Drafts

The goal of the research is to compare the two mutants of the rhodopsin mutation, P23H, and S15N. Specifically, the researchers were interested in how the mutations in the N terminus domain affect their invitro biochemical properties, their UV visible absorption, Fourier transform infrared, circular dichroism and metarhodopsin II fluorescence spectroscopy properties.  While this was not a formal goal of the research, the study also found that chlorin e6have the ability to rescue mutated rhodopsin protein that had its stability affected.

In the experiment, the proteins were first prepared through PCR mutagenesis technique. The genes were then placed into HEK293S cell lines and the P23H and V15S opsins were established. The cells were harvested and the proteins were purified using immunoaffinity chromatography. 

In the first experiment, the proteins were subjected to UV spectrophotometry, which would establish the degree of misfolding of the mutated protein. For the second experiment, both of the mutant proteins had their oligosaccharide chain from rhodopsin glycosylation sites cleaved using PNGase F. The resulting immunoblot was used for densitometric analysis of the immunoblot was done using Image J, then the Box plot was used to summarize the result of the analysis. This method would establish the amount of aggregation that may occur in cells, which would disrupt their protein function. For the third experiment, MetaII fluorescence was measured for three proteins to measure the amount of aggregation that may occur in the cell. For the fourth experiment, Fourier transforms infrared spectra were collected for COS-1 cell rhodopsin to gain structural insight into the degree of misfolding in the two mutants. For the fourth experiment, circular dichroism spectra were measured with the samples of rhodopsin and Ce6 at 2.5 and 100 micro M this would indicate the stability of the presence and absence of Chlorin e6. 

While studying for my Biology 287 class, which is introduction to Ecology, I have realized a lot of predators have broad diets. Most predators eat prey in relation to their availability without showing a preference for any particular prey species. A predator, like a lynx will be specific if it eats tat species more often than would be expected based on that prey's availibility. That is why it will choose the snow hare bunny. They are considered specialists. Same goes with the coyote. This constitutes 60-80% of the coyotes diet. Some predators concentrate teir foraging on what ever prey is most plentiful, which to me makes the most sense. The more they fill you up the better! In a research conducted, guppies were given fruit flies that float on the water surface or worms that sink, and they chose the one that is more abundant. They will switch to either, whichever is most abundant. 

The overall objective of our proposal is to identify a subject that contains a highly conserved gene that can be characterized by the amount of expression. One specific aim the proposal has is to identify highly conserved genes that are present in the Animalia kingdom. In preliminary work, we found that one example of a highly conserved gene is the BOP1 gene present in ribosomal DNA. Further research done on HOX genes has noted that “[the duplication of HOX clusters] are shared by all vertebrae.”1 The statement motivated our group to propose that the HOX genes express little variation in vertebrae, and test the statement by knowing how consistent this data would be if we were to expand our subject of interest to a whole kingdom, rather than specific subjects used for their evidence (chicken, zebrafish, Xenopus, and newt). We will now look into HOX genes to see how similar or different expression of the HOX gene is throughout the Animalia kingdom.

Now, while there have been many studies that are done on hox genes, there have been relatively few studies that were done on HOXC genes specifically. Also, the scope of the study has been quite narrow, with the study often limited to studying a few well known HOX gene in one specific family of an organism or how the mutations in hox genes often affect disease, specifically cancer. In both cases, the studies were mostly conducted through studies where specific hox genes are mutated in a model organism, and the resulting developmental structures are looked at or studying HOX genes in specific animals and using genetic data analysis using specific cancer cells. Because these two studies are the most common studies done on HOX genes, there are very few experiments done on how the hox genes differ between a wide range of species, and how they interact with each other. This experiment would aim to fill some of the gaps in the knowledge through the use of online genomic analysis, which would make it possible to generate a large quantity of predictive data that can be the basis of other experiments which can establish a more concrete interaction between different protein interaction that hox gene would have with other transcription factors.

