Les cellules endothéliales (CE), monocouche de cellules hexagonales jointives situées à la face interne de la cornée, assurent la transparence de ce tissu essentiel à la vision. Peu avant la naissance, ces CE perdent leur capacité proliférative en restant bloquées en phase G1du cycle cellulaire. Le non remplacement des cellules mortes est responsable de certaines pathologies cécitantes dont la cornea gutatta et les dystrophies bulleuses du pseudophaque sont les prototypes et comptent parmi les premières indications de greffe de cornée. Les mécanismes moléculaires impliqués dans l’arrêt de la prolifération de ces CE restent très partiellement expliqués. La première partie de cette thèse a pour objectif d’identifier si des changements d’expression transcriptionnelle des gènes régulateurs du cycle cellulaire interviennent au cours de l’organoculture (OC) et de la culture in vitro, afin de définir au mieux les cibles potentielles à inhiber ou celles à surexprimer pour re-déclencher une prolifération cellulaire contrôlée. Pour la première fois, nous avons mis en évidence des profils transcriptionnels variables en fonction de l’environnement des CE, avec une activation globale de l’expression des gènes en OC de routine et en culture primaire et l’expression accrue de gènes impliqués dans l’arrêt du cycle cellulaire en différents points comme DIRAS3, GADD45A, p15 p16, p18 et p19 ou impliqués dans la régulation du cycle des CE comme le complexe ubiquitine/protéasome (culines, APC...), laissant supposer que les freins antiprolifératifs sont encore plus complexes. Dans la seconde partie, nous avons développé une méthode d’analyse de la viabilité de l’endothélium pan-cornéenne afin d’évaluer au mieux la viabilité des cellules endothéliales sur les greffons. Nous avons développé un outil innovant de mesure combinant un triple marquage Hoechst/Ethidium/Calcéine AM à l’analyse pan-endothéliale permettant d’évaluer de définir la notion originale de densité en cellules viables d’une cornée. Cette technique peut être appliquée à l’analyse de n’importe quel procédé chirurgical ou non, susceptible d’altérer directement ou indirectement l’endothélium cornéen / Corneal endothelial cells (EC) form a monolayer of hexagonal contiguous cells located at the inner surface of the corne and are responsible for maintenance of its transparency, and therefore essential for vision. Just before birth, they lose the proliferative capacity and remain blocked in the G1 phase of the cell cycle. The absence of cell replacement by mitosis induces is responsible for certain blinding diseases such as cornea gutatta and pseudophakic bullous keratopathy, two prototype endothelial diseases that are among the first indications of corneal graft. The molecular mechanisms involved is the non-proliferation of EC remain only partially explained. The first part of this thesis aimed to identify whether changes in transcriptional expression of cell cycle regulators genes occurred during organ culture (OC) and during in vitro culture, in order to better define the potential targets to inhibit or to overexpress necessary to trigger a controlled cell proliferation. Forthe first time we have highlighted variable transcriptional profiles depending on endothelial environment, with a glolal activation of gene expression in routine OC and in primary cultures, especially with an increased expression of gene involved in cell cycle arrest at different points like DIRAS3, GADD45, p15 p16, p18 and p19 or involved in cycle regulation as the ubiquitin/proteasome complex (Culines, APC ...), suggesting that the antiproliferative brakes are even more complex than previously reported. In a second part we developed an original method of pan-corneal endothelial viability assessment. This innovative measurement tool combines a triple Hoechst/Ethidium/Calcein-AM labeling with a part endothelial image analysis allowing to define the new concept of viable endothelial cell density, that represent the reaviable cell pool of a cornea. This technique can be applied to the analysis of any procedure (surgical or not), likely to directly or indirectly alter the corneal endothelium

The characterization of the mechanical behavior of soft living tissues is a big challenge in Biomechanics. The difficulty arises from both the access to the tissues and the manipulation in order to know their physical properties. Currently, the biomechanical characterization of the organs is mainly performed by testing ex-vivo samples or by means of indentation tests. In the first case, the obtained behavior does not represent the real behavior of the organ. In the second case, it is only a representation of the mechanical response of the indented areas. The purpose of the research reported in this thesis is the development of a methodology to in-vivo characterize the biomechanical behavior of two different organs: the breast and the cornea. The proposed methodology avoids invasive measurements to obtain the mechanical response of the organs and is able to completely characterize of the biomechanical behavior of them. The research reported in this thesis describes a methodology to in-vivo characterize the biomechanical behavior of the breast and the cornea. The estimation of the elastic constants of the constitutive equations that define the mechanical behavior of these organs is performed using an