the heat stress as a model for leaky gut in embroiled I have to tell you we have one limitation here we cannot distinguish between the effect of heat stress and the depression in feed intake we missed we didn't use perfect control in that experiment to compensate that and to understand the molecular mechanism we used an additional in vitro model we used high-tech j2 cells from intestinal ports in episodes obtained from the genome and we use the trans well coda gene coated insert with two chamber here a pecan and basolateral chamber we have the cells here we have some medium on the apical and some medium in the basolateral and we monitor the formation of the mono layer on a daily basis using fluorescent microscopy and also teal transit material electrical resistance day 10 I don't know if you see well from there at bay then we used a double staining for clothings clothing one and clawed and five gloden one was in green dye and the clothing 5 red dye when you combine red and green you have that beautiful yellow orange color you see here we have the beautiful the blue dot a are the nucleus and the orange does the tight Junction approach we have a beautiful mono layer and beautiful mosaic and this also is supported by the tear that reach maximum at day 10 and stay constant for seven days and then we exposed the cells to the heat stress and as you see with heat stress till significantly dropped after fifteen minutes this indicate that heat stress also directly the tide junction we repeated the same experiment and in this case we add in green hair the third seed extreme and we expect if there's alteration in the tight Junction we will find that third seed extreme and the basolateral champ and sure enough dick Smith fits in the bio lab rattler chamber significantly increased after 16 minutes as I said the gut integrity is under the control of ty Junction addition Junction and gap Junction protein then next we used immunoblotting technique and we measure the expression of claudin v expression in the three segments of duodenum jejunum and ileum and we found that heat stress significantly increase or up regulate the expression of cloth and v and this is these results are in agreement with one paper from plus one from 2015 where they found that heat stress increased the chicken the clothing fighting the chicken you genome and ileum but that paper used only the mRNA the transcript level not the protein level and also we found that heat stress articulate the expression of uploading and closing v in ITEX cells we use here a PTH as housekeeping protein and again thus data are in agreement with the results from 2017 in scientific report where they used the brain cells and heat stroke and also from toxic on in vitro 2015 when they used sertoli cells and heat shock protein then from here we can conclude that heat stress after the gut integrity may be we are dysregulation of tie junction of protein on the right side here we used immunofluorescence and confocal microscopy and you see the TN is thermal neutral condition we also staining for occluding clothing 5 and clothing 1 we have a beautiful mono layer and mosaic structure however with heat stress if you look well here that tie junction is completely dysregulated now what is the mechanism how heat stress alter that tie Junction and this regulate tie junction of protein expression I found this paper in the American Journal of pathology from 2008 where they show that heat directly effect heat shock factor 1 and heat shakhter factor 1 is activated and translocated in the nucleus bind to the heat shock response element in the uploading promoter and then use the transcription of uploading and then the proteins entities of uploading and then effect the tight Junction for that we merged several known heat shock proteins including the heat shock protein 60 70 90 and GRP 75 and we used the pd h as housekeeping in all the three segments but what we found that heat shock protein expression seems to be tissue specific and i will give you an example here if you look here in the middle here a choc protein 60 is up regulated by heat stress in the duodenum and the ilium but not in the genome similarly there relate to transcription factor each of factor 1 2 3 & 4 seems also to be regulated in a tissue specific manner if you take each of factor 1 is also up regulated by heat stress in the duodenum and the ileum but not in the jejunum the only protein that seems to be consistently up regulated here is the GRP 75 and similarly for the ITEX head GRP 75 was significantly up regulated by heat stress and even the amplitude if you look here it's more and is higher compared to other heat shock protein then we shift a little bit our attention instead we still working with each of protein and heat shock factor but we focus on hit on GRP 75 a few words about GRP 75 it has been characterized in 1989 they found it belongs to the heat shock protein family 70 but in my million tissue but they found that it is regulated by glucose and that's why