Rozprawy doktorskie na temat „Scaffold based models”

La compréhension des organismes vivant a été une quête pendant longtemps. Depuis les premiers progrès des derniers siècles, nous sommes arrivés jusqu'au point où des quantités massives de données et d'information sont constamment générées. Bien que, jusqu'au present la plupart du travail a été concentré sur la génération d'un catalogue d'éléments biologiques, ce n'est pas que récemment qu'un effort coordonné pour dé- couvrir les réseaux de relations entre ces parties a'été constaté. Nous sommes intereses à comprendre non pas seulement ces réseaux, mais aussi la façon dont, à partir de ses connexions, émergent des fonctions biologiques. Ce travail se concentre sur la découverte, la modélisation et l'exploitation d'un de ces réseaux : le métabolisme. Un réseau métabolique est un ensemble des réac- tions biochimiques interconnectées qui se produisent à l'intérieur, ou dans les limites d'une cellule vivante. Une nouvelle méthode de découverte, ou de reconstruction des réseaux métaboliques est proposée dans ce travail, avec une emphase particulière sur les organismes eucaryotes. Cette nouvelle méthode est divisée en deux parties : une nouvelle approche pour la modélisation de la reconstruction basée sur l'instanciation des éléments d'un modèle squelette existant, et une nouvelle méthode de réécriture d'association des gènes. Cette méthode en deux parties permet des reconstructions qui vont au-delà de la capacité des méthodes de l'état de l'art, permettant la reconstruction de modèles métaboliques des organismes eucaryotes, et fournissant une relation détaillée entre ses réactions et ses gènes, des connaissances cruciales pour des applications biotechnologies. Les méthodes de reconstruction développées dans ce travail, ont été complétées par un workflow itératif d'édition, de vérification et d'amélioration du modèle. Ce workflow a été implémenté dans un logiciel, appelé Pathtastic. Comme une étude de cas de la méthode développée et implémentée dans le pré- sent travail, le réseau métabolique de la levure oléagineuse Yarrowia lipolytica, connu comme contaminant alimentaire et utilisé pour la biorestauration et comme usine cellulaire, a été reconstruit. Une version préliminaire du modèle a été générée avec Pathtastic, laquelle a été améliorée par curation manuelle, à travers d'un travail avec des spécialistes dans le domaine de cette espèce. Les données expérimentales, obtenues à partir de la littérature, ont été utilisées pour évaluer la qualité du modèle produit. La méthode de reconstruction chez les eucaryotes, et le modèle reconstruit de Y. lipolytica peuvent être utiles pour les communautés scientifiques respectives, le premier comme un pas vers une meilleure reconstruction automatique des réseaux métaboliques, et le deuxième comme un soutien à la recherche, un outil pour des applications biotechnologiques et comme un étalon-or pour les reconstructions futures.

