LabLife: the final week

LabLife: the final week

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Since my first blog post “Are you what your mother ate?” I’ve been working like crazy in the lab to finish my final experiments for my entire PhD (absolute madness!), and after over three years of PhD life and hard work the final lab week is here!

One of my reasons in making this blog is to give others an insight into the life of a PhD student. When I applied for my PhD I was currently doing my masters project but knew a PhD was going to be a big step up. I wanted to understand more about what it was like to do one, but couldn’t find many people’s experiences documented online. So here is is, one day in the life of one PhD student…

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Wakey wakey

After pressing snooze on my alarm a good few times and being too cosy to jump out of bed, a typical day for me starts at 6:45am. I get up, and as it’s December I go and find my advent chocolate which makes getting up that little bit easier! Porridge is always my staple breakfast as it gives me a good amount of energy to start a day in the lab. My walk into work is about 20 minutes which is great – not too far away but enough time to get fresh air and clear my mind before I’m deep in antibody calculations, immunofluorescence staining, PCR analysis and microscopy. A LOT to do!

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Work begins

Works starts nice and early at 8am (got to be productive before the necessary tea break)! First things first, check my to-do list that I made at the start of the week. Top tip – being organised and planning your week is a massive help to keeping on track and being productive amongst the craziness of a PhD. The last couple of months I’ve been doing a lot of immunofluorescence staining so I check my plan and grab the right set of samples out of the freezer and let them air dry before I start the experiment.

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Getting prepped

Whilst waiting for the samples to dry I check emails, calculate antibody dilutions and prepare the necessary buffers to use for my immunofluorescence staining.

What is immunofluorescence staining?

So my project is all about looking at how the offspring’s skeletal muscle development and function is affected by a maternal high-fat (first study) or vitamin D deficient (second study) diet. Research shows that diet can alter the type of muscle fibres that make up an individual’s muscle, so this is what this experiment is for. As a brief insight, I have cross sections of muscle samples previously cut and put onto glass slides (as you can see in the previous picture). Different muscle fibres express different types of the protein myosin heavy chain (MHC). Adding antibodies specific for these proteins and then adding a fluorescent tag to each one allows me to see which fibres are what type under the microscope, and ultimately see how the number of each type of muscle fibre changes with a normal or altered diet.

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Primary antibodies on and two hours to wait!

These primary antibodies are what attach to the specific types of MHC protein. I make two different cocktails of antibodies which are mixed with either of the two different buffers made above. One muscle sample will be incubated in three out of five primary antibodies, and another section of the same muscle will be incubated with the other two antibodies. I also have to include ‘negative controls’, these are sections of the same sample which are incubated in just the buffer without any antibodies. As there are no antibodies applied to these samples, in theory they should not fluoresce when I image the samples, and it shows that any fluorescence is due to the primary antibody attaching to the correct molecule.

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Tea break o’clock

The 11am tea break is a fundamental part of my day! My friends in the lab and I pretty much always have this without fail. Bit of caffeine and a catch up means a boost of energy and ready to go… until lunch break!

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Catch up take one: PCR analysis

Whilst waiting for my two-hour incubation to finish I catch up on other bits of work. Here I’m analysing a PCR I did a couple days before. PCR (polymerase chain reaction) is a technique that allows me to quantify how much of a specific gene is expressed in my tissue samples. Here I was looking at the gene troponin 1 – a gene important for the expression of a protein involved in muscle contraction regulation. I’m seeing a few significant differences (yay!), a point where all PhD students have a feeling of relief!

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Primary antibodies off, secondary antibodies on

After two hours, the primary antibodies are washed off with the buffers and the secondary antibodies are applied for one hour. The different secondary antibodies recognise their corresponding primary antibody and bind to them. They have a fluorophore attached and this is what makes the muscle fibres a fluorescent colour when I look at the samples down the microscope. It’s important that these are incubated with a non-see through lid as exposure of the fluorophore to light will ruin the fluorescent signal. Too much exposure to light = experiment fail.

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Catch up take two. Making pretty pictures

After my secondary antibodies have been applied, washed off after an hour and stored away ready for image analysis the next day, I catch up on some remaining image analysis from the previous day. This is all done in a dark room – how wonderful! This picture shows a typical muscle cell stained for the fibre type IIa. Using the fluorescent microscope, I change the filters to view the other fluorophores applied to the sample and therefore see the other fibre types which fluoresce in different colours. I use green, red and blue, which means pretty looking pictures!

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Me time = crossfit time

Lab work for the day done and off to crossfit I go.

A work/life balance is extremely important to me, and exercise is essential. If possible I go every weekday evening. Working out helps me stay healthy and gives me that time away from the lab to focus on something else, and relax.

After crossfit, it’s time to meet up with friends or go home and chill with some food. Day done!

