Vaisseau sanguin reconstruit par génie tissulaire : Développement d'une nouvelle approche pour la reconstruction de la media et interaction avec les microparticules

La media vasculaire est au coeur des processus physiopathologiques qui entraînent le développement de l’athérosclérose. L’utilisation d’une media reconstruite par génie tissulaire permet d’étudier les cellules musculaires lisses (CML) humaines dans un environnement plus physiologique que les cellules en culture monocouche. Les travaux présentés dans cette thèse sont orientés autour de la media vasculaire reconstruite par génie tissulaire comme modèle d’étude pharmacologique et prothèse vasculaire autologue. La première partie des travaux porte sur l’étude des interactions de cette tunique avec les microparticules (MP) circulantes. D’abord, nous avons montré que la présence de l’adventice modifie la réponse de la media aux MP produites in vitro à partir des lymphocytes T. Ensuite, l’étude de l’effet des MP isolées du sérum de patients en choc septique sur la media humaine a démontré que ces MP sont en mesure d’augmenter la contraction de la media par un mécanisme impliquant une diminution du NO et une augmentation de l’expression de l’ARN messager de l’interleukine-10. L’incubation de la media reconstruite avec cette cytokine anti-inflammatoire bloque l’hyporéactivité induite par les lipopolysaccharides. Le même phénomène a été reproduit in vivo, chez le rongeur. Ces résultats suggèrent que les SMP auraient un effet protecteur sur la fonction vasculaire, en potentialisant la contraction de la media. Ensuite, nous avons optimisé l’approche de reconstruction de prothèses vasculaires par auto-assemblage proposée initialement pour l’adapter au contexte particulier des CML. L’objectif principal était de permettre l’étude physiopathologique de la media à partir de toutes les lignées de CML; indépendamment de leur capacité de synthèse de matrice extracellulaire. Pour ce faire, nous avons développé un échafaudage de matrice extracellulaire produit par auto-assemblage à partir de fibroblastes humains. L’utilisation de cet échafaudage génère une media plus résistante et plus contractile que la technique initiale. Enfin, une anisotropie a été créée dans cet échafaudage pour permettre une orientation physiologique des CML. La media reconstruite devient ainsi plus résistante et plus contractile. Ces améliorations permettent de reconstruire des media à partir des cellules de plus de patients et mèneront à des études pharmacologiques plus représentatives de la population. Cet échafaudage facilitera la translation clinique de ce modèle de media reconstruite par génie tissulaire.

Supporting Cubane’s Renaissance: Metathesis reactions on 4-iodo-1-vinylcubane and Stetter reaction on 1-iodocubane-4-carboxaldehyde

Cubane is a peculiar cube-shaped alkane molecule with a rigid, regular structure. This makes it a good scaffold, i.e. a molecular platform to which the substituents are arranged in a specific and fixed orientation. Moreover, cubane has a body diagonal of 2.72 Å, very similar to the distance across the benzene ring, i.e. 2.79 Å. Thus, it would be possible to use cubane as a scaffold in medicinal and material chemistry as a benzene isostere 1,2. This could lead to advantages in terms of solubility and toxicity and could provide novel properties. For this purpose, the possibility of performing “modern organic chemistry” on the cubane scaffold has to be studied. This project was entirely carried out in the framework of the Erasmus+ mobility programme at the Trinity College (Dublin, IRL) under the supervision of prof. M. O. Senge. The main goal of this project was to widen the knowledge on cubane chemistry. In particular, it was decided to test reactions that were never applied to the scaffold before, such as metathesis of 4-iodo-1-vinylcubane and Stetter reaction of 1-iodocubane-4-carboxaldehyde. These two molecules were synthesized in 10 and 9 steps respectively from commercially available cyclopentanone, following a known procedure. Unfortunately, metathesis with different olefins, such as styrene, α,β unsaturated compounds and linear α-olefins failed under different conditions, highlighting cubane behaves as a Type IV, challenging olefin under metathesis conditions. Even the employment of a specific catalyst for hindered olefins failed in the cross-coupling with linear α-olefins. On the other hand, two new molecules were synthesized via Stetter reaction and benzoin condensation respectively. Even if the majority of the reactions were not successful, this work can be seen as an inspiration for further investigation on cubane chemistry, as new questions were raised and new opportunities were envisioned.

