Hello. My name is Sofia Blanchard and I'm a student at the University of Southern Maine and an intern in the brown lab at Maine Medical Center Research Institute. And I will be presenting on photo inducible energy expenditure as a treatment for metabolic disease. Obesity as a prevalent and serious health issue. And our world since the 19 sixties, obesity has increased over 40% and is projected to reach 50% by 2030. Obesity is of such concern because it leads to a multitude of other health problems such as diabetes, heart diseases, and even death. Obesity's onset by the accumulation of white adipose tissue, which contain large lipid droplets and few mitochondria. There is however, another type of fat known as brown fat, that is activated by exposure to cold and burns energy to release heat and prevent hypothermia in a process known as thermogenesis. Brown fat contains smaller lipid droplets and more mitochondria. Brown adipose is quite abundant in newborns and hibernating animals. Studies have shown that activation of brown adipocytes can also lower body fat and decreased symptoms of metabolic diseases, thus making it a target of recent attempts at combating obesity. In vivo induction and maintenance of UCP one expression and accompanying proton leak linked respiration is triggered through cold induced beta adrenergic signaling, which leads to activation of adenylyl cyclase and cyclic AMP production. Uncoupling protein one, or UCP One is an important protein and brown adipocytes and functions by uncoupling oxidative phosphorylation and increasing proton league across the inner mitochondrial membrane, resulting in increased thermogenesis and energy expenditure. However, studies have shown that trend planted brown fat may not become innervated. And even if it does, it would be unable to sustain activation over time without cold exposure, which would be very uncomfortable for potential patients for this type of therapy. One potential alternative to cold exposure is to generate cell-based therapies to supplement obese patients with additional brown adipose tissue. The brown lava, MMC, RRI, has generated functional brown adipocytes from the somatic cells of obese patients using induced pluripotent stem cells or iPS see technology and a novel differentiation method. These cells are renewable, thermogenic, AMD patient-specific, making them a good source of material for cell-based therapies. These brown adipocytes can be generated from somatic cells, including urine dried cells that are isolated from obese or diabetic patients in a noninvasive manner. Ipac derived brown adipocytes show high-energy expending metabolic activity and secrete anti-diabetic factors when activated in cell culture, making them a good source of cells for regenerative therapies. This additionally bypasses any issues associated with obtaining samples from obese patients in an invasive way. However, studies have shown that brown adipose tissue transplants Undergo a process of involution and trends differentiate into energy storing white adipocytes due to a lack of sympathetic innervation and cold. This leads to the work we have been doing and seeking novel ways to keep brown fat and the activated state after transplantation. This method involves harnessing the power of photo inducible adenylyl cyclase is or packs, which came from the genetic material of the bacterial genus. Beg you tell her what is unique about these bacteria is that they react to light by producing the second messenger, cyclic AMP, to essentially fire up their systems to him to areas of nutrients. The packs contain a blue light sensing FAD protein domain that when activated by blue light, causes a conformational shift to activate its adenylyl cyclase domain, thereby converting ATP to cyclic AMP. We hypothesize that by genetically modifying IPAC derived brown adipocytes to express pack genes, we can keep these cells in the activated state after blue light stimulation due to increase cyclic AMP, high levels of cyclic AMP induce thermogenic gene expression and results and continuous activation of brown adipocytes. First, we determined which pack of the free available allowed optimal activation of cultured brown adipocytes cells. And we found that to be Deepak as 27 a. We use an immortalized Brown adipocyte precursor cell line known as thermal mass cells. To test the pacs, we were able to generate a pack and cyclic AMP glow sensor, stable thermal mouse cell line, thermal mass, a dip agenda priest cursors were transfected with linearized BY pack as 27 a plasmids, which is neo mice, and resistant and glow sensor plasmids, which is hiker mice and resistant from their cells, were selected on g four 1A and hybrid mice in for seven days to select for stable transfect ends. Not all the cells were positive for red fluorescent protein, or RFP, as determined by fluorescence imaging seen