Hox genes are a family of genes that are vital to an animal’s embryonic development. The HOX genes belong to the homeobox gene family, which is the second-largest gene family which all encodes for 61 amino acid sequence that forms a helix turn helix structure. The expression pattern of HOX genes dictates the body plans of organisms. In vertebrates, HOX genes are organized into clusters that are on different chromosomes named abcd which individually each contain 9 to 11 hox genes  In this study, we propose to study HOXC genes for this experiment. 

HOXC  is a gene cluster that is located on the chromosome. There are 9 HOXC genes;HOX 4,5,6,8,9,10,11,12, and 13.  All of the genes are located close to one another and in some cases share a noncoding exon. HOXC 4,5,6 shares an exon. HOXC4 is involved with the development and involved in the stimulation of androgen receptors. The function of HOXC5 is unclear. However, the gene is associated with Lymphoma. The function of HOXC6 is similarly unknown. HOXC8 is involved in cartilage differentiation and a malfunction of the gene gives rise to cartilage disorders.  HOXC9 is involved in the differentiation of white and brown adipose tissue. The function of HOXC10 is not clear but it is highly expressed in cell differentiation and proliferation. HOXC11 is involved in the development, specifically the mesodermal commitment pathway. The function of HOXC12 is not known but is associated with a club foot. HOXC13 is involved in the development of hair and nails.

In the evolution of childbearing in humans, there is a conflict that occurs both between the mother and father of the child but as well as between the mother and child.  In order for a fertilized egg to be implanted, it has to "shout" loud enough to the mother so the uterine lining is not shed.  This is done by pushing itself further and further into the uterus while releasing hormones as well.  Once the fetilized egg is implanted, another battle begins between the mother's and father's genes to decide how large the baby will grow in the uterus.  The mother would want to keep the baby small to limit her personal investment while the father wants the baby to grow as large as possible to increase its change of survival.  The conflict between the mother and fetus is also brought to light because only half the DNA of the child is the mother's.  

To start the lab, turn the hot plate on (225 °C) and weight out nutmeg (1.002 g) into a microscale round-bottom flask (RBF).  To the RBF, add tert-butyl methyl ether (TBME 3 mL) and 2-3 boiling chips.  Attach the black plastic connector to the RBF as well as the air condensor column.  Using the three-prong clamp, lower the RBF into its appropriate hot well on the aluminum block of the hot plate and allow the mixture to gently boil.  If the solution boils too violently, lift it up slightly out of the aluminum block to avoid bumping.  After 10 min, remove the flask from the heat and allow the solids to settle at the bottom of the RBF.  While the solid settles, set up a microscale filtration system using a glass pipet and small wad of cotton. Below the pipet, place a tarred Erlenmeyer flask. With a clean pipet, remove the solution from the RBF, holding the RBF at a slight angle, and allow the liquid to drain through the filtration apparatus.  If the solution becomes stuck in the filtration system, attach a pipet bulb to the top of the filtration pipet and gently squeeze it to create pressure.  Be sure to remove the pipet bulb before releasing the pressure on it.  After all the liquid has been filtered, add TBME (2 mL) to the RBF and warm briefly (2-3 min) with the column reattached.  Again, let the solids settle and filter the solution as done previously.  In the fume hood, warm the filtered solution in the Erlenmeyer flask using body temperature and pass air over the top to evaporate off the TBME.  The resulting solid will be crude trimyristin.  After letting the crude trimyristin to dry (5 min), determine its mass (0.484 g).  For every 100 mg of crude product, use 1 mL of acetone during recrystallization.  During the first recrystallization of crude trimyristin, acetone (5 mL) was used as the solvent.  The resulting crystals were white and powdery. The melting point (54-55 °C) and mass (0.163 g) of the first recrystallized trimyristin.  For the hydrolysis of trimyristin, set aside some of the first recrystallized trimyristin (0.061 g) into a clean RBF.  To the flask add NaOH (2 mL, 6 M), ethanol (2 mL, 95%) and boiling chips.  For 45 minutes, reflux the solution on the hot plate (250 °C).  While the hydrolysis is being refluxed, perform a second recrystallization using the remaining trimyristin (0.102 g) with the same methods as before.  The final mass (0.066 g) and melting point (57 °C) of the second recrystallized produce was determined.  When the 45 minutes of refluxing was a complete, allow the solution to cool in the RBF to room temperature (rt).   Once cooled, add the solution to a 50 mL beaker containing water (8 mL) then drop-wise add HCl (2 mL, 6 M).  The addition of HCl should cause myristic acid to precipitate out. If a gelatinous solid forms, the mixture needs to be stirred more and more HCl should be added if it does not break up.  On ice, cool the beaker for 10 min with stirring.  Using a vacuum filtration, collect the solid and wash it three times with ice cold water.  Allow the solid myristic acid to dry at least overnight the determine the melting point (54 °C) and mass (0.035 g).

