SF2UF Ms. Lauren Roberts, For the Love of Pathogens!


Morning everyone. My name is Loren Roberts Thank you so much for having me this morning. I’m really really excited to kind of share with you my journey from high school all the way to where I am now and the challenges that I faced how to overcome them and just what to kind of expect as a Graduate student or someone that’s thinking of pursuing or doing something in research. So To get started. I just wanted to go over the overview of my project So or the overview of my talk? Which will just be to tell you a little bit about my journey to graduate school And then once I tell you about my journey I will go into what I’m actually studying what I’m actually doing here at the University of us Okay, so to start with I want to give you some background about Who I am I am a home schooler I Am from New York? Not this, New York which everybody tends to think when I tell them but I’m actually from upstate New York up here a Lot of people told me I was Canadian I’m not um, but I am very very close to Canada like an hour and a half away but Being a home schooler kind of presented several challenges That I didn’t foresee when I was becoming a college student So they might not be exactly challenges that you might have faced, but just to Give you some context of what I what I had to overcome in order to become successful in my undergraduate career, so With that being said I want to start with my journey. Um, so I was always fascinated as a kid with Microbiology and just biology in general but I was raised by parents who were engineers so my parents loved math. Um, way more than I did and didn’t really understand my fascination with Looking at leaves are looking at bark or while I was trying to catch butterflies. I would look at their wings, so I was a very odd in the way that I view the world compared to my parents. I Was just yeah I wanted a microscope by the time I was six to look at things more closely to see like if I could see little compartments in cells And when I was eight, I finally convinced my parents to get myself a microscope it might surprise you, but this is actually pretty normal for graduate students in my at least of my program BMS almost everybody in my program had a Microscope when they were 5 years old or eight because they were just so fascinated with what was around them But when I was a kid if you had asked me what I wanted to be when I grew up it would not have been A PhD student. I had no idea what that even was as a kid. Um he would ask me I probably would’ve said astronaut because I loved the Stars or a doctor because I liked helping people or a Cook because I liked cooking in the kitchen with my mom, but it was definitely I did I definitely did not foresee I’m becoming a PhD student But I became more and more interested in biology and just life sciences in general as a high school student and Loved anatomy and physiology and cell biology. So I had a feeling that I would end up somewhere in the health field But I just didn’t really know where As an undergraduate I Really really wanted to go to a four-year university And I thought that’s what was going to be the best choice for me that it would help me later in life But my parents really thought that the greatest art for me would be a community college especially with my history of being a home school student and it probably would have been a Little bit more of a shot going from being homeschooled from first grade to 12th To just jumping with both feet into a four-year university like somewhere far away that I didn’t know Um, so I ended up going to Community College called Monroe Community College, which was 30 minutes from where I grew up So I was pretty close to home. I’m in close to a support system when I started college I It was the best experience of my life. I absolutely loved it And if it was a four-year college, I would have stayed in a heartbeat I learned To like where I was wanted to be in research at this school and even what research was at this school As well as becoming a student athlete which came with those challenges of trying to learn how to balance and time manage Everything with studies as well as games and traveling So Monroe Community College, what is um, yeah, one of the greatest experiences of my life and something I always carry with me So, although I absolutely love my experience at MCC. I Underwent a lot of challenges and a lot of People I guess questioning my ability When I before I even enrolled in classes when I was just applying to the college itself No one believed that I’d actually earned my grades which is a common problem that you seem to have with home school students nobody really truly thinks that what you’re putting on paper is what you’re doing because they think it’s really easy for you to fudge your grades or That your parents can write them in for you. And unfortunately, there are cases where people will do that And I do know people that have done that but my family was not one of them So it was very frustrating to be To have been assumed that what I did was not what I I had actually earned so It started with this woman um Mary Lynch in the administrative office when I first apply it and just came in to