Archive for the 'Molecular Biology' Category

DNA provides a compact means of data storage. 1 gram of dehydrated DNA (about the mass of a paperclip, or the amount of sugar in a sugar packet) contains 10^21 bits of data. That’s over 100 billion gigabytes. A basic computer hard drive is about 250 GB and is about 3.5 inches wide, 5 inches long, and 1/2 of an inch thick. If You stacked 250 GB hard drives on on top of each other, you would need 400 million of them to store that much data, creating a stack 3,157 miles high (approximately the distance from Orlando, FL to Seattle, WA). All that data from 1 gram of DNA. Wow.

So I was reviewing my website statistics generated by a Python script I wrote when I noticed a peculiarity so bizarre that it made me questin the very purpose of my life. Okay maybe it wasn’t that bizarre, but it was interesting. The python script (which is automatically run every hour) downloads my latest access.log and saves it to its own folder. It then analyzes the data, creates some charts and graphs, and dumps out a bare-bones results file displaying some of the information I found useful. Of note is the number of times each page is hit.

This is where things get funny. Outperforming my home page by nearly double was indexOld.php (now indexOld22.php) – a simple webpage I tossed of for about a year before I put my big blog back online! Why were people still going to this page? Further investigation (from the referring sites section of my stats page) revealed a lot of hits from Google image-searches. I started looking at the actual requests and realized that many of these hits were people searching for the term Dwarf Gouramis “a type of freshwater aquarium fish) which was mentioned on that old webpage. The ironic part about it is what happens when you google image search for dwarf gouramis there is a picture of an extremely rare zebra pleco which is actually a link to my website! However the link APPEARS to be to wallpaperfishtalk.com because on my page I just linked to their image.

My conclusion: People are Google image-searching for ‘dwarf gouramis’, and an amazing picture of a zebra pleco is coming up which links to my site (due to the fact that months ago I talked about dwarf gouramis but posted a photo of a zebra pleco) and people (in their awe at this amazing fish) are clicking on it. So what did I do? I pulled a bait-and-switch! You bet I did. Now when you go to indexOld2.php it just forwards you to my current website – mua ha ha ha ha

PS: I’m appending to this entry at 2:17pm to note that I made a wonderful breakthrough in the lab today. Due to intellectual property protection blah blah and the fact that I don’t want anyone else to beat me to my research goal I will not describe what this is, I’ll just say that it took months of preparation and today – presto! It worked beautifully =oD

*As if threat of impending doom doom weren’t already haunting my nightmares* (possibly due to the intimidating threat of overlooked graduation requirements preventing my career from moving forward in a timely manner, but more likely due to terrifying possibility that my entire life will be spent stuck graduate school), yesterday I had two separate wrenches thrown into my academic gears. First, although I succeeded in confirming my legal rights with respect to being bound only to program policies that were already enacted prior to my admission, I came across a disturbing amendment to the thesis policies which claims that no student can propose, submit, or defend their thesis in the same semester. If this ruling applies to me, it will be devastating. I’ve spent a year and a half figuring out a way to do something no body has ever done before (looking at something no one has ever seen before, developing a method to quantitatively measure it that was never used before, and drawing conclusions about a pathological condition that was only previously theorized), and now that I’ve perfected my technique I want to use the subject matter for my thesis, propose the thesis, formally write and submit the thesis, and formally defend it (in rapid succession), thus satisfying my thesis requirements for graduation.
*So, I’m gathering preliminary data together and forming an experimental plan when, oh my, do I need to write more Python code?* Yeah, I know I feel that I look for excuses to find reasons to program, but the truth is that Python is so versatile and my working knowledge of it is thorough enough that I can’t imagine what I’d do without it! My department has an animal facility where they house literally thousands of mice of many different strains. These mice are housed in cages (maximum 5 mice per cage), and each cage contains only one (occationally two) types of mice with respect to sex, age, strain, and heredity. The project I’m working on will compare OVE26 (transgenic diabetic) mice with FVB (normal) controls. I also have a small collection of GFP (fluorescent) mice which I plan to use on a separate project.
*So, what’s the problem?* These mice are randomly distributed in giant racks with 6 stacked rows and 7 columns. Each rack has 46 cages, and racks are double-sided. Thankfully, all of the FVB, OVE26, and GFP mice are located on two large racks for a total of 184 cages. Unfortunately, interspersed are C57BL and other strains of mice I don’t plan to use. Another issue is that I only plan to use male mice in my experiment. A real pain is the fact that one rack is underground in the transgenic animal facility “headquarters” (for lack of a better word), and the other rack containing my mice is three stories up. Not only are these mice seemingly randomly distributed in location, but there appears to be a total lack of species/strain/heridity/sex specific inventory. The only inventory methods appear to be a total animal count. So when I went to my boss to ask how many diabetic mice we have and of what ages, he talked to another person who works in the laboratory and they concluded that they don’t really know. Do they have enough diabetic mice for my experiment? Are they old or young? Can we produce age-matched diabetic and control mice? Are there enough mice for my experiment that I don’t have to ruin the project of another laboratory worker planning on using the same mice? These are all questions that could not be answered, only speculated.

