Although nano-sized metal colloids are used in industrial and medicinal applications, little is known about the potential liver toxicity of these materials after occupational or intentional exposures. To begin to resolve some outstanding hepatotoxicity concerns, the inflammatory response of hepatocytes after exposure to metal colloids was assessed. Four ~30-nm-sized metal colloids, including silver (nano-Ag), copper (nano-Cu) and nickel (nano-Ni) were examined in an effort to understand the induced cytokine expression in a murine liver cell line (AML12). Here we also utilized another system, co-cultures of hepatocytes, Kupffer’s cells, and lymphocytes isolated from C57BL6 mice. Cells were exposed to the materials over dose-response (0.1mg/L to 1000mg/L) and time-dependent (4 h, 48 h, and 1-week) studies. Cytotoxicity was measured via metabolism of resazurin and validated via MTT assay and cell counts. Inflammatory response was determined by cytokine profiles (TNF-a and IL-6), as well as by mRNA and protein expression of heat shock protein (Hsp70). Results from cells exposed to nano-Ag to doses of up to 100mg/L exhibited no significant changes in cytotoxicity, IL-6, or TNF-a production, or Hsp70 expression. Both nano-Cu and nano-Ni exposed cells exhibited decreased metabolism, increased Hsp70 induction, and increased inflammatory responses (IL-6 and TNF-a). Dynamic light scattering and electron microscopy were used to characterize particle size and surface charge. All three metal colloidal systems demonstrated different particle size distributions, agglomerated sizes, and surface zeta potentials. Furthermore, each metal colloid system elicited different inflammatory biomarker responses and stress protein expression.

Adipose-derived stem cells (ASCs) have been extensively investigated for their applicability in the field of tissue engineering due to their multi-lineage differentiation potential and the convenience of cell acquisition. To date, conventional inductive media have been used to induce lineage-specific differentiation of ASCs; however, this general approach has limitations in terms of high costs and unstable differentiation responses. In native tissues, mesenchymal stem cells (MSCs) interact with their surrounding cells (i.e. mature cells) through paracrine and autocrine signaling, which can regulate their metabolism and cell function. Therefore, the author developed in vitro co-culture models to study the interactions between ASCs and three different mature cell types: adipocytes, chondrocytes and osteoblasts. In this work, bovine ASCs (bASCs) from the interdigital fat pad were first isolated and characterized, in terms of in vitro proliferation and multi-lineage (bone, cartilage, fat) differentiation with conventional inductive media and culture conditions. Doubling time calculations and gene expression analysis of stem cell markers indicated a threshold existed for stem cell degeneration at passage 5 (P5) for bASCs when expanded extensively in vitro. The multi-lineage differentiation potentials were compared between passage 2 (P2) and P5. Interestingly, while the P5 bASCs presented significantly lower levels of adipogenesis and chondrogenesis, osteogenesis was maintained or even improved with serial passaging. In the designed indirect co-culture systems, adipogenesis and chondrogenesis were investigated in growth medium without key differentiation factors, whereas osteogenesis was induced in conventional osteogenic medium, to maintain the stable phenotype of the mature osteoblasts in culture. The results were used to demonstrate the general feasibility of mature cell-induced or -enhanced bASC differentiation through soluble, cell-secreted paracrine signaling. When compared to growth factor (GF)-stimulated differentiation, the bASCs in co-culture presented an earlier, but potentially stronger, level of differentiation. Among these paracrine factors, Wnt proteins are known to play essential roles in mediating stem cell self-regulation and fate determination. In the current thesis, the Wnt inhibitors WIF-1 and DKK-1 were used to explore the involvement of the Wnt canonical and non-canonical signaling pathways in the designed co-culture environments. The data showed a strong correlation with the literature, indicating the canonical pathway was upregulated during osteogenesis, but inhibited during adipogenesis. The inhibition of chondrogenesis through the canonical pathway was suggested on a genetic level. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-03-09 13:39:14.553

