All posts by in2scienceuk

Supporting students from low income backgrounds progress to STEM careers to become the next generation of scientists and pioneers

Shannon Edwards – UCL Genetics Institute

“I am grateful to have gained so much in such a short space of time.”

During my placement at UCL, I was fortunate enough to gain an insight into Computational Biology under the supervision of Jack Humphrey. Having been informed of the functions FUS and TDP-43, I soon learnt that as with many proteins, the change in expression of the genes that code for such RNA- and DNA-binding proteins, respectively, can be argued to characterise many neurodegenerative diseases. In this case, the diseases looked at were ALS and Frontotemporal Dementia. The efficiency with which the analysis of the regulation of genes took place using programming software such as R Studio was apparent from the beginning and this became the main method to manipulate sample data.

Shannon Edwards 1As a practice for writing code, I made use of a publicly available differential expression dataset that compared the gene expression between mice brains that were treated with two antisense oligonucleotides, (ASO’s). One of the ASO’s was a random sequence and the other was specific to the FUS transcript. I found the process for plotting the resultant graphs particularly complex, considering that I am a part of a generation that is said to be ‘tech-savvy’. I believe that practice through an online course teaching coding, as well as creating sample plots whilst at UCL is enough to show the depth of understanding required to make the most out of one week, let alone a PhD or career. Nevertheless, after altering the R script several times I was able to comprehend that the greater the log10 (Base Mean) value, the closer the Log2FoldChange value was to zero, indicating a smaller quantity change, although the areas of clustering can suggest similarities between sub-sections of data. The resulting plot is shown below.

Shannon Edwards 2Arguably the most complicated task was set towards the end of the week. The proposition was that mutations in the TDP-43 gene would impact the RNA-binding ability of the TDP-43 protein, and I used pre-existing data to analyse whether it would act as a knockdown. I found that the hypothesis was difficult to support based on this dataset, and as expected in science; more data would need to be analysed. Using skills within R such as vector arithmetic helped to reach this judgement as it gives rise to additional data such as log10(gene length) which was calculated from given data to produce a plot with fewer points. Also; the ‘for()’ loop function loaded the data into one plot using the same commands, but did so in such a way that each dataset was still uniquely identified with the assistance of different colours, as you can see in the picture above.

My placement allowed me to witness how science calls for patience and the ability to ask the right questions to manipulate and evaluate the data that could add another piece to the puzzle. With thanks to Jack for giving up his time, everyone at UCL for their hospitality (and smoothies) and In2scienceUK for providing me with the placement, I am grateful to have gained so much in such a short space of time.

 

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Shakera Begum at MRC BNDU – Oxford

 

“My impending aspirations have been transformed since this placement and I look forward to what the future holds for me thanks to in2scienceUK”

My placement was with the University of Oxford in the Brain Network Dynamics Unit, alongside Petra Fischer and Eduardo Martin Moraud. I don’t think ‘passion’ is a strong enough word to describe their love for the infamous Basal Ganglia and its role in Parkinson’s disease.

Yes, I too first thought Basal Ganglia was an Italian dish and I couldn’t have been further away from the truth!

During my placement, I participated in several experiments and observed methods for recording or stimulating brain activity during different behavioural tasks. An EEG procedure was one of these, where Petra designed a programme to record brain activity during rhythmic movement to investigate how this activity changes with cognitive load.

In case you were wondering, this cap cannot be purchased anywhere on the high street – I know, what a shame!

The cap was connected to an amplifier in order to record the signals. The same amplifier can also be used to record activity of the basal ganglia from Parkinson’s disease patients to understand their involvement in movement related or cognitive tasks. We were shown different types of oscillations and readings to expect and how to filter a signal, which really just makes the data look clean and pretty.

We also had some fun controlling Edu’s movements with TMS, a tool that is not only used for research but also for example to treat depression. TMS relies on electromagnetic induction to stimulate a focal region of the brain. The procedure involves placing a magnetic field generator or coil near the head of the person receiving the treatment.

Researchers use TMS to measure the connection between the brain and a muscle to evaluate damage from stroke, multiple sclerosis, movement disorders, motor neuron disease and other disorders affecting the spinal cord.

