2011-2018 End of Year Report

Investigating the Role of the Caenorhabditis elegans Gene M05D6.2, an Ortholog of Human T- Complex Protein (TCP11), in Sperm Function and Fertility

 

During the 2016-17 academic year, I have been conducting research with Dr. Matthew Marcello. Our research lab looks into different genes that could potentially affect fertility. Over the course of this undergraduate-faculty research program, I have continued my investigation on the gene M05D6.2 in C.elegans, whose ortholog in humans is known as Tcp11 (human t-complex protein 11). The Tcp11 gene is necessary for proper sperm morphology as well as fertility. This gene has been associated with the coiling of a sperms flagella and has been found to localize in both a sperm’s head and flagellum. The goal of our research is to establish the role that the C.elegans ortholog of Tcp11, M05D6.2, plays in regards to fertility and ultimately, determine the molecular role of the TCP11 gene domain, which is still unknown. Previously, we were able to conclude that the M05D6.2 gene is necessary for male reproduction in C. elegans. After conducting research for the past few months, we have not been able to add much new data.

I mentioned in my most recent blog post that obtaining males can be a very time consuming process. Since males naturally occur so rarely we are force them by inducing reproductive stress onto L4 worms with knocked out genes of interest. The way we induce this stress is to subject the worms to increased temperatures which is supposed to give an increased yield of male progeny in ~2 days.

Previous to me, undergraduate researchers that started the project were able to determine that M05D6.2 is essential for sperm activation and fertilization because hermaphrodites with the gene knocked down produced less progeny. After they visualized this, the worms underwent DAPI staining which is a fluorescent dye that stains DNA. This staining revealed that the worms were still able to produce the same amount of sperm although they were unable to reproduce at the same rate. Next various mutants of M05D6.2 were created with the entire gene knocked down, one to resemble the same single nucleotide polymorphism (SNP) that is seen in infertile male humans, and one with a GFP tag on the proteins N-terminus. The fertility of the worms was then analyzed and visualized under a microscope where nothing was seen due to weak fluorescence staining. We were able to travel to William Patterson University in Wayne New Jersey to present this work at a poster session. This was my first time presenting in a professional setting which I enjoyed very much. Usually I get very nervous when presenting but this time was much different from what I expected. The setting was professional yet also very casual and it was really exciting to be able to talk with other scientists who were very obviously interested in our work.

For future directions, hopefully next semester I will take over this project and I will repeat the analysis of the mutant hermaphrodite’s fertility since the data cannot be considered conclusive yet since it was only conducted once. Secondly, I will re-do the GFP staining to hopefully get a good visualization of where the protein localized within the worm. Then hopefully I will conduct the same experiments with male mutants to really get a good idea of how this can possibly be related to male infertility in humans and possible bring many couples happiness. It would be beneficial if I could continue  this work over the summer but since I live out of state I will be forced to resume next semester and hopefully have better luck obtaining males.

 

End of year report

Inflammation is the human bodies natural response mechanism to relieve damage to a living tissue. There are four main groups of tissues including muscle, epithelial, connective and nervous tissue; with functions ranging from helping us perform daily functions like walking, to more complex functions like allowing signals to reach the brain. In order to relieve any stress that a soft tissue injury or break may endower, an inflammatory response proceeds as a defense mechanism. Through this defense mechanism, the ultimate goal is to confine and remove the opposing agent so that the healing process may occur. To do this blood flow may change as well as the permeability of the blood vessels and the movement of fluid, proteins and white blood cells to the location of the injury. Although a human body with a strong immune system will begin this process almost immediately, there are chemical factors that stimulate the changes outlined previously; including macrophages. Inflammation mediators, like endotoxin or lipopolysaccharide, are microscopic molecules which could be found inside or outside of the body. These molecules coat the outside cellular membrane of certain bacteria and interact with immune cells, causing an increase in inflammation.

