My Career Plans

July 18th, 2007 by Kalen Riley

            As the Howard Hughes Fellowship comes to an end, I am glad to say that I have more insight into what my future plans will be.  All throughout high school, I believed that I wanted to attend medical school and be a practicing physician.  However, there was always a lingering voice in my head that said that research was what I really wanted to do.  Having had zero research experience prior to Howard Hughes, I faced a great difficulty in deciding what was best for me.  Fortunately, I had the opportunity to do research this summer, and although I still am not one hundred percent sure of anything yet, I am definitely heading in the right direction.

            One thing I have learned this summer is that research can be very, very tedious at times.  Often a researcher spends more time performing redundant but necessary experiments than contemplating an actual research question.  One may notice from all of my posts that I talk about western blots quite a bit.  Well, the reason for that is not so much that I just love western blotting, it’s that I have to do so many!  This     aspect of life in research definitely forces me to pause and think if a career in research differs from what I actually want to do.  I do not know if I want to be pipetting for years to come! However, the one aspect of research that I truly believe diverges from my true interests is how narrow and specific research projects usually are.  For example, much research has been done in the area of determining molecular/signaling pathways for cells.  While such knowledge is certainly interesting and necessary to advancing overall scientific knowledge, I am not so sure that it is what I want to study.  I believe that I would be more interested in studying organisms on a larger scale, such as a specific organ system, versus what specific molecules or chemicals are involved in one specific process in the brain.

At this point I feel that I am more inclined to pursue a medical career as clinician, clinical researcher, or possibly both.  The reason for this is that I believe that results from this type of job are more tangible and easily appreciated, although no less valuable than the results of researchers.  That said, I still do not know for sure if what I feel know will still apply in two years.  These eight weeks of research I will complete, while a great experience, are hardly sufficient to truly grasp what research is all about.  Many beginning researchers such as myself are given simpler tasks to do at first before they can move on the more advanced techniques.  So, I hope to continue doing research, hopefully in the same lab, throughout my undergraduate years.  More experience in the lab will help me decide for sure what I want.  Who knows, maybe I will decide that both a medical and a biological research career (M.D./Ph.D are in my future, although the many years of schooling required may just be enough of a discouragement to prevent that from happening!  Hopefully in three years time all of my questions will be answered.

           

Status of the Rac

July 13th, 2007 by Kalen Riley

Once again I must apologize for not being prompt with my blog posts. It seems like every day I say to myself, “I will blog today,” but then two weeks go by without anything being written. Anyway, last time I discussed how I intended to measure the activation of the protein Rac in Hela cells via cell stimulation with EGF. Well, the cells did finally grow to sufficient confluence and I made my cell lysate samples. I don’t think I have yet mentioned cell lysates, so basically they are samples made by destroying the cells but retaining all of the proteins and components in the cell. This experiment was run by doing what is called a Rac pulldown assay, in which the cell lysates are mixed with a substance called GST-PBD. GST-PBD selectively binds to activated Rac, so in this way we are able to isolate the active Rac and run the samples through a western blot (this I know I have mentioned!) to measure the activation.

Unfortunately, all the time spent making the lysate samples and running western blots proved to be fruitless, as the phosphor imaging scanner showed dirty protein membranes with absolutely no Rac bands. I have yet to determine what error caused this, as the scanner is in fact functioning correctly. This was a most frustrating occurrence, as it seemed that several days of work went down the drain.

After the unpleasantness, Hideji and I regrouped and decided to perform a simpler version of that experiment using 3T3 cells, which are fibroblast cells from mice. In some ways I am glad that I had to redo some of this experiment because I am finding that each time I make the lysates, along with doing the western blot, my technique gets a little better. This time the scans came out much better and I will be obtaining data next week by quantifying the Rac bands from the scans. Although I still have to “officially” analyze the scans, they look promising and I am very glad to end the week in this way.

Once again thank you for reading and although there are only two weeks left in the program I will give it my best effort to update my blog reguarly (well, maybe semi-regularly!).

Ending the Drought

June 30th, 2007 by Kalen Riley

So, it’s been a while since I last posted.  I admit that this is partly explained by me simply not writing, but luckily it’s not all my fault.  Several days ago when I sat down to make another post,  I attempted to view the main Research Blogs website and the screen came up completely blue except for the top where it would say “Duke University” and “Student research” and all that.  I was very confused by this, and was unable to write anything.  Luckily today I find myself able to log on and I am hereby ending the blog drought.

Life in the lab is going well.  I am still essentially doing the same things (western blots) over and over, although each time is a different variation.  Right now I am trying to measure activation of the protein Rac1 in HeLa cells, and western blotting is a very good way to quantify this activation.  Basically, I will be stimulating my cells with Epidermal Growth Factor (EGF) to promote Rac activation for varying times and measure the activation.  Of course there will also be control cells with no activation involved as well.  The big problem this past week was that my cells grew extremely slow.  I will be able to make my cell lysate samples on Monday, but that is almost a week later than originally planned.  It has been very frustrating because I did not get much done this past week all because some cells in a 10 cm dish did not feel like growing!  Oh well, I guess its better that they grew slowly instead of becoming contaminated with bacteria.  On Tuesday I checked my cells and started panicking because I thought that I had contaminated them.  I told Hideji, so then he came and looked and told me that they were fine.  What a relief!  Anyway, hopefully things will proceed smoothly from Monday on and there are no more roadblocks.

Once again, it is time for my discussion of non-research related subjects.

1.)    This is late, but who predicted that the Spurs would crush the Cavs?

