Week 8: Department of Anatomy and Cell Biology, University of Iowa

August 15th, 2013

Brianna Christensen ’16, Dimensions Fellow in Research

This week was absolutely wonderful. I showed my poster and practiced my talk in front of those in those in the FUTURE Program. Though it wasn’t stellar, I know that the more I practice, the better I will be at speaking in public. On Tuesday, I printed my poster off. It’s the first time I’ve done this, and I’ve heard horror stories about coloring issues. Thankfully, everything turned out perfectly fine. It soon became my baby, and I probably became overly protective of it…

My baby

I discovered how much fun it is to present my poster. It’s so much more conversational than standing up in front of a crowd. I think that I am able to explain things better this way especially since questions can be asked along the way. It was also exciting to see all the posters at the Summer Undergraduate Research Conference and interact with new people. This conference allowed me to see all the different kinds of research that took place over the summer. Sometimes I was so focused on my project that I forgot that there are so many other projects going on.

A couple of the people from the FUTURE Program that I had the pleasure of being with!

On my last day, I had the FUTURE Research Symposium. I was fortunate to have my family come, along with my boyfriend. This program was just phenomenal. I found out that I really do like research, and I’m glad that I found this out early on. Now I have a little more time to figure it out if I want to try to get into a dual program or just medical school. Everyone that I have met has been so supportive, and I appreciate all the help that I received in and out of lab. I feel more prepared to tackle what’s ahead of me. I have my foot in the door for more opportunities, and I think I’ll be able to perform better in lab at Cornell. I feel more confident with certain equipment and procedures, and I able to understand scientific papers with more ease.

There is so much going on in my mind, and I can’t put it into words. I will never be able to thank the FUTURE Program, Barbara, Rob Cornell and his lab, and the Fellows Program enough to allow me to have this opportunity. I can’t express how much this experience meant to me, and I will never take it for granted.

John and me

My grandparents and me

 

Showing my sister the lab

 

Week 7: Department of Anatomy and Cell Biology, University of Iowa

August 1st, 2013

Brianna Christensen ’16, Dimensions Fellow in Research

Week 7 was wrapping up my experiment as much as I could. I finished running qPCRs using the cDNA from a NFN trial. After that, I compiled that data to determine if my results were significant or not. I was extremely pleased to find out that NFN significantly decreases TH1 and TH2. Yay!! However, the next thing we became concerned about was if NFN was affecting dopaminergic neurons specifically or if the whole brain was just fried. I then ran a few qPCRs looking at Brain Derived Neurotrophic Factor (BDNF) and Glutamate Decarboxylase (GAD). BDNF would allow us to assess the brain overall, and GAD would allow us to look at neurons that are glutamate specific. By the end of the week, I had analyzed that NFN does not significantly affect either of these expressions. Our results suggest that NFN affects dopaminergic neurons specifically.

The other thing that I’ve been working on is my TH2 probe. I haven’t had any success getting good expression with my embryos which is a little disappointing. I’ve adjusted the protocol which I was hoping would help. The embryos now overdevelop really easily, but putting them in 100% methanol is supposed to help clear up background, so maybe by Monday of next week, they will look good. It would be nice if I could have pictures for my poster!

I also injected the RNA I synthesized from the gene I cloned (vmat2) into embryos. I’m really excited that I had a higher survival rate this time around, and I think I understand what I’m doing wrong. Hopefully, I will be able to be successful if I need to inject again.

Barbara and I set up another experiment with pargyline, so we can assess their behavior next week.

Otherwise, I’ve just been working on my poster. Right now, I’m working on the wording for the introduction, methods, and conclusion. I’m planning on having that portion done by Friday.

Week 6: Department of Anatomy and Cell Biology, University of Iowa

July 19th, 2013

Brianna Christensen ’16, Dimensions Fellow in Research

My poster now has a title!!! The title is ‘The Inhibition of Aldehyde Dehydrogenase Affects Melanocytes and Dopamine Neurons.” Once Barbara and I finished figuring out the title, I realized that I need to make a poster. To make the poster, I need something to show for all the work that I’ve done over the summer. I need results. This sends me into a thinking frenzy questioning whether or not I actually have something to show the people that come to view my poster. I soon realize that I shouldn’t really be asking myself if I have results or not because I do have results. That’s not a problem. I should be asking what results should I use. I need the right combination of results to best tell others what I need and how it went. As my PI puts it, I need to be able to tell a story.

