Icon for: Stephanie Luff

STEPHANIE LUFF

University of Delaware
Years in Grad School: 2
Judges’
Choice

Judges’ Queries and Presenter’s Replies

  • May 20, 2013 | 04:10 p.m.

    Hello,

    how is the IGERT structure helping your research? How is your work related overall to your IGERT’s goals?

  • Icon for: Stephanie Luff

    Stephanie Luff

    Presenter
    May 20, 2013 | 05:30 p.m.

    Thank you for your question.

    Beyond the significance of general interaction with others in multiple disciplines, as part of my IGERT acceptance, I need to do a rotation in computational systems biology. This past semester, I took a systems biology course to prepare me for the rotation. The course has taught me to think more critically about the microarray data I’ve presented here, as well as helped me to think about the types of systems biology questions pertaining to my system. Also, as part of our work with the Innovation rotation, I’ve learned how to build biological systems, all while maintaining technical feasibility and economic restraint. This is related to the overall IGERT goals because our program focuses on systems biology and engineered environments. My research has always had a more engineered environment bias but the IGERT program has helped me immensely to focus more on systems biology.
  • May 21, 2013 | 10:55 a.m.

    Hi!
    If shear stress is much required for megakaryocyte maturation, how will you combat apoptosis caused by the same?

  • Icon for: Stephanie Luff

    Stephanie Luff

    Presenter
    May 21, 2013 | 11:57 a.m.

    I didn’t talk about this in the video because of the time constraints and desire to keep the video as simple as possible, but the other focus of my project addresses this concern. My other focus involves how the transcription factor p53 is involved in the maturation process, especially in response to shear stress (you can take a peek at my poster for more details). Previous work in our lab has shown that by knocking out p53, we can significantly decrease apoptosis in megakaryocytes. However, this would only be necessary for ex vivo platelet production IF we find increased apoptosis to be a hinderance to platelet production. There is a lot of disagreement in the field about the role of apoptosis for platelet production – some think it’s necessary, some think it’s merely a side effect. As a biologist, I could see how apoptotic processes could encourage platelet production (in particular – the process of membrane blebbing). So my reasoning for focusing on apoptosis isn’t to make a statement that it’s a good or bad thing, rather that this process which is normally seen after platelet production is encouraged by shear stress – indicating that shear stress can speed up the maturation process. Normally in culture, apoptosis isn’t seen until after day 10. My treatments were on day 9, further indicating that shear stress can accelerate maturation. I hope this addresses your question, thank you.

  • May 21, 2013 | 12:32 p.m.

    Did p53 expression levels change in your screen?

  • Icon for: Stephanie Luff

    Stephanie Luff

    Presenter
    May 21, 2013 | 01:17 p.m.

    At the transcriptional level, I did surprisingly see some downregulation. However, between donors (we use primary hematopoietic stem cells), there was some variability. At the translational level, we’ve shown through preliminary western blot analysis that p53 expression doesn’t significantly change in response to shear stress, but I’d like to confirm this in the immunofluorescence assay I’ve been using with the acetylation investigation. My prediction is that its activity is more regulated through post-translational modification (i.e. acetylation, my poster explains in greater detail) than through gene regulation, as this is quite common for p53 regulation.

  • Icon for: Zhaomin Yang

    Zhaomin Yang

    Judge
    May 21, 2013 | 08:24 p.m.

    Has the effect of shear stress on other cell types been investigated and if so, how do the different data sets compare?

  • Icon for: Stephanie Luff

    Stephanie Luff

    Presenter
    May 22, 2013 | 10:11 a.m.

    Great question! Shear stress has been much more thoroughly studied in three cell types (this isn’t an inclusive list, just the three biggest areas of shear stress-related research):

    1) Osteoblast: I’m not too familiar with the data sets of this field (other than it promotes differentiation) as it’s a bit outside my scope.

