Week 10

Thankfully, I decided to move back to doing Wes plates this week, as my supervisor Sarah deemed it necessary.

On Monday, we both worked on a Wes plate centering on the antibodies TSP-1 and a mulitplex of TSP-1 and rabbit action or mouse actin. We went through all the procedures as carefully as we could. But in the end, the procedure did not work too well, as the expression of TSP-1 in the samples from the S2BF( barrel cortex) region of the rat brain.

On Tuesday, we then ran a Wes plate comparing the S1BF and S2BF samples from the same animals to see if there was an issue with the tissue along with checking the multiplexing issues. After we completed the procedure, I worked on data entry and called it a day afterward.

On Wednesday, we ran a similar plate as the one on Tuesday, again checking the mulitplexing issues. After that procedure, I labeled tubes to be used for tissue collection and afterward I called it a day.

On Thursday, at the lab meeting, we discussed a lab article on Glutamate amount in relation to a drug, MS-153.

Till next week, signing off.

Week 9

Well, this week has been kinda of a doozy.

Ever since I figured out along with my supervisor Sarah that the issue with the Wes machine may be with the tissue, we have decided to check every individual Wes file for flaws in every plate to send it to Protein Simple, the company that manufactures the machines, to aid them in diagnosing the issue.

So for the past 4 days, I have been entering Wes files into an excel spread sheet by documenting each file based on what antibodies they displayed. In each folder, I then assessed what region of the brain we were testing, the date it was ran, whether the run worked, what peak did the antibody fall under etc. We are about 50%-60% through in accessing all the files.

Along with working on the files, I have been working hard on my literature review that can go into more detail on my project as such. When I am finished with the literature review, I will send it out through the blog. 

Hopefully by tomorrow or next week, we can get back to working on Wes plates and continuing our study.

Till next week, signing off.

Week 8

This week has been an unique experience as well as a lesson in the finer points or research.

On Monday, I started work on my SRP presentation with the help of my adviser Sarah. After a few hours, I attended a meeting with Dr.Thomas and Sarah where we discussed previous medical school applications and talked about what it takes to become a successful pre med student. I found the meeting a great lesson on the trials and tribulations of the medical profession and what it takes to rise above other pre med candidates. After that, I called it a day.

On Tuesday, I ran a Wes plate testing many different regions, reagents, and antibodies to determine problems with the Wes machine. After that, I worked on my presentation before calling it a day.

On Wednesday, I arrived to see that the plate was completely messed up due to the fact that we used an expired Wes plate. I then helped run a redo of the same plate again to assess the same issue again. After that, I helped organize a few papers and worked on my presentation.

On Thursday, in the lab meeting, we examined a publication and assessed its major flaws. After that, Sarah assessed the data and gathered that the region was messing with the data. Then, as she meet with Dr.Thomas and did some work for the rest of the day, I shadowed other researchers at the lab and called it a day.

Till next week, signing off.

Week 7

Well, the good news for this week is that I learned a lot about how to really create a study design for my SRP and gained an unique understanding for the importance of preparation and direction in research.

The bad news: I couldn't run Wes plates this week, as Sarah and Dr.Thomas want to still trouble shoot the problems that have been plaguing the machine.

On Monday, I, along with Caitlin, worked to find and locate samples from the hippocampus region of the brain. The MPFC region was not producing great results for the most part so Sarah decided that I should instead focus on the hippocampus region and to gather the samples we need. After accomplishing this, I went to work on my study design and called it a day.

On Tuesday, I worked again on sample collection as well as working on my study design.

On Wednesday, since Caitlin was not here, I could not work on the Wes. Instead, I helped Samdeet on his project, working on data collection.

On Thursday, our lab discussed the need for accurate study stipulations in research, such as randomization etc. I then helped clean out the freezer as well as working on my study design.

Till next week, signing off.

Week 6

This week was a very engaging and instructive experience on how patience is a virtue.

On Monday, I ran Wes plates on the mid pre frontal cortex region of the brain. Unlike all other days running Wes plates, I ran four plates on the same samples but for both neuroligin1 and glypican4 to determine protein amounts of both in those Wes plates. After running those plates, I did some work on my study design as well as some housecleaning work.

On Tuesday, I checked the Wes runs that we had ran the day before. The result was unexpected. All of the four runs were all off because there was no peak for our biological control, GAPDH. I was shocked and alarmed at the scenario presenting itself. After managing some sample work as well as some data analysis, I called it a day.

On Wednesday, Caitlin and I ran our optimization plate for the MPFC region to ascertain the flaws in the samples. After this, I worked extensively on my study design and analyzed some data.

