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Last posting – a forward-thinking retrospective
Dear readers,
It is both happily and sadly that I inform you that this will be my last posting for the makingbones blog. This is because I have now completed my DPhil (happy) but will now be moving on to other stages of my life (sad, but happy). When I first set out to do my DPhil I had no idea that it would be the emotional and intellectual roller coaster that it has been. Here are some of the ideas I had going into the process, compared to my actual experiences.
1) I thought I would be done in 3.5 years. It took 5. Research from 3.5 years of the total featured only in the appendices of the thesis.
2) I thought my experiments would look a bit like this:
Instead, they looked like this (actual flow chart from a poster I presented at a conference):
3) Up until grad school, my academic achievements were a source of happiness and confidence. Scientific research as a career (starting in grad school) often has a different effect. I’ve learnt to not attach my confidence to the results of experiments – after all, if we knew the answers it wouldn’t be called “research”.
4) Throughout your time in grad school you may work with one or many supervisors. Know yourself and what kind of help you need from these people – if they do not give you the support you need, look elsewhere, and sooner rather than later. But be kind to them.
Thanks to all of you who have read my blog over the past year! Best of luck with your decisions on grad school! And if you do decide to undertake this adventure, go in with an open mind, stay confident in yourself, and see where it takes you!!
Noteworthy: Are Genes Patentable?
From a scientist’s standpoint, the Supreme Court’s ruling this week in the case of the “Association for Molecular Pathology et al., v. Myriad Genetics, Inc., et al. is perversely logical. For the molecular biologists reading this, frustrated by the ambiguity in the mainstream media, the ruling can be summarized into 3 bullet points:
- DNA sequences are not patentable
- mRNA sequences are not patentable
- cDNA sequences ARE patentable
DNA sequences are not patentable because they exist in nature. The act of identifying the gene using common techniques does not enable to discoverer to patent either the location of the gene or its sequence so that others cannot utilize this information because she did not create the sequence or cause it to be in that location of the genome. This protection from patent coverage applies to all variations of the gene, including sequences with specific mutations linked to disease states.
mRNA (both as transcribed and once spliced) is not patentable because it is found in nature. If a scientist isolates mRNA, she did not create it, and therefore, she is not creating a new product.
It should be noticed, that the courts have ruled that if a scientist discovers a new method to isolate either DNA, mRNA, cDNA, or any similar scientific tool, then this new method would be patentable, even though the sequences discovered using the method might not be.
However, cDNA is created in a lab, even if naturally occurring enzymes and chemicals are used to create it. The theory behind this part of the ruling is that in the absence of human intervention, these specific sequences of DNA would not exist (the gene with the absence of introns). Thus the scientist has created a new a new patentable product. I’ll be discussing this ruling over a few posts, from different angles. In my next post I’ll discuss the impact that this ruling has for molecular biologists.
The official ruling can be found here.
Getting Back on The Bike of Research: the Basics of Science Research
This week I moved back to the United States to work at my lab at the NIH for two months before my thesis defense in July. Not only was I in Oxford for 6 months, I was also concentrating on writing my thesis, and didn’t go into the lab except to meet with supervisors. So now I’m back in the lab on a daily basis and doing science. That said, I ‘m currently in the stage of simply accumulating supplies before I can do actually do any experiments.
Originally, the plan had been to simply write up, have my viva, and graduate, without returning back to the NIH. However, my examiners were busy, and couldn’t schedule by defense until July. This gives me some time now to return and try to generate more data. If the data is good, this means that I will have more work to support both my thesis defense and also to help make more coherent papers for publication. Since I’m only going to be back for 2 months, this really limits the scale of experiments that I can do, and certainly rules out completing any new transplant experiments since they take a minimum of 8 weeks to run, with the preparation and analysis times extra. However, I’ve come up with an experiment plan that closely matches what I’ve already done, but to take it a step further in some cases, or to simply do a more sophisticated study to glean better results.
I’m not concerned about coming back into the lab having been away for 6 months. With the exception of one major technique, all others are methods that I’ve done before. And I find that for planning and executing experiments, the logic and the process never change. It’s in line with belief that once you learn to ride a bike, you never really forget. I’ve been seriously studying science for 10 years now, 6 of which have been spent doing genuine research (instead of planned coursework experiments), and over time on learns the basics of experimental logic:
1) How to choose an assay that will allow you to observe the specific factor you wish to
2) How to minimize distraction by other factors by designing more simplistic experiments
3) How to use proper controls to make sure that your experiment design is correct, even if the results you get are negligible or puzzling
4) How to manage resources and time
5) How to minimize human error (this involves knowing your personal limitations)
Over the next two months I’ll keep you informed about what I’m doing, the techniques and reasoning, and also my adventures into paper writing, along with updates on developments in tissue engineering and bone research.