    2) Endothelial cells: The type of flow greatly impacts the effect of the resulting shear stress. Steady, laminar flow is protective against athlerosclerosis and is vital for general endothelial cell function. However, turbulent flow results in a loss of the protection and increased apoptosis. (A great review can be found here: doi:10.1001/jama.282.21.2035)

    3) Platelets: For platelets, shear stress is more straight forward. From all that I’ve read, it appears that rate and type of flow isn’t as critical in determining its effect. All levels of shear stress induce some degree of aggregation and prime the platelets for activation. (A couple good articles are: 10.1007/s11239-009-0397-0 and 10.1007/s12573-011-0039-y)

    All of this makes logical sense. Endothelial cells and platelets see shear stress throughout the entirety of their life. It makes sense that it plays a significant role in their normal cell processes. With megakaryocytes, it’s not clear at all. Different Mk cells see different degrees of shear stress at different time points. Because of this, we’ve had to take the time to decide what levels of shear stress and at which time points are most appropriate for the greatest number of Mks. The effects of shear stress at day 9 with a medium level of physiological shear stress may not be identical to the effects of a premature Mk that enters the blood stream and travels to the lung, for example. This is why the implications of shear stress in Mks is such an interesting thing to study – it’s not as obvious or clear cut as it is with other cells.

  • May 21, 2013 | 08:53 p.m.

    HI Stephanie,
    Have you looked (look in the future) at phosphorylation levels of the proteins active at this checkpoint?

  • Icon for: Stephanie Luff

    Stephanie Luff

    Presenter
    May 22, 2013 | 10:22 a.m.

    That’s very interesting that you ask this! I actually have very preliminary immunofluorescence data that doesn’t show a significant difference in the phosphorylation levels of Chk1 at S345. So this could mean a few things:

    1) 2 hours of shear stress isn’t enough time to see upregulation plus subsequent protein production and activation (I’m not sure if this is the case, but I have some future work that could help address this)

    2) The Serine residue that is responsible for shear-induced activation may be S317 instead of S345 (This is a very simple thing to check, just buy a new antibody!)

    3) The upregulation, while real in those samples, may be donor-specific responses. I’m doing more microarray analysis and will be able to determine if this is a conserved response or donor-specific. (We use primary hematopoietic stem cells to derive our Mks, so we see a lot of variability between donors. That’s unfortunately very common with primary cells)

  • Further posting is closed as the competition has ended.

Presentation Discussion
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    Matthew Ralston

    Guest
    May 20, 2013 | 12:30 p.m.

    This video is very informative. The animation is choice and her figures so clear. Excellent work Stephanie!

  • Icon for: Stephanie Luff

    Stephanie Luff

    Presenter
    May 20, 2013 | 03:42 p.m.

    Thank you Matt!

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    Mohab Al-Hinai

    Guest
    May 20, 2013 | 10:36 p.m.

    Well done!! Nicely made.

  • Icon for: Stephanie Luff

    Stephanie Luff

    Presenter
    May 21, 2013 | 12:12 a.m.

    Thanks Mohab :)

  • Icon for: Tony Reames

    Tony Reames

    Trainee
    May 21, 2013 | 12:10 a.m.

    Great video and research! Eye catching intro.

  • Icon for: Stephanie Luff

    Stephanie Luff

    Presenter
    May 21, 2013 | 12:12 a.m.

    Thanks a lot Tony!

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    Daniel Nasko

    Guest
    May 21, 2013 | 01:31 p.m.

    Informative, clear and concise.
    Awesome video Stephanie!

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    Stephanie Luff

    Presenter
    May 21, 2013 | 01:47 p.m.

    Thanks Dan .

  • Icon for: Eamonn Walker

    Eamonn Walker

    Trainee
    May 22, 2013 | 11:36 a.m.

    Hi, Stephanie.
    Great video! You did a good job of both visually and verbally explaining the processes involved. I would be interested in hearing how you generated your animations.
    And as with most good scientific presentations, it made me want to learn more about the topic, so I hope you don’t mind a few technical questions.
    What were the end applications associated with this research? Are you interested in knowing how to efficiently generate platelets artificially, or more in understanding how the body generates them, and if the process can be affected in vitro?
    Also, in your poster I see that you compared a static and a constant-shear state in the cells. Have you investigated what affect, if any, the magnitude of the stress has on the various biological processes? Can you increase platelet production, for example, by increasing the flow rate?
    It’s also been quite a few years since I studied blood flow in the circulatory system. How does the shear experienced by vessel walls compare in larger blood vessels versus capillaries? Are more capillaries produced in one level of blood vessels than another?
    Thanks.

  • Icon for: Stephanie Luff

    Stephanie Luff

    Presenter
    May 22, 2013 | 01:07 p.m.

