Articular cartilage represents a primary target for tissue engineering strategies as it does not functionally regenerate within the joint. Many tissue engineering approaches have focused on the in vitro generation of neo-cartilage using chondrocyte-seeded scaffolds. Several studies have reported the morphological appearance of native cartilage, although its functional competence has not been demonstrated. Accordingly, mechanical conditioning has often been introduced to enhance biosynthetic activity of chondrocytes within 3D constructs. However although this strategy has significantly up-regulated proteoglycan synthesis, its effects on the synthesis of the other major solid constituent, type II collagen, has been modest. Analyses of normal joint activities reveal that cartilage is subjected to shear superimposed on uniaxial compression. This complex mechanical state has motivated the design of a biaxial loading system intended for use in vitro to stimulated bovine chondrocytes seeded in agarose constructs. This necessitated the redesign of the construct from cylindrical morphology to accommodate shear loading. The experimental approach was complemented with the development of computational models, which permitted prediction of both cell distortion under biaxial loading regimens and nutrient diffusion within the 3D constructs. An initial study established the profile of proteoglycan and collagen synthesis in free swelling cultures up to day 12. The introduction of dynamic compression (15% strain, 1 Hz for 48 h) enhanced proteoglycan synthesis significantly. In addition, when dynamic shear (10%, 1 Hz) was superimposed on dynamic compression, total collagen synthesis was also up-regulated, within 3 days of culture, without compromising proteoglycan synthesis. Histological analysis revealed marked collagen deposition around individual chondrocytes. However, a significant proportion (50%) of collagen was released into the culture medium, suggesting that it was not fully processed. The overall biosynthetic activity was enhanced more when the biaxial stimulation was applied in a continuous mode as opposed to intermittent loading. The present work offers the potential for a more effective preconditioning of cell-seeded constructs with functional integrity intended for use to resolve defects in joint cartilage.

The biodegradability and when functionalised biocompatibility of aliphatic polycarbonates (APCs) makes them an attractive class of materials for biomedical applications such as tissue engineering scaffolds and drug-delivery carriers. One route to accessing a wide-range of well-defined and functional APCs is the controlled ring-opening polymerisation (ROP) of cyclic carbonates. In turn, these would ideally be prepared by the direct coupling of CO2 with diols to give water as the only by-product. In this way, the combination of CO2 and sugar-derived diols draws upon two natural renewable building blocks for the construction of polycarbonates that are anticipated to show good biocompatibility properties. Chapter 2 develops a simple and mild alternative to the traditional use of phosgene derivatives for the synthesis of six-membered cyclic carbonates from 1,3-diols and CO2. DFT calculations highlighted the need to lower both the CO2-insertion and ring-closing kinetic barriers to cyclic carbonate formation. Organic superbase, 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU) enabled the formation of carbonate species at 1 atm CO2 pressure whereas, the introduction of a leaving group strategy lowered the cyclisation barrier. Mechanistic considerations suggested a kinetic preference for ring- closing via a nucleophilic addition-elimination pathway rather than a SN2-like intramolecular cyclisation. Chapter 3 applies the procedure with CO2 to the preparation of a novel monomer from natural sugar, ᴅ-mannose. ROP was carried out via an organocatalytic approach and a preference for head-tail linkages in the polycarbonate backbone indicated by NMR spectroscopy and supported by DFT calculations. Chapter 4 utilises CO2 to invert the natural stereochemistry of sugars and create a thymidine-based monomer. The thermodynamic parameters of the ROP with 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) catalyst are determined and the properties of the polycarbonates investigated to include preliminary cell attachment studies. Finally, chapter 5 details the synthesis of cyclic carbonates from 2- deoxy-ᴅ-ribose and the investigation into the different ROP behaviour of the α- and β- anomers. The ability to tune the polymer properties through copolymerisation with trimethylene carbonate (TMC) is also discussed.

