Finishing Up...

Today, I moved home from Harvard. I know I've said this a million times by now, but I can't believe how fast these 8 weeks have gone!

In the lab, I did minor things around the lab, and ran into last minute obstacles that kept me from finding any quantitative results on the Hippo project. However, Dirk is going to continue the project, and he promises to keep me updated. I also had a chance to see another surgery performed by Dr. Thayer. The surgery was supposed to be a normal hernia case where the small intestine slipped through a woman's muscle. However, it turned out it was her fallopian tube that had slipped through the muscle. This basically meant that the woman was at high risk for ectopic pregnancy because the fallopian tube was so damaged. Luckily, the patient decided she was already finished having children (she already had two healthy boys). I was really sad to leave Mass. General, but I left knowing I learned a lot... even more than I expected.

I spent my last few days at Harvard enjoying myself, and finding some time to study for my psych. final. I think the final went well, but what was also of high importance to me was enjoying my last few days at Harvard with my SSP friends. They're some people I will never forget, and I know we will always keep in touch. I will definitely miss Greenough Hall, and all of my other friends from outside the dorm as well.

Many thanks to Brooks, Mass. General, Dr. Dunn, Dr. Thayer, Dirk, everyone else in the lab, Harvard SSP, and Mr. Palm. This was an incredible summer, and it couldn't have happened without you all. I had fun, learned a TON about science and even more about myself, and had an overall amazing experience. 

Good luck to all of the future SFS-ers!

-Mike

Week 6

I still can't believe how fast the time is passing...

This week in the lab we ran into a lot of obstacles. At the beginning of the week we thought we were ready to compare PDAC and normal pancreas samples. However, when we ran our first comparison PCR, we did not get the results we expected. In fact, we got basically no results. So, I spent the week troubleshooting the PCR and figuring out what went wrong. I ran another reverse transcription cDNA synthesis to make sure it wasn't a problem with the cDNA. However, I believe that it was a problem with the primers used in the PCR. So, I ordered new primers and will hopefully be able to move on next week.

I spent Tuesday shadowing Dr. Thayer, who is the P.I. of the lab I work in. Dr. Thayer is also a surgeon at MGH, and specializes in treating cancer patients. I first followed her around in the clinic where she met with several patients. It was interesting to see the patient-doctor interaction as compared to when I was in the OR and the patients were under anesthesia. Later, I observed her perform a surgery in which she injected radioactive isotopes into a woman with breast cancer. She then examined her lymph nodes to see whether the cancer had spread/how it spread. This was all done in order to see if the patient was a candidate for chemotherapy or not. This was the most interesting part of the day because Dr. Thayer taught me a lot about breast cancer and how each and every patient has to be treated differently depending on things such as their age, personal beliefs/preferences, body type, etc. She also talked about the psychological effects breast cancer has on women. I loved hearing about this because breast cancer is so prevalent and effects almost everyone, whether they have breast cancer or not. It was also great to see Dr. Thayer working in the OR because before Tuesday I had only seen her when I was presenting my work to her at our weekly lab meetings (which can be quite intimidating).

Again, another great week!

Week 5

This week went really well, yet again!

In the lab, I finally finished finding our optimized conditions for the RT-PCR. The final step in finding these conditions was to run an RT-PCR with GAPDH primers (reliable, known primers we have) and the Hippo Primers in parallel. I needed to run different ratios of these primers to find a baseline ratio where the PCR could produce enough and equal amounts of each product (Hippo and GAPDH). This will allow me to compare the differences between normal Pancreas and Pancreatic Adenocarcinoma down the line. After the PCR, I found the correct ratios for three of the five Hippo primers. As for the other two, I found that the annealing temperature of 52 degrees was not producing accurate products from the cDNA. By using another temperature gradient during the PCR, I found that 61 degrees produced much more accurate products. I also tried increasing the magnesium (Mg2+) concentration in the PCR, which typically increases specificity of the primers binding to the cDNA. However, this didn't help out in the end. Now I officially have all of the optimized conditions for each Hippo primer, and next week I will finally compare PDAC and normal pancreatic samples. I may also compare chronic pancreatitis samples as well.

Although it has taken me five weeks to find these conditions, and I still don't have any conclusions on the Hippo project, this process has taught me a LOT about science. First of all, things rarely go the way you expect them to. However, you have to do your best with what you have and troubleshoot your way through the problems so that you can find answers to what is actually going wrong. I've also learned that research is just that: RE-search. You have to do experiments many times over and over. And many times, you are wrong. While this is often frustrating, it all has to be dealt with. I have also learned more and more about Pancreatic Cancer, lab techniques, and lab procedures.