iterative search algorithm which optimizes these parameters. The search is based on the iterative variation of the elastic constants of the model in order to increase the similarity between a simulated deformation of the organ and the real one. The similarity is measured by means of a volumetric similarity function which combines overlap-based coefficients and distance-based coefficients. Due to the number of parameters to be characterized as well as the non-convergences that the solution may present in some regions, genetic heuristics were chosen to drive the search algorithm. In the case of the breast, the elastic constants of an anisotropic hyperelastic neo-Hookean model proposed to simulate the compression of the breast during an MRI-guided biopsy were estimated. Results from this analysis showed that the proposed algorithm accurately found the elastic constants of the proposed model, providing an average relative error below 10%. The methodology was validated using breast software phantoms. Nevertheless, this methodology can be easily transferred into its use with real breasts. In the case of the cornea, the elastic constants of a hyperelastic second-order Ogden model were estimated for 24 corneas corresponding to 12 patients. The finite element method was applied in order to simulate the deformation of the human corneas due to non-contact tonometry. The iterative search was applied in order to estimate the elastic constants of the model which approximates the most the simulated deformation to the real one. Results showed that these constants can be estimated with an error of about 5%. After the results obtained for both organs, it can be concluded that the iterative search methodology presented in this thesis allows the \textit{in-vivo} estimation the patient-specific elastic constants of the constitutive biomechanical models that govern the biomechanical behavior of these two organs. / Lago Ángel, MÁ. (2014). A new approach for the in-vivo characterization of the biomechanical behavior of the breast and the cornea [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/44116 / TESIS

Dans un contexte d’augmentation du nombre d’amétropes dans la population mondiale, et en conséquence, de l’accroissement du recours aux techniques de corrections chirurgicales, la compréhension et l’amélioration de celles-ci sont un enjeu crucial. Nous avons cherché à améliorer la prédictibilité de certains résultats postopératoires dans le cas d’un LASIK (Laser-Assisted In-Situ Keratomileusis), d’une PKR (Photorefractive Keratectomy) ou d'une chirurgie de la cataracte, et ainsi formuler des recommandations pratiques qui contribueraient au développement de stratégies de traitement davantage personnalisés. Pour cela, nous avons utilisé prospectivement des méthodologies de « contrôle de qualité » des chirurgies sur de larges échantillons de patients. Dans un premier temps, nous avons étudié la dynamique pupillaire dans le cadre de chirurgies au LASIK et notamment le rôle du centre pupillaire, point de référence important dans les stratégies de centrage. Nous avons également évalué la dynamique du diamètre pupillaire et les modifications du segment antérieur sur des yeux subissant une chirurgie de la cataracte. La seconde partie du travail s’est focalisée sur le rôle de l’épithélium dans la topographique cornéenne. Nous avons comparé les topographies spéculaires de l'épithélium et de la couche de Bowman sur des cornées saines et des cornées kératoconiques, présentant une myopie faible à modérée corrigée par PKR. Enfin, dans la dernière partie de notre recherche, nous nous sommes intéressés aux changements de paramètres anatomiques de l'oeil, des performances visuelles et de la qualité de vision subjective survenant dans un échantillon d’yeux myopes après un LASIK réalisé avec le laser WaveLight® Refractive Suite (Alcon® Laboratories Inc., USA). / While the number of ametropic eyes in the world’s population and consequently the use of surgical correction techniques is increasing, understanding and improving these techniques is a crucial issue. We sought to improve the predictability of certain postoperative results in the case of LASIK (Laser-Assisted In-Situ Keratomileusis), PRK (Photorefractive Keratectomy) and cataract surgery, and thus to formulate practical recommendations that would contribute to the development of more personalized treatment strategies. To achieve this objective, we have prospectively used "quality control" methodologies to assess surgeries performed on large samples of patients. First, we studied the pupillary dynamics in LASIK surgery and in particular the role of the pupillary centre, an important point of reference in the centration strategies. We also assessed the dynamics of pupillary diameter and anterior segment changes on eyes undergoing cataract surgery. The second part of the work focused on the role of the epithelium in the corneal topography. We compared specular topographies of the epithelium and Bowman's layer in healthy and keratoconus corneas with mild to moderate myopia corrected by PRK. Finally, in the last part of our research, we were interested in the changes in anatomical parameters of the eye, visual performance and subjective quality of vision occurring in a sample of myopic eyes after LASIK performed with the WaveLight® Refractive Suite (Alcon® Laboratories Inc., USA).

