Massive congratulations on the official moulding of PhD and MSc by Research to our promising young scientists: Rabia Saleem, Dr Ciara Gallagher and Dr Ellen King! Great accomplishments!
Three different journeys, with two through the COVID-19 pandemic. The full range of ups and downs. Who said that the PhD is a straight line? It has never been. It is more like the Irish weather: some days are sunny and bright, and some have scattered showers, gale winds and stormy snow, with sunshine developing elsewhere. The journey was spiced up with publications, conferences, travels, days out and fundraising events with the team.
It is a proud moment for me as well. đ Three PhD and one MSc by Research students graduated within the last 12 months.
Of note, Ellen was behind our Twitter activities in the past, making our team visible!
Wish you all the best of luck on your new adventure!
Few advancements in biomedical sciences hold as much promise for revolutionising cancer research as CRISPR-Cas9. This ground-breaking gene-editing tool has sparked a wave of innovation, offering precision and efficiency in manipulating the human genome in the fight against cancer.
Now, what is it? CRISPR is basically an acronym for a very long name Clustered Regularly Interspaced Short Palindromic Repeats Associated Protein 9 or CRISPR-Cas9 for short. It was found in simple organisms such as archaea and bacteria. Interestingly, this is a component of bacterial immune systems that can cut DNA. So, this feature was proposed for use as a gene editing tool, a kind of precise pair of molecular scissors that can cut a target DNA sequence. So, the CRISPR-Cas9 scissors allow us to precisely edit the DNA sequence of living organisms by adding in (knock-in) or removing (knockout) a gene of interest.
For cancer research, for example, the CRISPR-Cas9 scissors can be used to introduce therapeutic genes or correct mutations associated with cancer predisposition syndromes. Meanwhile, those scissors can also disrupt genes involved in treatment resistance, sensitising cancer cells to existing therapies.
Jennifer Doudna and Emmanuelle Charpentier have won the 2020 Nobel Prize in Chemistry “for the development of a method for genome editing.”. A nice accompanying piece was published in The Conversation, highlighting the history of these scissors and the politics behind it.
Jennifer Doudna explains this revolutionary genetic engineering tool in a TED lecture. However, she warns:
“All of us have a huge responsibility to consider carefully both the unintended consequences as well as the intended impacts of a scientific breakthrough.”
How do researchers study cells? How do we get the nitty gritty?
We use many methods to tag and chase various cell components. One of my favourites is fluorescent microscopy. It allows the use of nearly all spectrum of colours from blue to purple in one go. However, we prefer to narrow it down to 2-3 colours and avoid their overlap.
How does it work? First, we use DAPI or Hoescht, which are blue fluorescent dyes used to stain DNA. This way, we tag the nucleus of the cell. Then, we tag a protein of interest. In our case, it was MYCN, a protein that acts as a transcription factor. MYCN amplification is associated with poor prognosis in neuroblastoma. As a transcription factor, it binds to genomic DNA and is located in the nucleus. We used a specific antibody that was labelled with a green fluorescent dye. Look at the image below. The green colour pattern overlaps with the blue colour. Then, we tagged the cytoskeleton, a complex of various proteins that hold the cell architecture and dynamics. We used phalloidin with red fluorescence. It is a highly selective bicyclic peptide and a popular choice for staining actin filaments.
Neuroblastoma organoids stained with DAPI, Phalloidin and anti-MYCN antibody. This work was done during the Fulbright journey to Ewald’s Lab at Johns Hopkins
Now, we can enjoy visualising cells and test different research questions. For example, how do cells respond to a drug? Or how do neuroblastoma cells spread?
In humans, NANOG, SOX2, and OCT4 are transcription factors that maintain the undifferentiated state of embryonic stem cells (ESCs). NANOG was first discovered in 2003 by Chambers et al. and Mitsui et al. as a transcription factor in ESCs responsible for cellular self-renewal. More importantly, it enables continuous self-renewal of cancer stem cells, leading to metastasis when the regulatory genes involved do not function normally. These have been identified as cancer stem cells, with NANOG being a marker of âstemnessâ. In multiple cancer types, NANOG has various effects, including cellular expression of mesenchymal phenotype, cellular invasion/migration, repressed apoptosis, drug resistance, and increased angiogenesis. In pathways, NANOG either promotes or represses the expression of other genes that lead to cancer-favoured cellular behaviour. Overall, a higher expression level of NANOG is usually indicated in cases of poor prognosis.