La compréhension des organismes vivant a été une quête pendant longtemps. Depuisles premiers progrès des derniers siècles, nous sommes arrivés jusqu’au point où desquantités massives de données et d’information sont constamment générées. Bien que,jusqu’au présent la plupart du travail a été concentré sur la génération d’un catalogued’éléments biologiques, ce n’est pas que récemment qu’un effort coordonné pour découvrirles réseaux de relations entre ces parties a été constaté. Nous nous sommes intéressésà comprendre non pas seulement ces réseaux, mais aussi la façon dont, à partir de sesconnexions, émergent des fonctions biologiques.Ce travail se concentre sur la modélisation et l’exploitation d’un de ces réseaux :le métabolisme. Un réseau métabolique est un ensemble des réactions biochimiquesinterconnectées qui se produisent à l’intérieur, ou dans les proximité d’une cellulevivante. Une nouvelle méthode de découverte, ou de reconstruction des réseaux métaboliquesest proposée dans ce travail, avec une emphase particulière sur les organismeseucaryotes.Cette nouvelle méthode est divisée en deux parties : une nouvelle approche pour lamodélisation de la reconstruction basée sur l’instanciation des éléments d’un modèlesquelette existant, et une nouvelle méthode de réécriture d’association des gènes. Cetteméthode en deux parties permet des reconstructions qui vont au-delà de la capacitédes méthodes de l’état de l’art, permettant la reconstruction de modèles métaboliquesdes organismes eucaryotes, et fournissant une relation détaillée entre ses réactions etses gènes, des connaissances cruciales pour des applications biotechnologiques.Les méthodes de reconstruction développées dans ce travail, ont été complétéespar un workflow itératif d’édition, de vérification et d’amélioration du modèle. Ceworkflow a été implémenté dans un logiciel, appelé Pathtastic.Comme une étude de cas de la méthode développée et implémentée dans le présenttravail, le réseau métabolique de la levure oléagineuse Yarrowia lipolytica, connucomme contaminant alimentaire et utilisé pour la biorestauration et comme usinecellulaire, a été reconstruit. Une version préliminaire du modèle a été générée avecPathtastic, laquelle a été améliorée par curation manuelle, à travers d’un travail avecdes spécialistes dans le domaine de cette espèce. Les données expérimentales, obtenuesà partir de la littérature, ont été utilisées pour évaluer la qualité du modèle produit.La méthode de reconstruction chez les eucaryotes, et le modèle reconstruit deY. lipolytica peuvent être utiles pour les communautés scientifiques respectives, lepremier comme un pas vers une meilleure reconstruction automatique des réseauxmétaboliques, et le deuxième comme un soutien à la recherche, un outil pour desapplications biotechnologiques et comme un étalon-or pour les reconstructions futures
Understanding living organisms has been a quest for a long time. Since the advancesof the last centuries, we have arrived to a point where massive quantities of data andinformation are constantly generated. Even though most of the work so far has focusedon generating a parts catalog of biological elements, only recently have we seena coordinated effort to discover the networks of relationships between those parts. Notonly are we trying to understand these networks, but also the way in which, from theirconnections, emerge biological functions.This work focuses on the modeling and exploitation of one of those networks:metabolism. A metabolic network is a net of interconnected biochemical reactionsthat occur inside, or in the proximity of, a living cell. A new method of discovery, orreconstruction, of metabolic networks is proposed in this work, with special emphasison eukaryote organisms.This new method is divided in two parts: a novel approach to reconstruct metabolicmodels, based on instantiation of elements of an existing scaffold model, and a novelmethod of assigning gene associations to reactions. This two-parts method allows reconstructionsthat are beyond the capacity of the state-of-the-art methods, enablingthe reconstruction of metabolic models of eukaryotes, and providing a detailed relationshipbetween its reactions and genes, knowledge that is crucial for biotechnologicalapplications.The reconstruction methods developed for the present work were complementedwith an iterative workflow of model edition, verification and improvement. This workflowwas implemented as a software package, called Pathtastic.As a case study of the method developed and implemented in the present work,we reconstructed the metabolic network of the oleaginous yeast Yarrowia lipolytica,known as food contaminant and used for bioremediation and as a cell factory. A draftversion of the model was generated using Pathtastic, and further improved by manualcuration, working closely with specialists in that species. Experimental data, obtainedfrom the literature, were used to assess the quality of the produced model.Both, the method of reconstruction in eukaryotes, and the reconstructed model ofY. lipolytica can be useful for their respective research communities, the former as astep towards better automatic reconstructions of metabolic networks, and the latteras a support for research, a tool in biotechnological applications and a gold standardfor future reconstructions

This thesis represents a step forward in the development of a pre-clinical model investigating a suitable substitute for host bone for use in human spinal fusion. By way of an animal model, it examines the biological performance of a novel bone graft substitute comprised of a combination of a custom-designed biodegradable material and biologics.

