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From the LabLit short story series

João Ramalho-Santos 4 December 2014

But after all that she still had nothing. It was one of the worst kinds of nothings you could possibly have.

Pedro sat down warily.

The student waiting on the other side of the desk sported a stoic look, had waited patiently for her turn even though Pedro had tried unsuccessfully to calm all sense of urgency via email. As far as he was concerned it was not as if there was too much at stake, really. The work had been done, it was all over. A project, a set of experiments, another Masters Thesis, an awaiting defense that should go smoothly, a degree. Didn’t get much simpler than that. In fact this was Thesis number twenty-five for Pedro this year, and he was getting tired.

So tired that he was mistaken. In front of him, neatly bound and sporting all the right University logos, was only Thesis number Nineteen, its author eagerly waiting for his input. It just felt like that many more.

Four years ago Pedro had signed a contract with the Company to blindly test some of their new compounds of interest. The money for consumables, maintenance and human resources (which the University had turned into the aforementioned endless stream of Masters students) was good enough, especially since Pedro had become one of the many good Basic Science Pharmacologists, with an additional interest in Toxicology, who had fallen on bad grant times.

Outsourcing some work made fiscal sense for the Company; in fact, it had allowed them to shut down one of their Research and Development units, as Pedro had recently learned, much to his dismay. In essence experienced scientists were being replaced with the cheaper alternative sitting in front of him, really interesting projects dropped in favor of the mere testing of, Pedro assumed, logical molecules for different (lucrative) human predicaments. And assume was all Pedro could do – he had no idea what his lab was testing or why. Each compound arrived coded, with a long list of instructions on how it was supposed to be assayed in mice, rats, or different types of cell cultures. Which kind of assays, in what organs, how often, for how long. Anything and everything, from the activity of a single enzyme, to whole organ ex-vivo experiments, to behavioral tests.

The end result were Theses that looked cloned from one other, and consisted mainly of Materials and Methods (those tended to be really repetitive) and Results. Pedro couldn’t exactly ask the students to write elaborate Introductions, when they didn´t know what problem they were investigating; or pointed Discussions, if they weren’t sure what their results meant. His solace was that at least the technical training was solid, actually well above average for the Masters, as cutbacks had forced many of his colleagues to ease up on the lab work they required of their trainees.

Not that the Department was very supportive, quite the opposite. The other Professors felt that he was (in varying order of priority depending on the individual): making a mockery of the Sacred Scientific Endeavor; further demeaning the Masters degree in general, and their Masters in particular; stealing the best students and transforming them into mindless technicians; pandering to crass blind commercialism. This could have led to tense departmental meetings, were it not that Pedro promptly agreed with all those criticisms, and could throw in a few more of his own, upon request. These were not the experiments he wanted to do, merely the ones he could do, lame (literally) as they were. Plus, if it had been up to Pedro he’d have hired a few trained technicians to breeze through the contract. Transforming human resources into cumbersome Masters students had been a University requirement, to help with dwindling enrollment, and make the contract work for the institution on another level, besides overheads. So when the Masters coordinator complained that 70% of her students were being “bribed” into servitude thanks to the meager scholarships only Pedro was able to provide, he merely pointed to the Dean’s office.

Of course jealousy could also serve as a powerful ingredient in the Sacred Scientific Endeavor, but that harsh and unpleasant reality Pedro kept for himself, mostly because he was somewhat ashamed of being able to trigger such reactions. Yes, there was a practical side to this; yes it was useful stuff worth doing, yes somebody had to do it; and yes it might as well be Pedro since no one was paying him to explore any of his extraordinarily original ideas. But was it Science? Often it felt like filling trays at a cafeteria line, not cooking.

However, in the past few years Pedro had learned to appreciate that on the other side of his desk sat eager young students for whom this was a shinning first time in a real lab, learning and doing their very own project; not Thesis number Twenty-Five. While the experiments may have been blind, their enthusiasm and dedication certainly were not, and they gradually made Pedro refuse to forget his role as mentor, guiding pilgrims through the swamps and deserts of Western Blots, qPCRs, enzymatic assays, metabolite quantifications and behavior tests. Guiding them, hopefully, to bigger and brighter things. These were individual Theses, made by individuals, not numbers. Individuals he had actively recruited and who had gleefully signed up, worthy of his full attention. In front of him right now was not Thesis Twenty-Five (actually, Thesis Nineteen) but...

Oh god, Pedro floundered hopelessly, what was her name again?

It was on the cover of the document he was holding. Duh!

Suddenly whatever problems the student wanted to discuss seemed solvable. Which was interesting, since she really didn’t have any problems, explaining why the Thesis was already neatly bound, not in the usual unruly draft form version desperate students tended to lay on his desk to die. She merely had a Thesis full of nothing. All the work had been diligently done, on schedule, with graphs and tables galore to prove it, plus repeats and statistics to shame the makers of statistics software. With so many students Pedro made sure that presentations throughout the year were tight, and after three years his lab was organized with a chilling and precise efficiency. Other than getting the compound into solution, the usual optimizing of antibodies or odd outliers there had been no serious issues with this Thesis, or he would have detected them. And the student agreed, or the Thesis would not have been bound.

But after all that she still had nothing. It was one of the worst kinds of nothings you could possibly have. Because that was exactly what the compound she had been given to test did.

Absolutely nothing.