 

As if all these new research skills were not already enough, we conducted a practical regarding muscle movement led by the King of the Spinal Cord himself. Edu showed us how to analyse muscle activity when movement change in speed or when they follow a template or shape in comparison to when freely performed. We observed greater muscle activity when movements were fast and more tightly constrained.

The past two weeks have not only been eye-opening for a new career path and a wonderful experience to learn new skills, but I genuinely feel it has been such a privilege to work with some of the world’s top scientists! I don’t think I have enough words to express my gratitude for the knowledge shared and the hospitality shown by the entire team. My impending aspirations have been transformed through this placement and I look forward to what the future holds for me thanks to in2scienceUK.

Molly Monk and Issy Bolton at the University of Bath

Pawan Jolly, Molly and Issy

Photo: Issy and Molly with Pawan Jolly in the department of electronic & electrical engineering 

Within the department we were working with the Biosensor group. During our time there we were given lots of opportunities organised by our supervisor, Pawan Jolly, to experience and learn about research science. We visited chemistry, electronics and the nanofabrication labs and were kindly shown around by other post graduates who explained to us their work. It was very interesting to know how the researchers were working on the development of diagnostic tools for cancer diagnosis where one of the main focus was Prostate Cancer (PCa) diagnosis. It was also fascinating how the different groups around the Europe has come together on a common platform to work on the diagnosis of PCa and the network was called PROSENSE (http://www.prosense-itn.eu/).

Within the PROSENSE project, we individually fabricated DNA based biosensor for PCa, observed the absorbance of light by gold nanoparticles using spectrophotometer and used localised surface plasmon resonance technique (LSPR) among other things.

On the 24th Aug 2015, we started with preparation of buffers namely trisma base and sodium acetate and a bovine serum albumin (BSA) as a protein for our biosensor. The next day we adjusted the pH of the buffer using a pH meter, which detects the ion concentration of a solution, to raise or lower the pH of the buffers  bases and acids were used.

As we were working with DNA, everything used must be extremely clean. So first we prepared our gold working electrodes by cleaning it using techniques like sonication, polishing with aluminium oxide in a figure of eight motion. As we wanted our surface to be very clean, electrochemical cleaning was performed in sulphuric acid as an electrolyte with a three electrode configuration using cyclic voltammetry. The silver reference electrode is kept in a salt bridge in order to protect it from the acid, the platinum counter electrode goes straight into the solution and the gold working electrode again goes into the solution. The electrode are connected up to a potentiostat that will cycle a voltage through them and causes oxidation and reduction of the gold surface and hence, removes dirt from the surface of the working electrode. In our experiment the next step was to fill caps with a light sensitive solution together with probe (receptors to capture DNA) and electrode was dipped in it, they were then left overnight.

Next we produced two separate electrolytes using potassium(II) hexacyanoferrate and potassium(III) hexacyanoferrate salts mixed with our prepared buffer solution. Then we placed our electrodes into the same buffer solution that was being used in our electrolytes. Firstly for each electrode we carried out cyclic voltammetry again. After the first run some of the electrodes had DNA attached and the others had a protein attached and depending on the length of time the electrodes were allowed to sit in the solution they should decrease the oxidation peaks as they interfere with the flow of electrons. We also carried out the programme electrochemical impedance spectroscopy (EIS) which measures the amount of resistance of the system  this is affected by not only the surface modification because of binding but also the ion concentration in the electrolyte. With capture of DNA, we observed the increase in resistance and also the decrease in the oxidation peaks in the CV. By the end, we successfully developed our own DNA based biosensor for detection of circulating DNA in blood for Prostate cancer.

All in all, we had a great time and can’t thank enough everyone who took the time to talk to us about their respective fields. Most of all a massive thanks to Pawan, for having the patience of a saint and showing us the “realities” of research in the field of science.

in2scienceUK blog by Adeolu Busari

Despite the warning at the introduction day that the labs we will be entering will not resemble ‘a giant room filled with new state of the art tech and machinery littered with scientists in their lab coats constantly pipetting’. I still had small hopes that some aspects of the description would be true.

How I was wrong.

On the first day I was nearly convinced that Dr Hristova’s lab was a kitchen. There were 3 fridges, 2 weighing scales, an oven and a microwave (definitely a kitchen I thought), but then I snapped back into reality once the fridge was opened and I saw it packed full with all these different chemicals and the huge bottles of methanol and ethanol in the cupboard with the bright orange flammable symbol on it.