*With all of the uncertainties already afflicting my thesis process, I didn’t want animal confusion to be a problem.* From what I heard, about a dozen diabetic animals (in the age range I wanted most for my experiment) were recently killed. I felt that this process needed to be organized before I could properly come up with a plan, and of course, Python would be involved. I spent the morning downstairs manually inventorying mice. I decided that a location-based system would be the most useful. Each cage got its own line on a sheet of notebook paper (actually more like 8 sheets), and on each line I noted the cage location (in story/row/column coordinates), and information about the 1 or 2 groups of animals housed in the cage (count, sex, and date of birth for each group). After a couple hours sweating beneath my disposable hairnet and lab coat, I completed the downstairs inventory. To the side is a picture I took of myself half way through the monotonous process. (I think blood was about to shoot out of my eyes) Later I ate lunch, then did the upstairs. After I finished, I went home and manually entered this data into the computer (open office spreadsheet -> XLS), so it looked like this:

*Then I saved this data as a CSV* (comma separated values) file (using either open office or microsoft excel). Note that this is done in such a way that it’s very easy to make modifications to the XLS file to reflect new cages, changing cage locations, or animal death. Anyway, once it was in a CSV format, I wrote a Python script to convert the CSV format to a custom easy-to-analyze format which basically boils down to one line per mouse (not one line per cage). This is the Python script:

f=open("data.csv")
raw=f.readlines()
f.close()
animals=[] # [cage,sex,type,dob,age]
cages=[]
def addAnimals(animal,cage):
        if len(animal[0])==0: return False
        if cage not in cages: cages.append(cage)
        count=int(animal[0])
        if animal[1] == 'm': sex="male"
        else: sex="female"
        strain=animal[2]
        dob=animal[3]
        age=int(round(float(animal[4])))
        animal = [cage,sex,strain,dob,age]
        for i in range(count):
                animals.append(animal)
for i in range(len(raw)):
        line = raw[i].replace('"','').replace('n',"")
        if "-" in line:
                line = line.split(",")
                addAnimals(line[3:8],line[14])
                addAnimals(line[8:13],line[14])
f=open("data.txt",'w')
f.write('['+str(animals)+"],["+str(cages)+']')
f.close()

*Running this script produced a file with content like this:*

[[['d-a1', 'male', 'fvb', '09/26/08', 3], ['d-a2', 'male', 'fvb', '08/12/08', 4], ['d-a2', 'male', 'fvb', '08/12/08', 4], ['d-a3', 'male', 'fvb', '12/24/07', 12], ['d-a3', 'male', 'fvb', '12/24/07', 12], ['d-a4', 'male', 'fvb', '11/16/08', 1], ['d-a4', 'male', 'ove26', '11/16/08', 1], ['d-a4', 'male', 'ove26', '11/16/08', 1], ['d-a4', 'male', 'ove26', '11/16/08', 1], ['d-a5', 'male', 'fvb', '10/26/08', 2], ['d-a5', 'male', 'fvb', '10/26/08', 2],...

