Thesis (Ph.D.)--Ohio University, November, 2004. / Title from PDF t.p. Includes bibliographical references (p. 186-195 )

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 105-120).

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 186-200). Also available in electronic version.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 116-131).

<p>High-throughput processing technologies hold critical position in biomedical research. These include screening of cellular response based on phenotypic difference and production of homogeneous chemicals and biologicals for therapeutic applications. The rapid development of microfluidics technology has provided an efficient, controllable, economical and automatable processing platform for various applications. In particular, emulsion droplet gains a lot of attention due to its uniformity and ease of isolation, but the application of water-in-oil (W/O) single emulsion is hampered by the presence of the oil phase which is incompatible with aqueous phase manipulation and the difficulty in modifying the droplet environment.</p><p>This thesis presents the development of a double emulsion (DE) droplet platform in microfluidics and two applications: (1) high-throughput screening of synthetic gene and (2) production of multicellular spheroids with adjustable microenvironment for controlling stem cell differentiation and liver tissue engineering. Monodisperse DE droplets with controllable size and selective permeability across the oil shell were generated via two microfluidics devices after optimization of device design and flow rates. </p><p>Next, bacterial cells bearing synthetic genes constructed from an inkjet oligonucleotide synthesizer were encapsulated as single cells in DE droplets. Enrichment of fluorescent signals (~100 times) from the cells allowed quantification and selection of functionally-correct genes before and after error correction scheme was employed. Permeation of Isopropyl β-D-1-thiogalactopyranoside (IPTG) molecules from the external phase triggered target gene expression of the pET vector. Fluorescent signals from at least ~100 bacteria per droplet generated clearly distinguishable fluorescent signals that enabled droplets sorting through fluorescence-activated cell sorting (FACS) technique.</p><p>In addition, DE droplets promoted rapid aggregation of mammalian cells into single spheroid in 150 min. Size-tunable human mesenchymal stem cells (hMSC) spheroids could be extracted from the droplets and exhibited better differentiation potential than cells cultured in monolayer. The droplet environment could be altered by loading matrix molecules in it to create spheroid-encapsulated microgel. As an example, hMSC spheroid was encapsulated in alginate or alginate-RGD microgel and enhanced osteogenic differentiation was found in the latter case.</p><p>Lastly, the capability of forming spheroids in DE droplet was applied in liver tissue engineering, where single or co-culture hepatocyte spheroids were efficiently produced and encapsulated in microgel. The use of alginate-collagen microgel significantly improved the long-term function of the spheroid, in a manner similar to forming co-culture spheroids of hepatocytes and endothelial progenitor cells at a 5 to 1 ratio. The hepatocyte spheroid encapsulated in microgel could be useful for developing bioartificial liver or drug testing platform or applied directly for hepatocyte transplantation.</p> / Dissertation

Dans la littérature, de nombreux types de synthèses de particules Janus ont été proposés. Nous montrons ici une nouvelle méthode de synthèse en continu afin d’obtenir des particules Janus inorganiques d’une trentaine de nanomètres. Nous associons pour cela deux techniques telles que la photodéposition laser et la microfluidique. Nous synthétisons au préalable par deux méthodes différentes des particules de dioxyde de titane de morphologies contrôlées. Ces particules sont ensuite dissymétrisées par photodéposition d'un sel métallique d'or ou d'argent, en écoulement continu. / In the literature, many types of synthesis of Janus particles have been proposed. We show here a new method of continuous synthesis to obtain inorganic Janus particles of about thirty nanometers. We associate these two techniques such as laser photodeposition and microfluidics.We synthesize by two different methods controlled morphology titanium dioxide particles. These particles are then dissymmetrised by photodeposition of metal salt of gold or silver, in a continuous flow.

L'objet de cette thèse fut d'utiliser l'avantage qu'offre la microfluidique couplée à la thermique, pour fabriquer des dispositifs de contrôle de température sur des projets/applications distincts en vue de leur valorisation. En biologie moléculaire, par la réalisation d'une PCR quantitative rapide, pour la détection d'agents pathogènes. Nous avons réalisé une nouvelle plateforme de détection d'agent pathogène capable de faire 30 cycles d'amplifications en moins de trois minutes. Nous avons également illustré la capacité du dispositif à quantifier par fluorescence en temps réel des agents simulant de l'Anthrax et d'Ebola. 7 minutes et 7 minutes 30 secondes suffisent pour amplifier/détecter ces bactéries et virus. Nous avons également prouvé qu'à ces vitesses ce dispositif rapide de qPCR/RT-qPCR ne dégradait pas l'efficacité, la spécificité et les cycles seuils de détection. Nous avons également démontré que la sensibilité du dispositif était de 100 copies d'Adn initiale. En biologie cellulaire, Nous avons réalisé un dispositif " universel " de contrôle dynamique de température pour l'imagerie cellulaire haute résolution. Ce dispositif de contrôle dynamique de température, permet l'étude spatiotemporelle de mécanismes cellulaires sous microscope haute résolution grâce à l'utilisation de mutant thermosensibles. De nombreuses validations biologiques ont ainsi été réalisées sur notre dispositif dont l'objet final est la commercialisation. / The purpose of this work is to take advantage of micro fabrication combined with heat and mass transfer to build potential commercialized temperature control devices in several domain. In molecular biology, for molecular pathogenesis detection we made a new PCR device able to able to perform 30 amplifications cycles in less than 3 minutes. We also demonstrated that our platform was able to quantify with fluorescence the presence of Ebola and Anthrax simulant agents. Respectively 7 minutes and 7 minutes 30 secondes are required to detect the virus and bacteria. In cellular biology we have made a universal temperature control for live cell imaging. It device allow to perform spacio-temporal study of cell mecanisms due to is fast ability to shift from a temperature to another in less than 10 seconds. Many biological validations have been performed. Thank to his universal adaptation to microscope we have also decided to commercialize this device by creating a new start-up.

