Student Research at Duke

The work is actually going pretty well! What I’ve done of importance:

Isolated and run rtPCR on the first of three RNA samples from the neural mutant and wild type strains of C. elegans, yielding (surprise!) insubstantial results. However, last Friday I isolated RNA from the second samples of C. elegans, and we’ll run these soon to determine the immune gene expression levels in both strains. This seems pretty exciting, and I’m looking forward to seeing where it goes.

The results from the first run may have been messy because of a possible screw-up in the mixing of samples in the first run, so I’ve been doing PCR and gel runs to determine if two of the samples were mixed. We used a primer we thought was specific for the experimental pathogenic species, and would then see if that primer amplified any of the DNA in the E.coli samples–if so, then the contamination would be confirmed. However,the primers were (unfortunately) not specific for the pathogen; in fact, they amplified DNA in all the samples we had, and then after I did another PCR and gel run, we determined that the primers definitely amplified DNA from the C. elegans, E. coli and pathogen. So we’re just waiting for the next results of rtPCR to compare them to the original, and will do a third and, for me, final RNA isolation and analysis to determine with some certainty the changes in gene expression.

I’m also choosing the best couple of transgenic mutants that produce green flourescence when their immune gene is expressed, crossing them with our mutant strain and will compare their levels of flourescence on pathogen and E.coli. No green flourescence, no gene production!

In other news, it’s the season of prelims in the MGM department, so we’re celebrating two of our grad students’ passing this week! Congrats to Ine and Mickey!

So I thought I’d finally add some pictures, now that the uploading is working for me! Although this most recent blog should deal with scientific ethics, I missed 2 of the 3 ethics programs while I was working in the lab…I promise I’ll do the ethics blog soon! In the meantime, like my grad student Katie told me, I’ll blog about the greatness of being in lab until 10pm! This is my lab bench, which I just cleaned today…

These are the stacks of media that take up an obscene amount of my time…and to the left is the worst contraption ever, the “pour boy” media pourer that honestly has ruined several of my afternoons. It has these tube sections that probably haven’t been replaced in at least 5 years that don’t work effectively whatsoever–in fact, one has a sticker saying “I’m old but I work,” and under this someone has written “at Wal-mart.” Amusing, and indicative of its frustratingly sporadic functionality. We have to hit it with heavy scissors when it stalls, and a media backup literally exploded all over the bench last Monday, making me miss Katie’s presentation on neural control on innate immunity.

This is my lab after celebrating a post-doc, Devin’s, birthday…Katie brought delicious cake, and we returned to our work much happier than usual.

And here are two pictures of me working at the lab…

When I was talking with my sister, Miranda, last week, she told me that she really has no idea what I’m doing here–I’m sure that anyone reading this blog would also like a little more detailed explanation on my work here, and I still need to pose my research question to the blogosphere.

While I have spent countless hours making and pouring media, I also have been setting up and gathering information for my main experiment, RNAi of C. elegans with a certain mutation rendering it much more susceptible to infection. Broadly, the experiment involves 4 conditions: the mutant and wild-type each grown in both E. coli and the pathogenic bacteria. Katie and I will take these worms, after they have been exposed for a set time, and extract and analyze their RNA. The RNA differences will show what genes are upregulated as a part of the immune response, and will help us determine the important factors in the immune response to this specific pathogen, which also infects plants and mammals, including humans. So I suppose my question, in one sentence, is this: what genes are upregulated in C. elegans when responding to infection by a certain pathogen?

Sorry for the lack of specificity; I don’t want to say too much about the project, as it could possibly be published at the end of the summer.

While, at first, I was a little nervous to interview and report on my PI, Alejandro, this was actually a great assignment! I enjoyed getting to know him better (as I really only talk to him maybe twice a week, during cake-time), and it was interesting comparing my life/interest in science with his and other mentors’ stories.

