Summarizing the Summer

I cannot believe that this summer is almost over. I am glad to say that my research went quite well. I was able to determine if CAV-1 and the TR mutant (S40T, K136R, L251P, V390A) are colocalized, and I was able to show the impact that one of those four TR mutations has on localization of the TR mutant. The next step could be observing the impact of another one of the four TR mutations to determine which mutation leads to the greatest mislocalization of this TR mutant; it will allow me to better understand the types of interactions that these mutations have in shaping the localization of the TR mutant. I am also interested in doing a time-lapse experiment using the confocal microscope to see when CAV-1 and TR mutant may be the most or the least colocalized. If there is a specific point of time at which they colocalize the most or the least, I would want to know the implications of those results. Although I have learned a lot about this TR mutant within the past two years, I know that there is so much more that can be understood about these TR mutations and that these TR mutations can help us better understand the functions of TR and its possible impact on the development of liver cancer. And, for that understanding, I will continue to conduct experiments on this topic as long as I am at W&M.

Ending with a Single Mutation

While I was working on determining how to best visualize and analyze colocalization of CAV-1 and the TR mutant (S40T, K136R, L251P, V390A), I began a new experiment. This experiment has been logistically much simpler because I am using a procedure that is similar to my previous experiment. So what exactly am I doing now? As you might have noticed, the TR mutant that I am observing has, not one mutation, but four mutations. I have no idea how each of the four mutation impacts the localization of TR individually because so far, I have been observing the impacts of all of the mutations together by transfecting a plasmid that has all four TR mutations. So, for this new experiment, I have been transfecting a plasmid that has one of the four TR mutations to observe that particular mutation’s impact on TR localization.

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Continuing with Confocal Microscopy

I have been researching over the summer for over two months now, and the research has been going quite well. One equipment that I have been using a lot during the past two months is the confocal microscope. I was very excited when I was getting trained to use the confocal because, until that point, I was using the inverted microscope, which only images cells in two dimensions. The confocal microscope, on the other hand, images cells in 3D because I can change not only the x- and y-axes but also the z-axis. When I looked at fixed cells under the confocal, the cells appeared to be much more clear and defined; the clarity of the images was oddly satisfying. I needed to use the confocal microscope because I wanted to look at colocalization of a mutated thyroid hormone receptor with caveolin-1. Colocalization cannot be determined by using the inverted microscope because since the z-axis cannot be changed, the proteins may simply be on top of each other (at a different z-value) rather than interacting with each other in the same plane. I used the confocal to ensure that the two proteins are actually colocalizing.

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Beginning with Transfection and Fixation

June 1:

Hello! My summer research has started just a few days ago, but I am already excited for what I will be doing during the next three months. In this blog post, I will be writing about transfection and fixation of cells. I have been transfecting and fixing cells since last summer, and they have been a major part of all of my experiments so I want to tell you a little bit about them.

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Abstract: The Impact of Thyroid Hormone Receptor Mutations on the Development of Hepatocellular Carcinoma

The thyroid hormone receptor (TR) is a nuclear receptor that is essential for the proper regulation of metabolism and development as it regulates gene expression. The amino acid sequence of TR contains nuclear localization signals (NLSs) and nuclear export signals (NESs) that allow for TR transport into and out of the nucleus, respectively. Previous research has shown that nuclear import, nuclear retention, and nuclear export of TR is associated with modulation of gene expression, the alteration of which can lead to various diseases, including Resistance to Thyroid Hormone (RTH) Syndrome, type II diabetes mellitus, and several types of cancer.

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