Friday, August 12, 2016

Lab Internship: Week 6 (Final Week)

So everything got deleted as I was writing, so I have to rewrite everything on Thursday, 8/11.  Not everything will be accurate since I am not writing on the day of.  I'll try my best though.

Monday 8/8
Victor is planning on doing another experiment with me.  Originally, it was supposed to be the time lapse experiment, but because of tricky scheduling, he decided not to do it.  Instead, we are doing an experiment with RELA, a transcription factor in the NF-kB family.  He wants to find the protein concentration in cells and how much translocates into the nucleus.  RELA is generally in the cytoplasm of the cell, bound by specific receptors inside the cell.  When perturbed, the RELA break away from the receptors and go into the nucleus to regulate transcription.  We are not perturbing the cells with any external stimuli, such as TNF, so we can accurately image the RELA concentrations in the cells.  Victor also wants to fix the cells so they do not escape the traps, like they've been doing in my TNF experiments.

Tuesday 8/9
I attended a thesis defense for a graduate student named Stacey Lawrence.  It was at the Kline Geology building behind the Peabody.  She presented her research on how G proteins affect the immune receptors/response (I think, I forgot since I accidentally deleted everything I wrote.  I also fell asleep halfway through the presentation, whoops).  The closed Q&A session with the thesis committee, which usually go on for about 15-20 minutes, according to every grad student I asked, lasted for over an hour.  The food from Thali was worth it, though.

I went back to lab and started on my experiment.  I did the first stain of NF-kB antibodies.  The antibodies will attach themselves to the RELA.  I also "fixed" the cells, meaning that I killed them so they become rigid.  It's not apoptosis.  I also had to permeabilize the cells to allow the stain to get inside of the cell.  I used ice-cold MeOH to permeabilize.  Don't know how it works, never asked (according to grad students, as long as it works, you don't ask questions).  I then put the device with the cells in a container and into the fridge to leave overnight.  Victor wrapped the container in parafilm to avoid evaporation.  This would cut off the exchange of gases, but since the cells are already dead, it does not matter.

Wednesday 8/10
I started work in the lab early, and by early, I mean I started as soon as I walked in.  I applied the 2nd stain to the cells.  The stain was called Alexa 488, a molecular probe that attaches itself to antibodies.  The devices had really good trapping efficiency, with over 80% of the traps filled with at least one cell.  Since the devices were in the fridge overnight, there was condensation on the devices, so it was somewhat difficult to see the channels at first.  I also added a Hoechst stain so I can identify the DNA and the nucleus in the cells.

There was a lab meeting today where Victor Bass, another graduate student, was presenting his progress on his research.  He was studying TNF and LPS stimulation.  I did not really understand everything because of the graphs, since they always mess me up.  Andres was supposed to present a paper, but he forgot, so it was a short meeting.

After the lab meeting, I imaged my cells in the devices.  I took 10 pictures of at least 15 cells, so at least 150 cells, max 200.  HIV was activated in some cells, making the cells appear as if they're from the Matrix.  Since they were so bright, I could not see the RELA concentrations in the cells, so I could not quantify those.  I could only quantify the cells without active HIV but had RELA, which was all of the cells.  I have to analyze the intensity of the light, or the grayscale intensity, the standard deviation of the light intensity, and the integrated density.  The integrated density is the area times the average mean gray value, or light intensity.  The integrated density is therefore the light intensity of the entire cell.  I will have to analyze each cell individually since the data also quantifies the cells with activated HIV, so that's a pain.

Thursday 8/11
I met with Kathryn, my P.I., in the morning to discuss how things have been going in the lab.  I told her about my RELA experiment and that I had a great time learning and doing experiments in the lab.  We talked about if I could come work in the lab throughout the year, but she did say scheduling would be tricky.  I also just realized that I will be busy throughout the year with college classes and extracurriculars, so I will have to tell her that.  She is planning on taking another high school intern next year, but also wants me to return so we can learn together.  I can help the high school student as well as learn in the lab.  Kathryn also told me about Victor's paper on how RELA regulates gene expression since it's a transcription factor, and how Victor is trying to determine a way to predict how RELA will behave.  They plan to use this work to treat disease and things like that.

I also went with Victor to West Campus since he had to use some tech there for his experiment. Victor asked if I wanted to go with him on Tuesday, so I said yes.  We ate tacos at the cafeteria and met with another graduate student there.  The microscope Victor used was extremely powerful, with magnification capabilities of up to 100x magnification.  Victor was going to use the microscope for a long time, so I left at 3:30 because I needed to go home.