Les cellules épithéliales sont des cellules polarisées qui forment l’un des types cellulaires le plus abondant dans le corps humain. Leur polarité apico-basale (A/B) est établie et maintenue par la ségrégation asymétrique de protéines adaptatrices hautement conservées. Cette polarité est essentielle pour de nombreuses fonctions cellulaires clés comme l’adhésion (jonctions intercellulaires) ou la signalisation et la prolifération par la localisation et la concentration des complexes de signalisation. Durant la cancérogénèse, un grand nombre de ces processus est dérégulé aboutissant à la sur-prolifération, la migration et/ou l’invasion des cellules cancéreuses. Une meilleure compréhension des mécanismes à l’origine de ces processus est indispensable pour trouver de nouvelles cibles thérapeutiques pour le traitement du cancer. Dans l’équipe, nous sommes particulièrement intéressés par les protéines adaptatrices à domaines PDZ (domaine de liaison protéine-protéine). De par leur structure modulaire et la diversité de leurs partenaires, ces protéines adaptatrices sont impliquées dans la régulation de très nombreuses fonctions et fournissent des plateformes où différents processus peuvent être intégrés. Durant mon doctorat, j’ai étudié deux protéines adaptatrices dans le système animal modèle Drosophila melanogaster, Bbg et Magi, impliquées dans deux processus cellulaires essentiels : la dynamique des jonctions et la prolifération. Grâce aux molécules d’adhésion, les cellules non seulement restent cohésives dans un tissu, mais c’est aussi à ce niveau qu’elles peuvent obtenir une information concernant la densité cellulaire d'un tissu. Cette information est alors relayée au cytosquelette d’actine via des protéines adaptatrices spécialisées dans le but de réguler la prolifération et la voie Hippo. Cependant, le contrôle de la voie de signalisation Hippo par certaines protéines adaptatrices et par le cytosquelette d’actine n'est que partiellement compris à ce jour. Dans le laboratoire, nous étudions notamment le rôle d'une nouvelle protéine adaptatrice apicale nommée Big Bang (Bbg) dans le disque d’aile de la drosophile. Nous nous sommes intéressés à Bbg car c’est une cible de la voie Notch chez la drosophile et son homologue humain PDZD2 (pour PDZ domain-containing 2 protein) est sur-exprimé dans plusieurs cancers (sein et prostate).Mes résultats montrent que Bbg est un nouveau régulateur du cytosquelette d’actine et de la voie Hippo. Une étude détaillée de la fonction de Bbg et de ses partenaires permet de mieux comprendre les relations existantes entre dynamique de l’actine et prolifération. Bbg induit une accumulation d’actine filamenteuse en augmentant l’activité d’Enabled et la phosphorylation de Myosin Light Chain (MLC). Cette régulation résulte en une augmentation de l’activité de Yorkie, effecteur final de la voie Hippo, pour soutenir la prolifération cellulaire.La régulation des jonctions adhérentes est une étape cruciale lors de l’évolution d'une tumeur solide. Malgré les récentes avancées dans le domaine, de nombreux aspects clés de la dynamique des jonctions restent peu caractérisés.Dans le laboratoire, nous recherchons de nouveaux régulateurs de jonctions et grâce au modèle de remodelage des AJs lors du développement de l’œil de pupe de drosophile. Nous avons identifié Magi en tant que protéine adaptatrice recrutant le complexe formé de RASSF8 et ASPP. Magi régule le recrutement de Bazooka à la membrane, le dépôt d’E-Cadhérine et des Caténines et finalement le remodelage des jonctions pendant la morphogénèse. J’ai identifié Echinoid, une protéine de type immunoglobuline impliquée dans l’adhésion cellulaire et la régulation de la voie Hippo, comme un nouveau partenaire responsable du recrutement de Magi aux futures zones de jonctions. / Epithelial cells are polarised cells that form one of the most abundant cell types in the human body. Their apico-basal (A/B) polarity is established and maintained by the asymmetric segregation of highly conserved scaffold proteins. Proper A/B polarity is critical for many key cellular functions such as intercellular junctions and therefore adhesion, or signalling and proliferation by localising and concentrating signalling complexes. During carcinogenesis, many of these processes are mis-regulated leading to the over-proliferation, migration and/or invasion of cancer cells. A better understanding of the mechanisms underlying these processes is really needed to find new therapeutic targets in cancer treatment.In the team, we are particularly interested in scaffold proteins with PDZ domains (protein-protein interaction domains). Due to their modular structure, the high number of interactions they engage in, and the variety of their binding partners, these scaffold proteins are implicated in the regulation of many key cellular functions and processes. During my PhD, I have studied two scaffold proteins in the Drosophila melanogaster animal model, Bbg and Magi, which are involved in two important cell processes: adherens junctions (AJs) dynamic and cell proliferation.Through adhesion molecules, epithelial cells not only remain cohesive, but can also sense cellular density in a tissue and relay this information through dedicated scaffolds to the actin