Everyone in my lab thinks that I'm a chemist by training. My degree is going to be in "biochemistry & molecular biology," but they all think I'm a chemist because, beyond a little bit of microbiology, I mostly do extractions, a little bit of synthesis, and a lot of staring at MS and NMR spectra. This is strange to me, because the synthesis I'm doing is basically a simple coupling reaction. It's a one-step synthesis to make substrates for my bacteria. Although purifying the shit on a column is always a bit of a pain (I fucking hate silica gel columns even though I do them basically every other day now), it's pretty much chemistry that anyone with a little bit of lab experience and someone to show them how to use the argon-line should be able to do. I mean I know how to set up organic reactions, work them up, purify them, and analyze them. I can follow a lit prep, but that doesn't seem so special to me. I'll be the first to admit that I'm more of a chemical biochemist than a biological biochemist, but I'm also pretty early in my training.

I guess in a sense I'm a bit of a weird biochemist in that I'm exceptionally interested in organic chemistry. I've done some organic synthesis research, and I guess I know about as much organic chemistry as any undergraduate interested in organic chemistry could be expected to know, but much much less than anyone who really calls themselves an organic chemist. I can make it through total synthesis, methodology, and mechanistic papers as long as I do a little bit of wikipedia-ing, I have a basic set of background knowledge of useful synthetic reactions, and I'm pretty decent at interpreting proton and carbon NMR spectra. But this doesn't mean anything. My undergraduate synthetic projects were trivial relative to what real synthetic chemists do. Christ, my synthetic target on my last project didn't even have any non-trivial stereocenters. And anyway, I also know how to do enzyme assays and Western blots and PCR and reverse-transcription and all that shit. It just doesn't happen to be what I'm doing for this particular project.

To me the borders of biology and chemistry are entirely trivial and artificial anyway, and being expected to regurgitate the derivation of the Boltzman distribution on an exam (which was required of me for my biochemistry degree) is far more "different" to me from protein biochemistry and molecular biology than being able to run a fucking silica gel column. I'm just not sure why it's so strange to everyone that I'm a biochemist by training who is also has decent organic chemistry lab hands. Yeah, okay, you transfer small volumes in molecular biology and large volumes in chemistry, but in a sense it's all the same. Transferring liquid, following preps, troubleshooting.

It's especially strange because it's a very interdisciplinary lab where everyone needs to do a little bit of everything. I mean, even if you're a biologist, you've probably seen an NMR spectra and even if you're a chemist you've probably done some bacterial culture in my current lab. Yeah, everyone is a specialist in something or another, but everyone also needs to be reasonably literate in fields outside of their own because the nature of the projects are so interdisciplinary and collaborative.

When it comes down to it, I think what's most telling about the fact that I'm not really a synthetic organic chemist is the fact that I consider synthesis to be one more lab tool to use investigate interesting questions rather than the intrinsic puzzle of making a complicated structure. To me, methods are methods. They are important, and it's important to learn how to do them well and how they work. But at this point in my training, there is always someone (a professor, PhD student, or post-doc) to teach me the methods. So whether the methods are chemical or biological makes no difference, really since I'm just at a stage where I'm learning how to think and troubleshoot anyway.

But damn, do people like their catagories. This is something I'm going to have to learn to deal with if I want to stay in interdisciplinary science.

Junior Qualifying Examinations

So at Reed you have to pass a junior qualifying examination before you can register for your senior thesis. As an interdisciplinary major, I had to take both biology and chemistry. Biology was an open note/book/internet exam basically based on my coursework. I wrote an essay about haploinsufficiency in Marfan's syndrome fibrillin-1 gene and the elastin gene in Williams' syndrome and another essay where I wrote about the growth hormone axis and how nutrition (both being malnourished and obese) affected this axis. I also did some quantitative questions like calculating Tms of DNA strands and making buffers. I should hear back about it soon, although I'm afraid I might have gotten a conditional pass from messing up calculating TRIS on that buffer question, eeek. It's not a big deal if that happens, I just need to fix what I got wrong.

The chemistry qual was an oral exam where I was given this paper and had to learn as much as I could about it in 48 hours. Then they asked me questions, specifically about the paper, but really probing my organic chemistry knowledge as a whole. I had two options, an organic option and a biochem option, and I decided to go with the organic option. The biochem option was interesting too, it was on nicotine binding in the brain and how it is different from its inhibitory effects on your muscles (if it did hit the receptor in your muscles you'd be dead). But ultimately I felt more comfortable with the sort of questions they would ask in an organic qual, especially since my advanced synthetic final last semester was in the exact same format. Luckily, my organic paper was a synthetic paper and not a mechanistic paper--because for some reason the long lists of data tables with numbered compounds in mechanistic organic papers followed by colums of de's and ee's result in my eyes glazing over a lot of the time.

I think it overall went well. A lot of the questions were definitional--what is the kinetic enolate vs. thermodynamic enolate, what is an allene functional group, what is "latent stereochemistry". The main places I got tripped up on were mechanisms. I had this divinyl oxy-anion cyclopropyl Cope rearrangement mediated by a Brook rearrangement, and I started off drawing the reaction in a different perspective than I had in my notes which kind of threw me. The paper went to great pains to discuss why this enolate attacked from the more sterically hindered side (because it was reversible and resulted in the more stable transition state for the subsequent Cope) but to demonstrate it only showed what didn't happen due to steric repulsions--so there was no picture to look at what did happen. Which I knew, but it's just sort of difficult to pull all of that together visually on the board with weird bicyclic systems and the boat transition state and so forth. It went ok, I just had a moment where I was like "wait Connie..."

