The End (Not Really)

My last post! Maybe. I have mixed feelings about this; this will be the last project I do for high school. I didn't think the day would come. It's weird to think that I won't be returning to BASIS, after going there since fifth grade. I suppose, though, you can take the person out of BASIS, but you can't take the BASIS out of the person.

I visited Tufts for a night and day. Even though I didn't really like the weather (though I suppose I'll have to get used to it. Cold weather actually exists - who knew?), the campus was nice. There was a great view of Boston from the library roof, and the dorms were quite spacious. And the food was good (for college food, anyways. I'll enjoy my home-cooked meals while I have them). I, however, always felt like I was walking uphill.

My grandma and I next to the new Jumbo statue. I think it makes me look even shorter.

You may be wondering why my blog post was late, and it's partly because of the Jumbo Day I went to. I also didn't want to post until I had made a final decision for my college. And now, six days before the deadline, Id like to say that, even though I'm not exactly tall, I'm proud to be a Jumbo! Going to the admitted students day really helped me become confident with my choice.

And now, drum roll please, what you've all been waiting for: my results!

I tested about six drug substances, with varying success.

Here's my data for AK11, the first drug that I tested:

Yay for pretty graphs.

The bars show the proton flux of the liposomes. You want the "blanks," the liposomes without M2 proteins, to have a low proton flux, because there should be no way for the protons to move into the liposomes when valinomycin is added. If there was proton flux, the liposomes would be "leaky" and I'd have to redo my whole entire experiment. You want the liposomes with M2 proteins to have a high proton flux, showing proton movement when valinomycin is added. And finallu, you want the liposomes with M2 proteins and drugs to have a lowered proton flux, showing that the drug blocked the protons from entering the M2 channels.

Although my bars on the graph look decent, my data was not the best. For one, I only did two runs of each type of liposome instead of three. (I know, I was lazy, but I only had one pH electrode. Can you blame me?) Also, the standard deviation bars, the vertical lines coming off the bars, are pretty big, showing that my data wasn't as consistent as it could have been. Finally, the proton flux is extremely low - partly because we weren't using the right concentration of M2 proteins to lipids, so the effectiveness of the experiment was compromised. But you live and you learn, right? (Except in this case, the learning took about a month, so I used to wrong concentrations for the first month I was there.)

I had one drug, AK40, that I didn't like that much. I couldn't make the drug dissolve that well, so some of my data was unexpected.

The graph style changed a bit. It was - surprisingly - updated. And - even more surprisingly - it worked better.

The standard deviation bars were even bigger on this graph, especially on the liposomes with drug. Since the drug easily precipitated out of solution, I think that sometimes I didn't add any drug into my liposome solution. So sometimes I got results for the M2 with drug that looked awfully similar to the results for just the M2. And, for anyone who is wondering why I just didn't repeat the experiment again, I'm just going to mention that this was my second attempt at testing this drug. (Ugh, I wasted like a week on just this drug alone.) So we decided that I should just test other drugs, and that maybe we'd come back to this drug.

If you thought that all my data was sub-par, then I'm sorry to disappoint you. (Not really.) I saved the best for last, although this graph will look a little bit different.

I'll give you a hint for the difference: the graph was not, in fact, "updated" again.

This graph actually shows two drugs that I tested. The first drug, AK13, was good at blocking the WT M2 channel, but only moderate at blocking the mutated S31N M2 channel. When I was testing, my mentor asked me to test the drug he had just purified, NAG107. Because I had extra S31N liposomes, I ran the experiment - and the block was pretty remarkable. (For clarification, the first six bars are the same as the earlier graphs. The last bar only shows the S31N M2 and the NAG107; I didn't rerun all the blanks.)

So, even though I didn't plan on testing the NAG107 in the beginning, I'm glad I did. I think that because it shows so much promise at blocking the proton flux, it's going to be tested on the oocytes. With any luck, it'll move on to animal testing as well. (And if it doesn't, well.... We have a lot more ideas for drugs. Some of them involve copper complexes. The AK drugs I tested were all amantadine derivatives.)

Overall, I got to see a bit of everything - good results, moderate results, results I wish I didn't get.... As my SRP journey draws to a close, I'm excited for a new journey to begin: college! I'm glad that I had the wonderful opportunity of working in the lab. I learned more than I thought I would. (Buses are pretty popular outside of Arizona, it seems like. Especially in Boston.) I think the lab is one of the few places that I can ask "why?" multiple times and not get annoyed looks. (Curiosity may have killed the cat, but the cat had nine lives.) Because of this experience, I want to get into research in college, and maybe after that. Life, it seems, is filled with uncertainties, and I just have to live it one day at a time.(Unfortunately, my time machine broke, so I can't live life backwards; I'm stuck living it forwards like everyone else.)