talk to her because she had a lot of questions when I was Trying to enroll um, and she was like, why do you expect me to believe that you got these like you have nothing to show that you actually Earned these grades and I looked around the eye and I was like I’ve taken Regents exams Which I don’t know if Florida has Regents exams But it’s a it’s a kind of milestone that you take her like whatever area you’re studying whether it’s history biology chemistry and You take an exam to kind of place you or show how much you’ve learned in this subject and how much you should have learned By whatever Grade you’re in So I took Regents exams to show my progress When I was in grade school as well as taking standardized tests which is also another method of how I Had to show I was actually progressing and doing what I needed to do and learn learning what I needed to learn in home schooling so Even with my Regents grades even with my standardized testing scores It was still a really big Challenge for people to believe that I had actually earned the grades that I did So I had to actually be placed And like take a placement test in order to show that I actually deserve to begin the classes that I said I wanted to take Which was really frustrating and kind of discouraging to be like, okay I’m not even in college yet and I thought it was going to be in a four-year college and even in this community college which I thought would be something that would be easier to get into which was a yeah a big misconception and on my part I was already Challenged with who I was and my ability to learn so During my freshman year I Thought I wanted to be a physical therapist. It was very convinced because I loved helping people. I loved being around people interacting with them but after playing one year as a Student athlete and becoming injured and having to go through the physical therapy. I realized I didn’t want to have to Mentor or treat somebody that would be like me in physical therapy I was very bad at doing exercises and would show up like every two weeks and be like, why am I not getting better? and it was because I wasn’t doing my exercises which would be Infuriating if I had to deal with that so I decided to switch my major From pre-med which is what I was taking over the track that I was taking to get to PT and went into biology so this is just kind of a diagram to Give you an idea of how I felt after I finished high school And if you haven’t heard this yet, I’m during undergrad You probably will hear it that your progress to your goal is not not linear at all There’s tons of Peaks tons of valleys. You will change your mind multiple times It’s yeah, not linear and honestly, I haven’t even gotten to these changes in my occupation or changes in my jobs I’ve only been in this first half and it’s been a crazy whirlwind so so I Joined my sophomore year. I was I Think I was actually in a biology class and I got a phone call from my mom saying that my sister and her round emergent Are on way to the emergency room because they just gotten attacked by a swarm of bees and it was pretty scary Because my parents my my family is pretty well renowned to have bad allergies So we thought that one of them was going to go into anaphylactic shock But as a result of the attack from the bees my sister developed an autoimmune disease And I don’t really know if any of you know What I mean autoimmune disease was because I had no idea what it was when the doctors told me Does anybody know what an autoimmune disease is by any chance? Yeah, so an autoimmune disease Is when your immune system which is supposed to be which is built into your body should protect you from diseases from pathogens Actually turns on you in a way and treats your body and your cells as something that it has to defend against So instead of looking at bacteria viruses and being like you don’t belong here Let me attack you and get rid of you. It actually looks at your own cells and it’s like you don’t belong here This is not me. I need to get rid of this. So Because my sister was diagnosed with an unknown autoimmune disease. They still don’t know what it is. I just know that her body’s attacking itself It raised a lot of questions for me on what autoimmunity was What that meant that my sister’s body was attacking her What was involved in that and what made the change in that so during my second semester of my sophomore year I ended up Taking a microbiology class with this professor called named professor Wickham Who is right here and? I Nagged him so much with the amount of questions that I asked him based on Bacteria based on viruses and based on the immune system I was just like I just don’t understand like why my sister is going through this and so Because of the amount of questions that I asked him He thought that I should do a summer internship and without me knowing he actually ended up signing me up for a symposium on the bridges to baccalaureate program and Start an application for me to the bridges to baccalaureate program at a school because he was like, this is really for you I think you would do great in research Even though I really had no idea what that was so after graduating with my AAS, which was my Associates in Arts and