Life in the Laminin Lab #5 – Examples

 

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Check back on Monday for #6

#4 available here (and links to the rest) or Meet the characters

Or, if you are feeling really keen, read about how Kevin actually did bring in the Mongol invasion of India in his PhD thesis and viva; here.

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They grow up so fast – MRes Jan 2018 edition

Another new year, another new “They grow up so fast” post. This time its the turn of four MRes Clinical Sciences students that have been with our lab for the past 3 months.

Today, the students were presenting their work at a poster session in the Institute of Ageing and Chronic Disease.

Four projects, all different in topic, direction and approach. My lab meetings have been not only large but also really stimulating. Much coffee required to stay on top of it all.

Lizzy Lourenco

20180112_123035(0)Picked up the next stage of the sunscreen studies we did last year (blog posts here, here and here). Using our UV sensitive camera and automated segmentation and analyses algorithms, Lizzy assessed the application habits of people using SPF moisturisers. Her project got lots of help; Gabriela Czanner for stats and design, Yalin Zheng and Harry Pratt for image analysis, Austin McCormick and myself for overall design and direction and Conro helped train Lizzy to get high quality images. Conro also took the pics in this post…. not sure how good a trainer he really was!).

Going in to this study, we expected less moisturiser to be applied in terms of volume compared to the sunscreens but that the users would be more thorough in terms of the coverage of the eyelids; our previous study showed the eyelids to be frequently missed/avoided with sunscreen. Turns out, we weren’t quite right; moisturiser application was actually even worse than the sunscreens. Intriguing. This leads to an important public health message- moisturisers are better than nothing but you still need eye protection. An abstract from this work is already submitted to the big dermatology meeting and we’ll write up the manuscript soon.

EDIT: Lizzy won joint second prize for her poster!

Laura Bowker

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Laura

Laura picked one of our molecular biology projects and worked with Thanos Papadimitropolous on part of his PhD project looking at an RNA therapeutic for a rare eye disorder called aniridia.

I think its fair to say that Laura experienced the full rollercoaster of emotions that accompanies most hypothesis testing scientific research; hope in the early periods, confusion as things don’t work, despair as the time ticks down and finally something close to joy (or perhaps just relief) as the final experiments finally start to deliver data.

Of course, at that point she had to stop. Genuinely, her poster and project write up changed in the last week from “this idea doesn’t work” to “actually, it’s got a chance”

EDIT: Laura won joint second prize with her poster!

Nikitha Pasunu

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Nikitha

Nikitha worked on one of our laminin projects, looking see if we can influence a “splicing switch” that some of our other work suggests goes wrong in squamous cell carcinoma. She worked with the help and guidance of two current PhD students and former MResers Conro Conro Sugden and Liam Shaw in trying to use an RNA approach to flip the switch back again.

Like Laura, Nikitha picked up a project early on its development and so a lot of her time was establishing the model, testing ranges of concentrations and timepoints. To be fair, this is what most time in the lab is spent doing so these 10 week projects are probably a fair reflection of that. Nikitha also ended up with some promising looking results that need lots of further confirmation but could be cool if they keep going the same way. This work will continue thanks to support from North West Cancer Research with Lee Troughton working on the project.

Elizabeth Attree

img_0566Lizzie #2 worked on a project with Dr Valentina Barrera that harks back to Vale’s time before the Hamill lab where she worked on Malarial Retinopathy in Malawian children. In this project Lizzie used immunohistochemistry and histological stains to investigate if and how a range of changes to vessels in the retina can predict disease severity, using the retina as a window to the brain. As always with Valentina involved, lots of really nice images of tissue staining were gathered and it turned into a well developed project.

It’s been fun, productive and hopefully enlightening for the students.

 

poster day

Lost in Durham – A Scouser’s tale of Science

Two blog posts in the space of a month? You lucky readers you!

Recently, as part of the BBSRC DTP experience, I went on lab rotation to the wonderful Durham University, where I spent 4 days working with my third supervisor, the legendary Prof (uncle) Roy Quinlan. Roy and his former PhD student, and my new favourite former PhD student, Fred gave me the opportunity to learn how to extract a lens basement membrane from a cow eye (see videos below). While super squeamish at first, with all the blood and what not, it was amazing to get the chance to learn such a cool and useful technique from one of the leading lens scientists in the world! “But why is this such a cool technique?” I imagine I’d hear you ask if it was in fact possible for me to hear you through a computer screen as you read this blog.

