Posted in Ph D posts

My roller coaster Ph. D project

I still remember sitting in my Ph. D supervisor’s office, way back in March 2016, being shown a figure depicting this peculiar Coxiella burnetii mutant.

Usually in a bacterial growth curve, you see something like this.

Bacterial growth, detachment and cell size control on polyethylene ...
Thanks, Google

There’s an initial lag (I like to think the bacteria are just revving their engines) followed by a logarithmic phase where the bacteria grow exponentially. This is then followed by the stationary phase, where the bacteria have essentially run out of nutrients, space, etc. and can no longer continue to multiply (they’ve hit ‘peak’ growth). Eventually, provided you don’t supplement them with more food or space, they will drop off and slowly die.

Anyway, this Coxiella mutant, missing a protein called CBU2072, couldn’t replicate inside host cells at all. The curve basically flat-lined after lag phase and didn’t go up significantly. The beauty of it was, when you gave the protein back to the bacteria (i.e. the mutant could once again make CBU2072), growth was restored to normal (wild type) levels, thus illustrating that the lack of growth was solely due to the absence of this protein.

On the left is normal (those big circles are hundreds of bacteria replicating inside the host cell), and on the right is the mutant. It just sits quietly on its own.

A protein so important that the bacteria can’t grow inside cells without it- sounds pretty funky and cool to me! is pretty much what I thought in that moment. I also got told that this project will be Metabolomics heavy, meaning I would get to learn how to study bacterial metabolism. It was a foreign concept (and associated technique/s), so I thought I should try and diversify my skill set and agreed to take on this project.

Four years of craziness followed.

This project has been with me since the beginning, and it’s been full of many ups and downs. We found out that the front portion of the protein contained a signal that was required for its function, and that the lack of growth was only seen during intracellular replication (i.e. growth inside the host cell). We found that you could ‘rescue’ growth of the mutant by infecting the same cell with a normal, ‘wild type’ strain, meaning whatever the protein was doing, it could allow replication to resume in strains that still couldn’t make protein themselves. I remember my brain just felt like it was melting all the time.

Usually when we find a novel protein, we run database searches to see if the protein is similar to other, perhaps more characterised proteins. Maybe it contains amino acid residues that are similar to a particular enzyme? Maybe it has a motif (i.e. barcode) that tells the protein where to go? Is it structurally similar to another protein? All of these things can be calculated computationally- so, I went ahead and checked all the databases, but, the rather unfortunate (but also cool) thing about Coxiella proteins is, most of them are highly unique, and are unlike proteins found in other organisms. It was just a huge, needle in a haystack situation.

When you’re at the centre of a project, it can feel like you’re at the centre of this crazy maelstrom of stress. It can affect your mindset, and ideas for experiments don’t come out naturally anymore. Especially at the beginning, I was just trying really hard to learn how to science, so things often felt really hard. Every failed experiment chipped away at your self-confidence, and every constructive criticism felt like a personal attack. Fortunately in my case, the vast majority of criticism was not personal, but I know that this isn’t always the case for others in my position.

So, what do you do when you just feel really drained, stressed, and genuinely depressed? Well, aside from learning how to dissociate your self-worth from your project, you just gotta keep crawling your way through. Try new approaches, consult experts, talk to your Ph. D committee and ask for advice… all that stuff that people tell you to do, that you kind of hand wave and dismiss at the time- turns out they help, little by little (and sometimes in leaps and bounds).

With this project, I tried so many different things. My Ph. D Thesis has more of these (inconclusive) experiments outlined, but eventually, they started to form a pattern.

It’s hard to describe everything without having a face to face discussion, but we discovered a couple years ago that when you don’t have CBU2072, even when you restored intracellular growth in the mutant, they still couldn’t pump out specialised bacterial proteins called ‘effectors’ through a bacterial syringe-like apparatus.

Pumping out effector proteins via the Dot/Icm type IV secretion system (T4SS) is absolutely essential for Coxiella to replicate inside cells

So if absence of CBU2072 meant effector proteins couldn’t get out, that suggests that there’s something wrong with the secretion system (T4SS in the diagram) itself… But unfortunately, by the time we bulked out the data with more experiments to try and support this claim, my Ph. D candidature was drawing to a close!

And I guess that’s the point of this post. Sometimes, the simplest conclusion (written essentially with a couple sentences) may take 3-4 years to become fairly solid, and even then, it might not answer your original question. I still have no idea what this protein really does- if I could just ask the bacteria, I would!

‘Excuse me, kind bug? Sorry to be a bother, but, WTF DOES THIS PROTEIN DO?!’

*shakes fist at sky angrily*

*Coxiella laughs and runs away into the distance, leaving me all sad*

There are plenty of other experiments that could be done to keep trying to answer that simple question, but it’s time for me to pass the baton on to somebody else. Maybe someone might figure it out one day, but at least I got to lay down the foundations for them to do so.

If you like closure, or a finality to things, science might not be a good fit. 99.99% of the time, experiments only churn out more questions. The story never really ends. It might curve around and take you on random tangents, but it’ll never come to a close.

So here’s to my last paper, and CBU2072, now named EirA (Essential for intracellular replication A). You’ve been a giant pain in the arse, but I’m glad I got to tell your story.

If anyone would like to read this paper, the link is here. Unfortunately this paper is not Open Access and will require purchasing to read the full text.

Posted in Careers, Ph D posts

How did I become #PhDLife? (part 4)

It’s been almost a year since my ‘How did I become #PhDLife? (part 3)‘ post, and given that I am now at that point where I’m just waiting around until my Thesis is accepted, I thought I’ll add a follow up post of what the latter part of my Ph. D was like.

I’ve written previously on the basic overview of a research-based Ph. D in a previous post, and another post on the basic overview of what is in a Ph. D thesis.

If you would like a more specific overview of what 2019 (i.e. fourth year of my Ph. D) looked like, I have another post from late December detailing all the major events.