Despite the fact that the atmosphere and the appearance of the lab was different from what I anticipated, I’m glad it was. The lab had a friendly, warm atmosphere and was hardly ever packed. The masters’ students and Dr Hristova were friendly and would often engage me in conversation and tell me about their project.

Now onto the actual science: The project outline at the lab I was placed at was to study the effects of pharmacological inhibition of TGF-β1 on microglial cells after mild neonatal hypoxic ischemic insult. The study comes from unpublished data which has found the deletion of TGF-β1 in microglia to be protective. The aim is to see if pharmacological inhibition of TGFR-1 would prove neuroprotective.

On the first day I observed images of stained mice brains underneath a microscope, these images showed areas of the brain that had some physical damage to it. This damage was due to hypoxic-ischemic event, meaning the brain was partially oxygen deprived and that brain cells died. We measured the amount of physical damage noticed in them, physical damage meaning that the brain matter had been fragmented due to the dead neuronal cells.

The remaining of my two weeks in the lab I helped in prep for staining and staining the brains, preparation consisted of taking out frozen sections of neonatal brains and spreading them. Spreading is a process where the brains are rehydrated with bidistilled water (ddH2O), then spread using fine brushes to become flattened and fan dried at room temperature.

Later the sections were fixed in 4% formaldehyde and washed in 0.1M phosphate buffer for 5 minutes. Then defatted in acetone for 2 minutes each, washed twice in 0.1M PB, and then in 0.1% bovine serum albumin/0.1M PB (PB/BSA). After, they were placed on wet chambers and blocked in 5% goat serum in 0.1M PB for 30 minutes. The block was removed, and 90µL of primary antibody was applied to be kept on overnight.

On the second day of fixing, the excess primary antibody was washed off using PB/BSA, PB, PB and PB/BSA and a few more washes took place to insure there were no more reactions taking place.

Finally the sections were ready to be stained either via nissl or tunel. Tunel is a stain that binds to fragmented DNA, so is a specific marker for cell death. Whereas nissl stains cell bodies. If a cell was damaged it would not stain much with the nissl, it may not even stain at all. This is why two stains are used whilst analysing the effects of hypoxic-ischemic even on neonatal brains.

In order to stain via tunel the sections were spread and fixed as described above and then incubated in 3% H2O2 in methanol and then washed in 0.1M PB. Sections were incubated with 0.1% terminal deoxytransferase and 0.15% deoxyuridine triphosphate for 2 hours at 37°C. The reaction was stopped in tunel stop solution for 10 minutes. Then lastly the sections were washed thrice times in 0.1M PB and incubated for 1 hour in ABC solution.Nissl staining is not that different to tunel but is also complicated, so I will not be describing it due to the word count limit!

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Overall, I really enjoyed my lab placement and I feel very fortunate to have been allocated it. It has allowed me to witness the research side of science, which is far more interesting and practical than I thought it would be. Thank you Dr Hristova and In2science for giving me the opportunity.

Laing O’Rourke engineering placement: C520 Custom House Station

by Aneesa Ali

1My engineering placement was with Laing O’Rourke; an engineering enterprise based in the UK who provide engineering, construction and management services to countries all over the world. One of the projects they are currently working on is Crossrail; a new rail service in and around London which will reduce congestion on train and lessen journey times. Once open in 2018 it will run from Shenfield/Abbey Wood to Reading/Heathrow, going through 40 stations; 8 of which are newly built. This includes the Custom House Station, which is where I was situated for my placement.

On the first day, before I could do anything I had to sit through an extensive induction which emphasised the importance of health and safety on site. I was also given my orange PPE (Personal Protective Equipment) which everyone is required to wear when on site. This includes goggles, hard hats, gloves, boots and hi-visibility jackets and trousers.

2The rest of the week was split between being on site and talking to people in the office. Being on site allowed me to see for myself what was happening rather than having someone tell me. When on site I shadowed different engineers who showed me different aspects of the station. The electrical engineer showed me around the plant rooms and explained how the electricity would get around the station, whilst the civil engineers showed me around and explained the actual building of the substructure and superstructure. The photos below were taken whilst I was shadowing a package manager who’s in charge of the subcontractors on the project. The subcontractors, in this case DELTA, were installing the glass on the sides of the VDR (Victoria Dock Road) bridge using a mini-spider crane.