*From there, I wrote an analysis python script.* Should I post it? [thinks about it] Sure why not. A warning though, the code is pretty rough. It works though =o)

import pylab

f=open('data.txt')
animals,cages=eval(f.read())
animals,cages=animals[0],cages[0]
f.close()
print "Processing data for %d animals in %d cages..." % (len(animals),len(cages))

##LIMITS######
sex="male" #'male' or 'female'
strain="ove26"
minage=3
maxage=9
##############

selected=[]

def passIt(animal): #['u-l7', 'male', 'ove26', '10/28/08', 2]
        global selected
        if not animal: #dscription
                title="Displaing "
                if sex: title = title+sex+" "
                if strain: title = title+strain+" "
                else: title = title+"all "
                title = title + "mice from "
                if minage: title = title+str(minage)+" months to "
                else: title = title+"birth to "
                if maxage: title = title+str(maxage)+" months."
                else: title = title+"death."
                return title
        if sex:
                if not animal[1]==sex: return False
        if strain:
                if not animal[2]==strain: return False
        if minage:
                if animal[4]maxage: return False
        selected.append(animal)
        return True

def histIt(strain="all",col='k',label=True,limitTest=False,lw=None):
        ages,xs,a=[],[],{}
        for animal in animals:
                if limitTest:
                        col='b'
                        if passIt(animal):
                                ages.append(animal[-1])
                                if not animal[-1] in a: a[animal[-1]]=0
                                a[animal[-1]]=a[animal[-1]]+1
                elif strain in animal or strain == "all":
                        ages.append(animal[-1])
                        if not animal[-1] in a: a[animal[-1]]=0
                        a[animal[-1]]=a[animal[-1]]+1
        for x in range(max(ages)+1):
                xs.append(x-.45)
                if not x in a: a[x]=0
        if not limitTest:
                pylab.title("Ages of %d (%s) Mice"%(len(ages),strain))
                pylab.xticks(range(1,max(ages)+1))
        if not label: col,lw='0.9',0
        else: pylab.title(passIt(None))
        pylab.bar(xs,a.values(),color=col,lw=lw)
        for x in range(max(ages)+1):
                if label:
                        if a[x]>0: pylab.text(x,a[x]+1,a[x],ha='center')

def showSelected():
        cages={}
        ids=[]
        m,f=0,0
        for animal in selected:
                if animal[1]=="female":f=f+1
                if animal[1]=="male":m=m+1
                if animal[0] not in cages:
                        cages[animal[0]]=0
                        ids.append(animal[0])
                cages[animal[0]]=cages[animal[0]]+1
        ids.sort()
        x=0
        disp="Total of %d mice (%dm/%df)nn"%(m+f,m,f)

        disp=disp+"Animal Cage Locations:n"
        for cage in ids:
                disp = disp+"%s(%d), "%(cage,cages[cage])
                x=x+1
                if x>7:
                        x=0
                        disp = disp + "n"
        return disp

def getLimits():
        global sex, strain, minage, maxage
        answer=raw_input("Sex [m,f]:")
        if answer=="": sex = None
        if answer=="m": sex = "male"
        if answer=="f": sex = "female"
        answer=raw_input("Strain [ove26,fvb,gfp]:")
        if answer=="": strain = None
        else: strain=answer
        answer=raw_input("Minimum Age [3]:")
        if answer=="": minage = None
        else: minage=int(answer)
        answer=raw_input("Maximum Age [9]:")
        if answer=="": maxage = None
        else: maxage=int(answer)
        print "nn"
        print passIt(None)
        raw_input("npress ENTER to start")
        return

getLimits()
fig = pylab.figure(figsize=(12,8))
histIt(label=False)
histIt(limitTest=True)
masterAxis = [0,pylab.axis()[1]+1,0,int(pylab.axis()[3]*1.15)]
pylab.axis(masterAxis)
pylab.figtext(.4,.85,showSelected(),va='top')
pylab.show()
#fname=raw_input("Enter a name for this image to save it or press ENTER to quit:")
#if len(fname)>1:
#       pylab.savefig(fname+".png")
#       print "nsaved as [%s]"%(fname+".png")
#       raw_input("npress ENTER to exit...")