Looking back, I can tell that I was much more excited and motivated by lab work at the beginning. I think it was a relatively gradual process, but over time I begun to resent the fact that you could put in so much time and effort into an experiment and not get anything useful back.

I’ve detailed this before, and anyone in science knows that sometimes, things just don’t work.

It could be user error- I’ve had my fair share of experiments that I could have done better if I’d just tweaked some things or asked the right questions earlier- but this is what experience and hindsight can bring you.

I know I had a lot of nudges from supervisor/s and committee to guide me in the right direction, but I found that sometimes, even when I thought I’d understood something, in reality I hadn’t grasped the whole concept. Eventually things clicked, but that took time and contemplation on my part. I wish I could make that process go faster, but sometimes I just couldn’t ‘get it’. If you don’t realise you haven’t understood something, you can’t even ask questions to clarify things, but sometimes that light bulb moment just couldn’t be rushed.

Issues with experiments could also just be the experimental procedure, or a combination of both user and procedure… sometimes you have no idea what went wrong, because so many things could go wrong at every step. Too many variables.

Some people are fine with this, because they can continue to persevere and stay motivated. Generally it’s fairly up and down, where you go from being motivated to mentally drained, sometimes within a couple of hours! But as I got over the half way point of my Ph. D, I realised that I was feeling more drained than motivated, and I started to tease apart what was still enjoyable and what wasn’t.

***

For me personally, I found that experiments themselves were still enjoyable. The mechanical action of pipetting something, or holding tubes in your non-dominant hand and opening the lids aseptically (i.e. without contaminating the contents)- that aspect of lab was still fun. I’ve always been dexterous, and I hope I always will be (well- until old age, I guess).

But… again, it was always when things went wrong that I’d feel really exhausted and sick of everything. E.g. something went wrong with the protocol so I have to start again (usually user error after an exhaustive day)… the experiment didn’t work (results aren’t coming up/are inconclusive) so I have to try again, while figuring out what went wrong so I can tweak the parameters appropriately… I don’t know about other lab people, but I always felt my gut clench and squirm uncomfortably as I waited for the results for an experiment, hoping that it’ll show something promising. Sometimes it would work, and things were good (stomach would relax), but most of the time it didn’t look good and you were left with this really sickened feeling. Given we do multiple experiments in one day, it would mean I would feel tense and uncomfortable all throughout the day- hell, the worst one is when you find out your experiments failed on a Friday evening. We’re also on a time crunch, with many deadlines coming up all the time. Maybe it’s for a publication, a review meeting, or just your weekly catch up with the supervisor. Either way, I begun to hate the feeling of disappointment in myself.

Don’t get me wrong- when experiments did work, I still got very excited about it. I would imagine it’s very similar to the rush of endorphins people experience when they’re playing slot machines and they win money (any money). For a brief moment, you feel elated, and can’t wait to tell your supervisor about the result. In the beginning, I had a lot of trouble trying to figure out what the result meant (i.e. how does it tie into the overall Biology of your organism?), but over time I got better at making good suggestions for discussion points. I felt like I understood the overall picture (albeit a fuzzy picture), and it was exciting to try to piece the puzzle together.

… and then something would happen that contradicts the hypothesis, and you had to change the narrative a little to suit this new finding…

… or you can’t repeat the original finding.

THEY KNOW

And that just meant more experiments- sometimes the same ones over and over again, until the results were relatively consistent, or they weren’t. If they were too inconsistent, the approach would have to be abandoned. URGH.

So over time, I realised that this wasn’t going to be for me anymore. The normal career trajectory for a Ph. D graduate is to find work as a Post-Doctoral researcher (Post-Doc). When I thought about doing this for the next five years… well- I didn’t want to. I didn’t want to have to grind through it for another few years and regret it, especially if I could have transitioned out of it earlier. I think there’s a weird mentality that you should suffer and toil as a researcher, and that you’re weak for seeking something easier (come on, fellow students, you’ve seen and heard this before). Well, f*** ’em. If they want to live like that, that’s their choice, but they don’t make that choice for your life. I decided that this wasn’t for me, because I realised it would be too much misery- you can, and should, prioritise your well-being.

***

Anyway, as I was beginning to hit this realisation that research might not really be for me, I was also doing a lot of work for my first manuscript. The first iteration of this manuscript (let’s go full Tony Stark and call it ‘Mark I’) was written during the latter half of 2018. The experiments were actually pretty good (they were working), and I found that writing the whole study up was really fun. I’ve said it before, but I’ve always loved story-telling. Usually it was fiction (Fantasy/Sci-Fi), but I would fill up notebooks with made up tales, usually based on dreams I would have. I’m not very cohesive when I talk (yeah, I know), but I always found that writing something down on paper let my ideas flow out in a much more ordered fashion. Turns out, this also worked with scientific writing. Not to say I was perfect at it (I had a lot of really good feedback), but I just enjoyed it a lot. It wasn’t tiring because I wanted to do it, and it didn’t leave me with stomach knots because I knew the feedback I would get would be constructive. Also- I knew the writing wasn’t shite! I was very lucky to have solid guidance from co-authors to help me improve my writing during this time.

We submitted Mark I for review at a relatively good journal (a pretty major Bacteriology journal) in late 2018. Unfortunately it was rejected, but they sent back the reviewer comments and we spent the first half of 2019 working to improve the manuscript for submission elsewhere. The experiments were annoying, but they worked in the end, and once again the write up was the best part.

This was around the time I started this Blog. I had been talking to some people who suggested that I start something like this, so while I kept working on my Ph. D, I also started writing about what it was like to do one.

I’d also well and truly started editing my Literature Review to form the Introduction to my Ph. D Thesis, and had begun compiling my Materials and Methods section. Part of it was due to anxiety (gotta start it now because I don’t want to leave it last minute and have all this anxiety and guilt looming over me), but I also just wanted to write. It gave me a sense of productivity that experiments just couldn’t.