Friday 8/12
I got gifts for Victor, Laura and the professor.   I also got donuts for everyone, but only Andres and Victor Bass showed up to lab.  I just stayed in lab and worked on my personal projects.

Internship Review

Reviewing my summer goals that I set at the beginning of this internship, I did fairly well and got comfortable with openly asking questions to my lab members when needed. This has helped me be more familiar with the feeling of talking out more, though I did not participate in any presentations so far. (So my practice with my presentation skills has been minimal.) I really put a lot of effort into what I was learning and I got to learn more about the process these research projects go through. This summer, I was able to properly pace myself through the long-term project of creating a poster to present what I had learned and worked on. My internship goals of pacing myself and speaking out were completed.

This summer, what I really enjoyed was watching how other lab members worked through their projects. I loved shadowing them as they worked on the equipment or listening as they presented their projects. I really wish we had more presentation opportunities and more chances to see the process that other lab members go through.

Through this internship I learned how to work an image processing program, how to more comfortably speak up and ask questions, how to pace myself on a long term project, how different electron microscopes work, how to properly organize a research poster, and more about the nanotechnology field and what it entails.

Thursday, August 4, 2016

Introduction and Procedure Poster

Bulk metallic glasses are amorphous solids that are created using metallic elements. The word “bulk” is simply used to refer to good glass-forming ability. A pure glass is a solid that bypasses crystallization, which means it has no structure whatsoever. Even the slightest bit of crystallization (in other words, order in the atomic arrangement) disrupts the purity of a glass. Crystallization is defined as the point at which the crystalline volume fraction within the melt reaches some small but finite value.
This suppression of crystallization in order to form a glass is equivalent to the formation of a super-cooled liquid. This research examines the distances between particles that share forces in a super cooled liquid in order to construct a neighbor list.

First, one needs to create a code that constructs a neighbour list, keeping the temperature constant (NVT). This code needs to be run in a compute node in one of the Yale HPC clusters. Along with this code, scripts containing source code need to be run in the clusters to create the necessary files for the code. The output data should be directed into a folder so that it is successfully stored.

Lab Internship: Week 5

Monday 8/1
I began to research TNF and HIV in an effort to comprehend my experiment and data.  So far, I found two hypotheses that I need to look into: TNF activates the NF-kappa B pathway, activating the NF-kB receptors which HIV then use to infect the cell; HIV attaches to and infects cell via TNF receptors.

I made some more devices for Laura.  The lab got two new master slides for the flow-patterning devices and Laura did not really like them.  She said that they were too sticky.  I found that they were the same, mainly because I did not really care.  I found that the area in which I poured the PDMS produced the most bubbles, so I focused on pouring PDMS in empty spaces so I could pop less bubbles.  I also punched holes for the inlets and outlets for some flow-patterning devices for Laura.  It took approximately 20-30 minutes (I used music as a method to keep track of time).

Tuesday 8/2
I did some more research on TNF, but I also asked Victor about why TNF activated HIV.  He said that the TNF pathway activates NF-kappa B, AP1, and SP1 receptors, as well as the NF-kappa B pathway and the HIV uses that pathway to infect the cell and proliferate.  However, I also found a paper from France that explains how HIV actually takes over the TNF pathway.  Victor asked me to send him that paper, so I did.  I need to read that French paper, but I also need to learn how HIV takes over the NF-kappa B pathway.

I also ran the TNF experiment again.  However, this time I changed the amount of media in the devices at the end since Victor believes that we lost the cells because flow was insufficient.  I ran the same procedure: I plasma bonded the devices, I put DI water in the devices to ensure flow can occur, I primed the devices with RPMI, loaded my cells, washed the cells with RPMI, stained them with Hoechst, then loaded the control devices with RPMI and the manipulated devices with TNF.  I left them to incubate with a humidifier for 24 hours.  The trapping efficiency in the devices were not too great, but most of them were above 80%, so it should be fine.

Wednesday 8/3
Victor did not show up today, and will not show up for the rest of the week.  He said he can come in tomorrow and prepare cells for another experiment that I can run on my own.  I will have to prepare the cells properly and without assistance (Ay, so much pressure, hopefully I don't break anything).

I learned that HIV cannot use TNF pathway to infect cells, only the NF-kappa B pathway.  I began to read a paper on how HIV infects the NF-kappa B pathway in an effort to better understand what is going on inside the devices.