*Running this code* asks some questions about what type of information I should display. if I have it display all male OVE26 animals (for example) the output looks like this (thanks to matplotlib):

*Here the gray bars are the total number of all animals, and the blue bars are the animals I searched for* (in this case, all male ove26 animals). Comparing FVB and OVE26 charts, I estimated that we had enough male 4-5 month old OVE26 and FVB animals to make the experiment work (about 12 of each group). Searching for male ove26 mice at least 4 months and no older than 5 months old produces this chart:

See how it lists the location of each of the cages and the number of animals I want from each *cage?* For example, “d-I2(1)” means that there is a cage downstairs, along row I, in column 2, which contains 1 male OVE26 mouse 4 or 5 months old. Awesome list generation! Thanks Python =oD

*But wait, couldn’t I have just gone down and looked for 4-5 month old mice?* Yes and no. Yes, it would be easy to find (and mark) these mice, but no because I would not have known to look for them. The goal of yesterday’s little python project was to be able to see at one time everything we have, so I could best determine the criteria of the animals I wanted. Before I made this program, I was planning on comparing mice between 5 and 7 months – something that appears would have been impossible based on the animals we currently have. I can also tell what other experiments I will be able to do in a few months, when some of the mid-age FVB mice will grow older. Additionally, this program is versatile and can be used again and again, for many different projects, with no modification to the code required. Yay Python!

In my program (the University of Central Florida’s Master of Science in Molecular Biology and Microbiology) there is a handbook distributed to each class during orientation. The handbook lists the requirements of the program. Presumably, when these requirements are met, one can graduate. It clearly states that you need 30 credit hours to graduate, of which 6 are thesis and 24 are non-thesis courses. Of the 24 hours of non-thesis coursework, there is a small list of required classes [core I (5hr), core II (5hr), Lab (4hr), Prac (2hr), Seminar I (1hr), and Seminar 2 (1hr)] totaling 18 hours. Therefore, since 24 hours are required, but required courses only total 18 hours, it’s assumed that one needs to take 6 hours of elective courses to make-up for this deficiency. I’m pulling my hair out today because my program advisor told me that I needed 10 hours of elective coursework to graduate – something that is not mentioned anywhere in the handbook. (To be accurate, there is a single passing mention of a single 3 hour elective course, but it’s to be taken to help reach the requirement of 24 non-thesis credit hours.) This change (requiring 10 hours of elective credit) was made after I began the program. I do not believe that the program has the right (ethically, or legally) to hold this change against me and prevent my graduation (preventing graduation means that even if I do get accepted into dental school, I could not attend, because acceptance is dependent upon completion of the program I’m in). I spent the last hour describing my plight to the members of the graduate office, in hopes that I can obtain legal documentation to support my case. This stuff is so frustrating. [sigh]

A couple days ago I finished writing my personal statement (4500 characters, ~1.5 pages single spaced) for the dental school application. After the blood shooting out of my eyes subsided, I was able to sit back and admire my work. There, in front of me, was a surprisingly eloquent description of my academic life from the perspective of why I think everything I have ever done makes me an incredible candidate for dental school. As far as the assignment goes, I’m content. I wrote it so well that I almost convinced myself that I wanted to go to dental school. Regardless, it’ll be submitted in a day or two once a few final things get processed. Once I’m accepted or rejected, I’ll have to remember to put a copy of it on here because it’s a real hoot. For now, here’s a snippet.

I know, right? Jeez. Moving on, I wanted to note something about my research. Things are progressing nicely, and I’m about to have my boss buy ~600 bucks worth of antibodies (a total volume of ~1100 microliters, or about 22 drops of a clear liquid). It’s crazy how expensive these things are. Yeah, I understand the concept behind the development of polyclonal antibody solutions – but I’ve never seen a detailed analysis of the costs involved along the way. When I spend $23 per drop of some chemical, where exactly does the money go? Anyway, I had a revelation. One of the most complicated aspects of my project is that the signal I’m trying to observe through the microscope is from a fluorophore that emits light around a λ of 498nm to 529nm, and that thick myocardial tissue is autofluorescent in this region. Yes, I might see a tad of labeled fibers, but it’s amidst a sea of background fluorescence! Today (since I needed to order some new secondary antibodies anyway) I decided to investigate exactly which regions of the spectrum were most affected by myocardial autofluorescence. I blasted some thick atrial tissue with the 405 UV diode and took a series of images with a 5nm-wide recording wavelength window shifted by 3nm each, then took the average intensity of each frame of the stack. The result (when graphed) was a pretty spectral representation of myocardial autofluorescence, which was incredibly string in the blue and green bands that I had been using all along. I’m going to order some far-red secondary antibodies, hoping that it’ll help.

