No description available.

No description available.

No description available.

No description available.

Se sintetizaron nanocompuestos híbridos en bloque de poli(etil metacrilato-co-hidroxietil acrilato) 70/30 wt%/sílice, P(EMA-co-HEA)/SiO2, con distintas proporciones de sílice hasta el 30 wt%. El procedimiento de síntesis consistió en la copolimerización de los monómeros orgánicos durante la polimerización sol-gel simultánea de tetraetoxisilano, TEOS como precursor de sílice. El TEOS se hidroliza eficientemente y condensa dando lugar a sílice, y presenta una distribución homogénea en forma de agregados inconexos de nanopartículas de sílice elementales en los híbridos con bajos contenidos de sílice (<10 wt%) o redes continuas interpenetradas con la red orgánica tras la coalescencia de los agregados de sílice (>10 wt%). La red polimérica orgánica se forma en los poros producidos en el interior de las nanopartículas elementales de sílice, y también en los poros formados entre los agregados de nanopartículas. Los nanohíbridos con contenidos de sílice intermedios (10-20 wt%) exhibieron las propiedades más equilibradas e interesantes: i) refuerzo mecánico de la matriz orgánica conseguida gracias a redes de sílice continuas e interpenetradas, ii) buena capacidad de hinchado debida a la expansión de la red orgánica no impedida todavía por un esqueleto de sílice rígido, y a un número alto de grupos silanol terminales hidrófilos (concentraciones inorgánicas en los alrededores de la coalescencia), y iii) mayor reactividad superficial debido a un contenido relativo bastante elevado de grupos polares silanol terminales disponibles en las superficies. La 'bioactividad' o capacidad de los materiales en bloque de formar hidroxiapatita (HAp) sobre sus superficies fue estudiada in vitro sumergiéndolos en fluido biológico simulado (simulated body fluid, SBF). La formación de la capa de HAp viene controlada por el mecanismo y el tiempo de inducción a la nucleación de la misma, que dependen a su vez de la estructura de la sílice. / Vallés Lluch, A. (2008). P(EMA-co-HEA)/SiO2 hybrid nanocomposites for guided dentin tissue regeneration: structure, characterization and bioactivity [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/3795

Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane. / <p>QC 20151217</p>

The relatively new and still growing field of 3D-printing has opened up the possibilities to manufacture patient-specific medical devices with high geometrical accuracy in a precise and quick manner. Additionally, biocompatible materials are a demand for all medical applications while biodegradability is of importance when developing scaffolds for tissue growth for instance. With respect to this, this project consisted of developing biocompatible and bioresorbable polymer blend and composite filaments, for fused deposition modeling (FDM) printing. Poly(lactic acid) (PLA) and polycaprolactone (PCL) were used as supporting polymer matrix while hydroxyapatite (HA), a calcium phosphate with similar chemical composition to the mineral phase of human bone, was added to the composites to enhance the biological activity. PLA and PCL content was varied between 90–70 wt% and 10-30 wt%, respectively, while the HA content was 15 wt% in all composites. All materials were characterized in terms of mechanical properties, thermal stability, chemical composition and morphology. An accelerated degradation study of the materials was also executed in order to investigate the degradation behavior as well as the impact of the degradation on the above mentioned properties. The results showed that all processed materials exhibited higher mechanical properties compared to the human trabecular bone, even after degradation with a mass loss of around 30% for the polymer blends and 60% for the composites. It was also apparent that the mineral accelerated the polymer degradation significantly, which can be advantageous for injuries with faster healing time, requiring only support for a shorter time period.