However, time passes much slower in TĂŹr na nĂg, making it precarious for humans to return to their own world. As is the fateful tale of OisĂn, who fell in love with the TĂŹr na nĂg goddess, Niamh. He travelled with her to TĂŹr na nĂg, where they lived happily in paradise. Upon a visit back to Ireland, OisĂn realized that all his family had died over the years. When OisĂn found a group of men who were struggling to move a giant rock, he stopped to lend them a hand while on his horse. However, the weight of the rock caused his saddle strap to snap. He fell from his horse, and when he touched the ground, he suddenly aged 300 years all at once.
Huge congrats to a newly minted Dr Ciara Gallagher!  She defended her PhD on March 8 – International Women’s Day. Your enthusiasm and perseverance are truly fascinating! May this be the stepping stone towards a brighter future, Ciara!
We thank examiners Dr Marie McIlroy (RCSI) and Prof Jan Ć koda (Masaryk Uni) for the time and expertise they provided.
We also thank the Irish Research Council for their generous support!
Dr Ciara Murphy (Chair), Dr Olga Piskareva (Supervisor), Dr Ciara Gallagher, Prof Jan Skoda (examiner), Dr Marie McIlroy (Examiner)
Researchers use various methods, but I employ gene knockdown in my experiments. Basically, I use small RNA molecules that specifically target and degrade the mRNA of my gene of interest. This leads to a decrease in the corresponding protein levels, enabling me to observe the effects on neuroblastoma cell behaviour.
I feel a bit like Sherlock Holmes, you know? I’m selectively putting my suspect protein â the one I’m eyeing â under the spotlight to see how it’s pulling the strings on the cell’s behaviour. It’s like I’m in a cellular mystery, complete with a gene knockout magnifying glass đđ§Źđ”
So, what I’ve been up to these past months is knocking down my protein and trying to find answers to the following questions:
Can neuroblastoma cells survive? And if not, how do they meet their demise? Do they go on a growth spree and start proliferating? Are they capable of migration? And here’s the twist â when my protein of interest takes a dip, do other proteins decide to change their expression levels?
The picture below can probably help you get an idea of what Iâve done so far. Do you see those brighter spots in Pictures A and B? Those are dead cells. Their number indicates the proportion of dead cells after a treatment. Picture A has just a few; the majority are healthy and well-spread cells. This is our negative control, a condition when we show neuroblastoma cells that have been transfected, but no gene knockdown happened. Transfection is the term for introducing small RNA molecules. Now, in Picture B, when we knocked down the protein, it caused the death of the cells, and you can clearly see that from all those many little bright spots.
We have found answers to many of the previous questions, but new questions have arisen, and we can’t wait to answer them!
For our next little series introducing a different thing in science and how it works every week, I decided to focus on classifiers. With artificial intelligence becoming more and more prominent in our daily lives as of late, I thought this would be a good lead into the explicitly science-focused topics to come. So, what is a classifier? How does it work? And why does it matter?
At their core, classifiers are algorithms designed to categorize input data into predefined classes or categories. They learn patterns and relationships from labelled training data to make predictions on new, unseen data.
Once features are extracted, identified and quantified from labelled or annotated input data, mathematical models are employed for pattern recognition and predictions.
These models can range from simple decision trees to complex neural networks, each with its own strengths and weaknesses.
Training these models is an iterative process. That means to produce one good classifier, lots of classifiers were created in the process: Every time the pattern recognition is run, the annotated data is categorised by the classifier and compared to the annotation class. Prediction errors are corrected, and performance is optimised. This whole process is one iteration. How many iterations are required for a well-trained classifier varies widely and is largely dependent on the input data and application. For my tissue classifiers, it took up to 20,000 iterations.
Classifiers use these models to categorise unseen data into categories the user-defined at the start. In the figure, you can see my annotated histological slides from which the classifier extracted patterns to then classify the rest of the slide and entirely unseen slides into tumour (red), stroma (green) and background (blue) classes.