After many more hours of writing code in python, I finally have some presentable data. Due to intellectual property reasons, I’m not going to include details about the method, units, or even samples I used. It’ll suffice to simply say that my method seems to be working well, and that the bottom line appears well outside of the upper band (and its respective standard deviation). ALL processing was completed ENTIRELY within Python. I used the python imaging library (PIL) to load data form the TIFF file into a HUGE array, and NumPY to assist with manging the array. Data was then graphed with MatPlotLib and placed directly into a PNG. Here’s an example!

Okay, it’s getting late and I have a project I have to prepare for tomorrow. I know my blog entries have become boring, dry, and overly scientific since I started writing again, but I have to admit that this is just the kind of person I’ve become. I don’t really write about relationships anymore because I’m kinda “set” in that department (with the whole marriage thing and all), and I don’t think it would be a very good idea to go about blabbing all of our personal life together anyway. Beside, these writings are an expression of my thoughts and (supposedly) not intended to be anything more. If my thoughts are boring, so are these writings. [sigh]

Last weekend I had a breakthrough with my research that may likely lead to a timely completion of my thesis and graduation. I know I’m projecting my wishes when I think this, but after the months of frustration I feel very excited about this breakthrough. What is this breakthrough I speak of? Sometime soon I’ll write a blog entry about it. None of my friends/family really understand what I do in the laboratory (with the exception of my wife I think) – perhaps a little blog entry would make it easier to comprehend. Anyway, I’m incredibly excited about it.

I’m also smugly satisfied by the amount of programming it took to accomplish this breakthrough! Working in a huge brick building with the giant words “Biomolecular Sciences” on the top of it, I wouldn’t be surprised if I’m the only one sporting a PC running Ubuntu Linux and writing software in python to process data with matplotlib ! I need to get going again, but I just thought I’d record the joy I had when this revelation transcended upon me ^_^

Sometimes it’s the smallest decisions that impact your life the most. “Come on Scott, just pick something”, I remember thinking to myself a year and a half ago as I browsed through UCF’s graduate catalogue website. “This one will work”, I told myself as I clicked the “Molecular Biology and Microbiology” link. I didn’t really care what I studied, and I’d only be fooling myself if I tried to act like I wanted to study anything. The fact is that the whole graduate school idea was a surprisingly haphazard academic crutch – something I had to rely on because my career was crippled when I received my rejection letter from dental school. It was never my intention to apply to the Molecular Biology and Microbiology program. Why did I? Because I didn’t know what it was.

When I perused the other options in the field, I was discouraged because I knew I wouldn’t be able to enjoy any of them. Marine biology, conservation biology, general biology, ecology – I know what these are, and they’re ridiculous. What am I going to do with a graduate degree in marine biology? Beside, there’s no way I could sit through any more classes listening to some professor tell me about why environmentalism is so much more important than constitutionalism. The Molecular Biology and Microbiology seemed interesting only because I’d only taken one microbiology class before, and had never taken a molecular class (I didn’t even know what molecular biology was!).

That careless mouse click will either become a horrible blunder or an incredible opportunity for my future but it’s still too early to tell. I don’t know where I’ll be able to go from here, but I hope it’s somewhere. I know one thing for sure – I have absolutely NO interest in molecular biology. In summary, molecular biology studies protein-level biological processes. A gene in DNA is transcribed into mRNA, then translated into a protein which goes off and does something incredibly boring in the cell. The monotony of studying the subject in any detail is brain-numbing.