Science from Monroe Community College, I Went to the bridges to baccalaureate program at Binghamton University, which is still in New York so during the summer of 2015 I Started this bridges to baccalaureate program It was in the Marquez lab, this is Claudia right here. This was my boss She was German and a little scary at times But she was a really great teacher. Um, and this is my bridges group that I did my summer internship with But my time at during the bridges to baccalaureate program I learned What graduate school could look like since I worked under a graduate student? I did a lot of research in biofilms. And the reason why I chose to do research in biofilms, they contain bacteria So all biofilms have bacteria. They’re like these little communities Of bacteria that kind of grow together and communicate together and since I was in a microbiology class with dr Wickham and he like that was part of the reason why I thought I wanted to go into research I wanted to start and want to see what Bacteria actually was like microbiology was great But what what do what do you do with it and research I didn’t know so I joined this lab and cloudiest lab was involved in trying to Affect effectively treat or cure chronic diseases that were caused by bacteria or bacterial biofilms because there’s a little part of the community that Will always stay there They’re called persister cells Like even if you treat somebody with a ton of antibiotic and wipe out all of the bacteria in that biofilm There’s always a little bit left behind that kind of hides from the immune system So they don’t clear it out. And then once it realized that all of the other cells in its community are gone. They Start to wake up and remake the biofilm So it’s really hard to get rid of a chronic disease. So that’s part of the reason why I was so interested in Trying to learn more about this disease but during this program I discovered that I really like to research I I had a ton of fun. It was only eight weeks. Um, but they flew by and I actually asked to stay longer So they let me stay four weeks longer to continue research just because I loved it so much I built a community through the bridges to baccalaureate program. I still keep in contact with a lot of these people. They’ve been great when I’m looking for advice or If I am NOT if I’m trying to look into an area that I’m not an expert in which I’m not an expert in anything right now but um They give they’ve been helpful in giving me guidance, even though were still kind of in the same area of life But I fell in love with microbiology through this program and it was just a overall fantastic experience for me um and really set my path and deciding to pursue a PhD So after the bridges to baccalaureate program I started at my four-year college and Completed my junior and senior years there and I went to Nazareth College, which was still in Rochester, New York Just like my real Community College and where I grew up but even when I transferred over to Nazareth I still received criticism and doubts from a lot of the faculty about Why I was there and if science was really the right path for me Which was kind of shocking and a little discouraging because I was like I have the grades to prove that I belong here So why don’t you believe I belong here? but in the summer were in the fall between Right before I started college at Nazareth I Actually went through a traumatic experience. So what the faculty did it not no and Nazareth College was that I was dealing with anxiety depression and a ton of other Effects from a traumatic event that happened during that fall semester and I wasn’t comfortable sharing that because I didn’t know these people So so when I would have to get up in the middle of class they thought it was because I was being lazy not because I was in the middle of a panic attack or Like little things like that. So it was just kind of Discouraging when I first got to the school because I was like, I’m really trying here but like To them it looked like I wasn’t because I was dealing with something on top of just my normal curriculum but this woman Lynn O’Brien she was the woman I did research with while I was at my four-year Was just really encouraging in a really good mentor And could see that there was something else like that was happening in my personal life that like even though I didn’t share the entire thing with her like she realized like, you know, you’re really trying here and I really think that you can Still progress and do well in research so I gained a lot of research experience and working with e.coli, which I don’t know if any of you are familiar with but that’s I mean like Chipotle and romaine lettuce like whenever there’s a outbreak like that’s it’s from ecoli, so but during my time at Nazareth College After graduating with my bachelor’s in science and biology As well as like minor in chemistry and philosophy. I realized I didn’t really want to work with bacteria. Like they were really cool but I Noticed that I was more interested in how our bodies were affected by the the pathogen. I’m Not necessarily the bacteria itself, so I knew I wanted to go to graduate school But I didn’t think that I had enough research experience or really