By the time manuscript Mark II had been submitted to another journal for review, I could tell quite clearly that I needed to find a job that was writing heavy. I also got introduced to this site that helps people figure out careers after Ph. Ds, and I think my results were all science writing related. I spoke to quite a few people in different professions- two medical writers, a research scientist (but with computers), patent attorneys, pharmaceutical representatives, lecturers, biotech/start up entrepreneurs… With all of their advice in mind, I’d started applying for different positions that were writing or editing related. Updating my resume (not updated since early undergrad) was an interesting exercise- almost like an archaeological dig.

But that isn’t to say I really knew what I wanted. For so long, I had these obvious aspirations: you do your Bachelors, then Honours, then Ph. D… so what do you do after that, if you don’t want to progress to a Post-Doc? I still don’t really have career aspirations. I never did. So many times in the past year, I had conversations with various people telling me to do/don’t do this/that, because it would or wouldn’t benefit my career. I didn’t go into science because I wanted to get a successful career out of it. I went into it because I wanted to know stuff, and I found it fascinating. I still do, but I also can’t tie it to a particular career path.

Anyway, I had so many rejections, because there are many people out there who have Post-Doctoral experience (i.e. even more experience than me) who want to get out of academic research, or just have better qualifications more appropriate to the field. Thankfully I wasn’t desperate for a job in late 2019 (I still had a ways yet with my Ph. D), so it was just a good exercise in writing CVs and cover letters. I’d accumulated quite a few different versions of each for various different roles I was applying for.

Manuscript Mark II was accepted in September, then published in October, but I had already moved onto working on my second manuscript. September was just… insane. So many experiments, and I’d over-committed myself with other stuff… it was terrible, but we managed to piece together everything so that we were able to submit in early December for review (which, given I was on holiday in October, means I did relatively okay). I think the writing side of things had sped up significantly, given I’d spent the previous twelve months working on my first one. I really enjoyed putting it all together into a cohesive story.

But again, publishing is never super easy, and when we got a response from the reviewers on Christmas Eve last year, there were many comments to address, some with words, but others with experimental data. This was another telling moment for me, because the fact I still needed to do more experiments made me feel really frustrated. I desperately wanted to be done with them, so that I could just spend my days writing. I’d also spent the majority of December working on my first results chapter, and wanted to work on the subsequent chapters quickly, so it put a lot of pressure on my overall timeline.

The next three months (Jan-Mar) were just horrendous- hopefully outlined in my blog posts during that time. I had experiments to do, a manuscript to edit, a thesis to write/edit, jobs to keep applying for, and teaching commitments. The crushing weight of all of these things made me very, very stressed and depressed very quickly. Eventually they started affecting my work output and my personal life, and I just felt like I was drowning. When I realised that this is what life in academia is like all the time (with a tonne more work), I knew I had to get out.

So, I did the only thing I could do at this point. I slowly crawled out of the hole and kept on crawling toward each milestone as best I could. I cut back on some planned revision experiments, which required negotiations with my supervisor and some eloquent rebuttals. I focused heavily on the writing (both manuscript and thesis), because that made me happier and gave me a sense of purpose. I revised my financial situation and paused job hunting momentarily so I could focus on my Ph. D. I also shared the workload for teaching so that I couldn’t over-commit, although it actually ended up working out okay.

Things started to slowly come together, and my work output picked up a little over time. I also got an interesting text from my supervisor mid-Feb about one of the divisions in our research institute- they were looking for someone to do some technical writing and data analysis part time. It seemed to be a steady job, and I was very keen. Thankfully my supervisor had already put my name forward, and I ended up having a few informal chats about the role in the coming weeks.

Speaking to all these people who were in various different roles within the scientific field, I found that it was very clear that there were opportunities out there outside of academic research. The only problem was they were very competitive.

But what I found most interesting was that almost all of the ones who had come from a similar background to myself would always reassure me that it was okay to leave academic research. It’s that stigma that I mentioned earlier. There’s a weird, underlying vibe that if you leave, you’re taking the easier/weaker path. The best part was when they would tell me how much happier they were for having left. Better pay, work-life balance, job security… These were ticking many boxes for me. On top of that, I found people who had the exact same feelings I did, in particular with their attitudes toward experiments. There were quite a few people who realised after their Ph. Ds that they didn’t really want to do anymore lab work, and had found success in other roles that were still within research. It seems silly, because it seems rather obvious now, but it was still really reassuring to hear it directly from those who once felt as I do now.

***

I pumped out what ended up being my last experiments all within the space of a week, and submitted my thesis the following Friday. I’d edited everything on the Tuesday, so I had plenty of time to sit and mull it over. I also submitted the rebuttals for this second manuscript the following Tuesday, and had it accepted two days later, so all of a sudden everything to do with my Ph. D just ended. Obviously the examination is still ongoing, but I literally couldn’t do anything else to make it progress faster. I had my job interview for the aforementioned technical writing role on the day my paper was accepted, so I felt really good for the first time in ages.

Then the following week, the lab shut down, meaning I’d just missed COVID-19 related chaos by a mere two weeks. Given my bug takes a week to grow up, I am so thankful I managed to get everything done before this all hit. I also managed to get the job (I found out late the following week), so that was also very fortuitous in the current climate.

So, basically in the space of a couple weeks, I’d submitted my Ph. D thesis, re-submitted my second manuscript and had it accepted, did a job interview and got accepted for the role, had my lab shutdown so I couldn’t see everyone anymore, and couldn’t visit friends and family due to restrictions…

***

To cut a very long story short-

If anyone else has hit that point in their candidature where they suddenly have a much bigger existential crisis where they don’t even know what to do with their life anymore, I hope this post helps. There was a lot of introspection and soul-searching involved, but I managed to figure out what I enjoyed the most and how I might be able to transfer those skills to a different role. Talking to people was really good, because they either confirmed your ideas or they don’t. Either way it’s still a learning opportunity. I think a lot of people saw the successes on the forefront, but hopefully you can appreciate that it came with a lot of internal struggle. Someone might look fine, but in private they might not be.

