No description available.

The local release of complexed siRNA from biomaterials opens precisely targeted therapeutic options. In this study, complexed siRNA was loaded to gelatin microparticles cross-linked (cGM) with an anhydride-containing oligomer (oPNMA). We aggregated these siRNA-loaded cGM with human mesenchymal stem cells (hMSC) to microtissues and stimulated them with osteogenic supplements. An efficient knockdown of chordin, a BMP-2 antagonist, caused a remarkably increased alkaline phosphatase (ALP) activity in the microtissues. cGM, as a component of microtissues, mineralized in a differentiation medium within 8–9 days, both in the presence and in the absence of cells. In order to investigate the effects of our pre-differentiated and chordin-silenced microtissues on bone homeostasis, we simulated in vivo conditions in an unstimulated co-culture system of hMSC and human peripheral blood mononuclear cells (hPBMC). We found enhanced ALP activity and osteoprotegerin (OPG) secretion in the model system compared to control microtissues. Our results suggest osteoanabolic effects of pre-differentiated and chordin-silenced microtissues.

Characterization of new pharmacological targets is a promising approach in research of neurorepair mechanisms. The G protein-coupled receptor 17 (GPR17) has recently been proposed as an interesting pharmacological target, e.g., in neuroregenerative processes. Using the well-established ex vivo model of organotypic slice co-cultures of the mesocortical dopaminergic system (prefrontal cortex (PFC) and substantia nigra/ventral tegmental area (SN/VTA) complex), the influence of GPR17 ligands on neurite outgrowth from SN/VTA to the PFC was investigated. The growthpromoting effects of Montelukast (MTK; GPR17- and cysteinyl-leukotriene receptor antagonist), the glial cell line-derived neurotrophic factor (GDNF) and of two potent, selective GPR17 agonists (PSB-16484 and PSB-16282) were characterized. Treatment with MTK resulted in a significant increase in mean neurite density, comparable with the effects of GDNF. The combination of MTK and GPR17 agonist PSB-16484 significantly inhibited neuronal growth. qPCR studies revealed an MTK-induced elevated mRNA-expression of genes relevant for neuronal growth. Immunofluorescence labelling showed a marked expression of GPR17 on NG2-positive glia. Western blot and RT-qPCR analysis of untreated cultures suggest a time-dependent, injury-induced stimulation of GPR17. In conclusion, MTK was identified as a stimulator of neurite fibre outgrowth, mediating its effects through GPR17, highlighting GPR17 as an interesting therapeutic target in neuronal regeneration.

Poor skeletal health results from aging and metabolic diseases such as obesity and diabetes and involves impaired homeostatic balance between marrow osteogenesis and adipogenesis. Tissue engineering provides researchers with the ability to generate improved, highly controlled and tailorable in vitro model systems to better understand mechanisms of homeostasis, disease, and healing and regeneration. Model systems that allow assembly of modules of MSCs, osteoblasts, and adipocytes in a number of configurations to engage in signaling crosstalk offer the potential to study integrative physiological aspects and complex interactions in the face of changes in local and systemic microenvironments. Thus, the overall goal of this dissertation was to examine integrative physiological aspects between MSCs, osteoblasts, and adipocytes that exist within the marrow microenvironment. To investigate the effects of intercellular signaling in different microenvironmental contexts, methods were developed to photolithographically pattern and assemble cell-laden PEG-based hydrogels with high spatial fidelity and tissue-scale thickness for long-term 3D co-culture of multiple cell types. This platform was applied to study effects of crosstalk between MSCs, osteoblasts and adipocytes on markers of differentiation in each cell type. Additionally, responses of MSCs to systemic perturbations in glucose concentration were modulated by mono-, co-, and tri-culture with these cell types in a model of diabetes-induced skeletal disease. Together, these studies provided valuable insight into unique and differential effects of intercellular signaling within the niche environment of MSCs and their terminally differentiated progeny during homeostatic and pathological states, and offer opportunities further study of integrative physiological interactions between mesenchymal lineage cells.

No description available.

