PBS Solution!

12.11.12

Last Tuesday (December 11th), I went over to RPI for another day of lab work! Today's work was preparing for a new experiment that is analyzing the size of particles that can normally pass through the blood brain barrier. As seen in the figure below, the set up for this experiment will be two wells (one inside the other) with a filter on the bottom of the inner one separating the two. On this filter, we will grow a monolayer of brain cells to represent the blood brain barrier.

We will have many of these setups in order to test solutions with different size molecules. We will be testing solutions with molecules that are 4 kDa, 10 kDa, 20 kDa, 40 kDa, and 70 kDa. All of these molecules are dextrans, or different size sugar molecules. For the normal type blood brain barrier, the permeability to size curve would look like the black line below. With the peptide modulator we are currently researching to find, we hope to make the curve look like the red line below.

In preparation, I prepared two Liters of phosphate buffered saline solution. This PBS can be used to dialyze protein solutions. To make this PBS, I combined 15.52 mL of 1M K₂HPO₄, 4.48 mL of 1M KH₂PO₄, 2 L of distilled water, and 11.6 g of NaCl. After mixing these contents, I had to raise the pH to 7.4 using 1M NaOH base. After many trials and test with a pH probe, the pH reached 7.4, and my PBS was ready to be filtered. To filter the solution, I used a bottle-top filter with a vacuum attached, like the one shown below.

My second job was to label the test tubes for the experiment. 10 mL samples will be taken from the bottom chamber 12 times over a 9 hour period. I labeled the test tubes needed for the first 5 data collections by writing the time on the cap and the time- kDa of the solution on the side of each tiny test tube.

I can't wait until next week to hear the results of the experiment!

Lab Work!

12.4.12

This Tuesday (December 4th), I went over to RPI for my first day working in the actual lab. The goal for today was to print 15 slides of peptide matrices to be able to run tests on in the future. There are 56 peptides that we are working with, and each peptide is printed in a 3X3 matrix on each slide. This means that there are 9 dots per peptide, and 504 dots per slide! Ben showed me how the computer program worked, and we checked the (x, y, z) coordinates of the printing needle on each slide so the machine would run correctly. The x and y values were mostly already correct from previous runs, but the z coordinates (height) needed some adjustment so that the needle was touching the slide. This placement of the needle allows the machine to dispense the correct amount of peptide on the slide. The printer works automatically after the programming is set, and the whole process takes about 3 hours! After double checking the coordinates, Ben showed me how to use the centrifuge to spin down the peptides. When the peptides were ready, we put them in the printer, and started the program. We had to watch it through its first cycle to make sure that it wasn't missing any dots because the machine often doesn't function correctly. Today the machine was running well, and we did not see any malfunctions!

I can't wait to return next week and see what's next!

Training

11.28.12

This Wednesday (November 28th) due to the school schedule change, I took my
trip over to RPI. After arriving late due to transportation confusion, I promptly
began my task for the day. I had to complete two training courses before I could
participate in actual lab work. This training consisted of about two hours
containing courses in both general lab safety and biohazard safety. The training
used an online course that consisted of text slides, audio, and video, and "tests"
were included along the way. After learning a ton about MSDS (material safety
data sheets) and different safety controls in the labs, I passed the final exam
(especially because the program made you retake it until you got a perfect score!).
After my training, JP took me to get my official RPI ID card. After giving many
forms of legal identification, the clerk took my picture and made me an official
part of the RPI system! I can now enter the building, office, and lab by
myself (finally!)! 

My ID!

Next week, I am hopefully going to participate in the lab because I have now 

completed all of my training. I can't wait!

Getting Started!

11.13.12

Last Tuesday (November 13th), I had my first real internship visit! I was extremely nervous, but also very excited because it was my first trip off campus alone. I met JP- the graduate student I am working with- and he gave me an introduction to his work. As seen in figure 1 below, the capillaries of the brain have a monolayer of endothelial cells that contributes to the blood-brain barrier. Between these cells, there are proteins called claudin-5 that serve as tight junctions between the endothelial cells and are semi-permeable. This semi-permeability allows some molecules to pass through the brain, but inhibits others. JP's research is looking for a substance that can stop the reactions between the claudin-5 proteins to allow the passage of medicines to the brain.

Figure 1

After this introduction, JP and I had an arts and crafts break, and we made models of endothelial cells out of cardboard boxes and pipe-cleaners. When we were done with this activity, he showed me the lab and introduced me to the other researchers. My first job was to help Ben (one of the other researchers) analyze the R-squared (R2) values of the most recent tests of different inhibiting proteins over a range of pH values. The R2 value measures the fit of the experimental data to the given best-fit curve. According to this data, we could determine the 'valleys' and 'peaks' of the data to determine the proteins that were working the best and worst. After examining a lot of data, we determined that we should remove the last two pH values to increase the R2 values of the data. I was taught to operate the computer program, and I accomplished this all by myself! The new graphs had much better R2 values.

Next week, I will take the safety course to get my access badge. I excitedly wait to see what next week has in store for me!

First Meeting!

11.6.12

This Tuesday (November 6th), I had my first visit to RPI to meet my mentor, Dr. K. The building I will be working in is a restricted-access building, so Dr. K met me at the door to let me in. After introductions and discussion about my interest in both chemistry and biology, he paired me with a graduate student named JP, who I will be working with in the lab. JP's research focuses on the biochemical interactions of the blood barrier in the brain, and how it inhibits the transport of drugs to the brain. This research will hopefully be applied to drug delivery for the treatment of diseases like Alzheimer's, Parkinson's, and brain cancer. This research aligns with my interests perfectly, and I can't wait to get involved!! I was not able to see the lab because I have to go through a bio safety course first, but Dr. K told me the main machinery that is used is a protein synthesizer, which does the work of human cells to create proteins. Next week I plan to take the bio safety course and receive a pass card that will allow me to access the building and the labs. I can't wait to start working and see this research in action!

Excitement

10.30.12

As I await the start of my internship, I am very excited to learn more about the interrelationship between science and medicine. The ability to engineer new materials to aid in new medical processes is something that I find intriguing, but I have never had the opportunity to experience it in action. Do terms such as biocatalysis, hybridoma cultures, and hydrophobic/hydrophilic interactions excite you? They do me! I hope to learn about things that I now have little to no knowledge of, and I look forward to doing this in an environment that is exciting, new, and unknown!