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Rupture of the anterior cruciate ligament (ACL) is one of the most common ligamentous injuries of the knee. Post rupture, the ACL does not heal on itself due to poor vasculature and hence surgical intervention is required to treat the ACL. Current surgical management of ACL rupture consists of reconstruction with autografts or allografts. However, the limitations associated with these grafts have prompted interest in tissue engineered solutions that combine cells, scaffolds and stimuli to facilitate ACL regeneration. This thesis describes a ligament tissue engineering strategy that involves incorporating biomolecules within fibers-based electrospun meshes which mimics the extra-cellular matrix microarchitecture of ligament. However, challenges exist with incorporation of biomolecules. Therefore, the goal of this research project was to develop two techniques to incorporate biomolecules within electrospun meshes: (1) co-axially electrospinning fibers that support surface-grafting of biomolecules, and (2) co-axially electrospinning fibers decorated with biomolecule-loaded microspheres. In the first approach, chitosan was co-axially electrospun on the shell side of poly caprolactone (PCL) and arginine-glycine-aspartate (RGD) was attached to the electrospun meshes. Bone marrow stromal cells (BMSCs) attached, spread and proliferated on these meshes. In the second approach, fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) loaded chitosan-alginate (CS-AL) microspheres were fabricated. The effects of cation to alginate ratio, type of alginate and concentration of CaCl2 on microsphere size, FITC-BSA loading and release were systematically evaluated. The CS-AL microspheres were then incorporated into the sheath phase of co-axially electrospun meshes to achieve microsphere-decorated fiber composite meshes. The results from these model study suggest that both approaches are tractable for incorporating biomolecules within fibers-based electrospun meshes. Both these approaches provide platform for future studies that can focus on ligament-relevant biomolecules such as FGF-2 and GDF-5. / Master of Science

Yes / More than 2 million people/year suffer a bone fracture in the UK1. Reconstruction of bone defects represents a major clinical challenge and is addressed using a number of medical devices. Although medical device compositions and applications may differ widely, all attract microorganisms and represent niches for medical device associated infections. For open fractures, the risk of infection can be 55%2. These infections are often resistant to many of the currently available antibiotics and represent a huge and growing financial and healthcare burden. The aim of this study was a fundamental understanding of how the presence of host defence peptides (HDPs)3 and/or RGD can influence the outcome of cell vs. bacterial viability and proliferation. / Presented at the conference: eCM XVI - Bone and Implant Infection June 24-26, 2015, Convention Centre, Davos Platz, Switzerland.

Yes / Alterations in integrin expression and function promote tumour growth, invasion, metastasis and neoangiogenesis. Head and neck cancers are highly vascular tumours with a tendency to metastasise. They express a wide range of integrin receptors. Expression of the αv and β1 subunits has been explored relatively extensively and linked to tumour progression and metastasis. Individual receptors αvβ3 and αvβ5 have proved popular targets for diagnostic and therapeutic agents but lesser studied receptors, such as αvβ6, αvβ8, and β1 subfamily members, also show promise. This review presents the current knowledge of integrin expression and function in squamous cell carcinoma of the head and neck (HNSCC), with a particular focus on the RGD-binding integrins, in order to highlight the potential of integrins as targets for personalised tumour specific identification and therapy.