Orientador: Ljubica Tasic / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T23:34:19Z (GMT). No. of bitstreams: 1 Awan_AlmasTaj_D.pdf: 4796874 bytes, checksum: 185a66c389aae68c266f385689030ef0 (MD5) Previous issue date: 2013 / Resumo: Os biocombustíveis de segunda geração surgiram como fontes energéticas promissoras, podendo ser obtidos a partir de vários tipos de biomassa que não seja utilizada para alimentos. Um tipo de biomassa que apresenta baixo custo além de apresentar níveis elevados de carboidratos, é a biomassa obtida após o processamento da laranja (Citrus processing waste from oranges, CPWO). Há um grande interesse na exploração desta biomassa em termos da produção do bioetanol (etanol da 2G). Nosso trabalho visa melhorar os processos de hidrólise do CPWO comparando o rendimento do processo clássico de hidrólise ácida com aplicação de enzimas comerciais ou provenientes do microrganismo Xanthomonas axonopodis pv. citri, cepa 306 (um fitopatógeno). Os resultados obtidos com a presente investigação evidenciam que ocorreu a conversão bem-sucedida do CPWO em uma mistura de açúcares. A posteriori, os açúcares redutores que foram obtidos foram convertidos em bioetanol por meio da fermentação em mono- e co-cultura. Para tanto, foi empregada a espécie Saccharomyces cerevisiae e duas cepas de Candida parapsilosis IFM 48375 e NRRL Y-12969, sendo que as duas últimas foram isoladas a partir do bagaço da laranja. Os rendimentos em termos de bioetanol obtido nas fermentações aplicando co-culturas estavam ao redor de 50 a 62%, constituindo valores muito maiores comparados com os obtidos por cepas usadas individualmente. Além disso, os açúcares foram consumidos mais rapidamente (6 h), tornando tais processos atraentes em termos de custo e aplicações comerciais / Abstract: Second generation biofuels from renewable resources have come forth as a result of energy security coupled with diminishing fossil fuel resources. Lignocellulosic biomass is a renewable resource, which can be converted in to liquid transportation fuels. Utilization of agro-industrial waste for the generation of biofuels makes it a cleaner production (Green Chemistry). Brazil is the world¿s largest producer of oranges. The current project deals with Citrus Processing Waste from Oranges (CPWO), and obtaining valuable products such as bioethanol, hesperidin, and essential oil. The process of hydrolyzing CPWO was improved and the classical way of biomass saccharification, i.e. acid hydrolysis, was compared with the enzyme hydrolysis. In enzyme hydrolysis, apart from applying commercial enzymes, saccharification was also investigated with protein extracts of Xanthomonas axonopodis pv. citri strain 306 (Xac 306), a potent pathogen that causes Citrus canker disease. Later, the obtained reducing sugars were converted into bioethanol by submerged mono- and co-culture fermentations that involved three yeast strains: Saccharomyces cerevisiae, Candida parapsilosis IFM 48375 and NRRL Y-12969, the last two being isolated from bagasse. Results demonstrated successful hydrolyses by Xac enzymes that released high levels of fermentable sugars. Also during co-culture fermentation processes, it was noticed that ethanol yield was improved from 50% to 62% w/w (calculated on the basis of total dry matter contents) and sugars were consumed faster. Thus by employing co-culture fermentation strategy, apart from getting better bioethanol yields, fermentation time is also reduced that makes it a cost effective technique / Doutorado / Quimica Organica / Doutora em Ciências

Les céréales et les produits issus de leur transformation sont susceptibles d’être contaminés par des espèces fongiques capables de produire des mycotoxines. L’Homme est ainsi exposé tout au long de sa vie à travers son alimentation à ces contaminants naturels, généralement à de faibles doses et en mélange. Cependant, l’incidence de la présence simultanée de ces toxines sur notre santé, à court terme comme à plus long terme, ainsi que les mécanismes responsables de leur toxicité sont encore peu ou mal caractérisés. L’utilisation de modèles cellulaires humains pertinents et adaptés est particulièrement importante pour de telles études. L’épithélium intestinal et le système immunitaire, qui constituent la première barrière de défense de l’hôte suite à l’ingestion de contaminants alimentaires, ainsi que le foie, de par son rôle majeur dans la biotransformation des xénobiotiques, représentent des modèles d’étude pertinents en toxicologie. Dans le cadre de cette étude, des modèles cellulaires humains d’origine intestinale (Caco-2), immunitaire (THP-1) et hépatique (HepaRG) ont été employés pour évaluer le risque associé à la co-exposition aux mycotoxines de Fusarium (appelées fusariotoxines) qui sont parmi les plus problématiques dans nos régions. Différents types d’interactions, tels