Purpose: Lectins play an important role in many biological processes. The aim of this work was to analyse mainly the expression of endogenic lectins, such as galectins and plant lectin, e.g. Dolichos biflorus agglutinin (DBA), and their glycoligands in the tear fluid, human corneal and conjunctival epithelium in physiological and disease conditions. Further, we studied the human natural antibody against Galα1,3Gal-R, which is mainly responsible for hyperacute rejection of xenografts transplants. We tried to investigate its localization in human corneal epithelium, lacrimal gland and tears. Material and Methods: Human tissue (lacrimal gland, tear fluid, conjunctiva, cornea, epidermis, keratinocyte and cultured corneal epithelium), as well as porcine tissue (cornea, liver and epidermis) were examined. Endogenous galectins (galectins-1, -3 and -7) were detected using immunohistochemistry methods. Binding sites for galectins, as well as binding sites for plant lectin Dolichos biflorus agglutinin, were localized by lectin histochemistry. Reverse lectin histochemistry was used for the study of binding reactivity of endogenous lectins using labelled (neo)glycoligands. Employing biotinylated natural human IgG anti -galactosides, as well as anti -galactosides, we detected reactive epitopes in human...

Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of many studies, the effects that environmental changes have on this form of movement are poorly understood. In certain pathologies where the cornea exists in a chronic hypoxic state, wound healing is delayed. The goal of this work is to examine the changes in corneal structure in hypoxic corneas that may affect migration and to determine the effects that changes in basement membrane stiffness and focal adhesion turnover have on epithelial cell migration. We analyzed migration after injury in organ cultures and determined that hypoxic corneas exhibited alterations in leading edge morphology. Under hypoxia, fibronectin localization to the apical epithelium and anterior stroma was reduced. Investigators have suggested that alterations in basal lamina composition may increase the stiffness of the membrane. To examine the effect that increased stiffness has on collective migration we performed traction force microscopy. Using multi-layered corneal epithelial sheets, we developed a novel method to analyze the generation of cellular traction forces and the directionality of sheet movement on polyacrylamide gels. We determined that the leading edges of corneal epithelial sheets undergo contraction prior to migration. Alterations in stiffness affected the amount of force exerted by cells at the leading edge. On stiffer surfaces, individual cells within sheets exhibited greater movement compared to cells on softer substrates. To further assess sheet dynamics, we examined the activation of focal adhesion proteins in hypoxic corneas and in human corneal limbal epithelial (HCLE) cells seeded onto soft and rigid substrates. Wounded hypoxic corneas displayed alterations in the localization of the focal adhesion proteins paxillin and vinculin. In HCLE cells cultured on stiff substrates, there was an elevation in vinculin pY1065 phosphorylation after injury, a reduction in vinculin-positive focal adhesions, and decreased actin bundle thickness. Our results demonstrate that changes in membrane stiffness may affect cellular tractions and vinculin dynamics, possibly contributing to the delayed healing response associated with certain corneal pathologies.