they're called GRP for glucose regulated protein and 75 because the molecular weight is 75 kilo Dalton at the same year there was another paper where they found that this protein also associated with cylinder motor phenotype in mammals and they called mortally if you look in the literature you will find this protein under different name ERP 75 mottling PBP 74 p66 MOT 1 etc what is interesting about this protein that it has a dynamic sub sub cellular distribution you can find in the nucleus but also in different organic mainly the mitochondria which explain its Platonic play tropic and versatile function from housekeeper to Guardian to killer it depend again on the physiological context and this rest type what is an intriguing it belongs to each of protein 70 family but is not induced by heat stress in mammalian in my lab we used IP amino precipitation coupled with the mass spec and we characterize GRP 75 in both species chicken and quail here I present only the chicken and we found that the sequence the amino acid sequence has higher homology more than 81% with different species from a swine - Mouse - rat to human bovine lizard frog and chicken and if you are interested about this sequence we it has been submitted to pride under the accession number P xd0 13591 feel free to check bio informatics and 3d structure prediction using P more modeling program showed that as for the other heat shock protein that the chicken GRP 75 has peptide binding domain and also ATP binding Toman and also showed that ATP binds to the chicken GRP 75 via designated ATP binding pocket we also found that chicken GRP 75 here in green the structure much perfectly the structure of the heat shock protein h HS p 5a and they share the same ATP pocket from here we can conclude that the chicken gr p 75 belongs to the heat shock protein family when we merged gr p 75 expression in order to show we also find as for the gut hates the significantly up regulate chicken gr p 75 in the liver in the muscle and in the hypothalamus from that we conclude also that the chicken gr p 75 in contrast to its homolog mammalian human homologue is heat stress responsive in terms of localization we used amino Florence staining the blue hair is duppy for the nucleus the green that is the gr p 75 the red is the micro tracker which is specific dye for the mitochondria and when we put them together the merge you see that orange yellow color that's indicate that the chicken gr p 75 is predominantly localized in the mitochondria we did the same for the Golgi C's and trans-golgi we used Eric 53 and TGN 38 died we also find to less extent than the mitochondria but gr p 75 is also localized in the Golgi and it is also localized here in the ER by using PR tracker and VIP todai specific for the er on the left on the right side here we also found that chicken GRP 75 is localized in the lysosome by using the lamp - and lies a tracker as for mammalian GRP 75 chicken GRP 75 has multiple subcellular localization the expression in the Golgi and in the earth suggest that this protein might be secreted and this is not surprising because recently we found that each of protein 60 is in the plasma of a human and the expression in the lysosome indicate that chicken GRP 75 might be involved in auto focus on formation and auto fashion auto-fire is a self eating or self digestion process and I will talk about it in a little bit this data are under revision in scientific report and the lab is now working on the role of the chicken GRB 75 in Thai Junction and epithelial integrity by using heat stress and also combination of genetic manipulation by over expressing or knocking down in 75 now I am going to switch gear to the second pathway and they're looking one beta and here in 2014 there was a paper in Journal of cell science where they show that enteric in when beta affect clothing five promoter and tie junction protein via protein kinase B or ATT fox one and also cuddling and beta catenin however the paper showed that this effect is mainly mediated by the via down regulation of protein file which is different than for us we found that there is an upregulation of tie junction they found a down regulation which make us thinking that maybe this is not the right pathway but we were still interesting what is the effect of heat stress on the other instruction cuddling and beta catenin what is the effect of heat stress on the entire looking one data expression we found that it says increase the expression of that Eric in one beta but also significantly now regulate the expression of cuddling then instead to go downstream of anti-black in one beta what are the upstream mediator of anti looking 1 beta how it is activated how heat stress activate and TELRIC in one bed we know that enteric in one beta and enter look in 18 are downstream of nlrp3 inflammasome complex another p3 contained three proteins another p3p car or ask and the pro caspase-1 which is involved in apoptosis this protein complex is about 700 kilo Dalton when it is an activated