I have a general idea of what I want to include, and now I’m working out the specifics. I’m still gathering some more results just to back up what I’ve already found for the most part. Since I only have about two weeks (one week of just research only), I’ve been working long hours usually 11-13 hours a day. It’s all been worth it because I know that I’m getting as much done as I possibly can. I feel like then I can be proud of what I did this summer no matter how my results turned out.

We started another experiment (I know it’s crazy). We are now testing to see if Pargyline will prevent NFN from affecting the dopamine neurons. NFN inhibits aldehyde dehydrogenase in dopamine synthesis which prevents DOPAL becoming dopamine. DOPAL, which is toxic, builds up causing the problems that we are noticing in the embryos. Pargyline is a monoamine oxidase B (MAO-B) inhibitor, so we are hoping that will prevent the buildup of DOPAL. We don’t expect a recovery in the melanocytes, but we are hoping to see an affect on the dopamine neurons.

Today I’m going to see a graduate student defend their thesis. When I started this research position, I found out that I enjoy researching, so I’ve been considering a MD/PhD dual program. I still need to think it over and do more research about it, but I have a few years left of college left to do that! Anyway, I think that this will be beneficial to see what this part of the process is like. This will allow me to get a bit of better idea of what I would have do for the PhD portion of the program.

Week 5: Department of Anatomy and Cell Biology, University of Iowa

July 7th, 2013

Brianna Christensen ’16, Dimensions Fellow in Research

Week 5 has passed, and I’m well into Week 6. Time has flown by, and I’m starting to realize that I only have about three weeks left until I’m done with the FUTURE Program. Three weeks to finish up as much of my experiment as I can, make a poster, and be ready to present it. Three weeks hardly seems like enough time, yet I start another project. Who doesn’t love a challenge?

For about a week, I have been going through the process of cloning a gene called vmat2. In the neurons, there are vesicles that help the neurotransmitters travel to the membrane of the neuron to be released. Vmat2 is found in the membrane of these vesicles and affects the ability for neurotransmitters to go inside. Another member of the lab is cloning the pink1 gene that I have mentioned in previous  posts. We are looking to see one of these genes will rescue the touchtone zebrafish mutant. After the cloning process is complete, we will inject the gene first into wildtype zebrafish to make sure that nothing is toxic to the fish. We will then inject the mutant zebrafish and let them develop. At 48 hours, we will perform a behavioral test that just involves touching the embryos a bit to see if they move. For wildtype embryos, they will zip all around the dish when probed. Touchtone mutants, on the other hand, won’t move during this test. After 5 days, we will run another behavioral test which will be like the one I showed in a previous blog. The embryos will then either be used for insitu or qPCR. Basically, we will be looking to see if pink1 and TH2 (and maybe TH1) levels have improved.

A diagram showing where vmat2 is and its relationship with dopamine

A diagram showing where vmat2 is and its relationship with dopamine

With this project, I’ll be able to use previous results. I have data showing that pink1 expression in down in the touchtone mutants, and I also have data showing that when fish are placed under oxidative stress pink1 expression is elevated (wildtype embryos were placed in hydrogen peroxide). This just allows me to have data to compare my future results to which will allow me to interpret if there is improvement or oxidative stress.

With the nitrofuran experiment, Barbara and I are trying to get as much done as we can. We are continuing more trials to repeat the insitu procedure, qPCRs, and behavioral tests to make sure that our results are reproducible. We have also started taking pictures of our insitu results which gives us a basic idea if the expression of a certain gene has changed. So far, our results have shown that movement is being affected which suggests that the nitrofuran is affecting dopamine in these fish. In my last post, I mentioned the very large insitu that we were working on which was going to show us the amount of melanocytes in the fish. One of the things that we noticed was that the wildtype embryos in nitrofuran looked similar to the touchtone control embryos. Another thing that we noticed the embryos that were in both nitrofuran and PTU had more melanocytes compared to the embryos just in nitrofuran. This suggests that the PTU is preventing the melanocytes from being killed. It will be interesting what else we see as we continue!

One of the pictures from the photo session last week

One of the pictures from the photo session last week

For the FUTURE Program, there was a reception held at Dean Schwinn’s house, and she is the Dean of the Carver College of Medicine. Current and past participants of the FUTURE Program, hosts of the current participants, and a few other administrative members. It was a great experience because I was able to interact  with people in different departments. Even though I did feel a little out of place, I think it was good that I gained exposure to a new experience like this.

On an unrelated note, I was able to see my family on the 4th. We went out to my grandpa and grandma’s house where we met up with my mom’s two brothers and their families. It was great to see everyone and relax a bit!