I hope you've enjoyed reading my blog as much as I've enjoyed writing it! (Is it bad that I've been highly amused by making songs into my blog post titles?)
- Lauren

She Said I Think I'll Go To Boston

T-minus two weeks, and counting. I wish I could have a moment of certainty and make a decision about the college I'll go to. But I suppose that would be too much to ask for. Instead, next week I'm going to attend an admitted students day at Tufts, so hopefully I'll closer to my decision soon. (But, let's be honest, May 1st is getting closer and closer whether I like it or not.)

Although it's been nice sleeping in, I miss going into the lab and doing something with my time. (Maybe I'll have to get a job soon so I have something to do. Yay...) All in all, I've had too much free time to sit around and think about the future. This includes the near future as well - it turns out I'm in the first SRP presenting group. I'll talk about my project and my experience on May 9th any time between 10am and 11am. At least I have something else to worry about now, besides college.

On a different note, my uncle took Dora to the vet this week. Since we're not the best people to take care of a wild dove, we gave her to the vet, who promised that she'd find a rehabilitation place for for her. It's nice to know that Dora will be returned to the wild, but she'll be missed all the same (except, probably, by my aunt).

I'm going to save my results for my final blog post (next week's post) and for my SRP presentation, so stay tuned. What will I talk about in this post then, you might ask. I asked myself this too, and after much discussion with myself (I'm not crazy; the voices in my head told me so) we decided to explain the graphs more. Before you get too excited (and yes, I can sense your enthusiasm), you should know that this means I'm guaranteed to add pictures along with the words, instead of just blocks of text.

That moment when it's easier to edit a picture in PowerPoint than Paint....

The pH on the side of the graph isn't exactly correct; the conversation from the voltage in the pH electrodes to the pH needs to be altered. The actual pH is around 6 to 7. We haven't fixed the conversion because we're looking for the change in pH, not the pH itself.

The first time I saw a graph like this, I was extremely confused. (And I didn't even have the arrows to help me.) I mean, sure, it looks cool - but it was just a bunch of peaks and valleys to me. If you're anything like me, you'd be wondering what, exactly, is this means in English. (And if you're not, I'm afraid you still have to put up with my questions and answers.)

Valinomycin is a potassium ion-selective transporter; it only moves potassium ions. It looks a little like this:

It looks so simple.... I've yet to figure out how it actually works.

The first graph I showed you is a blank, meaning it has no M2 protein channel, and it also has no drug added. The liposomes (hopefully) have more potassium ions than the external solution, while the external solution (in theory) has more protons than the liposomes. This sets up a gradient; the potassium ions want to leave the liposomes, and the protons want to enter the liposomes. However, without a channel, the ions can't move.

This is where valinomycin comes in. (Literally.) It's first added to depolarize the liposomes (meaning that protons enter the liposomes). It allows the ions to move - but only if there is a M2 channel. Therefore, in the blanks, we expect there to be no valinomycin signal (and by "signal," I mean change in pH). And, if you look back to the graph, you'll see that this is true. You could argue that there is, in fact, a small change in pH, but I'd tell you to look at the second addition of valinomycin. Both changes in pH are the same. We can assume that the pH change is from something else, instead of the uptake of protons into the liposomes. And it is; the valinomycin is in an ethanol solvent, which is basic. Adding ethanol to this experiment would produce a similar pH change.

Conversely, we would expect a valinomycin signal in a liposome with M2 channels.

Yay, the valinomycin signal is different. We're done now, right?

I'm afraid that there's more to the graphs than the valinomycin signal. (There're three different colored arrows; you should have expected this.) If you compare the graphs, you'll notice that not only did the valinomycin signal increase, the CCCP signal decreased.

CCCP is a proton-selective transporter, and it acts like a M2 channel. It allows protons to come into the liposomes, resulting in a small change in the pH of the external solution. In the blanks, we expect a CCCP signal, because the liposomes are not depolarized by the valinomycin. The CCCP depolarizes the liposomes in the blanks, instead of the valinomycin. In the liposomes with M2 channels, there is no CCCP signal, because the liposomes are already depolarized by valinomycin. (Again, the CCCP "signal" is due to the ethanol, instead of proton flux.)