by stress or any type of an salt that inflammasome is activated lead to cleavage of caspase-1 which in turn and use the production of pro-inflammatory cytokine enteric in 18 and enter the King 1 beta few years ago we published two paper in nature and cell in collaboration with dr. on body from the University of Kentucky and we found that interleukin 18 is downstream of another p3 activation which is induced by Dyson 1 this regulation and one last one is deregulated and use accumulation of double strand RNA you repeat element and that accumulation of cytotoxic double strand RNA and use mitochondrial ross few words about die so die sir is a key enzyme involved in non non-coding RNA biogenesis if you have for example micro RNA genes when it is a transcribe is processed by Pasha or digi cr8 and the Trojan in the nucleus to give pre micro RNA this B micro RNA is exported to the cytoplasm we are exporting Phi it dice it or cleaved by dice or to give mature that micro RNA and that mature micro RNA duplex entering the risk and are gonna machinery and target mRNA regulation that's for the micro RNA for the double strand RNA they came from bite dyke bi-directional transcription from repeat element and once they are transcribed I must stay in the nucleus we call nuclear retention because they are cytotoxic if they go to the cytoplasm they induce apoptosis and they kill the cells and that's what we saw in the world that we did in ink attack I found a few papers showing that I'll you repeat element and double cylinder and a bind Hedgehog factor and also they are involved in heat shock response and we have one paper here from molecular said from my collaborator Google and Goodrich from the University of Colorado where they clearly show that heat shock on the right side here significantly increased the expression of audio RNA by northern blotting first we measured the protein expression of Dicer one in the three segments of the gut and as expected we found that heat stress down regulate the expression of Dicer one and on fire on the right side we developed also analyze affordable strand RNA by using specific antibody that recognized any type of double strand RNA longer than forty BP in sequence and dependent manner and we found that heat stress increase the level of double-stranded RNA in all those segments the same for the IKEA sells on the left side here heat stress significantly increase the level of double strand RNA and as heat stress down regulate Dicer we used si RNA and on the antisense oligonucleotide we knockdown Dicer and we found also that Dicer knockdown increased double strand RNA level in the itec cells both of which reduced the cell viability if you look on the right side here in the thermal neutral condition we found those double strand RNA are mainly in the nucleus you see here they are many i can mainly in the nucleus under heat stress those double strand RNA diffuse in the cytoplasm and this was associated by the green one here the the SG is for stress granule it seems like double strand RNA and stress granule are Co localized far on the right side we also see that heat stress reduce the expression of nuclear protein P 54 and RB and the down one here we used electron microscopy and as you see under thermal neutral condition we have a beautiful nucleus with nucleolus with nuclear envelope and also with nuclear pore however under heat stress the nuclear envelope become thinner and we lost those nuclear pore to me it seems that the key got before the leaky gut there's a leak in nucleus and that why those dubs and RNA instead to maintain in the nucleus they go to the cytoplasm and then use whatever type of no stress and death pathway what we did here we used that repeat element and we made a plasmid and we overexpress we force to be over expressed in the cells and we use double staining amino fluorescence we used micro socks which is specific for rows from the mitochondria and also micro tracker which is specific for the mitochondria on the upper level here there is nothing but if you look we have a lot of mitochondria you put them together they give that yellow color which means that double strand RNA and use ROS which comes mainly from the mitochondria on the right side we used also electron microscopy and and the third monetary condition we have the healthy mitochondria however on the right side that mitochondria change the shape it is elongated from here we can conclude Oh to the double strand RNA and use micro control or loss and double strand RNA or heat stress and use mitochondrial maybe dysfunction or change of the morphology I found this paper where they showed that mitochondria and use inflammasome activation just a few word about the life cycle of mitochondria the cycle of mitochondria is dynamic that why we called mitochondrial dynamics it is under fusion and fission event the fusion here is a fusion of two mitochondria to make a new network and to mix their content few minutes later it under enter fish where the mitochondria split