A anóxia neonatal, considerada problema clínico mundial, é importante causa de lesão encefálica em neonatos que pode apresentar consequências graves e permanentes, como déficits cognitivos e comportamentais. O objetivo deste estudo foi analisar longitudinalmente possíveis alterações na morte, proliferação e diferenciação neuronais no hipocampo de ratos submetidos à anóxia neonatal. Para tanto, utilizamos modelo adaptado e validado em nosso laboratório. Os resultados mostraram que a anóxia neonatal causa morte neural em CA1 e CA2-3, detectadas pela maior quantidade de células TUNEL+ em CA1 e CA2-3 e FJB+ em CA2-3, além de diferentes tipos de morte neuronal em CA1 e GD, 24 horas após a anóxia,observadas por microscopia eletrônica. Houve aumento do volume de CA1 em P14 no grupo anóxia, porém o padrão de proliferação na zona subgranular não foi alterado. Enfim, a anóxia neonatal promoveu diminuição da neurogênese em animais adultos, o que poderia estar associado aos déficits de memória espacial e aprendizagem descritos em literatura para modelos similares. / Neonatal anoxia, considered a worldwide clinical problem, is a major cause of brain injury in neonates and may present serious and permanent consequences such as cognitive and behavioral deficits. The aim of this study was to analyze possible changes longitudinally in neural death, proliferation and neuronal differentiation in the hippocampus of rats submitted to neonatal anoxia. We used an adapted model validated in our laboratory. The results showed that neonatal anoxia cause neural death in CA1 and CA2-3 detected by the TUNEL+ cells in CA1 and CA2-3 and FJB+ in CA2-3, and different types of neuronal death in CA1 and GD 24 hours of anoxia, observed by electron microscopy. There was an increase in the volume of CA1 in the P14 anoxia group but the pattern of proliferation in the subgranular zone was not changed. Anyway, neonatal anoxia caused decrease in neurogenesis in adult animals, which could be associated with deficits in spatial memory and learning described in the literature in similar models.

Paracoccidioidomicose é adquirida pela via respiratória e a enzima indolamina-2,3-dioxigenase (IDO) e o catabolismo do triptofano estão envolvidos no controle da imunidade inata e adaptativa contra patógenos. Investigamos o papel da IDO na doença em animais suscetíveis (B10.A) e resistentes (A/J). Caracterizamos o efeito do tratamento com 1-Metil-DL-Triptofano (1MT) no fenótipo e comportamento de células dendríticas (DCs) e T reguladoras (Tregs) de A/J e B10.A quanto à expressão de IDO. IDO controla a carga fúngica de A/J e B10.A, reduzindo a imunidade de TCD4 e TCD8 e aumentando Tregs. Constatamos que IDO diminuiu a migração de DCs para o pulmão de A/J e B10.A e a inibição da atividade anti-inflamatória de IDO por 1MT tem um efeito deletério somente em B10.A cuja suscetibilidade é ligada à excessiva atividade pró-inflamatória. A infecção de A/J e B10.A parece induzir as funções catalítica e sinalizadora de IDO.Grande parte da função de IDO nos mecanismos imunorreguladores na paracoccidioidomicose se faz através da modulação da função de DCs em A/J e B10.A. / Paracoccidioidomycosis is acquired by the respiratory route and the enzyme indoleamine-2,3-dioxygenase (IDO) and tryptophan catabolism are involved in the control of innate and adaptive immunity against pathogens. We investigated the role of IDO in the infection in susceptible (B10.A) and resistant (A/J) mice. We characterized the effect of treatment with 1-Methyl-DL-Tryptophan (1MT) in the behavior and phenotype of dendritic cells (DCs) and regulatory T cells (Tregs) from A/J and B10.A for expression of IDO. IDO controls the fungal load in A/J and B10.A, reducing the immunity of CD4 and CD8 T cells and Tregs increased. IDO decreased the migration of DCs to the lung of A/J and B10.A and inhibition of anti-inflammatory activity of IDO by 1MT has a deleterious effect only in B10.A whose susceptibility is linked to excessive proinflammatory activity. Infection of A/J and B10.A appears to induce catalytic functions of IDO. Much of the function IDO immunoregulatory mechanisms paracoccidioidomycosis is done by modulating the function of DCs in A/J and B10.A.