The corneal wound healing process is a complex process, which often leads to the development of scar tissue with loss of transparency. When the cornea is wounded, some of the viable keratocytes are activated by growth factors to proliferate, and repair the wound by the production of a new extracellular matrix (ECM) that is either normal or is disorganized (fibrotic). The first part of this dissertation aims to show that the growth factors IGF-I, TGF-ß, FGF-2, and PDGF stimulate keratocytes to synthesize different levels of collagens and proteoglycans and are therefore responsible for initiating the wound healing repair process. FGF-2 stimulated keratocytes to proliferate but did not stimulate collagen production; IGF-I and PDGF stimulated keratocytes to proliferate and produce a collagenous ECM that could restore transparency; while, TGF-ß stimulated keratocytes produce a fibrocollagenous ECM that is opaque. The second part of this dissertation aims to evaluate collagen fibril content, distribution, and orientation in the ECM deposited by keratocytes cultured in IGF-I, TGF-ß, FGF-2, and PDGF under a layer of agarose: a culture modification that enhances the formation of ECM in vitro. FGF-2 agarose cultures had little ECM and the keratocytes were in close cell contact while IGF-I, TGF-ß, and PDGF agarose cultures had the least cell contact with an extensive fibrillar ECM. This newly developed agarose overlay cell culture system increases ECM formation with the cell layer only when the synthesis of collagen is stimulated and that the ECM morphology is growth factor specific. Cell culture has proven to be a reliable technique to study the keratocytes response to trauma and disease; however, limitations exist. Primary keratocytes that possess quiescent phenotype are unable to be rapidly expanded or subcultured without the addition of a mitogen(s). Most commonly, keratocytes are cultured and passaged in the presence of fetal bovine serum (FBS), which activate the cells to proliferate and differentiate into fibroblasts or myofibroblasts as well as lose expression of their unique transparency enabling gene products. The third part of this dissertation aims to develop a defined culture media that can be used to expand and subculture keratocytes that express keratocyte specific markers. Culture medium supplemented with FGF-2 combined with ITS was used to: expand and subculture primary bovine keratocytes while maintaining their expression of keratocan and to restore keratocan expression to bovine keratocytes expanded and subcultured with media containing 2.5% FBS. This dissertation shows the significance of signaling molecules in vitro to produce keratocyte cultures useful for understanding normal stromal biology and its repair process.

Syfte: Studiens syfte var att kontrollera repeterbarhet och jämförbarhet hos tre kliniska instrument som används för att mäta hornhinnans tjocklek i friska ögon. Instrumenten som användes har flera olika användningsområden men fokus i denna studie ligger specifikt på pakymetri: mätning av hornhinnetjocklek. Metod: Det var 64 personer som deltog i studien. Alla var över 18 år och befann sig på Linnéuniversitetet i Kalmar vid tidpunkterna för mättillfällena. De tre optiska pakymetri instrumenten som användes var Topcon TRK-2P, Bon Sirius samt Essilor Wavefront Analyzer Medica 700+. Tre mätningar utfördes vid varje instrument på deltagarnas båda ögon. All data sammanställdes i Google Kalkyl och analyserades vidare i Graphpad Prism (version 9). Resultat: Repeterbarheten testades med ICC vilket var över 0,98 på samtliga instrument, CR var mellan 5,28-8,80 μm och CV var mellan 6,29-6,74%. Jämförbarheten visade att det finns en signifikant skillnad mellan de tre instrumenten (One-way RM- ANOVA,  p<0,0001). Slutsats: Samtliga instrument som användes inom studien visade på en utmärkt repeterbarhet vid mätning av friska ögon, däremot förekom signifikanta skillnader dem emellan. Detta innebär att man bör hålla sig till ett och samma instrument vid uppföljning av patienter. / Purpose: The purpose of this study was to examine the repeatability and comparability of three clinical instruments used to measure the corneal thickness in healthy eyes. The different instruments all have various fields of usage but the focus in this study is pachymetry: measurement of the corneal thickness. Method: In total, 64 people participated in this study. Everyone that participated were above the age of 18 and were located at Linneaus university in Kalmar, Sweden at the time the measurements took place. The three optical pachymetry instruments used were Topcon TRK-2P, Bon Sirius and Essilor Wavefront Analyzer Medica 700+. Three measurements were taken on each instrument on the participants’ both eyes. All collected data was collated in Google Sheets and analyzed in Graphpad Prism (version 9).  Results: The repeatability was tested with ICC that was above 0,98 on all the instruments. CR was between 5,28-8,80 μm and CV was between 6,29-6,74%. The comparability showed that there was a significant difference between the three instruments (One-way RM- ANOVA,  p<0,0001). Conclusion: All three instruments that were used during this study showed an excellent repeatability when measuring healthy eyes. However, there were significant differences between the instruments themselves. This means that you should stick to the same instrument while following up patients.