Previous Studies show that fatty acids mediate these interactions, reducing inflammation, as well as act as an antimicrobial resistors. Such fatty acids include, oleic acid, stearic acid, linoleic acid and alpha-linoleic acid. Past chemical researchers hinting the ability of these acids to compete as a natural approach to resist bacteria as well as improve the inflammation/healing process led us to our proposed experiment in testing against the gram-positive bacterium Staphylococcus aureus. S. aureus was chosen because of its pathogenic property, ultimately causing an infection to turn into a disease. This bacterium typically causes skin infections and is at a higher risk to individuals including: neonates, breastfeeding mothers, injection drug users, patients with transplants or open wounds, and patients with chronic bronchopulmonary disorders.1 Through model organisms, research also shows that alpha-linolenic acid obstructs ultraviolet radiation, impeding the expression of cancers.UV rays contain more energy than visible light but less energy than x-rays. As the energy increases within the UV portion, it increases its ability to ionize, remove an electron, from an atom or molecule turning it into ionization energy.3 This ionization energy can then produce mutations in our DNA which can lead to health complication, one of which was noted above. In order to decrease the chances of UV rays to penetrate our skin, different inorganic and organic substances may be applied as a topical treatment. Inorganic compounds works as a sunblock in which they reflect UV light, while organic compounds absorb the UV light into the molecules themselves, instead of passing through the skin. In the case of our experiment, organic compounds were used as natural sunblock approaches.

In total, 4 different exotic butters and 21 different essential oils were used in the creation of 54 sample plates that were tested for their antimicrobial strength against S. aureus as well as their ability to decrease penetration of UV radiation through the skin. Antimicrobial results were classified upon their ability to clear bacteria around their spot of placement in the testing Petri dish. The threshold between being a positive vs. negative antibacterial plate was at 1.0cm, with anything higher than this being positive, and anything lower being negative. All four butters experiences zones of inhabiting of about 1.0cm but greatly increased when oils including vetiver, black cumin, carrot seed, and olive oils were added. Our most successful plate to date cleared 1.8cm of bacteria, with its formulation being composed of hemp butter and carrot seed oil. UV radiation results were classified upon their ability to decrease the amount of color on color changing beads. When no UV light was shined on the beads they were clear, but once UV light hit them without any barrier, they would immediately turn dark red. The results were classified on a 0-10 scale with 10 being the most positive in acting against UV penetration, comparable to the SPF 70. No butters experienced positive results on their own.The oils that were classified as positive included black cumin, vetiver, wheat germ and carrot seed oil. The results classified under okay (4-7) were comparable to the SPF 15. This classification included illipe and hemp butter. The negative results (0-3) did not aid in UV penetration at all or were less active than the SPF 15; including tacuma butter, mowrah butter,  macadamia nut, avocado and red raspberry oils.

This experience has been extremely humbling to be able to conduct such high level research under my undergraduate career. It has provided insight on what the research world looks like and let us really dive in critiquing our results. It is important to note that our research takes on a natural approach to antimicrobial treatments as well as sunscreens. The increase in the usage of strong, chemically man made antibiotics for non-life threatening bacteria has directly produced a greater abundance of antibiotic-resistant bacteria. As antibiotics are increasingly used, bacteria gets more and more accustomed to the drugs and develops its own system to fight off the drug, which produces inverse results. This has become an epidemic and it has been noted that 2 million people in the United States get infected with antibiotic resistant bacteria and of those, thousands die because the drugs that are supposed to fight off deadly infections, are now not doing any damage to the bacteria. In fact, antibiotics were found to kill off microbes in the gut by their chemical interaction, introducing chemical imbalances, which can lead to health problems including damage to the immune system and the reduction of brain cell growth.2 Finding natural approaches and eliminating the abuse of man made drugs is the future for chemistry and medicine and I am thankful for the support of Pace University to promote their students in making a change for the future.

 

References:
1. Bush, Larry M., and Maria T. Perez. “Staphylococcal Infections – Infectious Diseases.” Merck Manuals Professional Edition, www.merckmanuals.com/professional/infectious-diseases/gram-positive-cocci/staphylococcal-infections.
2. Nield, David. “Antibiotics Found to Cause Immune System Damage And Reduce Brain Cell Growth.”ScienceAlert,www.sciencealert.com/side-effects-from-antibiotics-include-immune-system-damage-and-fewer-brain-cells-study-shows.
3. “What Is UV Radiation?” American Cancer Society, The American Cancer Society Medical and Editorial Content Team,

End of Year Report

Kate Becker

UGR Final Report

Mycobacterium bovis-BCG is the organism responsible for Tuberculosis (TB), an infection that can cause coughing, fever and chest pain (CDC, 2017). Tuberculosis can be characterized by two distinct states of infection, active and latent. In the active infection, the infected individual displays signs and symptoms of infection, while the latent infection hides from the host’s immune response and does not express any signs or symptoms. A latent infected individual can go long periods of time without knowing they are Tuberculosis, until the infection is reactivated. The infection will usually be reactivated when the host’s immune response is no longer strong enough to suppress the infection and when they have become immunocompromised (CDC, 2017).

