WISE journal entry #23

Name: Leah Gerald

Entry: #23 (5/15/2012)

WISE Week:12

Written on Date:5/15/2012

Mentor: Sagar

today was a presentation day for sagar and a handful of other students in the lab. it was a chance for them to present what they've been working on and whatever results they've found. my mentor had a poster that talked about what hes working on, and after I went back to the posters several times because today they were trying something very different. Sagar explained to me how in clumps of cells, the outer cells have  access to oxygen, but the inner cells don't, therefore they are in a hypoxic condition, and they cannot reach oxygen, so in cells that are in this condition, they have a gene that expresses this protein called Hif1 alpha. Hif1 alpha allows these cells another means to survive as a substitution for oxygen. they're not sure in which area in the chain of command where hif1 alpha is expressed, so they knocked down several different levels using antibiotics. they weren't able to do much today though except to quantify the cells that had the antibiotics in them and make sure enough viable cells remain. they also did something today they call licing the cells. the purpose of licing the cells is to open them up, which is how they extract the hif1 alpha protein, which is how they can see how much expression of it is going on within the cells

WISE journal entry #22

Name: Leah Gerald

Entry: #22 (5/10/2012)

WISE Week:11

Written on Date:5/13/2012

Mentor: Sagar

today, sagar was planning on using a flow-cytometry machine. we needed to use this machine because they needed to look at specific protein the cells produce (one in particular, but I cant remember the name) that will help them classify/ know/ find stem cells in the cancer cells. this protein is found in cells that self regulate (a stem cell-like behavior), which is why they are trying to spot out the cells that produce it. the first part of the day was spent quantifying the cells, because we needed at least half a million cells for the machine. after they were  measured out and spun down a couple times, we added antibodies in the cells which should attach to the protein of interest and the machine will light up the cells with the antibodies attached, thus proving if or if not the cells express the protein of interest. the cytometer does this basically by sorting the cells. it takes them all, one at a time and sorts them into cells populations. with this information, the machine created several graphs which had many clustered areas that my mentors sectioned and noted the differences in the different samples. there was a control, and for all the other samples, if it differed from the control, that showed how much it expressed the protein of interest

WISE journal entry #21

Name: Leah Gerald

Entry: #21 (5/9/2012)

WISE Week:11

Written on Date:5/13/2012

Mentor: Sagar

today, the first thing we were to work on was an RNA isolation procedure, which I have never seen before. basically, the RNA isolation procedure is, as the name denotes, to isolate RNA. to do so, we had to pour a bunch of buffers through the RNA and spin it down several times to wash it of contaminants so when looking at the RNA later, nothing will get in the way, like DNA and contaminants. cells don't always use all their genes, so looking at the RNA shows us which genes they are ACTIVELY using. this procedure didn't take very long, and for the rest of the day, there were a lot of cells that needed to be re-fed new media, so I spent the most of the day in the cell culture room doing just that. I probably added media to 20-30 flasks. also, Sagar showed me some recently acquired xeno-grafted cordoma cells. they are called this because foreign cordoma cells were placed in a mouse, and then extracted. because the cells were placed in a body, they are mixed up with many other cells, so the plate with the xeno-grafted cordoma cells had a mixture of more than just cordoma cells.

WISE journal entry #20

Name: Leah Gerald

Entry: #20 (5/2/2012)

WISE Week:10

Written on Date:5/6/2012

Mentor: Sagar

today, I had the hood to myself for the first time ever! Sagar had set aside a time and some lung cancer cells that had become too confluent for me to play with that day. I cleaned the station, got out what I would need for my lab, and got to work. I was to take the cells out, count them, dilute them properly, and replate them in 6 different dilutions. first, to get the cells out, I washed them with PBSO, which helps remove calcium, which slows down the trypsin. then I added the trypsin and waited 5 minutes for the trypsin to disconnect all the cells from the bottom of the plate. then I added some media to the mixture to help calm the trypsin down, and spun the cells down in the centrifuge. I then removed the trypsin and media, and mixed the cells with some new media. then I took a small section of this new mixture and counted the cells in the cell counter. I found out how many cells were in one mL, and then had to create 6 different dilutions so that I would have 1 million, 500,000, 50,000, 10,000, 1,000, and 100 cells in each separate well. this part proved very difficult for me, because the mental math was kind of rough for me, and also I never did very well with dilutions in math. so I messed that part up several times and had to redo my dilutions more than once. in the end, I'm not sure I even got it right, but the fact that I did it was enough I guess