cytoskeleton to ultimately regulate the Hippo pathway and proliferation. However, many aspects of the control of Hippo signalling by apical scaffolds and the actin cytoskeleton are still poorly understood. In the laboratory, we are interested in the study of a new conserved apical scaffold, Big Bang (Bbg). Bbg is a new and quite unknown protein expressed in a variety of Drosophila epithelia, and appears as a potential Notch target in Drosophila. Its’ human homolog called PDZD2 (PDZ domain-containing 2 protein) has been shown to be over-expressed in several types of cancers (breast and prostate cancers).My results show that Bbg is a new regulator of the actin cytoskeleton and of the Hippo pathway in Drosophila. A detailed study of Bbg function and of its associated partners have helped to better understand the intricate relationships between actin dynamics and proliferation. My results suggest that Bbg promotes accumulation of filamentous actin (F-Actin) through the increase of the activity of Enabled (Ena) and the phosphorylation of the molecular motor Myosin Light Chain (MLC). This regulation leads to the increase of Yorkie activity, the final effector of the Hippo pathway, to promote cell proliferation.The regulation of adhesion, and in particular of Adherens Junctions (AJs), is a critical step during the evolution of solid tumours. A better understanding of how these structures are regulated will provide valuable insights into different phases of the disease. Despite the recent advances, many key aspects of AJ dynamics remain poorly understood. In the laboratory, we are interested in the identification of new AJs regulators. Using the remodelling of AJs during the development of the Drosophila pupal eye as a model, we have identified Magi as a scaffold recruiting a complex formed by RASSF8 and ASPP, regulating Bazooka membrane recruitment, E-Cadherin and catenins deposition, and ultimately AJs remodelling during morphogenesis. I uncovered Echinoid, an immunoglobulin-like protein involved in cell adhesion and in Hippo pathway regulation, as a new binding partner responsible for the recruitment of Magi at future AJ sites.

Functional integration of bone with soft tissues such as tendon is essential for joint motion and musculoskeletal function. This is evident in the rotator cuff of the shoulder, which consists of four muscles and their associated tendons that connect the humerus and scapula. The cuff functions to stabilize the shoulder joint, and actively controls shoulder kinematics. Rotator cuff injuries often occur as a result of tendon avulsion at the tendon-bone interface, with more than 250,000 cuff repair surgeries performed annually in the United States. However, these procedures are associated with a high failure rate, as re-tears often occur due to the lack of biological fixation of the tendon to bone post-surgery. Instead of regenerating the tendon-bone interface, current repair techniques and augmentation grafts focus on improving the load bearing capability of the repaired rotator cuff. Biologically, the supraspinatus tendon inserts into bone via a biphasic fibrocartilaginous transition, exhibiting region-dependent changes in its compositional, structural and mechanical properties, which enables efficient load transfer from tendon to bone as well as multi-tissue homeostasis. Inspired by the native tendon-bone interface, we have designed and evaluated a biomimetic bilayer scaffold, comprised of electrospun poly (lactide-co-glycolide) (PLGA) nanofibers seamlessly integrated with PLGA-hydroxyapatite (HA) fibers, in order to engineer tendon-bone integration. The objective of this thesis is to explore the key design parameters that are critical for integrative tendon-bone repair using this biphasic scaffold as a model. Specifically, intrinsic to the scaffold, effects of fiber alignment, fiber diameter, mineral distribution, and polymer composition on integrative rotator cuff tendon-bone healing were evaluated in vivo using a rat model. Results indicated that an aligned, nanofiber-based scaffold with a distinct order of non-mineralized and mineralized regions will lead to insertion regeneration and integrative tendon-bone repair. Additional tissue engineering design parameters such as healing time and animal model were also tested. It was observed that the biphasic scaffold exhibited a stable long term response, as the mechanical properties of rat shoulders repaired by this scaffold remained comparable to that of the control at 20 weeks post-surgery. This scaffold was also evaluated in a large animal model (sheep), in which a clinically-relevant rotator cuff repair procedure was implemented with the biphasic scaffold. Results demonstrated the scaffold lead to integrative rotator cuff repair through the regeneration of the enthesis in both small and large animal models. In summary, through a series of in vivo studies, the work of this thesis has identified the critical tissue engineering parameters for integrative and functional rotator cuff tendon repair. More importantly, the design principles elucidated here are anticipated to have a broader impact in the field of tissue engineering, as they can be readily applied towards the regeneration of other soft-hard tissue interfaces.