I have no idea what the next few years hold for me (especially given this current pandemic has cancelled my teaching commitments), but I will continue to post bits and pieces from my Ph. D life. Otherwise, stay safe, stay well, and 2 m please (yes I know Australia is 1.5 m- just watch the video, it’s hilarious).

Posted in General, Ph D posts

Tips for writing

Given that a lot of people are stuck at home with nothing else to do but to write their thesis/manuscript/Literature Reviews, here’s a short post on what I do to ensure I get some level of productivity in a day.

1.Have a rough plan/layout.

It might sound a bit silly, but it really does help to have an idea of what you’re supposed to be writing (and how to structure it). Go over a dot point plan of an outline for the chapter/manuscript with your supervisor. All you need to do is to literally write it out on a Word document and email it to them. Ask for feedback, and that way you’ll both have a clear understanding of what you need to write about (and how to do it).

2. Know yourself.

Do you tend to get distracted easily? Do sounds bother you? Are you addicted to social media (myes!)? Do you love playing the Sims 4 on your laptop (also yes)??

If you know that you struggle a bit with certain activities, then try and address them before you get started. It’ll take some discipline (like uninstalling the Sims 4 from your laptop… *sob*), but hopefully it’ll do you good in the long run.

I personally love writing, so provided I have a desk and chair I could probably write in most places- although I tend to avoid my bedroom, because I have no desk and chair in there (and I find writing while lying down or sitting cross-legged hurts my frail back).

But- I do know that I can’t stand talking/human noises. If it’s a whole classroom and the noise is constant, it’s fine (because your brain sort of drowns it out as background noise), but if it’s infrequent conversation, or spontaneous laughing, I’m very easily distracted. So, to address this, I saved up and bought myself some noise-cancelling headphones. I generally find loud noises and such to be quite startling, so these have become my go to wherever I may be. Downside is people can sneak up on you, but… at least I can concentrate when I need to. I just need to pick the right sound to play. When I was writing things that required a bit of concentration (e.g. discussion), I tended to listen to soundtracks and classical music, or even nature sounds, just because I found that music with lyrics was distracting- also I tend to sing along spontaneously and no one really needs to hear that.

Thankfully no more smokey air, but now people are wearing P2 masks for different reasons!

3. Be in the right environment.

Obviously prior to the ‘stay at home’ period, you had the choice of either writing at home or writing at work. I was fond of the writing at home option, because I found work was too chaotic and distracting to concentrate. We’re in an open plan office, with lots of people coming and going. Any visitor to my supervisor had to walk behind me and my desk to see them, and the Post-Doc office was right behind me so I was in full view of pretty much every single individual. I found that really difficult to deal with, so I would often opt to stay home and write.

Others find that the work environment helps them concentrate more, because they’re surrounded by colleagues. The pressure from them helps drive the writing, because they feel compelled to work (and not get distracted).

Unfortunately for now, most people don’t have the option of going into the lab to write, so… you’ll just have to make do with what you’ve got.

If you have a room you can hole up in, that’s also a good way to get your concentration up. If you can designate that room as your work place, it might be easier to train your brain into realising it’s time for work. These things don’t happen overnight, but with constant repetition, you might be able to trick your brain eventually.

I like wearing PJs or trackies for as long as physically possible (because I am a lazy slob and enjoy the freedom of being able to do this), but if you find that getting properly dressed for work is helping you concentrate- just do it. Whatever helps, right?

The ideal writing environment?

4. Schedule your work day.

If you find that it’s harder to concentrate because you don’t have a set schedule- make one! If you’re a lab student, you have to plan and schedule your day out anyway (or at least be in the process of trying to do this), so why not just do the same thing at home? Set a timer and blitz it out for a certain amount of time (Pubmed-ing, writing, editing, etc), then set another timer for a short break. In one of my previous posts, a student talks about doing this on/off method to stay focused. Organise yourself appropriately.

5. Have realistic goals.

Sometimes, a paragraph is the cumulative result of hours upon hours of literature searching. Especially in the introduction or literature review. Other times, you can write a whole page in 15 minutes, because you’re just interpreting results. The point is, different texts require different amounts of time to formulate.

Also, if you write super duper fast, good on you, but if you’ve always known you’re not a fast writer, then don’t feel bad that someone else drafted something way more quickly than you did. Comparing yourself against other people is a good way to start feeling really bad about yourself- so try to stop yourself from doing it.

Just write as much or as little as you can. Regularly writing small portions is better than occasionally writing a chunk and forgetting about it.

6. Take breaks.

And by breaks I mean proper breaks (not half-arsed ones). If you’ve only worked for an hour and you take a two hour break… that’s not appropriate. But, if you’re taking a half an hour break after a couple hours of solid work, don’t then start scrolling through literature- that’s not a break! Leave the desk, go for a walk (exercising counts as a reason to leave the house!), make a cuppa, go and pee- physically leave the work space and do something completely different.

#Exercise

7. Unwind as necessary

If you find you’re not getting enough sleep, or you’re not feeling well-rested of a morning, maybe work on the schedule more so that you have time to unwind of an evening. If you find that post-dinner writing sesh is stressing you out- stop it. Exercise instead, or stream something. Working right up until bed time isn’t really good for you in the long run. Prioritise proper rest and sleep.

Much hobby

Also- we’re all stuck at home, so catch up with your mates! Call them, or do a video conference call! It’ll be good to see other human beings.

I don’t know why but all my colleagues are starting to look the same…

***

Writing purely about myself, I tended to have a writing day like this:

  1. ~8-9am: Get up, sit at laptop and start writing/editing. Put on headphones as necessary to avoid distractions.
  2. ~11am: Realise I’m starving and eat breakfast/brunch while watching Netflix. Have a shower and finally change out of PJs.
  3. Keep writing/editing and realise you’re stuck in a time loop- where if you leave, you’ll lose your train of thought. Now you are desk-bound forever, or until the next section break.
  4. ~2-3pm: Realise enough is enough- I need to pee, I’m also kinda hungry again, and I haven’t drunk anywhere near as much water as I should’ve. Eat lunch and take bathroom break. Make a cuppa that I’ll inevitably forget about. Keep writing/editing after a lengthy Netflix sesh while eating.
  5. ~7-8pm: Realise that I haven’t really moved around much all day and make a hasty attempt at fitness by going for a walk/run. Make dinner and eat it while streaming something.
  6. ~9-10pm: Either keep working like a lunatic or start some weird hobby (see numerous, previous posts on #hobbies).
  7. ~12-1am: Realise I should’ve gone to bed hours ago, and start making way toward bed.
  8. ~1-2am: Go to bed and stream something like an idiot. Eventually decide to go to sleep.
  9. Repeat from step 1.