For my clinical rotation this week, I ventured into the SICU (Surgical Intensive Care Unit). I first saw Dr. Bigatello (an attending in the SICU) give a short lecture on Acute Lung Injury and Acute Respiratory Distress Syndrome. This was very interesting (and confusing!) because treating these patients is a HUGE balancing act. There was a lot that went over my head, but what I did understand was very interesting. I spent the majority of the morning on rounds with a SICU team comprised of attendings, residents, anesthesiologists, interns, and more. They all worked together on discussing what treatments the patients have received  and are going to receive. This was incredible to observe for two reasons. The first was that it was amazing to see all the different types of patients in the SICU. I saw everything from a 91 year old woman who had a hernia to a morbidly obese woman who had recently had gastric bypass surgery and couldn't breathe on her own because of her weight. Every patient had to receive majorly different treatments, and each of these treatments had to be examined extremely carefully. The second reason this was so incredible was that I got to see how rounds actually works. I was very interested in seeing the team dynamic between the attendings, residents, interns, etc.: Everyone had an equal say, and while Dr. Bigatello clearly had authority, their teamwork was outstanding.

Overall the week was great. Harvard is still awesome. Although I miss home, I don't want to go home because there is not much time here left, and I'm trying to savor every moment.

-Mike

Week 4

It's crazy to think I'm halfway done with my SFS program!

Everything in the lab is going very well. I'm still working on the long, slow and steady process of finding the optimized conditions for the Hippo PCR experiment. This week I ran a lot of RT-PCR's. The most interesting one I ran though was one that involved a temperature gradient to find the best/most efficient annealing temperature for each of the primers. We got pretty good results from the gradient, and found that 52 degrees was the best temperature for most of the primers. This process also taught me the importance of making master mixes when preparing PCR's. When you have to pipet around 50 different PCR tubes each with slight variations, master mixes can save LOADS of time.

I also began isolating RNA from Pancreatic Adenocarcinoma (PDAC). This proved to be more finicky than expected, because PDAC simply doesn't have as many cells as normal pancreas, and therefore there's not as much RNA to be isolated.

For my clinical rotation, Dr. Dunn brought me down to the PACU (Post-Anesthetic Care Unit) to see Electroconvulsive Therapy treatments (ECT's). ECT is a treatment for people with severe depression in which small, controlled seizures are induced on the patient. This treatment was used in the early 1900's, but quickly lost popularity because it was seen as torture. However, with the advancements of anesthesia, the patients are now unconscious throughout the procedure, and have no recollection of the treatment after it is finished. Most patients go for treatments quite regularly (some once a week for months or even years). Though this may seem extremely taxing, it is by far one of the most effective treatments for severe depression. Just as I was in the OR last week, I was thoroughly amazed at what I saw in the PACU. The procedure took a total of 20 minutes per patient from the time their bed rolled into the room to the time it rolled out, and it literally can save their lives. It was incredible!

As for Harvard life, I couldn't be happier. I'm getting closer and closer with my new friends, I'm really enjoying Cambridge, and my psychology class keeps getting more and more interesting. This week we're learning about personality and intelligence. These two areas of psychology can be really hard to grasp - how can you define intelligence anyway? Is it knowledge? Natural brain power? If someone has a below average IQ score, does that mean that they aren't intelligent even if they are a genius at playing some musical instrument or sport?  Some of these questions are hard to answer, but it's definitely interesting to learn about!

All in all, everything is going great! I'm so glad I have this opportunity.

-Mike

Weeks 2 and 3.

These past two weeks have gone by so quickly! There's SO much going on, and I'm still loving it all.

The lab has been pretty much the same for the past few weeks. Right now, we're still working on setting up optimal conditions for the Hippo experiment. I've done a LOT of RNA isolations from different types of tissue (mouse liver, human pancreas) and cells. I have also changed some of the isolation conditions and made minor alterations to the isolation protocol. This is starting to make the isolations more reliable in giving us sufficient amounts of uncontaminated, pure RNA. Some things we have altered are the amounts of tissue used, and the ratio of amount of tissue and lysis buffer. If there is too much of one or the other, the RNA will degrade. We've also changed little parts of the protocol to speed up the entire isolation process.