La medicina regenerativa és una disciplina que ha guanyat reconeixement en les últimes dècades pel fet que moltes malalties no són tractables amb fàrmacs convencionals. Molts grups de recerca i empreses inverteixen temps i diners en la producció de nous paradigmes per curar malalties com el Parkinson, l'artrosi o la regeneració de medul·la espinal. Aquestes propostes es basen en l'ús de teixits biomimètics per reparar òrgans danyats. En aquesta tesi es presenta un nou model experimental per estudiar la formació d'os i cartílag i eventualment la reparació d'aquests teixits. Hem utilitzat Fibroblasts Embriònics de Ratolí (MEFs) combinats amb diferents materials biomimètics per estudiar os i cartílag in vitro i in vivo. Aquests MEFs es van cultivar in vitro i in vivo en RAD16-I, un pèptid auto-ensamblable amb estructura similar a matrius extracel·lulars genèriques, amb l'objectiu d'estudiar l'evolució dels fibroblasts en aquestes dues condicions. També s'han recobert superficialment micropartícules de hidroxiapatita obtenint càrregues inorgàniques similars a l'os i biològicament actives per a utilitzar-les com a substituts d'os o cartílag. Amb la idea de millorar els recobriments superficials, hem desenvolupat una plataforma que permet dur a terme proves combinatòries amb factors de creixement i altres compostos biològicament actius. Cultius in vitro de MEFs han mostrat que quan fibroblasts embrionaris primaris de ratolí es cultiven en RAD16-I, estableixen una xarxa intercel·lular que causa una contracció cel·lular organitzada, proliferació i migració cel·lulars i culmina amb la formació d'una estructura bilateral i simètrica amb un eix central distingible. Durant aquest procés morfològic, augmenta l'expressió d'un grup de gens mesodèrmics (brachyury, Sox9, Sox5, Sox6, Runx2). L'expressió de brachyury està localitzada primer en l'eix de simetria central i després s'extén als dos costats de l'estructura. Per acabar, la formació espontània d'un teixit similar al del cartílag acompanya l'expressió de Sox9 i Runx2.L'estudi in vivo de MEFs es va fer gràcies a la tècnica de presa d'imatges no invasiva basada en bioluminiscència (BLI) que ha desenvolupat en el grup de recerca del dr. Jerónimo Blanco. Aquests experiments han mostrat que el RAD16-I és una matriu molt permissiva per a la supervivència i proliferació cel·lulars in vivo. A més, sembla que les pobres propietats mecàniques que té el RAD16-I no li suposen cap desavantatge en termes de creixement cel·lular in vivo. Finalment, hem desenvolupat diferents tipus de micropartícules de hidroxiapatita (HA) no recobertes, i recobertes mitjançant polimerització assistida per plasma. Els recobriments permeten afinar les propietats de la HA i produir partícules que satisfacin les necessitats de diferents aplicacions mèdiques en reparació d'os i cartílag. També hem desenvolupat un mètode per produir plataformes basades en plaques de 96 pous que permetin fer proves combinatòries amb compostos biològicament actius per vàries aplicacions en medicina regenerativa. En conclusió, hem aportat noves idees i eines que permetran trobar teixits regeneratius basats en l'ús de fibroblasts i materials biomimètics. / La medicina regenerativa es una disciplina que ha ganado reconocimiento en las últimas décadas porque muchas enfermedades no son tratables con fármacos convencionales. Muchos grupos de investigación y empresas invierten tiempo y dinero en la producción de nuevos paradigmas para curar enfermedades como el Parkinson, la artrosis o la regeneración de médula espinal. Estas propuestas se basan en el uso de tejidos biomiméticos para reparar órganos dañados. En esta tesis se presenta un nuevo modelo experimental para estudiar la formación de hueso y cartílago y tal vez la reparación de estos tejidos. Hemos utilizado Fibroblastos Embrionarios de Ratón (MEFs) combinados con diferentes materiales biomiméticos para estudiar hueso y cartílago in vitro e in vivo. Estos MEFs se cultivaron in vitro e in vivo en RAD16-I, un péptido auto-ensamblable con estructura similar a matrices extracelulares genéricas, con el objetivo de estudiar la evolución de los fibroblastos en estas dos condiciones. También se han recubierto superficialmente micropartículas de hidroxiapatita obteniendo cargas inorgánicas similares al hueso y biológicamente activas para utilizarlas como sustitutos de hueso o cartílago. Con la idea de mejorar los recubrimientos superficiales, hemos desarrollado una plataforma que permite llevar a cabo pruebas combinatorias con factores de crecimiento y otros compuestos biológicamente activos. Cultivos in vitro de MEFs han mostrado que cuando fibroblastos embrionarios primarios de ratón se cultivan en RAD16-I, establecen una red intercelular que causa una contracción celular organizada, proliferación y migración celulares y culmina con la formación de una estructura bilateral y simétrica con un eje central distinguible. Durante este proceso morfológico, aumenta la expresión de un grupo de genes mesodérmicos (brachyury, Sox9, Sox5, Sox6, Runx2). La expresión de brachyury está localizada primero en el eje de simetría central y después se extiende a los dos lados de la estructura. Para terminar, la formación espontánea de un tejido similar al del cartílago acompaña a la expresión de Sox9 y Runx2.El estudio in vivo de MEFs se hizo gracias a la técnica de toma de imágenes no invasiva basada en bioluminiscencia (BLI) que han desarrollado en el grupo de investigación del dr. Jerónimo Blanco. Estos experimentos han mostrado que el RAD16-I es una matriz muy permisiva para a la supervivencia y proliferación celulares in vivo. Además, parece que las pobres propiedades mecánicas que tiene el RAD16-I no le suponen ninguna desventaja en términos de crecimiento celular in vivo. Finalmente, hemos desarrollado diferentes tipos de micropartículas de hidroxiapatita (HA) no recubiertas, y recubiertas mediante polimerización asistida por plasma. Los recubrimientos permiten afinar las propiedades de la HA y producir partículas que satisfagan las necesidades de diferentes aplicaciones médicas en reparación de hueso y cartílago. También hemos desarrollado un método para producir plataformas basadas en placas de 96 pozos que permitan hacer pruebas combinatorias con compuestos biológicamente activos para varias aplicaciones en medicina regenerativa. En conclusión, hemos aportado nuevas ideas y herramientas que permitirán hallar tejidos regenerativos basados en el uso de fibroblastos y materiales biomiméticos. / Regenerative medicine is a discipline that has gained recognition in the last decades because many diseases are not treatable with traditional drugs. Many research groups and companies invest time and money in the production of new paradigms to cure conditions such as Parkinson's, arthrosis or spinal cord injuries. These approaches are based in the use of biomimetic tissues to replace damaged organs. In this work we present a new experimental model to study the formation of bone and cartilage and eventually to repair these tissues. We have used Mouse Embryonic Fibroblasts (MEFs) combined with different biomimetic materials to study bone and cartilage formation in vitro and in vivo. MEFs have been cultured in vitro and in vivo in RAD16-I, a synthetic self-assembling peptide with structure similar to generic extracellular matrix milieu, to study the evolution of these fibroblasts in both conditions. Also, hydroxyapatite microparticles have been surface coated to produce biologically active bone-like inorganic charges for use in cartilage or bone substitutes. In order to improve the particles' coatings, we have developed a platform that allows us to perform combinatorial testing of growth factors and other biologically active compounds. In vitro cultures of MEFs has shown that when primary mouse embryonic fibroblasts are cultured in a soft nanofiber scaffold, they establish a cellular network that causes an organized cell contraction, proliferation, and migration that ends in the formation of a symmetrically bilateral structure with a distinct central axis. A subset of mesodermal genes (brachyury, Sox9, Sox5, Sox6, Runx2) is upregulated during this morphogenetic process. The expression of brachyury was localized first at the central axis, extending then to both sides of the structure. The spontaneous formation of cartilage-like tissue mainly at the paraxial zone followed the expression of Sox9 and Runx2.In vivo study of MEFs was facilitated by a non-invasive bioluminescence imaging (BLI) technique to detect luciferase-expressing cells, developed by Dr. Blanco's research group. These experiments showed that RAD16-I is a very permissive scaffold for cell survival and proliferation in vivo. Furthermore, it seems that the poor mechanical properties of RAD16-I are no disadvantage in terms of cell growth in vivo.Finally, we have developed different types of coated and uncoated hydroxyapatite (HA) microparticles by plasma polymerization. The coatings permit to tune the properties of HA and produce particles that suit the needs of different medical applications in bone and cartilage repair. Moreover, we have developed a method to produce platforms based on 96-well plates that allow the combinatorial testing of biologically active compounds for various applications in regenerative medicine. In conclusion, we have supplied new insights and tools that will enhance the finding of new regenerative tissues based on fibroblasts and biomimetic materials.