The len’s basement membrane is quite a unique basement membrane, as it comprises only one cell type, in addition to being one of the thickest basement membranes in the body. This is particularly useful as we believe we can decellularise this basement membrane and use it to culture our Pierson syndrome cell models on, providing an ex vivo model of the basement membrane. Another cool aspect of using cow eyes for this model is that the animals were already being used for meat, so no part of the animal has went to waste!

After many, many goes, and the wonderful patience of the lovely Fred, I successfully managed to extract the basement membrane from the cow lens. I was also able to decellularise the lens surprisingly easily! I’d like to thank the lovely Quinlan lab for helping me out while I was in Durham and helping me take some really useful skills back to Liverpool, with special thanks to Roy (for organising the trip), Fred (for showing me the ways of cow dissection), and Alexia (for being super friendly to a weird scouser and showing me around, and also being camera woman for the videos above!).

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Another Brucey Bonus point, I couldn’t have went to Durham on a more Christmassy weekend! I drove up in the middle of a snow blizzard, so Durham was a winter wonderland for the weekend I was there. While there, I purchased the newest member of the Hamill Lab at the Durham Christmas Markets. I know Derek the Reindeer will play a huge role in keeping me sane during my PhD!

Derek the Reindeer

Welcome to the lab Derek!

So that was my weekend in Durham! It was a fabulous five days or so, and I look forward to many more days in the Quinlan lab!

Thanks for reading science homies!

And so it begins…

Hi!  I’m Conro Sugden (aka Conor), and I’ll start my PhD in the Hamill Lab in October investigating  the mechanisms through which age and disease associated alveolar epithelial matrix changes drive development and progression of pulmonary fibrosis. This isn’t my first time being a part of the Hamill Lab; Dr. Hamill was my supervisor for 2 x 12 week projects during my MRes.

I studied my undergraduate degree in Biochemistry here in Liverpool, taking the chance to complete a sandwich placement at the University of Navarra in Pamplona, Spain (see below). Here I got my first taste of what it was like to be a research student, where I was part of a group developing novel plasmids to analyse Leishmania parasites.

Three Firsts

Three firsts: (Left to right) The first picture I took in Pamplona; My first conference poster; The first image I captured of Leishmania major parasites expressing our pXG-mCherry plasmid (kinetoplasts appear blue after staining with DAPI).

Following my undergraduate degree, I completed an MRes in Clinical Sciences, completing 3 x 12-week projects (2 of these projects, here and here, were supervised by Dr. Hamill!) in the Institute of Ageing and Chronic Disease, and this is where I fell in love with matrix biology. My work in the Hamill Lab so far has involved (with the help of Lee Troughton) using minigene constructs to investigate intron retention with alternative polyadenylation (IRPA) in the LAMA3, and then following up this research testing ways we can manipulate this ratio. The data from these projects has been exciting and encouraging, and I’m glad that I get to carry on being a part of this.

During my time in the Hamill Lab I’ve attended my first Burns night, an awesome ‘Assembly, Dynamics and Organisation of Filaments and Cellular Responses’ workshop at Durham University, and I’ve helped with the ‘Sunscreen Challenge’ at Meet the Scientists and other outreach events.

Time in the Hamill Lab

From my time in the Hamill Lab so far: (Left to right) Project 2 presentation; project 3 poster; Sunscreen Challenge preparation.

I’m in the process of becoming a STEM ambassador, and I’m planning to bring the ‘Sunscreen Challenge’ to my old high school so that I can talk to the students about what it means to be a research student. Alongside my PhD research I’ll continue with the outreach activities, and I’ve also enrolled onto a Spanish language course (can’t let myself forget everything I learned when I was over there – I will be fluent one day!). Recently I wrote for The Biochemist blog about the link between inflammation and fibrosis, you can find that here.

Going forward, I couldn’t be happier to begin my PhD in the Hamill Lab. The atmosphere is great, as are the lab members. The research currently being carried out is extremely exciting, and I can’t wait to get stuck in and make my contribution to the field.

Thanks for reading,

Conro 

 

You can see a lot by looking – TIRF

Time for a new series. “You can see a lot by looking” basicaly cool images from our research + a little explanation. Microscopy in its various forms has always been one of my favourite things to do, especially when trying out new modalities that open up opportunities to ask new/deeper questions, so I figured we might as well show off some of our pretty images.