The most exciting part of the past two weeks was when we ran a RT-PCR and got positive results without any degradation or contamination! This was exciting because it meant we had reliable RNA and cDNA (after the Reverse Transcription step). This allowed us to move on and use the primers in a PCR step that showed us the genes from the Hippo pathway. The PCR results were pretty good. We got some strong bands on the gel, and some not-so-strong bands, so we need to optimize the conditions of the PCR. So, we are working on finding the correct annealing temperatures, and amount of cycles for the PCR in order to amplify sufficient amounts of the Hippo genes. Once we find these conditions, we'll be able to actually run the experiment to compare the Hippo genes in cancerous and non-cancerous pancreatic tissues. There is still a lot more to be done in this optimization process, and it could be a few weeks before we actually run the experiment. Though this is very frustrating at times, it's great to learn how scientific research actually works: There is a lot of error, but we learn from those mistakes and unexpected results to help us reach our final goal.

On Friday, I went on my first clinical rotation. I followed Dr. Dunn (a Brooks alum. and father) around Mass. General's OR. We started the day talking a bit about the administration and organization of the OR. Considering the entire OR is comprised of seven different buildings, keeping things organized and functioning takes a LOT of work. After that, we talked a bit about some of the procedures we were going to see, and then got dressed in our scrubs and went down to the OR.

The first procedure we saw was a Carotid Artery case. Basically, this procedure is used to clean plaque out of the Carotid Artery. Plaque can build up in the artery as a result of many things such as smoking, hypertension, high cholesterol, genetics and more. This procedure can be very dangerous because minor mistakes can cause the patient to stroke. However, in many cases this procedure is necessary in order to prevent an inevitable stroke.

After this, Dr. Dunn pulled me aside and told me about the next case. He told me that the patient had a tumor behind his nose, and that the procedure involved making an incision across the patients hairline, and then essentially peeling down his face so they can reach the tumor from above. He then said that if I was uncomfortable seeing this or if I felt woozy or nauseous at any time, to let him know. Though his description of the procedure made me a bit nervous I said I thought I'd be alright, so we went in. As it turned out, I wasn't at all uncomfortable in the watching the procedure - I was simply amazed. It was incredible to see this unique procedure, and it was awesome to see a procedure that you never really think of when you think of surgery.

The final case we saw was a trauma case. The patient was a motorcyclist and was in a horrible accident in which he ended up in the back of the car in front of him. Though the trauma team didn't know exactly what injuries he had, they had to move quickly, so they basically had to open him up and look around for what they needed to fix. Though this may seem a bit unprofessional, they had a very methodic system and as far as I know, the operation was a success.

Finally, at the end of the day, Dr. Dunn taught me a lot about anesthesiology. This was a great opportunity because I knew nothing about it, and this is one of Dr. Dunn's specialties. We talked about the different drugs, gases, and treatments used during surgeries, and we also talked about anesthesia awareness - when a patient wakes up during a surgical procedure. This can occur for a number of reasons, and is also sometimes done on purpose to save the patients life. This, of course, was really interesting, and although there are a lot of small details I won't understand for at least a few years, I was very intrigued by it all.

Though there's a lot going on, I've also had a chance to go to Nantucket with some friends, and go home for a weekend. I love life in Cambridge/at Harvard, but it was definitely nice to get away for a bit to see family and friends and do a bit of relaxing.

We're approaching midterms at Harvard. Luckily, I only have one, whereas most of the other 8-weekers have two. There's still a lot of studying to be done, so I'm going to do that right now!

Thanks for reading!

-Mike

p.s. I'm hoping to get some pictures up of the lab, Harvard, etc.... So stay tuned!

Week 1

I took my first trip over to MGH on Tuesday, and was immediately welcomed and folded into the work at Dr. Sarah Thayer's lab. Dr. Thayer is also a surgeon, so she hasn't been around the lab much. However, I'm working udner Dr. Dirk Bausch who is from Germany and just moved tot he States just under a year ago to do research at MGH. Prior to his research work, he was a surgeon as well.

The main goal of the lab is to research the HIppo Kinase Signaling Pathway. This is a complicated pathway which still has a lot of holes in it that need to be researched. The Hippo Pathway has been extensively researched in Drosophila Melanogaster (the fruit fly), and mutation sin the pathway are largely connected to tumorgenesis in Drosophila. This pathway also parallels a very similar pathway in mammals. Hippo seems to be extremely important to cancer, especially Pancreatic Adenocarcinoma. At the lab, we are in the process of beginning to compare normal and cancerous pancreatic cells. We are going to try to detect whether the components of the Hippo pathway are over or under expressed in the cancerous cells.