Breast cancer is one of the most common types of cancer affecting women in the world today. To better understand breast cancer initiation and progression modeling biological tissue under physiological conditions is essential. Indeed, breast cancer involves complex interactions between mammary epithelial cells and the stroma, both extracellular matrix (ECM) and cells including adipocytes (fat tissue) and fibroblasts (connective tissue). Therefore, the engineering of in vitro three-dimensional (3D) systems of breast tissues allows a deeper understanding of the complex cell-cell and cell-ECM interactions involved during breast tissue development and cancer initiation and progression. Furthermore, such 3D systems may provide a viable alternative to investigate new drug or drug regimen and to model and monitor concurrent cellular processes during tumor growth and invasion. The development of suitable 3D in vitro models relies on the ability to mimic the microenvironment, the structure, and the functions of the breast tissue. Different approaches to develop a novel 3D breast model have been investigated. Most models use gel scaffolds, including Matrigel® and collagen to generate breast tissue-like structures. However, the physicochemical, mechanical, and geometrical properties of these scaffolds only partially meet the mechanical, physical, and chemical parameters of the breast tissue matrix. In the present studies, we investigated the overall hypothesis that electrospun SF-derived scaffolds promote mammary cell growth and the formation of mammary-like structures depending on the composition and/or coating of the scaffolds with ECM proteins. Through an extensive literature search (1) the importance of 3D modeling of tissues and organs in vivo, (2) 3D modeling of the mammary tissue and currently available models, (3) the properties and applications of SF in tissue modeling and regeneration were reviewed (Chapter 1). Our studies provide evidence of the effects of various concentrations (Chapter 2) of SF along with different electrospinning techniques (Chapter 3) on the structure of electrospun scaffolds and whether those scaffolds provide suitable microenvironments for mammary epithelial cells as determined by MCF10A cell attachment, viability, and structure formation. Further, we investigated the effects of the key ECM proteins collagen I (Chapter 4) and laminin (Chapter 5) used to blend or coat, respectively, SF scaffolds on the attachment, viability and structure formation of mammary epithelial cells. Our studies first highlight the mechanical and physical properties of the different SF-derived scaffolds through various SF concentrations and electrospinning techniques. Second, the biocompatibility of these SF electrospun scaffolds was defined based on MCF10A cell survival and adhesion. Third, our data indicate that scaffolds derived from blended and/or coated SF with collagen I also promoted human mammary cell survival and adhesion. Lastly, our observations suggest that on laminin-coated SF scaffolds MCF10A mammary cells, in the presence of lactogenic hormones, differentiated forming acinus-like structures. Overall, these studies provide evidence that SF electrospun scaffolds closely mimic the structure of the ECM fibers and allow many advantages such as; physical and chemical modification of the microenvironment by varying electrospinning parameters and addition of various proteins, hormones, and growth factors, respectively. Further, coating these SF scaffolds with essential ECM proteins, in particular laminin, promote cell-ECM interactions necessary for cell differentiation and formation of growth-arrested structures, through providing cell integrin binding sites and appropriate chemical cues.

2016 - 2017
In this thesis, new possible applications of electrospun scaffolds are presented. Besides, the interaction of human dermal fibroblasts (HDFs) with the materials has been investigated using multiphoton microscopy (MPM) coupled with fluorescence lifetime imaging microscopy (FLIM), using a non-invasive, marker-free approach. In the first part of the thesis, pure poly-L-lactide (PLA) scaffolds were obtained and characterized as delivery systems for Diclofenac sodium salt (DCF) and a synthetically obtained prodrug of it for the treatment of actinic keratosis. The Diclofenac prodrug was obtained via solid phase peptide synthesis using a versatile, clean, high yielding procedure. Besides, the drug encapsulation and its release from the scaffold could be imaged using MPM. Moreover, when working with the unmodified DCF we were able to control the release profile by adding small amounts of dimethyl sulfoxide. The DCF-loaded scaffold was used as a delivery system to induce in vitro cell death in HDFs. The target cells were imaged using MPM coupled with FLIM, using a non-invasive, marker-free in vitro model to investigate drug effects. In the last part of the thesis, we produced and characterized different hybrid gelatin/PLA scaffolds. In this case, the goal was to obtain well-blended scaffolds with tunable properties, such as porosity, hydrophobicity and wettability... [edited by Author]
XXX ciclo

