1-28-2010: Last day of the internship

Today was a really good day. It was pretty relaxed, all I did was dry samples that Dr. Nuss will run on a western blott next week. I also talked to Dr. Brown about my experience in the lab and he helped kind of connect the small experiments we have been doing into the whole grand scheme of all the research that has been done on invertebrate insulin-like peptides. During my off time I worked on my paper some more and started putting photos together for the presentation.

Tomorrow I will be shadowing Dr. Harper, a general surgeon.

1-28-2010: Results Day

I woke up this morning and realized that I did not post a blog yesterday... But we had a great day yesterday because we got really positive results back. The western blot that we have been running for the past three days with bloodfed vs. nonbloodfed showed phosphorylated tyrosine in the bloodfed mosquitoes and it was not seen in the non-bloodfed mosquitoes which is what was expected.

It was also good because I took a lot of pictures for my presentation next week and everyone thought that was fun. I was also able to take some pretty cool pictures of dissections through the microscope lense which I didn't think was going to work. This was my favorite of the dissections. From the left to right is the head, thorax, and abdomen of an Aedes aegypti mosquito. We seperate these parts in order to test the presence of peptides in certain areas of the mosquito.

1-26-10: paper feedback

Today I showed Dr. Nuss some parts of my paper that I had finished. I had written the western blott procedure for the materials section with a lot of my own notes and steps on how to do the procedure, but he said for a more scientific paper you only need to put the ingredients for the buffers, temperatures and more conditions than actual instruction on how to do the experiment. I also did a lot of dissections - 250 Aedes aegypti heads. We also did added the primary antibody to our blott and it is being washed on a rocker in the cold room right now.

I am working on my paper some more tonight and tomorrow we will be finishing the last western blott I will be here for and getting our results.

1-25-2010: Western blott and dissections

Today I basically did the same thing I have been doing the past two weeks. We started another western blott today with our bloodfed dissected mosquitoes and the non-bloodfed mosquitos in order to compare the amount of phosphorylated insulin receptor in the two groups. Today was more exciting because I did a lot of the western blott on my own. I set up and ran the gel and prepared the nitrocellulose paper and most of the western blott sandwich for running. I also took the gel and the paper out, which is one of the hardest parts of the procedure because the gel can stick to the paper and if it dries then you can't read it and it's important not to tear the gel and the paper. I did mess up a little and couldn't get a small piece of the gel off, but luckily it was over a part of the paper that didn't matter too much and it was ok that it didn't come off there. I also did a lot of dissections for the next western blott we will run that I probably will not be able to see.

Over the weekend I got my TB test and background check so that I can shadow in the OR on Friday and I will be shadowing a general surgeon, Dr. Harper. I also worked on my paper a bit over the weekend and Dr. Nuss said that he would read it tomorrow to make sure it's accurate and organized well.

1-22-2010: Shadowing Dr. Larrimore

Today I shadowed Dr. Larrimore who is an OBGYN in Covington, GA. I arrived at her office a little before 9:00 because she was doing a procedure at 9. However, her patient did not sign the consent form to allow me to view the procedure, but I was able to follow a midwife who works in her practice. The midwife did an ultrasound on a woman who is pregnant with triplets and showed me how to read an ultrasound. That was really cool because Dr. Larrimore said that she has been in practice since 1996 and this was only the second case she has had in which a woman was going to have triplets. The midwife also checked the heartrate of the baby of a woman who was about 15 weeks pregnant. By the time we had done that, Dr. Larrimore had finished the procedure. She had removed a benign tumor from a young pregnant woman and showed it to me.