Like the nerd I am, I thought the interview was meant to be written formally, so I have a nice report typed below…however, if you’re like me, you’d enjoy the cliffnotes and highlights I’m going to mention here:

Dr. Alejandro Aballay was born in Argentina, where he remained until obtaining his Ph.D. in Biology for cell signaling. He then came to the Washington U. in St. Louis to fulfill his post-doc in genetics, during which time he helped to pioneer the organism C. elegans as a research tool by proving it’s susceptible to the same pathogens as humans and other mammals. He began studying host-pathogen interactions and has worked in the Microbiology and Molecular Genetics department at Duke for 4 years.

Alejandro seems to have taken a fairly linear path through school to his current position–from the onset of his education, he knew he wanted a career in academics and (quite successfully) has achieved his position as a professional scientist.

One reservation I have about becoming a researcher is the level of isolation a lab seems to have, as compared with other scientific professions (namely, medicine). However, when I asked Alejandro whether he feels isolated, he responded strongly, saying, “No, not by any means. It’s the way that science actually works, by interacting with new people and discussing new ideas. It’s true that scientists spend a fair amount of time in their offices writing grants and business-related emails, but it’s all the time discussing with members of the lab, a network of other colleagues—it may seem isolated but it’s not by any means.”

If you’re interested, here’s the long format of my interview:

Biographical Information

Dr. Alejandro Aballay, my P.I., is a member of the Molecular Genetics and Microbiology department at Duke, where he heads a lab and teaches classes in the medical and graduate schools on microbiology, molecular genetics and innate immunity. After receiving his Ph.D. certificate for his work studying intracellular trafficking in Argentine university, he completed his postdoctoral training at the Washington University in St. Louis. He has worked at Duke for 4 years, and is an esteemed member of the international scientific community. He is an esteemed scientist in the US and in Argentina, his native country, who often invites him to review grants, give talks and advise the government scientists.

Ph.D. and Post-Doc Work

As mentioned above, Alejandro earned his Ph.D. in Argentina for his work studying cell biology and intracellular trafficking. When deciding where to continue his education, he came to Wash U. to strengthen his knowledge of genetics. Although he began training with the idea of developing C. elegans as a mechanism to study trafficking, for a variety of reasons the project did not work as it was supposed to; instead, he began studying host-pathogen interactions. He helped pioneer C. elegans as a model organism for genetic studies by showing its use with pathogens with a narrower host range, finding that similar virulence factors from the pathogen Salmonella affect C. elegans and mammalian hosts, demonstrating C. elegans’ potential as a model for human disease. He identified the programmed cell death pathway as part of the immune response in C. elegans, and then looked for his independent pathway at Duke University.

Current Research

The Aballay lab studies host-pathogen interactions using “genetic and functional genomic methodologies to study the genetic bases of innate immunity,” infecting the worm C. elegans as a model for mammalian disease. Currently, he is very excited about studying the communication between the nervous and immune systems in innate immunity, particularly in studying cellular signaling to determine the neurological control of the inflammatory response.

On Being a Scientist

Explaining how he first became interested in science, Alejandro says, “There is no one particular memory, but during my first classes of biology in high school…it was clear that I wanted to follow a career in academics from the very beginning.” After considering doing his Ph.D. in Physics, he decided it was “too dry” and decided instead to obtain his Ph.D. in Biology. When asked if the life of an academic scientist is isolated, he responds strongly “No, not by any means. It’s the way that science actually works, by interacting with new people and discussing new ideas. It’s true that scientists spend a fair amount of time in their offices writing grants and business-related emails, but it’s all the time discussing with members of the lab, a network of other colleagues—it may seem isolated but it’s not by any means.”

That’s a long post for now, and I’ll be sure to post pictures soon!

I just wrote a ridiculously long post that deleted itself because I didn’t have sufficient security privileges to post it. This really sucks, and my post is now going to be less witty and more bitter because of it.

To rewrite what I just spend 45 minutes doing:

Since my last post, I’ve continued scoring and transferring worms with the same mutation but at different alleles that have died after exposure to a pathogen. I expected them to all have died by now, but, surprisingly, some have remained alive! I mean, only about 10% are alive at best, but they have been living in and eating pathogenic bacteria, so I’m impressed by them. Interestingly, the worms have pretty complex immune systems, producing antibacterial agents as part of their response to infection.