Orientador: Kleber Gomes Franchini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T03:46:54Z (GMT). No. of bitstreams: 1 Pereira_AnaHelenaMacedo_D.pdf: 4791935 bytes, checksum: 1afe275f0fa4f53003b18816bc5c27cb (MD5) Previous issue date: 2013 / Resumo: Os fatores MEF2 (Myocyte Enhancer Factor 2) pertencem à família MADS Box (MCM1-Agamous-Deficiens-Serum response factor) e foram descritos pela primeira vez como fatores de transcrição que se ligam a sequencias de DNA ricas em A/T nos promotores de vários genes músculo específicos. Existem 4 genes da família MEF2 que foram identificados em vertebrados: MEF2A, B, C e D que são expressos de forma distinta durante a embriogênese e nos tecidos adultos. Estudos anteriores do nosso laboratório demonstraram que o fator de transcrição MEF2 é ativado por estiramento mecânico e influencia a expressão de genes relacionados à hipertrofia cardíaca. Utilizando a tecnologia de siRNA para MEF2C (siRNAMEF2C) demonstramos a atenuação da hipertrofia cardíaca induzida por coarctação da aorta nos animais que receberam o siRNAMEF2C. Por outro lado trabalhos demonstraram que animais transgênicos com a superexpressão de MEF2A ou de MEF2C e submetidos à sobrecarga de pressão por coarctação da aorta, não apresentam hipertrofia cardíaca compensatória. Nesses animais a superexpressão de MEF2A ou de MEF2C no coração está associada à deterioração cardíaca funcional e estrutural e o desenvolvimento de cardiomiopatia dilatada. Contudo, a caracterização fenotípica e os mecanismos moleculares envolvidos na superexpressão de MEF2C em miócitos cardíacos ainda são desconhecidos. Da mesma forma não é conhecido o papel do fator de transcrição MEF2C na resposta hipertrófica do miócito cardíaco após coarctação da aorta. No presente trabalho foi demonstrado que a superexpressão de MEF2C em miócitos cardíacos de ratos neonatos (NRMV), com o uso de partículas adenovirais, induziu a desdiferenciação celular e a ativação de mecanismos envolvidos na progressão do ciclo celular. Esses resultados foram obtidos por meio de experimentos de microarranjo de DNA, proteoma, PCR em tempo real e western blotting. A análise do fenótipo celular por microscopias de luz, confocal e eletrônica de transmissão demonstra que NRMV possuem aumento na binucleação e desorganização sarcomérica, alterações coerentes com o quadro de desdiferenciação celular e ativação da progressão do ciclo celular. Por meio da técnica de incorporação de iodeto de propídeo e citometria de fluxo confirmamos o aumento de células em ciclo celular. Para confirmar os achados nos cardiomiócitos neonatos passamos a investigar o efeito da superexpressão de MEF2C em cardiomiócitos de ratos adultos. Para isso padronizamos a técnica de isolamento destas células e tratamos com AdMEF2C. Sendo assim o tratamento com AdMEF2C em miócitos cardíacos de ratos adultos resultou em aumento da expressão de MEF2C após 48 horas de tratamento. O efeito observado foi semelhante ao encontrado em cardiomiócitos neonatos, sendo que os adultos apresentaram aumento da expressão de genes relacionados ao ciclo celular e diminuição dos genes estruturais. O nível ultraestrutural observado por microscopia eletrônica de transmissão no tempo de 48 horas de tratamento não observamos diferenças na estrutura sarcomérica das células tratadas com AdMEF2C. Por fim demonstramos que o silenciamento de MEF2C pela injeção de lentivírus no coração demonstrou ser capaz de impedir o desenvolvimento da hipertrofia cardíaca em camundongos coarctados por 15 dias. A hipertrofia do coração foi avaliada por meio da espessura da parede posterior do ventrículo esquerdo e gravimetria do ventrículo esquerdo e dos pulmões. O conjunto de dados demonstra que a superexpressão de MEF2C leva a alterações estruturais no miócito cardíaco compatíveis com quadro de deterioração e insuficiência cardíaca e que o silenciamento de MEF2C no coração impede o desenvolvimento da hipertrofia cardíaca decorrente da coarctação da aorta / Abstract: The factors MEF2 (myocyte enhancer factor 2) belong to the family MADS box (MCM1-Agamous-deficiens-Serum response factor) and were first described as transcription factors that bind DNA sequences rich in A / T in the promoters of multiple muscle-specific genes. There are four MEF2 family genes that were identified in vertebrates MEF2A, B, C and D are expressed differently during embryogenesis and in adult tissues. Previous studies from our laboratory demonstrated that the transcription factor MEF2 is activated by mechanical stretch and influences the expression of genes related