Conservation narratives in Peru: envisioning biodiversity in sustainable development

In the mega-diverse country Peru, a resource intensive development model collides with the interest of conserving biodiversity. Peruvian biodiversity experts have developed different lines of argumentation as to how to integrate conservation into the sustainable development of their country. Applying grounded theory, I define five groups of conservation narratives based on the analysis of 72 qualitative interviews with experts working in areas of biodiversity conservation. I have labeled them: biodiversity protectionists, biodiversity traditionalists, biodiversity localists, biodiversity pragmatists, and biodiversity capitalists. These groups are each discussed in connection with what they have to say about biodiversity in relation to human life, valuation and knowledge systems, participation and leadership, substitutability of natural capital, and its predominant political strategy. In a second step, a comparative analysis of the dominant and diverging political perspectives is made. I argue that by deconstructing underlying premises and ideologies, common ground and possible opportunities for collaboration can be identified. Moreover, although the presented results can serve as a discussion scaffold to organize conservation debates in Peru, this example demonstrates how the terms biodiversity and sustainability are operationalized in conservation narratives.

Substrate and catalyst effects in C-H insertion reactions of α-diazoacetamides

Intramolecular C–H insertion reactions of α-diazocarbonyl compounds typically proceed with preferential five-membered ring formation. However, the presence of a heteroatom such as nitrogen can activate an adjacent C–H site toward insertion resulting in regiocontrol issues. In the case of α-diazoacetamide derivatives, both β- and γ-lactam products are possible owing to this activating effect. Both β- and γ-lactam products are powerful synthetic building blocks in the area of organic synthesis, as well as a common scaffold in a range of natural and pharmaceutical products and therefore C–H insertion reactions to form such compounds are attractive processes.

Augmentation de l'immunogénicité de la nucléoprotéine de l'influenza par multimérisation in vitro

Les travaux effectués au cours de ce mémoire ont permis de développer une alternative aux vaccins présentement utilisés contre le virus de l’influenza. Nous avons utilisé la nucléoprotéine (NP) de l’influenza comme base vaccinale puisque cette protéine est conservée chez les souches d’influenza A et qu’elle possède un potentiel de protection croisée. Nous avons montré que la multimérisation de la NP grâce à un gabarit d’ARN permet d’augmenter son immunogenicité. Cette multimérisation en pseudo-nucléoparticule virale (NLP) a augmenté la réponse humorale et cellulaire spécifique à NP et l’ajout d’un adjuvant (PAL) a permis d’amplifier davantage la réponse humorale contre NP. Une dose du vaccin candidat NLP-PAL n’a pas réussi à protéger des souris contre une infection létale avec une souche homotypique d’influenza. Cependant, des résultats avec un régime de deux immunisations montrent des résultats encourageants qui permettent d’espérer une protection envers une infection virale.

The bovine pericardial patch in breast reconstruction: a case report

In the last years there has been a growing demand of plastic surgery for soft tissue reconstruction. In response to this, many biological and synthetic devices have been produced, aiming to allow wide and complex body reshapings. Acellular dermal matrices are one of these devices, and are made of human or animal tissues made acellular after their sampling. They are used for cervical, breast and abdominal wall reconstruction. Tutopatch®, generally used for face reconstruction or neurosurgery, is made of acellular bovine pericardium, and its high amount of collagen allows a fast tissue healing and a scaffold for the surrounding tissue rigeneration. In our case report Tutopatch® has been used in immediate breast reconstruction after mastectomy. This device has been used to close laterally the subpectoral pocket, allowing a bigger volume prosthesis to be placed We have not experienced particular postoperatory complications, and after 12 months of follow up we have found a valid functional and aesthetic result. We consider Tutopatch® as a valid alternative to other acellular dermal matrices specifically designed for breast reconstruction.