Mitochondrial dysfunction has been implicated for a wide range of human diseases. One of the major biosynthetic processes in human mitochondria is the biogenesis of Iron-Sulfur (Fe-S) clusters which primarily involves in electron transfer reactions during oxidative phosphorylation (OXPHOS). Defects in Fe-S cluster biogenesis process leads to mitochondrial dysfunction and that eventually results in various human mitochondrial disorders. One of the major mitochondrial disorders associated with Fe-S cluster biogenesis impairment is exercise intolerance disorder ISCU myopathy, which is a result of loss of function of Fe-S cluster scaffold protein ISCU. Our biochemical results using yeast model system and HeLa cells lines suggests that ISCU Myopathy results in defective Fe-S cluster biogenesis in mitochondrial compartment. As a result, electron transport chain (ETC) complexes demonstrate significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, in ISCU Myopathy, mitochondria display enhancement in iron levels and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. On the other hand, in mammalian mitochondria, the initial step of Fe-S cluster assembly process is assisted by NFS1-ISD11 complex, which delivers sulfur to the scaffold protein ISCU during Fe-S cluster synthesis. In humans, loss of ISD11 function leads to development of respiratory distress disorder, Combined Oxidative Phosphorylation Deficiency 19 (COXPD19). Our study maps the important ISD11 amino acid residues critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these critical ISD11 residues to alanine leads to its compromised interaction with NFS1, which results in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Moreover, our findings highlight that, COXPD19 associated R68L ISD11 mutant displays reduced affinity to form a stable sub-complex with NFS1, thereby fails to prevent NFS1 aggregation, resulting impairment of Fe-S cluster biogenesis. The prime affected machinery is the ETC complex which demonstrates compromised redox properties, causing diminished mitochondrial respiration in COXPD19 patients. In summary, our findings provide compelling evidence that respiration defect due to impaired biogenesis of Fe-S clusters in ISCU myopathy patients, leads to manifestation of complex clinical symptoms. Additionally, our study highlights the role of ISD11 protein in Fe-S cluster biogenesis and maps the surface residues of ISD11 protein that are involved in interaction with sulfur donor protein NFS1. Moreover, we have demonstrated the molecular basis of disease progression of COXPD19 as a result of R68L ISD11 mutation.