to cardiac hypertrophy. Using siRNA technology to MEF2C (siRNAMEF2C) demonstrated attenuation of cardiac hypertrophy induced by aortic coarctation in animals that received siRNAMEF2C. On the other hand studies have demonstrated that transgenic mice with overexpression of MEF2A or MEF2C and subjected to pressure overload by aortic coarctation show no compensatory cardiac hypertrophy. In these animals the overexpression of MEF2A or MEF2C in the heart is associated with structural and functional cardiac deterioration and development of dilated cardiomyopathy. However, the phenotypic and molecular mechanisms involved in the overexpression of MEF2C in cardiac myocytes are still unknown. Likewise, there is known the role of the transcription factor MEF2C in cardiac myocyte hypertrophic response after aortic coarctation. In the present study it was shown that overexpression of MEF2C in neonatal rat cardiac myocytes (NRMV) with the use of adenoviral particles, and cellular dedifferentiation induced activation mechanisms involved in cell cycle progression. These results were obtained by DNA microarray experiments, proteomics, real time PCR and western blotting. The analysis of cell phenotype by light microscopy, confocal and transmission electron shows that NRMV have increased binucleation and sarcomeric disorganization, changes consistent with the framework of cellular dedifferentiation and activation of cell cycle progression. By means of the propidium iodide incorporation technique and flow cytometry, confirmed increasing cells in the cell cycle. To confirm the findings in neonatal cardiomyocytes we investigate the effect of overexpression of MEF2C in cardiomyocytes of adult rats. For this standardized technique and isolation of these cells treated with AdMEF2C. Thus treatment with AdMEF2C in adult rat cardiac myocytes resulted in increased expression of MEF2C after 48 hours of treatment. The observed effect was similar to that found in cardiomyocytes neonates, adults who showed increased expression of genes related to cell cycle and decreased structural genes. The ultrastructural level observed by transmission electron microscopy in the time of 48 hours of treatment showed no difference in sarcomeric structure of cells treated with AdMEF2C. Finally we show that MEF2C silencing by lentivirus injection in the heart has been shown to prevent the development of cardiac hypertrophy in mice after 15 days of pressure overload. The heart hypertrophy was evaluated by the thickness of the posterior wall of the left ventricle and the left ventricle gravity and lungs. The data set shows that overexpression of MEF2C leads to structural changes in the cardiac myocyte compatible framework of deterioration and failure, and MEF2C silencing of the heart prevents the development of cardiac hypertrophy due to aortic coarctation / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutora em Ciências

Un patient peut souffrir d’une perte de substance osseuse de taille critique suite à des accidents ou des pathologies. Aujourd’hui, le traitement le plus fréquent consiste en la greffe du tissu osseux (autogreffe ou allogreffe). Compte tenu des complication rencontrées (réponse immunologique, morbidité du site donneur), la recherche actuelle s’inscrit dans la recherche de synthèse d’un biomatériau bioactif favorisant la régénération osseuse. Ces matériaux devraient imiter les qualités de la matrice extracellulaire osseuse pour stimuler la formation osseuse.Les cellules souches mésenchymateuses jouent un rôle important du fait de leur capacité de prolifération et différentiation en ostéoblastes. Pour profiter de ce potentiel des cellules souches, il est nécessaire de comprendre comment contrôler leur comportement et mesurer l’impact du microenvironnement cellulaire sur la différenciation ostéogénique de ces cellules. Comme les cellules souches mésenchymateuses sont capables de différencier en plusieurs phénotypes différents, il est indispensable de les diriger dans la direction désirée. Plusieurs facteurs qui influencent le devenir cellulaire ont été identifiés, comme certains peptides bioactifs, des facteurs mécaniques comme la rigidité, ou la topographie de surface de matériaux.Dans la matrice extracellulaire naturelle du tissu osseux, les cellules souches mésenchymateuses sont entourées d’une variété de principes actifs, dont le plus abondant est le collagène I. Cette protéine s’assemble pour former des nanofibres qui présentent une nanomorphologie périodique avec une périodicité bien définie. La question posée au début de ce