DISP3 protein, also known as PTCHD2, belongs to the PTCHD family of proteins, which contain a sterol-sensing domain in their structure. The expression of the Disp3 gene is high in neural tissues and is regulated by thyroid hormone. The DISP3 gene is associated with development and progression of certain types of tumors, as well as with development of some neural pathologies. Neural stem cells also display high expression of the Disp3 gene. Neural stem cells are defined by their capability to self-renewal and capacity to differentiate into the basic types of neural cells - neurons, astrocytes, and oligodendrocytes. Precise regulation of the balance between proliferation and differentiation of neural stem cells is crucial for development of the central nervous system and its subsequent proper functioning, and disruption of this balance may lead to development of various pathologies. In this work we mainly focused on describing the function of the DISP3 protein in neural cells and tissues. We have shown that during differentiation of neural stem cells, the expression of the Disp3 gene is significant decreased. Furthermore, we have found that in neural stem and progenitor cells, the increased expression of the Disp3 gene promotes their proliferation. Moreover, when Disp3 expression was disrupted, the...

L'objectif de ce travail était de mettre au point un système de culture tridimensionnel compartimenté pour la différentiation de cellules neurales et la croissance des neurites en 3D. Les matériaux biomimétiques permettent l'élaboration de microenvironnements contrôlés qui peuvent orienter la réponse cellulaire. Ils sont particulièrement intéressants pour les études fondamentales visant à étudier des voies de signalisation impliquées dans des processus physiologiques ou pathologiques. Nous nous sommes intéressés à la maladie d'Alzheimer, où l'on observe des neurites dystrophiques associés aux plaques amyloïdes. Aucune relation n'a été réellement établie entre l'interaction neurites - agrégats, leur dystrophie et la mort neuronale. Dans un premier temps, nous avons décrit et caractérisé la structure et les propriétés de matrices de collagène fibrillaire d'épaisseur calibrée. Ensuite, nous avons mis au point la fonctionnalisation de ces matrices avec des facteurs de croissance neurotrophiques (NGF et BDNF). Deux techniques ont été étudiées : l'imprégnation/libération et le couplage covalent. Ces matrices fonctionnalisées ont été validées comme support pour la différenciation de cellules nerveuses (PC-12 et SH-SY5Y) par des études de la morphologie cellulaire. Enfin, nous avons caractérisé des agrégats amyloïdes (Aβ) formés à l'intérieur des matrices de collagène par co-précipitation du peptide Aβ avec le collagène et nous avons étudié leur toxicité sur les cellules neurales. / The objective of this work was to develop a 3D compartmented cell culture set-up that allow the differentiation of nerve cells and the growth of neurites in the matrix depth. Biomimetic materials enable the formation of controlled microenvironments that orient cell behavior. They are particularly interesting for fundamental studies that aim to study signaling pathways involved in physiologic or pathologic processes. We focused on Alzheimer's disease, in which dystrophic neurites are associated to amyloid plaques. No direct relationship has yet been established between Aβ aggregates-neurite interaction, neurite dystrophy and cell death. First, we described and characterized the structure and properties of fibrillar collagen matrices with adapted thickness. Then, we adjusted functionalization of these matrices with neurotrophic growth factors (NGF and BDNF). Two methods were studied: impregnation/release and covalent coupling. Cell morphology studies confirmed that these functionalized matrices were efficient supports for nerve cells differentiation (PC-12 and SH-SY5Y). Finally, we have characterized Aβ aggregates that were formed inside collagen matrices by coprecipitation of amyloid peptide and collagen and we studied their toxicity on neural cells.