 

Week 4: Department of Anatomy and Cell Biology, University of Iowa

June 28th, 2013

Brianna Christensen ’16, Dimensions Fellow in Research

I am pleased to say that this week went better than the previous week! For my experiment, I am pretty much repeating what I have done in the past. I am making cDNA from RNA that I’ve isolated from previous trials with the NFN and/or PTU. I’ve held back on making this earlier because I wanted to make sure that I could obtain significant data using qPCR first. Our most recent trial is by far our largest yet. It includes not only wildtype fish but mutant zebrafish called touchtone (tct) resulting in 24 test tubes that contain approximately 360 zebrafish embryos. Due to the large number of test tubes, the insitu that will perform on the embryos will be a challenge. I think that this will be a test at how well I can perform this procedure, so I’m excited tackle this challenge!

All of the test tubes!

All of the test tubes!

This week I also learned about collaboration. My PI, Barbara, and I met with another PI of a different lab to discuss about working together. Someone in his lab will be working with cell lines to gather more data for the project that I’m working on. I think that this is a valuable experience for me because not only have I learned how to work with other people in my lab but with other people from different labs as well.

For the FUTURE program, I had to have my picture taken with Barbara and my PI for their yearbook. It was interesting because I haven’t taken candid pictures before only posed ones. I have to say it was a little awkward, but I’m excited to see how they turned out!

I’ve been reflecting this past week on how attending Cornell has prepared me for my research position. One of the things that I’ve noticed is that I am able to really focus on projects especially more long-term projects. For example, my lab in Cell and Molecular Biology was the same project for the entire block. This has helped me gain the ability to not focus on a project for several weeks but also keep the main goal of the project in mind. As I’m mentioned in a previous post, I’ve had to read a lot of scientific literature. For Cell and Molecular Biology, I had to select two scientific papers and provide a summary over them. By gaining exposure to this type of material, I know what I have to look for and what I need to do to ensure that I understand the content. I hope that as I practice this will only get easier for me.

Week 3: Department of Anatomy and Cell Biology, University of Iowa

June 18th, 2013

Brianna Christensen ’16, Dimensions Fellow in Research

I’ve learned a lot this week. Yes, I’ve learned a lot during the other weeks too, but this time it’s a different sort of knowledge. Of course I’ve learned more procedures and tips on how to perform them better. I’m sure that I will continue to learn those things every week. However, the big thing that I learned this week is it’s okay to be wrong and mistakes happen.

Now it may seem like a surprise to some of you that I haven’t realized this. After all, we have been told that since elementary school it seems, but I’m a perfectionist. I want things to work out the first time. I feel like I have failed when things don’t go the way I wanted them to. This made Thursday a fairly stressful day for me. I have been looking at the level of expression of pink1 in trpm7 mutant zebrafish compared to their wildtype siblings. This is done using the qPCR machine, and it takes quite a bit of time to prepare it, run it, and then analyze the data.

My results were too inconsistent, and they were turned out insignificant.

I’ve also worked on making a TH2 and a DCT probe for insitu for later trials with some of the other projects I’m working on.

Those results suggested that they wouldn’t work.

A few other things weren’t turning out right that day too, and I felt frustrated. However, I was then reminded that in scientific papers, they don’t tell you how many times they had to run the experiment to get their results consistently or how many times they made errors. It’s true, and I know at least for me that’s forgotten.

Things improved over the rest of the week. After a bit of tweaking, I got the probes to work, and I am now putting one of them to use. I’m continuing the trials with the qPCR. I haven’t been incredibly successful, but I’ve been trying a couple of different techniques to see if that improves my results. The most recent one seems to be helping so far! I also ran a behavioral test with a recent trial from the nitrofuran and PTU experiment. We had some really interesting results, and I’m excited to see what the next trials show.

When things don’t seem to be working from now on, I’m going to think about what I can try differently instead of instantly getting frustrated…oh and chill out with some RNA at my desk.

pic 9

 

Week 2: Department of Anatomy and Cell Biology, University of Iowa

June 10th, 2013

Brianna Christensen ’16, Dimensions Fellow in Research

Week two has been pretty exciting for me! I’ve started a few more projects, and I’m beginning to see the results! As I mentioned in my previous post, I did start a project involving NFN and PTU. Unfortunately, some of the embryos that I had left to look at their movement, all of the ones in NFN died, so I was left with the control embryos and the ones only in PTU. However, it turned out to be okay because I used those to test out the behavioral machine. The machine is able to detect their movement through the embryos pigmentation. The embryos in PTU no long have pigmentation, and there lies the problem. We placed them in red dye to see if they would absorb the color and if the machine would be able to detect the color. The embryos did absorb the color, but the results of their movement make it difficult to determine if the machine was actually able to see them.