Submitted by Marta Toyoda (1144061@mackenzie.br) on 2018-05-14T18:21:27Z No. of bitstreams: 2 André da Silva Siqueira.pdf: 3776282 bytes, checksum: 6603b366db594cfde0276b33c03a1968 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Paola Damato (repositorio@mackenzie.br) on 2018-05-21T13:34:35Z (GMT) No. of bitstreams: 2 André da Silva Siqueira.pdf: 3776282 bytes, checksum: 6603b366db594cfde0276b33c03a1968 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-21T13:34:35Z (GMT). No. of bitstreams: 2 André da Silva Siqueira.pdf: 3776282 bytes, checksum: 6603b366db594cfde0276b33c03a1968 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-02-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The present work aims to obtain and characterize a composite filament based on poly (lactic acid) (PLA), hydroxyapatite (HA) and graphene oxide, to process it by fused deposition modeling (FDM), and then make scaffolds for bone tissue growth and order to evaluate the properties of the obtained structure. To achieve these goals composite of PLA/HA, PLA/GO and PLA / HA / GO with concentrations of 0.05 wt %, 0.1 wt % and 0.3 wt % GO and 30 wt % HA were obtained by melt state blending and subsequently processed by FDM. The graphite oxide was obtained by modified Hummers method and characterized by X-ray diffraction (XRD), thermogravimetric analysis and Raman spectroscopy. The PLA and composites were characterized by molar mass measurements, infrared and Raman spectroscopy, tensile strength tests, contact angle measurements (surface energy), differential scanning calorimetry (DSC) and rheological tests. The insertion of GO into PLA and PLA/HA composites led to improvements in the mechanical properties (tensile) and also modified significantly the surface properties of the materials and the composition with 0.05 wt % of GO has shown the better results in both characteristics. These improvements occurred due to the strong interaction of the GO sheets with the PLA matrix that indicates the process of obtaining the composites via the melting state was correctly conducted. All PLA / HA / GO compositions presented rheological characteristics compatible with of scaffolds production process via FDM. The insertion of the GO into the PLA matrix and the PLA / HA composite has been shown to be extremely promising, and possibly to increase the variety of PLA / HA based biomaterials application. / O presente trabalho visa obter e caracterizar um filamento compósito baseado em poli(ácido lático) (PLA), hidroxiapatita (HA) e óxido de grafeno, processá-lo por Fused deposition modeling (FDM), fabricar scaffolds para crescimento de tecido ósseo. Para alcançar esses objetivos fcompósitos de PLA/HA, PLA/GO e PLA/HA/GO com concentrações de 0,05%, 0,1% e 0,3% de GO e 30% de HA (em massa) foram preparados por meio de mistura no estado fundido e posteriormente processados por FDM. Comprovou-se a obtenção do óxido de grafite por técnicas de difração de raios-X (DRX), análise termogravimétrica e espectroscopia Raman. O PLA e os compósitos foram caracterizados por medidas de massa molar da matriz polimérica, espectroscopia no infravermelho e Raman, ensaios mecânicos de tração, medidas de ângulos de contato (energia de superfície), calorimetria exploratória diferencial (DSC) e ensaios reológicos. A inserção do GO no PLA e no compósito PLA/HA conduziu a melhorias das propriedades mecânicas (tração) dos materiais e também modificou significativamente as propriedades de superfície dos materiais estudados, sendo a concentração de 0,05% em massa a que apresentou melhor desempenho em ambas as características. Essas melhorias aconteceram devido à forte interação das folhas de GO com a matriz de PLA, o que indica que o processo de obtenção dos compósitos via estado fundido foi corretamente conduzido. Todas as composições PLA/HA/GO apresentaram características reológicas compatíveis com o processo de produção dos scaffolds via FDM. A inserção do GO na matriz de PLA e no compósito PLA/HA demonstrou-se ser extremamente promissora, e possivelmente aumentarão a variedade de aplicações dos biomateriais baseados em PLA/HA.