Confocal microscopy demonstrated its value in the diagnosis of Acanthamoeba and fungal keratitis which considered sight-threatening corneal diseases. However, it can be difficult to find and train confocal microscopy graders to accurately detect Acanthamoeba cysts and fungal filaments in the images. Use of an automated system could overcome this problem and help to start the correct treatment more quickly. Also, response to treatment can be difficult to assess in infectious keratitis using clinical examination alone, but there is evidence that the morphology of filaments and cysts may change over time with the use of correct treatment. An automated system to analyse confocal microscopy images for such changes would also assist clinicians in determining whether the ulcer is improving, or whether a change of treatment is needed. This research proposes a fully automated novel system with GUI to detect cysts and hyphae (filaments) and measure useful quantitative parameters for them through many stages; Image enhancement, image segmentation, quantitative analysis for detected cysts and hyphae, and registration and tracking of ordered sequence of images. The performance of the proposed segmentation procedure is evaluated by comparing between the manual and the automated traced images of the dataset that was provided by the Manchester Royal Eye Hospital. The positive predictive values rate of cysts for Acanthamoeba images was 76%. For detected hyphae in Fusarium images, many standard measurements were computed. The accuracy of their values was quantified by calculating the percent error rate for each measurement and which ranged from 23% to 49%.

La cécité cornéenne touche 12,7 millions personnes globalement. Il y a une pénurie des cornées de donneurs humains (CDH), et donc les tissues disponibles sont implanté préférentiellement dans les patients avec des troubles cornéens à faible risque comme le kératocône et la dystrophie endothéliale de Fuchs. Les patients qui ont un risque élevé d’inflammation, comme ceux avec des brûlures acides, alcalines et thermiques, des infections et des ulcères, ne reçoivent pas de greffes pour leurs maladies cornéennes. Les biomatériaux offrent une alternative aux CDH en permettant le développement de solutions de régénération cornéenne avec une longue durée de conservation, une thermostabilité pour un déploiement en zone rurale, et biocompatibilité chez les patients à haut risque. Les biomatériaux peuvent être développés sous forme d’implants cornéens solides à greffer dans des opacités cornéennes ou sous forme de liquides gélifiants injectables qui peuvent sceller des petites perforations cornéennes. Les implants cornéens solides conviennent aux chirurgiens ophtalmologiques, mais les produits de comblement liquides peuvent être utilisés par les prestataires de médecine d’urgence ou le personnel médical non spécialisé dans les zones où les chirurgiens ophtalmologistes ne sont pas disponibles. Cette thèse explore les formulations de biomatériaux pour les cornéens solides et gélifiants in situ, leurs performances en tant que dispositifs composites, l’ajout de la stérilisation terminale à la fabrication d’implants cornéens solides et le développement de futures protéines mimétiques du collagène pour la formulation d’hydrogel. Le premier objectif de cette thèse était de développer un implant cornéen solide adapté à l’implantation chez les patients cornéens à haut risque. Les implants cornéens peptide-mimant-le-collagène-polyéthylène glycol-phosphorylcholine (PMC-PEG-MPC) et les implants recombinants de collagène humain de type III-phosphorylcholine (RCHIII-MPC) ont réussi à régénérer les cornées de mini-porcs et de lapins, respectivement. La phosphorylcholine présente dans la formulation PMC-PEG-MPC a diminué l’inflammation et fourni une alternative cornéenne viable dans les brûlures alcalines à haut risque. Des nanoparticules d’argent coiffées de peptides étaient fabriquées avec succès à la surface d’un implant cornéen solide de collagène porcin de type I. Ces implants ont inhibé P. aeruginosa, S. aureus et S. epidermidis in vitro et empêché la formation de biofilm à l’interface air-liquide. Ces implants cornéens solides élargissent la gamme d’efficacité pour