travail était: Cette structure a-t-elle un impact sur la différenciation des cellules souches?Pour étudier l’impact de la périodicité nanofibrillaire, nous proposons dans ce travail de recherche l’utilisation d’hélices modèles qui miment en partie la morphologie du collagène. Les hélices nanométriques auto-assemblées des surfactants gemini peuvent avoir un pas d’hélice et un diamètre similaires à ceux du collagène. La modulabilité de ces paramètres et la possibilité de modifier ces structures par des molécules bioactives permettent de moduler les caractéristiques des nanoobjets et d’étudier l’impact de ces nanomatériaux sur les cellules souches mésenchymateuses. / Tissue engineering is a field related to regenerative medicine which aims at replacing or regenerating a patient’s tissue, usually using a combination of cells and bioactive material designed to influence cell behaviour. In approaches for bone regeneration, human mesenchymal stem cells (hMSCs) are a common choice because of their ability to proliferate and differentiate into osteoblasts. Harnessing this potential requires biomaterials which promote osteoblastic differentiation, for example by mimicking the conditions in natural bone. Collagen I is a common protein in human bone; it forms fibrils with a characteristic periodic structure, which raises the question whether this morphology has in impact on stem cell fate. Collagen-mimicking nanomaterials can help investigate this question: Gemini surfactants with chiral counterions form twisted bilayers the morphology of which can be tuned by variation of enantiomeric excess, time and temperature. The self-assembled helical nanoribbons which are obtained by this process can be transformed by a sol-gel condensation to form silica nanohelices the size and twist pitch of which resembles that of collagen fibres. The objective of this study is to prepare 2D culture environments featuring these nanomaterals (with and without bioactive peptide functionalisation) to explore the effect of these materials on hMSC differentiation.Silica helices are fabricated by synthesis of surfactants with tartrate as counterion, and organic-inorganic transcription using a silica precursor compound. They can be modified by reaction with APTES and an N-hydroxysuccinimide ester and covalent immobilisation of a peptide. Two peptides were used in this study, one adhesion-promoting peptide and the active domain of the osteogenesis-inducing peptide BMP2. Helices with or without this bioactive functionalisation were covalently grafted to glass substrates using APTES and EDC/NHS-coupling. The presence of peptides on helices was shown by the absorption of helix-grafted peptides bearing the FITC-fluorophore. Successful peptide grafting onto glass surfaces was verified by XPS and fluorescence microscopy. The morphology of helices was monitored with TEM and SEM. SEM images were used to determine the amount of helices on surfaces. HMSCs were cultivated for four weeks on surfaces modified with APTES, peptide(s) or left- or righthanded nanohelices, functionalised or not with bioactive peptide(s). After fixation, the quantities of the osteogenic markers Runx2 and Osteocalcin (OCN) in the cells were evaluated. The results show that BMP2-functionalised surfaces exhibited an elevated level of Runx2 and OCN expression. Some helix-grafted materials exhibited a significantly higher Runx2 and/or OCN expression than the corresponding homogeneous materials, but these differences were not consistent across samples of the same chiral orientation or bioactive functionalisation. Therefore, conclusive general statements about differences in osteogenic effect between helix functionalisations and handednesses aredifficult to make. A potential reason for this is the variability of surface coverage of helix-grafted materials: As the quantity of helices that are immobilised onto the surfaces is lower than expected and varies greatly between the samples, the number of cells that are not in contact with the helices might change as well, which can lead to false negatives.The results of a proteomic experiment have shown which proteins are differentially expressed in cells cultured on helices with or without BMP-functionalisation, compared to bare glass. Comparison with other proteomic studies shows that proteins which are known to be upregulated during osteogenic differentiation are overexpressed most frequently in cells cultured on BMPmodified helices. The proteins that were identified with this method might serve as starting point for future investigations.