This is the program used to detect the embryos' movement. When they move, green lines appear following their movement.

This is the program used to detect the embryos’ movement. When they move, green lines appear following their movement.

Since the embryos in NFN died, we decided to try again. We added a petri dish with a lower concentration of NFN, and we also added another dish with a lower concentration of NFN and PTU. We also thought that maybe they had to be out of their embryonic sacs for the drug to actually affect them which would possibly explain why they died after 48  hours. We also made a couple of other adjustments to either keep the embryos alive or to make sure that NFN was working.

We also placed embryos in hydrogen peroxide, and this is suppose to create oxidative stress which will become more useful later on. As of right now, it doesn’t look it it had much of an effect on the embryos, so either we new hydrogen peroxide or we need a higher concentration.

After learning some of the basic procedures, I have been instructed in how to perform a qPCR. I am learning why they perform so many repetitions with this procedure. I have completed four trials, and my results have changed. I have really good results, some okay results, and some results where I sit there thinking, “what on earth…”. The point of this is to compare the levels of gene expression for pink1, TH1, and TH2 between a mutant zebrafish’s cDNA and its wildtype sibling’s.

This is the qPCR machine.

This is the qPCR machine.

This is the program used to collect the data, so I can analyze it.

This is the program used to run the machine and collect the data, so I can analyze it.

I’ve been able to get to know those in my lab fairly well, and today we are getting frozen yogurt ! I’ve made plans outside of the lab as well. I will be donating plasma for the first time tomorrow. I’m really excited for that! One of my housemates also asked me if I wanted to go to the farmer’s market with her next week, so I’ll have pictures of that for next time!

This is shows the development of the embryos. It's a lot cooler in person!

This is shows the development of the embryos. It’s a lot cooler in person!

Fun fact: it takes about a month for a zebrafish to become fully grown!

 

Week 1: Department of Anatomy and Cell Biology, University of Iowa

June 3rd, 2013

Brianna Christensen ’16, Dimensions Fellow in Research

My first week has been super busy! I am working in the Department of Anatomy and Cell Biology under Rob Cornell. His lab is looking at neurodegeneration in zebrafish and how it specifically relates to Parkinson’s disease. I am also a part of the  FUTURE program, so my time will be divided between the research I complete in lab and this program. I moved in a few days before I actually started. I am subletting a room in a house that is not too far from the building I’m working in. I will be living with six other people, and it’s been really fun getting to know them!

This is the room that I am staying in.

This is the room that I am staying in.

I had orientation on Wednesday through the FUTURE Program, and I met the other students and faculty involved in the program this year. Orientation included tours of the DNA facility and the Microscopy lab. We had our pictures taken both in a large group and with our respective faculty member and PI. I will be attending weekly meetings on Mondays, and they also offer seminars on Thursdays.

Fun fact: Some people say that this building was built to resemble DNA.

Fun fact: Some people say that this building was built to resemble DNA.

Most of my time in lab has been spent getting acquainted with where everything is and completing basic procedures. This was just to insure that I could accurately perform these tasks because I will be using them often as I conduct my research project. One of my favorite parts has been breeding the zebrafish and watching their embryos develop.

This is me collecting wildtype zebrafish. We had them breed, and we would use their embryos.

This is me collecting wildtype zebrafish. We had them breed, and we would use their embryos.

One of the basic procedures was to test primers used for qPCR.

One of the basic procedures was to test primers used for qPCR.

I have also been in the process of reading A LOT of scientific literature including 9 articles, 1 thesis paper, and 1 other paper, but who’s keeping count? We have weekly lab meetings on Fridays, so I just recently went to my first one. I was a little intimidated, but it was really interesting to hear about everyone’s project that they are working on and how that contributes to the overall goal of the lab. I’m very thankful that they have been so patient  with me and are willing to explain various concepts to make sure that I understand the point of the projects.

I have started one project that I am very excited about. Essentially, it is testing the affect of a combinations of drugs (PTU and NFN1) to see how they affect the melanocytes in these embryos. Due to how these drugs work, we are hoping to see PTU block NFN1′s ability to cause melanocyte death. For this project, I had to tear open a few of the embryonic sacs that the embryos are in, so I could fix them in a solution. These embryos are super tiny making this extremely hard to do. I can’t wait to see the results during the second week!

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