Drafts

Fruit flies (Drosophila melanogaster) go through a 4-stage life cycle. A life cycle that is 11-14 days long providing an efficient option for observations in a laboratory setting (Potter 2000-2016).  This cycle can be altered simply by exposing the larvae to different hormones. We were eager to see the effect varying concentrations of Juvenile Hormone, a hormone essential to fruit fly development, inhibitor had on the life cycle of a fruit fly in a lab setting (Yamamoto R1, Bai H, Dolezal AG, Amdam G, Tatar M. 2013). Because of the fact that the juvenile hormone is essential to fruit fly development we hypothesize that the overall development will be stunted and predict to see a larger percentage of larvae in trials that contain a higher concentration of inhibitor and a lower percentage of pupae in trials that contain a higher concentration of inhibitor. We mixed 10ml of water (Control) or various concentrations (0.01, 0.1, and 1) Juvenile hormone (JH) Inhibitor with 2 grams of dehydrated fly media. We then added 2 male and 2 female flies to each tube. For a week we let the flies mate in the media. In the second week we removed all the adults from each trial and placed the tubes back into the incubator. After the third week we scored the vials for: number of larva, number of pupa, number of males and female adults, average length of adults. As you can see in Figure 1 there is an upwards trend seen in the percentage of larvae as the concentrations increase, as supported by the fact that 14% of flies in the 0.01 concentration were larvae and in the vial a concentration of 1 approximately 38.4% of the full population is larvae. Also in Figure 2 a downward trend is seen in the percentages of pupae, as supported by the fact that 63.4% of the population in 0.01 concentration were in the pupae stage, while the population with a concentration of 1 only had 35.1% of the population in the pupae stage.

Throughout the process of this lab many mechanisms of evolution were observable, most prominently the mechanism of natural selection. In order for evolution by natural selection to occur three requirements must be met. The first is that individuals in the population must vary in trait that is being considered. Also the trait be considered must also be heritable, meaning it must be able to be passed down from generation to generation through germline cells. Lastly there must a selection differential, which simply means that the trait being considered must either increase or decrease the likelihood of the organism to survive and reproduce. This idea of evolution through natural selection was discussed throughout the SimUText Lab and was displayed during the Flat Periwinkle snail activity. In the activity the trait of Periwinkle shell thickness was shown to be variable, heritable and provided a selection differential, in the case of this trait a thicker shell resulted in a greater likelihood to survive and reproduce in an environment that contained predation by crabs. This trend of thicker shells leading to a higher likelihood of survival and reproduction in Flat Periwinkle populations was the driving force behind the constructed hypothesis for the effects of crab predation on the Dogwinkle snail population. The Dogwinkle snail species is the species under observation in the experiment, the Dogwinkle species exists in two distinct factions, an Eastern and a Western. In the Eastern population there is a predation pressure due to the presence of Rock Crabs and the average shell thickness of the Eastern population is thicker than the Western population in which there is no Rock Crabs present. With all of this information under consideration the hypothesis reads as follows, if there is predation by crabs present and the trait of shell thickness is being observed, then evolution by natural selection will occur and a shift to a larger average shell thickness in the experimental tanks containing the Dogwinkle snails can be expected.

Dr. Chao’s current research involved how the mitochondria gets its shape and factors that influence mitochondrial organization. F1F0 ATP synthase dimers play a pivotal role in the formation of creases and folds in the mitochondria needed for increasing surface area. Dr. Chao showed this by increasing the amount of ATP synthase dimers, which in turn increased the number of creases and organization in the mitochondria observed. Dr. Chao also wanted to understand how dynamic membranes are regulated. This dynamic membrane and formation of cristae is regulated by a number of proteins included Mfn1/2 and OPA1, both of which are in the dynamin family of GTPases. Dr. Chao is currently looking to into the relationship between these proteins and membrane conformation.

Also importantly, they discovered the pre-fusion state envelope protein to be dimeric, while the post-fusion state envelope protein was trimeric. Based on these findings, Dr. Chao and Harrison conducted where they discovered yield of fused membranes increased if they increased the size of the contact patch, and yield decreased as the pH of buffer increased. They also were able to show that the trimerization of envelope proteins is a kinetic bottleneck, limited by the availability of monomers required for fusion. This is important because of its possible application in drug development. The kinetic bottleneck that Chao and Harrison propose means that full saturation would be needed in order to block entry, as opposed to drugs targeting SNAP/SNARE vesicle fusion, which fires and fuses rapidly in the presence of calcium.