While small bone defects heal spontaneously, critical size defects may exceed the body’s regenerative capabilities, and require the use of bone substitutes and implants. To date, in vitro and in vivo testing of implants remains the gold standard for rigorous mechanical stability and biological safety checks. Current 2D in vitro testing is limited by a lack of dynamic environment and an inability to investigate mechanical strength of the attachment between the bone-matrix and implant surface. 3D in vivo tests are also limited by differences in the behaviour and structure of human and animal cells, high costs and difficulty of replicating human ageing effects. The aim of this thesis is to develop biocompatible and osteoconductive polyurethane-based scaffolds with optimal mechanical and biological properties that can be used as 3D in vitro bone models for bone regeneration and implant testing. 17 Plain-PU and PU-HA scaffolds were fabricated from three different medical grade polyether-urethanes, namely, Z1A1, Z3A1 and Z9A1. The polymer’s ability to dissolve in graded concentrations of DMF/THF solvents was assessed as part of this study. Composite scaffolds containing nano or micro HA particles were fabricated in a ratio of 3 PU: 1 HA by doping PU solutions with HA particles. Electrospinning, freeze drying, freeze extraction and particulate leaching were the main fabrication techniques explored for creating scaffolds. Electrospun scaffolds with non-aligned fibres were spun at 300 rpm whilst those with aligned fibres were spun at 1300 rpm. Particulate leaching using NaCl particles optimized 3 novel fabrication protocols namely, the layer-by-layer, homogenized or physical-mixing techniques for creating highly porous PU-based constructs. Investigation of non-aligned electrospun scaffolds showed that the choice of solvents, on their own or in combination, strongly influences the final properties of solution, hence the fibre morphology of scaffolds. Reducing the amount of DMF contained in the solution, increased fibre diameter, eliminated beads in fibres and led to scaffolds with a more uniform morphology. Moreover, reducing the DMF solvent content led to lower tensile properties of electrospun scaffolds, whilst incorporation of nano and micro HA particles reinforced the mechanical properties of both aligned and non-aligned electrospun composites. RAMAN and FTIR spectroscopy confirmed the presence of HA in all composites. Xylenol orange staining showed that composite mHA scaffolds supported a higher percentage of mineral area coverage compared to plain-PU scaffolds. SHG imaging identified that collagen deposition appeared to be guided by the alignment of the scaffold fibres in the matrix deposited near to the fibres, but changed orientation with an increase in distance from the originally deposited layers. Layer-by-Layer particulate leached scaffolds made from all the three types of PU had a highly porous 3D structure. 3:1 PU:nano-HA composites had the highest Young’s Modulus and yield strength in the Layer-by-Layer group and there was no significant difference between the mechanical properties of 3:1 micro-HA composites and 2:1 micro-HA composites. A novel physical mixing fabrication protocol shortened fabrication time by about 90% and was used to mass produce particulate leached scaffolds in a shorter period of time. Physically mixed particulate leached scaffolds had an interesting and contrasting mechanical profile compared to previously fabricated scaffolds. Physically mixed PU scaffolds without HA had the highest mechanical properties in this group and the inclusion of neither nano, micro nor combined micro and nano-HA particles enhanced their mechanical properties. Similar to the Layer-by-Layer particulate leached scaffolds, the inclusion of HA particles in physically mixed PU-only scaffolds did not support a higher cell viability. Osteoid bone formation was present in only nHA composites by D7 of the in vivo studies, but present in all scaffolds after D45. Collagenous matrix deposition increased over the 56 day period in all scaffold types, however, this increase was more pronounced in PU-only scaffolds. Finally, mimicking push-out and pull-out tests by inserting titanium screws into particulate leached scaffolds, showed that inserting the screws during cell seeding is a better method than inserting them after a D28 culture period. PU-based scaffolds that serve as a novel biomimetic in vitro 3D bone model for testing of small orthopaedic implants have been developed.

碩士
文藻外語大學
國際事業暨跨文化管理研究所
106
Construction industry is highly labor-intensive and technically professional. The work environment is complicated and variable, therefore, the construction site often suffers from unsafe environment, poor operation methods, and unsafe personal behaviors, which result in high occupational disasters incidence. Scaffolder is clarified high-risk work in construction industry, because scaffolder needs to carry heavy materials and climing on the heights. According to the pioneer of the American occupational safety, W.H. Heinrich, who proposed the famouse “domino theory”, claiming that the factors of accidents have causality and the unsafe personal behaviors are more likely to cause accidents and injuries than unsafe environment. Also, unsafe behavior is mostly caused by the lack of corresponding knowledge and skills. Thus, the purposes of this study are to discuss and development the competency model and competency-based training course of scaffolder in order to help raise scaffolders’ competency and decrease incidence of occupational disasters. This study uses secondary data and expert focus group meeting as data collecting methods to confirm the labor and course requirements and use ADDIE model to develop the competency model whick contains 8 elements, 35 performance indicators, 32 kinds of knowledge, 21 kinds of skills, and 9 kinds of attitude. Also, the competency-based trinaing course is developed based on the competency model.

To this day, cancer remains the leading cause of mortality worldwide1. A major contributor to cancer progression and metastasis is tumor angiogenesis. The formation of blood vessels around a tumor is facilitated by the complex interplay between cells in the tumor stroma and the surrounding microenvironment. Understanding this interplay and its dynamic interactions is crucial to identify promising targets for cancer therapy. Current methods in cancer research involve the use of two-dimensional (2D) monolayer cell culture. However, cell-cell and cell-ECM interactions that are important in vascularization and the three-dimensional (3D) tumor microenvironment cannot accurately be recapitulated in 2D. To obtain more biologically relevant information, it is essential to mimic the tumor microenvironment in a 3D culture system. To this end, we demonstrate the utility of poly(ethylene glycol) diacrylate (PEGDA) hydrogels modified for cell-mediated degradability and cell-adhesion to explore, in 3D, the effect of various tumor microenvironmental features such as cell-cell and cell-ECM interactions, and dimensionality on tumor vascularization and cancer cell phenotype.