2.)    Wimbledon is underway, and once again Roger Federer is the favorite.  I like Federer a lot, but I would like to see someone else breakthrough here, such as Andy Roddick.  Roddick is playing well right now, and is one a collision course with Federer in the semi-finals.  Roddick’s record against Federer is an abysmal 1-13, so chances are Federer will beat him again, but we can always hope.  I would have like to see fellow American James Blake go deep in this tournament also, but he found a way to lose in the 3rd round to a Spanish clay-court specialist.  Notice I did not list Rafael Nadal as someone I would like to see win here.  He may have a shot sometime down the road, but not now.  Other than Roddick, there is a small list of other players I would like to see win, but Roddick is the only one with a slight chance.

3.)    You may have been able to figure this out after my last long post, but I strongly, strongly dislike the New England Patriots.  It’s really tough to put my finger on the exact reason, but for now I will say Bill Belichick.  What kind of coach wears a hoodie with cut off sleeves on the sidelines?  There are many other reasons, but this is the first one that came to mind.

P.S.

June 12th, 2007 by Kalen Riley

I hope some people like my previous post, because you have no idea what kind of hell I had to go through to get it to work.  Thanks for reading!

A Third Attempt.

June 12th, 2007 by Kalen Riley

        So, last night I sat down to make a post, and before I was done writing I had to get up to get my laundry.  Instinctively, I hit the “save” key to safeguard my work from deletion.  Apparently, I did not hit “save” in the correct way, because my computer interpreted the command as “delete.”  Clearly frustrated, I quit for the night.  So, about one hour ago I sat down and attempted to post again.  After completing a thoughtful and insightful post (in my opinion), I pressed the “publish” key, which the computer again interpreted as “delete.”  Anyway, I cannot say that I am particularly pleased.  So I will try one more time, although it will be touch to reproduce the gold that was my second attempt!  

  I began this week in the lab by splitting the line of cells that I have been taking care of, and followed that up by finishing the western blot I started last week.  One annoying aspect of the western blot (and many lab procedures) is that it takes a very long time.  For example, yesterday I first had to incubate my protein membrane with a primary antibody for one hour, change solutions three times and incubate for three ten minute intervals, then incubate with a secondary antibody for another hour followed by three ten-minute incubations with solution changes.  During those steps, there is not much that you can do other than sit and wait.  I almost wish that there was more work to do!  Anyway, after that was done I began to image the protein membranes.  If the previous steps had been done correctly, the antibodies of the previous steps should have bound to the protein of interest (Rac1) such that bands representing the protein would show up on the images.  I imaged the protein membranes using two different techniques: a specialized scanner and film.  Using the scanner was not particularly interesting, but imaging using the film was an experience.  I first had to go into a dark room.  The entrance to this little room was not like the usual door, but instead like a revolving door that was a small capsule that let in absolutely no light.  Most of the time we had to work in almost complete darkness in order to not destroy the films.  Anyway, the end result of all of this imaging showed that I had indeed isolated and marked Rac1, which was the main goal of this experiment.  Other aspects of the experiment could have been performed more efficiently/better, but it was for practice and I am sure that I will become completely proficient in the necessary lab procedures over the course of this program.  At this point in the program I feel pretty well adjusted to working in a lab.  Every day I am becoming more familiar with lab protocol and simply where everything is.  I am also used to walking to the lab every day and the hot and very, very humid conditions that make up the North Carolina climate. 

I realize that the purpose of this blog is to share my experiences in the Howard Hughes program, but there are some unrelated subjects that I feel I must discuss.  I believe that these outside topics will both make my blog more interesting and increase the readership, which we all know is key.   

1.) I am very dissapointed that Roger Federer once again failed in his bid to win the French Open and the career Grand Slam.  He was thwarted by none other than the clay-court king Rafael Nadal for the third time in a row.  I have nothing against Nadal, but for the time being he is nothing but a clay-court specialist (perhaps the best ever) whereas Federer is likely the best around player in the history of the game and deserves to have the career Grand Slam.  Barring injuries, Roger Federer will most likely shatter Pete Sampras’ record of 14 Grand Slam titles.     

2.) The San Antonio Spurs will crush the Cleveland Cavaliers.  Lebron is very good, but Tim Duncan and Tony Parker are too much and I predict the Cavs will lose at home tonight.    

3.) I am so sick and tired of hearing people say how good the New England Patriots are.  They haven’t even gone to training camp yet, much less played a game and so many people are ready to hand them the Super Bowl.  People, if you must crown them so prematurely, at least watch a regular season game or two.     Thanks for reading my blog.  Pictures will be coming soon, assuming the upload glitch was  fixed.

June 8th, 2007 by Kalen Riley

Well, this is my first post.  I never imagined that I would ever write a blog, but this was inevitable (The program told us we would all be writing blogs, and here I am), so I will try to do a good job.  All this week I have been working in Dr. Ryohei Yasuda’s lab in the neurobiology department, working under a post-doc named Hideji.  My research this summer will involve imaging two proteins called Rac1 and RhoA in living cells.  Doing this requires one to be able to exploit and understand a phenomenon called fluorescence resonance energy transfer, or FRET.  When I was first told this is what I would be doing, I was completely shocked as I had never even heard of this “FRET”, and it was supposed to be one of the principle concepts behind my project.  As I read more about the topic, I became very interested and I am glad that this is what I will be doing.

Unfortunately, there was no FRET this week.  Most of my time thus far has been spent becoming proficient in elementary lab techniques.  so far, the things I have learned are how to split cell cultures, make electrophoresis gel by hand, run an electrophoresis machine, and carry out western blotting, to name a few.  Hideji has been great showing me how to do all of these things, and I’m sure he’s had to take a good amount of time away from his own research to help me.  Anyway, its been a pretty good first week and I look forward to learning more and getting involved in the actual project.