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Characterization of tissue mechanics and cell cycle during wound healing : when software meets biology
Publication . Pereira, Telmo Ribeiro Ramos; Jacinto, António, 1965-; Almeida, Luis Manuel Lopes Neves de; Rino, José, 1976-
Wound healing is essential to all living organisms. All organisms need robust mechanisms to overcome injury, starting from a single cell wound to large chronic wounds in the human skin. Inflammation processes, tissue movement and proliferation along with tissue remodeling are critical in order to close and restore what once was a tissue discontinuity. All these events need to be extremely well regulated and coordinated: if one step fails wound healing may be compromised. The way all these processes are interconnected and how tissues activate the needed repair mechanisms is not yet fully understood. Our aim was to address how tissue mechanics affects wound healing and how the cell cycle is regulated during this process in the context of a living organism – in this case Drosophila melanogaster. To study these mechanisms in vivo, advanced imaging techniques are required, thus our first aim was to design new software tools to facilitate processing and analysis of the large amounts of data that are generated. On this note, we developed three open source software Matlab™ toolboxes that are easy to use and work fast: PIVBio allows us to successfully track wound closure movements without segmentation making it a perfect tool for a global analysis of cellular behaviors; CellSeg is a modular and integrated segmentation and analysis toolbox that, by implementing a new segmentation algorithm, helped us getting new insights on a novel phenomenon on the initial stages of wound healing, the actin flow; and CellDivTracker_fucciEdition that allows us to track cell division at single or whole tissue level over time. Additionally, the latter was successfully employed as a screen scoring tool that has proven to be useful to uncover new links between the cell cycle and wound healing. Our next aim was to understand how tissues responded mechanically to the wound insult. Upon detailed analysis of all the gathered data, we revealed that the edge curvature plays a fundamental role in the way the wounds close and in determining where cells assemble the actomyosin cable and filopodia and lamellipodia. To further analyze this discovery, we built a mathematical model taking into account all the previous knowledge. This model allowed us to understand what are the physical properties of the tissue that contribute significantly to wound closure in the Drosophila notum epithelium. We found that viscosity was the dominant property in the Drosophila tissue in detriment of a friction dominant tissue. This implies that cell migration and movement depends more on cell-cell interactions than on cell-substrate interactions, and thus shows that cell-cell adhesion complexes play a crucial role in wound closure in vivo. We took the model one step further by using it to investigate the role of myosin regulators on the tissue properties and wound closure. The model allowed us to understand that Stretchin-MLCK downregulates the actomyosin cable, which corresponds to a lower cable tension. Despite this tension loss, filopodia and lamellipodia are able to compensate for this deficiency in the actomyosin cable, albeit by engaging a different geometry. The third goal of this thesis was to investigate how the cell cycle is regulated and coordinated with the wound closure process. Interestingly, we found that the cells that surround the wound temporarily arrest at the G2 phase of the cell cycle, only resuming cell division after the wound has successfully closed. This type of arrest has not been described before and is accompanied by misregulation of key cell cycle players, such as Cyclin B, Cdc25 and chromatin condensation. Finally, we identified novel pathways that link the cell cycle arrest to the cellular behaviors occurring during wound healing, namely the Toll/NF-kB pathway, by performing a screen. Strikingly, when this pathway is knocked down, cells at the wound edge overcome the typical cell cycle arrest, dividing faster than wild type cells. Together, our novel software tools helped us to uncover new biological mechanisms by using new algorithms for data extraction and analysis. Additionally, the novel mathematical model developed revealed that the wound edge curvature gives an important contribution to wound closure and deepened our understanding of how the tissue and cells interact at a global level. Finally, we identified a new link between wound closure and the cell cycle: The Toll pathway. This unexpected connection has both expanded our knowledge and raised new and challenging questions that will be worth to explore in the future.
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Fundação para a Ciência e a Tecnologia
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SFRH
Número da atribuição
SFRH/BD/74745/2010