que de l’antagonisme et du synergisme, ont pu être observés sur la viabilité cellulaire en fonction de la nature du mélange, des doses testées, de la lignée cellulaire étudiée et du modèle mathématique utilisé pour prédire les effets combinés. Des interactions ont également été observées à l’échelle moléculaire, les effets des mélanges étant très différents de ceux induits par les toxines individuellement sur le protéome des cellules. D’autres résultats obtenus interrogent sur la façon dont les mycotoxines déclenchent réellement la réponse cellulaire. De plus, les interactions entre cellules cocultivées semblent capables de modifier la réponse cellulaire suite à l’exposition à ces toxines. Ces résultats soulignent l’importance de développer des modèles in vitro de plus en plus sophistiqués et s’approchant des conditions in vivo pour permettre une meilleure caractérisation du risque « mycotoxine ». / Cereals and cereal-based products are susceptible to be contaminated by mycotoxin-producing fungi.Thus, through their diet, humans are exposed throughout their life to these natural food contaminants, mostly at low doses and in mixture. However, the health impact of the simultaneous exposure to these toxins, in acute and chronic conditions, as well as the mechanism related to their toxicity, are still poorly characterized. The use of relevant and suitable human cell models is therefore of particular importance for such studies. The intestinal epithelium and immune system, which constitute the first host defense barrier following the food contaminant uptake, as well as the liver, due to its major function in xenobiotic biotransformation, are relevant for toxicity studies. In the framework of study, the intestinal (Caco-2), immune (THP-1) and hepatic (HepaRG) human cell models were used for risk assessment associated with co-exposure to Fusarium mycotoxins (called fusariotoxins) which are the most problematic in our regions. Different type of interactions, such as antagonism and synergism, were observed on cell viability depending on the nature of the mixture, tested concentration, studied cell line and used mathematical model to predict the combined effects. Interactions were also highlighted at the molecular level, the effects of mixtures being very different from those induced by the toxins alone on the cell proteome. Other results raised the question about how mycotoxins actually trigger the cellular response. In addition, cell-cell interactions in co-cultured systems appeared to modify the cellular response following exposures to these toxins. Overall, the obtained results highlighted the relevance of developing in vitro models increasingly sophisticated and closer to in vivo conditions to allow for a better characterization of the "mycotoxin" risk.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Pseudomonas aeruginosa is a gram negative opportunistic pathogen with the capacity to cause serious disease by forming biofilms, most notably in the lungs of cystic fibrosis (CF) patients. Biofilms are communities of microorganisms that adhere to a solid surface, undergo global regulatory changes, secrete exopolysaccharides, and are innately antibiotic resistant. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified including the protein, MgtE. MgtE has recently been discovered and has been implicated in virulence modulation, as an isogeneic mutation of mgtE leads to increased cytotoxicity. To further elucidate the role of MgtE in P. aerugionsa infections, transcriptional and translational regulation of this protein following antibiotic treatment has been explored. I have demonstrated that mgtE is transcriptionally upregulated following antibiotic treatment of most of the twelve antibiotics tested utilizing RT-PCR and QRT-PCR. A novel model system was employed, which utilizes cystic fibrosis bronchial epithelial (CFBE) cells homozygous for the ΔF508 mutation for these studies. This model system allows P. aeruginosa biofilms to form on CFBE cells modeling the P. aeruginosa in the CF airway. Translational effects of antibiotic treatment on MgtE have been attempted via Western blotting and cytotoxicity assays. Furthermore, to explore the possibility that mgtE is interacting with a known regulatory pathway, a transposon-mutant library was utilized and the regulatory proteins, AlgR and NarX, among others have been identified as possibly interacting with MgtE. Lastly, an MgtE homologue from Staphylococcus aureus was utilized to further demonstrate the virulence modulatory effects of MgtE by demonstrating the expression of the homologue results in decreased cytotoxicity, exactly like expression of the native P. aeruginosa MgtE. This research explores a newly discovered protein that impacts cytotoxicity and biofilm formation and provides valuable information about P. aeruginosa virulence.