(Although the lab is researching the mammalian version of this pathway, for clarity, I will describe it all in terms of the Drosophila version). In Drosophila,  the Hippo Pathway is essentially a pathway of kinases that regulates organ growth. When cells are growing, the pathway is "off," and the protein "Yorkie" binds to DNA and regulates the trancription of genes that are involved in cell proliferation and apoptosis (programmed cell death). When the organ reaches its "adult" size, an unknown signal activates the Hippo pathway. The kinase "Hippo" then activates the kinase "Warts" by way of phosphorylation. Warts then phosphorylates Yorkie, and thus inactivates Yorkie. This essentially causes the organ to stop growing because Yorkie is no longer able to regulate growth. HOwever, mutations in this pathway can lead to sustained activation of Yorkie and thus, uncontrolled growth and cancer. So, in the lab we are hoping to find overexpression of Yorkie (known as YAP in mammals) and underexpression of Hippo (MST1 or 2 in mammals) and Warts (LATS 1 or 2 in mammals) in the cancerous pancreatic cells. To see a more detailed description and diagrams see http://www.mshri.on.ca/mcneill/fat.html .

However, we have not gotten that far yet. My first task of the week was to test a RT-PCR (Reverse Transcription - Polymerase Chain Reaction) for contamination. It is important to test the kit for contamination so that we can be sure we are getting accurate results later on down the road. RT-PCR essentially converts RNA to cDNA, and then amplifies (or creates many copies of) the cDNA. Then, we use gel electrophoresis to see if everything went as planned. The kit tested fine, so we were able to move on!

On Wednesday, Dirk and I isolated RNA from pancreatic tissues (Thank you, organ donors!!). This process essentially involves grinding the tissue into a homogeneous liquid, and then filtering out everything but the RNA. The RNA is then mixed into a solution used in the RT-PCR.

On Thursday, we used our isolated RNA in another RT-PCR to test if we had contaminated the pancreatic RNA. After performing the gel electrophoresis, it was clear there was a decent amount of contamination. However, we did not know where this contamination came from. It was possible we had mixed something wrong, something had made it through the filter, or some other contamination occured during the RNA isolation procedure.

So, on Friday, I performed yet another RT-PCR test for contamination. In the gel electrophoresis step, we not only looked at the cDNA we had created in the RT-PCR, but also the diluted RNA, the DEPC-treated water used in the isolation, and straight RNA. It turned out that the source of contamination was the DEPC-treated water! Although this process literally took up an entire extra day of work, it saved a lot of work and confusion down the road, as well as prevented any false conclusions.

Next week, I believe we are going to begin actually comparing cancerous and non-cancerous pancreatic cells. There is also word that I may be able to go down to the mice lab and see what goes on down there, as well as go to PAthology to observe the retrieving of donated pancreases. I will also begin my clinical "rounds" in two weeks. I will be following doctors around the OR, ICU, ER, etc. once a week from then on. I can't wait!

Overall, everything is going extremely well! Life at Harvard is still great (even though the thought of taking a class during summer vacation isn't very attractive, I love it so far!). I decided not to participate in the choir, just because there isn't enough time in the day! Finally, although I was and still am a bit overwhelmed at the lab, I am learning a TON every day and am loving every minute of this new challenge.

-Mike

I'm at Harvard!

Today I moved into my dorm at Harvard's Secondary School Summer Program (SSP). I'm living in a 3-man suite in Greenough Hall, which is a dorm just outside of Harvard Yard. The suite has two rooms, both quite spacious,  so my roommates and I decided to put the beds in one room, and our desks and everything in the other room. It should feel like home soon enough!

Everyone here seems great (I just hope I'll be able to remember their names!). There are a lot of people from a LOT of different places. My roommates are from New York and Texas, and other guys living on my hall are from all over - Athens, Dubai, etc. Being just a short drive away from home myself, this is pretty exciting.

Along with my internship at Mass. General, I will be taking an Intro. to Psychology class at Harvard, which should be pretty interesting. Classes are Monday and Wednesday nights. Depending on my work schedule, I'm also thinking of auditioning for Harvard's Choir, which will meet on Tuesday and Thursday nights. Looks like I'm going to be quite busy, but I couldn't be more excited!

Well, that's all I have for now. I'll get to the actual MGH part once I get started later this week. I can hardly wait!

-Mike