Tips for Submitting a Thesis, A Retrospective: After the First Draft
Image from http://www.phdcomics.com
Phew! You’ve done it! You’ve just finished writing your first draft and sent it to all your supervisors. Now what? I was actually really surprised by just how long it took me between finishing my first draft, and submitting it to the examiners.
1) Keep feedback expectations realistic
After several years of working together with your supervisors, most graduate students have a good idea about each of their supervisor’s styles. Some supervisors can issue vague instructions, others give advice down to the smallest detail, and still others will never give any advice at all. Feedback will come in all forms (see my post on February 22) but try and work with what you’re given. Generally, your supervisor will want to help you produce the best thesis possible, because your performance reflects back on them.
When sending the supervisors draft material, set deadlines for when you want to review it with them. Just as in my last post I recommended setting deadlines for your writing with your supervisors, the same is now reversed for setting deadlines for your supervisors feedback during a meeting with you. Make sure the deadlines you give them are reasonable, but do set a meeting time and turn up, regardless of how much you hear back from them in the meantime. If they repeatedly miss deadlines, assess the situation, and either apply more pressure or look for feedback from a different source.
2) Keep turnaround time minimal
Your supervisors are going out of their way to make sure that not only did your experiments go well, but that your entire PhD does, including the thesis. An elegant PhD project with good results is only as good as the thesis your examiner reads. Because your supervisor is taking their time out to help with the thesis, it is important that they never have to edit the same thing twice. This is particularly true if you have several supervisors reading your thesis at the same time. It’s only fair to them, that you are not wasting their time, by having them correct something you’ve already corrected based on feedback from a different supervisor. Incorporate their changes, and distribute the latest draft as soon as possible. Obviously, rename the file so they know it’s a new draft.
3) The Tedious Details: A Checklist
- Check the margin requirements set by the university.
- Check the line spacing.
- Check the word count.
- Check the font and formatting (particularly important for Microsoft in order to generate an automatic Table of Contents).
- Set the thesis language and use spell-check. As a scientist I confess that I often ignore the Microsoft spell-check, because a lot of the time it doesn’t know scientific terms. Go through and use it anyway, because it will catch common words that you’ve misspelled. And teach it the scientific terms (“add to dictionary”) – but make sure that you teach it the correct way to spell the word, otherwise it will forevermore be misspelled in your thesis.
- Check your spacing. I spent 4 hours one very tedious afternoon going through all 200 pages of my thesis and checking there were two spaces between all the sentences.
- Clarify any sentence that starts with the word “this”.
- Find synonyms for every time you’ve used the word “important” – your thesis is not that important.
- Find synonyms for, or simply remove all references to something “interesting” – your thesis is not that interesting.
- Take a deep breath and count to ten.
4) Take care of the paperwork
Several weeks in advance of when you think you’re going to submit, check the paperwork requirements set by the University. You might find, as I found at Oxford, that some paperwork is due to be submitted 6 weeks prior to submitting the thesis and that this can delay the process if you haven’t submitted it with enough time. Also, check any paperwork that needs to be submitted at the same time as the thesis. Also, check for any further impediments to your thesis being accepted (as per my post on April 4th), and remove any obstacles.
5) Assess the printing, binding, and submitting situation
One of my flat-mates who recently submitted her thesis warned me that the process on the day of submission takes longer than you might think it should. That is certainly true. One of the things to decide is where to print the thesis. Crucially for this is the decision of how many, if any, pages should be in color and also the cost of printing vs. photocopying. Then, figure out how the thesis is going to get bound (again, check the university regulations), and if using a company to professionally bind the thesis, find out their opening hours. Most importantly, figure out the opening hours of the university office that is going to be accepting the bound thesis. Buy any envelopes etc. needed.
6) The final day
If you have not yet done so, reread the entirety of the thesis to check for any last-minute corrections. Make sure to do this while fully awake. Update the bibliography. Update the table of contents. Convert the document to a PDF. Print using the PDF. Partway through the printing, check the printed pages to ensure that your printer has not run out of ink. I printed about 50 pages that had to be reprinted from a different printer because the printer had run out of ink and the quality was unacceptable. Hand the thesis to the binder then wander around the city in purgatory until you can return to pick up your finished product. Take the thesis, with any necessary paperwork, to the appropriate university office and submit it.
7) Book a holiday
At some point during the time between writing the first draft and submitting the thesis, book a holiday to start a few days after you’ve submitted. By then you’ll have recovered from both the exhaustion of the last few days, and from the partying after submitting. Go somewhere different and relax. If you’re lucky, get engaged.
