UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
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Item MECHANICAL MAPPING OF NEURAL TUBE CLOSURE IN LIVE EMBRYOS USING BRILLOUIN MICROSCOPY(2022) Handler, Chenchen; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neurulation is a process that serves as the precursor to the spinal cord in vertebrates. Neural tube closure (NTC), part of primary neurulation, involves the extensive coordination of cellular, molecular, and mechanical events to transform the flat neural epithelium to a luminated epithelial tube. Neural tube defects (NTD) are the result of mechanical failures that arise during neurulation. Recent research has focused on understanding the molecular mechanisms underlying neurulation but has difficulty correlating them to physical mechanisms. To better understand how physical mechanisms are integrated and responsible for neurulation, several techniques have been applied to study NTC in a range of in vitro environments. However, many of these techniques have been limited due requiring the specimen to be fixed and/ or being invasive and requiring physical contact with the specimen to extract the modulus. As such, there is limited resolution and only the superficial layer of the sample is measured making assessing 2D/3D tissue mechanics inside a growing organism is highly challenging. In this dissertation, we aim to quantify the mechanical state of the neural tube without disruption to development. To do this, we adapted Brillouin microscopy, a non-invasive, label- and contact-free imaging technique, to allows us to probe thelongitudinal modulus of the neural plate at every step of NTC with cellular resolution. This quantification is performed as the embryo develops in real time using time-lapse Brillouin and an improved ex-ovo culture method. We observed an increase in the Brillouin modulus of the neural plate as the embryo develops from Hamburger-Hamilton stage (HH)-6 to HH-12. This increase in modulus is consistent with previous data from other vertebrates such as Xenopus and Mouse embryos and demonstrates the process of neurulation is driven by mechanical forces. Time-lapse Brillouin imaging depicted stiffening and thickening of the neural plate during NTC, suggesting these are coordinated events for NTC. Here, we show that tissue stiffness plays an integral role in NTC and directly quantifying tissue mechanics during neurulation should allow us to better determine the biomechanical nature of NTD.Item Mechanical Adaptability of Ovarian Cancer Tumor Spheroids(2021) Conrad, Christina Barber; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A major obstacle in ovarian cancer treatment is the onset of ascites, an abnormal build-up of fluid in the peritoneal cavity. Using in vitro perfusion models, ascitic flow has been shown to drive epithelial-mesenchymal-transition (EMT) biomarker expression, promote epidermal growth factor receptor (EGFR) downstream signaling, and upregulate chemoresistance. Given the close ties between cell mechanics and behaviors, it is of interest to establish if mechanotransduction serves a role in cell signaling dysfunction. Here, we identified the mechanical behavior of tumor spheroids subjected to flow using Brillouin confocal microscopy, a non-contact optical method based on the interaction between incident light and microscopic mechanical waves within matter. We validated this technique by establishing a relationship with the traditionally derived Young’s modulus measured using atomic force microscopy and a parallel-plate compression device. Following characterization, we used Brillouin confocal microscopy to map mechanical properties of tumor spheroids embedded in a microfluidic chip and found that continuous flow for 7 days caused a decreased Brillouin shift (i.e., stiffness) compared to tumors in a static condition. Another physical phenomenon related to ascites is dysregulated osmolality. Maintaining cell water homeostasis is driven by the transport of water to balance solute concentration and can have direct consequences on mechanics and biochemical signaling in cells. Recently, it was demonstrated in single cells that cell volume correlated with mechanical properties; but the effects in tumor spheroids which exhibit multi-cellular interfaces has remained unclear. Here, we derived relationships between osmolality and nuclear volume, tumor cell density, and Young’s modulus, and found the correlations in spheroids resembled single cell relationships previously described in literature. Additionally, we looked at the impact of osmotic shocks on E-cadherin junctions and found aggregates formed with a unique timescale compared to morphology. Lastly, we observed reversibility of the mechanical, morphological, and molecular properties which showed the tumor’s dynamic ability to respond to physical cues. Altogether, this work demonstrated how flow and osmosis associated with ovarian cancer ascites can trigger phenotype transformations. These findings warrant future investigations into how the regulation of mechanotransduction pathways can be harnessed to prevent chemoresistance and signaling dysfunction.