Tissue engineering: technological advances to improve its applications in reconstructive surgery

Background. Tremendous advances in biomaterials science and nanotechnologies, together with thorough research on stem cells, have recently promoted an intriguing development of regenerative medicine/tissue engineering. The nanotechnology represents a wide interdisciplinary field that implies the manipulation of different materials at nanometer level to achieve the creation of constructs that mimic the nanoscale-based architecture of native tissues. Aim. The purpose of this article is to highlight the significant new knowledges regarding this matter. Emerging acquisitions. To widen the range of scaffold materials resort has been carried out to either recombinant DNA technology-generated materials, such as a collagen-like protein, or the incorporation of bioactive molecules, such as RDG (arginine-glycine-aspartic acid), into synthetic products. Both the bottom-up and the top-down fabrication approaches may be properly used to respectively obtain sopramolecular architectures or, instead, micro-/nanostructures to incorporate them within a preexisting complex scaffold construct. Computer-aided design/manufacturing (CAD/CAM) scaffold technique allows to achieve patient-tailored organs. Stem cells, because of their peculiar properties - ability to proliferate, self-renew and specific cell-lineage differentiate under appropriate conditions - represent an attractive source for intriguing tissue engineering/regenerative medicine applications. Future research activities. New developments in the realization of different organs tissue engineering will depend on further progress of both the science of nanoscale-based materials and the knowledge of stem cell biology. Moreover the in vivo tissue engineering appears to be the logical step of the current research.

Crime and Punishment.

The study of Victorian crime and punishment is a rich area of research that has attracted the interest not only of literary scholars but also of social historians, legal historians, and criminologists. Related scholarship therefore often situates itself at the intersection of traditional disciplinary boundaries, facilitating interdisciplinary conversation. Crime and punishment was a pressing issue for the Victorians and provoked a wealth of responses from contemporaneous commentators in literature, culture, and science. As a new phase of industrialization brought immense wealth for some and abject poverty for others, Victorian urban centers in particular were afflicted by crime. Without an effective system of social welfare in place, social inequality and deprivation drove women, men, and children into petty crime and more serious offenses, resulting in severe punishment ranging from incarceration via penal transportation to hanging. Public executions, not abolished until 1868, attracted huge crowds of spectators, including authors such as Charles Dickens and William Thackeray, who wrote about these experiences. A forerunner of the popular press, street literature conveyed and illustrated these events for a broad audience. Execution broadsides of famous cases, printing the alleged last lamentations of convicts on the scaffold in verse, are estimated to have sold by the million. As the legal system was undergoing reform (comprising changes in legal evidence procedure, divorce law, women’s property rights, and punishment for sexual offenses, for example), sensational trials caused furor and stimulated commentary in literature and the media. Crime and punishment was discussed in a range of literary and popular genres, poetry, and reformist writing. The “Newgate School” of fiction was accused of glamorizing crime, and the popular penny dreadfuls were feared to corrupt public morals. Sensational fiction in the 1860s, which often drew on real-life criminal cases and newspaper reports, depicted the supposedly respectable middle-class family home as a center of transgression. Similarly, detective fiction typically focused on crime in the world of the middle classes. For the student new to the subject of crime and punishment, this area’s interdisciplinary nature can pose an initial challenge.

Chitosan fibrous scaffolds for cartilage tissue engineering

The biocompatibility of chitosan and its similarity with glycosaminoglycans make it attractive for cartilage engineering despite its limited cell adhesion properties. Structural and chemical characteristics of chitosan scaffolds may be improved for cartilage engineering application. We planned to evaluate chitosan meshes produced by a novel technique and the effect of chitosan structure on mesenchymal stem cells (MSCs) chondrogenesis. Another objective was to improve cell adhesion and chondrogenesis on chitosan by modifying the chemical composition of the scaffold (reacetylation, collagen II, or hyaluronic acid (HA) coating). A replica molding technique was developed to produce chitosan meshes of different fiber-width. A polyglycolic acid (PGA) mesh served as a reference. Constructs were analyzed at two and 21 days after seeding chondrocytes with confocal microscopy, scanning electron microscopy, histology, and quantitative analysis (weights, DNA, glycosaminoglycans, collagen II). Chondrocytes maintained their phenotypic appearance and a high viability but attached preferentially to PGA. Matrix production per chondrocyte was superior on chitosan. Chitosan meshes and sponges were analyzed after seeding and culture of MSCs under chondrogenic condition for 21 days. The cellularity was similar between groups but matrix production was greater on meshes. Chitosan and reacetylated-chitosan scaffolds were coated with collagen II or HA. Scaffolds were characterized prior to seeding MSCs. Chitosan meshes were then coated with collagen at two densities. PGA served as a reference. Constructs were evaluated after seeding or culture of MSCs for 21 days in chondrogenic medium. MSCs adhered less to reacetylated-chitosan despite collagen coating. HA did not affect cell adhesion. The cell attachment on chitosan correlated with collagen density. The cell number and matrix production were improved after culture in collagen coated meshes. The differences between PGA and chitosan are likely to result from the chemical composition. Chondrogenesis is superior on chitosan meshes compared to sponges. Collagen II coating is an efficient way to overcome poor cell adhesion on chitosan. These findings encourage the use of chitosan meshes coated with collagen II and confirm the importance of biomimetic scaffolds for tissue engineering. The decreased cell adhesion on reacetylated chitosan and the poor mechanical stability of PGA limit their use for tissue engineering.