knew where I wanted to go So I decided to take a year off and started looking for opportunities to gain that research experience and What I found was this prep program at the University of Rochester So perhaps stands for a post-baccalaureate research education program It’s something that’s offered through the University of Rochester again still, Rochester, New York So still in the same general area of where I grew up But it’s simulated the first year of graduate school. So they had you take grad courses You were responsible for working full-time in a lab and doing research and whatever lab you chose but on top of the First-year graduate school simulation. I was also responsible for studying for the GRE taking the GRE as well as hosting symposiums hosting events and a lot of other administrative stuff that I liked at the time was like well This is so much but it ended up being coming a really big help for me where I am now but When I was at the prep program I ended up Researching in this guy’s lab Steve Dewhurst I did not want to go in a virus lab. I thought that viruses were boring And I didn’t really see any point. I wanted to be in an immunology lab And so I was very adamant. I had to make a list of people I wanted to Research with and Steve was at the very bottom and somehow I ended up in his lab and the Dewhurst lab studies flu viruses, so They work on host-pathogen interactions Where we’re trying to or where they were trying to develop a new vaccine That was more effective than the vaccine that we usually get so there is a live attenuated vaccine which is what you get shot up your nose like through a nasal spray and then there’s the inactivated vaccine which is what you Actually get in your arm. So my work in Steve’s lab was to try to develop a vaccine That would be more effective but safer that was inhaled because in the past like the reason why we still get the in arm shots is because the LA IV the live attenuated vaccine was like still – like it wasn’t as safe as it needed to be so my job was to try to figure that out and It was in Steve’s lab that I realized I wanted to Go into biology. I fell in love with viruses they were just Really fun puzzles, like constantly moving constantly mutating, and it was just a constant challenge And I really enjoyed it. So I decided to pursue a PhD neurology and So I applied to several different colleges for graduate school kind of all over the place Especially since I had been born and raised in Rochester, New York, did my undergrad in Rochester, New York? Did my post baccalaureate research program in Rochester New York and the bridge to baccalaureate program was still in New York? So I just I felt like I needed to get out of New York to be able to grow Personally, but also to show that I could be successful and still do well in a place that is not my home so When I was accepted to both University of Rochester and University of Florida It was a it was a really really hard choice I trying to decide whether or not I was going to stay in this lab that I fell in love with with the Flu virus research that we were doing or if I wanted to kind of go very far out of my comfort zone Down south to a place of where I didn’t really know many people. I had some family but not tons but I When I applied to University of Florida, I also applied for the bridges to doctorate program. They kind of brought it up in my application because I was on minority but I applied for it and once I got accepted to the University of Florida, I actually got the NSF fellowship award as well And I was only eligible because I did the bridge two baccalaureate program in my undergrad So I was very thankful to dr. Wickham for that opportunity But I ended up choosing to come to UF because I believed it was the place that I would grow the most and So far I have grown a lot Wow So my first year as a bridge to baccalaureate or bridge to doctorate um fellow um, It really helps you grow professionally. Um, so if you guys decide that you want to go into research and think that you might want to go in to Get your PhD then or even your masters this is a really good opportunity Just because they they’re really invested in making sure that you are ready professionally as well as personally to enter whatever world you’re going to go into post PhD so They offered several different career development seminars to navigate graduate schools So like how to even get through graduate school, but also in my first year they were still giving us Symposiums and workshops on what to do now to prepare yourself for actually going out of graduate school after you exit with your PhD They really encourage you to get involved in your community as well. Um since the bridge to doctorate Fellowship requires that you apply again After your two years of funding, they ask you to reapply for this new fellowship called the NSF GFR P which is funding for your last three years of your PhD and So when you’re in your first couple years with this fellowship they really ask you to Start working up your CV and really starting to get involved and figuring out where you want to be Do you want to be in academia? Do you want to be an industry like to try to get yourself? Acclimated to where you want to be