step 2: check!

completing a PhD is kind of like completely an epic backpack - a Pacific Crest or Appalachian Trail backpack. it doesn't proceed as one would expect and years can be spent struggling without achieving any appreciable progress. there are heady moments when one finishes writing a grant or gets results back from the statistician, but more often days are filled with abusive reviewer comments*, incomprehensible experimental technique failures, and long periods of slow slogging.

to keep from getting lost in the hugeness of it all, i made a 'To Do' list when i started the PhD. this list broke down everything for every project in play and planned and i update it regularly. it is this list that has kept me going, because a PhD is huge, but 'Finish literature search' and 'Follow-up with statistician' are manageable. recognizing this, i have taken the same approach to the Defense. step 1 was getting it to my boss. step 2 is getting it to my committee. step 3 - prepare slides for my talk, study and plan follow-up experiments so i can field potential questions best. step 4 - defend. step 5 - either celebrate or fall in front of a truck (or perhaps one too many margs to temporarily drown my sorrows). the steps definitely help, especially given my new friends Insomnia and Probable Gastric Ulcer.

yesterday morning i turned my dissertation in to my committee. to celebrate with me, Mom sent me these lovely flowers. they really brighten up the place and my spirits. i started my slides yesterday, but didn't push myself too hard. i was pretty fried and figured it would be better to recuperate than work inefficiently. the weather was nice enough that i was also able to fit in a 7 mile bike ride in doing errands. today i have an appointment to recheck my 'niece' Zane and then home to work on slides and future research 'directions'.

Rainbow trouts
Originally uploaded by jacquichris.

last night i did finish the fish socks. they are Knitty Pomatomus pattern using Marks and Kattens Clown on US 1s. i think they turned out very nicely and take pleasure in thinking of them as 'Rainbow trout' - even though no one else seems to appreciate the almost humor. cast on the yarn for a sock swap sock. pretty excited about it, and i think i ended up with just the right yarn.

have to get back to work now... sigh.

*anonymity can release otherwise normal people from social constraints, allowing them to say things both undeserved and beyond the pale.

speed socks

Cowgirl slipper socks
Originally uploaded by jacquichris.

Monday night i made the other cowgirl sock while puzzling through a problem. they are in fact quite yummy. they are the Cowgirl slipper socks from Interweave Winter 2006 edition knit in Ry Classic Silk Wool DK in Brownstone. in the non-digital world, they really are the color of New England brownstones. keep your fingers crossed that they wash well.

also, sorry about the missing info on the country socks! i forgot to transfer it over from the photo file! those socks were made with adapted use of the Country Socks pattern from Nancy Bush's Folk Socks. i knit them using Ecco Extrafine merino in #201.

been working on the fish socks. once you get your head around the pattern, they are actually quite fun to knit. a little hard on the arms maybe, but that probably is more that i am obsessively over-knitting. eventually i will sleep again and maybe not look like the undead snowpeople on Kylie's Christmas stocking, but right now i knit.

got the first comments from my boss re the dissertation. not too bad. only two things that i will need to sort out. won't get the comments on the three remaining chapters until Friday (at the earliest). it is due to the committee Tuesday. things may be tight...

also got the galleys for a scientific article that has been in press for almost a year and is a chapter in the dissertation. one thing particularly entertaining about peer-reviewed publications is that the journal takes 2-3 months to get back to you about anything (at best) but you have to respond within 48 hours when it is your turn. [grouse grouse grouse.]

...

you are right. i tend to respond at that speed anyway. hate having things on the To Do list. but still, it is the principle of the matter.

ambiguous? maybe not.

so, my boss has started reading my dissertation. i had told him i considered the protein results huge but i would not share them in advance. i, of course, have since worried that i overstated things and he would be disappointed in the results.

thus far today, i have received three emails from him. the first:

HOLY SHIT....you are going to be famous! WOW!

later dudettee....perseverance.....pays......wahoo

then three minutes later:

Will you be able to withstand the impulse to say I TOLD YOU SO...to Dr. AA?

and finally two minutes after that:

I am going to ask the person that asked me to speak to the nephrology group to let you and I do it together......I will give the lead in on the work that preceeded the protein stuff and then introduce you to DROP THE BOMB!

the question is, do you think i overstated things?

[insert picture of me smiling]

Step 1: Check

last night i got the first complete draft of my dissertation in to my boss. now i study and wait for his feedback. then frenetic revisions and dissemination to the committee March 20th.

for a variety of reasons, it is a relief to have it in...

chasing the high

yesterday, something wonderful happened. not quite as wonderful as falling in love but close... i got the results back from my protein isolation.

things that are wonderful about my results:

• even though i did not manage to get all three bands, all three proteins were still present in sufficient quantity in the sample to be sequenced [which really says alot about the specificity of Western blots if you think about it].


• the proteins identified thus far appear to make sense, not that i want to jinx anything here.