In neurobilogy we leared about LoxP sites. These are areas in the DNA that correlate to each other. Thye work in the presence of CRE recombinase in order to flip the DNA sequence between the paired Lox sites, or cut it out completely. When the LoxP sites are facing the same direction, they loop the DNA between the two sites and cut the sequence between the two sites, and one of the lox sites, out of the genome. This results in two strands of DNA, each with one of the paired lox sites. CRE recocmbinase can only work on one pair of LoxP at a time, so cutting out of Lox of a pair is a perminant and irreversable change. On the other hand, if the two paired LoxP sites are facing opposite directions, they form a loop in the DNA and flip the DNA in between the LoxP sites around. For example, if the DNA inbetween the LoxP sites is in the anti-sense direction, the flipped DNA would then be in the sense direction. This is a method used by neurobiologists who want to insert a sequence of DNA into specific cells. The neuoscientist would insert a sequence of DNA in the antisense direction, between two inverted LoxP sites. The DNA sequence would be taken up by all cells in the area, but only cells expressing CRE recombinase would be able to flip the DNA sequence into the sense direction in order to express it. 

In psychology we learned that there are some cultures that do not have words for certain, or any numbers. These societies do not crumble because they don't have our version of numbers, but it is hard to imaging a society that could function without numbers. It has been proven that all human babies are born with an innate way of thinking about quantities and numbers. Innatly, we think of numbers logarythmically. We think that the jump between 1 and 2 is infanitely bigger than the jump form 8 and 9 because 1 to 2 is doubling the number while 8 to 9 is adding just a fraction of the number. This is how some of the societies without words for discrete numbers oporate. They use the logarythmic numbers that we, in our society, are much less comfortable with than our version of intervaled discrete numbers. 

Overall, this is a very organized and informative poster. The color scheme looks attractive, there are not too many words or figures, information is spread out nicely. This is a great example of a model poster.

            The design of the poster is nicely put together. The flow of information and spacing between sections is well thought out. The poster is consistent with formatting, and the typography looks good on the poster.

            The organization of the poster is satisfactory. There is a nice flow, the information is easy to find and to read. The important information is clearly displayed and explained in each section. This poster did not get full credit for organization because it does not include an abstract or a discussion section. There is a background information section that could serve as an introduction, but the abstract and discussion are left out.

            The writing of the poster is very clear and concise. The information is presented in paragraphs for the background and methods sections. There are no grammatical or spelling errors and the typography is appropriate and uniform. The poster is written in scientific writing, and the word count is appropriate for a poster.

            The content of the poster is informative and well presented. The data is presented appropriately, showing graphs with final numbers and conclusions, not raw data or statistics. All graphs have high resolution and are crisp and sharp. There does not seem to be a problem with the copyright. Overall, the poster is persuasive in that utilizing current public health information and databases does not cost more, or take time away from other patients, for the treatment of TB.