Victor forgot to put me in the schedule for the microscope...
Someone else scheduled the microscope for the whole day (11-6).  Now, I have to use another microscope, the Nikon.  I need to remember to put myself on the schedule for the EVOS microscope when I do the experiment again tomorrow.  This could've been really bad.  Thank god for Laura, saving my butt again.

Speaking of Laura, I cleaned some of her devices and punched holes in 6 of them now.  It used to be 4, but since we got the new master slide for the flow-patterning device, we have 6.  Hooray.  More work.  (punching holes in those devices take forever, like 30-45 minutes.  Thank god for music.)

Thursday 8/4
Victor did not come again today.  He said he has a family emergency.  Laura will be preparing my cells, but instead of stimulating them with TNF, we will be stimulating them with LPS.
LPS (lipopolysaccharides) are large molecules found in the outer membrane of gram-negative bacteria (bacteria that don't have much peptidoglycan--substance that forms the cell wall of bacteria).  It acts as an endotoxin and induces an immune response in humans when it binds to receptors, such as the CD14 receptor.  Downstream, LPS activates NF-kappa B via CD14 pathway, therefore it is likely that LPS will activate HIV in the cells.

While doing experimentation, I was supposed to make a solution of LPS and RPMI so the cells can be stimulated by LPS while living in a favorable environment.  However, I accidentally put in pure LPS into two devices and killed the cells.  Laura helped me get more LPS, and I made the solution for the devices.  I had to take out everything from the two devices I accidentally messed up.  The devices were still intact and undamaged, so that was good.  I redid the procedure for the two devices while the rest of the devices were still fine.  I constantly checked on the devices under each microscope to ensure that cells were still trapped and the device was functioning properly.  I then put a humidifier into the container with the devices at 1:25 pm, so by 1:25 pm tomorrow, I will need to image my cells.

Also, I realized that no one taught me the cleaning procedure for experiments.  I asked Laura, and she taught me what to toss out, what I can immediately throw in the trash and what I have to bleach first, and how to sterilize the workbench properly.

Friday 8/5
Most of the cells in my devices disappeared... once again.  After looking at my devices, I realized why the cells were disappearing.  The flow pressure from the large amount of fluid in the inlet caused cells to escape the traps and end up in the outlet.  I would like to try decreasing the amount of liquid in the inlets and outlets, but I do not want to put in so little that it could equilibrate so easily, which would disrupt flow.  Laura did not prepare the Hoechst stain properly, so I could not see the cells under the DAPI channel (filter to see stain).  I imaged what I could, but it is likely that I will not even bother to gather data from these images.  I did notice that LPS slightly activated HIV in some cells, while inducing apoptosis in others.

Friday, July 29, 2016

Lab Internship: Week 4

Tuesday 7/26
I just discovered that I was not supposed to culture cells, according to the safety advisor for the building.  So technically, I was not doing something that I was supposed to do.  Victor will now have to prepare all of the cells needed for experimentation.  Victor is planning on doing an experiment testing how TNF (tumor necrosis factor) affects cell behavior.  TNF is a cytokine that regulates many cell functions, such as proliferation, apoptosis, coagulation, etc.  In this experiment, Victor explained that the TNF will excite the cells and may cause them to jump around and possibly out of the traps.  I prepared the cells in four devices with Hoechst stain so we can identify the cells and I introduced TNF into two of the four devices, yet the trapping efficiency was not 100% in all devices.  Victor said we will have to image specific regions of the channel to see how the TNF affected the cells.  We just need to see the cells, so the trapping efficiency is irrelevant.

Wednesday 7/27
Victor asked me to accompany him to the stock room to get some supplies.  I assumed the stock room was in the basement of the building, but it was really in KBT.  The stock room was reminiscent of a corner store, like a drug dealer's grocery store.  Walter White would probably love the stock room.  There are two on Yale's primary campus: one at KBT and one in the med school.  There is also one at West Campus, but that's far away (at like West Haven or something).

There was also a lab meeting where one of the labmates, Linda Fong, presented her progress on her project on how p38, a mitogen-activated protein kinase, affects HIV latency and apoptosis of HIV infected immune cells, which could lead to more effective forms of therapy.

I also began to learn ImageJ to analyze the images of the devices I took with Victor.  I need to learn how to use ImageJ and develop a macro to count the amount of cells in the image and differentiate between the cells.  CellProfiler is another program I can use, but I do not know how to use it.  I asked AndrĂ©s for help, but he was busy with his project.