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Actuellement, la chirurgie représente la première indication pour la thérapie du cancer. Néanmoins, la résection complète du tissu tumoral, la détection des micrométastases et la préservation des tissus sains pendant l'intervention représentent un enjeu majeur et influencent fortement le pronostic du patient. Les récents développements technologiques en imagerie pour la chirurgie guidée des cancers ont conduit à des résultats précliniques prometteurs et les premiers essais cliniques utilisant des traceurs non-spécifiques confirment déjà le potentiel de ces systèmes pour l'amélioration de la chirurgie. De plus, le diagnostic précoce des tumeurs, ainsi que le développement de thérapies ciblées sont également des axes majeurs de recherche en cancérologie. Dans ce contexte notre équipe a précédemment développé un vecteur synthétique ciblant un récepteur cellulaire l'intégrine αVβ3. Ce vecteur est constitué d'un châssis décapeptidique cyclique RAFT (Regioselectively Addressable Functionalized Template) et présentant deux domaines indépendants permettant de séparer les deux fonctions du vecteur. Sur un domaine, la fonction de ciblage est assurée par la présentation multivalente de ligands -RGD- spécifiques du récepteur. L'autre domaine du vecteur porte les molécules d'intérêt à vectoriser, agents thérapeutiques ou de détection pour l'imagerie médicale. Dans la première partie de ces travaux, nous avons évalué la combinaison de ce vecteur couplé à un fluorophore avec une sonde portative pour imager et guider le chirurgien pendant la chirurgie des fibrosarcomes spontanés chez le chat. Cette étude représente une preuve de concept pour la translation clinique chez l'homme. Les résultats ont montré que l'injection du traceur ne provoquait pas d'effets toxiques chez le chat et permettait un marquage spécifique de la tumeur avec un bon ratio tumeur/tissu sain, qui devrait améliorer la qualité de la résection tumorale en aidant le chirurgien à mieux délimiter les marges du tissu tumoral. Dans la seconde partie de ces travaux nous avons développé un nouveau vecteur bi-fonctionnel dérivé du RAFT-RGD. Au composé d'origine a été ajoutée une séquence peptidique clivable par la matrixmetalloprotease-9, une enzyme surexprimée dans la tumorigénèse. Cette molécule à fluorescence activable a montré une amélioration du ciblage tumoral in vitro et in vivo comparée au RAFT-RGD suggérant un effet additionnel lié au double ciblage. Ces résultats préliminaires encouragent la poursuite de sa caractérisation pour son potentiel de « pro-drug » mais également pour l'étude des interactions entre l'intégrine et l'environment tumoraux. / Cancer surgery is still the gold standard therapy in most cancers. Nevertheless, total tumor resection and metastasis detection while preserving healthy tissues represent a crucial point for further prognosis. Development of imaging technologies for intra-operative guided surgery provided promising results and efficient application in preclinical studies and first clinical trials using non-specific tracers already confirmed the improved out-come in surgery. Moreover early and precise diagnosis and targeted therapies are major domains of cancer research. In this context our team previously developed a synthetic vector based on a cyclic decapeptide scaffold RAFT (Regioselectively Addressable Functionalized Template) which allows the independent functionalizing of two domains: a targeting domain with multivalent RGD-ligand targeting the cell receptor integrin αVβ3, and a vehicle domain grafted with a pro-drug or an imaging agent. One part of this work consisted in the evaluation of the combination of this molecule carrying a fluorophore with a portable fluorescent imaging device for image-guided surgery of natural occurring feline fibrosarcomas. This study represents a proof of concept for further translation into human clinics. No toxic effects in cats after administration of the tracer could be reported. Furthermore the tumors were specifically labeled showing a good tumor-to-healthy tissue ratio. This should improve tumor resection by helping the surgeon to delineate tumor margins. In parallel we developed a bi-functinal derivative of the RAFT-RGD. Therefore we engrafted a peptide sequence flanked by two fluorophores, which is activatable by matrixmetalloprotease-9, an enzyme overexpressed in tumors. This molecule showed an improved tumor labeling in vitro and in vivo compared to the conventional RAFT-RGD, suggesting an additional effect of the double targeting. These preliminary results encourage further caracterisation for its potential as pro-drug vehicle, as well as for studying interactions between the integrin and the tumor environment.

