Members of the Runx family of transcription factors play essential roles in the differentiation and development of several organ systems. Here we address the contribution of the osteoblast-related Runx gene, Runx2, to the osteogenic and chondrogenic differentiation of mesenchymal stem cells. Using a transgenic mouse model, we observe Runx2 transcription through one of its two known promoters (designated P1 in pre-cartilaginous mesenchymal condensations as early as E9.5. Runx2 gene activity is later repressed at the onset of cartilage formation, both in vivo and in vitro, necessitating examination of the regulation and function of Runx2 in mesenchymal stem cells. We demonstrate that Runx2 gene activity is repressed by the direct interaction of the homeodomain transcription factor Nkx3.2 with the proximal Runx2 P1 promoter. This repression was found to be required for the progression of BMP-induced chondrogenesis, thereby identifying Runx2 as a modulator of BMP activity in the chondrogenic as well as osteogenic differentiation program. To further understand the regulation of the Runx2 P1 promoter and to determine the contribution of P1-derived gene product, Runx2 Type II, to the formation of mineralized tissue, we have generated a Runx2 Type II-LacZ gene replacement mouse model in which the initial coding sequences and splice donor sites of the Type II isoform are replaced with the LacZ reporter gene. Activity of the endogenous P1 promoter can therefore be monitored by β-galactosidase production. Analysis of Runx2 Type II-LacZ mice demonstrates that the P1 promoter is transcriptionally most active in mature osteoblasts, but its product, Runx2 Type II is dispensable for embryonic skeletal formation. Lastly, we examine the link between growth control and osteogenic differentiation by tissue-specific deletion of the Mdm2 proto-oncogene in developing skeletal tissues of the mouse embryo. Loss of Mdm2 results in impaired bone formation, with skeletal elements exhibiting lower bone mineral content and higher porosity. Ex vivo cultures of calvarial osteoprogenitor cells exhibit severely decreased osteoblastogenesis and bone nodule formation accompanied by a failure to activate Runx2 gene activity. These findings suggest that Mdm2 is required for inhibition of p53 activity that ultimately allows for post-confluent proliferation and induction of Runx2 during maturation of the osteogenic phenotype. Taken together, our findings suggest that Runx2 modulates the commitment of progenitor cells to the osteogenic and chondrogenic lineages, and that Runx2 activity is inextricably linked to mechanisms that control cellular proliferation.

The aim of my study is to evaluate the impact of seed density on yields of hemp, which was cultivated in two areas, and subsequently to assess biological efficiency of infusion from hemp on fibroblastoidal line of mammalian cells L292. Afterthat in the case of above mentioned biological test we want to find out, if the density and different parts of plant will have an impact on efficiency of extract.

Made available in DSpace on 2015-07-22T20:49:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-09-30. Added 1 bitstream(s) on 2015-08-11T03:25:27Z : No. of bitstreams: 1 Publico-00264.pdf: 1668171 bytes, checksum: dbdf365617164c11a9a209e69d18f997 (MD5) / O ƒÑ-tocoferol tem sido o foco das pesquisas dentre os demais componentes da vitamina E por ser a forma predominantemente encontrada nos tecidos de mamiferos, e por possuir uma extensa gama de atividades biologicas. O ƒÑ-tocoferol pode atuar como regulador de enzimas especificas e de fatores de transcricao de forma a influenciar estruturas celulares como membranas e dominios lipidicos, desencadeando respostas celulares que muitas vezes se mostram independentes de sua funcao antioxidante. No sistema hematopoetico, seus efeitos foram favoraveis em casos de anemias hemoliticas, aumentando a resistencia dos eritrocitos a lise. Tambem foi observado que a suplementacao previa com ƒÑ-tocoferol a irradiacao resulta em aumento da sobrevida de camundongos por induzir aumento no numero de unidades formadoras de colonias (CFUs). Entretanto, os mecanismos biologicos ativados pelo ƒÑ-tocoferol nas celulas hematopoeticas ainda nao foram descritos. Assim, os bjetivos deste trabalho foram verificar os efeitos do ƒÑ-tocoferol na hematopoese murina e os mecanismos intracelulares relacionados a estes efeitos. Para tal, camundongos foram tratados intraperitonealmente com doses de 40 mg/kg/dia de ƒÑ-tocoferol durante 2 semanasem dias intercalados, sendo sacrificados 24 horas apos a ultima dose; condicoes estas que nao causaram toxicidade as celulas da medula ossea. Amostras