The previous graph has liposomes with M2 channels, and the next graph has liposomes with M2 channels and a drug. Kudos to the person who can guess what kind of "signals" we're looking for. (Hint: A good drug will block proton flux.) (Another hint: CCCP will depolarize the liposomes, whether or not there are M2 channels present.)

Am I the only one who would look at the graphs and this painstakingly detailed explanation, and ask what the HCl is for?

If you guessed that a good drug would reduce the valinomycin signal, you win! (If you didn't, at least you tried, right?) My whole project was based on the comparison of these graphs. I put my data into an Excel spreadsheet, and somehow they got an estimate for proton flux during the addition of valinomycin. Unfortunately, the numbers were pretty subjective, since we changed the numbers for the graphs ourselves by moving the red lines near the valinomycin signal. Fortunately, someone in the lab is working on a program that will automatically take the differences in the pH before and after the addition of valinomycin into account, so the numbers will be less subjective. Naturally, this program will only be used after I've completed my project. (I suppose that's the way the cookie crumbles.)

 

Thank you to anyone who even attempted to read this! I appreciate your efforts. (And if you just skipped to the end, and saw the word "cookie" - well, no cookies for you, then.) Feel free to ask me any questions.

Until next time!

- Lauren

It's the Eye of the Tiger, It's the Thrill of the Sight

A few weeks a go, whenever someone asked me where I wanted to go to college, I replied, "I don't know." I didn't want to make a decision until I knew everything - whether I was accepted, what the financial aid was, etc.. I wanted to make a fully informed, logical decision, so I didn't let myself get too attached to any one college. However, I'm not entirely sure this was a good idea (although, if I could redo everything, I'd do the same thing), because now if you asked me where I wanted to spend the next four years of my life, I'd still say, "I don't know." I've discovered that I'm a pretty indecisive person (at least I learned something from this process). I have 20 days to make a decision (but who's counting?), yet I hope I'll have a decision next week.

Even though I've now spent almost six weeks in Utah, I still managed to learn new life lessons this week. My aunt went away this week, and she left some meals for me to bake for dinner, so I learned how to not burn food (Yay me). Four meals down, one meal to go. I also learned that there's something more awkward than walking through a crowded college campus as a high school student: walking through an empty college campus as a high school student. It turns out that UVU, the campus I walk through to get back to the house, was on spring break this week, and I didn't realize it until I had walked halfway through the building. (It's amazing how self-conscious you can become even when there aren't any people around.) But the doors were open, so I don't think I was doing anything wrong. Finally, I learned that I really shouldn't be surprised when random things happen. For instance, I woke up on Tuesday to be greeted by this sight:

Say hello to my new pet dove, Dora.

My uncle had apparently found her on his morning walk. Her wing was injured, and she flew right in front of him. She even let my uncle carry her back to the house and put her in this cat carrier. We took her to the vet on Wednesday, and we're trying to heal her wing, which looks like it was scratched by a cat. We're not sure if she's domestic or wild, but I think, if her wing heals, we'll put her back outside. (And by "we," I mean my uncle, since I'm leaving on Sunday. Sadly. I've become to primary caretaker of her.) She seems fine besides her wing, but I don't think she's eating much. Hopefully she'll be okay.

This week was bittersweet, since it's my last week in the lab (but, (un)luckily for you, I still have a few more weeks of blog posts, so stay tuned). Fortunately, I finally learned the liposome-creation-and-testing process well enough so I can test three to four drugs per week, if not more. Unfortunately, it's a bit late to apply that knowledge, but better late than never, I suppose. Looking back at my first month here, my results just seem so slow.  Half of the results I've obtained have come from this week alone; the other half come from my previous five weeks here. On the bright side, I'm glad that none of my tests went awry yesterday. (I was told that apparently everything goes wrong when you're leaving soon. It's like the liposomes know you're leaving.... My gloves, at least, knew I was leaving; I used the last of my extra-small gloves today.)

This week, I  had - not one, not two - but three pH electrodes to work with. ( I never thought this day would come.) It was, needless to say, very exciting to finally test liposomes in sets of three, instead of one test at a time. (This is also probably why I was able to test so many more possible drugs. Maybe if they had fixed the pH electrodes sooner, and I would've tested more possible drugs.) I'm sure you're all wondering how pH electrodes can be "fixed," so here is our wonderfully high-tech solution (as usual) to the pH electrode noise/artifact problem.

Yes, that's tinfoil on the pH electrodes. Welcome to the 21st century.

Something with the metal-to-metal contact must have worked, because all three pH electrodes now have no noise, and very minimal artifacts. I'm not entirely sure this is a good permanent solution, though. The tinfoil seems to easily break and sever the metal contact.