While the infection is known as being latent, the organism behind the infection has entered a state called non replicative persistence (NRP). The NRP state is characterized by an active metabolism, without bacterial replication (K Patel et al., 2011). NRP allows the bacterium to live inside the body and successfully avoid any attempts the immune response makes to destroy the infection. With little information about NRP and its mechanisms, it is very difficult to treat latent Tuberculosis. The rise of antibiotic resistant strains of TB reinforces the difficulties of treating the infection.

According to the World Health Organization (WHO), one third of the world’s population is infected with Mtb. Of these infections, 50% are multidrug resistance and 10% are in the latent stage (WHO, 2017). As the population increases and the infection becomes harder and harder to treat, it is very important to find new ways to prevent and treat the infection. In order to do this, the mechanisms and metabolism behind NRP Mtb must be understood.

When TB is in the latent stage, Mtb has been sequestered in a granuloma, a cluster of cells that has absorbed the infection. The interior of the granuloma maintains a very harsh environment. Needing a carbon source to survive, the sequestered Mtb only has cholesterol available to it. Quigley and colleagues have shown that cholesterol is mandatory for the bacterium to enter NRP, leading to the latent state. It has also been shown by Pandey and colleagues that Mtb can completely break down cholesterol, using it for nutrients and the virulence factors that enable the bacteria’s ability to make a host sick.

Due to the fact that Mtb can use cholesterol as a sole carbon source for nutrients and persistence, it is necessary to understand Mtb’s cholesterol metabolism to create effective treatments and vaccinations for TB. The goal of this study is to determine the best cholesterol media that the bacterium can thrive on, allowing us to conduct energetics studies on the organism when they are only exposed to a minimal cholesterol media. Due to its near identical genome, and the deletion of its virulence factors, Mycobacterium bovis-BCG (BCG) will be used to determine the best cholesterol rich media for Mtb. BCG will be grown in two cholesterol medias, known as 7H12 ad 7H12T, where they will be tested for their ability to grow and if they remain viable after being exposed to the cholesterol. By determining the best media to grow these organisms, future metabolism studies will be able to be conducted.

When I first began this study, I did not foresee the cholesterol media being so difficult to make. Although I knew the fastidious nature of BCG, I did not understand how difficult it would actually be to culture. Before realizing this, my goal was conduct an NAD+ GLO assay by promega to understand how cholesterol was being metabolized within the organism and if this metabolic shift would enable active BCG to become resistant to glutathione induced reductive stress killing. After finding out how difficult cholesterol media can be, I shifted my main goal to just finding the best way I could get mycobacteria to grow in a nutrient depleted environment. After creating three different types of of media, 7H12, 7H12T1 and 7H12T2, I finally had success culturing the BCG. After conducting a growth trial, I found that my second attempt of making 7H12T media was actually working. Although the only difference between my attempts of making 7H12T was the way the cholesterol was dissolved, it seemed to make all the difference. By heating the cholesterol stock slightly above room temperature, I found that I was able to prevent the cholesterol from precipitating out of the base of the media, and therefore can remain in the media even after filter sterilization. I believe that in my first attempts, the inability to dissolve the cholesterol completely was causing the only available carbon source to be filtered out of the media.

Now that I have found a way to culture BCG in cholesterol, I can continue on with my experiment. In my final weeks of this semester I am planning on conducting a growth trial to understand how glutathione will effect BCG treated with and without cholesterol. I hypothesize that I will see decreased reductive stress killing and increased growth in the BCG treated with cholesterol, and decreased growth and viability in the active untreated BCG sample. Although I was not able to carry out my initial experiment, I think that this was a valuable lab experience. I think that this experience helped show me what working in a BSL2 laboratory is actually like, and that bacteria does not always grow like you want it to. Throughout my UGR experience I was able to learn about dealing with contamination, trial and error of making media and learning new techniques that I would not have come across if it wasn’t for this project.

 

 

 

OBOR and the Petro-Yuan: Shanghai’s Yuan-Denominated Oil Futures Contract Arrives This Month!

In the course of our research we have studied how the One Belt One Road Initiative (OBOR) is part of an effort by Beijing to “internationalize” the yuan.  By setting up new dispute mechanisms and by providing nearly $1 trillion in loans and currency swaps to 65 countries[1], China is making a full court press to see the RMB actively used in trade settlement throughout Eurasia and Africa.

An additional facet of this push is the yuan-denominated crude oil futures exchange that opens in Shanghai later this month.