Protein biomarkers are important diagnostic tools for detection of non-communicable diseases, such as cancer and cardiovascular conditions. In order to be used as diagnostic tools they need to be detected at very low concentrations in biological samples (e.g. whole blood, serum or urine). This has been currently performed in central laboratories using expensive, bulky equipment and time consuming assays.

Heat shock response (HSR) is an ancient and highly conserved signaling pathway in cells that regulates the expression of heat shock proteins (HSPs) in the presence of thermal and other environmental stresses. HSPs function to prevent the formation of non-specific protein aggregates and to assist proteins in acquiring their native structures. Although HSR has been extensively studied, key aspects of this pathway remain a mystery. In particular, how HSR is activated and regulated by the master transcription factor HSF1 is not well understood. The broad goal of this thesis is to develop a quantitative framework aimed at elucidating the HSF1-mediated activation of HSR in yeast cells. Understanding this process has important implications for development, physiology and disease. Indeed, HSF1 is conserved from yeast to human, has been shown to play an important role in stress resistance, health and disease, and is a therapeutic target for neurodegenerative diseases. Broadly, there are two putative (not mutually exclusive) models for activation in response to heat shock: (1) HSF1 dissociation from chaperone proteins and (2) hyper-phosphorylation and the subsequent activation of HSF1. However the relative contribution of each of these events in the activation process is not characterized. Thus far, there is no direct evidence linking either of these two events to activation, and the relative contribution of each mechanism to the activation process has not been quantitatively characterized. To address these issues, we develop a quantitative model of HSR in yeast cells. We use the model to make a series of quantitative predictions and, in a collaborative effort, experimentally test these predictions in a yeast model of HSR. Critically, we provide the first direct evidence for chaperone dissociation of HSF1 in response to heat shock. Moreover, we find that HSF1 phosphorylation is dispensable for activation of HSR, but is able to modulate its activity. Taken together, our work leads to a model for two “orthogonal” mechanisms regulating HSR in yeast, in which chaperone dissociation acts as an ON/OFF switch, whereas phosphorylation functions to tune the gain of the response. Finally, to complement and further test this quantitative model, we develop a novel microfluidic system to explore in more depth the behavior of individual cells in the presence of heat shock inputs. This includes (1) a microfluidic device with microscale on-chip heaters enabling programmable thermal perturbations and (2) a custom image analysis platform to follow single cells through heat shock time courses. In preliminary single-cell studies, we find a relationship between HSF1 phosphorylation state and cell-to-cell variability in HSR activation level (as measured by a transcriptional reporter). These preliminary results suggest that HSF1 phosphorylation may be generating and tuning noise in the HSR in order to promote phenotypic plasticity and increased survivability of a cell population in the face of stress.

It has been repeatedly proven that cell therapies can address many current unmet clinical treatment needs and also improve on current treatment options for various diseases, from neurological disorders to bone repair (Rosset et al. 2014; Corey et al. 2017). Though the potential of cell therapies has been demonstrated at a relatively small scale, the realisation of bringing cell based treatments to a larger market is hindered by the complexity of the product along with safety concerned and high production cost. Safety concerns can be informed with more in-depth analytical analysis of the product, however this in turn increase the costs involved in producing a cell therapy (Davie et al. 2012). Consequently the cost of analytical techniques also needs to be reduced, to address this need the area of microfluidic based bioanalytics holds much promise (Titmarsh et al. 2014). The culturing of human mesenchymal stem cells (hMSC) was used as a proof of concept model to demonstrate where improved bioanalytical and bioassay methods could be utilised in the production of cell therapies. Cells from four donors were cultured under three different oxygen environments and the conditioned medium assessed for pro-angiogenic capabilities using a tube formation bioassay and a proportion of the cytokine secretome profile measured using Luminex technology. Thorough secretome analysis it was shown that predicting cytokine levels based solely on the donor was not possible as the handling of the cells also had an influence on the secretome profile. The donor expression profiles did not behave in the same manner across all oxygen environments, for example in some donors IL-8 levels increased per cell at lower oxygen where as other donors showed a decrease per cell. While the tube formation assay showed some differences between donors in pro-angiogenic capabilities it also highlights the challenges with interpreting large data sets. The feasibility of using a microcapillary film (MCF) based enzyme-linked immunosorbent assay (ELISA) to detected two relevant cytokines, IL-8 and hepatocyte growth factor (HGF) was investigated. Following on from this work the development of a combined MCF ELISA assay with hMSC cell culture to produce a fully closed cell screening system was initiated. It was shown that it was feasible to measure IL-8 and HGF using the MCF ELISA platform but further work would need to be done to make the system more compatible with the manufacturing environment. In order to adapt the MCF to also be an hMSC culture platform the first challenge was to functionalise the Fluorinated Ethylene Propylene (FEP) surface of the MCF. It was concluded that a poly (vinyl- alcohol) (PVA) and gelatin mixture produced a homogenous coating to which a consistent level of hMSC would attach. This work was carried out on a flat surface; therefore steps were taken to adapt this knowledge into the MCF, while there was evidence of hMSCs present inside the MCF more work will need to be done to bring this concept to an established platform.