or where you think you want to end up so Another great thing about this fellowship is that it just sets you apart from several different applicants Like there’s very there’s several people that apply for this fellowship, but only a few get chosen So it’s really really helpful when you’re applying for grants fellowships or a job application It just it shows that Or so I’ve been told by post fellows or alum that like they’ve been Even before they show up for their interview They are people already have an image of you as like a hard worker as somebody that goes back to their community as somebody that is Really driven in a certain aspect whether it I be going to academia or industry So it’s just it’s a really great program if you guys decided that you would like to go to graduate school for your PhD So this kind of brings me now to my research to what I’m doing now at the University of Florida So I work in the Toth lab and our lab studies Kshv, which I’ll go into what KSHB is but it is a virus and it is a herpes virus so Before I get into what kshv is I wanted to start with a little bit of background I’m just because I know not all of you are probably biologists or cell biologists even so every cell in your body contains DNA so this is like a diagram of a cell and then inside that cell there’s a nucleus and that nucleus contains all of your genetic information that is stored as Chromosomes, which is just another word for very very very tightly wound DNA So if you were to write unwind the chromosome, which is what this diagram is showing You have different regions but that doesn’t really matter all I really want you to get is that when you unwrap a winder chromosome, it is made up of just very very very tightly wound DNA so So DNA is double-stranded in ourselves and In DNA, there’s these specific messages called genes So like each of these regions you might consider a gene and our cells take these genes and they Read it and they decide what to do with that message. So The issue is is that in the form of DNA. It’s not the It’s not a message that your cells can understand. So it has to be transcribed into something else so the best way to describe it is if you were trying to If you had a cookbook and you were trying to cook something, but the cookbook was written in Hebrew. And so you had to Sit down and try to figure out how on earth am I supposed to make a salad? I don’t know something from a Hebrew cookbook when I can’t understand words. So that’s kind of what DNA is and Transcription would be if somebody who was able to speak or read Hebrew And write Hebrew was able to look at the cookbook and transcribe it from Hebrew to English so that you were able to understand what you had to do to make whatever meal so That message will get transcribed in the sells case from DNA to RNA and So that RNA turns into this code that the cell can now understand and there’s certain proteins in your cell that are able to read this message and Produce protein so this would be like you finally being able to make your salad or whatever your meal because you finally understand What you have to do in order to make it so My my lab works a lot on DNA RNA and protein but in the case of KSHB, which is another name for a Kaposi sarcoma associated herpes virus It’s a large double-stranded DNA virus so it has the same kind of DNA that our cells are made up of And it has to undergo the same processes to actually become this. It has to transcribe its DNA into RNA so that our cells turn its message RNA into the protein that it needs to make more of itself so, um Kshv is part of this herpes verde family. So it’s a herpes virus And there’s three families or three sub families of herpes every day There’s the alpha subfamily Which you guys might be familiar with somebody having it or you having it yourself because it’ll show up as a cold sore So it’s HSV one or herpes simplex virus one that gives you a cold sore Or if you haven’t had a cold sore know someone that has a cold sore You probably knew somebody that had chicken pox, which is also a herpes virus So that’s a subfamily alpha herpes virus and then there’s a beta herpes virus which is CMV or side a metal cytomegalovirus? And that can cause congenital defects our birth defects so it can cause deafness or blindness or other kind of complications and development in Utero, um, if you’re infected with this virus and then my virus or what my lab studies Our gamma herp is but gamma herpes viruses which are kshv EBV so Kaposi sarcoma herpes associated virus or modern Iran epstein-barr virus and so Gamma herpes viruses are known to either severely Debilitating immune system or cause cancer so gamma herpes viruses are also called oncogenic viruses because if you’re infected with them and If your immune system weakens or shuts down Then you will likely develop the cancer Kaposi sarcoma from this virus so the virus is transmitted sexually and non sexually so through the saliva and That’s actually the main route is orally through the saliva. Not sexually But this is true of all other herpes viruses as well And then there are several diseases that my virus causes Kaposi sarcoma Is one of them and then primary fusion in fusion lymphoma Which is the only two