A eletrofiação é uma celebrada técnica de processamento de polímeros, capaz de produzir fibras de diâmetro nanométrico. A montagem comum do sistema de eletrofiação permite a captação de fibras aleatórias sob a forma de um não-tecido. Diversas modificações nessa montagem permitem a obtenção de diferentes morfologias de fibras. Tais modificações são revisadas e discutidas neste trabalho. Na produção de suportes de crescimento de células neurais, é interessante que seja incorporada alguma anisotropia no meio. Assim, um aparato de eletrofiação, capaz de produzir fibras alinhadas, foi construído e a variação dos parâmetros de seu processamento permitiu a obtenção de diferentes qualidades de alinhamento das fibras para dois polímeros biodegradáveis. Diversos parâmetros influenciaram a qualidade desse alinhamento, porém a velocidade de captação das fibras mostrou ser o mais impactante, em acordo com dados reportados na literatura. A morfologia das fibras foi avaliada quanto ao seu diâmetro, com o auxílio de micrografias de MEV e do software de edição de imagens ImageJ. Adicionalmente buscou-se avaliar a qualidade do alinhamento de tais fibras. Para tanto, foi desenvolvida uma metodologia de quantificação de qualidade de alinhamento de fibras, baseado nas micrografias e na ferramenta de FFT do ImageJ. A metodologia proposta foi capaz de ordenar de maneira objetiva e consistente a qualidade do alinhamento das fibras obtidas, mesmo quando a análise visual (usada como referência) se provava ineficiente. A metodologia proposta foi incorporada num plugin para ImageJ, via algoritmo computacional escrito em Java. Com o uso do plugin, foi possível processar diversas micrografias, obtidas em diferentes pontos das malhas eletrofiadas e com variadas magnificações, a fim de se criar uma estatística dos resultados obtidos para qualidade de alinhamento das fibras, algo inédito na literatura. Malhas eletrofiadas com diferentes qualidades de alinhamento de suas fibras foram utilizadas como substrato na cultura de células precursoras neurais, provenientes de neuroesferas. Foi feita a cultura de células progenitoras neurais, provenientes de neuroesferas, tendo como substrato malhas eletrofiadas com diferentes qualidades de alinhamento, a fim de se avaliar o impacto dos contatos físicos das fibras sobre a migração e diferenciação de tais células. / Electrospinning is a celebrated technique of polymer processing, able to produce fibers with nanometric diameter. Common assembly of electrospinning apparatus allows collection of random fibers in a non-woven matt. Several modifications on this assembly enable different fiber morphologies to be obtained. Such modifications are revised and discussed in this work. In the production of cell growth scaffolds, its interesting that some anisotropy is incorporated in the medium. Therefore, an electrospinning apparatus capable of producing aligned fibers was constructed. Variation of processing parameters of said apparatus enabled different alignment qualities of fibers to be attained for two biodegradable polymers. Many parameters influenced on the quality of said alignment; fiber collection speed, however, proved more impacting, in accordance with literature data. Fiber morphology was assessed in regard to its diameter with the aid of MEV micrographs and ImageJ software. Furthermore, assessment of fiber alignment quality was sought. For this matter, it has been developed a quantification methodology for fiber alignment quality, based on micrographs and ImageJ\'s FFT tool. The proposed methodology was able to objectively and consistently rank fiber alignment quality, even when visual analysis (used as reference) failed to do so. This methodology was incorporated in a plugin for ImageJ, via Java script algorithm. With the aid of this plugin it was feasible to process several micrographs, taken from electrospun mats at different spots and magnifications. This helped create statistics about obtained results of fiber alignment quality, on an unprecedented approach in written literature. Electrospun mats with varying quality in fiber alignment were used as substrate in the culture of neural precursor cells from neurospheres to assess the influence of contact guidance on migration and differentiation of such cells

