Drafts

It's really interesting that fusion proteins can be expressed. I assumed that Abl did something to the protein that it fused with in the cytosol, like affect its noncovalent bonding and consequently change its conformation/function. But, this implies that the fusion protein is expressed so the mutation begins at the DNA before transcription.

Does the Abl kinase act as a phosphatase? It states that one of its functions is to inhibit tyrosine kinases. Maybe it acts as a competitor for phosphorylation as a mechanism of inhibition instead?  

This is fascinating because I assumed that all SH2 domains functioned as phosphotyrosine binding partners. But in the case of Abl, it seems that the SH2 domain functions to bind to the SH3 domain and thereby maintain its autoinhibition. If the SH2 domain is not able to bind to a phosphorylated tyrosine, then its normal function will no longer work.

This facet of Abl 1b coincides with its cancerous tendencies. If the kinase activity is deregulated then the implications for the cell as a whole are not good. Cancerous cell utilize these kinds of mutations for grow and proliferate. Without normal regulation, the kinase can promote this. Therefore, it makes sense that the Abl 1b mutant is commonly a part of the cell that when mutated can lead to cancer.

Since Gab/Grb2 fulfill such a large role in the cell, it makes sense that its deregulation by Bcr-Abl results from the mutation. Gab/Grb2 bind to epidermal growth factor receptors (EGFR) which is a commonly mutated in various forms of cancer. Furthermore, because Gab2 serves a number of other functions in the cell like being the binding site for the assembly of SHP2 and potentially coordinating STAT5, its deregulation can have serious consequences for the longevity of the cell.

I took Resource Economics 212 to fulfill my statistics requirement. We learned about common statistical notation. For example P(A) stands for probability of event A. P(A U B) stands for the probability that A OR B will happen, whereas P(A ∩ B) stands for the probability that BOTH A and B will happen. P(A I B) is the probability of event A, given that event B occurred. We also learned about standard deviation, which tells you, on average, how much the values as a whole are different from the average, i.e. determining whether they all right around the middle, or are there a lot on the lower end and a lot on the higher end that just average out to a middle value.  We learned how to calculate permutations and combinations as well. Permutations are the number of different ways a set of things from a group of things can be arranged, paying attention to their order, while combinations are also a set selected from a group but not paying attention to the order.

The cellular cytoskeleton has a large number of functions, due to the varying structure of cytoskeletal proteins and filaments, and their various interactions. Two major proteins in the cytoskeleton are actin and tubulin, which comprise microtubules and microfilaments respectively. Actin and tubulin have different structures and functions, and interact with each other to create emergent properties. They have very different stiffnesses. When together in high concentrations, they have steric interactions that conform to the “reptation” or “tube” model. Each filament is spatially restricted to a tube-like area, which is formed by the constraining filaments around it. To relax and decrease the straining forces on it, it reptates, (sliding curvilinearly) out of its tubular space. There are other methods by which the filaments can partially relax, such as bending fluctuations. The interactions that occur in cells between actin and tubulin provide controlled, structured support of the cytoskeleton. Their interactions are also important in cytokinesis and cell motility. Microtubule strength is also reinforced by the presence of supporting actin; they can withstand greater forces without buckling in the presence of actin than they can on their own. Studying the interactions of these filaments has different potential applications. Potentially, one could learn to identify an ideal ratio of soft to rigid rods to be used in the synthesis of a material that effectively combines light weight with durability. Combinations of actin and tubulin also provide the possibility for increased control over large-scale mechanics. Previous research studies have shown that actin is compressible in the presence of microtubules, and that low concentrations of microtubules added to cross-linked actin cause strain-stiffening in the actin, as opposed to the normal strain-softening that usually occurs in cross-linked actin. These previous studies, however, were limited in a few ways. The parameter space of the composite matter was limited, so differences between varying concentrations and ratios of actin and tubulin were not measured to be contrasted. The studies also measured large-scale strain and micro-scale strain, but not mesoscale strain which would be more useful based on the mesh size of actin and tubulin. These studies also used microtubules that were pre-polymerized before they were added to the actin, which oftentimes encourages actin bundling, preventing the possibility of a truly isotropic composites. In contrast, the study “Co-Entangled Actin-Microtubule Composites Exhibit Tunable Stiffness and Power-Law Stress Relaxation”methodically varies the relative concentrations of tubulin and actin and characterizes mesoscale mechanics of the filaments by displacing optically trapped microspheres by 30 µm at a rate that is very high compared to the normal relaxation rate of the filaments, and measuring the restoring force that the composite applies to the sphere. These measurements disrupt the equilibrium of the composite and allow exploration for possible buckling, rupture, and rearrangement. 