Though China recently surpassed the US in oil imports (and more broadly Asia has surpassed both America and Europe in crude consumption), the Pacific region lacks a benchmark of any great recognition.  Many analysts believe the Shanghai’s crude futures will emerge as the dominant price benchmark in the region, becoming part of the 24-hour global trading system, alongside the Brent and WTI futures.

Trading in these crude oil futures contracts will start at 9 a.m. on March 26 at the Shanghai International Energy Exchange, or INE, according to a recent press release the organization.

The trading margins for the futures have been set at 7% of the contract value, with the trading limits on the first trading day set at 10% of the benchmark prices. (The INE will release the benchmark prices on March 25, one trading day before the launch of the futures).

Will there be hiccups — liquidity issues, contract settlements concerns, etc — at the start of this new exchange? Of course.  China actually tried and failed with a futures market back in 1993; it was simply overcome by the unanticipated volatility.  But much has changed since then.

Though the yuan remains a minor currency in worldwide trade settlement, China recently surpassed the US in oil imports and GDP on a PPP basis.[2]  Its financial institutions are far more robust.

Ultimately this futures exchange is designed to give China much greater pricing power over the oil it consumes, and in the long term it will likely succeed.   One question that remains for the American investor is whether this new Sino-centric oil benchmark (and thus a new era of multi-currency pricing for oil) will have ramifications for both the demand for dollars and US interest rates.

“Petrodollar recycling” has existed as a major aspect of the international financial markets since Nixon’s famous pact with the Saudis in July 1974 to have them only sell oil in US dollars.   International trade settlement was critical for dollar demand after his administration closed the “Gold Window” and essentially defaulted on the long wavering Bretton Wood’s promise to exchange one ounce of gold for $35.

Since that time any oil importing country worldwide needed dollars to make its transactions. The vast volumes of petrodollars earned from oil producers were cycled back into US treasury securities and invested through the major US and European commercial banks. By 1977, Saudi Arabia alone had accumulated about 20% of all Treasuries held abroad.  The petrodollar process was also responsible for the massive growth of the Eurodollar market as it was less-regulated rival to US monetary markets, adding to the ubiquity of the dollar.

In the long term, it is clear that a new settlement of China’s oil contracts in yuan –especially one that is accepted by the Saudis –will reduce demand for dollars, though it is not clear whether other Asian countries will quickly follow suit.

There is certainly much to distrust about a futures exchange and price benchmark so firmly under Beijing’s control.  Its history of state intervention, capital controls, and favoritism toward Chinese companies could keep non-Chinese players from any initial involvement.  The main assumption is that Beijing will push its national oil companies onto the exchange and that the benchmark will greatly improve the risk management capabilities of the country’s “teapot” refiners,[3]  small and mid-sized firms that were just given oil import licenses in 2016.

Last year China’s total oil refinery capacity exceeded China’s oil needs by about 3 million barrels a day.[4] Additional refining capacity under construction is expected to surpass 18 m b/d in the next few years, so clearly the country will be facing a glut.   Whether this will winnow out the small players or whether the oversupply will be actively sold to the foreign markets now involved in OBOR remains unclear.

Most financial counterparties in a typical marketplace don’t like contracts with only a few dominant buyers or sellers and a government role, but perhaps this futures exchange is simply emblematic — like sovereign wealth funds– of the new institutions of the emerging “authoritarian capitalist” paradigm.[5]  According to Juerg Kiener, managing director and chief investment officer of asset manager Swiss Asia Capital: “Iraq, Russia and Indonesia have all joined in non-dollar trades . . . and as China is an importer it will push harder to get yuan contracts.”[6]  Pace University researcher  Banyan McGuire has explored how Pakistan –a major trade partner and emerging geopolitical ally of the PRC– has recently explored doing all of its bilateral trade in yuan (CYB).

March 26, 2018 will likely covered by China’s state media in a low key way, much like it did the formation of China’s first sovereign wealth fund –the CIC– back in 2007.  But its March date may eventually be seen by historians as an essential milestone in China’s hegemonic rise.   Though Nixon’s closing of the gold window in August 15,1971 is well-remembered, it was actually in March of 1973, exactly 45 years ago this month, that the West German government finally allowed its Bundesbank to stop buying US dollars. Germany’s exit from the system of fixed exchange rates sealed the fate of Bretton Woods.

