Determining the Role of Alpha-Catenin in the Cell's Resistance to Dissociation Induced by Autoimmune Disease

Joseph Broadway Author

08/03/2021 Added

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UCARE Summer 2021 Submission for Joseph Broadway. Special thanks to the MME department, Dr. Ruiguo Yang, and Xiaowei Jin

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[00:00:00.980]Hello, my name is Joseph Broadway.
[00:00:02.630]And today I'm going to be telling you about my project,
[00:00:05.750]determining the role of alpha ketamine in the cells,
[00:00:08.390]resistance to dissociation induced by auto immune disease.
[00:00:12.560]That was a mouthful,
[00:00:13.430]but I think you'll understand it here within the next five minutes. We,
[00:00:17.840]as a species take for granted how well our cells do their job.
[00:00:22.640]I mean, why wouldn't we, they make up our entire bodies function autonomously.
[00:00:26.870]And for as far as most of us can tell,
[00:00:28.550]they function perfectly day in and day out. Unfortunately, for those afflicted,
[00:00:33.050]with pemphigus vulgaris or PV for short,
[00:00:36.320]their own antibodies begin attacking the proteins that pine their very cells
[00:00:40.580]together. This disease has a high mortality rate to five to 15 minutes,
[00:00:45.410]which makes sense because by destroying these proteins,
[00:00:48.110]these disease effectively rich yourselves, apart from each other,
[00:00:51.920]this separation of cells leads to the formation of harmful blisters.
[00:00:56.210]And this affects skin cells like on your arm or face the internal mucus
[00:01:00.320]membranes like in your mouth.
[00:01:02.060]And these blisters can even form fatally on the heart here at the university of
[00:01:06.920]Nebraska Lincoln research was already underway to,
[00:01:10.130]for a cure to this troubling disease researchers under Dr. Yang.
[00:01:14.330]My professor found that by repeatedly stretching a mano layer of cells,
[00:01:19.250]the cells became more resistance to the effects of antibodies.
[00:01:23.450]Essentially when cells exercise their ability to hold on to each other,
[00:01:27.200]they get better at it. Go figure my research the summer,
[00:01:31.550]and to develop the computer program capable of measuring the tension within the
[00:01:35.840]cells. The internal polling by the proteins in the cells were,
[00:01:40.580]was hoped to be related to the cell health and the effect of the antibodies by
[00:01:45.290]using genetically engineered cells that produce fluorescent versions of their
[00:01:49.610]proteins, specifically alpha captaining.
[00:01:52.490]We can scan cells under a microscope and relate the brightness of these
[00:01:56.390]fluorescent proteins to how much tension the proteins are under this
[00:02:01.220]process is called fluorescence resonance, energy transfer or fret for short,
[00:02:06.890]when being scanned under a microscope energy is being given to the proteins in
[00:02:11.750]the form of light and the proteins emit back light to the scanner
[00:02:16.640]at different intensity and wavelengths based on the amount of force within the
[00:02:20.330]molecules, a cell,
[00:02:22.040]essentially the proteins look bright green when they're under high tension and
[00:02:26.390]they look bright red when they're under load tension and by taking just a photo
[00:02:30.680]of the red proteins and just a photo of the green proteins,
[00:02:33.830]we can actually calculate how much tension is in the cell.
[00:02:37.640]This metric we use is called the fret ratio,
[00:02:41.390]and it is calculated by dividing the matrix of red
[00:02:45.860]intensity by the matrix of green intensity.
[00:02:53.000]Everything I have explained up until this point has already been established by
[00:02:56.810]my research group and other researchers in the field.
[00:02:59.980]My job this summer was to create a mat lab program capable
[00:03:04.390]of doing hundreds of fret ratio calculations automatically for
[00:03:09.070]multiple samples. Mat lab is a programming platform,
[00:03:12.880]specifically designed for scientists and engineers it's as its name implies.
[00:03:17.530]The coding environment is very good at handling matrices.
[00:03:20.830]It just so happens that images are basically matrices of color and brightness.
[00:03:25.150]So they work well together for the project, the code.
[00:03:28.210]It also needs to be able to automatically group these sample images and perform
[00:03:32.380]statistical analysis on the groups, including removal of outliers,
[00:03:37.510]finding the significance of the difference of their means or P value, uh,
[00:03:42.790]between each dataset to conduct the experiments.
[00:03:47.470]First,
[00:03:47.950]a single layer or mono layer of genetically modified cells were cultured in a
[00:03:52.210]flask. The cells were then treated according to the experimental plan.
[00:03:56.500]One act as, as, as a control with nothing done to it.
[00:03:59.790]One was given mouse antibodies that simulated PV,
[00:04:03.260]another was giving the McCarren a mechanical therapy.
[00:04:05.980]And the last one was given both antibodies and the therapy next,
[00:04:10.120]the cells were scanned multiple times per sample.
[00:04:13.000]So this led to hundreds of pairs of images per sample,
[00:04:16.090]one red and one green per pair. These images were saved into a folder,
[00:04:20.230]according to a strict naming convention that allowed for the code to work.
[00:04:26.080]The first thing the analysis script needed to do was reduce the noise in the
[00:04:30.640]microscope photos and isolate the pixels along the boundary.
[00:04:34.510]Since that's where the protein on antibody action is happening.
[00:04:38.740]This one actually sounds easier than it was because,
[00:04:41.320]because how do you teach a computer? What is,
[00:04:43.420]and isn't a cell boundary to accomplish this.
[00:04:46.360]The program actually had to use shapes within the pixel matrices.
[00:04:50.890]So it would make a cross or a four by four square to compare the relative
[00:04:54.850]concentration of pixel spacing and intensity to a preset
[00:04:59.320]threshold.
[00:05:00.670]It also iterated this process until the photos average intensity stopped
[00:05:04.660]changing. Once the images showed a clear boundary,
[00:05:08.290]the program divided the red matrix by the green matrix,
[00:05:12.520]and this yielded the fret ratio matrix all of the fret ratio
[00:05:17.320]matrices were then grouped all into samples sets according to the naming
[00:05:22.210]convention,
[00:05:23.470]all the non-zero elements in the matrix are statistically analyzing compared
[00:05:28.600]the mean for each sample is calculated. Outliers are removed.
[00:05:32.290]And the one way a Nova test for means as administered,
[00:05:35.650]which supplies the P value between each dataset,
[00:05:39.400]the result is an Excel sheet full of the means and others statistical analysis,
[00:05:44.680]uh, for each sample paring. And finally the photos,
[00:05:48.970]both the process cell boundary photos and the fret ratio
[00:05:53.380]matrices were saved.
[00:06:00.710]The analysis program is the final step in a long chain of research,
[00:06:04.550]all focused on learning about the inner mechanisms of the human cell,
[00:06:08.240]its proteins and how we can treat in combat PV.
[00:06:11.570]The completion of this program will now allow researchers to mass produce cell
[00:06:15.560]samples, scan them in bulk and quickly analyze all the data at once.
[00:06:20.510]Future work will also include numerous experiments to determine the roles of
[00:06:24.260]other proteins in the cell and explore how that affects the treatments of
[00:06:29.090]PV. Thank you very much for your attention.

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Determining the Role of Alpha-Catenin in the Cell's Resistance to Dissociation Induced by Autoimmune Disease

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