Tips for Submitting a Thesis, A Retrospective: Getting to the First Draft
Previously in my posts I’ve outlined my feelings on the general structure of writing a science thesis, and what the style and substance of each section should be. In this set of posts I’ll discuss my retrospective advice on the easiest way of writing a thesis.
1) Write a Literature Review your first year
Quite a large portion of my literature review/introduction section of my thesis was written in the first year of my PhD. In my second year this was easily cut, pasted, reformatted, tweaked, and with the addition of a few paragraphs made up the introduction section to my transfer report (standard mid-way thesis in England).
In the third and fourth years, I updated the original literature review to include the latest papers and to incorporate changes in the experiments that had since been made. For example, genetically modifying cells to overexpress growth factors had originally been planned, but later changed to simply adding growth factors to the system. This meant that while the overall gene transformation section was scrapped, the section on each individual growth factor could still be used.
This preparation throughout the course of the PhD not only means that less writing needs to be done at the end, but also gives context on where your experiments stand in the context of the research community. This can both give you ideas for new experiments and also lead to the abandonment of experiments, which have been shown to be futile (though usually the former).
2) Set aside time at the end of the PhD to write and not do any experiments
Having time to focus on organizing the thesis and simply writing, without simultaneously carrying out experiments is a luxury. Some supervisors will allow you to do this, and others will not. Try and set a deadline when experiments will stop and stick to it. I was lucky in that I had a plane ticket to leave the country, and go into a different environment, but I know many students who either feel pressure from supervisors to concurrently work on experiments, or who, of their own volition, keep striving to get “just one more” piece of data to put into their thesis.
Continuing to carry out experiments leads to two main problems. The first is that research takes up time and focus. This necessarily draws time and focus away from writing and disrupts writing momentum. I made all the results figures for my thesis within 5 days. On a separate hot streak, I sat down and wrote the wording for the results section in about 3 days. That simply cannot be done if you’re spending hours out of every day working in the lab. Even when you sit down to write, either anxiety about the potential outcomes of those experiments, or plans for the next day’s work are going to be running through your head.
The second main problem with continuing to work on experiments when writing up, is that it detracts from the solidity of the thesis itself. If one is constantly trying to figure out where the current experiment is going to fit into the thesis, or even worse, where future experiments are going to fit into the thesis, then the format of the thesis never solidifies. It is much more difficult to write when the contents are not set. Particularly when working on a piece of literature, such as a PhD thesis, where all the components need to fit together, it’s important to be able to step back and see the larger picture; difficult when experiments, and sometimes whole sections may be missing.
3) Set deadlines and find people to hold you to them
It’s often easy to set a deadline and then watch it slip past. It’s harder to set a deadline and watch it slip past, if that deadline involves a meeting with your supervisors to go over a specific goal you had set to accomplish by that deadline. If you disagree with that statement, find someone who’s not as nice as your supervisor, someone you care about not disappointing, and set a deadline with him.
When I moved back to Oxford, my lab offered to set me up with a desk in the office where I could write. I decided against having any space in the lab, because I wasn’t doing any experiments, because the lab is outside of the city center and would involve a long commute, and because I felt like I needed a different writing environment (see Tip 4). This meant that the only time I came into the department was every 2-3 weeks, on a date I’d set with my supervisors at the previous meeting, to discuss my progress.
If you’re working in an office, and people see you sitting at your desk 9-5, then they automatically assume that you’re being productive during that time. If however, you’re working out of the office for 2-3 weeks without anyone seeing you, then the pressure is on you to turn up with product at the end of that time. Otherwise, they’ll assume you were sitting around in your pajamas all day catching up on BBC television shows.
Find someone, set a goal, and a date to review your progress on that goal. Turn up for the meeting on that date, having met your goal, and receive their approval. Repeat.
4) Find an environment that stimulates your writing
I do not write well in my dorm room. I procrastinate very well in my dorm room. I will tidy. I will wash the dishes. I will cook. I will surf the web. I will reorganize my entire closet and take pictures of each item to organize my wardrobe using an app on my iPad (yes, I did do that).
When I lived in DC I had to figure out the environment I needed for the literature review. I found that if I was trying to read a lot of papers in one day, that I would get easily distracted, no matter where I was, if I could check my email, etc. on the internet. I figured out that what worked very well for me was going somewhere without internet, with a stack of printed papers, and a highlighter, and even with my computer to write notes on, but somewhere without internet access. The Smithsonian Institutions museums worked very well for this – particularly the National Gallery and the National Portrait Museum. Both even have a café where you can get lunch and coffee, but no Internet access.