[ES] El desarrollo de la sociedad ha estado siempre ligado a los avances científicos y tecnológicos. Sin embargo, algunos de estos avances han supuesto la aparición de nuevos problemas, sobre todo de tipo ético, dando lugar a nuevos movimientos asociativos contrarios a ellos. Un ejemplo de ello es el uso de la experimentación animal, fundamentalmente en el campo de la medicina, y más concretamente en el ensayo de fármacos y dispositivos implantables en contacto con medios biológicos. En este sentido, desde el ámbito de los biomateriales y la ingeniería tisular se trabaja para buscar alternativas a la experimentación animal. Una de estas alternativas es el desarrollo de modelos in vitro a partir de soportes poliméricos para el crecimiento celular in vitro. Estas estructuras, además, podrían emplearse no sólo en ensayos de fármacos o investigación in vitro para reducir el uso de animales en experimentación, sino también para regeneración tisular, simulando desde tejidos simples en los que se tienen un único tipo celular, a tejidos más complejos a partir del co-cultivo celular. Así pues, en esta tesis se ha desarrollado un sistema tridimensional con estructura porosa estratificada que permite tanto el co-cultivo celular indirecto, como la realización de ensayos de liberación de fármacos. Para ello, se ha obtenido soportes porosos (scaffolds) por medio de la técnica de solvent-casting particle-leaching empleando como porógeno sal, cuyo tamaño de poro permite albergar células en su interior, sobre los que se han dispuesto, formando una estructura tipo sándwich, membranas electrohiladas. Estas membranas forman una estructura de fibras entrecruzadas, dejando entre ellas espacios de tamaño muy inferior al tamaño celular, de modo que permiten el paso de nutrientes y moléculas a través de ellas, pero actúan de barrera para las células impidiendo su migración a otras zonas del sistema tridimensional. Este tipo de estructuras permiten simular, por ejemplo, la arquitectura tubular renal, con la zona porosa intersticial central y las dos capas epitelial y endotelial externas, que estarían en contacto con la orina y la sangre, respectivamente. Para obtener estas estructuras, se ha optado por emplear polímeros de la familia de los poliésteres, en particular ácido poliláctico y poli(¿-caprolactona), así como su mezcla y copolímeros con ácido poliglicólico. Estas combinaciones permiten ajustar la hidrofilicidad, y por tanto la biodegradabilidad, la cinética de liberación de fármacos y el comportamiento biológico, según interese. Además, el uso de la técnica de electrospinning para el desarrollo de las membranas, permite obtener diferentes diámetros de fibra a partir de la modificación de los principales parámetros del electrohilado, permitiendo también regular las propiedades de estos materiales. Finalmente, para estudiar la liberación de fármacos desde el sistema anterior, las membranas electrohiladas se han cargado con curcumina mediante dos métodos diferentes: a través del método de electrohilado en disolución y con electrospinning coaxial, para obtener así diferentes perfiles de liberación. / [CA] El desenvolupament de la societat ha estat sempre lligat als avanços científics i tecnològics. Malauradament, alguns d'aquests avanços han suposat l'aparició de nous problemes, sobretot de tipus ètic, donant lloc a nous moviments associatius contraris a ells. Un exemple d'això és l'ús de l'experimentació animal, fonamentalment al camp de la medicina, i més concretament en el testatge de fàrmacs i dispositius implantables en contacte amb medis biològics. En aquest sentit, des de l'àmbit dels biomaterials i l'enginyeria tissular es treballa per a buscar alternatives a l'experimentació animal. Una d'aquestes alternatives és el desenvolupament de models in vitro a partir de suports polimèrics per al creixement cel·lular in vitro. Aquestes estructures, a més a més, podrien emprar-se no sols en testatge de fàrmacs o investigació in vitro per a reduir l'ús d'animals en experimentació, sinó també per a regeneració tissular, simulant des de teixits simples en què es té un únic tipus cel·lular, a teixits més complexos a partir del co-cultiu cel·lular. Així doncs, en aquesta tesi s'ha desenvolupat un sistema tridimensional amb estructura porosa estratificada que permet tant el co-cultiu cel·lular indirecte, com la realització d'assajos d'alliberació de fàrmacs. Per a això, s'ha obtingut suports porosos (scaffolds) mitjançant la tècnica solvent-casting particle-leaching emprant com a porògen sal, amb una grandària de porus que permet albergar cèl·lules en el seu interior, sobre