• antibodies against one of the proteins identified have been associated with multiple immune-mediated diseases in humans, including nephritis. this supports the protein's identity and provides an independent degree of support for my hypothesis that these antibodies are associated with immune-mediated disease in cats.

overall, the results appear to be everything a girl could possibly want. they validate that my research hasn't been total bunk (which i was starting to secretly wonder) and indicate it may in fact be pivotal work. the kind of work that changes things. again, not that i want to jinx anything.

as you can imagine, i felt incredible yesterday. by ten last night though, the high had mostly dissipated. i am recapturing it a bit this morning as i read the human literature on my proteins, but it certainly isn't the same.

i find myself thinking, 'A year of struggle to purify the proteins and the high can't last a bit longer? Sheesh.'

that clanging from the ocean floor, its me

today in my committee meeting, we set the date for my PhD defense.

as soon as i left the meeting, i started to feel nauseous.

one would think this might have something to do with the two sets of letters... one of my committee members will be attending by teleconference. because the Final Examination Results form has to be turned in basically within a day, i have been instructed to send two sets of forms to Marty. he will then sign one set in the pass column and one set in the fail column and return them for use after the defense has been completed.

Tim, one of my other committee members, said we can celebrate if i pass by burning all the 'Fail' forms. i said, 'Sure. And then we can go scout around in the hallway for my stomach lining.'

realistically, i don't think this is why i feel the walls closing in on me. it is the combination of a ticking clock with outstanding lab results. the committee agreed on a Plan B dissertation structure in case the protein isolation is unrewarding, which is nice. it doesn't change my inability to write up the results and overarching conclusions until i know *something*. [did i mention that the machine used to sequence the protein is currently on the fritz?]

all in all, it is good to have a plan, good to have a framework of expectations...

still, i feel like a naval officer in a top secret nuclear submarine stranded on the ocean floor with 96 hours of oxygen and no one knowing that i am even missing.

[so that you can play along at home, i have installed a countdown timer in the right sidebar.]

a break in my lab luck?

as some of you may know, i have basically spent the last year trying to purify 1-3 proteins so they can be identified. this is the last lingering torment of my PhD. to date, i have tried 3 completely different methodologies and something like 20 combinations of reagents, incubation conditions and antibody preps each. early in the fall i got one methodology to yield one of the bands, but that particular technique yielded a huge overweighting of immunoglobulin. this became my back-up, my Plan B.

when all other options were exhausted, i conceded defeat and decided to harvest the one band from Plan B. my world was completely rocked when i discovered that the protein was not present in great enough density to visualize using the less sensitive stain necessary for protein isolation. efforts to concentrate the solution further actually caused the band to disappear.

these results left me teetering in shock, wondering whether perhaps i am not PhD material.

with no other ideas and nowhere to turn, i decided to call the woman to whom i would be submitting a successful sample to see if perhaps she had any ideas. Jessica is fabulous and, after validating that this is hard to do and that many people don't succeed, came up with several great ideas. since one involved learning a fourth methodology entirely and one might compromise the sample quality, i decided to start with the easy option. i reduced my antibody concentration to 3% of the original recommendations. (note: this was not a scientific level of decrease but instead reflected the most i could decrease the antibody concentration without having to make a separate stock dilution. basically, i was disheartened and lazy.]

A protein road map
Originally uploaded by jacquichris.

this magically led to a 1:1 ratio of antibody to protein, which is ideal but makes no sense given the initial starting concentrations and initial band densities. after magic autosubstraction using Photoshop, we are left with this clear illustration of three bands in the right lane. the top is a bit of a mystery band, the middle is the antibody band and the bottom band is my proteinaceous holy grail.

of course the concentration of protein in my sample was far too minute to show up using the cursed (pronounced cur-sed in the style of movie villains) Coomassie stain.

so i spent three days running the column over and over and over and over and over and over and over and over and over again...

...on the same lysate sample since i knew there was plenty of protein in the sample, just a persnickety recalcitrance on the part of the antibody to bind it. yesterday afternoon, knowing i was headed to a committee meeting today and uncertain of how hard to campaign for a failed experiment chapter in my dissertation, i decided to run what i had. before i started, i instructed Jen (my patient, supportive and awesome labmate/friend) to remind me i expected it not to work when it ended up failing. by the time Jen left for the night, i had 20 minutes destaining to go and it was a pretty obvious failure, so she consoled me and i took it well.

twenty minutes later, this is what i slid out onto the glass in preparation to through it in the trash.

A break in my lab luck
Originally uploaded by jacquichris.

and there it was. my sweet, sexy, playful, perfect friend - the ~40 kD band.

you are looking at the blob of slimy goo and thinking, 'She is truly unhinged. There is nothing there.'

but it was obvious when held at an angle with the light shining through it. i dragged my advisor down to witness the glory of the band before i cut it out to save for Jessica. unfortunately, to avoid potential keratin contamination (read failed experiment), i could not risk taking it to the image capture system for a photo. so i did the best i could with my camera through the plexiglass hood and without a mount.

if you squint your eyes up till you feel a headache coming on and use the membrane at the top of the post as a guide, you can almost believe what you are seeing on this photo, annotated for your viewing pleasure...

Annotated: A break in my lab luck
Originally uploaded by jacquichris.

i could cry. i could laugh. i could laugh until i cry.

Jen and i are going for margaritas at the Rio on Saturday to to celebrate. you are welcome to join us.