Playing for Real: Designing Alternate Reality Games in Learning Contexts

Alternate Reality Game (ARG) represent a new genre of transmedia practice where players hunt for scattered clues, make sense of disparate information, and solve puzzles to advance an ever-evolving storyline. Players participate in ARGs using multiple communications technologies, ranging from print materials to mobile devices. However, many interaction design challenges must be addressed to weave these everyday communication tools together into an immersive, participatory experience. Transmedia design is not an everyday process. Designers must create and connect story bits across multiple media (video, audio, text) and multiple platforms (phones, computers, physical spaces). Furthermore, they must engage with players of varying skill levels. Few studies to-date have explored the design process of ARGs in learning contexts. Fewer still have focused on challenges involved in designing for youth (13-17 years old). In this study, I explore the process of designing ARGs as vehicles for promoting information literacy and participatory culture for adolescents (13-17 years old). Two ARG design scenarios, distinguished by target learning environment (formal and informal context) and target audience (adolescents), comprise the two cases that I examine. Through my analysis of these two design cases, I articulate several unique challenges faced by designers who create interactive, transmedia stories for – and with – youth. Drawing from these design challenges, I derive a repertoire of design strategies that future designers and researchers may use to create and implement ARGs for teens in learning contexts. In particular, I propose a narrative design framework that allows for the categorization of ARGs as storytelling constructs that lie along a continuum of participation and interaction. The framework can serve as an analytic tool for researchers and a guide for designers. In addition, I establish a framework of social roles that designers may employ to craft transmedia narratives before live launch and to promote and scaffold player participation after play begins. Overall, the contributions of my study include theoretical insights that may advance our understanding of narrative design and analysis as well as more practical design implications for designers and practitioners seeking to incorporate transmedia features into learning experiences that target youth.