els que s'han disposat, formant una estructura tipus sandvitx, membranes electrofilades. Aquestes membranes formen una estructura de fibres entrecreuades, deixant entre elles espais de grandària molt inferior a la grandària cel·lular, de mode que permeten el pas de nutrients i molècules a través d'elles, però actuen de barrera per a les cèl·lules impedint la seua migració a altres zones del sistema tridimensional. Aquest tipus d'estructures permeten simular, per exemple, l'arquitectura tubular renal, amb la zona porosa intersticial central i les dues capes epitelial i endotelial externes, que estarien en contacte amb l'orina i la sang, respectivament. Per a obtindre aquestes estructures, s'ha optat per emprar polímers de la família dels polièsters, en particular àcid polilàctic i poli(¿-caprolactona), així com la seua mescla i copolímers amb àcid poliglicòlic. Aquestes combinacions permeten ajustar la hidrofilicitat, i per tant la biodegradabilitat, la cinètica d'alliberació de fàrmacs i el comportament biològic, segons interesse. A més, l'ús de la tècnica d'electrospinning per al desenvolupament de les membranes, permet obtindre diferents diàmetres de fibra a partir de la modificació dels principals paràmetres de l'electrofilat, permetent també regular les propietats d'aquests materials. Finalment, per a estudiar l'alliberació de fàrmacs des del sistema anterior, les membranes electrofilades s'han carregat amb curcumina mitjançant dos mètodes diferents: a través del mètode d'emulsió i amb electrospinning coaxial, per a obtindre així diferents perfils d'alliberació. / [EN] The development of society has always been linked to scientific and technological advances. However, some of these advances have led to the appearance of new problems, especially of an ethical nature, giving rise to new associative movements opposed to them. An example of this is the use of animal experimentation, mainly in the field of medicine, and more specifically in the testing of drugs and implantable devices in contact with biological media. In this sense, the field of biomaterials and tissue engineering is working to find alternatives to animal experimentation. One of these alternatives is the development of in vitro models based on polymer supports for in vitro cell growth. These structures, moreover, could be used not only in drug testing or in vitro research to reduce the use of animals in experimentation, but also for tissue regeneration, simulating from simple tissues in which there is a single cell type, to more complex tissues from cell co-culture. Thus, in this thesis, a three-dimensional system with a stratified porous structure have been developed to allow both indirect cell co-culture and drug release assays. For this purpose, porous supports (scaffolds) have been obtained by means of the solvent-casting particle-leaching technique using salt as porogen, whose pore size allows cells to be housed in its interior, on which electrospun membranes have been arranged, forming a sandwich structure. These membranes form a structure of cross-linked fibers, leaving spaces between them that are much smaller than the cell size, so that they allow the passage of nutrients and molecules through them, but act as a barrier to the cells, preventing their migration to other areas of the three-dimensional system. This type of structure allows us to simulate, for example, the renal tubular architecture, with the central interstitial porous zone and the two outer epithelial and endothelial layers, which would be in contact with urine and blood, respectively. To obtain these structures, we have chosen to use polymers of the polyester family, in particular polylactic acid and poly(¿-caprolactone), as well as their blend and copolymers with polyglycolic acid. These combinations allow adjustment of hydrophilicity, and thus biodegradability, drug release kinetics and biological behavior, as desired. In addition, the use of the electrospinning technique for the development of membranes allows to obtain different fiber diameters by modifying the main electrospinning parameters, allowing also to regulate the properties of these materials. Finally, to study drug release from the previous system, the electrospun membranes have been loaded with curcumin by two different methods: through the emulsion method and with coaxial electrospinning, in order to obtain different release profiles. / Herrero Herrero, M. (2022). Desarrollo de andamiajes con porosidad estratificada basados en poliésteres como soportes en co-cultivo celular indirecto [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/190919