here. And this may be indicative of incorrect integration of the pack construct in many of the cells. We sorted the cells for RFP by flow cytometry to increase the purity of cells containing the desired genetic integration. We then assembled the 24 well, micro plate photo irradiation system based upon a paper by cats at all with help from the US I'm College of Science, Technology and health. Next, we measured the stimulation of cyclic AMP and real time using glow sensor technology with aluminum. And then we collected RNA and protein samples and measured fossil crab and target genes by western blot and qPCR Our initial experiment ourselves for transfected with a biker Shauna backpack, S10 7A, and RFP reporting plasmon, as well as a CMB promoter, which was cloned upstream for high mammalian expression of B pack as 20 7A. And a diagram of that can be seen here. We pulse transfected cells at 463 nanometers for 0.66 or 60 seconds, which are times that previous studies have shown to be optimal are positive. Control was forced colon, which is a known drug that activates cyclic AMP and brown adipocytes. Prior to light treatment or forceful in treatment, the cells were incubated with Glo sensor cyclic AMP reagent for two hours. This allowed for us to carry out a cyclic AMP detection assay. The alumina commoner ten minutes after light. Cosine close enter technology is a luciferase based biosensor that allows for real-time detection of signaling events in live cells. Essentially, more signaling events within cells leads to higher luminescence activity. When measured 15 minutes after the raw luminescence data that we gathered can be seen here above that, our graph shows that six seconds and 60 seconds of light gave statistically significant increases. And cyclic AMP equivalent to R or higher than the observed with the force colon. While the glow sons are assays shows the immediate effect of blue light on cyclic AMP production. We are interested to determine if we can cause sustained activation of the cells over a longer period of time by measuring transcriptional activity downstream of cyclic AMP. For our most recent experiments, our focus has been on finding the optimal light intensity and exposure time for thermal mass predict sites with a newly constructed EF one alpha promoter in the backpack as 278 cells. The cells were transfected, transiently, selected for stable lines, and then flow sorted by RFP get a more pure population. Our data shows that this promoter is more active throughout creative site differentiation. Preliminary results show that a high dose of either for Scollon or light for ten seconds doesn't do much in term of gene expression. This suggests that sustained signaling through adenylyl cyclase is necessary to activate brown adipocytes. When comparing this to our results from the first experiment we did, we saw that only a few seconds were needed to increase cyclic AMP, but sustained activation, the ally is still needed to express UCP one and an R4, A3, which are two important target genes. Through our experimentation, we found the sustained intensity light heated the culture medium to levels that would be unacceptable for high cellular viability. So next we tried lower doses of light that could be shined continuously without heating the culture medium and found that point form and one milliwatt did not heed the culture medium appreciatively. Here we saw that UCP went up in the pre adipocytes somewhat comparable to force colon. Six hours at one milliwatt per centimeter squared seemed to be the optimal time and intensity for light exposure from what we have tested thus far. With those results being here in our first colon here, as well as in our Fourier three. We're looking into possibly pulsing 15 seconds on and 15 seconds off for six hours with a higher light intensity. And comparing those results to our six, our continuous results, our initial experiments have demonstrated that a photoactivatable adenylyl cyclase can be used to increase cyclic AMP production after a short burst of blue light stimulation. Our most recent experiments have shown that sustained light stimulation might be needed to express important genes and brown adipocytes. Overall, we conclude the optogenetics stimulation of packs has the potential to increase activation of brown adipocytes and therefore can be used one day to increase caloric burn an obese patients. If this proves successful transplantation of light inducible brown adipocytes alongside a remote controlled light device will first be tested in obese and diabetic mouse models for proof of principle experiments to determine if these mice become more metabolically active and lose weight. This could be potentially applied to humans. One day as well, I would like to acknowledge the support of my colleagues and special thanks to my mentor, Dr. Aaron Brown. Thank you.