Ces travaux ont pour objet l’élaboration de nouvelles structures poreuses non tissées antibactériennes. Différentes stratégies ont été développées : l’une a consisté à élaborer des mats poreux par electrospinning en utilisant un polymère biosourcé et biocompatible et l’autre voie consistait à modifier un support fibreux provenant d’un masque de protection respiratoire commercial. La méthode des assemblages par interactions ioniques en superposant de façon alternative les couches de polymères cationiques et les polymères anioniques à la surface du filtre médian en polypropylène (PP) a permis d’élaborer de nouvelles structures ayant de bonnes propriétés antioxydantes et antibactériennes. Le polymère anionique, dérivé du polymère de cyclodextrine présente l’avantage de pouvoir encapsuler un agent antimicrobien biosourcé, le carvacrol. Une autre approche a consisté à modifier des supports en PP avec de l'acide tannique, un polyphénol d'origine naturelle. Dans cette étude, deux stratégies ont été mises en place afin de fonctionnaliser le PP avec de l’acide tannique (AT). La première est l’extrusion réactive du PP avec l’AT en présence (ou non) de peroxyde de dicumyle (DCP) pour greffer directement l’acide tannique sur le PP. La deuxième stratégie consiste à polymériser l’AT au travers d’une couche poreuse de PP extraite d’un masque de protection commercial, afin de permettre l’immobilisation physique de l’AT à la surface du mat fibreux en PP. Le greffage en surface via un procédé “grafting from” a également été étudié. Ces matériaux ont montré de bonnes propriétés antiradicalaires. / The purpose of this work is to develop new antibacterial non-woven porous structures. Different strategies were developed: one was to develop porous structures by electrospinning using a biosourced and biocompatible polymer, the other was to modify a fibrous support from a commercial respiratory protection mask. Assembling materials using ionic interactions by alternatively superposing cationic polymer layers and anionic polymers on the surface of the polypropylene (PP) median filter allowed to develop new structures with good antioxidant and antibacterial properties. The anionic polymer, derived from the cyclodextrin polymer, has the advantage of being able to encapsulate a bio-based antimicrobial agent, carvacrol. Another approach was to modify PP filters with tannic acid, a naturally occurring polyphenol. In this study, two strategies were implemented to functionalize PP with tannic acid (TA). The first is the reactive extrusion of PP with TA in the presence (or not) of dicumyl peroxide (DCP) to directly graft tannic acid onto PP. The second strategy consists in polymerizing the TA through a porous layer of PP extracted from a commercial mask, in order to allow the physical immobilization of the TA on the surface of the PP fibrous mat. Surface grafting using a "grafting from" process was also studied. These materials have shown good anti-free radical properties.