One cue for determining position is using magnetic parameters. Birds may process such information using the ophthalmic branch of the trigeminal nerve (V1). The goal of this study was to test if V1 plays a role in the navigation of migratory Eurasian reed warblers after being displaced 1,000 km toward the east from their breeding ranges during spring migration. The hypothesis was that V1-sectioned birds would use vector navigation and behave like intact birds in Rybachy, while sham-sectioned birds would readjust their orientation because an intact V1 plays a role in true navigation.

In the Fall of 2018 at the University of Massachusetts Amherst during Biology 284- General Genetics Lab, my partner and I designed an experiment to mutate Saccharomyces cerevisiae, or baker’s yeast, and categorize the resulting mutations.  

Baker’s yeast has two almost identical mating types, MATa and MATɑ, which can sexually reproduce with each other and asexually reproduce themselves.  If the environment they are in is nutrient poor, the yeast cells can exist in a haploid form of MATa or MATɑ. A colony of haploid cells can be maintained by asexual budding.  If the environment they are in is nutrient-rich, the different mating types will become shmoos, a nodule of the original cell that the cells use to join together. Once they become an a/ɑ diploid, they can bud to asexually reproduce two yeast cells, the new cell being exactly identical to the first. If a diploid cell is starved of nitrogen and also on a carbon-poor source, it will sporulate to form four ascospores within an ascus.  Those spores can be released from the ascus membrane and become 4 haploid yeast cells, two a and two ɑ cells.

Mutations come about by mutagenesis, which is a relatively rare event in nature.   DNA replication is a highly regulated event that rarely lets imperfections slip by. Even when a mutation occurs in DNA, it does not always lead to a change in phenotype. Mutagens such as UV light, as used in this experiment, X-Rays, and chemicals are often used to increase the frequency of mutations for scientific study. In order to successfully study mutations, the cells must live and be able to reproduce through the mutagen exposure and contain a non-lethal mutation.  

Cesium -gluconate Preparation

Add 35 mL of stock CsOH to a clean beaker, while the solution is stirring add gluconic acid until the pH is 7.2, this will be around 65-95 mL and will take a while to stabilize ( this step is an exothermic reaction and you will be able to feel the beaker warm up) the solution will be light brown

Evaporate to approx. half of the total basic volume

Add methanol p.A.(~50-60 mL)slowly while stirring until you can see crystallization(they look like stringy clumps falling to the bottom)

Let it sit overnight at around 4 degrees C / put it in the fridge

Crystals should be white and about ¼ inch thick on the bottom and side of beaker. Use the ground frit to separate as much of the saline as possible from the crystals

Transfer crystals to a 600 mL beaker and clean the frit filter out, dissolve the crystals with as little H2O as possible ~ 30 mL ( solution should turn light brown)

Add 2 spatulas of activated carbon, stir and heat to 80 degrees C

Filter immediately with the filter for activated carbon, the filtered solution should be light brown again

Pour the filtered solution into a clean beaker and add methanol p.A. (~35 mL) slowly while stirring until you can see crystallization( this time it will take less methanol to crystalize)

Overnight at 4 degrees C

Use frit filter to separate crystals from saline

Transfer crystals to a 600 mL beaker and clean the frit filter out, dissolve the crystals with as little H2O as possible ~ 20 mL ( solution should turn light yellow/clear)