WISE journal entry #19

Name: Leah Gerald

Entry: #19 (5/1/2012)

WISE Week:10

Written on Date:5/6/2012

Mentor: Sagar

today, I spent the first part of the day researching fixation, immuno-florescence, and H & E staining, and their connection, because earlier that week, my mentor had recieved some mouse brains from germany which he was staining with H & E. basically, first they had to fix the brains, which causes them to not deteriorate. then, once they are fixed, they stain them with H &E, which colors the nuclei of the cells blue and the other parts red. after this, I went into the hood with Nate, who was replated cells once again, but something else exciting was going on that day, so I was between two things. in the other room, some of the other lab members were preparing to inject some mice with some cancer cells, so my mentor told me I could watch if it interested me. I went to and from the mouse surgery, because It freaked me out a little bit. they pinned the mouse down, cut their skin open, counted down a couple of ribs to the point in the mouses body where its heartbeat was the greatest and injected the cells. at one point, we could see the mouses beating heart under the muscles. I went back to the hood several times because I couldn't watch the whole process. when I did, there were some issues because Nate was replating these cells that showed an irregularity I couldn't quite note.

WISE journal entry #18

Name: Leah Gerald

Entry: #18 (4/24/2012)

WISE Week:9

Written on Date:5/6/2012

Mentor: Sagar

today my mentor was planning on performing a transduction, but unfortunately we never got to it. a transduction and a transfection have basically the same results, but the processes and results differ slightly. with a transfection, the process is quick and the results also do not last forever, because we add simply add plasmids to the cells that we wish to see the replication in, whereas in a transduction, the results are permanent because it takes much longer, and also because we must add virus to "infect" the cells with the plasmids so that it will permanently change the DNA of the cells and their future lines. to start this process, the first thing we needed to do was a mini prep, which is basically the process in which we isolate the plasmids (DNA) from the bacteria. unfortunately, something got in the way this day, and it was more important that some cells get replated, so instead of doing all of this, we talked about it, and I replated a set of lung cancer cells that had become too confluent.

WISE journal entry # 17

Name: Leah Gerald

Entry: #17 (4/18/2012)

WISE Week: 8

Written on Date:4/23/2012

Mentor: Sagar


today my mentor planned to stain the mice brain cells we worked with yesterday using immunoflourescence. this type of staining helps show human cells, and since the cancer they inserted in the mice was human based cancer, it would make the cancer cells stand out. unfortunately, I wasn't around for this part, but i was around when they performed the transfection for another experiment. they added some plasmids to some gbm (glialblastoma) cells they had been working on for a while. next, they needed some cells to be re-seeded into a smaller plate for them to photograph their movements. these containers they put them in contained very small, controlled areas that they would put under the microscope with a camera and photograph its movements over time to see how it migrates.

WISE journal entry # 16

Name: Leah Gerald

Entry: #16 (4/17/2012)

WISE Week: 8

Written on Date:4/23/2012

Mentor: Sagar


today was the day that a large accumulation of my mentors works came together. we looked at the brain slices they had taken from the mice they injected tumors in after months of preparation. first they had to accumulate and culture the cells, then once they had enough, they had to inject them into the mice, then, BEFORE the mice died, they had to extract the brains and freeze them. then another member of the lab had to take those mice brains and meticulously slice them into tiny slices to put under the microscope. then they had to add different types of staining, which they had to deduce which type of staining they used (different for different cell types) after all of this, they had to then sort the sections of the brain they took into slides with cancerous cells on them, slides without, and find the slides where the point of injection is obvious, because that area should have the largest concentration of cancer cells, and also, it helps as a reference point. ONLY after all this can they make deductions about the cells they are working with, and even then, they weren't fully sure of everything. there were sections of the brain that looked very strange to me, so I asked my mentor about them, but they didn't know what they were. so there are still things they do not know.