yes / Abstract Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT-1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co-localization of GLT-1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5-tagged GLT-1, pDsRed1-Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co-localization was quantified using Volocity software. Image analysis of confocal z-stacks revealed no co-localization between mitochondria and GLT-1 in pure astrocyte cultures. Co-culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT-1. This co-localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K+. In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT-1/mitochondrial co-localization, however, in co-cultures, Y27632 abolished mitochondrial: GLT-1 co-localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT-1 distribution or GLT-1: mitochondrial co-localization. We conclude that the association between GLT-1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments. Mitochondria have limited co-localization with the glutamate transporter GLT-1 in primary astrocytes in culture. Few mitochondria are in the fine processes where GLT-1 is abundant. It is necessary to culture astrocytes with neurones to drive a significant level of co-localization, but co-localization is not further altered by depolarization, manipulating sodium ion gradients or Na/K ATPase activity.

No description available.

No description available.

The inability to accurately reproduce the complexities of the in vivo tumor microenvironment with reductionist-based two-dimensional in vitro cell culture models has been a notable deterrent in identifying therapeutic agents that reliably translate to in vivo animal and human clinical trials. In an effort to address this, a growing number of three-dimensional (3D) in vitro tumor models capable of mimicking specific tumorigenic processes have emerged within the last decade. This concept stems from the understanding that cells cultured within 3D in vitro matrices have the ability to acquire phenotypes representative of the in vivo microenvironment. The objective of this project was to apply a tissue engineering approach towards developing a 3D in vitro tumor angiogenesis model. Initially, different scaffolds were investigated for supporting 3D tumor growth, including bacterial cellulose, electrospun polycaprolactone/collagen I, and highly porous electrospun poly(L-lactic acid). However, cancer cells cultured on these scaffolds demonstrated poor adhesion, sufficient adhesion with poor infiltration, and increased but still inadequate infiltration, respectively. Collagen I hydrogels were chosen as an appropriate scaffold for facilitating 3D in vitro tumor growth for two reasons -- cell-mediated degradation and immediate 3D cell growth. It was hypothesized that cancer cells cultured within collagen I hydrogels could be encouraged to recapitulate key characteristics of in vivo tumor progression. MDA-MB-231 human breast cancer cells were shown to experience hypoxia and undergo necrosis in response to limitations in oxygen diffusion and competition for nutrients. Upregulation of hypoxia-inducible factor-1" resulted in a significant increase in vascular endothelial growth factor gene expression. To capitalize on this endogenous angiogenic potential, microvascular endothelial cells were cultured on the surface of the designated "bioengineered tumors." It was hypothesized that paracrine signaling between tumor and endothelial cells co-cultured within this system would be sufficient for inducing an angiogenic response in the absence of exogenous pro-angiogenic growth factors. Endothelial cells in the co-culture group were shown to invasively sprout into the underlying collagen matrix, forming a capillary-like tubule network. This project culminated with the establishment of an improved in vitro tumor model that can be used as a tool for accurate evaluation and refinement of cancer therapies. / Ph. D.