Flavivirus is a genus of viruses that includes west Nile virus and cause severe disease such as yellow fever. Flavivirus genomic RNA replication occurs on the rough endoplasmic reticulum, in membranous compartments, and the focus of Dr. Chao and his advisor Dr. Stephen Harrison’s research at Harvard was the fusion of the viral and cellular membranes. Cellular entry by the virus requires this membrane fusion and in order to do so, viral fusion proteins undergo dramatic conformational rearrangements. These proteins, which exist on the surface of the viral envelope, must undergo conformational changes to create a thermodynamically favorable chemical reaction and overcome the energetic barriers to merge the two membranes and create hemisfusion (lost him a bit in the biochemistry on this part).  They discovered that the envelope protein comprised of three main domains, one largely hydrophobic involved in the fusion loop. In its pre-fusion state, the hydrophobic domain is tucked in the protein, however the hydrophobic domain extends into cell membrane then collapses to bring membrane together and create hemifusion and full pore opening.

The research seminar I attended was given by Dr. Luke Chao who is the head of a laboratory at the Department of Molecular Biology at Massachusetts General Hospital and the Department of Genetics at Harvard Medical School.  Dr. Chao received his B.S/M.S in Biochemistry from Brandeis University and received his Ph.D. in Molecular and Cell Biology from UC-Berkeley where he worked with John Kuriyan doing structural studies of calcium/calmodulin-dependent protein kinase II. The majority of Dr. Chao’s seminar discussed his work as a postdoctoral fellow in the laboratory of Stephen Harrison at Harvard investigating the mechanism of flavivirus membrane fusion, however, he also discussed briefly research he is currently conducting regarding the assembly and maintenance of cellular ultrastructures such as organelles.

If 3 UV active spots were in the crude material and co-spot TLC plate, and only one spot had the same R_f as the starting material and the other 2 are very different, predictions for the 2 non-starting material spots could be something in the middle of the mechanisms. There are four parts to the mechanism, so the second and third part of the mechanism would be the 2 non-starting material spots. This is because of the rate of the hydride attack on the carbon double bonded to the oxygen. It depends heavily on the R group present, and the more electron deficient it is, the faster the hydride can attack. But because they are different from the starting material spot, it would mean that it was caught between and could not fully reduced and go to completion.

            First, add benzoin (0.5g) and ethanol (4mL) to an Erlenmeyer flask (25mL) swirling at room temperature until it is all dissolved. Add sodium borohydride (0.1g) using a micro spatula in small amounts for 5 minutes (swirl for addition 20 minutes at room temperature). Cool the mixture using ice bath, add water (5mL) after and 6M HCl (0.3mL). Wait 15 minutes to add more water (2.5mL). Then collect the product using vacuum filtration (reserve 1-2mg for TLC analysis) after 15 minutes. Recrystallize from acetone, using 25 mL flask. Let it all dry and come back for evening hours. MP, yield %, and mass needs to be determined. Dissolve a small amount of benzoin, using recrystallized product and reserved crude product in ethyl acetate. Spot 2 TLC plates, with starting material, reserved crude product, recrystallized product, and a spot that contains both in the middle. Run the TLC plates in 9:1 CH­­₂Cl₂: ethanol. Add eluent to TLC developing chamber, use tweezers to carefully put the TLC plate in the chamber and screw the cap. Allow the solvent to run from the baseline to about 1cm from the top. Remove the TLC plate when it is ready marking the solvent from it and allow it to dry. Use UV light and mark them once it is all dry. Tape the plates on a sheet of the lab notebook paper or take a picture and draw into the lab notebook. 

The three known substances naphthalene, urea, and sulfanilamide were all provided with its relative MP ranges. Naphthalene was 79 degrees and I found it to be 80 degrees. Urea was given 132-134 but mine did not completely melt until it hit 137. Lastly, sulfanilamide was 165-167 but mine was 1 degree higher to be 168. For the two unknowns, I wasn't given the MP range so I had to start from 0 degrees and slowly raise the temperature. Unkown 8 that I was given which I did for my first unknown came out to have melting point of 95 which is why I thought it was acenaphthene with melting point of 94-96. Unkown #7 which I used for my second unknown came out to have MP 166 which is why I concluded that it was sulfanilamide once again because it had 165-167 degrees.

The kidneys of the stellar river otter look very similar to the American river otter Lutra Canadensis. This is a multi-lobed kidney which is commonly found in many marine and aquatic mammals. The kidneys are multi-lobed and are fully equipped to remove toxins however they are not specialized for water conservation. Because the stellar river otter lives in a freshwater environment it does not need kidneys adapted for the conservation of water. Water is extremely prevalent in the habitat of the river otter and it has little need to conserve it when there is a river just outside its back door. The river otter also has a large multi-lobed liver, which supports the idea that its body is more adapted to purify toxins than it is to conserve water.