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Research Seminar Summary - Mitochrondria Shape

Submitted by jonathanrubi on Sun, 04/22/2018 - 18:57

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.

 

Research Seminar Summary - Viral Fusion pt2

Submitted by jonathanrubi on Sun, 04/22/2018 - 18:57

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.

 

Research Seminar Summary - Viral Fusion

Submitted by jonathanrubi on Sun, 04/22/2018 - 18:56

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.

Research Seminar Summary - Intro

Submitted by jonathanrubi on Sun, 04/22/2018 - 18:56

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.

 

Esterification Discussion - PP

Submitted by jonathanrubi on Fri, 04/20/2018 - 12:12

In this lab a Fischer esterification reaction was performed using the reagents n-propyl alcohol and propionic acid to produce an ester product of n-propyl propionate in the presence of sulfuric acid. The ester product was characterized using odor and IR spectroscopy and was retained with a yield of 68.9%. The odor of the starting reagent propionic acid was described as unpleasant and similar to body odor. The odor of the product was described as fruity and sweet, which is in agreement with the characteristic odor of an ester. This indicates that the reaction went to completion and yielded the desired ester. IR spectroscopy also indicated characteristics of the ester product. Esters are characterized by a sharp, strong peak at 1740 cm-1, indicated a C=O, and one was seen at 1741 cm-1. In addition, a sharp, strong peak was described at 2972 cm-1 , which is typical of an alkyl C-H bond. In addition, the broad peak at 3000 cm-1 indicated the presence of a carboxylic acid O-H bond and a broad peak with medium to strong intensity at 3300 cm-1 indicating an alcohol O-H were not seen on IR spectroscopy. The odor and IR spectroscopy indicate successful completion of the esterification reaction to yield n-propyl propionate.

Esterification Discussion - Rough Draft

Submitted by jonathanrubi on Fri, 04/20/2018 - 12:12

In this lab a Fischer esterification reaction was performed using the reagents n-propyl alcohol and propionic acid to produce an ester product of n-propyl propionate in the presence of sulfuric acid. The ester product was characterized using odor and IR spectroscopy and was retained with a yield of 68.9%. The odor of the starting reagent propionic acid was described as unpleasant and similar to body odor. The odor of the product was described as fruity and sweet, which is in agreement with the characteristic odor of an ester. This indicates that the reaction went to completion and yielded the desired ester. IR spectroscopy also indicated characteristics of the ester product. Esters are characterized by a sharp, strong peak at 1740 cm-1, indicated a C=O, and one was seen at 1741 cm-1. In addition, a sharp, strong peak was described at 2972 cm-1 , which is typical of an alkyl C-H bond.

Esterification Procedue

Submitted by jonathanrubi on Fri, 04/20/2018 - 12:11

To a round-bottom flask add n-propyl alcohol (0.82mL, 11mmol) and propionic acid (0.97mL, 13 mmol). Add four drops of concentrated sulfuric acid and mix. Connect the rb-flask to a reflux condenser and heat to a gentle boil. Reflux the solution for 45 minutes. Cool the solution sufficiently. Transfer the cooled contents into a centrifuge tube containing water (1mL) and wash the solution. Perform two subsequent washes with saturated aqueous sodium bicarbonate (1mL) and saturated aqueous sodium chloride (1mL). Pipet organic layer into a vial and add anhydrous CaCl2 (5 spheres) and swirl. Pipet contents into dry tared capped vial. An IR spectrometry was performed to determine properties of obtained product.

Isolation of trimyristin from nutmeg discussion part 2

Submitted by jonathanrubi on Thu, 04/12/2018 - 17:25

Melting point of the once recrystallized product was determined to be 53-55oC. The melting point of the twice recrystallized product was determined to be 54oC. In literature, the melting point of trimyristin was 57oC. The narrowing melting point range after each recrystallization indicated a relatively pure product formed from recrystallization. The slightly lower melting point obtained versus in literature indicates some soluble impurities present. The product of the hydrolysis and acidification of trimyristin was determined to be myristic acid using melting point analysis. Melting point of the myristic acid was determined to be 51-52oC and the literature value of expected melting point of myristic acid was 54oC. The slightly lower melting point obtained as well as the narrow melting point range indicate subtle impurities.

Isolation of trimyristin from nutmeg discussion part 1

Submitted by jonathanrubi on Thu, 04/12/2018 - 17:24

In this lab trimyristin was isolated from the organic compound nutmeg in the presence of tert-methyl butyl ether. The crude trimyristin product was then recrystallized in the presence of acetone and hydrolyzed in the presence of sodium hydroxide and 95% ethanol and then acidified in the presence of hydrochloric acid. Recrystallized trimyristin was recrystallized a second time in the presence of acetone. The recrystallized product was determined to be trimyristin using melting point analysis with a yield of 23.7% after the first recrystallization and 6.0% after the second recrystallization based on the original amount of nutmeg used in the reaction.

Isolation of trimyristin from nutmeg procedure

Submitted by jonathanrubi on Thu, 04/12/2018 - 17:23

         To a round-bottom flask was added nutmeg (1.00g) and tert-butyl methyl ether (3mL). Heating at boiling point was performed to the solution for 10 minutes. Micro-scale filtration was performed on the solution. Crude product was recrystallized from acetone (2mL) and cooled. To a round-bottom flask was added recrystallized trimyristin (60mg), 6M NaOH (2mL) and 95% ethanol (2mL). The solution was refluxed for 45 minutes. The solution was allowed to cool and poured in a beaker containing water (8mL). To the solution was added HCl (2mL) dropwise and stirred while cooled. Remaining recrystallized trimyristin was recrystallized from acetone (2mL). Melting point of product and both recrystallized trimyristin was determined.

 

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