La levure Candida shehatae est le microorganisme modèle d’étude choisi. Cette levure peutconvertir le xylose et le glucose en éthanol, contrairement à Saccharomyces cerevisiae, levuretraditionnellement utilisée dans les procédés industriels, qui ne peut convertir le xylose.L’optimisation des performances de production d’éthanol à partir de xylose passe par unemaximisation des trois critères suivants : la productivité volumique, le titre final et lerendement éthanol/xylose. Pour diriger le flux de carbone vers la production d'éthanol defaçon optimale, le paramètre majeur qu’il faut contrôler est le degré de limitation en oxygène.Les cultures sont réalisées sur milieu minéral en mode fed-batch et conduites en deux phases :aérobie puis limitation en oxygène. Une valeur moyenne de la vitesse spécifique derespiration (qO2) de 1,19 mmolO2/gX/h permet de maximiser les trois critères deperformances sur xylose : le rendement en éthanol (0,327 gETOH/g-xylose), la productivitéspécifique maximale (0,22 gETOH/gX/h) et le titre en éthanol final (48,81 g/L). Pour lafermentation du glucose, le rendement en éthanol le plus élevé (0,411 gETOH/g-glucose) estobtenu lorsque qO2 est faible et a pour valeur moyenne 0,30 mmolO2/gX/h; tandis que laproductivité spécifique et le titre en éthanol final atteignent les valeurs maximales de 0,35gETOH/gX/h et 54,19 g/L pour respectivement qO2 de 1,7 et de 2,5 mmolO2/gX/h.Pour la consommation simultanée des deux substrats, un phénomène de répression du glucosesur le xylose est démontré par expérience en chemostat de pulse glucose en régime stabilisésur xylose. La simple présence intra-cellulaire des enzymes de la voie du xylose (XR andXDH) ne permet pas la co-consommation efficace des deux sucres et le glucose estpréférentiellement consommé.Afin de structurer la connaissance acquise sur le métabolisme de C. shehatae et pouvoiroptimiser par simulation les co-cultures C. shehatae / S. cerevisiae pour la productiond’éthanol à partir de mélanges xylose/glucose, un modèle cinétique de C. shehatae estconstruit. Ce modèle est validé avec des cultures sur substrats purs (xylose et glucoseséparés). Un modèle cinétique de co-culture est ensuite développé de manière à simulerdifférentes stratégies de fermentation pour l’optimisation de la production d’éthanol surmélange xylose/glucose de type hydrolysats de paille de blé / The yeast Candida shehatae was the model microorganism of the study. This yeast canconvert xylose and glucose into ethanol, unlike Saccharomyces cerevisiae traditionally usedin industrial processes, which cannot convert xylose. Performance optimization of ethanolproduction from xylose is performed through maximization of the following three criteria:volumetric productivity, final ethanol titer and yield of ethanol over xylose. To direct thecarbon flux towards ethanol production, the major parameter which must be controlled is thelevel of oxygen limitation. Cultures are carried out in fed-batch in mineral medium andperformed in two phases: the first one is not limited in oxygen and the second one is oxygenrestricted. A mean value of qO2 equal to 1.19 mmolO2/gX/h maximizes the three criteria ofperformance on xylose: ethanol yield (0.327 gETOH/g-xylose), the maximum specificproductivity (0.22 gETOH/gX/h) and the final ethanol titer (48.81 g/L). For glucosefermentation, ethanol yield is the highest (0.411 gETOH/g-glucose) when qO2 is low as anaverage value of 0.30 mmolO2/gX/h, while the specific productivity and the ethanol final titerreach maximum values of 0.35 gETOH/gX/h and 54.19 g/L for respectively qO2 of 1.7 and2.5 mmolO2/gX/h.For the simultaneous consumption of the two substrates, a phenomenon of glucose repressionover xylose is observed in chemostat experiment with glucose pulse on xylose steady state.The presence of intracellular enzymes of the xylose pathway (XR and XDH) is not sufficientfor efficient co-consumption of both sugars and glucose is preferentially consumed.In order to structure the knowledge obtained on the metabolism of C. shehatae and tooptimize by simulation the co-culture C. shehatae / S. cerevisiae to produce ethanol fromxylose/ glucose mixtures, a kinetic model of C. shehatae is built. This model is validated withpure substrate cultures (xylose and glucose separated). A kinetic model of co-culture is thenbuilt in order to simulate several fermentation strategies to optimize the ethanol productionfrom xylose/glucose mixture similar to wheat straw hydrolysates

