Wound infection is a major clinical burden that can significantly impede the healing process and cause severe pain. Prolonged wound infection can lead to long-term hospitalization or death. Pre-clinical research to evaluate new drugs or treatment strategies relies on animal studies. However, animal studies have several challenges including interspecies variations, cost, and, ethics question the success of these models. Recent advances in tissue engineering have enabled the development of in vitro human skin models for wound infection modeling and drug testing. The existing skin models are mostly representative of the healthy human skin and its normal functions. However, to study the wound healing process and the response of skin to the infection, there is still a need to develop a skin model mimicking the wound infection. This work presents a simplified functional infected epidermis model, fabricated with enzymatically crosslinked gelatin hydrogel. The immortalized human keratinocytes, HaCaT cells, was successfully cultured and differentiated to a multilayer epidermis structure at the air-liquid interface, and expressed terminal differentiation marker, filaggrin, in the outer layer. The barrier function of the epidermis model was studied by measuring the electrical resistance and tissue permeability across the layer. The results showed that the developed epidermis model offered a higher electrical resistance and a lower drug permeability compared to the cell monolayer on gelatin and cell-free gelatin. To show the capability of the developed epidermis model in wound modeling and drug, the model was infected with Escherichia coli and the inflammatory response of keratinocytes was studied by measuring the level of proinflammatory cytokines, including IL-1β and TNF-α. The results demonstrated the proinflammatory response of the epidermis model to infection by producing a higher level of TNF-α and IL-1β compared to the control group. While treating with antibiotic ciprofloxacin terminated the proinflammatory response and reduced the level of TNF-α and IL-1β. The robust fabrication procedure and functionality of this model suggest that this model has great potential for wound modeling and high throughput drug testing. / Graduate

La lysyl oxydase (LOX) est une enzyme extracellulaire dont le rôle canonique est de catalyser la réticulation des fibres de collagènes et de l’élastine, assurant ainsi l’intégrité des tissus conjonctifs. La LOX agit également dans plusieurs types cellulaires comme régulateur de différents processus biologiques, soulignant des fonctions à la fois extra et intracellulaires. Ces travaux de thèse ont contribué à améliorer la compréhension du rôle de LOX dans l’épiderme, et plus précisément au cours de la différenciation des kératinocytes. Le développement d’un modèle de culture en monocouche à confluence, et d’un modèle tridimensionnel d’épiderme reconstruit ont permis d’aborder l’étude de LOX au cours de l’induction du programme de différenciation des kératinocytes et du processus de différenciation terminale conduisant à la formation d’un épiderme pluristratifié, cornifié et fonctionnel. L’expression de LOX est induite au cours des premières étapes de la différenciation de kératinocytes primaires ainsi que d’une lignée de kératinocytes immortalisés. Grâce à l’établissement de lignées de kératinocytes éteignant l’expression de LOX de façon stable, nous avons mis en évidence l’implication de la protéine LOX, indépendamment de son activité enzymatique, dans la régulation des premières étapes de différenciation des kératinocytes. En absence de LOX, l’initiation du programme de différenciation est perturbée, affectant la différenciation terminale et fonctionnelle des épidermes reconstruits. Ainsi, une régulation fine de l’expression de LOX est nécessaire au déroulement normal du processus de différenciation des kératinocytes, et donc au maintien de l’homéostasie épidermique / Lysyl oxidase (LOX) is an extracellular enzyme that catalyzes the cross-linking of fibrillar collagens or elastin, thereby regulating the structural integrity of connective tissues. Moreover, LOX displays multiple roles in different cell types, acting as a regulator of various biological processes at both extra and intracellular levels. The aim of the present work was to shed light on LOX functions in the epidermis, especially during keratinocyte differentiation. The development of culture models, consisting of confluent monolayers or reconstructed-epidermis allowed us to study LOX functions during the induction of the differentiation program, and furthermore during the terminal differentiation process leading to the formation of a pluristratified, cornified and functional epidermis. LOX expression is induced at the onset of the commitment to differentiation, both in primary and immortalized keratinocytes. Stable silencing of LOX expression affects the induction of the differentiation program and strongly impairs terminal and functional epidermal differentiation in reconstructed-epidermis. Therefore, LOX protein acts during the first steps of keratinocyte differentiation, independently of its enzymatic activity, and is implied for subsequent commitment into terminal differentiation. Taken together, these results suggest that a finely regulated expression of LOX is required for normal keratinocyte differentiation, and thus for maintenance of epidermal