that are like really important to what I study but commitee sarcoma is What I’m showing here you get multiple lesions and nodules under your skin from the virus but also primary effusion lymphoma You know, I talked about our immune system and how there’s like certain cells that identify like what’s not you and what is you and Primary effusion lymphoma is when one of those cells which are these b-cells end up becoming cancerous because they’re infected with the virus So primary effusion lymphoma is a b-cell lymphoma. It’s a b-cell cancer So one of your immune cells is pretty much down for the count So like all herpes viruses my virus has a biphasic life cycle Which means there’s two different phases of its life cycle. It’s either latent, which means that it’s dormant pretty much like our immune system can’t detect it or it’s lytic which means it’s actively replicating and actively making more of itself And like all herpes viruses kshv also Causes lifelong infection and to this date there are no vaccinations Available for any of the herpes viruses except for chicken pox. So I wanted to just bring this back because I’m going to start talking about Proteins and my research isn’t mainly based on proteins right now so It’s it’s going to become more apparent in the next slide But I just want you to guys guys to keep this in mind that DNA turns to RNA which is the message that ourselves can read to turn our RNA into proteins so Kshv infects a decent amount of people. Um, it’s not it’s not super prevalent like Hsv-1, like herpes virus that causes the cold sores But it’s still like a lot of people are still exposed to KSHB Even if they don’t actually end up having Kaposi sarcoma later in their life And it’s most prevalent in Africa Where you see a ton of people like sixty percent of the population are exposed to KSHB But not everybody that has kshv will also develop Kaposi sarcoma So it’s still very prevalent, even though it’s not as widespread or what we call ubiquitous. So not as common as Hsv-1, it’s still pretty widespread So I’m going back to compose the kshv by a basic life cycle I said that it has a dormant state so of a state where it’s not replicating where I body can’t detect it But there is a way if you want to Sorry reactivate or cause the virus to become actively replicating either in a lab setting you can use chemicals to kind of wake the virus up from its dormant state or in an actual human being your immune system would become weakened either by HIV or AIDS or a different cancer or some other kind of really? Massive stressor that’s going to cause your immune system to really really weaken so that it’s not able to defend against any kind of pathogens So and when that happens the virus will become Awakened and it’ll start replicating and making tons of itself to go on and infect other cells And this usually takes two or three days from when you awaken the virus to when it actually starts becoming produce So my lab like I said study is kshv and Recently in our lab we were asking the question in every cell At the gene level so at the DNA level What happens to the cell when the virus becomes? Awakened like what gets does are there genes that get turned on or their genes that get turned off? What happens when this virus gets we activated or when it becomes actively replicating so one thing that they found in this humongous screen where they were looking at like every single gene in a cell and seeing whether or not it was up or down based on when the virus was asleep or dormant and when it was active and they found that this protein or this gene id2 was really quickly down regulated by six hours post induction which when I say induction I mean chemically forcing the virus to wake up in a cell line that’s latent ly infected or has dormant virus in it You don’t you don’t take human samples I’ll go into a aside with like actual like how I did this But yeah, it’s not human samples that we’re using. This is in a lab setting with What we call pel cells. So I told you that KSHB causes primary effusion lymphoma or pel so we use Cells from primary fusion lymphoma that is from a patient but is adapted to a lab that we grow up Like we don’t take live human samples constantly. It’s just a cancer cell line that were able to maintain and we look at what happens to the virus in those cells because it’s clinically relevant to the Since lots of patients tend to develop the disease So, um Before I get into the results that I got The one of the main assays that I use are one of the way main ways that I analyze my data is through Western blots So I wanted to go with you and like show you guys what Western blots are but this is taking the protein so after the DNA message has been transcribed into RNA and Then that RNA message has been made into protein like this This assay will look at the protein that has been made based on the gene level that’s present So what you end up doing is isolating cells And these cells again are usually lab adapted cells. So some type of cancerous cell line that we have in our lab That is usually latent ly infected with kshv. So something that has dormant virus in it and what you end up doing sorry is you’ll end up liking these cells with this detergent