Moral of the story: don’t be like me. It’s super bad for your health!

Posted in Ph D posts

What it was like to write a Ph. D Thesis

In a previous post, I detailed the general structure of a Ph. D thesis, complete with a basic guide on what each chapter contains. But when you get down to it, how do you actually start writing such a massive chunk of text? Here I’ve outlined the general process I followed for each Chapter- hopefully they might help someone who is stuck writing at home during this pandemic.

***

As a general workflow, writing text has always worked like this for me:

  1. Plan and structure the chapter (manuscript) with my supervisor. Write it all out in dot point format so you get a general idea of where things need to go and what needs to be said. This is where we discuss the general flow and tone required.
  2. Write a rough draft of the chapter (or a manuscript subsection).
  3. Send it to my supervisor, who will then edit said draft.
  4. I get the edits back, and I will go through each comment and address them. If I don’t agree with or understand a particular comment, I’ll discuss it with my supervisor and figure out how to move forward with it.
  5. I send this new draft back, and the editing process continues, or…
  6. I keep this edited file as my final draft for said chapter (or manuscript subsection), and move onto the next chapter, without my supervisor looking at it again. Repeat steps 1-5 until all chapters (or subsections) are finished, and compile into the one document (for a manuscript it’s already in the same document).
  7. (Optional) Have this fully compiled document present with so many formatting issues you want to throw the laptop against a wall. Fight through it. Google is your friend.
  8. Send this full version back to my supervisor, who will do a final edit.
  9. Incorporate all comments, and now I have a final, full draft!
  10. (Optional) Read the text again and discover more spelling/grammatical errors, and drive yourself insane trying to fix them all.

My supervisor and I generally rely heavily on Microsoft Word. I draft a chapter/manuscript, I save it onto a shared folder or send it to them. They can then either print it out (used to do this) or use Track Changes to edit and leave comments as necessary (we do this more now). Track Changes is handy because any edits done are highlighted and coloured, and any comment left can be addressed and responded to within the document. Technology is a beautiful thing.

***

In terms of what Chapter to start with, while I’ve previously made some comments, I’ll summarise it again here.

The easiest one to start is the Methods section. Hopefully you’re keeping up to date with your Lab notebooks so that somewhere you have a detailed and well written protocol for your experiments. If written well, these can directly form subsections for your Methods. Ideally you want to just be doing this as you do experiments, because you don’t want to leave this last minute. Thankfully I had an electronic Lab notebook, so for some experiments, all I had to do was copy paste from these straight into my Word document. Sure, I had to change some symbols, but the bulk of it was already written, which saved me a lot of time. For the most part, though, you’ll have to re-write a protocol to better suit a Methods section, but at least you’ll have a template with your Lab notebooks.

When you do a Ph. D (or Honours), you will inevitably have to write a Literature Review. In Honours, I had to write one and send a full draft to my supervisor in early July. For a Ph. D, you had to write one for your Confirmation meeting (12 months after you start). A Literature Review will become your Introduction, so having to write one early is a nice way to get started with the writing process. Obviously this will need to get updated when it’s actual Thesis writing time (projects change, the literature is constantly being updated- you have to keep on top of it), but it’ll be good to have some foundations laid sooner rather than later.

Next are the Results Chapters. These usually start with the making of Figures. Every time you do a big experiment, and you get some useful (or maybe not so useful but it’s what you gotta run with) results, you should begin compiling it into a publication format figure. For instance, here’s a graph I made previously:

This is actually from my latest publication in Infection and Immunity (I’ll write a post on it once it’s available in print).

On its own, the graph is a little useless. No one else knows what’s being depicted, and a figure will typically contain multiple graphs and/or graphs accompanied by images. One graph on its own isn’t quite enough.

Generally speaking, I’ll discuss what the overall figure layout is going to be with my supervisor, before I actually go ahead and make it in Adobe Illustrator.

We usually discuss and jot down ideas for how each figure is going to be placed. This one is from my first publication, in Biochemical Journal. Also- I really need to work on my handwriting. It’s terrible!!

Once you compile all the figures, it’ll look something like this…

Growth curves of WT (solid black circle), 0265::Tn (white square box) and 0265::Tn pFLAG-0265 (solid square box) C. burnetii in HeLa CCL2 cells (A) and THP-1 cells (B), n = 3. Error bars represent standard deviation. Immunofluorescence (IF) images at 3 days post-infection for HeLa CCL2 (C) and THP-1 (D) cells. Cells stained with LAMP-1 (green), C. burnetii (red) and DAPI (blue). Scale bar = 10 µm. * indicates CCV. (E) Survival of G. mellonella following infection with WT (solid black circle), 0265::Tn (white square box), 0265::Tn pFLAG-0265 (solid square box) C. burnetii at 106 GE. A PBS control (white circle) was also included. Results are shown as a representative of two replicates, each with 12 larvae per condition.
You can see how the earlier sketches contributed.

Now you can write in a figure legend. The figure legend should contain enough information that the reader can discern what is being presented, without having to look over the bulk text (because who has the time?).

***

Okay, so you’ve been writing out your protocols, written a Lit Review (and are somewhat keeping up with current literature), and are compiling results figures (it’s a slow process sometimes). You’ve got around twelve months to go before your thesis submission date- now is the time to really start writing.

I was quite lucky in that I had a manuscript written and underway, but pretending that you don’t, this is how you would tackle the thesis.

