Results from Trial 1/ Starting Trial 2

At the beginning of this week, I looked at my Trial 1 slides for no treatment/ chloroquine that I made the week before. I examined the twelve chloroquine slides and took pictures of those slides. I need to perform statistics on the slides but I will do that throughout this week. In general, the majority of the cells on the chloroquine slides were not viable and the cells that were alive were scarce. One explanation for this could have been forceful washing during the cell fixation process. This week when I do cell fixation, I will make sure to be careful when washing the cells using the autopipette. I have also done cell culture for Trial 2 of no treatment/chloroquine. I will add my DNA conditions to my cells today and add the autophagy inhibitor drug, chloroquine, to the cells tomorrow.

Starting my independent project

This week, I have started my first trial of my independent project. As a reminder, I am trying to determine if pseudophosphatase, MK-STYX is reducing stress granule formation via the autophagy (cell recycling pathway). To tackle this question, I have to culture mammalian cells, transfect the cells with the different DNA plasmid conditions,  induce and inhibit the autophagy pathway via pharmaceutical drugs, fix the cells, and examine the cells under the fluorescence microscope.  This week I am starting my first trial of my experiment looking at the results of added no drug and the autophagy inhibitor drug. Thus far, I have cultured and transfected mammalian cells with my different DNA plasmid conditions. Later in the week, I will add autophagy inhibitor drug, fix the cells, and examine the cells using fluorescence microscopy. I am excited to see preliminary results of this experiment.

Nesting Ecology of Diamond Back Terrapins at the Cattlett Islands

The object of the study is to test a GIS-based model of terrapin occurrence in the Chesapeake Bay (Funkhouser et al. 2019). This model predicts the Catlett Islands in the York River as a suitable nesting habitat for Diamondback Terrapins. Terrapins are known to occur in the waters around Catlett Islands but to date, no one has studied their nesting on this island complex. The Catlett Islands are largely protected from human intervention and development, so this project is a good location to not only test one prediction of the GIS model, but also to give a more holistic view of the nesting ecology of terrapins on the island complex. Throughout their range in Virginia the status of terrapin populations is uncertain, and currently few studies describe the nesting ecology of terrapins in Chesapeake Bay. My study will address the following research questions: What is the location, number and success of terrapin nests on the Catlett Island complex?
Each day during the nesting period is to paddle out in a canoe from a point in Cedar Bush Creek (adjacent to Catlett Islands) and land the canoe on the beach. We will mark the location of intact nests or record the area. Once nesting is completed by active females, we will measure and mark the nesting female prior to release on-site.

End of Summer

This last week has been surprisingly busy. There were a lot of loose ends to tie up before we left for 3 weeks. We finished another round of plasmid preparation so we have enough supplies to last us through the Fall semester. Plasmid preparation can be tedious so getting a head start on that will make our lives easier in the coming months. We also had to freeze down our cell lines. Since we won’t be here for the next weeks, we need to ensure our cell lines will be taken care of. Instead of replacing medium and flasks, we will freeze the cells down in nitrogen to keep them viable for experiments. We have also taken care of various lab maintenance issues like ordering more equipment to restock the lab. I also took the time to score a previous successful trial. Results were interesting, and I definitely was surprised at how much time it took me. Overall it has been a relatively smooth week in lab. I look forward to continuing my research in the Fall.

End of an Era

Today is my last day lab, and this last week has been packed. We finished up several trials, one of which we’ll actually be able to score for data since the cells look amazing. We also figured out the concentration of retinoic acid that we want to use for N2As since we were having some issues with actually seeing neurites and cell differentiation. When we fixed the cells, we didn’t see neurites and the cells looking very unhealthy. We realized that this was likely due to serum starving the cells the entire time we stimulated them (which was what was suggested to us by the provider of the new cell line) since we had been adding the RA to RPMI medium with 0.5% FBS instead of the normal 5% FBS, 10% HS. We made up a sample six-well plate of different concentrations of RA with normal RPMI after serum starving the cells overnight, and after 24 hours we were already starting to see neurites in each of the wells and the cells looked healthy. We found that the concentration of RA we had already been using was most effective, we just needed to stop serum starving for such a long period of time. Our future trials of N2As are sure to look much better.

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