A eletrofiação é uma celebrada técnica de processamento de polímeros, capaz de produzir fibras de diâmetro nanométrico. A montagem comum do sistema de eletrofiação permite a captação de fibras aleatórias sob a forma de um não-tecido. Diversas modificações nessa montagem permitem a obtenção de diferentes morfologias de fibras. Tais modificações são revisadas e discutidas neste trabalho. Na produção de suportes de crescimento de células neurais, é interessante que seja incorporada alguma anisotropia no meio. Assim, um aparato de eletrofiação, capaz de produzir fibras alinhadas, foi construído e a variação dos parâmetros de seu processamento permitiu a obtenção de diferentes qualidades de alinhamento das fibras para dois polímeros biodegradáveis. Diversos parâmetros influenciaram a qualidade desse alinhamento, porém a velocidade de captação das fibras mostrou ser o mais impactante, em acordo com dados reportados na literatura. A morfologia das fibras foi avaliada quanto ao seu diâmetro, com o auxílio de micrografias de MEV e do software de edição de imagens ImageJ. Adicionalmente buscou-se avaliar a qualidade do alinhamento de tais fibras. Para tanto, foi desenvolvida uma metodologia de quantificação de qualidade de alinhamento de fibras, baseado nas micrografias e na ferramenta de FFT do ImageJ. A metodologia proposta foi capaz de ordenar de maneira objetiva e consistente a qualidade do alinhamento das fibras obtidas, mesmo quando a análise visual (usada como referência) se provava ineficiente. A metodologia proposta foi incorporada num plugin para ImageJ, via algoritmo computacional escrito em Java. Com o uso do plugin, foi possível processar diversas micrografias, obtidas em diferentes pontos das malhas eletrofiadas e com variadas magnificações, a fim de se criar uma estatística dos resultados obtidos para qualidade de alinhamento das fibras, algo inédito na literatura. Malhas eletrofiadas com diferentes qualidades de alinhamento de suas fibras foram utilizadas como substrato na cultura de células precursoras neurais, provenientes de neuroesferas. Foi feita a cultura de células progenitoras neurais, provenientes de neuroesferas, tendo como substrato malhas eletrofiadas com diferentes qualidades de alinhamento, a fim de se avaliar o impacto dos contatos físicos das fibras sobre a migração e diferenciação de tais células. / Electrospinning is a celebrated technique of polymer processing, able to produce fibers with nanometric diameter. Common assembly of electrospinning apparatus allows collection of random fibers in a non-woven matt. Several modifications on this assembly enable different fiber morphologies to be obtained. Such modifications are revised and discussed in this work. In the production of cell growth scaffolds, its interesting that some anisotropy is incorporated in the medium. Therefore, an electrospinning apparatus capable of producing aligned fibers was constructed. Variation of processing parameters of said apparatus enabled different alignment qualities of fibers to be attained for two biodegradable polymers. Many parameters influenced on the quality of said alignment; fiber collection speed, however, proved more impacting, in accordance with literature data. Fiber morphology was assessed in regard to its diameter with the aid of MEV micrographs and ImageJ software. Furthermore, assessment of fiber alignment quality was sought. For this matter, it has been developed a quantification methodology for fiber alignment quality, based on micrographs and ImageJ\'s FFT tool. The proposed methodology was able to objectively and consistently rank fiber alignment quality, even when visual analysis (used as reference) failed to do so. This methodology was incorporated in a plugin for ImageJ, via Java script algorithm. With the aid of this plugin it was feasible to process several micrographs, taken from electrospun mats at different spots and magnifications. This helped create statistics about obtained results of fiber alignment quality, on an unprecedented approach in written literature. Electrospun mats with varying quality in fiber alignment were used as substrate in the culture of neural precursor cells from neurospheres to assess the influence of contact guidance on migration and differentiation of such cells

Proteomic techniques were used to analyze the protein expression profile of the early-stage differentiation of pig embryonic stem cells (ES cells). The pig ES cells were induced to develop to neuronal cells by all-trans retinoic acid (ATRA) in vitro by Tainan Livestock Research Institute. The ES cells were cultured with ATRA and collected at time intervals of 0, 1, 2, 4, 8 and 10 days. The cell lysates were analyzed by two-dimensional electrophoresis, and the differentially expressed proteins are identified by MALDI-TOF. Our data shows that the expression profile of pig ES cells is similar to other mammalian models but with some differences. Preliminary pig ES cells 2D database was set up. Six spots each with up or down-regulation in neurogenesis were identified by MS. These proteins may become the good markers of pig ES cells into neural cells by RA. Among those proteins, vimentin, prohibitin and annexin A10 were up-regulated, zinc finger protein 482 (ZNF482), fyn-related kinase (FRK) and annexin A1 were down-regulated during differentiation of pig ES cells to neural cells. Addtionally, we ultilized RT-PCR technique to investigate mRNA expression during neurogenesis, vimentin and prohibitin was up-regulated, anxa1(annexin A1) was slightly down-regulated, neuroD1 and neurogenin 2 were high expression on day 10, beta-catenin was high expression on day 8 to 10.

