51ÉçÇø

Course:&²Ô²ú²õ±è;µþ¾±´Ç±ô´Ç²µ¾±³¦²¹±ô&²Ô²ú²õ±è;²¹²Ô»å&²Ô²ú²õ±è;°ä³ó±ð³¾¾±³¦²¹±ô&²Ô²ú²õ±è;³§³¦¾±±ð²Ô³¦±ð²õ&²Ô²ú²õ±è;–&²Ô²ú²õ±è;µþ¾±´Ç²õ³¦¾±±ð²Ô³¦±ð&²Ô²ú²õ±è;±ð³æ¾±³Ù&²Ô²ú²õ±è;°ù´Ç³Ü³Ù±ð&²Ô²ú²õ±è;

Supervisor: Prof. Elizabeth Ryan

Name of Research Project/Activity: Modelling Innate Immune Responses to Microbial Stimuli: A Preclinical Approach Toward Understanding Immune Compromise in Blood Cancer Patients

Q) Can you tell me a bit about yourself and why you decided to study Biological and Chemical Sciences at UL?

I am progressing into my second year of the course LM123 (Biological and Chemical Sciences) next month, pursuing the BSc Bioscience exit route. I grew up helping my Mom take care of my grandparents, whether that meant helping them at home or going on trips with them to hospital appointments. My interest in biology surfaced when my nan had precursors for a type of blood cancer. I sat through appointments where her blood cell counts were discussed, and it was in those moments I knew I wanted to learn more about what the numbers meant or how they affected her health. After my first lesson of blood cells in secondary school, I quickly realised I had a keen interest to continue my studies in that area, especially having seen firsthand the effects if these cells aren’t working properly. As I went through my teenage years, this passion grew within me, which subsequently led me to pursue the Biological and Chemical Sciences course to study Bioscience.

Q) What motivated you to apply for the Summer Bursary Programme?

Throughout my first semester, I was trying to find a job that would help me gain skills and experience early in my career. After attending the careers fair in UL and discussing internships with industrial biotechnology companies, I learned that I cannot apply for anything along those lines until the summer after I complete my second year. During lectures in my second semester, I was told about the Summer Bursary Programme and I was instantly intrigued to find out more information. When I learned that Prof. Elizabeth Ryan was currently researching immune compromise in blood cancer patients, I approached her and she motivated me to submit my application which was accepted.

Q) What are you doing as part of your research here at UL?

My research project is a preclinical approach towards understanding the immune compromise experienced by Chronic Lymphocytic Leukaemia (CLL) patients. CLL is a B- cell cancer, most commonly diagnosed in geriatric patients. These patients experience a reduced capacity to fight off infections, therefore I focused on the innate immune system throughout the duration of my research to acquire an insight into why this is. The innate immune system is commonly regarded as the detection arm of the immune system as it recognises conserved microbial signatures. When microbes enter the body, neutrophils can quickly respond and try to destroy them. In CLL, neutrophils are seen to have altered behaviour, causing a reduced immune response. I am interested to see what these neutrophils are able to respond to. Therefore, I cultivated a model of neutrophils using the immortalised HL-60 cell line to optimise procedures before using whole blood samples. This project was completed with help from fellow summer student Jessica Keogh, MSc student Elzbieta Krupa, and PhD candidate Brian Gleeson. I carried out a mini literature review using PubMed and Covidence to discover which methods have been used to detect the function of neutrophils. I cultured the HL-60 cells in a flask, keeping them in an environment ideal for their growth. Due to the ideal conditions for microbial growth, it is imperative to use aseptic techniques to avoid the growth of other microbes which would cause contamination. 

When the cells were strong enough, I set up experiments to differentiate them under different conditions. One condition, which was the control, was using complete RPMI medium, which in theory allows the cells to proliferate. The other condition was using 1.25% DMSO which matures the HL-60 cells into neutrophils. After understanding all the antibodies used in previous studies, I decided which markers are best for evaluating the maturity of the cells and which ones to check for activation of the neutrophils. Samples were taken from the plates on days 0, 3 and 6 to show the trend over time of antibody expression. It was shown that DMSO had no significant impact on the cell's maturity, therefore I continued onto the activation of the cells using fresh, healthy cells from culture. After noticing unusual projections from the healthy cells, I dug into some more reading and discovered an interesting paper showing HL-60 cells growing with odd string/ needle like projections, which I would have hypothesised were Neutrophil Extracellular Traps (NET) if I had activated them. I therefore carried out an interesting experiment using PMA, TNFα and Pam3CSK4 to activate the cells I had cultured. Surprisingly, I discovered most of the neutrophils had these projections after an overnight incubation, which suggests that HL-60 cells could have spontaneous NET formation before activation. 

Unfortunately, I was not able to confirm this observation as I didn’t have access to the enzyme myeloperoxidase, but I am planning to order this and try the assay on whole blood samples. I collected the supernatant off the cells to use for an ELISA assay using IL-6, a cytokine involved in inflammatory response. I stained the cell pellets with antibodies to detect if they are mature/ immature and if they are activated/ inactivated. The samples were acquired using flow cytometry, a laser-based lab test that can detect chemical and physical differences of cells. The main antibodies that showed a difference, after validating them by flow cytometry, was CD49d which showed a decrease and CXCR2 which showed an increase when the cells were treated with PMA. Based off these results and the confirmation that PMA is a good stimulant, these assays will be continued using whole blood from CLL patients.

Q) What skills have you developed over the summer?

I have developed numerous skills over the summer, from working in the lab to understanding results. A skill that I developed, which will stand to me in the future, is working aseptically in a Class II laboratory. This involved high standards of work every day to ensure contamination was avoided. This work involved tissue culture, which was the backbone of my research because if I didn’t have healthy cells, I could not carry out experiments on them. I also gained confidence in designing experiments and analysing my results using Fiji Image J and Floreada. The bioinformatic skills I have developed will be especially useful to me as I continue my studies and will aid me in future projects. A very underestimated skill that people push to the side too often is teamwork, teamwork is a key skill required in the laboratory to keep it running smoothly.

Q) What has this experience taught you and why would you recommend it to others?

This experience has taught me that most experiments will not work, but the key thing is to not let it get me off track. You need to persevere and push forward. Eventually something will work and when it does, it will give you the excitement to keep going. This is the first thing that everyone needs to learn, because nobody sees how long it takes to get nice results, and it can take a long time to adjust to that truth. It is because of this that I would recommend this programme to others.

Q) What are your future career plans, would you consider a career in research?

For now, I must focus on getting my bachelor’s degree in Bioscience, but I plan to stay working in the lab in my free time during the semester to try out assays on whole blood from CLL patients, especially the NET assay. I would love to apply to complete a PhD after I graduate in 2028, but I have a long time before then to build up my skills, and to refine my interest in the different areas of Bioscience. I am currently leaning heavily towards a career in research and this project has added weight to that. I cannot wait to see what the future holds for me.

Learn more about the BSc in Bioscience