WISE journal entry #15

Name: Leah Gerald

Entry: #15 (4/11/2012)

WISE Week: 7

Written on Date:4/11/2012

Mentor: Sagar


today went by very fast because I was able to do a lot in the hood. we started out by checking on the pancreatic cells I set yesterday. Sagar said they looked very good, and then I got a chance to look at them

they looked very healthy, and somewhat more different from all the other cells they culture, but because they express that common gene (brachiary) they share at least one similarity. after checking on this, they needed to put some new media in a large amount of the Cordoma cells, and they needed to culture some other cells because they had become too confluent. first, I aspirated all the containers and then pip-petted the new media into each container. it was a long process because there were a lot that needed new media, and as I did this, I didn't realize how much time had passed. the process was very menial and boring, but time just flew by. next, there were some cells that were getting cramped in their container, so we aspirated the media, added trypsan so the cells would un-stick from the bottom of the plate, spun down the cells, mixed them with new media, and re-plated them. the process is one I have watched done several times, except this time, I was able to take part in it as well. at this point, it was time to go, so my day was done.

WISE journal entry #14

Name: Leah Gerald

Entry: #14 (4/10/2012)

WISE Week: 7

Written on Date:4/10/2012

Mentor: Sagar


today, we were using the western blots previously formed to make one of these:

in order to produce these images, we have to go through a photo-like procedure. first, I washed the membranes in a buffer three times to make sure they were clean. then, we mixed in rabbit antibodies and mouse antibodies, one in each of the trays that corresponded with whichever type of cells. we let those sit for a little while, and while we did that, we went upstairs to get some pancreatic cancer cells. my mentors had found out that there is a gene, Brachiary, seperate from the hippo gland, that can also have an affect on YAP proteins. they found this gene was present in Cordoba cells, which they have in the lab, but recently, they found out that it is also present in pancreatic, colon, and some forms of lung cancer. because of this they were hoping to begin to observe this gene in action in the other types of cells, so I cultured the pancreatic cells into its proper media, and stored it with the others. after this, the western blots were ready to be developed. we poured out the antibodies, and washed them in a sort of photo-developing chemical that brings out light where proteins are present when developed. we then went into another lab, where there is a room to develop westerns, placed them in a machine that does all the developing work for you, and make a couple of images to compare the proteins. at this point in time, I was late for pickup, and I had to go.

WISE journal entry #13

Name: Leah Gerald

Entry: #13 (4/4/2012)

WISE Week: 6

Written on Date:4/10/2012

Mentor: Sagar


today I was able to fully witness, until the last step of course, the process of making a western block. a western block is something my mentors use to compare the amount of proteins given off by certain cells in certain conditions.

the different rows (horizontal) show how much protein appears in different conditions. the lines (vertical) show the different sizes of proteins and where in the spectrum your protein of interest falls.what we had to do first, was mix the protein with a dye so it would be easy to see. then we had to put the protein in a hot bath, because proteins take a string-like shape, and when they are in the mixture they are all jumbled up. heating them up makes them spread out somewhat and become un-jumbled. after that, they are poured into a gel, which is in a container that can send an electrical current through the gel.
once all the proteins are sorted into their specific compartments in the gel, we then turn on the electricity, and the lighter proteins travel down the gel through the current. after about an hour, the protein has shifted all the way to the bottom, and at this point, my time is up, and I must go once again.

WISE journal entry #12

Name: Leah Gerald

Entry: #12 (4/3/2012)

WISE Week: 6

Written on Date:4/3/2012

Mentor: Sagar


today was an exciting day because I finally got to work under the hood! the main things that they had to get done today was, Sagar needed to do an RNA synthesis, but first, we needed to culture some cells. the breast cancer and breast cells from a while back were becoming confluent (basically there were too many in one container) and they needed to move some from one container to another so the original ones would have room to grow and expand. because it was a task that didn't require too much precision, Sagar let me work with him under the hood. to start with this process, we sucked all the existing media out of the containers. then we added an enzyme called trypsin, which makes the cells disconnect from the base to the flasks. with that done, we shook the containers and made sure all the cells disconnected before we added calcium, which acts as an inhibitor, and stops the trypsin from acting on the cells too much. next, we took the cells in the flasks, and put them in tubes. we then put the tubes in the centrifuge and spun them until all the cells were in a pellet at the bottom. we took the tubes back to the hood and sucked all the trypsin and calcium out of the tubes. once they were all without trypsin, we filled the tubes with media and mixed the cells with the media.we then got new flasks, and put part of the cells in the tubes in the flask, along with some new media and put them back in the incubator.