 

 

 

[1] https://www.nytimes.com/2017/05/13/business/china-railway-one-belt-one-road-1-trillion-plan.html?mtrref=www.google.com&gwh=E41DC199913A9F9C0237D7D356F4C68C&gwt=pay

[2] https://www.bloomberg.com/view/articles/2017-10-18/who-has-the-world-s-no-1-economy-not-the-u-s

[3] https://www.ft.com/content/5a78b594-a365-11e7-9e4f-7f5e6a7c98a2

[4] https://www.ft.com/content/5a78b594-a365-11e7-9e4f-7f5e6a7c98a2

[5] https://www.foreignaffairs.com/articles/china/2007-07-01/return-authoritarian-great-powers

[6] https://www.cnbc.com/2017/10/24/petro-yuan-china-wants-to-dethrone-dollar-rmb-denominated-oil-contracts.html

Final Blog Post

Over the summer of 2017 the research I have been conducting involved the Cryspovirus, which is found within the parasite Cryptosporidium parvum. The overall outcome of my project was to insert a green fluorescent protein, GFP, into the virus to make it glow and be easier to see the virus and how much of it there is.

The first thing I had to do before beginning anything else was to extract the RNA from the Cryspovirus. Breaking the oocysts, or shells, of the Cryptosporidium parvum, which is where the Cryspovirus is contained, did this. This was a success for me as I was able to extract the RNA using this method. With this RNA I was able to do a polymerase chain reaction with it, PCR, using different reaction conditions. The PCR was done to copy a specific portion of the virus RNA. After the PCR was complete I then made an agarose gel and injected the PCR samples into the gel and let it run for a half hour. This was done with intentions to see a specific band on the gel that would mean that the PCR had worked and the RNA was successfully copied. Unfortunately this came out with negative results.

The reason why I think there was no band showing up for the RNA is because the hb, ns, and cpv primers I used were not cutting the RNA correctly and therefore the PCR was copying the wrong sections of the RNA. I think the primers are the problem because I have used them to do PCR with many different times using different reaction conditions and all of them came out with negative results when running a gel. So, to fix this problem I designed new primers that would hopefully bind to the correct sequence of the RNA and therefore the correct sequence will be copied when running the PCR. Just last week these primers arrived. They consisted of two hb primers and two ns primers whereas before I was only using one ns primer and one hb primer. These primers were used to do a PCR using a new set of reaction conditions and thankfully the results came out positive for the ns primers. As for the hb primers the results still came out negative. This means that I am going to have to repeat PCR using the hb primers with different reaction conditions to see if I can get positive results. If that does not work after doing multiple different PCR’s then I will most likely need to design and order new primers.

To be able to extract GFP from the plasmid I had to swab E. coli onto an agar plate containing ampicillin. These plates were left to grow overnight and once they had grown, one colony was picked off of the plate and put into a tube containing agar broth and left to grow more overnight by being incubated with shaking. When the tube turned cloudy is when I knew it had grown enough to extract. The GFP DNA was extracted using the QIAprep Spin Miniprep Kit and then the extracted DNA was cleaned using the QIAquick Gel Extraction Kit. After the DNA was cleaned, I ran a gel with it to make sure that there was DNA present. Unfortunately the gel came out negative indicating that there was no DNA present. So to see if there was any GFP present at all I used vecotrs and restriction enzymes to see if the DNA was the correct size. These results came out negative as well meaning that there was no GFP present in my sample at all. I am still not sure why there is no GFP present in the sample.

Along with the GFP I also ran a gel using cas9, CRISPR-associated protein-9 nuclease, which is an enzyme that will help insert the GFP into the virus RNA so that it can fluoresce. I used vectors and restriction enzymes to make sure that there was cas9 present and these results came out positive. Since the cas9 is used to help the GFP be inserted into the virus we had to be able to extract the GFP successfully before being able to use the cas9 so I just froze down the sample until it is ready to be used.

Moving forward, I will hopefully be able to get the PCR to work for the hb primers. From there, then I will try to extract the RNA from the gel again and hopefully it will work since I have the new primers that work. If that works then the GFP will be inserted into the Cryspovirus with the use of the cas9 to help it be inserted properly. If this step works then the virus will fluoresce green and my research project will be complete.

Overall, I am very thankful to have had this opportunity as I learned a lot. First of all, I have learned to be very meticulous while working in the lab because it is very easy to make small mistakes that can cause your research to be affected. I have also learned that there are not always straight forward answers as to why something went wrong and that it takes a lot of time and thinking to solve an issue. However, even if you think you have found the answer as to why something went wrong you may not always be correct. There is a lot of patience, critical thinking, and trial and error that goes into research.

