Development of a cell motility characterization system for industrial biotechnological applications
Villella, Valeria Rachela (2010) Development of a cell motility characterization system for industrial biotechnological applications. [Tesi di dottorato] (Inedito)
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Cell migration is a very complex mechanism linked to the inflammatory response as well as to several processes of cell biology and development. The movement of cells in response to a chemokine gradient is the most popular form of interaction between cell environment and surface and specific surface receptor triggers an intracellular signaling pathway. This phenomenon is known as chemotaxis and is characteristic of neutrophil movements in response to the chemokine Interleuchin-8 (IL8) in a 3D space. Another paradigm of dynamic interaction between cells is the mechanism underlying the confluency of cells cultured in a Petri dish. This process is particularly studied in growing epithelial cell lines, and is influenced by still undefined external stimuli as well as by cell proliferation and cytoskeleton reorganization, that allow the contact and interaction between cells in 2D space. Chronic inflammation is an ideal condition for the study of cell migration and its de-regulation; understanding the mechanisms involved in cell motility and their putative modulation, is the first step in setting new appropriate models of study. During PhD project, different cellular models have been used: IB3 cell lines belonging to patients with Cystic Fibrosis (caused by mutations of cystic fibrosis transmembrane regulator (CFTR)), and T84 cell lines, a popular model of study of Coeliac Disease, a common intolerance to proteins of wheat. Both disease are characterized by a pro-inflammatory milieu with high level of tissue Transglutaminase (TG2), a multi-functions enzyme with a defined role in several human pathologies. Cystic Fibrosis is the prototype of diseases in which an uncontrolled production of IL8 leads to a dysregulated neutrophil recruitment. The IB3 cell lines have been used to understand a molecular mechanisms of enhanced IL8 production and their modulation. The results evidence i) the role of TG2 as a new pathogenic factor in Cystic Fibrosis; ii) the role of post-translational modifications of TG2 as a link between genetic defect of CFTR ad inflammation iii) the mechanisms of autophagy inhibition via ROS-TG2 axis, in epithelial airway cell line and mice model of CF. The consequence is a fine modulation of IL8 production. The system allows the design of a 3D model to study cell migration under IL8 diffusive flux in qualitative and quantitative ways. Another important pathological system with chronic inflammation is celiac disease and the analysis of interactions of alimentary peptides with the frontline gut epithelium is a useful model of study. The epithelial cells are pivot in the innate immune activation in CD and cytoskeleton rearrangement is the earliest event in such a response to gliadin peptides. The data in T84 cell model show a gliadin peptide-driven pro-inflammatory environment as a consequence of its impaired lysosomal degradation. Moreover, alterations of motility and cytoskeletal reorganization emphasize the “toxic” effect of peptide. Therefore, CD offers an ideal opportunity to set up a 2D model of study of cell motility. Understanding the mechanisms of migration and the identification of appropriate target of modulation of cell recruitment could allow wide industrial applications in biotechnology and will be successful to provide a useful tool to test potential therapeuthic molecules.
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