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There it is, a snapshot of one day in the lab. Admittedly I am in my last week but this has been a pretty typical day for me over the last couple months in between other experiments going on. Of course a day in the lab can be completely different to this. I had days where I’d be doing long 18-hour days due to animal studies, or shorter days in my first year when there wasn’t as many bits and pieces to do. But for those who don’t know what kind of thing us PhD students get up to, I hope this gives you a bit more of an idea!

So my lab time is up, and off I go to enjoy my Christmas holidays before I start the mammoth task of writing my thesis.

Merry Christmas everyone!

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Are you what your mother ate?

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Adapted from DOHaD

It’s near impossible to escape the news headlines concerning the rise in obesity, but despite this it’s hard to ignore all that delicious sugary and full-fat goodness lining the supermarket shelves. Obesity is known to increase the risk of developing health complications such as type-II diabetes and cardiovascular disease. On other end of the nutritional spectrum, undernutrition and deficiencies in various vitamins and minerals (e.g. vitamin D, calcium, iron) can also be detrimental to an individual’s wellbeing. However, how your body develops and functions is not necessarily all about your lifestyle and what food you put in your own body. There is increasing evidence that the mother’s diet before and during pregnancy can also affect the child’s health in later life.

Developmental Origins of Health and Disease

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The Developmental Origins of Health and Disease (DOHaD) hypothesis suggests that the environment a baby is exposed to during pregnancy affects the long-term health of the offspring via physiological adaptations during pregnancy, which carry on into adult life. Interesting right?! So this hypothesis arose from numerous epidemiological studies in human populations worldwide, which found that a low birth weight was a risk factor for developing various diseases in adult life. Results from the Hertfordshire Cohort Study found that a low birth weight was linked to an increased risk of high systolic blood pressure [1], coronary heart disease [2], type-II diabetes [3] and osteoporosis [4].

Skeletal muscle is key

Skeletal muscle is one of the main sites for glucose handling. Having good skeletal muscle health is important for whole body metabolic function, strength and also mobility/stability. What a mother ate whilst she was pregnant may impact on her baby’s muscle function in adulthood, and so my PhD is exploring how various diets during pregnancy can affect skeletal muscle development and function in the offspring.

Maternal diet and the life trajectory of skeletal muscle

The intrauterine environment is believed to be important for the development and growth of skeletal muscle, which consequently determines peak muscle mass and strength in adult life, and as a result impacts on its decline with ageing – see the figure below!

Studies have found a link between birth size and adult muscle mass and strength. Low birth weight (an indicator of poor maternal conditions during pregnancy) and a reduction in muscle strength was found in a study of 717 elderly men and women born in Hertfordshire between 1920 and 1930 [5]. This same relationship has also been observed in other studies of varying age groups, and all of these findings suggest that a suboptimal maternal environment during pregnancy may increase the risk of muscle wasting and impaired skeletal muscle function in later life.

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Life course model of muscle growth and decline with ageing. Adapted from here.

Studies investigating the effect of maternal overnutrition or undernutrition have observed differences in the offspring muscle. A research group based at The Royal Veterinary College in London found that feeding female rats a high-fat/high-sugar diet during pregnancy impaired offspring muscle development. In 21-day old pups they found a decreased number and size of muscle fibres, and an increase in the amount of fat deposited within the muscle [6]. Another group based at King’s College London found that feeding an obesogenic diet in pregnancy caused reduced activity levels and decreased muscle mass in young mouse offspring [7]. On the other end of the scale, researchers from my lab group at The University of Southampton found that nutrient restriction in the mother before implantation or in the later phase of pregnancy also coincided with a reduction in the triceps brachii muscle fibre density in the fetal sheep [8].

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But what nutritional drivers might alter this trajectory?

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To explore this whole concept further my PhD is mainly focusing on how vitamin D deficiency in the pregnant mother affects offspring muscle. Vitamin D deficiency is highly prevalent in pregnant women, as well as the rest of the population. It is known to be important for various physiological processes such as calcium handling and bone formation, but research suggests that this vitamin is also important for skeletal muscle. Interestingly, vitamin D deficiency is associated with obesity as the increased fat tissue is thought to sequester vitamin D so it can’t be used by the body. So, to make it a little more complex (because PhDs are never straight forward!) I’m also looking at a high-fat pregnancy model to explore the relationship between obesity, vitamin D and muscle.

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Well there you have it, the overall concept of my PhD! In future posts I’ll be divulging into the more specifics of the experimental models I’ve been using and revealing some of my findings so far, so watch this space!

Take home message: you might not be the only one who’s affected by what you eat and the lifestyle you choose!

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Hello world ♥

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Welcome to In a Science World, a blog dedicated to showing my journey through science. I’ll be giving useful tips and tricks in completing a PhD – after all, I’m currently in my last year and although there are great things that come with a PhD, it’s not without the stressful moments!

This blog will also put exciting, novel research in the spotlight. One goal of mine is to communicate science effectively, so I’ll be explaining the science in a way for everyone (scientists and non-scientists) to understand and enjoy.

I am so excited to have this blog to document my life as a scientist and welcome any comments or feedback. To find out a little bit about me click here.

Time to start writing!

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