On Thursday, I looked at our data and ascertained that the biological control was still not present and therefore we needed to run a total protein assay to map out the protein involved. After prepping that plate, I called it a day.

Till next week, signing off.

Week 5

After a week off for spring break, I felt ready to get back to work on my project and maybe gather a lot of useful data for my project as well.

But this week was again not only unique but also a very informative experience.

On Monday, I finished the two Wes plates we needed to collect all the data for the S1BF region of the mice brain. For the plates themselves, I ran two plates testing for Glypican 4 in S1BF samples from day1-7 post injury to complete that region.

During that day as well, I had a meeting with Dr. Theresa Thomas and my senior advisor, Sarah. There, we discussed the challenges facing students who pursue the medical profession and the characteristics that are needed to truly elevate yourself from the pack. All in all, the meeting was very informative and an unique learning experience.

On Tuesday, I started running Wes plates for the MPFC (mid pre frontal cortex) region of the mice brain. After that, I gathered samples from the MPFC region that need to be used for future plates and put them into one box for safe storage in the freezer. I then called it a day after that.

On Wednesday, my senior advisor, Sarah, advised me to work with the Compass software that I use to interpret the Wes results and learn more about the units and values I measure in my experiments. After a few hours tinkering with Compass, I aided our lab manager, Bret, in his study on cage dividers and their effect on mice aggression.

I hope that tomorrow's lab meeting will as informative and unique as this week has been.

Till next week, signing off.

Week 4

This is an incomplete blog entry as I spent two days in Kansas City for a medical school interview.

But I will give as much information as I can over events that occurred on Monday and Thursday.

On Monday, I ran some more Wes plates with my secondary adviser Caitlin. I ran plates for samples day 14 to day 56 from the S1BF region in mice brains. My results indicate that the run was completed successfully as compared to the disastrous run of the same plate a few days ago. After I completed the Wes plates, I took a break for lunch and organized my notebook and my materials. After that, I called it a day and I prepared for my medical school interview.

Tuesday and Wednesday can be summarized in four words: the interview was difficult. And that is all I am going to say.

On Thursday, after a red eye flight and arriving in Phoenix at 8 in the morning, I caught the light rail to the Arizona Biomedical Collaborative. At the weekly lab meeting, I learned again what not to do in lab studies along with learning some new facets of the medical industry. Overall, I had a very light work day, and after doing some organizing and setting a wes plate for my senior advisor, Sarah, to run, I called it a day.

I hope that on Friday, I can do some more organizing as well as helping others in the lab.

Till next week. Signing off.

Week Three

The past week at the Arizona Biomedical Collaborative has been quite a unique and enlightening experience.

But it started normally though.

On Monday, Caitlin, our secondary advisor, and I ran some more Wes plates. Since we had finished running Wes plates testing for Neuroligin-1 and Glypican 4 in the VPM (ventral posteromedial nucleus) region of mice, we then proceeded to run Wes plates for the S1BF region. After running two plates, I then did some housekeeping work and proceeded to call it a day.

On Tuesday, Caitlin was not in the lab, so my main supervisor Sarah Ogle decided that I assist the Research Lab manager, Bret, on his study involving cage dividers and the reduction of aggression on mice in their cages. So, for the rest of the day, I assisted Bret by accounting for the amount of time a specific rat in the cage spent in a tunnel created by the cage divider.

On Wednesday, Murphy’s Law reared its head.

At 9, Sarah told me to gather samples that we needed for protein extraction of sample tissues from certain regions. But the whole process soon turned into a fiasco due to both mistakes on my part as well as misplacement of sample tissues in previous studies. Truth be told, it took us nearly two and a half hours to find all of our samples top run a protein extraction and assay. After a nerve racking rest of the day, I retired at my usual time.

On Thursday, in lieu of the recent developments (see Wednesday), Sarah assigned me the task of noting down which samples we could find for the hippocampus region of the brain. The region had been used by many different people and as a result were scattered across the lab for me to find. After a few hours of noting down, I then had to leave early to make it back to school.

Till next week, signing off.

Week Two

My second week at the Arizona Biomedical Collaborative can be best described as a wacky and unique experience.

The week started normally enough.

On Tuesday, I, with the help my research supervisors Sarah and Caitlin, ran some more Wes plates. That day, I tested for the presence of neuroligin-1 in the prepared samples we were given. This procedure, compared with the procedure for glypican-4, takes considerably less time. The glypican-4 denaturation takes place at 37 degrees Celsius for 30 minutes plus heating up the heating bloc to 95 degrees Celsius for the biotinylated ladder control for 5 minutes with all the other standard procedures in place. In contrast, the neuroligin-1 denaturation process takes place at 95 degrees Celsius along with the ladder for 5 minutes. But technicalities aside, the runs were successful and I have completed quite some progress in two weeks.