Type 1 Diabetes (T1D) is an autoimmune disorder in which the pancreatic β-cells are destroyed by the body's immune system. The reduced number of β-cells leads to inadequate insulin secretion and high glucose levels in the body. The requirement of insulin injection throughout life and lack of donors for islet transplantations has prompted a search for more accessible and available sources of insulin producing cells that can be transplanted in T1D patients. To that end, the discovery of induced pluripotent stem (iPS) cells has provided a potential source of precursors for cell therapy for T1D. iPS cells are reprogrammed somatic cells which can be transplanted back into the patient from whom the somatic cells were initially derived, thus potentially avoiding immune rejection when transplanted. As a potential therapy for T1D, we aim to derive insulin producing cells (IPCs) from human iPS cells. In contrast to the conventional two dimensional (2D) cell culture systems used in many iPS derived IPC studies, the inner cell mass (ICM) from which various organs differentiate during embryogenesis is a cluster of cells that enables signaling crosstalk between cells of different types. Three dimensional (3D) cell culture systems allows cells to form cell clusters that promote cell - cell signaling. Hence, we hypothesized that 3D cell culture systems will yield better efficiency of differentiation to functional IPCs in vitro than 2D cultures. Initially, the synthetic polymers sodium alginate and matrigel were analyzed for their ability to enable cell clustering to establish 3D cell culture systems. The 3D cell environment established using matrigel was used for the differentiation of human iPS cells to Insulin Producing Cells (IPC). The cells were first converted to endodermal cells. A mixture of growth factors then induced the differentiation of endodermal cells to pancreatic cells. The pancreatic cells were converted to IPCs that resemble pancreatic β-cells. Our 3D differentiated IPCs strongly expressed pancreatic endocrine transcription factors and pancreatic hormones. The IPCs also produced insulin when exposed to a high glucose environment. But the number of IPCs obtained at the end of the differentiation was low. Hence, our results demonstrate that 3D differentiation generates functional IPCs in vitro unlike 2D differentiation. In the future we aim to improve the percentage of IPCs that we generate from the 3D differentiation. Our expectation is that these cells will be able to cure hyperglycemia in diabetic mice more rapidly compared to the 2D differentiated cells owing to their proven insulin production in the presence of a high glucose environment in vitro.

This study examines and describes the nature of questioning moves used by two exemplary fourth-grade teachers during reading instruction. Questioning moves are defined in this study as the ways in which teachers use scaffolding questions to engage students in talk about text. Another point of interest in this study was to determine how teachers perceive the influence of instructional materials on the language they use to engage students in talk about text. This study was situated within a constructivist paradigm of inquiry and drew from the case study tradition for its design. Naturalistic methods of data collection were employed including transcripts of teacher and student talk, field notes, videotapes, and interviews with the teachers. Data analysis was conducted in two stages. First data were analyzed separately within each case to locate emerging patterns to build each teacher's profile. Then data were juxtaposed for the purpose of comparison to illuminate similarities and differences in patterns that cut across cases. In general, results show that while questioning moves used by exemplary fourth-grade teachers are different, they are simple and subtle. The questioning moves used provided scaffolding for the purpose of increasing the students' responsibility for constructing meaning from text and signaled teachers' high expectations in their students' ability to read and interact with text. Teachers' use of questioning moves was determined by the instructional focus and hinged on the nature, intensity, and support of their professional development opportunities and experiences. Additional findings, indirectly related to teachers' use of questioning moves, and the influences on their use, were themed around the nature of attention that teachers gave to their classroom environment and instructional design. Implications of the results of this study for reading teachers and educators are themed around issues of professional development and time.

There are not enough donor lungs available to meet the increasing demand for lung transplantation. To compound the problem, transplant recipients have a projected survival time of only 5.7 years despite life-long immunosuppression. An alternative approach for acquiring transplantable lungs and reducing post-operative complications may be possible through tissue engineering. Perfusion-decellularization generates natural, three-dimensional extracellular matrix (ECM) scaffolds of an organ that are apt for tissue engineering. Decellularization of the heart, lung, liver, kidney, and pancreas has been reported in animal models and from human tissue. Decellularization of fibrotic and emphysematic lungs indicated that this technique can efficiently remove cells from diseased tissue—a potential source of materials for engineering of transplantable lung tissue. Pulmonary hypertension (PHT) is a vascular disease characterized by increased pulmonary vascular resistance leading to right heart failure and death. Lungs damaged by PHT are unsuitable for transplantation; however, decellularization of these organs may provide