Final Report

Kate Becker

UGR Grant Final Report

Summer 2017

In the beginning of the summer, I aimed to understand the intracellular response of Mycobacterium tuberculosis when attacked by the detoxification molecule, Glutathione. Using Mycobacterium bovis-BCG (BCG) as our model organism, our previous researched showed that non replicative persistent (NRP) BCG was resistant to glutathione induced reductive stress killing, while normal BCG succumbed to detoxification. This shows us that NRP BCG has an extra line of defense against the usual way the body would try to kill the bacteria. This data, combined with research from other laboratories stating that the presence of cholesterol in the granuloma can act as an electron sink helping to overcome reductive stress, gave way to my hypothesis.

I hypothesized that a cholesterol induced metabolism will protect M. bovis-BCG from GSH induced reductive stress killing similar to how NRP BCG is able to resist GSH. In order to test this, I planned to use an NAD/NADH-GloTM Assay in hopes of seeing BCG accumulating more NAD+/NADP when associated with cholesterol. These markers would indicate a metabolic shift towards an oxidative environment in the bacterial cytoplasm and would prevent glutathione induced reductive stress killing.

Before I could use the NAD/NADH-GloTM Assay, I had to create a media that contains enough cholesterol to induce a metabolic shift while also ensuring that the organism is not nutrient deprived. A recipe was created for 7H12 media using information from other labs who also worked with MTB and cholesterol. This recipe created our first obstacle because in order to fully dissolve the cholesterol, we had to heat 200 proof ethanol to 80C; which is highly dangerous. Ultimately, with much trial and error, we were able to create a media with the dissolved cholesterol without heating ethanol to 80C, called 7H12T. Additionally, a detergent to prevent the clumping of these naturally sticky organisms was needed. Originally, we attempted Tyloxapol, which was very hard to use due its thick consistency and a large amount was necessary to be effective. This made the media too bubbly when we had to filter sterilize it. Instead we found that we were able to use Tween80, a detergent that we use in our regular 7H9 media when we culture BCG without cholesterol.

Once we finished creating the media, we conducted growth trials to ensure that we would be able to culture BCG with it. When conducting growth trials, 1 mL of frozen BCG suspended in glycerol, then added to 4mL 7H9 media and was allowed to incubate for 24 hours. Then, 1 mL was added to 24mL of 7H12T media and incubated for three days. The growth was monitored each day by measuring optical density using a spectrophotometer. After the first day, we suspected contamination because of the doubling time. BCG usually takes three days to reach mid-log stage with an optical density of 0.6-0.8 nm. But, this culture reached 1.1 nm by day two. With other contamination issues being observed in the lab, by using a crystal violet stain we were able to observe three different types of contamination including yeast spores. The contamination was traced backed to our original frozen stock, forcing us to purchase fresh BCG.

The need for new BCG was a huge setback for the project, because we could not continue without it. Being a biosafety level 2 organism, it was difficult to obtain, and the shipping process took some time. After a few weeks, we finally received our new BCG and are currently working on culturing it to create new frozen stocks. Although I was not able to complete the goals that I had set, I still benefitted from this process. I was able to learn a lot about contamination; when to be suspicious, how to find it and how to get rid of it. A large part of the process included staining samples and observing them under microscopes, which was very interesting. Although the bacteria was damaging to my research it was fascinating to see all the other microbes living around us and how easily something could become infected. I also think that the many setbacks in creating the media was helpful to me as a researcher and scientist. With guidance from my advisor, I was able to learn how to go about making media that is suitable for a specific experiment and it really expanded my ability to comprehend scientific literature.

For the remainder of the summer and into the first semester, once the new BCG has been cultured; I will continue working on obtaining a successful growth curve which will lead to performing a NAD/NADH-GloTM Assay. Although scientific research does not always go as planned, I am incredibly grateful for the opportunity to work with BCG and further my knowledge in the field of microbiology.

Final Report

Through this research process I have learned a lot in regards to my subject and also in terms of the type of time and organization necessary to carry out research. It is also very important to stick to specific questions in order to keep your research focused and coherent. It has made me think a lot about academia and all that is necessary to pursue it. I have enjoyed learning more in depth about the topic and finding new ways and platforms to access data, journals, and articles on this.