I was also able to see how slip testing (testing the resistance of the ground we walk on) is carried out and go on a site quality inspection with people from Crossrail and the quality manager on site. What I enjoyed most on site was surveying because it was more hands on; We used specialist equipment such as theodolites to measure the levels of the ground.

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In the office I worked with document control, commercial (quantity surveyors, procurement etc.), quality control and planning management. I was also able to talk to the environmental manager on site which I found to be very beneficial as the area of engineering which interests me most is environmental/energy engineering.

Something I feel like I’ve gained is the appreciation for all the work that goes into something we use every day, like stations. Whenever I’m at a train station now I can’t help but think of all of the engineering and construction that went into it.

Overall, it was an amazing experience and I’m really grateful to have had the opportunity to see what happens on a construction site. Most importantly I gained exactly what I wanted out of this experience, which was to see what engineering involves first hand.

Primers, DNA and enzymes – it’s a PCR! by Leighvi Pilcher

A PCR – Polymerase Chain Reaction – is a reaction that is used to amplify the genes from a sample of DNA. In my PCR, we were trying to identify the normal “wild type” samples, and the SOD samples. I ran 12 samples in total – 2 controls (1 wild type, 1 SOD) and 10 random samples.

The first step in a PCR (aside from acquiring the DNA – this is done by taking a sample, and digesting it in Proteinase K, which is an enzyme, and heating it at a high temperature) is making the reaction mixture. Within the reaction mixture, 4 primers are used. 2 of these primers are complementary to the wild type DNA, and the other 2 are complementary to the SOD DNA. dNTPs (free nucleotides) are also added. These bond to the newly amplified single DNA strands in order to make them double stranded again. A buffer is added to maintain the pH of the reaction mixture, with magnesium chloride added in order to remove the additional phosphate molecules from the dNTPs. Taq polymerase is added, which copies the target DNA. All of the reagents of the PCR mixture are added to sterile water, and enough is added to make 14 samples. After each addition, the tube is vortexted, which ensures that the mixture has been evenly mixed.

Once the reaction mixture is made, it is separated into 12 Eppendorf tubes. After this, the samples are added. Again, these are vortexted, which ensures an even distribution. The samples are labeled, and added to the PCR machine. In the PCR machine, the samples are heated. The heat “melts” the hydrogen bonds in the DNA. They are also cooled in the machine, which subsequently allows the hydrogen bonds to reform. After this, the samples are heated to 75 Celsius, which is the optimum temperature for taq polymerase, so the target DNA can start to be copied.

After running the PCR machine, a process called electrophoresis is carried out. Agarose powder is added to a buffer solution so that 10ml of a solution is formed. 50ml is used in the gel, and it is heated to approximately 100 Celsius before cooling to 60 Celsius. This is added to the electrophoresis chamber, which has slides put in it to help identify the holes that the PCR samples will be added to. Once the gel has set, the combs are removed, and the remaining buffer is placed on top. Bromophenol blue is added to each of the 12 samples, as this helps to identify them when placed under UV light in the transillumiantor. A control ladder is added to the first chamber in the gel, which is used to show the approximate placement of the DNA bands. The remaining chambers are loaded with the 12 samples. Once this is done, the gel is “run”. In this process, electricity is passed through the gel (50 volts) for 30 minutes. This stimulates the movement of the samples. The denser bands rest closest to the chamber, with the lighter bands resting further away from the chambers.

pcrNow that the hard part is out of the way, the samples are viewed in a transillumiantor. Here, a UV light is beamed from under the samples, which shows the movement of them. As you can see, the far left of the image is the ladder. Next to it is the wild type control, with the SOD control next it it. Samples 1, 2, 4, 6, 7 and 9 were all wildtypes, and the remaining samples were SOD samples.

When I was first given all of this information, I screamed internally, because it is such a lot to take in. It’s not necessarily a complicated procedure, however it is long and fiddly. So, I summarised in the following steps:

  • Reaction mixture
  • Addition of samples
  • PCR machine
  • Gel preparation
  • Electrophoresis
  • Transillumination

If only it was that simple!

Leighvi undertook her placement in UCL’s Sobell Department of Motor Neuroscience and Movement Disorders, at the Institute of Neurology, supervised by Dr Bernadett Kalmar in the group of Professor Linda Greensmith.