WISE Journal entry #11

Name: Leah Gerald

Entry: #11 (3/28/2012)

WISE Week: 6

Written on Date:4/3/2012

Mentor: Nathaniel


today was different because Sagar had to take a test, so he was not at the lab today. Nate acted as my mentor in his place for a day. there wasn't much to be done, because they hadn't been able to do much recently, so Nate took me to the hood to feed the cells new media. basically, all of the cells they culture live in little bottles filled with a substance that contains the nutrients they need to survive. as the cells eat up the nutrients, the pH levels in their food change, and they change from red to yellow. Nate took out all the cell cultures that were yellowish, aspirated out the food, and refilled it with more media. Its OK for them to suck out the substance like so because the cells are attached to the bottom of the plate, and when the new media is added, they were hungry for only a short time so they survive. on another note, I finally met Dr. Q today. he didn't have much time, and it was truly a coincidence he was here, but I saw him, shook his hand and said a few words, and then he was off again, taking care of some crises in the research lab. that was the highlight of my day.

WISE Journal entry #10

Name: Leah Gerald

Entry: #10 (3/14/2012)

WISE Week: 5

Written on Date:4/3/2012

Mentor: Sagar


Today was a roughly slow day, because once again , there was nothing yet for me to get my hands on and "do" but rather a lot of watching to be done. Nate was working on making a control comparison of the DNA/RNA levels in a sample of some proteins they had been working with. To make this control, he first wanted to find out the concentration of plasmids (the shape DNA takes for bacteria. like a circle instead of the double helix) that is he needs to get an average concentration, which is 230/260. He tested them in the protein assay machine, and found out the amounts he needed of 3 different strands: one was the first part of the plasmid, then the second part, and then the full strand of the two together. This way he could see different areas of expression in the genes. after this, I watched as Sagar transfected the cells in the hood. He used the plasmids Nate had set up earlier to transfect the cells. Its hard for the plasmids to bind with the cancer cells, because plasmids take on a circular shape, and the cancers DNA is in the double-helix shape, but overtime it does bond and starts to express whatever genes were in the plasmids combined with its own. and with this, I had to leave, and my day was over

WISE Journal Entry #9

Name: Leah Gerald

Entry: #9 (3/13/2012)

WISE Week: 5

Written on Date:3/15/2012

Mentor: Sagar


Today was a somewhat slow day. It was slow because there was a lot of very specific things that had to be done, so I wasn't able to actually do anything with my own hands, so I spent most of my time watching. Nathaniel and Sagar were both working on separate parts of the same experiment. Nathaniel was prepping some proteins to act as a sort of "control" for what Sagar was doing, and Sagar was working on extracting proteins from 2 different cell plates, one with YAP (the gene that controls a cells ability to stop or start making more cells) and one with YAP knocked out. Each cell plate had an array of different dilutions of a chemical to see how it will be affected. Later, once the cells were all properly treated, he would test the protein levels in these cells in comparison to what Nathaniel was doing. Nathaniel was basically setting up a standard curve created by the DNA of the same cells, but without the chemicals in it to compare. To see the difference, since we cannot see DNA, what they do is compare the proteins the DNA creates. YAP makes a specific protein, and they can tell if they were able to actually knock down YAP and to what extent by looking at the proteins created. to see the proteins, they use a process called PCR (polymerase chain reaction) that replicates the DNA enough times to create enough proteins and a visible difference between the different amounts. I didn't get to see all of this, because I left before they were able to see the proteins, but I did get to watch most of the progress. It was fairly slow because it was a lot of pip-petting and very specific, small measurements that needed to be made. near the end, they also had a mouse they had infected with the cordoma cells (cancerous cells that infect the area of the brain nearer to the spine) that had recently died, and they needed to open it up and extract the cells. Sagar showed me the mouse, and how the cordoma caused the mouses back to become crooked and a large growth to appear on his back, but by this time, it was already 4:30, and I had to leave before the action.