When I started writing in Oxford, I first tried to work in the Radcliffe Camera, one of the library buildings at the university. It’s an absolutely gorgeous building, and full of studious scholars, but I quickly found that the atmosphere was too stifling. I ended up writing the vast majority of my thesis in the café at Blackwells Bookshop on Broad Street in Oxford. This café offered me the four things I found necessary to stimulate my writing: free wi-fi, plug sockets for my computer (so that I wasn’t limited by battery life), coffee, and ambient noise. I’ve found that I actually find background noise comforting, and surprisingly less distracting than silence. In the library I felt pressured to work, in the café I feel relaxed and able to work. And the coffee doesn’t hurt either – after all, it’s buy 10 get 1 free!
Image from http://www.phdcomics.com
Thesis Writing: A Hectic March
Image from http://www.phdcomics.com
I submitted my thesis on March 8th. Submitting the thesis actually much more stressful than I thought (discussed more in my next 2 posts: Tips for Submitting a Thesis). The final few weeks of thesis preparation were very stop and start as I waited for supervisors to get feedback to me and then rushed to get the newest version out, to ensure that they weren’t wasting their or my time by editing sections which had already been changed. Finally, however, I was able to print the thesis, get it bound, and officially submitted it to the university.
Throughout this process I had to file a lot of beaurocratic paperwork with the University including permission to receive dispensation for being in residence. Oxford requires that graduate students be “in residence” in Oxford for 6 trimesters of their DPhil. This generally does not pose a problem, because most students are here for the whole of their studies, but as I was traveling back and forth to the NIH in DC, this meant that I was only in residence for 5 full academic trimesters (they don’t count all the time spent working outside of term time). Additonally, I officially set who my examiners were to be, and officially requested that my viva (thesis defense) occur by the end of April (which I thought reasonable given that it was seven weeks after the thesis was to be submitted). However, it became clear that the examiners weren’t available within these time constraints, and neither were the back-up examiners, and therefore the thesis defense has now been set for July 8th – exactly four months after I submitted the thesis.
As an international student this posed a large problem because the question became one of what to do during the interim period. I discussed this with my supervisor at the NIH, and we came to the solution that I would stay and write papers/plan experiments in Oxford until the April (I’d already paid for the rent) and then move back to DC and work at the NIH for 2 months prior to returning to England for the viva. I’m really lucky to have a supervisor who’s willing to pay for the extra months that I didn’t think I would require to get everything taken care of.
Right after I submitted my thesis my boyfriend came into town for a few days and we took a short vacation to Paris, where he proposed. So now I’m aflutter with wedding planning as well as viva preparation and continued science writing. Currently I’m still living in college in Oxford. I’ve just finished the first draft of my first paper, the topic of which is the syngeneic murine model of ectopic bone formation.
Technique: Chemotaxis Revisited – the Boyden Chamber Assay
Chemotaxis, introduced in my post on January 26, is the process by which cells migrate along a gradient of growth factor. Researchers study chemotaxis for many different reasons. First, there’s the biological study of how these cells move. Then, there’s the study of why these cells move. In my post from September 28, I describe how random movement of cells in response to an even distribution of growth factor can be measured. However, the next step is to measure whether this response can be seen in a directional manner in response to a gradient.
The easiest method, perhaps, for studying chemotaxis is the Boyden Chamber assay. The concept of the Boyden Chamber assay is straightforward. Known as a transwell system, a smaller well is suspended over the main well of a tissue culture plate. However, the bottom of this insert is not solid plastic, put a permeable membrane. Cells are added to the top of this membrane, in solution, and their migration across the membrane to the other side in response to stimuli can be measured simply by counting the cells that have passed through. A great product for this are the FluoroblokTM inserts from BD Biosciences. Cells can be fluorescently labeled, and light from cells on the top side of the membrane won’t shine through to the bottom. The layout of a basic Boyden Chamber Assay is shown (blue dots are cells, green dots are stimuli):
The Boyden Chamber Assay may be simple, but requires a high level of finesse. The first main concern is the use of proper controls. Not only are negative and positive controls important, but also we need to measure the directionality of response to control for random movement. I.e., although, for example, growth factor might be added to the bottom well, and cells to the top, we need to show that the a gradient is actually maintained and that it is not simply a case of growth factor being evenly distributed on either side of the membrane (equilibrium) and cell movement increased in general.