In aim 1, PEG hydrogels were utilized to evaluate the effect of cells in the tumor stroma, specifically cancer associated fibroblasts (CAFs), on endothelial cells (ECs) and tumor vascularization. CAFs comprise a majority of the cells in the tumor stroma and secrete factors that may influence other cells in the vicinity such as ECs to promote the organization and formation of blood vessels. To investigate this theory, CAFs were isolated from tumors and co-cultured with HUVECs in PEG hydrogels. CAFs co-cultured with ECs organized into vessel-like structures as early as 7 days and were different in vessel morphology and density from co-cultures with normal lung fibroblasts. In contrast to normal lung fibroblasts (LF), CAFs and ECs organized into vessel-like networks that were structurally similar to vessels found in tumors. This work provides insight on the complex crosstalk between cells in the tumor stroma and their effect on tumor angiogenesis. Controlling this complex crosstalk can provide means for developing new therapies to treat cancer.

In aim 2, degradable PEG hydrogels were utilized to explore how extracellular matrix derived peptides modulate vessel formation and angiogenesis. Specifically, integrin-binding motifs derived from laminin such as IKVAV, a peptide derived from the α-chain of laminin and YIGSR, a peptide found in a cysteine-rich site of the laminin β chain, were examined along with RGDS. These peptides were conjugated to heterobifunctional PEG chains and covalently incorporated in hydrogels. The EC tubule formation in vitro and angiogenesis in vivo in response to the laminin-derived motifs were evaluated.

Based on these previous aims, in aim 3, PEG hydrogels were optimized to function as a 3D lung adenocarcinoma in vitro model with metastasis-prone lung tumor derived CAFs, HUVECs, and human lung adenocarcinoma derived A549 tumor cells. Similar to the complex tumor microenvironment consisting of interacting malignant and non-malignant cells, the PEG-based 3D lung adenocarcinoma model consists of both tumor and stromal cells that interact together to support vessel formation and tumor cell proliferation thereby more closely mimicking the functional properties of the tumor microenvironment. The utility of the PEG-based 3D lung adenocarcinoma model as a cancer drug screening platform is demonstrated with investigating the effects of doxorubicin, semaxanib, and cilengitide on cell apoptosis and proliferation. The results from drug screening studies using the PEG-based 3D in vitro lung adenocarcinoma model correlate with results reported from drug screening studies conducted in vivo. Thus, the PEG-based 3D in vitro lung adenocarcinoma model may serve as a better tool for identifying promising drug candidates than the conventional 2D monolayer culture method.

Dissertation

This mixed methods study compared distance and blended undergraduate environmental students at Royal Roads University (RRU) as they participated in online asynchronous PBL case discussion forums as part of an Ecotoxicology course. This study examined the differences between distance and blended teams in their activity, approaches, and levels of critical thinking in an online PBL activity. Critical thinking was evaluated using the cognitive presence indicators of the community of inquiry framework developed by Garrison, Anderson and Archer (2001). An organization indicator was added to the framework to capture posts that organized the discussion forum layout or the team and the distribution of work. The use of the organization indicator in the thread map analysis revealed that teams adopted one of two approaches to the online PBL activity, either an organic approach or an organizational scaffold approach. An open coding approach to content analysis of the posts was used to develop two coding schemes to capture the use of learning scaffolds and degree of online collaboration respectively. These coding schemes were used to compare scaffolding and collaboration behaviours of distance and blended students during the online PBL activity. The study found that whether teams used the online discussion forums or face-to-face discussion as their primary communication method influenced both the timing and the critical thinking content of the online discussion forums. Student moderators’ choices influenced the structure and approach to the PBL activity, as well as the form of document assembly that was observed in the online discussion forums. The learning scaffolds coding scheme demonstrated that both distance and blended students were reading beyond the assigned reading list. Both distance and blended students appeared to develop skills in identifying information gaps over the progression of the PBL case problems as their observable level of critical thinking remained consistent as the problem scaffolding was faded. Although both environmental and non-environmental work experience may be used to scaffold team learning, they are used differently. Online PBL is a good fit for the Royal Roads University Learning and Teaching Model and may be used to provide some consistency across blended and online course content.
Graduate