Wood-derived biomaterials for biomedical applications

Driven by the global trend in the sustainable economy development and environmental concerns, the exploring of plant-derived biomaterials or biocomposites for potential biomedical and/or pharmaceutical applications has received tremendous attention. Therefore, the work of this thesis is dedicated to high-value and high-efficiency utilization of plant-derived materials, with the focus on cellulose and hemicelluloses in the field of biomedical applications in a novel biorefinery concept. The residual cellulose of wood processing waste, sawdust, was converted into cellulose nanofibrils (CNFs) with tunable surface charge density and geometric size through 2,2,6,6-tetramethylpiperidinyloxy (TEMPO)-mediated oxidation and mechanical defibrillation. The sawdust-based CNFs and its resultant free-standing films showed comparable or even better mechanical properties than those from a commercial bleached kraft pulp at the same condition, demonstrating the feasibility of producing CNFs and films thereof with outstanding mechanical properties from birch sawdust by a process incorporated into a novel biorefinery platform recovering also polymeric hemicelluloses for other applications. Thus, it is providing an efficient route to upgrade sawdust waste to valuable products. The surface charge density and geometric size of the CNFs were found to play key roles in the stability of the CNF suspension, as well as the gelling properties, swelling behavior, mechanical stiffness, morphology and microscopic structural properties, and biocompatibility of CNF-based materials (i.e. films, hydrogels, and aerogels). The CNFs with tunable surface chemistry and geometric size was found promising applications as transparent and tough barrier materials or as reinforcing additive for production of biocomposites. The CNFs was also applied as structural matrices for the preparation of biocomposites possessing electrical conductivity and antimicrobial activity by in situ polymerization and coating of polypyrrole, and incorporation of silver nanoparticles, which make the material possible for potential wound healing application. The CNF-based matrices (films, hydrogels, and aerogels) with tunable structural and mechanical properties and biocompatibility were further prepared towards an application as 3D scaffolds in tissue engineering. The structural and mechanical strength of the CNF matrices could be tuned by controlling the charge density of the nanocellulose, as well as the pH and temperature values of the hydrogel formation conditions. Biological tests revealed that the CNF scaffolds could promote the survival and proliferation of tumor cells, and enhance the transfection of exogenous DNA into the cells, suggesting the usefulness of the CNF-based 3D matrices in supporting crucial cellular processes during cell growth and proliferation. The CNFs was applied as host materials to incorporate biomolecules for further biomedical application. For example, to investigate how the biocompatibility of a scaffold is influenced by its mechanical and structural properties, these properties of CNF-based composite matrices were controlled by incorporation of different hemicelluloses (O-acetyl galactoglucomanan (GGM), xyloglucan (XG), and xylan) into CNF hydrogel networks in different ratios and using two different approaches. The charge density of the CNFs, the incorporated hemicellulose type and amount, and the swelling time of the hydrogels were found to affect the pore structure, the mechanical strength, and thus the cells growth in the composite hydrogel scaffolds. The mechanical properties of the composite hydrogels were found to have an influence on the cell viability during the wound healing relevant 3T3 fibroblast cell culture. The thusprepared CNF composite hydrogels may work as promising scaffolds in wound healing application to provide supporting networks and to promote cells adhesion, growth, and proliferation.

Shaping surface acoustic waves for cardiac tissue engineering

The heart is a non-regenerating organ that gradually suffers a loss of cardiac cells and functionality. Given the scarcity of organ donors and complications in existing medical implantation solutions, it is desired to engineer a three-dimensional architecture to successfully control the cardiac cells in vitro and yield true myocardial structures similar to native heart. This thesis investigates the synthesis of a biocompatible gelatin methacrylate hydrogel to promote growth of cardiac cells using biotechnology methodology: surface acoustic waves, to create cell sheets. Firstly, the synthesis of a photo-crosslinkable gelatin methacrylate (GelMA) hydrogel was investigated with different degree of methacrylation concentration. The porous matrix of the hydrogel should be biocompatible, allow cell-cell interaction and promote cell adhesion for growth through the porous network of matrix. The rheological properties, such as polymer concentration, ultraviolet exposure time, viscosity, elasticity and swelling characteristics of the hydrogel were investigated. In tissue engineering hydrogels have been used for embedding cells to mimic native microenvironments while controlling the mechanical properties. Gelatin methacrylate hydrogels have the advantage of allowing such control of mechanical properties in addition to easy compatibility with Lab-on-a-chip methodologies. Secondly in this thesis, standing surface acoustic waves were used to control the degree of movement of cells in the hydrogel and produce three-dimensional engineered scaffolds to investigate in-vitro studies of cardiac muscle electrophysiology and cardiac tissue engineering therapies for myocardial infarction. The acoustic waves were characterized on a piezoelectric substrate, lithium niobate that was micro-fabricated with slanted-finger interdigitated transducers for to generate waves at multiple wavelengths. This characterization successfully created three-dimensional micro-patterning of cells in the constructs through means of one- and two-dimensional non-invasive forces. The micro-patterning was controlled by tuning different input frequencies that allowed manipulation of the cells spatially without any pre- treatment of cells, hydrogel or substrate. This resulted in a synchronous heartbeat being produced in the hydrogel construct. To complement these mechanical forces, work in dielectrophoresis was conducted centred on a method to pattern micro-particles. Although manipulation of particles were shown, difficulties were encountered concerning the close proximity of particles and hydrogel to the microfabricated electrode arrays, dependence on conductivity of hydrogel and difficult manoeuvrability of scaffold from the surface of electrodes precluded measurements on cardiac cells. In addition, COMSOL Multiphysics software was used to investigate the mechanical and electrical forces theoretically acting on the cells. Thirdly, in this thesis the cardiac electrophysiology was investigated using immunostaining techniques to visualize the growth of sarcomeres and gap junctions that promote cell-cell interaction and excitation-contraction of heart muscles. The physiological response of beating of co-cultured cardiomyocytes and cardiac fibroblasts was observed in a synchronous and simultaneous manner closely mimicking the native cardiac impulses. Further investigations were carried out by mechanically stimulating the cells in the three-dimensional hydrogel using standing surface acoustic waves and comparing with traditional two-dimensional flat surface coated with fibronectin. The electrophysiological responses of the cells under the effect of the mechanical stimulations yielded a higher magnitude of contractility, action potential and calcium transient.