Abstract Type 1 neurofibromatosis (NF1) syndrome is caused by a mutation of the NF1 gene. NF1 protein (neurofibromin) contains a domain which is related to the GTPase activating protein (GAP) and accelerates the switch of active Ras-GTP to inactive Ras-GDP. The clinical symptoms of NF1 patients include e.g. the formation of benign neurofibroma tumors and hyperpigmented lesions of the skin. The NF1 protein has been referred to as a tumor suppressor since cells of malignant schwannomas of NF1 patients may display loss of heterozygosity of the NF1 gene. In the present study, the expression of the NF1 gene was investigated during tissue repair in human skin. Elevated NF1 protein levels were seen in a fibroblastic cell population of healing wounds. In vitro studies were designed to investigate NF1 expression in dermal fibroblasts under the influence of growth factors that are operative during wound healing. Platelet-derived growth factor (PDGF) isoforms AB and BB and transforming growth factor β1 (TGFβ1) elevated NF1 mRNA levels in cultured dermal fibroblasts. In further studies, histological examination on apparently healthy skin of NF1 patients revealed frequently small masses of neurofibromatous tissue at the vicinity of hair follicles. Thus, action of the NF1 gene appears to be an integral part of normal tissue repair. Enhanced NF1 tumor suppressor expression may serve to limit excessive fibrosis in wound healing. As Ras proteins play a role in the regulation of cell differentiation and formation of cell junctions, the functional expression of NF1 protein was elucidated using differentiating keratinocytes as an in vitro model system. The results demonstrate that an intense NF1 tumor suppressor signal on intermediate filaments was temporally limited to the period in which the formation of desmosomes takes place. In analogy to NF1 protein, a rapid elevation of NF1 mRNA level was detected following initiation of differentiation. Interestingly, NF1 mRNA hybridization signal polarized towards cell-cell contact zones. This finding recognizes a potential way for post-transcriptional modification of NF1 expression and targeting of translation to subplasmalemmal location. The results demonstrate that the function of NF1 protein is associated with the formation of cell junctions, and thus to cellular communication.

O epitélio pulmonar é formado por uma grande diversidade celular, que incluí: células secretoras, ciliadas, basais e neuroendócrinas (NE). A distribuição balanceada destes tipos celulares é crucial para a função pulmonar e pode ser dramaticamente alterada em doenças como a asma. Neste trabalho, estudamos o papel de Notch no pulmão em desenvolvimento ao inativar condicionalmente Rbpjk ou Pofut1, componentes críticos da sinalização Notch. Pulmões mutantes apresentaram-se superpopulados por células ciliadas e NE, além da ausência de células de Clara. Nossos dados sugeriram que Notch suprime os programas de diferenciação de células ciliadas e NE para permitir a diferenciação de células de Clara, através de um mecanismo de inibição lateral. Identificamos também genes associados com a diferenciação de células secretoras e ciliadas através de microarrays. A heterogeneidade no padrão de expressão gênica sugeriu que a via de sinalização Notch estabelece múltiplos subtipos de células ciliadas e secretoras no epitélio pulmonar em desenvolvimento. / The airway epithelium comprises a diverse population of secretory, ciliated, basal and neuroendocrine cells (NE). The proper balance of these cell types is critical for normal lung function and can be altered dramatically in conditions, such as asthma. We studied the role of Notch in airway progenitor cell fate by conditionally inactivating Rbpjk or Pofut1, two critical Notch pathway components in mouse mutants. This resulted in airways overpopulated with ciliated and NE cells and absence of secretory Clara cells. We found that Notch suppresses the ciliated and the NE cell programs to allow secretory cell differentiation through a lateral inhibition mechanism. We also identified genes associated with the differentiation of secretory and ciliated cells through a microarray gene profiling experiment. The great heterogeneity of gene expression patterns suggested that Notch plays a role in establishing multiple subsets of secretory and ciliated cells in the developing lung.