The other place I got tripped up due to nerves was the mechanism for oxidizing an enolate with a Davis oxaziridine. Again, it was ultimately ok, just a little bit of nerves. Also drawing 7 and 8 membered rings on the whiteboard was tricky!

The spectroscopy portion was easy since they had already assigned the peaks in my paper and my profs didn't ask me anything too difficult. Just like, why methyl groups attached to silyl groups were more upfield than methyls attached to carbons and whether this one set of diastereotopic methylnes were chemically equivalent or not, and why they had different J-values coupling to a neighboring proton that sort of thing.

It also struck me last night that wow, college has crammed a lot of chemistry into my head in the past 2 years.

So I should be hearing back about whether I passed, conditionally passed, or failed those shortly.

Trials and Tribulations of undergrad projects

So at Reed we do independent projects in biology classes. They're usually kind of fun, and it's kind of a unique part of the curriculum here. However they get annoying for the following reasons:

1) we have 6 weeks to do them, so we are discouraged from doing time consuming or complicated techniques (because you just can't)
2) We aren't allowed to do something expensive. If we need to buy something expensive (like, say, a $300 antibody), we better damn have a reason for purchasing it, like someone else in the department will use it later. It can't just be your pet antibody for your pet protein. We used to have the resources to write a grant for expensive materials for independent projects, but one of our big teaching grants has run its course, so the department is tighter on cash than it has been for a while.

This makes sense; we're a small liberal arts school with limited resources. Our science program is solid, but as a small liberal arts school we don't have a lot of fancy schmancy equipment nor do we have a large budget. The opportunity to do independent projects is great for gaining lab experience, and a great opportunity, but at the same time, more of the money is spent on the faculty's research and people's undergrad theses. As it should be.

Depending on the class there is more or less guidance. Animal Physiology, Bio 381, happens to be a junior/senior class. Thus, there is little guidance at all and we are encouraged to think like scientists and figure out our own problems based on issues we are interested in. Good, great, I'm liking that. But it's hard because...shit son, it's hard enough to find an experiment that hasn't been done before, period. Add that on to a no more than 30 dollar budget and a 4-6 week time scale, and you have really slim pickings.

So we searched and searched and found this interesting pro-apoptotic signalling molecule, ceramide. It's kind of neat because it's a structural membrane lipid as well as being a pro-apoptotic signalling molecule, which is unusual. An experiment in the past showed elevated amounts of ceramide in the prescence of ionizing radiation (well in this case gamma radiation), in similar fashion to the way it was elevated in the prescence of TNF alpha (tumor necrosis factor) and chemotherapeutic drugs. Well, Reed has a nuclear reactor, kind of one of those odd little anomolous things--I'm actually a licensed operator, it's nearly completely run by undergrads--so it seemed like a good resource to take advantage of. Why not repeat the experiment looking for ceramide levels, but look for it with a greater variety of types of ionizing radiation (neutrons, gammas, and betas) in the reactor? Well cool.

Except then the issue of how to quantify the ceramide comes up. There's a kit that uses a radioactive phosphorous and a kinase, but that kit is expensive. No dice. Using a HPLC is probably the easiest method, but to my knowledge, both the HPLC in Arch's phyisology lab and the HPLC in the organic/biochem reasearch labs are not up and running. Okay. Oh here's a method, you can 1) extract the lipids out of the cells 2) run some silica gel chromotography (we found an elution plan in the lit) 3) functionalize it with benzyl chloride 4) do absorbance studies. Oh wait, forget that silica gel chromotography is a pain in the butt, that would take a shit ton of cells, we'd need to check to see if we actually purified it with the GC-MS/NMR, and...well....that's not exactly happening in 6 weeks on top of coursework. Okay, nix that.

Well, it turns out that a molecule in that pathway, spingomyelin (well, it's actually made from spingosine, but it's in the same pathway) turns into ceramide. So it would just be easier to take a look at upregulation or downregulation of the spingomylenase. We found a paper (which I don't have on hand and am too lazy to look up) about how TNF alpha and chemotherapeutic drugs lead to an upregulation of singomylenase, just as they lead to elevated levels of ceramide. BUT, no experiment was done with ionizing radiation. So, bingo. Primers are cheap. These are even published, so we don't have to worry about our primers not working (even if primer 3 input has done me well in life for designing primers for arbitrary genes, it's always nice to be assured that they have worked in the past).

Okay, problem. We asked if we could culture some fibroblasts to do the experiment. And got a "no, it's too complicated to do in 6 weeks". But I mean, if we take arbitrary dead tissue, that's confounding the experiment. Ischemia causes apoptosis and we're studying apoptosis! Not cool. I mean I see what he's saying, but damn. He suggested looking into using, say, sheep's blood. That shit is cheap. He also said using qPCR was unnecessary. Well, everyone uses qPCR now. Band brightness is a shitty way of determining upregulation vs. downregulation. I mean, I know that the qPCR machine is in heavy use by people doing their theses and the cytogreen dye is expensive, but it's really hard to make any sort of real interpretation of anything by band brightness and no one really does it anymore.

So here we are. We tried to come up with a cheap, fisable experiment and got shut down. All due to culturing a few fibroblasts. Maybe we can still do this experiment with sheep's blood? Or maybe the cell bio prof has a few extra cell lines floating around her incubators?

In a similar fashion, anyone who wants to do a Western Blot pretty much, well, can't unless they are studying a protein that other people in the department are studying.

This shit is hard.

What are men good for anyway?

"Males are kind of like pasteur pipettes from an evolutionary point of view. You need something to drop the reagent and when you're done, you can dispose of the receptacle."

This was my animal physiology professor's take on the fact that males are conceived in higher number than females, but also have a higher rate of post-natal death.