histologicas dos femures dos animais que receberam o tratamento com ƒÑ-tocoferol apresentaram hiperplasia medular. O tratamento com ƒÑ-tocoferol induziu aumento na porcentagem das celulas progenitoras hematopoeticas (Lin-Sca-1+c-Kit+ e Lin-Sca-1-c-Kit+), assim como o aumento do estado proliferativo destas populacoes (com mais celulas primitivas na fase S/G2/M do ciclo celular), com o consequente aumento da capacidade de formar CFUs de granulocitos e macrofagos. Dentre as distintas populacoes de celulas maduras da medula ossea, houve um favorecimento da linhagem granulocitica/monocitica (Mac-1+Gr-1+) em detrimento das linhagens eritrociticas (Ter119+) e linfociticas (B220+ e CD3+). Como forma de avaliar o mecanismo de acao do ƒÑ-tocoferol, investigou-se tambem a ativacao das proteinas relacionadas com a sinalizacao das celulas hematopoeticas. Assim, observou-se que as populacoes primitivas medulares apresentaram uma menor ativacao da cinase regulada por sinais extracelulares 1/2 (ERK1/2), da proteina cinase C (PKC), do ¡§ativador de transcricao e transdutor de sinal ¡V 5¡§ (STAT-5), mas nao da proteina cinase B/Akt. Tambem foi verificado que a diminuicao do estado fosforilado da ERK1/2 ocorreu desde os primeiros dias de tratamento. Interessante destacar quer o ƒÑ-tocoferol potencializou o efeito da interleucina-3 (IL-3) sobre a ativacao da ativacao da ERK1/2 nas celulas primitivas hematopoeticas. O inibidor da MEK (PD98059) foi capaz de restabelecer as porcentagens normais das linhagens eritrocitica e granulocitica/monocitica, assim como os niveis normais da fosfo- ERK1/2, alem da resposta da ERK1/2 ao estimulo com IL-3. Entretanto, o PD98059 nao restabeleceu as porcentagens normais das celulas primitivas hematopoeticas, nem da linhagem linfocitica. A quantificacao das especies reativas de oxigenio nas diferentes populacoes da medula ossea mostrou que, nas condicoes de tratamento estabelecidas, o ƒÑ-tocoferol nao exerceu funcao proou antioxidante, pois nao houve alteracao significativa dos niveis de especies reativas de oxigenio entre os grupos controle e tratado com ƒÑ-tocoferol. Desta forma, foi mostrada uma nova propriedade do ƒÑ-tocoferol independente de sua acao redox: a inducao de hiperplasia na medula ossea pelo aumento dos progenitores hematopoeticos e favorecimento da diferenciacao destes em granulocitos e macrofagos, pela potencializacao da resposta da ERK1/2 ao estimulo com IL-3. / TEDE / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2015-07-22T20:50:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Introducao: Um dos objetivos da Medicina e superar os danos causados ao organismo por doencas, senilidade e traumas, restabelecendo um equilibrio normo-funcional. Nas perdas teciduais, inumeros autores afirmam que o substituto gideal h e o proprio tecido saudavel, de mesma origem ou o tecido produzido por Engenharia Tecidual (ET). Porem, ainda sao necessarias muitas pesquisas para a utilizacao in vivo. Objetivo: Avaliar tres suplementos hemoderivados, empregados em meios de cultivo, quanto a proliferacao celular e o dano celular de celulas-tronco de origem dentaria. Metodos: Foram realizados cinco experimentos a partir de dentes terceiros molares em desenvolvimento. Apos a digestao enzimatica, as celulas-tronco adultas foram cultivadas em quatro diferentes meios de cultivo. Meio I, isento de suplemento hemoderivado; meio II, suplementado com FBS (heterologo); meio III, suplementado com soro humano homologo; meio IV, suplementado com soro humano autologo. Essas culturas foram analisadas comparativamente quanto a proliferacao celular, submetidas a testes com marcadores Von Kossa e Alizarina Vermelha durante quatro semanas (avaliadas semanalmente) e a cada duas semanas quanto as unidades formadoras de colonias (UFCs). No 28o dia as quatro culturas foram submetidas ao teste do gcometa h, analisando-se possivel dano no DNA celular. Os resultados foram submetidos a analise estatistica de Variancia de Friedman, estabelecendo-se significancia para (p) . a 5%. Resultados: O meio de cultivo IV atingiu uma proliferacao celular superior ao Meio I, demonstrando um resultado significante (p*=0,0074). O meio de cultivo II, mostrou proliferacao superior ao meio I e desenvolvimento semelhante ao meio III, porem nenhum demonstrou significancia em relacao ao meio IV. Os resultados do teste do cometa evidenciaram um menor dano celular nas culturas do meio IV em relacao ao meio II e meio III. As UFCs foram numerosas nos meios IV e III respectivamente, havendo maior indice de mineralizacao no meio IV do que nos meios II e III. Conclusao: O meio de cultivo suplementado com hemoderivado autologo favoreceu significativamente a