Sana’s Immunobiology blog
By Sana Hussain
King’s College London-Department of Immunobiology at Guy’s Hospital
25th July- 5th August
My placement combined observing and working in the lab, alongside my supervisor Dr Susan John; meetings, visiting museums and watching PhD students’ presentations (i.e on Blue Tongue Virus and Ebola). The lab studies TCR and cytokine induced signalling mechanisms that regulate T cell activation and differentiation in healthy T cells, and the dysregulation of these molecular mechanisms in inflammatory diseases and cancer. The research carried out uses molecular and cellular biochemical techniques.
Here are a couple of techniques I observed.
Pipetting: Attach a tip to the pipette, place thumb on the operating button until the first stop and then place it into the liquid; releasing your thumb slowly. This causes the liquid to be taken up into the tip. Now press the operating button to the second stop, to dispense the liquid completely.
Pipettes, pipette tips but more fascinating was observing the multi- chamber pipette, skilfully, being used.
Another, more intriguing technique was western blotting; used to determine the relative quantity of protein present in a sample of proteins or other molecules. Firstly electrophoresis is used to separate the proteins within the sample, and then the protein array is transferred from the gel to a support membrane by blotting. Finally for the detection of the protein, involves the binding of an enzymatically labelled antibody specific to that protein.
However the most exciting was when I worked on my own lab project, in week 2. The overall aim was to understand how mutation of transcription factor STAT3 contributes to T cell malignancy; but I did have a more precise question to focus on, which I will state later. I was provided with a textbook to read in order to have a base understanding of the JAK-STAT signalling and ATLL, from which I could progress from.
JAK-STAT signalling mechanism diagram
I gathered that ATLL is an aggressive malignant T cell cancer, with a poor prognosis and poorly understood pathogenesis profile. It originates from T cells infected by HTLV-1 at a very young age, followed by a long latency period of 30-50 years before ATLL develops.
In ATLL patients, mutant STAT3 proteins: STAT3Y694F and STAT3D66IY were found. My investigation focused more specifically on answering if it was the constitutive activity of the tumour specific STAT3 mutants and their dependency on tyrosine phosphorylation of the tyrosine residue in the SH2 domain, that allows for tyrosine induced dimerization and activation. A quick overview of the investigation: culturing of the mammalian cells, mutated STAT3 proteins were introduced into them and a genetic reporter (Renilla) was used to monitor gene expression. Through the values obtained by the luminometer I recorded them into my results table, calculated the relative luciferase activity and plotted a graph on Excel.
From these results I concluded that the answer to the focus question is YES.
Overall, this placement has allowed me to gain a more all-rounded glimpse into the life of a scientist and has confirmed my aspirations of pursuing a science course in the future. Thank you to in2scienceUK for this opportunity and Dr Susan John, and the PhD students, for an invaluable experience!