While other students in the program are memorized by concepts similar to the one I described, I sit in class listening to rotating professors ramble in a manner reminiscent of incessant static on an empty radio station wondering if anyone would clap a few times if I stood up and screamed “I hate DNA!”. On second thought, quite a few of the students are heavily invested (intellectually) into the subject, and I’m sure they’d become quite hostile toward me. I’m not exaggerating that example either. Just google image search for molecular biology signaling mechanisms. This is an example result (the type that students like me are expected to memorize in large numbers). See how every little circle (representing a protein) has a random-seeming name? Try memorizing 100s of those little abbreviations (if you’re lucky enough to have to memorize proteins whose names actually stand for something) in bulk. I can just look this stuff up if I ever need to study it, why do I have to memorize this?

Don’t get me wrong – I admit and agree that the field can be important for medical research. It’s easy to convey how the study of molecular signaling pathways that influence disease is important. Yes, this field is a major component of medical research, especially cancer and neurodegenerative diseases. It’s just… [sigh] why does it have to be this boring. If this were my life – my future – perhaps I could learn to enjoy it, but it’s not. I don’t want this. I’d rather work at Mc. Donalds (Burger King, actually) than peruse a job studying molecular mechanisms of biological processes. Knowing this isn’t my future makes it that much harder to endure it. What is my future? When I was in high school and in the beginning of college, I was good at computers, electricity, engineering, programming, and logic-type stuff. I know that if I pursued this path, I would have excelled quickly, and gotten a job I enjoyed. Somewhere along the line I got screwed up thinking I had the grades to get into medical school. I have conversations with undergraduates at UCF in the cafeteria occasionally, and I’m sure I come across as mean when I tell them how hard it can be to get into and how it’s never a bad idea to have a backup plan. I wish someone firmly told me the same thing when I was in their position. My classmates, parents, friends, and even academic advisors were all like “yeah you can do it reach for the stars” blah blah.

A major component of my graduate work is thesis-based research. I’m incredibly thankful that the classroom-style component of the degree was finished after about a year. From here, the major component that stands between me and graduate is the completion of my thesis research. Basically, I have to discover something novel (and relevant) by designing an experiment, gathering results, drawing accurate conclusions, and presenting my work (in the form of a manuscript) for review by the scientific community (randomly selected reviewers from many universities). There are ~20 labs in the molecular biology and microbiology program I could have joined, and I was lucky enough to find the one that didn’t study either molecular biology or microbiology! I’m doing higher-level (pathology/physiology) studies investigating the effects of diabetes with the cardiovascular neural system. Yeah – the heart as brain cells on it, weird huh? Actually I should call them neurons, because a brain cell is a neuron in the brain, and I study the neurons on the heart.

Graduate school is like 3D video rendering. It’s hours and hours of work (like designing 3D models for a computer animation) followed by awkward periods of extreme boredom (waiting for the 3D animation to render). In actuality, my “work” is reading-type research study, confocal time, and immunohistochemistry (IHC) solution preparation. My “extreme boredom” comes from the short periods between experiments when I’m waiting for a batch of tissue to complete IHC. Yeah, I know I could always use my free time to start new experiments, but then when the original experiments begin to become more labor-intensive, the newly-started experiments will also require this labor-intensive work, and I’ll perform poorly on both of them (I try to maintain only about 4 simultaneous projects). Anyway, the reason I mentioned this is because I do have some free time (in blocks of 20 or 30 minutes a few times a day) and I would like to be able to write again. I’ve written ~1.4 million words over the years (about 7), but stopped abruptly 2 years into college (my undergraduate work was overwhelming).

I’m really sad that I stopped writing though – many amazing things happened over those two years that I wish I documented. I moved to another state, studied biology (which is INSANITY to most of the people who knew me prior to the move, who would have put big money that I would have continued a live of computers and engineering), met a cute girl, and even got married. Is this time lost? I wrote in a pen-type journal, not sporadically and in no where near the detail of this web log –the greatest accomplishment/creation/manuscript of my entire life (thus far).

Although I would desire to write regularly, I have so many other obligations. Coursework, thesis work, marriage, and planning my future are all hovering around me at the same time. Whenever I take time out of my schedule to work on one of these, it’s at the detriment of all the rest. I feel like a small piece of saran wrap trying to cover a wet bowl – It’s being stretched tightly, to the point where it seems like it might break, but at the same time the edges aren’t sticking very well, and there’s a good chance the whole thing will have to be wadded up and thrown away so the whole process can start over.