proliferacao celular. O hemoderivado humano mostrou-se viavel como suplemento nas culturas de celulas-tronco dentarias humanas. / Introduction: One among many aims of medicine is to overcome injuries inflicted to the organism by diseases, aging and trauma, re-establishing the usual functions. About tissues losses, several authors claim that the ideal replacement is the healthy tissue itself, originated from the same source or developed by Tissue Engineering (TE). However, much research is needed before in vivo application. Objective: To evaluate three different kinds of sera supplies used in stem cell culture media, as to cellular proliferation and cellular injuries on dental stem cell. Methods: Five experiments were made utilizing incompletely developed third molar teeth. After enzymatic digestion, the adult stem cells were cultivated in four different kinds of culture media. Medium I, serum free (SF); medium II, supplied with FBS (heterologous serum- HeS); medium III, supplied with homologous human serum (homologous serum- HoS) and medium IV, supplied with autologous human serum (autologous serum . AuHS).These cultures were analyzed comparatively as to cellular proliferation; they were submitted Von Kossa (VK) and Alizarin Red (AR) markers tests for four weeks (checked weekly), and each two weeks checked for Colonies Forming Unities (CFUs). On the 28th day, all four cultures were submitted to comet assay, and were inspected for possible cellular DNA injuries. The results underwent a non-parametric statistical Friedman fs variance test, with significance (p) . 5%. Results: Culture medium IV reached a cellular proliferation rate higher than medium I, showing a significant result (p*=0,0074). Culture medium II presented a superior proliferation result than medium I, and similar to medium III, although neither of them presented significant result when compared to medium IV. The comet assay fs results showed minor cellular DNA injury in the medium IV cultures, when compared to medium II and III cultures. The CFUs were numerous in the media IV and III cultures, respectively, and there was higher mineralization rate in the medium IV than in the media II and III. Conclusion: The culture medium supplied with AuHS significantly improved cellular proliferation. Human sera proved to be a viable supply to human dental stem cell culture. / TEDE / BV UNIFESP: Teses e dissertações

Bien que les plantes et les animaux diffèrent largement par plusieurs aspects, certaines fonctions biologiques sont extrêmement conservées entre ces deux règnes. Au cours du développement d’un organisme, la mise en place d’un organe possédant une forme, une taille et une fonction précise résulte de la coordination de plusieurs processus cellulaires tel que la prolifération et l’expansion cellulaire. Translationally Controlled Tumor Protein (TCTP) est une protéine très conservée chez tous les eucaryotes. Sa mutation entraine une létalité au stade embryonnaire, démontrant son importance dans le développement de l'organisme. De plus, il a été montré que TCTP contrôlait la croissance des organes en régulant la progression du cycle cellulaire et plus particulièrement la transition G1/S chez les plantes et les animaux. Chez les animaux, les voies moléculaires par lesquelles TCTP contrôle la prolifération cellulaire commencent à être de mieux en mieux décrites. En revanche chez les plantes, ces mécanismes restent très peu connus. Afin de comprendre plus précisément comment TCTP contrôle la prolifération cellulaire et le développement chez Arabidopsis thaliana, les intercateurs potentiels de TCTP ont été identifiés. Parmi eux, CSN4, une sous-unité du complexe COP9 Signalosme (CSN) a été trouvée. CSN est connue pour être impliquée dans le contrôle de l’état de neddylation des CULLINES (CUL) et donc influencent l’activité des complexes CULLIN-RING ubiquitine ligases (CRLs). Les CRLs, par leur activité d’ubiquitination, sont connus pour contrôler l’accumulation de certains acteurs clés du cycle cellulaire, tel que les Cyclines ou les Kip Related Proteins. Au cours de ma thèse, j’ai donc étudié l’interaction entre TCTP et CSN4, afin d’évaluer si le complexe CSN pouvait être l’intermédiaire moléculaire entre TCTP et le cycle cellulaire. Via des approches génétique, biochimiques et cellulaires j’ai pu montrer que TCTP interagissait physiquement avec CSN4 dans le cytoplasme. De plus, par la caractérisation phénotypique de plantes et de cultures cellulaires sur- ou sous-exprimant ces deux gènes, j’ai pu mettre en évidence que TCTP et CSN4 interagissaient génétiquement et que ces deux protéines contrôlaient la transition G1/S du cycle cellulaire. Dans le but de comprendre si