WISE journal entry #8

Name: Leah Gerald

Entry: #8 (3/7/2012)

WISE Week: 4

Written on Date:3/11/2012

Mentor: Sagar


today was a day for watching. According to my mentor today we were extracting RNA. first, we had to take the cells, wash them, and then put trizol on them. the trizol would seperate all the parts of the cell. I got to see it in action under the microscope and it was pretty cool. I could see all of the parts of the cell breaking into tiny little pieces. once all that was done, we had to put a little chloroform in each of the viles, and then put it in the centrifuge. after that, we put a little isopropyl in them, and they were to sit in the freezer for a while, so I didnt get to see the final results. its sad when I dont get to see things to the end, and when I miss things. sometimes I wish i could just come in for a second so I could see the results, because I really wanted to see the RNA.all I was able to actually do that day was wash out the containers, because they had to make sure everything was done very precisely. It was a little reminiscent of my first days, because I spent most of the time watching, and following, and not understanding the humor.

WISE journal entry #7

Name: Leah Gerald

Entry: #7 (3/6/2012)

WISE Week: 4

Written on Date:3/11/2012

Mentor: Sagar


today was a very short day, partially because we left early, partially because I ended up not doing very much. I was in the lab for an hour less, plus my mentor had a meeting, so I ended up listening a lot. the trays I pipetted last week showed an inconsistency that proved to be problematic. we did 2 sets of each dilution, and basically one of the set of dilutions showed two different results, which doesn't make sense, so they had to re-pipette the whole thing. the week before, my mentor asked me to look up trypan blue, and today, I was quizzed on it. trypan blue is basically a dye they use that colors dead cells blue, because the dead cells allow it to pass through their membrane whereas the live cells do not. they weren't using it today, but in the future they will.

WISE journal entry #6

Name: Leah Gerald

Entry: #6 (2/29/2012)

WISE Week: 3

Written on Date:3/4/2012

Mentor: Sagar


today I did not expect what happened to happen. I walked in, and my mentor was dissecting a little mouse on what looked like an operation table to me. I'm a bit squeamish, and I didn't see this coming, so that was a real shocker for me. these mice were part of there experiment though, he wasn't just doing this for fun. they had mice which they inserted some cancer cells in earlier, and they needed to take the brains out so they could look at the results. they hadn't planned on taking them out on that day, but because some of the mice were dying before they expected, they wanted to extract the brains from the still alive mice before they became useless. so that day consisted of a lot of mice deaths, which took a great deal of time. first, they had to weigh the mouse so they could know how much anesthesia to give them. then, once they gave the mouse the anesthesia, Sagar would put them on a foil table in this section off to the side and pin them down. once pinned down, he has to pump saline solution into the right atrium (sorry if I'm wrong) and drain the blood from the left atrium, so there will not be any blood in the brain when they take it out, and they can clearly see what they are looking for. once that is done, they add some formalin so that the cells will preserve and can sit for a while without decomposing and stuff. they had to repeat this process with several mice, so this day was very much a watching day. I didn't want to have any part with the process with these mice, so I didn't look at the mice as much as I could, but every once in a while I'd try to sneak a peak to help desensitize myself. after a while I could look at the mouse without feeling sick, so I'd say that was my progress for the day.

Wise journal entry #5

Name: Leah Gerald

Entry: #5 (2/28/2012)

WISE Week: 3

Written on Date:3/4/2012

Mentor: Sagar

Today was a special day. Like last week, we went see another speaker speak. This time it was about from what I understood how cells tend to try to organize themselves. He used some words I remembered from Chemistry like "entropy" and I was excited I could actually understand that part, but for the most part, it was fairly boring. Many people in the room fell asleep. In fact, one of my mentors fell asleep. It wasn’t because the material itself was boring, but because it was a fairly complex concept that didn’t interest many people there, therefore they couldn’t relate to it. The exciting part about that day was, before we went to see the speaker, I got to work under the hood for the first time! There was some pipetting that needed to be done. They just needed to change the medium for some of the breast cancer cells. Though it was something so menial, it was hard for me. I believe I took almost twice as long as my mentors. Even though it was my first time, I was shocked at how difficult it was. I had been watching them do it every day for a long enough time now; I just assumed it would come easily to me.

