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!