On Wednesday, Caitlin did not show up to help us with the Wes procedure. So to pass the time working in the lab, I managed to double down slides by putting slides from one box in the freezer into another box to save space in a 4 degree Celsius room for three hours. It was not a job I expected to do, but one that I learned was vital to the organization of the lab.

But the highlight for the week was Thursday.

On Thursday, the labs hold their weekly lab meetings, where they review orders of business along other items on the agenda. This lab meeting, after all the items were taken care off, one of the lab regulars gave a presentation on an article that a reputable neuroscience journal had published related to the research in the lab. The lab regulars then proceeded, to much hilarity, rip the article into pieces regarding its factual assumptions and premises. Though the experience in general was hilarious, it taught a valuable lesson in what NOT to do in a research study.

On Friday, I hope to resume running Wes plates again, albeit for a different region.

Till next week, signing off.

Week One

My first week at the Arizona Biomedical Collaborative at the Translational Neurotrauma Lab can be wholesomely described as an entirely new and alien experience.

Before joining the research efforts at the ABC, I had a very preconceived notion about the nature of research. I thought that the professor controls everything within the lab and dictates every responsibility to high school interns out of a sense of untrustworthiness. Also, I thought that to pursue research was to throw yourself into the fire immediately with some training and you proceeded under the watchful eye of the professors and lab interns.

All of these notions were proven a mix of truths and untruths.

But anyway, updates on the past week.

On Monday, I learned all about the materials needed to properly run the Wes machine and where they were all stored, which is important information to consider for experiments.

On Wednesday, I ran a sham plate for the samples from the VPM region of the brain for Sprague Dawley rats to ascertain which samples would be appropriate to run with the injured samples.

On Thursday, I participated in a lab meeting with all the personnel in the lab. There, we discussed specific projects that other lab personnel were pursuing as well as addressing problems and solutions to many issues in the lab. Afterwards, we watched a presentation on a study on the reduction of aggression in mice after the addition of a cage divider, where it was ascertained that a cage divider heavily affected aggression levels in mice. Then, lab personnel offered other avenues to pursue as well as improvements to make on the experiment. After the lab meeting, my advisor then again extensively reviewed the steps needed to run the Wes machine again and I wrote the steps in my lab notebook.

I hope that on Friday, I can finally run Wes plates on our own and be well on my way to maximizing our data.

So that’s the update for the week.

Till next week, see you all later.

Project Summary

It is common knowledge that traumatic brain injury (TBI) leads to increased synaptogenesis, which can amplify the traumatic sensations caused by diffuse TBI due to the increase in nerve impulses. The traumatic sensations that can be triggered by increased synaptogenesis initiated by diffuse TBI into memory related areas (Hippocampus, PFC, Amygdala) include loss of memory and difficulty of memory retrieval. Using a streamlined version of the western blot technique, I will measure the impact of diffuse TBI on the levels of neuroligand-1 and glypican4, protein factors essential in the regulation of synaptogenesis in CNS neurons, present in memory related brain areas over a two-month time period. The results can prove to be essential in determining factors that play a vital role in stimulated synaptogenesis while also proactively aid in finding a solution for post -TBI symptoms in the memory related areas of the brain.

Terms to know

Wes machine: A machine that has streamlined the traditional Western blot process in our Translational Neurotrauma Lab. For those who are not familiar with a Western blot procedure, it is a widely used  technique for protein analysis that utilizes both gel electrophoresis to separate out the denatured proteins by the length of the polypeptide as well as the transfer to a membrane that stains the protein with an antibody specific to the target protein. Usually, the Western blot process takes about 2 days to complete, taking into account the  time needed to create the gel among other things. This Wes process shortens the time frame to about 6 hrs to gather data from one plate.

Neuroligand-1: protein factor in the brain that stimulates the production and redirection of synapses after traumatic brain injury using thrombospodin 1.

Glypican-4: astrocyte secreted signals sufficient to induce functional synapses between ganglion cells in the human brain. Have similar effect like neuroligand-1

Sham: Designates a sample from a region from an uninjured animal. Acts as a baseline for comparison to injured animal samples.

Project Goals

• Examine amount of protein factors in select tissue samples from Sprague Dawley rats at specified days after TBI
• Hopefully aid in research into factors controlling increased synaptogenesis after TBI.