    Thanks for the questions! The animations were made in Final Cut Pro, based of animations I use in my powerpoint presentations.

    The end applications are mostly focused on two areas.
    1) To make better therapies for those with bleeding disorders
    2) To make platelets ex vivo (aka in vitro) to give to people who need platelet transfusions (those with the bleeding disorders or cancer – chemotherapy greatly decreases platelet counts). Transfusions are very expensive and platelets can’t be frozen (they lose functionality when frozen). So obtaining platelets from donors isn’t ideal. My lab is a Chemical Engineering lab (even though I’m a biologist), so this is the long term goal of our lab. Because I’m a biologist, I focus on understanding how Mks generate the platelets, but once we have this knowledge, the hope is that we can manipulate this information to increase platelet production in culture.

    Great question about the magnitude. The data I presented here was with the lowest (1.0dyn/cm2) physiological shear stress (determined to be about 1.0-4.0 dyn/cm2 in the marrow vasculature) . My goal for the summer is to do microarray analysis on cells with stronger shear stress (2.0 dyn/cm2), so I’m interested in seeing how the data differs.

    In terms of platelet yield from shear stress, my lab mate found that greater shear stress resulted in more platelets produced. Static cells made relatively few platelets, low shear rates were much higher than static, and high shear rates had even more than the low shear.


    Large blood vessels (arteries, veins, vena cava, aorta etc.) have much lower shear rates. Smaller vessels like arterioles and cappilaries (and to a lesser extent, venules) have much higher shearing rates. While the marrow sinus vasculature is smaller than large blood vessels, the physiology of the region is quite different, and shear rates are much lower in these vessels. About the amount of capillaries, I’m not quite sure what you’re asking, I apologize.

  • Icon for: Eamonn Walker

    Eamonn Walker

    Trainee
    May 22, 2013 | 02:21 p.m.

    Sorry, that was a typo on my part. I meant to ask if more platelets were produced in veins or arteries versus capillaries. Of course, I now realise the last two questions were irrelevant, as I forgot my high school biology and was imagining this process taking place in the blood vessels instead of the bone marrow. But thank you for answering, anyway.

    Still an interesting project. I wish you luck in your further research.

  • Icon for: Stephanie Luff

    Stephanie Luff

    Presenter
    May 22, 2013 | 04:26 p.m.

    No need to apologize, it’s understandable for information to be tucked away when you haven’t used it in a while :)

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    Prathamesh

    Guest
    May 22, 2013 | 06:47 p.m.

    Supercool animation! Very informative work, Stephanie.

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    Stephanie Luff

    Presenter
    May 22, 2013 | 07:12 p.m.

    Thank you very much!

  • Icon for: Kathryn Furby

    Kathryn Furby

    Trainee
    May 23, 2013 | 01:05 a.m.

    Hey Stephanie, just wanted to say great job again. Did you use Flash to make the animated parts?

  • Icon for: Stephanie Luff

    Stephanie Luff

    Presenter
    May 23, 2013 | 09:55 a.m.

    Thanks so much! I used Final Cut Pro actually. They were mostly based off animations I use in my powerpoint presentations, so I didn’t really have to create new graphics or plan how they would move, I just had to make the animation happen!

  • May 23, 2013 | 01:12 a.m.

    That introduction was amazing! You mentioned in an above response that one goal is to make platelets ex vivo for patients. Would the platelets need to be customized for each individual, and how long would it take to make enough for a patient?

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    Stephanie Luff

    Presenter
    May 23, 2013 | 09:51 a.m.

    That’s actually the beauty of ex vivo platelet generation – you can customize it to each individual. With the current platelet donation-transfusion method, alloimmunization of the donated platelets can occur in the transfused individual.

    With ex vivo platelet production, as long as the individual doens’t have cancerous progenitors, we would be able to harvest their own stem cells and make platelets from them! The time it would take to make enough is truly dependent upon the system. Transfusions add ~3.5^11 platelets to the patient’s circulatory. Current ex vivo production methods aren’t efficient enough to make this many platelets in a cost effective manner (meaning, they need a LOT of expensive stem cells!) If you’re curious, an excellent review recently came out about this issue: DOI 10.1182/blood-2012-09-455428

  • Further posting is closed as the competition has ended.