L’épithélium intestinal, siège de l’absorption des (micro)-nutriments est aussi le premier système de défense de l’organisme. Un déséquilibre dans l’homéostasie peut être à l’origine d’une réaction inflammatoire associée à des défauts de la barrière intestinale et de la fonction immunitaire, ainsi qu’une malabsorption des nutriments, comme rencontré dans les MICI (Maladies Inflammatoires Chroniques de l’Intestin), dans les stratégies de fortification en micronutriments et les pathologies non transmissibles (obésité). Il est donc important de trouver des moyens d’action, via l’alimentation par exemple, pour prévenir ou au minima réduire, les conséquences nutritionnelles et pathologiques de l’inflammation intestinale, et de comprendre les mécanismes impliqués. Parmi les modèles d’études de l’intestin, les modèles in vitro de culture cellulaire sont de plus en plus utilisés et permettent d'évaluer les mécanismes moléculaires d'une manière simple et reproductible et de réduire l'expérimentation animale.Dans ce contexte et dans le but d’étudier l’interaction de composés bioactifs de l’alimentation avec l’intestin en état d’inflammation, le premier objectif de ce travail de thèse a été la mise au point d’un modèle in vitro d’intestin enflammé associant en co-culture deux lignées intestinales humaines : les Caco-2 TC7 (entérocytes) et HT29-MTX (cellules caliciformes) et une lignée immunitaire de macrophages (THP1). Plusieurs marqueurs d’inflammation ont été évalués et nous avons pu montrer que le modèle de tri-culture répondait à un stimulus inflammatoire (LPS/IFNγ), par une augmentation de la production de cytokines pro-inflammatoires (TNF-α, IL6 et IL8) et d’enzymes (INOS et COX2) ainsi que l’expression de leurs gènes. Par ailleurs, une augmentation de la perméabilité épithéliale via une altération des jonctions serrées (TJs) a également pu être mise en évidence ainsi qu’une surproduction de mucus, lesquels sont des caractéristiques reconnus d’inflammation.Le deuxième objectif était d’étudier l’interaction de la β-cryptoxanthine (BCX), caroténoïde des agrumes, lipophile et anti-oxydant, avec le modèle enflammé. Nous avons utilisé pour solubiliser la BCX deux types de micelles (artificielles et physiologiques) et étudié les marqueurs d’inflammation. Bien qu’il semble d’après les résultats préliminaires que les micelles de BCX montrent une tendance à diminuer la production de certaines cytokines (IL6 et IL8), le rôle des constituants des micelles (Tween 40 ou sels biliaires/phospholipides) dans ce phénomène observé et dans la perméabilité épithéliale reste à clarifier par la suite. / The intestinal epithelium, main place of the absorption of (micro)-nutrients is also the first body's defense system. An imbalance in homeostasis can lead to an inflammatory reaction associated with defects in the intestinal barrier and immune function as well as malabsorption of nutrients, as seen in IBD (Inflammatory Bowel Diseases), in micronutrient fortification strategies and noncommunicable diseases (obesity). It is therefore important to find ways of action, for example through diet, to prevent or at least reduce the nutritional and pathological consequences of intestinal inflammation, and to understand the mechanisms involved. Among intestinal models, in vitro cell culture models are increasingly used and allow to evaluate the molecular mechanisms in a simple and reproducible way and to reduce animal experimentation.In this context and in order to study the interaction of dietary bioactive compounds with the intestine in state of inflammation, the first objective of this work was the development of an in vitro model of inflamed intestine combining in co-culture two human intestinal cell lines: Caco-2 TC7 (enterocytes) and HT29-MTX (goblet cells) and an immune cell line of macrophages (THP1). Several inflammation markers were evaluated and we were able to show that the tri-culture model responded to an inflammatory stimulus (LPS / IFNγ), by increasing the production of pro-inflammatory cytokines (TNF-α, IL6 and IL8) and enzymes (INOS and COX2) as well as the expression of their genes. In addition, an increase of epithelial permeability via tight junctions (TJs) alteration has also been demonstrated, as well as overproduction of mucus, which are recognized inflammation characteristics.The second objective was to study the interaction of β-cryptoxanthin (BCX), a lipophilic and antioxidant carotenoid of citrus, with the inflamed model. To solubilize BCX, we used two types of micelles (artificial and physiological) and studied markers of inflammation. Although it appears from the preliminary results that BCX micelles show a tendency to decrease the production of some cytokines (IL6 and IL8), the role of micelle constituents (Tween 40 or bile salts / phospholipids) in the phenomenon observed and in the epithelial permeability remains to be therefore clarified.