Again add methanol p.A. (~30 mL) slowly while stirring until crystallization

Overnight at 4 degrees C

Use frit filter to separate crystals from the saline

One may repeat this procedure once, but by now Cs-gluconate crystals of sufficient quality and bright white color should be obtained

If the solution before you add methanol is yellowish then do another round of precipitation. If the solution is more clear and barely yellow then go ahead to the drying process

To dry the precipitate put the Cs-Gluconate in a 10 cm petri dish, (weight the dish before) and put the dish in an exsiccator with the dish top slightly ajar

When the precipitate is fully dried take a mortar and pestle and crush the precipitate until it looks like flour

Now weigh the precipitate and record it on the petri dish

I took ResEcon 220 my second semester freshman year to fulfill the statistics requirement for biology majors.  I remember the first thing we learned was how to use excel in the most basic way. The next class was simple review from high school math class about ratios and other basics for statistics.  We then learned basic statistic material such as variables and what it means to have a hypothesis and a null hypothesis. I then remember we started to learn about standard deviation and learned how to calculate it, how it is used, and why it is used.  After we learned all about standard deviation we learned how to use it in excel and how to write formulas to do out the work for us. Pretty early on into the semester we were assigned a group project looking at football statistics. This was my first group project in college so not only did I learn about statistics but also how to work in a group setting and facilitate a 7-8 member team to develop a research paper about football statistics.  This project also taught me about how to write a lab report for a subject other than biology, physics, or chemistry, as those were the only classes I had to write reports for yet. At the time It was my least favorite class but I can honestly say I learned alot from Professor Wayne and his ResEcon 220 class.

Part of the reason that wildtype Abl and the Bcr-Abl fusion act so differently is the change in form that occurs. The structure of a protein relates to the function of the protein, so any minor changes could end up changing the function completely. The change expressed in the fusion is what disrupts the regulation that wildtype Abl usually provides and increases the level of signaling beyond wildtype Abl. Wildtype interacts with multiple signal adaptors, phosphatases, transcription factors and cell cycle regulators.  It is tightly regulated and has nuclear import and export signals so it is capable of interacting in the nucleus and cytoplasm. It is regulated by intramolecular interactions and phosphorylation. When Abl kinase becomes Bcr-Abl, the new N-terminus creates a binding site for other proteins, causes the loss of the CAP domain (which is important for cell regulation) and causes the localization to be purely cytoplasmic. Overall, Bcr-Abl has binding sites for cell proliferation signaling pathways that Abl doesn’t have and has a greater kinase activity.

This video contained two mare and two foals of about 6 months. Throughout the half hour of video, you could watch them interact with each other as well as the environment in a number of ways. We grouped the behaviors into a different groups that included: feeding, playing, communicating, and other. The other contained behaviors that did not match another group well. Behaviors found under play included the foals running, jumping, prancing, and nudging one another. The foals would nuddge one another with their heads and this would entice a reaction out of the other foal, so this one could also be placed under communication. Many of the acts fall somewhere in between categories and they are not completely concrete. Feeding was put as its own category because the horses were often observed to be grazing. Even at points where they may not have been eating, they often had their heads toward the ground nudging around the grass. They looked to be continuously grazing almost. Communication was another big category of traits observed. Horses can be seen nudging other horses and neighing. Communication is observed when a stimulus from another horse that entices a reaction from another horse. When a reaction is observed of the second horses behavior is alterede as a result of the stimulus. Some behaviors under the other category include the horses scratching along the fence and other random movements. 

Some key principles that I remember from statistics include probability, regression, standard deviation, and the empirical rule. Probability is the chance that something will happen given certain conditions. Regression an estimation of the relationship between variables (for example X and Y). Standard deviation is a measure that is used to quantify how far a value is from the mean of the population and/or sample. The empirical rule states that 68 percent of data on a standard distribution will fall within one standard deviation from the mean, 95 percent of the data will fall within two standard deviations of the mean, and finally 99.7 percent of the values fall within three standard deviations of the mean.