scaffolds appropriate for ex vivo lung engineering. Monocrotaline-induced PHT (MCT-PHT) is a well-established model of this disease in rats closely resembling the clinical presentation of PHT in humans. Thus, decellularization and recellularization of hypertensive lungs was evaluated using the MCT-PHT model. Decellularization of control and MCT-PHT Sprague-Dawley rat lungs was accomplished by treating the lungs with Triton X-100, sodium deoxycholate (SDC), NaCl, and DNase. The resulting acellular matrices were extensively characterized by molecular, mechanical, and structural analyses revealing that decellularization was able to remove cells while leaving the ECM components and lung ultrastructure intact; however, the vasculature of MCT-PHT acellular lung scaffolds was narrower than control scaffolds—a hallmark of PHT. To evaluate the effect of narrowed vasculature on the use of hypertensive lungs for tissue engineering, an optimal vascular recellularization technique was developed. Gravity-based seeding of endothelial cells followed by bioreactor-based whole-organ culture resulted in efficient vascular recellularization of control lung scaffolds. However, this method led to heterogeneous re-endothelialization of the vasculature of MCT-PHT matrices suggesting that additional manipulation or optimization is required. / acase@tulane.edu

In this thesis, I explore how design scaffolds, or (i.e., intellectual supports) can assist learners engaging with computational making processes. Computational making combines programming with artifact production. Due to the complexity of tasks involved in computational making, there is an increasing need to explore and develop support systems for learners engaging with computational making. With $3,000 funding from Utah State University’s College of Education and Human Services, an undergraduate researcher and I, who both have experience with youth and computational making research, explored how design scaffolds impact youth engaging with computational making processes. To do so, we held a workshop where 11 learners (11 female, ages 11-16) used ARIS, a platform designed for non-programmers to create mobile games. In addition, we interviewed five ARIS designers who were able to evaluate our design scaffolds. We provide insights for improving the use of design scaffolds in computational making with ARIS specifically that also apply broadly to computational making processes. Moreover, we developed an ARIS course that teaches educators to use a design scaffold tool for ARIS. This research provides immediate benefits for educators who access the ARIS course and researchers seeking to improve upon design scaffold research for computational making processes.

Orientador: Gustavo Franco Barbosa / Resumo: Há décadas, a engenharia de tecidos passou a ser considerada em diversas aplicações, um tratamento médico adequado, devido suas excelentes vantagens, além da escassez de órgãos e disponibilidade de tecidos para serem transplantados. Conhecida como regeneração de novos tecidos, esse ramo da engenharia biomédica fundamentada nos conhecimentos de Biologia, Química e Física, torna-se uma grande alternativa quando tratamentos farmacêuticos convencionais não são mais aplicáveis, utilizando-se de três tipos básicos de ferramentas: célula, scaffolds e fator de crescimento. Dessa forma, esse trabalho tem como propósito principal a manufatura de scaffolds, utilizando a tecnologia de impressão 3D a partir de polímeros termoplásticos biodegradáveis e fosfatos de cálcio (em escala micrométrica), com o objetivo de se obter estruturas 3D complexas e porosas que apresentem propriedades mecânicas adequadas (em relação a ossos) e interconectividade estruturada entre os poros. Com os modelos 3D dos scaffolds projetados, e a seleção e preparação dos materiais envolvidos, foram realizados ajustes de parâmetros para o processamento dos scaffolds e posterior fabricação dos mesmos, mediante o uso da tecnologia de manufatura aditiva com bioimpressora de microextrusão que utiliza sistema de distribuição pneumático para extrusão contínua do material. Por fim os scaffolds foram caracterizados por técnica de análise de propriedade mecânica por ensaio de compressão e as amostras avaliadas pelo método de M... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: For decades, tissue engineering has come to be considered in several applications, an adequate medical treatment, due to its excellent advantages, in addition to the scarcity of organs and the availability of tissues to be transplanted. Known as regenerating of new tissues, this branch of biomedical engineering grounded in the knowledge of biology, chemistry and physics, becomes a great alternative when conventional pharmaceutical treatments are no longer applicable, using three basic types of tools: cell, scaffolds and growth factor. Thus, the main purpose of this work is the manufacture of scaffolds, using the technology of 3D printing from biodegradable thermoplastic polymers and calcium phosphates (in micrometric scale), with the objective of obtaining complex and porous 3D structures that present properties mechanical (in relation to bones) and structured interconnectivity between the pores. With the 3D models of the scaffolds designed, and the selection and preparation of the materials involved, adjustments were made to the processing parameters of the scaffolds and their subsequent manufacture, using the technology of additive manufacturing with microextrusion bioprinter that uses pneumatic distribution system for continuous extrusion of the material. Finally, the scaffolds were characterized by technique of mechanical property analysis by compression test and the samples evaluated by Scanning Electron Microscopy (SEM) method. / Mestre