Esta tesis aborda la actividad biológica de la fibronectina (FN) como proteína de interfase en la interacción célula-material. La tesis investiga la respuesta de la proteína, en términos de cantidad adsorbida y conformación, ante diferentes propiedades físico-químicas del material. Además, se correlaciona la respuesta celular temprana y la funcionalidad celular con el estado de la proteína adsorbida sobre el material. Para ello se prepararon diferentes series de materiales con propiedades físico-químicas controladas. La distribución de FN sobre las diferentes superficies se caracterizó mediante el uso de la microscopía de fuerza atómica (AFM) y la densidad superficial adsorbida fue cuantificada mediante técnicas de marcado radioactivo y western blot. La respuesta celular se evaluó en términos de la adhesión inicial a las superficies, así como los procesos posteriores de diferenciación, proliferación, reorganización y producción de matriz extracelular. Se investigó el efecto de la nanotopografía en la adsorción de la FN y el comportamiento celular sobre una serie de topografías controladas en la escala nanométrica, obtenidas mediante el spin casting de soluciones de ácido poli(L-láctico)/poliestireno (PLLA/PS) de distintas concentraciones. La migración del PLLA hacia la superficie del film durante el proceso de spin coating proporciona superficies de PLLA con nanopicos de diferentes tamaños (14, 29 y 45 nm). El tamaño de la nanoestrutura afecta a la densidad de FN adsorbida, siendo mayor en la superficie de menor nanotopografía. En cuanto a la respuesta celular inicial, se observan adhesiones focales más desarrolladas y mejor reorganización celular de la capa de FN adsorbida en las superficies de mayor topografía (29 and 45 nm), lo que resulta en una mayor producción y organización de nueva matriz. Por otra parte se empleó una familia de materiales con sutiles variaciones en la composición química: polímeros acrílicos (polimetil, etil y butil acrilato -PMA, PEA y P / González García, C. (2012). BIOLOGICAL ACTIVITY OF FIBRONECTIN AT THE CELL-MATERIAL INTERFACE [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/17701 / Palancia

Abstract While most of the in vitro cultures are carried out on bi-dimensional (2D) substrates, most of the in vivo extracellular matrices are threedimensional (3D). Consequently cells behave differently on 2D substrates as a way to self-adaptation to a non-physiological environment. This fact has encouraged the development of more relevant culture conditions seeking to provide more representative models for biomedicine (e.g. cancer, drug discovery and tissue engineering) and further insights into any dimension-dependent biological mechanism. Different 3D culture systems have been established though their variability and complexity hinder their standardisation in common cell culture procedures. So, this thesis deals with the dimensionality issue in cell/material interactions and introduces sandwich-like microenvironments as a versatile tool to study cell behaviour. Cells cultured within this system use both dorsal and ventral receptors to adhere and spread, undergoing important changes with respect to the 2D cultures and approaching to 3D conditions. Stimulation of dorsal receptors has been previously addressed by overlaying a protein gel on cells already attached on a 2D surface. Here we propose a sandwich-like system that consists of two 2D surfaces so that wider spectra of conditions can be investigated by changing the nature of the substrate (material, topography…) and the protein coatings of both ventral and dorsal sides. Since sandwich culture provides an altered cellular adhesion compared to the traditional 2D substrates by the excitation of the dorsal receptors, changes in the intracellular signalling are expected, which might alter important processes such as proliferation, morphology, migration and differentiation. Hence this thesis evaluates the effect of different sandwich culture parameters in cell behaviour. First, cell fate upon adhesion was evaluated in terms of morphology, proliferation and adhesion. Different conditions were studied such as materials with different properties or protein coatings (dorsal and ventral substrates), as well as the effect of sandwiching cells just after seeding or after been allowed to adhere to the ventral substrate. Interesting results were obtained such as the relationship between the ability of cells to reorganise the ECM with cell morphology, proliferation and adhesion, similarly as observed in 3D hydrogels (degradable vs nondegradable systems). Then, cell migration within sandwich culture was studied by live imaging of a wound healing assay. Results revealed the key effect of both ventral and dorsal substrates in determining the migration rate as well as the migration mode used by cells. Moreover cells within the sandwich culture migrating in the wound healing assay adopted an elongated cell morphology that resembled cells migrating in other 3D systems. Beyond differences in cell morphology and migration, dorsal stimulation promoted cell remodelling of the extra-cellular matrix (ECM) over simple ventral receptor activation in traditional 2D cultures. Finally the effect of sandwich culture on cell differentiation was evaluated. First we showed an increase in C2C12 myogenic differentiation when cultured within the sandwich system. This enhancement was shown to be dorsal stimulation dependent and related to an alteration of the signalling pathway and the growth factor release. To determine if sandwich culture leads only to myogenic differentiation or whether it allows differentiation to other lineages, 4 different human mesenchymal stem cells (hMSCs) lines were cultured under the same conditions. Results showed the same sandwich environment triggered different cell differentiation. This points out the importance of the microenvironment cell niche in vivo, which highly influence cell fate, and thus the need of mimicking it properly in vitro. Overall, sandwich-like microenvironments switch cell behaviour towards 3D-like patterns, demonstrating the importance of this versatile, simple and robust approach to mimic cell microenvironments in vivo. / Ballester Beltrán, J. (2014). Sandwich-like systems to engineer the cellular microenvironment [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48166 / TESIS

Cultivation of cells in bioreactors with mechanical load simulates the physiological conditions to which cells in the body are exposed. This technology has been used to induce the differentiation of stem cells from adipose tissue towards the phenotype of vascular smooth muscle cells, which can further serve to form vascular replacements. At present, there is no established strategy for cultivating stem cells while being exposed to mechanical stress. The main aim of this work was therefore to optimize the cultivation strategy and determine the ideal load parameters. Differentiation was analyzed by immunofluorescence of specific smooth muscle cell markers, α-actin and h1-calponin, which were quantified by Western blot. Extracellular matrix production was also detected by immunofluorescence staining. The outcome of this work is the establishment of ideal conditions of cell culture in a bioreactor with mechanical load, during which they differentiate into smooth muscle cells. Three types of scaffolds were used for cultivation; plasma treated glass, fibrin-coated glass and decelularized pericardium. Preliminary results show that smooth muscle differentiation was succesfully induced in human and porcine adipose tissue stem cells. Cells were analyzed after 3 and 7 days of culture. Developing a stem cell...