#1 in the series is from today; Lee and I used the new Total Internal Reflection Fluorescence (TIRF) microscope at the University of Liverpool’s Centre for cell imaging. This was the first time we have used this microscope and these are just a couple of a set of really nice images, acquired thanks to the expert help of Dave Mason (@dn_mason).


Why is it cool? Well, TIRF is pretty awesome. Basically, whereas normal microscopy involves illuminating the sample directly and then collecting either the transmitted light or the reflected light depending what you are looking for, TIRF involves illuminating the sample at a shallow angle and collecting the light that is internally reflected  (the same principle that fibre optics work by). The practical upshot of this is that when you image at the critical angle, you effectively limit the illumination to near the cell substrate boundary, the bottom ~75nm (1/750,000 of a mm!). You can see the difference this makes in the images below; left = TIRF, right = conventional imaging. Without the TIRF it is much harder to see the fine organisation of the protein at the bottom of the cell.

How are we using it? Well in the magenta and green image, we have imaged live corneal epithelial cells where we have induced expression of the LaNt alpha31 protein with a green fluorescent tag and laminin beta3 with a mCherry tag (shown in magenta) and while the whole cell expresses these proteins, we have limited what we are looking at to just the point where the cell is touching the glass. So here, where we see codistribution of signal i.e white, it’s showing the LaNt and the laminin are close together at the bottom of the cell. The green only signal, e.g. on the left middle, is where there is lant but limited laminin or vice versa for the magenta. In the image below, I have split one of our other images into its component parts (left=LaNt, right=laminin), in this cell there is a much closer match up in the patterns of the two proteins. Proximity alone doesn’t mean interaction but these data add to the other pieces of the story that we are building about how these two proteins influence each other. FYI the scale bar in the image below is 10 micrometers, 1/100th of a mm, you are looking at just a small part of one cell)

TIRFcr
This ‘scope itself pretty amazing, not only can you do TIRF but also Atomic force microscopy at the same time. Looking forward to the next set of experiments…

Valentina en route to viva!!!

Hello readers!

It’s Valentina here, the first PhD student to have joined the Hamill’s lab. I am writing my first article on a very special day…on my way to Dundee to defend my thesis! It’s been a really cool adventure, full of incredible discoveries. 

When I have started, the only available data on LaNts were related to the skin; after three years and many exciting experiments we now know the effects of LaNt a31 in the cornea…which may seem only a little organ of the body, but how amazing is it to be able to see the world?! We have loads of beautiful videos of live laminin’s deposition from corneal epithelial cells and we have established a LaNt a31 overexpression animal model, the first one ever! 

Working in this lab has been fantastic and hopefully today I will be able to transmit my excitement to the examiners 🙂 If my work could open at least one more door to future projects and ideas, then I’ll be doubly proud. 

Going on, the Hamill lab, laminins and LaNts are waiting to be completely undercovered 🙂  much more to follow on this exciting topic…

Soon to be (hopefully) Dr. Vale 

They grow up so fast – NWCR symposium

Today was the Northwest Cancer Research annual symposium held in the stunning, Harry Potter-esque, Victoria Gallery on the IMG_20130714_180621
University of Liverpool campus.

Lots of excellent, inspiring talks including two of my long-term favourites; actin remodelling and RhoGTPases, and integrin signalling. The notebook is full of the next set of experiments and grant ideas! (Thanks Mark)

There was also a thoroughly impressive poster session, displaying the diverse, excellent cancer research work going on in Liverpool, Bangor and Lancaster.

Included in that  poster session, Lee Troughton and Valentina Barrera

C-fSHW1XoAI6tn2.jpg-largepresented our new splice regulation story in LAMA3 that is beginning to really take off. Unfortunately, I missed the opportunity to grab a photo with Vale so its just Lee on this edition of the obligatory “they grow up so fast”.

Lee moaned extensively about his quads hurting while squatting down to take this picture so I made him pose for 10 more, saying it was out of focus…

I also captured his response to finding out about the unnecessary extras. Totally worth it

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Mini (or squatting) Lee

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They grow up so fast – MRes Spring 2017

April time again and the end of another block of MRes Clinical Sciences projects. This time the Hamill lab hosted three students; Kareem Hassanin, Conro Conro Sugden and Tobi Oyewole. Today the boys had their presentations/poster session.