On another note, I have found the one working new pieces of equipment that we have in the lab (besides, of course, the building itself). I'm not entirely sure how expensive it was, but I think it was a few grand, so I'm glad it actually works (though it took a few weeks to be calibrated).

And you thought your dishwasher was expensive.

I was going to talk about my most recent results, but I think this post is already a decent size (wow, the words go by fast). So, you're welcome; I won't confused you any more (for now...).

Until next time!
- Lauren

Why You Gotta Be a Fool? Don't You Know I'm Human Too

Wow, a lot has happened this week. Although I got rejected from my "reach" schools, I still have quite a few options for colleges. I'm having a tough time deciding what to do. I wish I could see into the future, but I suppose life is more fun when it's surprising.

Anyways, it's amazing how much can change in a few weeks - and how much stays the same. I'm glad to be back in the lab, actually doing something. (As an added bonus, it keeps my mind from over-stressing about college decisions.) But, early Monday morning, still groggy from getting up at 4am, I walked into the lab hallway to be greeted with a sign. You know it's a Monday when you see this:

How, exactly, you can close a hallway, I'll never know.

I didn't want to walk through that hallway, anyways. Thanks for asking.

I tried to make more liposomes on Monday, but in hindsight, I should've just waited. I spent at least an hour preparing my lipids and M2 proteins, and everything was going fine. I was even told that we got more internal buffer (it was running out before I left. I actually thought we weren't going to have any when I returned). Unfortunately, somebody didn't put the red tape back onto the internal buffer.

So I may or may not have put 1M NaOH into my lipids instead of internal buffer. Whoops. (I'm sorry, liposomes. I failed you.) How can such a small change of tape color make such a difference.... (I know, I know, I should've read the label on the bottle, but I wasn't expecting it to change.) At least this mistake shows that I am, indeed, human.

I was also told that we had three working pH meters now. Sadly, I ended up doing my tests - or at least trying to do them - on April Fool's day. I feel like the world wanted to prank me, so, naturally, all three pH meters weren't working. I mean, I know computers don't like me, but can't pH meters at least tolerate me? (Is that too much to ask for?) So Wednesday was spent trying to make them less noisy and less drifty, with moderate success.

On the bright side, some of the pranks I saw were highly amusing. Especially this one.

Notice how there are posters and even a lamp.

What are the handrails doing in an office, you might ask. This "office" is actually an elevator, and the teacher spent his day greeting unsuspecting students as if they had all come to his office hours. (I suppose that's one way to get a higher turn-up for office hours....)

On Thursday, only one pH meter was working (I really shouldn't be surprised), which meant it was a long day. One of the pH meters had almost no noise, and I was excited to use it. It wasn't meant to be, however. I discovered that if I leaned on the table, the two new pH meters would exhibit "artifacts," basically spikes in the pH readings that are not from pH changes in the solution. I thought I was onto something important, until my uncle informed me that this effect was common in pH meters.

Basically, my body was acting as magnetic field, which interfered with the voltage readings from the pH meters. To solve this, we have to ground ourselves, or the pH meters. (One solution involved me wearing a metal bracelet, which was strapped to some kind of "ground." I felt like I was chained to my experiment....) I don't think we've found a permanent solution yet, but I hope that we're on our way. It's odd that only the new pH meters are affected by my proximity. (Such is new technology, I suppose.)

Speaking of new technology, when I was gone, my lab decided to update the Windows 2000 computer, which is the computer that records all the pH changes. This lab must be allergic to new technology or something, because apparently the new program crashed, and brought down the program I used with it. Thankfully, we had another similar - and older - program that I now use.

Don't worry, though - this means that the Computer Says game just became more interesting! Only now, the correct information isn't even displayed on the computer; I had it written in my lab notebook somewhere.... The computer beeps one sometimes, usually before it thinks I'm supposed to do something, then kindly beeps three times ten seconds later. I think I'm becoming more adept at ignoring the beeps. (And this is coming from someone who rushes to get everything out of the fridge before the fridge beeps as a reminder that it's been left open.)

In other news, my results from testing AK40 again were inconclusive. Sometimes, it seemed like I had drug, and sometimes the drug was suspiciously absent. I suppose I should've seen this one coming; the drug was hard to get into solution. My mentor thinks that there was a very small amount of drug precipitate that wasn't in solution. So, only one thing to do from here: make more liposomes.

On the bright side, at least my graph from AK11 (the first drug I tested) still looks nice.

Until next time!
- Lauren