I learned a lot about the history of New York City and how policy and a lot of actions undertaken by the government in the latter half of the 20th century led to the creation of New York as we know it today. There is a lot to be said and looked into about the relationship between private interests and government work. Certain policies may seem like a good idea in order to boost an economy or to create a housing boom but what are the real affects on the average citizen? We also discovered information on which the ways neighborhoods may be manipulated into a situation where they can then be “flipped” and gentrified. It’s very interesting to see how a city like New York, which has been long praised for it’s diversity and being a liberal haven, has partaken in discriminatory housing practices, unequal distribution of resources, and placing the needs of corporate interest over the needs of people. One of my favorite parts of this has been connecting the dots of all of this and truly understanding the way in which institutional racism functions and what it entails. Studying the origins of the debt crisis and what the actual factors were and how the Emergency Financial Control Board was instrumental in the rebuilding of New York and what the consequences of that decision were has also been an interesting part of this study for me.

 

For my work with Dr. Lavariega on Voces it has been much less eventful but just as interesting. I have been coordinating with peers to write their essays on their experience as a Latinx in this country and it has been incredible to hear such a diverse range of stories. I have loved delving into this topic and learning more about the varied experiences of my community. It has helped me to get a better feel of the sources of some issues within our community and also helped me form a deeper understanding of the ways in which bi-culturalism functions in the United States.

We have a wide range of stories that tell an important narrative that is usually missing from the greater conversation of race and identity in mainstream media. Often times, the experiences of Latinos in this country is homogenized and we are viewed as foreigners in a country over half our population was born in. Voces aims to change that narrative and humanize Latinos, especially in the current political climate. I have been able to further explore my own identity as a mixed-race Latina and what that means for me and my identity, my peers have been able to explore their experiences with machismo culture and pressures to appear hyper-masculine and dominant, or being Latino and unable to speak Spanish and the complex feelings that accompany it, and so many other experiences that challenge the current notion of Latinos in this country.

Final Report


Overview

The Human T-cell leukemia virus (HTLV) is a retrovirus that is known to cause cancer and other diseases in adults who have contracted it. The goal of the research this summer was to generate a reporter cell line that would then be used to study viral gene expression under various experimental conditions. Different methods were used to try to generate the reporter cell line.  PCR was performed using a plasmid containing HTLV-LTR (promoter) as the template. The PCR product was then digested, ligated to an appropriate luciferase reporter vector, transformed and mini-preps performed on subsequent colonies.  A 1.5 kb band appeared consistently on the gels that did not match the size of the insert. After several failed cloning attempts, we developed an alternative cloning procedure-using adaptors to add restrictions sites to our vector. There was only time for one attempt at this new technique that yielded no results.  For the upcoming school year, this technique will be performed once again in an attempt to yield colonies that are positive for the insert.

As part of our final project for the spring semester, immunofluorescence of Tax and TORC proteins was carried out in HEK-293T cells.  It is known that Tax activates HTLV through transcription factor and that it physically interacts with all three members of the TORC family.  In previous studies, it was shown that TORC could inhibit Tax activity.  In order to further explore this, the 293T cells were transfected with HTLV Tax and human TORC proteins, then stained with two different antibodies to the proteins.  Two different secondary antibodies then were used (one from mouse and the other one from a rabbit) to identify Tax from TORC using the fluorescence microscope.  TORC was stained with a 488 nm fluorescent antibody (GFP), Tax with a 594 nm  (Texas Red) antibody, and a DAPI stain was used to visualize the nuclei   Eight different experimental conditions were viewed under the microscope 1) negative control 2.) Tax 3.) TORC1 4.) TORC2 5.) TORC3 6.) Tax + TORC1 7.) Tax + TORC 2 and 8.) Tax + TORC 3.  By using the fluorescence microscope the localization of TORC with regards to Tax was hoped to be observed however the TORC staining was weak and further studies will be performed during the fall semester to obtain better staining and further understand the relationship between the two proteins. Furthermore, we did immunofluorescence to test the mutants created by one of my colleague and confirm that the mutants were working properly. She generated several TORC mutants and I was able to confirm her hypothesis about their localization. I was also able to test out a rabbit polyclonal antibody that was not very successful with the cells. This antibody produced a great amount of background and therefore, different options have to be consider to carry out a co-localization for TORC and Tax.

Reflection

Working in the lab during year on a one-to-one basis with Prof. Isaacson was an exciting and valuable experience. I was able to gain knowledge but most importantly gain independence and confidence in my own skills.  Doing calculations on a day-to-day basis to split the 293T cells and prepare/modify recipes was a great way to practice my math skills. I was responsible for maintaining my own cell line and I learned how to bring them up, freeze them down, and became comfortable enough to make decisions on my own about them. I also learned how to count cells and how to plate them as well as, how to trouble shoot some of the problems we ran into with the cells. Many protocols became second nature to me and I was able to complete them more efficiently.