  1. Start finalising your Lit Review to turn it into your Introduction. It’s usually been a while since you’ve even looked at it (for me it was like, 1-2 years), so your project direction has probably changed, and new papers have been published that you need to incorporate. This can be quite dry, because it’s just a case of combing through the literature again (hello, Pubmed) and bulking out your first chapter. I went through the chapter subheadings with my supervisor again so that we were both clear on what I was going to write about (and how it would be ordered)- just so I didn’t end up writing lots, only to have them go, ‘actually… I don’t like the way it’s set up’. It’s good to have direction. Get a draft done sooner rather than later, and just send it to your supervisor for them to edit. That way you can get the ball rolling and move on to the next chapter. They can give you feedback about what’s missing or where you’ve written too much.
  2. Methods. You can actually work on this while you do your Introduction, or you can do this before. Entirely up to you, but I did this after the Intro just so I could be productive while being brain dead from all the reading I did for the Intro. Same deal as the Intro in terms of discussing the ordering and content in dot point format prior to writing it out. Then you can simply write out everything- because you’ve got a template protocol to go off, right?? Because you’ve been writing down your experimental procedures somewhere…? Right? You just need to make sure you put a no-break space (ctrl+shift+space for PC) between the number and the unit measurement symbol. E.g. 10 uL as opposed to 10uL, and all that stuff. Formatting… Yay. Once it’s drafted, you can send it back to your supervisor again for review.
  3. Results chapters. For a Ph. D thesis, this chapter, as mentioned previously, is like a manuscript draft. It has a mini introduction, the results section, then a discussion. The best way to gauge the content for these chapters is to look at published manuscripts. You need an adequate introduction to give enough information for your reader to understand the results (because by now they would have forgotten everything from the actual introduction- mostly because it was two chapters ago and also it’s ginormous). The results themselves will hopefully be about writing the bulk text of a subsection for each figure (which you’ve been making already…?) in the order that you and your supervisor agreed on. The discussion is directly in relation to your results, and not a broad one like what is contained in the perspective chapter of your thesis. Again, a manuscript is a good way to figure out what to put in here. For things like Honours, though, all you need is just the results subsections. Much easier to handle.
  4. Broad discussion/perspective. Once again, would highly, highly recommend discussing the outline with your supervisor, prior to actually attempting to write this. Given the contents of your thesis, what do the results mean? How does your work contribute to the broader field (and society)? Is this thesis, and therefore you, worth granting a doctorate to?? Usually involves a brief summary of the results chapters and the broad conclusions drawn, and then delves further into what that means. Hopefully these concepts are things you’ve already thought about and have been discussing with your supervisor/colleagues/strangers in the night throughout your entire degree. These big picture questions and answers are what makes a thesis, so it’s good to ponder on this chapter for as long as you can, and write bits and pieces before tying it all together.
  5. Addressing editorial comments. Hopefully by this stage you’ve accumulated quite a few chapters with a large number of comments that need to be addressed. Every time you submit a chapter for review to your supervisor, and you get bored of writing the next one, you can always go back to these edited chapters and start fixing it all up. But if you didn’t get a chance to do that until you wrote all the chapters out first (like me), now is the time to edit. It’s usually a lot of back and forth-ing between you and your supervisor, but hopefully you can fix everything and address every comment.
  6. Compile each chapter into the one document. This is where all hell breaks loose with the formatting. All I can say is Google is your friend, and good luck. Hopefully your citation manager (I use Endnote) doesn’t go berserk. Make sure all the Appendices are in order.
  7. Write an abstract. Now that everything is compiled, it’s much easier to write the abstract. I always leave the abstract until the very end, because I can’t summarise something that’s only partially written. Better to have a good grasp of the overall tone and conclusions before you write a summary.
  8. Add all the administrative stuff. Thesis title page, preface, declarations, table of contents, table of figures, table of tables… All the fun stuff.
  9. Read the whole, compiled draft and edit as necessary, then send to your supervisor. Waiting patiently for the edits can feel super weird, because you literally aren’t allowed to work on your draft until you hear back from them (because what would be the point?).
  10. Incorporate all final suggestions. Discuss any comments you’re not sure about. Fix everything. Read it, re-read it. Read it aloud. Get someone else to read it.
  11. Submit for examination. Pop the champagne and relax.
Posted in Ph D posts

Post-Ph. D hobbies part 4

With this ‘stay at home’ period being extended for the foreseeable future, I’ve been doing all sorts of things to pass the time. My last post was my first foray into a proper cooking blog post, but I’m not rich enough to be cooking all day every day (also I’d probably go insane).

So here are some other things I’ve been doing to pass the time.

Some of you may remember a post from last year, where I wrote about a little miniature house making kit. Well, I’ve got the time again now so I’ve whipped that out and have been building small bits and pieces for the last couple weeks. My dexterity has definitely gone up a few levels *ding* so this type of activity is pretty fun. I have a feeling I’m starting to miss the finicky, hands on experiments you got to do in the lab, and my hands need something to keep them occupied. 😂

For those that are interested, the kit itself is showcased quite extensively on this website. There’s quite a lot of different ones available.

About a year ago, I wanted to get a birthday card for someone, but I completely left it ’til last minute, and I ended up stealing (well, they offered) a card from my housemate. The card ended up being super cool because you had the option of drawing/colouring in the cover. I always liked colouring, so I thought now would be the perfect opportunity to get back into it with some designs I actually liked. Thankfully I found some on Amazon so I bought a box to colour in at a leisurely pace. I’m quite fond of plant motifs (probably because I like plants) and these are full of it!

So yes, while I haven’t quite delved into books and video games like I normally do, I’ve still been distracting myself from the current situation with these sorts of arts and crafts hobbies. Still doesn’t make up for not being able to see people (physically), but it’ll have to do for now. Highly recommended hobbies in general if you’re after some easy procrastination that gives you a good sense of accomplishment!