From identifying fraudulent transactions, filtering out junk mail, targeted advertising, and facial recognition to unlock your phone or diagnosing diseases, classifiers play a vital role in automating decision-making processes and driving advancements across a wide range of industries. Keep your eyes peeled, and you can find more classifiers in action all around you.
In the world of scientific research, Dr. Pittman’s name stands out. She was a bacteriologist and one of the founders of vaccines against infectious diseases such as typhoid, cholera, whooping cough and meningitis. Dr. Pittman achieved another incredible feat as the first woman to give rise to a National Institutes of Health Laboratory in the US. This accomplishment dispelled the persistent gender stereotypes that had long prevented women from pursuing careers in STEM and showcased her extraordinary credentials and experience.
Dr. Margaret Jane Pittman was born on January 20, 1901, and grew up in Prairie Grove, Arkansas. Dr Margaret Pittman graduated magna cum laude from Hendrix in 1923. In 1925, she took her first course in bacteriology. At the time, it was propitious. Later in the nineteenth century, bacteriology became a distinct biological science. She got her Ph.D. from the University of Chicago in 1929 and started working for the National Institutes of Health in 1936. She developed, assessed, and standardised immunisation programmes against cholera, whooping cough, typhoid, and other diseases during most of her career. She continued to work at NIH as an unpaid guest. She occasionally served as a consultant for the World Health Organisation and as a guest scientist in several countries, including Spain, Scotland, Egypt, and Iran.
Her work and relentless efforts have revolutionised the world of vaccines and immunisation. Dr Pittman’s Haemophilus influenzae study has significantly influenced the medical field. Based on her research, numerous lives have been saved due to the creation of potent vaccinations, which has led to a notable drop in diseases linked to Haemophilus influenza. Pittman’s collaborations with esteemed scientists like Drs. Jonas Salk and Albert Sabin were essential in creating vaccinations against polio. They collaborated to carry out a great deal of research and clinical testing, which led to the development of the first effective polio vaccine. This enormous accomplishment was a game-changer in the global battle against polio, saving countless lives.
CREDIT: NATIONAL MUSEUM OF AMERICAN HISTORY Margaret Pittman and fellow NIH physician Sadie L. Carlin are âreadingâ an agglutination reaction, part of the test for potency of commercially prepared anti-meningitis serum during the meningitis epidemic of 1935-1937 (1937).
As a woman in a male-dominated field, Margaret Pittman faced numerous challenges throughout her career. In an era when gender bias was pervasive, women struggled with standardisation and opportunities for advancement in scientific research. Pittman’s journey was the same, as she faced challenges that would have prevented her from moving further. The legacy of Dr. Margart Jane Pittman endures today, as her contributions continue to influence the direction of immunisation research. She stated that revolutionising problems with pertussis immunisation makes this work one of my best accomplishments that âdespite the problems that have occurred with the pertussis vaccine⊠I consider this work one of my best accomplishments.â. Her partnerships and contributions have established a standard for upcoming generations and cleared the path for additional developments in vaccine research. By honouring her outstanding accomplishments, we can ensure that her influence on public health continues to be a driving force in the battle against infectious illnesses.
November 22, 2023 â Catherine was officially coined Dr Catherine Murphy. A Big Day for Catherine, her family and me.
Catherine joined our team in July 2019 to carry out a research project funded by Neuroblastoma UK. In this project, she aimed to use 3D culturing to engineer a novel experimental model and study the biology and immunology of neuroblastoma, an aggressive childhood cancer. There was the full spectrum of challenges and hard work spiced up with the uncertainty of the COVID-19 restrictions!
The PhD journey is never a straight line. It has a range of colours with 50+ shades for each. There are black alleys and hidden cul de sacs. Between July 2019 and June 2023, some days were sunny and bright, and some had scattered showers, gale winds and stormy snow, with sunshine developing elsewhere. The journey was spiced up with publications, conferences, travels, days out and fundraising events with the team.
Of note, she was behind our Twitter activities and blogging #AskCat, making our team visible! All these together have moulded into a new multi-skilled professional â Dr Catherine Murphy!
Well done to Catherine! Wish you the best of luck in your new adventure!