This project put me in the position to present at the Eastern Colleges Conference, poster presentations, and for my own research with the school. This forced me to speak to others about my project and practice my communication skills. I learned how to present and how to communicate effectively when people would ask me questions. I would constantly get bombard with really challenging questions but because I became so comfortable with my own project, I was able to answer these. This project prepared me beyond the laboratory and gave me a taste of the real world. Moreover, I became proficient at reading and understanding scholarly articles in the science field. Prior to working in the laboratory, I did not feel comfortable reading scholarly articles as I found them really hard to understand but as part of our weekly lab meetings we were required to read scholarly articles and than present them to the team. This was extremely useful as I was learned how to better interpret the data and process what I was reading. Furthermore, I enjoyed being part of a team of students who would look out for each other and became friends that were quick to help when I had a question.

Monitoring with Optical Spectroscopy the Interactions of Hemoglobin with Nanomaterials: Post 1

The title of my project is Monitoring with Optical Spectroscopy the Interactions of Hemoglobin with Nanomaterials. The goal of this project is to determine the interactions between hemoglobin and various nanomaterials using spectroscopy. I know from previous classes that hemoglobin has binding properties with oxygen and I expect for there to be a similar correlation with our different nanomaterials. All of the nanomaterials have oxygen in their compounds, this leads me to believe that all of them will bind to some degree and the different metals associated with each nanomaterial will show how tightly it binds.

 

The methods used to create this research will involve preparing hemoglobin solution to be added with increasing amount of nanomaterials. The hemoglobin will have a fixed concentration. A hemoglobin solution without the nanomaterials will be used as a point comparison with hemoglobin solutions added with nanomaterials. The nanomaterials will then be added to the hemoglobin mixture and then I will run them in the two spectrophotometry machines that we have available, UV-vis and the spectrofluorometer. The resulting graphs will show the differences and from there we can draw conclusions.

Note: This was written in October, due to the technical issues of this blog, it is being posted at a later date.

End of the Year Report

This research project was a lens into the real world of research and data collecting. In our American history, a lot of global issues have progressed simply from the misinterpretation of data. The United States involvement with Iraq is a prime example of such where one data collector finds that a Middle Eastern country found a source of uranium and then begins the domino effect of war. This is a real world instance where data has made a huge effect on international diplomacy. Granted, my data collecting did not have that exact impact, it still gave me insight into the present issues in our policing system.

My research was about encounters between the mentally ill and the police when deadly force is used. When I began researching I found a wealthy amount of literature on the topic. There were also hundreds of webpages with statistics about how many encounters were “deadly” and the circumstances behind each situation. I also had to inform myself more on the specifics of mental illnesses in order to fully comprehend the situations and policies that were within the research. I also needed to refresh myself on police procedure so when reading I could identify mistakes made when handling cases with mentally ill people.

It was within this research that I found that people with an existing mental illness are 16 times more likely to be killed by the police. The Treatment Advocacy Center really stands up for those who suffer with mental illness. They often fight for better/ more available treatment centers and for specialized police task forces for cases with mentally ill in order to save their lives.

The paper writing was the hardest part for me personally. I had never written a literature review before and actually struggled really hard with doing so. In addition to my heavy course load this semester I definitely could have been a better research assistant for Professor Arslan. I tried my best to devote as much time to writing the paper as possible but many obstacles from my classes and other curricular came my way. The research was more of a time commitment than I had originally anticipated, however, when Professor Arslan depended on me to complete a data sheet I worked day in and day out to do it.

A data sheet is an excel spreadsheet that has many different cells consisting of details about the incident of deadly or excessive force being used on a person. There is a cell for the persons name, gender, race, location of the incident, and especially whether or not the person is mentally ill (including much more). This excel spreadsheet makes up the S.H.O.T. Database for Police use of Deadly Force. This database looks at the issue of police use of deadly force in its entirety. My job as the researcher was to focus in on the mentally ill column of the spreadsheet and then fill out any other information as it came my way. I had experience working on a similar project from my Terrorism and Society class (which is amazing, highly recommend it) with Professor Arslan. It was exciting to contribute to the database and leave my mark on it as it progresses to be something even more amazing.

There were trials and tribulations within this research work. There were times where it overwhelmed me and I shut it out, but there were also times where I buckled down and enjoyed the work I was doing. I am soon having my final meeting with Professor Arslan to discuss the charts and poster board for our Show Case. This research topic will go far and I can’t wait to see what it develops into moving forward.