Also, there are several steps in which human inaccuracy can lead to deviation between wells, ultimately resulting in lack of statistical significance because of too much variability. The assay is indeed incredibly sensitive to both the number of cells and the amount of stimulus, both of which tend to be very low, meaning that everything must be incredibly accurate. The first year of my PhD I conducted a gigantic Boyden Chamber assay, but got no significant results, just trends, because the error bars were too large.
The third part of the assay which requires delicacy is the counting of the cells. As I mentioned, the cells are fluorescently stained. However, in order to calculate fluorescence, because the desired cells are on the bottom of the membrane, in order to use spectrophotometry, you’d have to have a bottom plate reader. Our spectrophotometer could not be reprogrammed to do this (although in theory it had the ability). This meant that the membrane had to be cut off and mounted upright before being imaged. The cutting of the membrane is tricky, because it becomes easy to crease, which can lead to problems when trying to mount it flat (it’s very difficult to image a crinkled sample due to focus issues).
The protocol for Boyden Chamber Assays:
- Trypsin cells, centrifuge, resuspend in desired medium at desired density
- Make sure this cell suspension is evenly mixed
- Place cells in the top of the transwell
- Place desired medium in the bottom chamber
- Make sure the volume of the bottom corresponds to the top volume so no fluid pressure is exerted across the membrane (manufacturer’s recommendations)
- Incubate for the desired amount of time (good idea to perform a time-course with controls as a preliminary study)
- Gently aspirate medium on top and bottom, wash 3x with PBS
- Incubate 15 minutes in 4% PFA in PBS in order to fix the cells
- Wash 3x with PBS
- Incubate with 1:10000 DAPI in TBS with 0.1% Tween for 5 minutes at 37°C.
- Wash 3x with PBS
- Carefully cut out membranes and place, bottom-up on slides
- Mount with fluorescence-stabilizing hard-set mounting medium
- Pressing down to eliminate any air bubbles and make sure membrane is flat
- Image at 4x magnification
- Use ImageJ to increase brightness/contrast to max, and convert to binary for quantification
How would you like your feedback today?
I have 6 supervisors. So far, I have received draft edits from 3 of them:
- Supervisor 1: read the thesis Intro, then reread the whole thing at a later draft version
- Supervisor 2: has read everything but the conclusion, but can’t seem to stomach reading 5 more pages
- Supervisor 3: has read the whole thing through, and reread the entire second half
- Supervisor 4: read 2 pages
- Supervisors 5 and 6 have not read anything as far as I know
And my mother was the first person to proofread the entire thesis.
So far I have edits in a variety of media:
- Supervisor 1: prints out the drafts, writes on them with red pen, and gives them to his secretary, from whom I pick them up
- Supervisor 2: has sent me “track changes” documents
- Supervisor 3: prints out the drafts, marks them up, scans them, and puts PDFs in a shared folder
And my mother sent me track changes.
So far the comment highlights have included:
- “I’m sorry, it’s really difficult when experiments don’t work out. (my mother)”
- “This looks weird.”
- “This is almost boring.”
- “Surely you didn’t perfuse the whole mouse?!”
- And “too verbose”.
I’m currently on draft #6. I haven’t yet labeled any documents as “final” yet, but I do have several “final figures” folders. I love this comic by PhDComics, but it doesn’t quite show the enormity of having two different computers open, one with edited documents, and one with the original document, constantly scrolling through and tweaking the thesis.
Ten Hundred Words of Science Challenge
I was recently told about the amazing Ten Hundred Words of Science challenge. The challenge is really simple. Try to explain what your scientific project entails, using only the ten hundred (thousand) most commonly used words in the English language.
That sounds easy enough, until you try to use the text editor, and realize that out of the paragraph above there are many many words that would not be allowed: recently, challenge, scientific, project, entails, thousand, commonly, English, language… and SCIENCE. Science is not one of the top 1000 words in English!
This is my project description I ended up writing:
My college paper is about a fix for when a human breaks a leg or other body part. Many people in the world are working on such things. The best way to do this is to come up with a thing, that turns into part of the leg when it is put into the person, instead of trying to make a leg outside of the person and then put it in. To do this we need to focus on having good blood roads quickly, and then good leg can form. We also need to give our pretend leg everything it needs so that it can become real leg over time.
Thank goodness leg and blood was allowed! Neither bone, nor transplant, nor vessels, were allowed, which are very key words in my thesis. In the current thesis draft I mention the word “bone” over 500 times.Above, “college paper” translates into PhD thesis, leg = bone, and blood roads = blood vessels. Oh, and “thing” and “pretend leg” translate to “transplant”.
Are you up for the challenge? Scientific or not, I dare you to attempt to explain what you do in only ten hundred words! The tool can be found here:
And the website to view other people’s scientific projects is here:
makingbones 