La Sclérose Latérale Amyotrophique (SLA) est une affection neurodégénérative affectant sélectivement les motoneurones et conduisant au décès en 2 à 4 ans. Des facteurs génétiques, ainsi que diverses hypothèses physiopathologiques, telles que l’excitotoxicité et le stress oxydant, ont été évoqués pour expliquer la dégénérescence des motoneurones, mais aucune étiologie n’explique aujourd’hui la survenue de cette pathologie. Afin d’améliorer les connaissances des voies métaboliques impliquées dans la physiopathologie de la SLA, nous avons développé un modèle in vitro de co-Culture de motoneurones et d’astrocytes sur-Exprimant la Superoxyde Dismutase (SOD1) humaine sauvage ou mutée (SOD1G93C) et exposée au stress oxydant. Nous avons étudié les modifications de métabolisme après traitement oxydant par une approche métabolomique utilisant la chromatographie gazeuse couplée à la spectrométrie de masse et une analyse statistique multivariée des résultats. Ainsi nous avons observé une modification de métabolites impliqués notamment dans le cycle de Krebs, la neurotransmission excitatrice et la synthèse du glutathion, dans un modèle in vitro de SLA exposé au stress oxydant. / Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder affecting selectively motor neurons and leading to death in 2 to 4 years. Genetic factors and various pathophysiological hypotheses, such as excitotoxicity and oxidative stress, have been suggested to explain the degeneration of motor neurons, but today no etiology explains the occurrence of this disease. In order to improve the knowledge of the metabolic pathways involved in the pathogenesis of ALS, we developed an in vitro model of co-Culture of motor neurons and astrocytes over-Expressing human superoxide dismutase (SOD1), wild-Type or mutated (SOD1G93C), and exposed to oxidative stress. We studied the changes in metabolism after oxidative treatment with a metabolomics approach using gas chromatography-Mass spectrometry and multivariate statistical analysis. Thus we observed a change in metabolites involved in the citric acid cycle, the excitatory neurotransmission and the glutathione synthesis, in an in vitro model of ALS exposed to oxidative stress.

La dermatite de contact allergique (DCA) est une réaction exacerbée du système immunitaire cutané vis-à-vis d’un allergène de contact. La prévalence de la DCA étant de 20 % au sein de la population mondiale, il est important d’identifier les composés allergisants. Différentes réglementations européennes, telles que le règlement REACh ou la directive cosmétique, interdisent l’utilisation de test sur l’animal. C’est dans ce contexte que différentes méthodes alternatives ont été développées pour évaluer la sensibilisation cutanée. La stratégie actuelle d’évaluation du potentiel sensibilisant consiste à réaliser un ensemble de tests alternatifs, chacun mimant un évènement clé du mécanisme : l’hapténisation, l’activation des kératinocytes ou des cellules dendritiques.Cependant, ces tests utilisent principalement des monocultures et ne prennent donc pas en compte les interactions cellulaires qui peuvent avoir lieu in vivo. De plus, les évaluations de la pénétration et du métabolisme cutanés sont négligées dans les tests développés.Afin de mimer la fine orchestration des événements intervenant lors de la sensibilisation cutanée, nous proposons un modèle d’épiderme humain reconstruit (RhE) co-cultivé avec la lignée cellulaire THP-1, servant de substitut aux cellules dendritiques. Nous avons caractérisé, et étudié la pertinence de ce modèle à l’aide de molécules chimiques de référence. Ce travail a permis l’identification de biomarqueurs, tels que CD54, IL-8 et CCL3, spécifiques à l’évaluation in vitro de la sensibilisation cutanée des xénobiotiques. / Allergic contact dermatitis is an exacerbated reaction of skin immune system toward contact allergen. The prevalence of DCA being 20 % among the world population, it is important to identify allergens. Different European regulations such as the REACh regulation or the cosmetic directive prohibit the use of the test on animals. It is in this context that different methods have been developed to evaluate skin sensitization. The current sensitization potential assessment strategy consists of a set of alternative tests, each of which reproduce a key event of the mechanism: the haptenation, the activation of keratinocytes or dendritic cells.However, these tests are mainly based on monocultures and therefore do not account for the cellular crosstalk that happen in vivo. In addition, the evaluations of skin penetration and metabolism are neglected in the developed tests.In order to mimic the fine orchestration of the events involved in skin sensitization, we propose a model of reconstructed human epidermis (RhE) co-cultivated with the THP-1 cell line, as a substitute for dendritic cells. We have characterized and studied the relevance of this model using reference chemical molecules. This work has enabled the identification of biomarkers, such as CD54, IL-8 and CCL3, specific to the in vitro evaluation of skin sensitization to xenobiotics.