Preparación y caracterización de biomateriales híbridos de hidroxiapatito con fosfonato (2-Hidroxi fosfonoacetato)

El hidroxiapatito es el biomineral de los tejidos duros de los mamíferos y por ello ha sido ampliamente estudiado como biomaterial en cementos óseos, recubrimientos de metales implantables y scaffold en ingeniería de tejidos. En este trabajo, se estudia un proceso síntesis y caracterización de un derivado de este material, donde el ion fosfonato: 2- hidroxifosfonoacetato sustituye parcialmente al ion fosfato [1-10%]. El proceso de síntesis de hidroxiapatito consiste en dos etapas: (1) una reacción a 100ºC entre el ácido fosfórico (añadido lentamente) y una mezcla de hidróxido de calcio + 2-hidroxifosfonoacetato de calcio (Relación Ca/P=1.67). (2) un tratamiento térmico de los sólidos obtenidos en (1) a 150ºC. Los productos finales se han caracterizado mediante análisis químico, análisis termogravimétrico, difracción de rayos-X con análisis de Rietveld, microscopía SEM y determinación de la superficie específica y porosidad. En las fases cristalinas, el grado de pureza de los hidroxiapatitos oscila entre 91-100%. Todos presentan estructura monoclínica, en contraste con la estructura hexagonal que habitualmente se obtiene aplicando métodos de síntesis similares. Esta variación se atribuye a la presencia de fosfonato. El contenido de éste también influye en la morfología y en la porosidad de los materiales, siendo las muestras con más fosfonato las más porosas. Los materiales, al menos aquellos con una cantidad menor o igual 5% de fosfonato, muestran una biocompatibilidad similar a un material de hidroxiapatito de referencia.

3D Porous Architecture of Stacks of β-TCP Granules Compared with That of Trabecular Bone: A microCT, Vector Analysis, and Compression Study

International audience

Synthetic Circuits for Feedback and Detection in Bacteria

Synthetic biology, by co-opting molecular machinery from existing organisms, can be used as a tool for building new genetic systems from scratch, for understanding natural networks through perturbation, or for hybrid circuits that piggy-back on existing cellular infrastructure. Although the toolbox for genetic circuits has greatly expanded in recent years, it is still difficult to separate the circuit function from its specific molecular implementation. In this thesis, we discuss the function-driven design of two synthetic circuit modules, and use mathematical models to understand the fundamental limits of circuit topology versus operating regimes as determined by the specific molecular implementation. First, we describe a protein concentration tracker circuit that sets the concentration of an output protein relative to the concentration of a reference protein. The functionality of this circuit relies on a single negative feedback loop that is implemented via small programmable protein scaffold domains. We build a mass-action model to understand the relevant timescales of the tracking behavior and how the input/output ratios and circuit gain might be tuned with circuit components. Second, we design an event detector circuit with permanent genetic memory that can record order and timing between two chemical events. This circuit was implemented using bacteriophage integrases that recombine specific segments of DNA in response to chemical inputs. We simulate expected population-level outcomes using a stochastic Markov-chain model, and investigate how inferences on past events can be made from differences between single-cell and population-level responses. Additionally, we present some preliminary investigations on spatial patterning using the event detector circuit as well as the design of stationary phase promoters for growth-phase dependent activation. These results advance our understanding of synthetic gene circuits, and contribute towards the use of circuit modules as building blocks for larger and more complex synthetic networks.