Introduction : La thérapie ciblée des cancers des VADS nécessite la mise au point de nouveaux vecteurs spécifiques. Ces vecteurs servent à acheminer des substances thérapeutiques, mais aussi ils peuvent être couplés à des fluorophores afin de les utiliser dans la chirurgie guidée par l'imagerie de fluorescence proche infrarouge.Objectifs : L'objectif de notre travail est de tester de nouveaux vecteurs des cancers des VADS et d'étudier l'apport de l'imagerie de fluorescence proche infrarouge dans la chirurgie des cancers des VADS chez un modèle animal orthotopique que nous mettons au point.Matériel et méthodes : La lignée cellulaire des cancers des VADS CAL33 est caractérisée in vitro et in vivo. De nouveaux vecteurs qui ciblent un ou plusieurs récepteurs des cellules CAL33 comme l'intégrine alpha v beta 3, l'EGFR et la NRP1, sont testés in vitro. Parallèlement, un modèle animal orthotopique des cancers des VADS est développé par implantation de fragments tumoraux des cellules CAL33, au niveau de la cavité buccale de la souris nude. La résection des tumeurs orthotopiques est guidée par l'imagerie de fluorescence proche infrarouge, après injection systémique du peptide RAFT-c[RGD]4 couplé à un fluorophore. Ce peptide cible l'intégrine alpha v beta 3 et est préalablement testé in vivo sur les cellules CAL33.Résultats : Nos résultats préliminaires montrent que certaines molécules bispécifiques présentent une liaison accrue in vitro aux cellules CAL33. Par ailleurs, la chirurgie guidée par l'imagerie de fluorescence proche infrarouge ciblant l'intégrine alpha v beta 3, présente un impact positif sur la survie sans rechute dans notre modèle orthotopique, à travers la détection de reliquats tumoraux qui pourraient passer inaperçus si l'exérèse tumorale avait été réalisée exclusivement d'une façon macroscopique. Elle permet aussi de détecter les adénopathies métastatiques.Conclusion : L'imagerie de fluorescence proche infrarouge améliore la qualité de l'exérèse tumorale dans notre modèle orthotopqiue optimisé des cancers des VADS. Cette étape préclinique est indispensable avant de tester cette technique chez l'être humain. / Introduction: Targeted therapy of head and neck squamous cell carcinoma (HNSCC) requires the development of novel specific vectors that can deliver therapeutic molecules. These vectors could also be coupled to fluorophores to be used in near infrared fluorescence imaging-guided surgery.Objectives: The aim of our work is to test new targeted vectors of HNSCC and to study the role of the near infrared fluorescence imaging-guided surgery in HNSCC resection in a novel orthotopic animal model that we develop.Materials and Methods: The HNSCC cell line CAL33 is characterized in vitro and in vivo. Novel vectors that target one or more receptors of this cell line such as alpha v beta 3 integrin, EGFR and NRP1, are tested in vitro. Meanwhile, an orthotopic animal model of HNSCC is developed by implanting tumor fragments of CAL33 cells, in the oral cavity of nude mice. Surgical resection of orthotopic tumors is guided by the near infrared fluorescence imaging after systemic injection of RAFT-c[RGD]4 peptide coupled with a fluorophore. This peptide targets alpha v beta 3 integrin and is previously tested in vitro.Results: Our preliminary results show that bispecific vectors would present an increased binding to CAL33 cells in vitro. On the other hand, near infrared fluorescence imaging-guided surgery has a positive impact on the recurrence-free survival rate in our orthotopic model, by detecting fluorescent cancer foci that could remain unidentified if resection was performed exclusively under visual guidance. Our results show also that near infrared fluorescence imaging can also help to detect metastatic lymph nodes.Conclusion: Near-infrared fluorescence imaging-guided surgery improves the quality of tumor resection in our optimized orthotopic animal model of HNSCC. This preclinical stage is essential before testing this novel technique in humans.