Then we went to the hospital and saw three patients. One woman had just had twins and was in a hospital bed recovering. She was having a sharp pain near her right hip and Dr. Larrimore said that she may have an infection and had a nurse do a urine test to check for infection. The second woman we saw was having contractions and about to have a baby. We just went in to check on her and see if she needed any pain medication. The last woman we saw had just had a baby and was leaving the hospital that day. We also went to the area of the hospital where they keep the babies right after they are born. I talked with the nurses that work there and they told me all about their job and how peaceful it is unless they have a baby that is going through drug withdrawl because they are always irritable from being weened off of whatever drug their moms were on while they were pregnant. We also met and spoke with a radiologist who had done an X-ray on one of Dr. Larrimore's patients. Dr. Larrimore said that they usually only work halfdays on Fridays and I left during lunchtime around 11:30.

Now, I am about to write in my shadowing log about what I did today and I am going to write up a rough draft of the western blotting procedure we have been doing in the lab for my paper.

1-21-2010: More dissections...

Today I and Dr. Nuss did a lot of dissections, but these were different from the previous two types of dissections we have done so far. For these dissections, we isolated the midgut of the mosquito and will test on a western blott to see if a phosphorylated insulin receptor is present in the midgut of bloodfed mosquitos. The results will be compared to the peptides we find in non-bloodfed mosquitos. We expect to see a lot of phosphorylated insulin receptor in bloodfed mosquitos because our hypothesis is that bloodfeeding starts the insulin pathway. We believe that proteins from the blood interact with a receptor in the midgut (which may or may not be present... we will see) which then sends a signal to the brain and causes neurohormones to be released and sent to the ovaries of the mosquito which signals egg production and ultimately controls the reproduction of the mosquito. So, in order to isolate the midgut in order to test for the phosphorylated receptor, we put about 100 mosquitos on ice in order to anethesize them and then decapitated them so that we could pull the midgut out of the mosquito from the tail (part of the midgut is attached to the head of the mosquito which is the reason for the decapitation). By pulling on the bottom tip of the abdomen of the mosquito, the outer layer rips and the attached ovaries, digestive tract, midgut, and salivary glands all come out in one connected string of organs. We then seperate the rest of the organs from the midgut and then put the midgut into solution (made up of ingredients that preserve the midgut and inhibit proteases from degrading our target peptide). We had to isolate the midgut from 40 bloodfed and 40 non-bloodfed mosquitos, so we did that and then prepared the extraction solution. After setting the midguts into dissection solution, centrifuged them and then resuspended them into homogeniztion solution (and I am not sure what the function of this solution is). We then ground up the pellet into the solution in order to release more of the target peptide. We then stored our samples in the -80 freezer.

In the midst of doing dissections, I also prepared the western blott for the primary antibody wash. I put the antibodies on and the nitrocellulose paper is soaking in the antibodies overnight.

Tomorrow I will not be shadowing Dr. Harper because I found out that I have to have a background check and a TB test in order to shadow in the operating room. However, I will be shadowing an OBGYN, Dr. Larrimore, tomorrow.

1-20-10: Gel Day

Today we started a new western blott with the samples we prepared yesterday. This morning, we first prepared the samples by adding ethanol to rehydrate the peptides and sample buffer. We then vortexed them to resuspend the proteins, poked holes in the top so that pressure would not build up in the tubes during heating and then put them in the incubator at 55 degrees Celsius. After the samples were prepared we put them on ice and loaded them onto the gel to be electrophoresed. We ran the current through the gel for about three and a half hours. After that, we prepared the western blott sandwich by cutting the blotting and nitrocellulose paper and soaking them in 10% methanol. Then we removed the gel and put the sponges, blotting paper, nitrocellulose paper, and gel into the sandwich. The proteins are being transferred from the gel to the paper now and will be ready around 4:05. After the paper is ready, we will remove it from the sandwich and set it out on our lab space to dry overnight.

Dr. Brown gave me some extra textbooks today on endocrinology that should help with the primary literature I have been reading. I have been reading one of the books during some of the down time today.

I also spoke to my dad last night and he said that he had arranged for me to shadow Dr. Harper, general surgeon on Friday.

Tomorrow we will label the paper and do the primary and secondary washes over our western blott.