Throughout the Results section, tables and graphs comparing Control and Drug experiments showed undistinguishable numbers, overlapping lines, bars with the same height, Western blots that looked like twins, p values impossibly far from 0.05. Although the student had tried to alternate representations for the sake of variety, the data stubbornly said exactly what it said. She was not here to ask for suggestions, but to be reassured about her Defense. Evaluating these particular Theses had proven a challenge, smartly resolved by the Examination Committee: the student may not have a clue as to what drug X-15 was or what it was being tested for, but if it down-regulated the expression of HIF1- alpha, increased the activity of the manganese superoxide dismutase, or improved rat performance in the Morris water navigation task, informed speculations could certainly be made. Any respectable candidate should be able to easily dive into the literature, show knowledge, intellectual ability. After all, there was a reason the Company asked for certain tests.

That particular conundrum, Pedro was told in no uncertain terms, was what had kept the author of this Thesis awake the past few weeks. Not the data, certainly not lack of confidence in her skills. It was just that the compounds tested by her colleagues had all shown differences to the controls. Sometimes many differences, sometimes just a few; sometimes huge differences, sometimes barely above statistical significance. But to a certain extent they all knew what kind of questions were coming on exam day, and could prepare accordingly. This type of Science, the student calmly informed her supervisor, was about differences, not similarities. So, what could she possibly expect? How do you prepare yourself to defend nothing?

Pedro was simultaneously marveled by the maturity (only smart and committed students ever had these sorts of issues), and stumped. The obvious answer was that the student could be asked questions on how she had performed any of the tests, what they monitored, and what they were generally used for; but he understood that she would not be satisfied with such an obvious offering. He picked up the Thesis and thumbed through it; it was impossible to remember all experiments in all Theses: perhaps there were a few here that the Examination Committee would be more interested in.

As the Figures and Tables went by Pedro was reassured by their familiarity; except for the very last pieces of data he had already seen most of it, but simply never noticed that all the results with this particular compound never strayed from the control. There was, however, something different. Compared to the Theses with similar experiments, this one seemed to have more data points. For a split second Pedro rationalized the thought away as yet further proof of dedication, but he quickly realized this did not make sense. The student hadn’t repeated experiments more than everyone else, what she had done was to prolong her assays for longer time periods. Pedro went to the shelf where other Theses comfortably rested and pulled out a few which had tested for similar parameters. He quickly found what he was looking for. For example, all other students had only managed to perform four time points of most behavioral tests following compound administration, and noted that the rats were too frail to do any more. This student had six.

Pedro suddenly realized what he had missed with this project. Where, he asked, was the raw data?

Pedro always told his students to present data with the compounds in terms of percentage of what had been obtained with parallel control experiments. If the idea was to test new drugs, what the company really needed to know was what compounds increased or decreased whatever was being monitored, and this was the quickest way to visualize any such effects. But always using direct comparisons instead of actual values meant it was also easy to miss important, and above all unexpected, results.

When the student pulled up her data Pedro could immediately see what it showed. While it was true that the drug experiments were exactly the same as controls, both sets of numbers were very different from control rats of the same age, or cell cultures treated in a similar manner in other Theses. The normalization of always considering the control 100% was hiding the fact that in this Thesis, all rats performed better in behavioral tests; all cells had increased activity of antioxidant defenses and upregulated autophagy markers; probably other things Pedro was missing at first glance. In these experiments there were huge differences; things always seemed to be getting “better”. In all rats, all cells.

Lord, Pedro realized. This had nothing to do with the Company drug.

It was the vehicle.

The first crucial lesson Pedro imparted on his naive trainees was the notion that a control experiment did not consist of doing nothing; but rather implied injecting the rats, or adding to cell cultures, the exact same amount of whatever was used to dissolve the test drug. The so-called 'vehicle'. In this case it was the only explanation for changes in both controls and experiments. What was it? Not water, none of the Company compounds was soluble in water. Also not the two obvious solvent choices, ethanol or dimethylsulfoxide; while the “miraculous” properties of the former were of a recreational nature, the unspecific effects of DMSO were well know, and never good. Funnily enough, this information was not even in the Thesis. But the student lab book had the answer: a mixture of several organic solvents and natural oils in odd percentages that read like the recipe for Portuguese Stone Soup, a traditional dish that basically used any meat or vegetables available. How had the student arrived at this formulation? By trial and error, using her previous background in Pharmacy. Dissolving the compound had been such a chore it had put her behind on the experiments, and she thought that getting the damned thing in a workable form was a mere detail, challenging though it had been.

Pedro smiled, barely controlling his excitement. There were so many things to say, he worried about saying them exactly right, and in the right order.

First of all, the Company would get their due report, saying what the data eloquently proclaimed: that this particular compound had no effect whatsoever. Second, while they would continue to test whatever the Company sent their way, Pedro’s lab was about to embark on a new exciting parallel project, its first in many years. The compound may have been an unknown Company property, but the vehicle was no such thing. The composition gave some hints as to why it did what it seemed to do, but he wasn’t about to jinx anything by overanalyzing.

Finally, the student should stop worrying about her unimportant Masters Thesis defense, and focus instead on the PhD position Pedro was about to propose. He wasn’t yet sure where the money was going to come from, but one thing was certain. The path had been odd, but he was finally coming back to the Sacred Scientific Endeavor, on the shoulders of a humble vehicle. And he was storming in with eyes wide open.

Related Information

© 2014 by the author.

For Sandra Amaral, because differences are often the ones we don’t see

This story is somewhat a follow-up to the previous story Blind, but can be read separately.

Other articles by João Ramalho-Santos