Posted in General, Ph D posts

‘Stay at home’ Ramen – post Ph. D hobbies

It’s the Easter long weekend, but we still need to stay at home as much as possible, so! In lieu of my usual Easter activities (which involves some family get-togethers), I’ve decided to pre-occupy myself with some cooking. Because I love cooking.

I had a thought about ingredients that people might not be buying so much of over the weekend, just to try and minimise the risk of inconveniencing anyone, and… they sort of aligned quite well with homemade Torigara/chicken broth Ramen, so I decided to run with this idea.

I’ve made Tonkotsu/Pork broth before- just once. It was one of those ‘things I want to cook before I die’ type things (I have a weird bucket list?), and while it tasted f***ing amazing, it also took me three days to make and it was very stressful.

Chicken, on the other hand, is way easier, and way quicker, so this is my second attempt at making it. I don’t have any lingering PTSD to worry about in this case!

Day 1: Chashu pork

Every good Ramen has a delicious porky topping, and it turns out this stuff is actually pretty easy to make! I’m just going to write in what I threw into the pot.

  • 2x Pork belly slabs
  • 3 stalks of spring onions
  • 1 whole bulb of garlic
  • A small (2.5 cm^3) bulb of ginger
  • And a make it up as you go along array of soy sauce, mirin, sake, and sugar- I feel like mine is closer to a 2.5:1:1:0.5 ratio.
  • Water to balance (i.e. cover the pork belly)
  • Salt to season pork belly

All you need on top of this is a slow cooker/multi cooker.

So, to prep your own Chashu pork, you start by removing the skin on the pork belly with a very sharp knife.

Be very careful! 🔪

Once this is done, you can just liberally cover all sides of the pork belly with salt, and then let it sit for a while (I feel like if you can leave it overnight it’ll be even better, but I’m not that organised so I only left it for like, 15 minutes). If it’s a bit wet, you can pat it down with paper towel.

At this point you can attempt to clumsily roll up and bind the pork belly. I’m sure you can take your time with this and make it look pretty, but it was like 10:30pm for me and I wanted to have a shower before bed, so… don’t judge.

Once the belly is bound tightly, then you can proceed to sear all sides in a frying pan, or in my case, in my new multi cooker (this is the test run).

After that, all you need to do is just throw everything into a slow cooker (or just chuck the rest of the ingredients into your multi cooker), and let it slow cook on low overnight. You can also throw the skin you removed earlier (because flavour).

You’ll be greeted the following morning with this magnificent product.

You can sift the contents but keep the soup!! It’s essential for Ramen.

All you need to do now is to let it cool, and then chuck it in the fridge.

Day 2: Ajitama/marinaded egg, fragrant oil, and Ramen broth

Once you’ve dealt with your Chashu- do not throw out the marinade. That’s gonna become your Tare/soup base. The final Ramen broth is a combination of fragrant oil, soup base, and broth, so you need to keep this stuff (it’s hella salty so don’t drink it straight- you’ll die).

One of the most popular Ramen toppings is the Ajitama, or the marinaded egg. It’s delicious stuff, but it’s also a bit of a pain to make.

  • Room temperature egg/s
  • Your Tare from earlier (that you made your Chashu in), semi-diluted with water

So, you have to get a needle/tack, and poke a hole through the shell at the bottom of the egg, where there’s a natural air sac- that way you don’t have any eggy bits oozing out. The bottom of the egg is the end that has a gentler curve and looks more flat. The top of the egg is more pointy- don’t stab that end.

Once the hole has been poked in all the eggs you want to marinade, you can gently place them in a pot with some cold water, and let the water come to a boil. Make sure the pot has a lid on it. As soon as the water starts boiling violently, kill the heat, and keep the lid on. Depending on how you like it, you can leave the eggs in there for 3 minutes (for a gentle oozing yolk), 5 minutes (for less ooze but still somewhat translucent), or whatever else you prefer. I’m a fence-sitter when it comes to recipes that have a range, so I just did 4 minutes.

Place the eggs in some ice-cold water, and leave it in there until it is cool to the touch, before gently removing the shell. Hopefully the hole you poked at the start makes it easier to peel off the shell.

Once the eggs have had their shells removed, you can throw them into the Tare/soup base you made earlier, and let it marinade overnight in the fridge.

***

For the fragrant oil, I just use lard (I had some left over cold smoked bacon fat so I just used that). Just throw in a hunk of lard in a pot with a few cloves of garlic, a bit of ginger, and some spring onions, let it heat and cook for a while until the fat smells divine. Let it cool before you extract the oil. It can sit in the fridge for now until you’re about to serve, at which point you can just microwave it and have it super hot.

***

Now the Torigara/chicken broth- I just had a mixture of chicken wings and chicken drumsticks (again, I figured no one really wants to eat those over Easter). You just need bones, because it’s the marrow that gives the broth flavour.

  • ~3 kg (600 g chicken wings, ~2.4 kg chicken drum sticks)
  • 8 small stalks of spring onions (they were super thin)
  • 1 whole bulb of garlic
  • A small (1.5 cm^3) cube of ginger (add more if you want!)
  • 1 red apple
  • 2 small bay leaves
  • However much Sake as you want
  • Water to balance (to cover the chicken)
  • A touch of soy sauce

So, you can simply throw all these ingredients in a pot and let it boil (a ‘rolling boil’) for a minimum of two hours, and it will give you a decent broth.

I had the equipment and the time, so I threw it all in to slow cook overnight instead- because, why not?

Day 3: Bringing it all together

I didn’t really have a lot to do, so I kinda just faffed about with the broth. I had it in the multi cooker (slow cooking overnight), so I tried to saute on high, then low, and realised the temperature was just too high (it kept burning the bottom), so then I sifted the broth and transferred it into a pot to boil more gently on the stove top (I threw some bones back in). Once I was ready to eat, I just sifted it all again to remove any bony bits.

For toppings, I just went with thinly sliced spring onion, sauteed cabbage and bean shoots, alongside the Chashu (sliced) and Ajitama. Pickled ginger is optional.