Dans cette thèse, deux dispositifs de culture in vitro de cellules ont été développés selon des technologies de microfabrication, qui offrent de nouveaux niveaux de contrôle sur le microenvironnement de la culture cellulaire. Les applications des dispositifs développés dans la recherche sur le cancer et la neurobiologie ont été démontrées, notamment pour l'étude fondamentale de métastases du cancer et le pathfinding axonal de neurones. La puce microfluidique dédiée à la transmigration comprend des microcanaux utilisées pour mimer les capillaires des tissus le long de la trajectoire des cellules cancéreuses lors de la métastase. La transparence optique du dispositif a permis une bonne observation de la déformation et de la migration des cellules dans les capillaires artificiels. Les résultats ont montré que la déformation du noyau de la cellule rigide était une des étapes les fastidieuses du processus de transmigration. Les restrictions physiques modifient la morphologie des cellules, mais elles affectent aussi de manière significative leur profil de migration. D'autres études sur le contenu moléculaire et les propriétés biologiques des cellules transmigrées ont montré que le blocage des modifications des histones par un médicament spécifique peut inhiber la transmigration des cellules cancéreuses dans le microcanal, ce qui pourrait avoir des implications sur la prévention et le traitement du cancer. La puce microfluidique peut également être utilisée pour évaluer la déformabilité de la cellule, qui est un marqueur pronostique potentiel pour le diagnostic du cancer. La puce de la culture de neurones permet la culture de cellules dans un microenvironnement au sein duquel de protéines sont imprimées selon des motifs géométriques précis. Les somas et axones des neurones mis en culture dans le dispositif peuvent être polarisés dans différents environnements fluidiquement isolés sur une longue période. L'extension des axones peut être guidée par des protéines immobilisées sur le substrat de verre. La croissance axonale orientée peut en outre être modulée par un traitement médicamenteux localisé. Les études sur le mécanisme moléculaire sous-jacent ont révélé que ces processus ont été étroitement associés à des protéines synthétisées localement dans les extrémités d'axones en croissance / In this PhD project, two in vitro cell culture devices were developed via microfabrication technologies, which provided entirely new levels of controls over the cell culture microenvironment. The applications of the developed devices in cancer and neurobiology researches were demonstrated, specifically for the fundamental study of cancer metastasis and neural axonal pathfinding. The microfluidic transmigration chip used microchannel structures to mimic the tissue capillaries along the path of cancer cell metastasis. The transparent optical qualities of the device allowed good observation of the deformation and migration of cells in the artificial capillaries. Results showed that deformation of the stiff cell nucleus were the most time-consuming steps during the transmigration process. The physical restrictions not only changed the morphology of the cells, but also significantly affect their migration profile. Further studies on the molecular contents and biological properties of the transmigrated cells showed that blocking the histone modifications by specific drug can inhibit the transmigration of cancer cells in the microchannel, which might have implications on cancer prevention and treatment. The microfluidic chip can also be used to evaluate cell deformability, which is a potential prognostic marker for cancer diagnosis. The neural culture chip integrated microfluidic cell culture and protein patterning techniques. The somas and axons of neurons cultured in the device can be polarized into different fluidically isolated environments for long period, and the extension of the axons can be guided by proteins immobilized on the glass substrate into specific patterns. The oriented axon growth can be further modulated by localized drug treatment. Studies on the underlying molecular mechanism revealed that these processes were closely associated with the proteins synthesized locally in the tips of growing axons

Human autopsy or biopsy tissue samples, mouse models and cell cultures of various types represent the most common materials in the investigation of cell pathogenesis of inherited diseases. This dissertation is devoted to all these approaches in the study of two X-linked lysosomal storage diseases, Fabry disease (FD,α-galactosidase A (AGAL) deficiency) and mucopolysaccharidosis type II (MPSII, idunorate-2- sulfatase (IDS) deficiency). The primary goal of the work was analysis of lipid blood group B antigens with terminal α-galactose (B-GSL) in the pancreas of FD patients with blood group B (FD-B).,In addition to the main glycosphingolipid (GSL) substrate, globotriaosylceramide (Gb3Cer), B-GSLs represent another minor substrate of AGAL. The deposition of undegraded B-GSL has been demonstrated in FD-B pancreas where it was significantly higher than in other organs such as the kidneys and lungs which accumulate mainly Gb3Cer. High concentration of lipid and non-lipid B-antigens was primarily confirmed in exocrine acinar epithelial cells of FD-B, accompanied by massive accumulation of ceroid (secondary sign of lysosomal storage). Unlike acini, the endocrine portion of the pancreas remained unaffected by accumulation of AGAL substrates. This interesting phenomenon of cell biology shows how a specific...