Anyway, scoring the worms is difficult as the dead ones look like nearly invisible sticks suspended in a thick lawn of pathogen; it takes an inordinate amount of time for me to spy them, and I commiserated with a fellow undergrad, Thomas, about how hard it is to find the dead ones. In addition to the invisible-stick dead worms, some crawl up the sides of the plate, burrow into the media, or just disappear somehow to escape the throes of death by bacteria (score these as ‘gone’). Hopefully my score sheet of alive/dead/gone worms isn’t too off and produces relatively pretty graphs.

Other than that, though, I’ve just poured and inoculated some more media for the upcoming RNAi experiments I’m doing with Katie, the grad student under whom I’m working. The basic idea for this experiment (which we’ll really begin next week) is to compare RNA in 2 different strains of worms, each in 2 separate environments, one pathogenic and one with regular E. coli. The RNA that is upregulated in the wild type but has no change in transcription levels in the mutated type will indicate the gene(s) involved in the specific immune response to infection and will tell us more about the mutated strain, trying to decipher the role of a specific gene that was mutated.

Thankfully, a new undergrad, Thomas, has just started working in the lab and is now making some of my media, leaving me lots of free time to blog.

While I really like the lab because it’s interesting working with mutated worms and determining their genetic activity/makeup, etc., I love the lab because of my co-workers, who are amazingly hilarious and intelligent…it’s kind of like having my own version of NBC’s The Office, except that Alejandro is a great boss, unlike like Michael in the show. Recent notable lab events include the lab’s intramural softball team, the Sex Panthers’, victory to secure a spot in the playoffs, and a birthday party (lunch, followed by a ridiculously large chocolate cake with about 1.5 in thick iced rainbows and clouds) for an MD-PhD student, Mickey. Another PhD student, Devin, is having his birthday tomorrow, so I’m looking forward to some more cake then.

So in my profile I say that I work in the molecular genetics and microbiology lab under Dr. Aballay…I guess here I’ll give a more detailed description of what I do daily and hope that it’s at least moderately interesting to someone besides my grandma and family.

In the lab::My first day was like any other first day in a job, I guess-spent learning the techniques and trying not to break too many things or make an absolute fool of myself. The other people in the lab are great and hilarious, more on them later, I guess, as this post needs some kind of scientific validity to pacify the Howie Hughers.

Ok, so I spent some time learning how to “pick the worms,” which is a technique to transfer the worms with this really tiny needle-like pointer…after killing about 10 of them, I got the hang of it and can now do it quite impressively. If anyone reading this ever has to pick worms, I’m sorry, but it’s easier if you take some of the E.coli or whatever bacteria they’re plated onto and scoop the worms on it, using their sticky coating as a kind of glue for the worms. Katie, a grad student I’m working with, has me transferring the worms with a mutation of the same gene but in different alleles onto plates with a pathogenic bacteria to see if they all die in the same pattern (I would tell you the exciting finding associated with this, but I don’t want it to be lost and taken advantage of in cyberspace).

Actually, that was a lie, there’s no exciting finding, but I’m amusing myself, so sorry.

So it’s supposed to only take a few minutes or something to plate the worms because I guess the sweet experienced post-grads and grad students have been doing it daily for the past 6 years of their science-saturated lives, but it took me about, hmm, maybe 4 hours to transfer the different strains. This has to be done daily, and sometimes twice daily, so I’m really looking forward to some late nights with the worms. Anyways, in addition to this worm transfer, I’ve spent a lot of time making and pouring media for the bacteria and worms to grow on. Today, I made the “slow kill” formula for the worms, which kills the worms more quickly than the other type of media we use.

My main project, though, which I’m looking forward to, and (possibly) could coauthor a publication of, involved a microarray from the C. elegans looking at a correlation between the nervous and immune systems. I really don’t know how much of the work I’m doing on this is going to be published, so I’ll leave it alone for now! I’m really impressed if anyone has read this far, and thanks!