First up; Conro told his story of developing our new minigene construct to investigate LaNt regulation and testing a few mutant versions of it to prove it works. Conor, with Lee Troughton’s help, did some excellent work; generating flo2017-04-13 15.38.50w cytometry,
RT-PCR, western blotting and fluorescence microscopy images that demonstrate that our new system is effective for studying intron retention and alternative polyadenylation and showing that the exon 9 splice site has a big role to play in determining splicing efficiency. His data has gone straight into a grant application and should form the starting point for the next stage of the LaNt project.

Next, Tobi under 20170413_120630the joint supervision of myself and Colin Willoughby and working alongside Fight For Sight student Thanos, worked on determining the optimal delivery mechanism and conditions for delivering small RNA molecules into corneal epithelial cells. This work is critical first step for the the next stage of Thanos’ PhD studies and, although Tobi was frustrated at times, makes a big difference to the lab. Optimisation is the biggest part of all experiments, without these steps we wouldn’t be able to ask the big questions.

Finally, Kareem working alongside Dr Valentina Barrera and using samples kindly provided by Prof Geor20170413_141809ge Bou Gharios, optimised our new rabbit anti-mouse LaNt antibody staining protocol and then did the first staining of embryos and various tissues in the adult. These are really short projects and filled with frustration as just at the very end, Kareem finally had conditions nailed down to get super clean, specific, staining that has opened up a whole range of new options and research questions. One thousand emails bounced back and forward yesterday as he tried to identify all the structures that were and were not stained. Although super cool, really this is just the beginning; knowing where the protein is automatically generates the question of what it is doing in that location as we predict that LaNt function is context specific, so these data too will lay the groundwork for the next grant(s) and next paper.

All in all, a really productive 3 months. Looking forward to the next block…

 

The wall of LaNt research

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Just back from another conference and Lee has added his recent poster to our growing collection. A quick rearrange and we have the wall of LaNt research!

Yes, we have gone too far, taking over a bunch of the poster boards on our floor but it is so cool seeing the work from the various members of my group coming together into a really big story. I recommend working from left to right as you look at them to get a picture from the gene to the protein to the whole tissue.

It starts at the transcript level with Lee’s poster showing LaNt regulation of laminin expression. Also including images of changing protein expression in squamous cell carcinoma from Valentina Barrera and  Conro’s minigene analysis of critical residues involved in splicing regulation.

Next we move on to some of our cell biology work looking at LaNt a31 in the front of the eye where Vale B demonstrates differential distribution accacross the corneal epithelium and Lee and Valentina Iorio show knockdown and overexpression affecting cell migration/spreading

After that its into the molecular biology, with Vale I using fluorescently tagged LaNts and laminins to study their interplay in corneal epithelial cells in culture and also the effects  LaNt overexpression has on junctional complex assembly and matrix organisation.

In Poster 4 Tobi and Vale B’s work brings us back to whole tissues, this time using limbal explants as a 3D model of corneal wound repair and includes our data demonstrating changes in LaNt distribution as wounds heal.

Finally Umar and Vale B show some quite preliminary work of LaNt roles in angiogenesis and the effect of overexpression/knockdown on HUVEC tube formation.

I will now be sending all new students to study all these posters before starting work in the group. Seems like an easy way to get up to speed!

They grow up so fast – BSID 2017 edition

This week PhD student Lee Troughton and I have been attending the British Society for Investigative Dermatology meeting in exotic Manchester.

Lee presented some of our recent findings in poster form. This is the first time we have rolled out this new story and, so far, the response has been very positive.

Loads of data including contributions from Valentina Barrera and Conro Sugden. If anyone in the IACD was wondering why Lee was frantically running 1000s of qPCRs last week, check out the graphs in the bottom left. Nothing like the impending deadline to give a sense of urgency! However, the findings are really interesting and have opened up some really nice questions.

It’s also cool to be back at the BSID after many years away. I think my first “real” conference presentation was at a BSID in Oxford during my PhD where I presented some of our very preliminary data describing the first identification of the alternative splice isoforms that we now call the LaNts. It’s really cool to be back and to see many of the people I know in the skin field and learn about all the excellent derm research going on in the UK.

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Lee looking chuffed at his bold colour choices