WISE journal entry #4

                                                                                                                                

Name: Leah Gerald

Entry: #4 (2/21/2012)

WISE Week: 2

Written on Date:2/27/2012

Mentor: Sagar


today was different because I got to see a lecture by a professor who discovered something important to the research my mentors are doing. as soon as I arrived, Sagar told me what we were going to do. he said the man we were about to see ( his name was DJ Pan I believe) was the person who found out about this Hippo pathway that they work with. We walked over to it (It was in the hospital) and along the way we had some issues because I forgot to carry my ID, but I will not be doing that again. Once we got there I was surprised to see so many people there. I expected a smaller number, because I figured this was something only a small amount of people are actually interested in, but I was wrong. the room was almost entirely full. I understood little to nothing of what this man said, partly because of his accent, partly because of the content, but I could understand the parts my mentor had explained to me before, about how the Hippo pathway then lead to the Yorkie or the yap, so for the most part, I understood the basics. I left feeling like I didn't quite get everything, but like everything else that just reminded me how much out of my element I was. when I'm down at Hopkins, I feel like the slowest person in the room, and today I finally started getting used to that feeling. So far, I've been trying to get up to speed with everyone in the lab, but that's just not going to happen, so I guess today is the day that I learned to accept the fact that I am probably not going to catch up to these people anytime soon.

WISE journal entry #3

Name:Leah Gerald

Entry: #3 (2/20/12)

WISE Week: 2

Written on Date: 2/27/12

I watched as Sagar collected data from last weeks cells and put them into their statistic charts. a lot of their work involves taking data and looking at a it in perspective with other data, so that day was more of a data day. I also watched Nathaniel take measurements for new samples of the virus he would be putting in with more cells. he explained to me what conversions he was doing, and how important it is to check your math, and also helped explain more about their project and the hippo and yap genes they are working with. basically, what they know from other's research is that the hippo gene and yap correspond with each other, and tell cells when and when not to continue to make more cells. what they're trying to do is figure out how to control this, and how to tell it to stop when its been making more cells too rapidly. after WISE, I was actually really excited to go back the next day, because I feel like I understand more whats going on and I don't feel like a complete idiot all the time

WISE Journal Entry #2

Name: Leah Rose Gerald

Entry: Entry #2 (2/15/2012

WISE Week:Week #1

Written on Date: (2/15/2012)

Mentor: Dr. Alfredo Quinones-Hinojosa, Sagar R. Shah

today I was actually really excited at the start. I read the pamphlets Sagar gave me, and I thought I got them for the most part and was excited to actually help somewhat today. sadly, I was not yet ready to work under the hood yet, but Sagar did allow me to look at some samples and "observe" the effects of whatever they were doing to it. It was all going well, but then I accidentally contaminated this other guys samples, and I felt like crap. Before, I told myself I was not going to be one of those people that messes stuff up, and then I messed it up so, I failed at that. I just hope I didn't set him back in his work at all. I also messed up on some basic conversions so that was a great confidence booster. either way, I did a lot more watching and listening today and then I went back home.                

WISE journal entry #1

Name:Leah Rose Gerald

Entry: Entry #1 (2/14/2012

WISE Week:week #1

Written on Date: 2/15/21021

Mentor:  Dr. Alfredo Quiniones-Hinojosa / Sagar R. Shah

so my first day was a mix of nervousness and exitement. When we first got to the luncheon, I was really excited to meet my mentor and ask them all about what they do, but I kind of had a lot of questions, so it was hard to focus. When I got to  the actual building I would be working in, I got lost, but the people there were nice enough, or at least they knew I was lost and showed me the way. once I finally found Sagar at the lab, he showed me around, and mostly was helping me get aquainted with the lab and every one there. I have yet to meet my mentor, but I have heard many great things about him. Sagar gave me some literature to read about what he was working on, which I still barely understand, but I'm trying!