called SDS as well as a chemical called beta mercaptan all but all you need to know is that it lysis the cells and it causes them to Denature when you add heat to the protein, so a lot of the proteins in our body have secondary and tertiary structures which just means that they’re very Globular, like they’re not just this linear form of a protein like there’s a structure to it in order for it to actually work but when you add heat you’re taking away that structure and making it linear so that when you run your samples on a gel you’re able to separate your protein by size and The reason why that’s important is because you want to be able based on What? Literature says so what people have already shown you want to be able to find whatever protein you’re looking at by its size to see What happens to it? If you treat it with a drug, or if you cause a stress to happen, like reactivating a virus in a cell so what you end up doing is you run your samples that have been donated on a gel separate your proteins by size and Then you will end up transfer your protein onto this solid membrane It’s kind of like paper but it’s a special kind of paper. It’s called a cellulose membrane and Then after that you’re able to analyze a protein and look at what happens to whatever protein of interest you have based on Certain treatment so a lot of people use it to observe changes in protein expression After you treat a certain cell with a drug After a certain stress you apply to a cell or if you actually cause a certain protein to be over expressed What happens to the other? parts or proteins of the cell So I’m going back to this the main question that we were asking after getting these results that when you wake the virus up from a dormant ly infected cell and Seeing that this gene ID 2 was rapidly downregulated Why does the virus have to do downregulated in order to reactivate like what is the importance of this gene? So in order to understand that we looked at what the proteins normal function was So I’m just bringing this back as a reminder of like what how protein is made But we looked at in healthy cells. What does this protein do so ID 2 is an inhibitor of DNA binding protein. So all you need to know is that This protein ID 2 this pink protein when DNA is trying to be turned transcribed or when a gene is trying to be transcribed from DNA to a messenger RNA to make protein this protein Will inhibit certain transcription proteins from being able to turn your gene? into a message that you’re a protein that your cell can read to make protein, so It’s really important for your immune cells So if you want to have healthy B cells or T cells you need this protein ID 2 to be functional in order for your immune cells and your body to be healthy, so The role of this protein though hasn’t been studied and texts of the virus I look at KSHB so We based on what we knew that ID to on this inhibitor a DNA protein tends to bind to other proteins and prevent them from Transcribing or turning other genes into proteins or being expressed We hypothesize that idea to prevents the reactivation of kshv By using this negative regulatory Function which negative regulatory just means like it’s it’s preventing something from happening instead of allowing something to happen So the way that we study this hypothesis was through asking two questions What happens during reactivation? So what happens when you wake the virus up from dormant ly infected cells? And then what happens if we overexpress this protein? So if we overexpress ID 2 what happens to the virus? Will it prevent the virus from being able to wake up and be able to ask actively replicate itself? What’s going to happen if we add so much protein that the virus isn’t able to shut it down so What happens when the virus and what happens when I D 2 or What happens to Chi ID – when KSHB is reactivated, excuse me so I wanted to bring this back but this is just showing the biphasic life cycle and I’m reactivating the virus and a latent ly Infected cancer cell and I’m using a drug to wake it up because it’s not in a human being it’s not like actual human samples and When it’s being reactivated and woken up again It’s it’s making more of itself to go on and infect other cells where it may become dormant or may become Activated again and make more of itself So how I did this was I took this cell line Called Trek’s BCPL thiefbug RTA, which is just another word for pel cells it’s a specific type of pulse cell that we use that has a Gene in it. That is Dependent on a drug that we add to the cells So if I add this drug over Chemical doxycycline to the cells? It removes a repressor or something that prevents the virus from being reactivated It removes it and allows the virus to become activated or wake up And so what I did was I used these dormant lis infected cells Added doxycycline or a drug to wake them up and then harvested protein samples, so to do Western blot and then RNA samples to look at genes to see what happens with my expression level of my gene to see what happened to my protein ID – like when or how or why does KSHB John regulate this protein and this is what I saw. So when you reactivate these late in the affected cells the first protein and the first