There are approximately 600,000 cases of delayed or aberrant fracture healing in people each year, with a small subset of these fractures experiencing disunion. Dietary phosphate deficiency has been shown to impair oxidative phosphorylation and decrease BMP-2 mediated chondrogenic differentiation during fracture healing. Prior studies using pre-committed chondro-progenitor ATDC5 cell line grown in phosphate deficient media showed that energy consumption was linked to protein production and collagen hydroxylation but inversely related to matrix mineralization. The goal of this study was to further define the relationship between energy consumption and BMP-2 mediated stem cell chondrogenic differentiation and further examine how dietary phosphate, and promotion of collagen hydroxylation via ascorbate availability effected these processes. C3H10T1/2 murine cells, a multi-potential cell line, were expanded in pre-differentiation growth medium (DMEM with 10% FBS and 1% Pen/Strep). Once cells reached 60% confluence (day 0), they were grown in differentiating media (α-MEM with 5% FBS and 1X insulin-transferrin-selenium) containing either 100% (1mM) or 25% (0.25mM) inorganic phosphate (Pi), ± 200ng/mL BMP-2(BMP), and ±0.2 mM L-ascorbic acid (AA). In total, there were 8 groups with varying combinations of these three substances. Intracellular lipid, total DNA, protein, and hydroxyproline (HP) content were examined. Chondrocyte gene expression (Col2a1, Acan, ColXa1) and adipocyte gene expression (Pparg, Plin1, Ucp1) were measured to check for cell lineage commitment and specific differentiation of the C3H10T1/2. All measurements were acquired at day 8. The +BMP differentiation media groups contained significantly less DNA content and more protein content than the –BMP differentiation media groups (both p<0.0001). There was also a significant interaction between phosphate and ascorbic acid treatment (p=0.0296), with 25% Pi +AA groups producing significantly more protein than 100% Pi +AA groups. Hydroxyproline production was not different in 100% Pi or 25% Pi conditions (p=0.2951). AA presence in culture media led to greater HP production than culture media lacking AA (p=0.0035) There was a trend of an interaction between phosphate content and AA availability (p=0.0744). 100% Pi ±AA groups produced significantly different amounts of HP while 25% Pi ±AA groups did not produce significantly different amount of HP. Col2a1, Acan, and ColXa1 expression were all increased in +BMP groups. Ascorbic acid treatment groups expressed significantly more Col2a1and Acan than –AA groups. 100% Pi media led to greater Acan expression over 25% Pi groups (p=0.0009), whereas 25% Pi media trended to lead to greater ColXa1 expression over 100% Pi groups (p=0.0734). Pparg and Plin1 expression were increased in the 25% Pi condition. There were no significant differences in expression of Ucp1. C3H10T1/2 cells were significantly affected by phosphate concentration, BMP-2 treatment, and ascorbic acid supplementation. Phosphate deficiency hindered maturation of early chondrocytes into proliferating chondrocytes while also promoting MSC differentiation into the adipocyte cell lineage. Hypertrophic chondrocyte expression was decreased in phosphate deficient media, which may coincide with increased protein production observed in low phosphate conditions. BMP-2 promoted chondrogenesis which resulted in increased protein production. Whereas, lack of ascorbic acid in cell culture media led to decreased hydroxyproline production.