Évaluation de l'efficacité de la pasteurisation à la vapeur pour le contrôle de la contamination microbiologique des carcasses bovines

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

Development of fluorescent biomarker for the in vivo diagnosis of infectious keratitis

La cornée est la partie la plus antérieure de l’œil et possède un rôle majeur dans la vision. La cécité par atteinte cornéenne est la 4e cause de cécité dans le monde et ces atteintes sont souvent d’origine infectieuse et causées par tous types d’agents pathogènes. L’objectif de la présente thèse consiste au développement de biomarqueurs fluorescent pour le diagnostic minute de ces kératites infectieuses in vivo. Cette recherche se subdivise en 3 axes principaux qui sont 1/ La mise en place de modèles d’infection in vitro et ex vivo de ces kératites infectieuses. 2/ La production de biomarqueurs fluorescents spécifiques des agents infectieux. 3/ Le développement d’un système d’imagerie ultra sensible et compatible avec l’imagerie après marquage de l’infection in vivo. Une autre partie traitée dans cette thèse concerne la capacité du sars-cov-2 à infecter la surface oculaire, mais également la recherche de sa présence dans les tissus et les milieux de conservation des cornées destinées à la greffe.The cornea is the most anterior part of the eye and plays a major role in vision. Blindness due to corneal damage is the 4th worldwide cause of blindness and these damages are often of infectious origin and caused by all types of pathogens. The objective of this thesis is to develop fluorescent biomarkers for the in vivo diagnosis of these infectious keratitis. This research is subdivided into 3 main axes which are 1/ The setting up of in vitro and ex vivo infection models of these infectious keratitis. 2/ The production of fluorescent biomarkers specific to these infectious agents. 3/ The development of an ultra sensitive imaging system and compatible with the imaging after marking of the infection in vivo. Another part of this thesis concerns the capacity of sars-cov-2 to infect the ocular surface but also the search for its presence in the tissues and conservation media of corneas intended for transplantation

A molecular chaperone dedicated to folding and translocation of reductive dehalogenases

Reductive dehalogenases (rdhA, RDases) are key enzymes involved in anaerobic organohalide respiration (OHR), during which bacteria are able to use chlorinated compounds as terminal electron acceptors. RDases are redox enzymes containing FeS clusters and a corrinoid as cofactors, and are translocated across the cytoplasmic membrane by the Twin-arginine translocation (Tat) system. In members of Dehalobacter and Desulfitobacterium spp., the product of an accompanying gene, generally named rdhT, was recently proposed to play a role as molecular chaperone in the folding of the reductive dehalogenase (1,2). Recently, this finding was applied to heterologously produce active RDases (3). However, the mechanism by which the molecular chaperone acts on the maturation of RDases is not yet solved. In this study, we investigate the diversity of RdhT chaperones in Dehalobacter restrictus and their interaction with the Tat signal peptide of their cognate redox component, as well as their specificity or cross-reactivity towards alternative signal peptides. To this respect, both in vivo and in vitro experimental approaches are conducted and will be presented. References (1) Morita et al., 2009. Appl. Microbiol. Biotechnol. 83:775. (2) Maillard et al., 2011. Microbiol. 157:2410. (3) Mac Nelly et al., 2014. Appl. Environ. Microbiol. 80:4313

FOLDING OF COMPLEX REDOX PROTEINS BY DEDICATED MOLECULAR CHAPERONES

Chlorinated compounds (so-called organohalides) are widespread soil and groundwater pollutants. Only few bacteria have the ability to degrade these compounds via organohalide respiration (OHR). Reductive dehalogenases (RDases) are complex redox enzymes involved in the reduction of organohalides, and contribute to the biodegradation of these pollutants. RDases need to be folded and loaded with iron-sulfur centers and a corrinoid cofactor prior to their transport across the cytoplasmic membrane via the Twin-arginine translocation (Tat) pathway. A new family of Tat molecular chaperones, named RdhT, was recently shown to participate in the maturation of RDases (1-2), and successfully applied for heterologous production of these complex redox enzymes (3). The present study focuses on the interaction of RdhT molecular chaperones with their cognate RDases. PceT (the paradigmatic member of the RdhT family) interacts as a dimer with the Tat signal peptide of PceA, its cognate RDase, as shown by isothermal titration calorimetry. When recombinant pceT and pceAHis genes are heterologously expressed in E. coli, both proteins co-purify on Ni-NTA chromatography which indicates that PceT binds to PceA also in vivo. Although recombinant PceA is not functional in E. coli, it is produced in a soluble form when pceT is co-expressed and represent the basis for reconstitution experiments. Currently, in vivo strategies are developed in E. coli to allow a rapid screening of interacting RdhT chaperones with the Tat signal peptides of RDases. This will further help evaluating the cross-reactivity of RdhT chaperones towards Tat signal peptides, and help identifying specific amino acids of the chaperones that are involved in the interaction event. References: 1. Morita Y, Futagami T, Goto M, Furukawa K. Appl Microbiol Biotechnol 2009;83:775-81. 2. Maillard J, Genevaux P, Holliger C. Microbiology 2011;157:2410-21. 3. Mac Nelly A, Kai M, Svatoš A, Diekert G, Schubert T. Appl Environ Microbiol 2014;80:4313-22