L’Opacification Capsulaire Postérieure (OCP) est la fibrose de la capsule développée sur la lentille intraoculaire implantée (LIO) suite à la dé-différenciation de cellules épithéliales cristalliniennes (LECs) subissant une transition épithélio-mésenchymateuse (EMT). La littérature a montré que l'incidence de l’OCP est multifactorielle, dont l'âge ou la maladie du patient, la technique de chirurgie, le design et le matériau de la LIO. La comparaison des LIOs en acryliques hydrophiles et hydrophobes montre que les premières ont une OCP plus sévère, médiée par la transition EMT. En outre, il est également démontré que l'adhérence des LECs est favorisée sur des matériaux hydrophobes par rapport à ceux hydrophiles. Une stratégie biomimétique destinée à promouvoir l’adhérence des LECs sans dé-différenciation en vue de réduire le risque de développement de l’OCP est proposée. Dans cette étude, les peptides RGD, ainsi que les méthodes de greffage et de quantification sur un polymère acrylique hydrophile ont été étudiés. La surface fonctionnalisée des LIOs favorisant l'adhérence des LECs via les récepteurs de type intégrine peut être utilisée pour reconstituer la structure capsule-LEC-LIO en sandwich, ce qui est considéré dans la littérature comme un moyen de limiter la formation de l‘OCP. Les résultats montrent que le biomatériau innovant améliore l'adhérence des LEC, et présente également les propriétés optiques (transmission de la lumière , banc optique) similaires et mécaniques (force haptique de compression, force d'injection de la LIO) comparables à la matière de départ. En outre, par rapport au matériau hydrophobe IOL, ce biomatériau bioactif présente des capacités similaires vis à vis de l’adhérence des LECs, le maintien de la morphologie, et l'expression de biomarqueurs de l’EMT. Les essais in vitro suggèrent que ce biomatériau a le potentiel de réduire certains facteurs de risque de développement de l’OCP. / Posterior Capsular Opacification (PCO) is the capsule fibrosis developed onto the implanted IntraOcular Lens (IOL) by the de-differentiation of Lens Epithelial Cells (LEC) undergoing Epithelial-Mesenchymal Transition (EMT). Literature has shown that the incidence of PCO is multifactorial including patient’s age or disease, surgical technique, and IOL design and material. Reports comparing hydrophilic and hydrophobic acrylic IOLs show the former has more severe PCO after EMT transition. Additionally, the LEC adhesion is favored onto the hydrophobic materials compared to the hydrophilic ones. A biomimetic strategy to promote LEC adhesion without de-differentiation to reduce PCO development risk is proposed. RGD peptides, as well as their grafting and quantification methods on a hydrophilic acrylic polymer were investigated. The surface functionalized IOL promoting LEC adhesion via integrin receptors can be used to reconstitute the capsule-LEC-IOL sandwich structure, which is considered to prevent PCO formation in literature. The results show the innovative biomaterial improves LEC adhesion, and also exhibits similar optical (light transmittance, optical bench) and mechanical (haptic compression force, IOL injection force) properties comparing to the starting material. In addition, comparing to the hydrophobic IOL material, this bioactive biomaterial exhibits similar abilities in LEC adhesion, morphology maintenance, and EMT biomarker expression. The in vitro assays suggest this biomaterial has the potential to reduce some risk factors of PCO development.

Doctor of Philosophy / Department of Psychology / Ronald G. Downey / Literature on work and family, the two important domains in an individual’s life, has focused heavily on the conflicts that could occur when individuals try to juggle between their responsibilities in the two domains. Lately, there has been enthusiasm to also study the facilitation aspects that could result from being engaged in both domains. This dissertation empirically tests the Resources-Development-Gain model (RGD), a recently developed work and family facilitation model, which include work and non-work factors that can bring facilitation. Over 500 academic faculty members from four universities completed an online survey comprised of demographic items, family and work variables, variables to measure facilitation, outcome variables in both domains, and personality variables. The hypothesized model (model 1) was analyzed using AMOS, and was found to be a poor fit. Personality factors included as moderators in the facilitation process were found to be non-significant and hence dropped from the modified model (Model 2). This was a significantly better fit. Model 3 was analyzed to see if a better fit would be obtained when personality variables were directly connected to outcome variables. As Model 3 did not add anything significant, Model 2 was accepted. The findings suggest that faculty tenure influenced their turnover intentions, with new academic faculty and full professors showing lower turnover intentions. Family support brought facilitation from one’s family to work and contributed to life satisfaction, while organizational support contributed to facilitation from one’s work to non-work life. No significant overlaps were found between work and family domains in the facilitation stage, but were observed at the outcome levels. Thus, job satisfaction in the work domain contributed to overall life satisfaction in the family domain. Satisfaction in one’s personal relations also tended to influence one’s turnover decisions. Future directions for research and recommendations are discussed.