like the first protein and the Only protein that’s responsible for turning on and causing the rest of the virus to reactivate. Is this protein RTA If you don’t have our ta the virus won’t be able to wake up. It can’t do anything. It’ll stay dormant so when I added the drug by six hours the most important protein and the virus was being transcribed and turned into a protein which meant that all the other genes were all the other genes or DNA and the virus were able to be transcribed so that the virus is able to make more of itself but by the minute that this Protein our Tia was being expressed. My protein ID too was completely gone Which was what we expected because we saw at the gene level that this was happening But we wanted to confirm that it was happening at the protein level so Once we saw this our next question was what happens to kshv When I d2 is overexpressed So what happens to the virus if I add so much ID to that it’s not able to get rid of it Like will the virus be able to still? replicate And what we and this is how I did this so I used a virus called a Lenti virus that expressed my protein so that had a code a DNA code in it for ID 2 and You infect with the Pala cells with this virus. It causes them to make a ton of my protein. So a ton of id2 and then after a couple days of adding like a like brute forcing the cells to make a ton of id2, I Used the drug doxycycline to wake the virus up in the same latent lee infected cells and look to see what happened So when we overexpress the virus Id2 was able to stay constant. So instead of being rapidly down regulated in wild-type So what usually happens when there’s no? Overexpressed id2 and what happens when there is over Express ID – ID – was not done regulated. It wasn’t gone But RT was still able to be expressed. So that’s telling me right away that the virus is still able to at least start reactivating but one thing that I did notice was this protein called KO point one and all you need to know is that this Protein is really important in virion assembly. So it’s really important in making new virus particles. So When we saw that Katy point one this protein that’s responsible for making more viruses Was being down regulated when I had a ton of my protein ID to present in the cells. Yes, sorry So these proteins are yes controls, but they’re also there to see whether or not To see whether or not when you use ID to overexpression if any part of the virus Reactivation is inhibited. So when kshv is reactivated, there’s like three different phases that happen There’s this immediately or immediate early phase than an early phase and then a late phase So I chose a protein from each phase to see if one phase was affected when I over expressed this protein So RTA is in the media early. So when I saw that RTA expression was not affected I Assumed it wasn’t the immediate early part of the reactivation that was being affected Then I chose an early protein of the virus called Ora 45 and It’s slightly reduced but it didn’t really seem to affect the early phase as well But when I looked at the late phase K 8 point 1 it was a pretty dramatic reduction of the protein from regular like Noah overexpress 82 to overexpressed ID 2 So it kind of led to the idea that maybe a d2 is preventing the virus from being assembled further So now what I’m doing is just invest investigating how ID 2 is down regulated So what’s causing the protein to being down regulated? And what happens if we remove ID 2 like if I take the protein away does the virus? Automatically wake up or does it still need to be induced by drug? And then does I do to prevent reactivation and other herpes viruses. So if I do a chickenpox like the Varicella-zoster or if I do herpes simplex virus 1 if I overexpress ID 2 does that affect the virus? I don’t know. So that’s what we’re working on in my lab now But just in summary of my journey and what I’m doing now It has not been linear whatsoever. And if you choose to do research your journey will probably not be linear. And that’s ok Don’t let any of them define who you are and tell you what you can or can’t do if I have listened to people telling me that I was not able or capable of doing anything or Doing undergraduate. I would not be here And I would not have been given the opportunity of the bridge to baccalaureate or the bridge to doctoral program And don’t be afraid to get out your comfort zone. It was very much So out of my comfort zone to move down to Florida But it is it has been absolutely amazing and really just helped me grow as a researcher and as a person in general And find what you love if you are really thinking of becoming a researcher thinking that you might want to do research Get out there and try to find opportunities So that you can start to narrow down or start to figure out what you really like So that you end up doing something you love So with that I just wanted to acknowledge my lab the tothe lab and then the PAP lab without also helped produce the data that I show and the programs that I was a part of so the bridge to baccalaureate prep program and the bridge to doctorate so With that I’ll take any questions and thank you for having me

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