Lifetime extension for large offshore wind farms: Is it enough to reassess fatigue for selected design positions?

Fatigue reassessment within the decision process of lifetime extension might be uneconomical when individually performed for each turbine of a large offshore wind farm. This paper analyses the possibilities to extrapolate results from fatigue reassessment of wind turbines at selected design positions to other wind turbines of the wind farm. Five monopile-based turbines placed in a generic offshore wind farm were assessed with integrated aero-hydro-elastic simulations. A fatigue assessment was performed for each out of the five selected turbines using site specific environmental data commonly available during the design process. The results were compared to a fatigue reassessment where environmental data were modified in order to account for changes in environmental conditions during the service lifetime of the wind turbines. Results indicate that an extrapolation is feasible for selected parameters when changes in environmental conditions are small. This is an important step towards an effective and efficient assessment methodology for lifetime extension of offshore wind turbines

Investigating the Role of TGF-β Signaling Pathways in Human Corneal Endothelial Cell Primary Culture

Corneal endothelial diseases are the leading cause of corneal transplantation. The global shortage of donor corneas has resulted in the investigation of alternative methods, such as cell therapy and tissue-engineered endothelial keratoplasty (TEEK), using primary cultures of human corneal endothelial cells (hCECs). The main challenge is optimizing the hCEC culture process to increase the endothelial cell density (ECD) and overall yield while preventing endothelial–mesenchymal transition (EndMT). Fetal bovine serum (FBS) is necessary for hCEC expansion but contains TGF-βs, which have been shown to be detrimental to hCECs. Therefore, we investigated various TGF-β signaling pathways using inhibitors to improve hCEC culture. Initially, we confirmed that TGF-β1, 2, and 3 induced EndMT on confluent hCECs without FBS. Using this TGF-β-induced EndMT model, we validated NCAM as a reliable biomarker to assess EndMT. We then demonstrated that, in a culture medium containing 8% FBS for hCEC expansion, TGF-β1 and 3, but not 2, significantly reduced the ECD and caused EndMT. TGF-β receptor inhibition had an anti-EndMT effect. Inhibition of the ROCK pathway, notably that of the P38 MAPK pathway, increased the ECD, while inhibition of the ERK pathway decreased the ECD. In conclusion, the presence of TGF-β1 and 3 in 8% FBS leads to a reduction in ECD and induces EndMT. The use of SB431542 or LY2109761 may prevent EndMT, while Y27632 or Ripasudil, and SB203580 or SB202190, can increase the ECD

Femtosecond Laser Cutting of Human Crystalline Lens Capsule and Decellularization for Corneal Endothelial Bioengineering

The bioengineering of corneal endothelial grafts consists of seeding in vitro cultured corneal endothelial cells onto a thin, transparent, biocompatible, and sufficiently robust carrier which can withstand surgical manipulations. This is one of the most realistic alternatives to donor corneas, which are in chronic global shortage. The anterior capsule of the crystalline lens has already been identified as one of the best possible carriers, but its challenging manual preparation has limited its use. In this study, we describe a femtosecond laser cutting process of the anterior capsule of whole lenses in order to obtain capsule discs of 8 mm diameter, similar to conventional endothelial grafts. Circular marks made on the periphery of the disc indicate its orientation. Immersion in water for 3 days is sufficient to completely remove the lens epithelial cells and to enable the seeding of corneal endothelial cells, which remain viable after 27 days of culture. Therefore, this method provides a transparent, decellularized disc ready to form viable tissue engineered endothelial grafts