in two mitochondrial daughter if the mitochondrial daughter is healthy and has a high membrane potential it will enter again in the fusions cycle but if it is not ready it will stay solitary and recovery if there is no recovery it will enter in the pre auto physique pathway it will be eliminated by autophagy I told you Auto fashion self eating or self adjusting process and it can be specific for each organelle in the case in the case of the mitochondria it is micro fashion and the case of ER it is reticular fashion in the case of a ribosome it is rebel fashion then if that mitochondria is not ready and is not healthy it will be eliminated by micro fudgie process on the right side and the stress condition and here starvation in our case is heat stress that mitochondria will stay in a post fusion state and that why it is elongated if you see elongated the self tried to be more efficient then we measure several genes associated with mitochondrial dynamics in this case we measured Dyna mean 1 DN M 1 and we made we measured microfiche in 1 & 2 they are they are key genes in mitochondrial fusion and we found that heat stress selectively increased the expression of microfiche in one which may explain that post Fusion state we also measure several genes involved in mitochondrial function including nerve - and sky and we found that heat stress increase the expression of sky which indicated heat stress affect mitochondrial function and also we found that heat stress significantly up regulate the expression of P G c1 alpha which is a key genes in mitochondrial biogenesis all this data indicate that heat stress my effect the junction protein via alteration of mitochondrial morphology mitochondrial dynamics function and biogenesis in term of mitochondrial function and bioenergetics we used here seahorse xf8 to measure basal respiration of the mitochondria ATP production proton leak and also maximal respiration as as you see here heat stress reduce all of those parameter for the reduction of ATP we expected and the heat stress you don't have enough energy right because the birth divert all the blood to the surface to eliminate that heat there's no destiny truant less oxygen to the internal organ than you know you have less ATP however the proton leak normally proton leak produce heat here as i said that post fusion state the micro congest or the cell try to be more efficient to maintain that low production of ATP ATP by reducing the proton leak in 2001 in collaboration with dr. Daniel Ricky from CNRS from France he is the first one who cloned UCP one in brown adipose tissue of a human and at that time he called thermo Jeanine we cloned the avian and copying protein we found in human there are four or five UCP in chicken there was only one and is highly expressed in the mass here we use two chicken line + + R - divergently selected for high or low residual feed conception by borders and murat the r-class line consume more than 40 percent then the our minds line but they are leaner because they are inefficient they eat more but instead of producing ATP that produce heat what we found that the avian UCP is highly expressed in the muscle of the our last line the muscle is the main site of thermogenesis for chicken a human they have brown adipose tissue a chicken they don't have that UCP the avian UCP is mainly localized in the inner membrane of the mitochondria here we have the five complex on genetic you have f0 f1 and ATP synthase if you have a proton it goes through that channel and produce ATP from ADP UCP if you have high expression of UCP that proton will leak and instead of making ATP it will produce heat in the gut and the heat stress condition we found that the UCP expression is down regulated that's explained also that reduction of a proton leak for conclusion here we identified the new gr p75 protein that may play a key role in gut and dignity and hit stress we also identified by Sauron and double strand RNA and mitochondrial dismissible ism we are planning to work and delineate those mechanism and we hope that in the future we may help to identify and develop strategy to improve gut and dignity and health and I don't think that I will be able to do that by myself and that way here looking for a collaboration with the colleagues from both University and Industry and both from applied and fundamental research and also I have few scholarship for good student self driven and motivated student I would like also to thank our sponsor I would like to thank my team especially dr. Elyse green and also my collaborator Billy Hargis and mimmo group Liz Libby and Michaela our department for helping sponsoring the student rohana from the mass spec core facility Nick Anthony and Sarah for sharing their genetic lines especially the quail Cena and Nasim Han from the biomedical engineering group Goodrich and Google from Colorado and Robertson Avril from Queensland Australia and thank you and I will be more happy to answer any questions that you may have [Applause] [Music]