Victor and I were about to image the devices with cells for the TNF experiment, but we found out that the RPMI evaporated in the incubator and the cells died as a result.  Many traps in some of the devices also disappeared.  Victor guessed that this occurred because there was not enough humidity in the incubator.  Tomorrow, we will begin repeating the experiment by preparing the devices.

I found a way to analyze an image and count the cells.  I just need to use the Color Threshold function to "select" the cells and analyze the particles with the Analyze Particles function.  I then get a table full of data with the measurements of each particle.  I already tested this method with images from the efficiency experiment.  The method was accurate.  I will use this method with my experiment to count the cells and further test the efficacy of this method.

Thursday 7/28
I prepared four flow-patterning devices for Laura and her experiment.  I cut them out into individual devices, then I cleaned them.  I also punched holes in the inlets and outlets (20 inlets, 20 outlets for each device).

I prepared four more devices for my experiment (take 2).  I used J65C 6.6 for my experiment, not 4.4 since 6.6 was thinner and less clumpy and dense.  I prepared two of the four devices as controls, and the other two as the manipulated group.  I will stimulate the cells in the manipulated group with TNF to see how TNF affects cell behavior.  According to Victor, the TNF will excite the cells in a way that will make the cells jump out of the traps.  I will leave the cells in the devices, and leave the devices in an incubator for 24 hours.  I will also put a humidifier (fancy term for water in a bowl) with the devices to ensure that the RPMI will not evaporate so the cells do not die.

Friday 7/29
Most of the cells from the devices all disappeared.  We do not know why.  It was perfectly fine when we incubated the device.  Maybe the humidifier loosened the bonding of the devices?  We checked the outlet and everything, but nothing was different--besides the fact the most of the cells were gone.  It's not like they planned a prison break or anything.  Cells can't do that--I think.  There were a few cells I could image in specific regions of the devices, so I imaged them.  The control devices were plain blue because of the Hoechst stain, but the TNF activated HIV in almost 100% of the cells, so that made for some pretty pictures.  It almost calmed me of the stress and shame of having almost another failed experiment.  Victor told me this happened all the time, which just made me feel bad for scientists everywhere.  Now, I have to understand my data and learn what it means.  It's likely that the TNF was an activator for transcription, allowing HIV to be transcribed and become active in the cell, but it could also be that TNF activated a specific MAPK (mitogen-activated protein kinase) pathway.  MAPK pathways are involved in many cellular activities, such as gene expression, proliferation, mitosis, apoptosis, etc.

The Transmission Electron Microscope

When compared to a light microscope, the transmission electron microscope (TEM) may seem similar in concept, but there are a few things that have been changed. The light source is replaced with an electron gun, and the glass lenses are replaced with electromagnetic lenses. The entire chamber through which the electrons pass is put under a vacuum, to avoid electron collisions with air molecules, and magnification variety is achieved through different currents in the electromagnetic lenses (as opposed to just moving the glass lenses in a light microscope).

The electron gun is usually composed of a heated tungsten filament, from which there is an electron cloud. This makes up the electron source, from which the electrons are pulled, accelerated, and then focused onto the sample. The sample has to be really thin (most are approx. 0.5 micrometers!) for the electrons to pass through and form an image. When the electron beam hits the sample, multiple things can happen. They (the electrons) can be absorbed, scattered, or even reflected; depending on the thickness of the sample and its composition. The image is formed on a fluorescent screen using the information gathered form the different actions of the electrons. This image can then be photographed for later use or documentation.

(see article link in "All You Wanted to Know About Electron Microscopy...But Didn't Dare to Ask!" post)

-Patricia Acorda

Lab Update: Week 4

7/25 Monday - Worked on poster, asking lab partner for feedback on conclusion and title sections. Completed these sections and started writing up a new Methods section. Added references to photos. Went home early because of a stomach ache.

7/26 Tuesday - Finished writing up Methods section and sent poster to Apple for feedback. Using feedback, reformatted Results section again, trying to remove bullet points and labeling sections. Made minor edits to the Methods section and sent back for feedback. Attended a presentation on the other lab members' projects and participated in the ImageJ quiz made by the teachers, winning first place.

7/27 Wednesday - Awaiting feedback from Apple. Went to Hillhouse to fill out the CRISP program survey.

-Patricia Acorda