inclure les personnes souffrant de brûlures alcalines et d’infections. Finalement, on a validé une méthode de stérilisation terminale des implants cornéens solides. Le RCHIII-MPC a été stérilisé en phase terminale avec succès à l’aide d’une irradiation par faisceau d’électrons, offrant une future voie pour la stérilisation terminale des implants cornéens solides à base de biomatériaux. Le deuxième objectif était de concevoir un hydrogel qui se solidifierait in situ pour sceller les perforations cornéennes. Le PMC-PEG était combiné avec du fibrinogène pour former “LiQD Cornea”, le premier produit de comblement cornéen liquide à être chimiquement réticulé avec succès in situ pour sceller les perforations cornéennes et les plaies chirurgicales chez le lapin et les mini-porcs. Pour le troisième objectif, ce projet fournit également une méthodologie future pour la production de protéines mimétiques de collagène personnalisées pour les futures formulations d’hydrogel. Dans l’ensemble, le collagène et les biomatériaux inspirés du collagène se sont révélés être des greffes et des scellants cornéens prometteurs avec des voies viables de fabrication commerciale. / Corneal blindness and opacities affect 12.7 million people globally. There is a shortage of human donor corneas (HDCs), which are prioritized for patients with low risk corneal disorders like keratoconus and Fuch’s endothelial dystrophy. Patients with high-risk inflammatory conditions like acid, alklai and thermal burns, infections and ulcers are often unable to receive transplants to treat their corneal disorders. Biomaterials provide an alternative to HDCs by allowing the development of corneal regenerative solutions with a long-shelf life, thermostability for deployment in rural areas and biocompatibility in high-risk patients. Biomaterials can be developed as solid corneal implants to graft into large corneal opacities or as injectable in situ gelling liquids that can seal small corneal perforations. Solid corneal implants are suited for use by ophthalmic surgeons, but liquid fillers can be used by emergency medicine providers or non-specialized medical personnel in areas where ophthalmic surgeons are not available. This thesis explores biomaterials formulations for solid and in situ gelling corneal biomaterials, their performance as composite devices, the addition of terminal sterilization to the manufacture of solid corneal implants, and the development of future collagen mimetic proteins for hydrogel formulations. The first objective of this thesis was to develop a solid corneal implant suitable for implantation in high-risk corneal patients. Collagen-like-peptide-polyethylene glycolphosphorylcholine (CLP-PEG-MPC) corneal implants and recombinant human collagen type III-phosphorylcholine implants were successful in regenerating the corneas of mini-pigs and rabbits, respectively. The phosphorylcholine present in the CLP-PEG-MPC formulation decreased inflammation and provided a viable corneal alternative in high-risk alkali burns. Peptide-capped nanoparticles were successfully fabricated on the surface of a porcine and S. epidermidis in vitro and prevented biofilm formation at the air-liquid interface. These solid corneal implants expand the range of efficacy to include individuals with alkali burns and infections. This thesis validated a method of terminal sterilization for solid corneal implants. RHCIII-MPC was successfully terminally sterilized using electron-beam irradiation, providing a future avenue for terminal sterilization of biomaterials-based solid corneal implants. The second objective was to design a hydrogel that will solidify in situ to seal corneal perforations. CLP-PEG was combined with fibrinogen to form LiQD Cornea, the first liquid corneal filler to be successfully chemically crosslinked in situ to seal corneal perforations and surgical wounds in rabbit and mini-pigs. For the third objective, this project also provides future methodology for the production of custom collagen mimetic proteins for future hydrogel formulations. Overall, collagen and collagen-inspired biomaterials were demonstrated to be promising corneal grafts and sealants with viable pathways to commercial manufacture.

No description available.