Matrix metalloproteinases (MMPs) are known to be the mediators of extracellular matrix remodeling. Increased levels of matrix metalloproteinases, particularly MMP-9, have been found in the aqueous humor of patients with glaucoma. However the exact role of MMP-9 in glaucomatous changes is not understood. Previous results from the West-Mays’ lab indicated that MMP-9 deficient (knockout - KO) mice exhibit elevated IOP, in the absence of distinct morphological changes in the anterior chamber. In the current thesis, I investigated whether the elevated IOP in MMP-9KO mice leads to RGC death. Wild type and KO littermates at different age groups: 2-3 months, 3-4 months, 6-8 and 9-12 months were studied. IOP was measured using TonoLab rebound tonometer. My results demonstrated that IOP was significantly increased in MMP-9KO mice compared to control littermates at all ages examined. To investigate if the elevated IOP was due to a difference in central corneal thickness (CCT), CCT measurements were made between WT and KO mice using ultrasound pachymeter. There was no difference in CCT demonstrating that the elevated IOP observed in MMP-9KO mice was not related to changes in corneal thickness. To determine whether the elevated IOP led to RGC death, the animals were sacrificed, eyes were enucleated and retinas (n=4) from both WT and KO animals were dissected and stained with Brn-3a antibody. Additional eyes were harvested from both WT and KO mice for histological and immunofluorescence studies. I found no observable difference in Brn3a+ RGC count between MMP9-WT and KO mice. Furthermore, no difference in retinal morphology, glial reactivity and laminin expression between WT and KO mice was observed. In the future it will be important to investigate whether elevated IOP in the MMP-9KO mice leads to optic nerve axonal loss and further investigate the possibility that the MMP-9KO retina is neuroprotected. / Thesis / Master of Science (MSc)

L'angiogenèse est une étape clef dans le processus de progression tumorale. Elle est caractérisée par une surexpresssion d'un grand nombre de métalloprotéinases matricielles (MMP). Parmis ces MMP, les gélatinases (MMP-2 et MMP-9) sont connues pour jouer un rôle important dans l'angiogenèse tumorale et la croissance de nombreux cancers. Les "Photodynamic Molecular Beacon" (PMB) sont des constructions moléculaires qui peuvent être utilisées dans le traitement de cancers en associant un photosensibilisateur (PS) de type chlorine et un inhibiteur d'états excités, aussi appelé "Quencher", liés par un peptide substrat des gélatinases afin d'inhiber la toxicité du PS dans les cellules non ciblées, et de restaurer sa toxicité uniquement à proximité des gélatinases. Nous avons donc cherché à déterminer le couple PS/quencher permettant la meilleure inhibition de la production d'oxygène singulet, principale source de la toxicité du PS, puis avons synthétisé une famille de PMB ciblant les gélatinases. Différents peptides et bras espaceurs ont été utilisés pour évaluer l'influence de la distance entre le PS et le quencher sur les propriétés photophysiques et l'activation enzymatique du PMB / Angiogenesis is a key step in the tumoral progression process. It is characterized by an over-expression of a number of matrix metalloproteinases (MMP). Among these MMPs, gelatinases (MMP-2 and MMP-9) are known to play a critical role in tumor angiogenesis and the growth of many cancers. Photodynamic Molecular Beacons (PMB) can be designed for cancer treatment by associating a chlorin-like photosensitizer and a black hole quencher linked by a gelatinase substrate peptide with the aim of silencing photosensitizer toxicity in non-targeted cells and restore its toxicity only in surrounding gelatinases. We investigated the PS/quencher pair allowing the best singlet oxygen production inhibition, and then we synthesized a novel family of PMB triggering gélatinases MMP-2 and MMP-9. Different lengths of peptide and spacers were used in order to determinate the influence of the distance between PS and quencher on the PMB photophysical properties and enzymatic activation.

Collective cell migration is a defining feature of many morphogenetic processes. Diseases such as congenital heart diseases and cancer arise due to mis-regulation of collective migratory behaviour and animal models have played a pivotal role in dissecting the molecular mechanisms which underlie this process. During embryonic heart development, cardiac precursors undergo a stage of collective migration in both vertebrates and invertebrates. We developed a paradigm to quantitatively assess collective cell migration of cardiac precursors in live embryos of Drosophila, which is the simplest genetic model organism with a heart. Therefore, we studied processes which are commonly observed in most collective cell migration models such as guidance signalling and extracellular matrix remodelling. Our results demonstrate that leading edge of migrating cardioblasts is highly active and that this behaviour is regulated by guidance cues, Slit and Netrin and their respective receptors Robo/Robo2 and Frazzled/Uncoordinated5. These molecules cooperatively promote leading edge motility and epithelial characteristics of the cardioblasts. Next, we determined that matrix restructuring around the cardioblasts requires proteases Mmp1 and Mmp2, which are members of the highly conserved Matrix Metalloproteinase family. We demonstrate that Mmp1 and Mmp2 have distinct roles during lumen formation, however, both Mmp1 and Mmp2 are required for collective motility of the cardioblast leading edge. Hence, we propose that embryonic heart development in Drosophila is an effective and amenable model of collective cell migration which can be applied to discover unique mechanisms which coordinate cell movement in groups. / Thesis / Doctor of Philosophy (PhD)

Matrix remodeling could be an important mode of action of multipotent mesenchymal stromal cells (MSC) in extracellular matrix (ECM) disease, but knowledge is limited in this respect. As MSC are well-known to adapt their behavior to their environment, we aimed to investigate if their mode of action would change in response to healthy versus pathologically altered ECM. Human MSC-derived ECM was produced under different culture conditions, including standard culture, culture on Matrigel-coated dishes, and stimulation with the pro-fibrotic transforming growth factor-1 (TGF1). The MSC-ECM was decellularized, characterized by histochemistry, and used as MSC culture substrate reflecting different ECM conditions. MSC were cultured on the different ECM substrates or in control conditions for 2 days. Culture on ECM increased the presence of surface molecules with ECM receptor function in the MSC, demonstrating an interaction between MSC and ECM. In MSC cultured on Matrigel-ECM and TGF1-ECM, which displayed a fibrosis-like morphology, gene expression of collagens and decorin, as well as total matrix metalloproteinase (MMP) activity in the supernatant were decreased as compared with control conditions. These results demonstrated that MSC adapt to their ECM environment, which may include pathological adaptations that could compromise therapeutic efficacy.