The easiest way to slice Ajitama is to get a piece of thread (for sewing) and essentially garrote the egg length-ways. Way better at preserving the oozy yolk than using a sharp knife.

You can just adjust the cooking time if you want a runnier yolk- but it’s that translucent colour that’s the best.

Provided the broth, soup base, and fragrant oils are boiling hot, and the bowl you’re putting them in have been warmed, the ingredients should warm up on their own, but… I still give them a quick zap in the microwave (especially the Chashu) beforehand.

The noodles I use are non-fry, meaning they haven’t been deep fried to dry the noodles out. You don’t have to use these- I just have them because my mum can’t eat the normal noodles without feeling ill.

Once the noodles are boiling, it’s go time. Assemble the broth (I think I ended up doing 2.5:0.75:0.1 broth:Tare:oil- but do taste to check), and have it ready to plonk the noodles in, because otherwise the noodles tangle and clump. I undercooked the noodles by a full minute because I eat slow and that way the noodles don’t overcook so quickly. I reckon in hindsight you could undercook it by two minutes.

Once you’ve got the broth and noodles, you can go nuts and put all the toppings on, and enjoy. We certainly did in our household.

It was way too much food, but it was amazing.

Hope you enjoyed my first foray into a cooking post.

Merry Egg-mas, wherever you are. Stay home, stay safe, and take care. 🍜 ☺️

Posted in General, Ph D posts

How has your life changed under COVID-19 (student edition)? – Vol III

I think we’re all varying degrees of both.

I’ve been writing up a series of posts with some students’ experiences on how life has been during this global pandemic. Here are a couple more responses.

Student number 6:

What degree are you currently completing?

Doctor of Philosophy.

How many months/years are you (with your degree)?

11 months.

What do you work on?

Trying to cure HIV through improving the immune response to kill HIV-infected cells.

What was your life like prior to lab shutdown?

A combination of designing experiments, performing experiments, analysing the data and reviewing the literature (and lots of writing)!

What is life like now, post shutdown?

How are you managing your degree?

Well my kitty is definitely loving seeing more of me and all the extra attention! I am now working from home indefinitely. Bad because it means I can’t perform experiments and generate data to progress the project. Silver lining – it means I have more time to read and to write. And the extra benefit of reducing the transmission risk helps to put it all into perspective.

What is a typical day like?

I’m trying to keep a sense of normality (despite spending all of my days now in the same apartment). I get up same time, get ready (out of pj’s/track pants!), and try to operate the same as if I were in the office. I do find it harder to focus at home because normally home is where I relax, so I’m relying a lot for the moment on the Pomodoro Technique (25 min off, 5 min off) to maintain focus.

What are your biggest concerns in a time like this?

My biggest concerns right now aren’t for my degree or data. The University, the Department and supervisors are being incredibly supportive and understanding that these delays will prevent generating new data (in my experience). My biggest concern is the safety and health of my family and friends. My partner is a doctor. My parents are both immunocompromised. Right now, they, and people in similar situations are the priority, so I’m okay with working from home right now. They’re worth it!

How are you managing your mental/physical health?

Facetime and Zoom catch ups really are fantastic! In addition, a few of us have a fitness group where a couple of times a week we link up through Facetime and do an hour of exercise at home. Apart from that, I’m trying to maintain eating well (and keeping a safe distance from the fridge at home!), trying to maintain regular sleeping habits, and keeping a schedule.

What are some some things you are looking forward to doing (post-shutdown)?

Brunch. Brunch. Brunch. I am all for getting out there and supporting local businesses once things return to normal. Also catching up with friends in person. Board games nights, Friday night after-work drinks, coffee breaks to House of Cards. All the things that help keep you grounded during a PhD. I cannot wait!

Student number 7:

What degree are you currently completing?

PhD

How many months/years are you (with your degree)?

4 years 2 months!

What do you work on?

I investigate how the immune system interacts with nerves.

What was your life like prior to lab shutdown?

I was already in my writing phase so was doing a lot of inside work anyway. I did have some casual work in an office and the gym which was a good break.

What is life like now, post shutdown?

How are you managing your degree?

Most of my work now is tidying up my thesis and organising my data from the past 4 years. Everything is a bit of a mess from having no set routine but very slowly chipping away at the work which I think is better than wallowing at not being at 100% efficiency.

What is a typical day like?

Wake up with some Animal Crossing, lunch, trawl through data a bit, exercise, cook, and finishing the day with more Animal Crossing (seriously why am I feeling more reward completing tasks in a virtual island).

What are your biggest concerns in a time like this?

I’m privileged to be well supported, both mentally and financially, but my heart goes out to people who are not in the same spot. I am a little worried about job prospects from this but it’s not something I can control so why stress over it now.

How are you managing your mental/physical health?

I’ve started doing more voice calls – used to find them annoying but it’s nice having people to chat with. Even if you are just sitting around doing your own thing. Home workouts also real good to get blood going again (LesMills on Demand is relatively cheap with a free trial start).

What are some some things you are looking forward to doing (post-shutdown)?

BRUNCH! and gym. Brunch lines will be so long though 😦

Any other comments?

It’s important to not feel like you have to be super productive in these current times. There’s a quote going around saying “If you don’t come out of this with some sort of skill/side hustle, you don’t lack time, you lack discipline” which works for some people but overall you should not be expected to have “maximum efficiency”. If you need to spend the day doing nothing to cope, then do it. Don’t let other people shame you into thinking you’re not doing enough. Times are tough already and we don’t need extra guilt piled up on you. As long as you are happy with what you are doing you’re off to a good start.

Also don’t forget to check in with your friends (even the low-maintenance ones) and reach out if you need it. No shame at all.

***

I feel like we’re just playing into stereotypes with all these brunch posts (not to say brunch isn’t awesome). #Melbourne

Once again, if you would like to participate in these posts, feel free to let me know, and I can send you the link to the questionnaire form.

Hope everyone has a good break over Easter. It can be lonesome and depressing at times, but I hope I can bring some level of entertainment and distractions with my Blog. Take care everyone.