l’interaction entre ces deux protéines pouvait interférer avec la fonction du complexe CSN, j’ai analysé par une approche biochimique l’état de neddylation de CUL1 dans les lignées transgéniques. Les données démontrent que la perte de fonction de TCTP accroit la fraction déneddylée de CUL1, alors que sa surexpression augmente la fraction de CUL1 neddylée. Ces données suggèrent que l’interaction est fonctionnelle et que TCTP interfère négativement avec la fonction de CSN. Ainsi, j’ai établi un modèle putatif pour expliquer comment TCTP régule la progression du cycle cellulaire via une interférence avec l’activité de deneddylation du CSN, et donc contrôle l’activité des complexes CRLs. Dans la dernière partie de ma thèse, afin de comprendre si le rôle de TCTP est conservé chez les animaux, j'ai par une approche biochimique évaluée la neddylation de CUL1 chez Drosophila melanogaster. Mes données montrent que comme chez Arabidopsis, la fraction déneddylée de CUL1 augmentait dans des larves de drosophile sous-accumulant TCTP, suggérant que ce mécanisme puisse être conservé entre l’Arabette et la drosophile / While plants and animals largely diverge in several major aspects, some biological functions are highly conserved between these kingdoms. During organism development, the correct implementation of organs with unique shape, size and function is the result of coordinated cellular processes as cell proliferation and expansion. Translationally Controlled Tumor Protein (TCTP) is a highly conserved protein among all eukaryotes. TCTP mutation leads to embryo lethality, indicating that it is mandatory for organism development. Moreover, it has been shown that TCTP controls organ growth by regulating the G1/S transition and cell cycle progression both in plants and animals. In animals, the molecular pathways by which TCTP controls cell proliferation are well known. However, in plants, the mechanism implicating TCTP in the control of development and cell cycle is less understood. To better understand how TCTP controls cell proliferation and development in Arabidopsis thaliana, the putative TCTP interactors were identified. Among them, CSN4, a subunit of the COP9 signalosome complex (CSN) known to be involved in the control of CULLINS (CUL) neddylation status and CULLIN-RING ubiquitin ligases (CRLs) activity, was identified. Through their ubiquitination activity, CRL complexes are known to control the accumulation of mains cell cycle regulators as Cyclins or Kip Related Proteins. Thus, during my PhD, I studied the interaction between TCTP and CSN4, in order to evaluate if CSN complex could link TCTP to cell cycle control. I used genetic, cellular and biochemical approaches to demonstrate that TCTP and CSN4 interact in the cytoplasm. Phenotypic characterization of plants and cell cultures down- or overexpressing these genes demonstrated that TCTP and CSN4 interact genetically to control G1/S transition. In order to understand if the interaction between these two proteins could interfere with the CSN complex function, I characterized CUL1 neddylation status in transgenic lines misexpressing TCTP and CSN4. Loss of function of TCTP increases the non-neddylated CUL1 fraction, while overexpression of TCTP increases neddylated CUL1 form. These data show that TCTP interferes with the role of CSN complex in regulating CUL1 neddylation. Accordingly, our data suggest that TCTP controls cell cycle progression via controlling CSN deneddylation activity, and thus influencing CRL activity. In the last part of my PhD, I addressed if this role of TCTP is conserved in animals. I used biochemical approach to evaluate CUL1 neddylation in Drosophila melanogaster downregulated for dTCTP. My data show that Drosophila larvae knockdown for dTCTP also leads to an increase of non-neddylated CUL1 fraction. These data suggest that the mechanism by which TCTP/CSN4 regulate cell cycle, is likely conserved between Arabidopsis and Drosophila

L'ingénierie tissulaire consiste à assembler de façon intelligente des cellules et des matériaux biocompatibles dans le but de créer des tissus artificiels. Pour la construction de tissus en laboratoire, il est indispensable d'élaborer des matériaux qui miment cet environnement. Dans ce cadre, la collaboration entre les scientifiques de différents domaines (matériaux, chimie, biologie, biochimie) s'avère nécessaire. L'ingénierie du muscle squelettique est prometteuse pour remplacer le tissu musculaire endommagé et pour le traitement des maladies du muscle, mais aussi pour les essais pharmaceutiques. Dans ce but, les matériaux avec les propriétés mécaniques et chimiques contrôlées sont requis -- pour l'amplification et la différenciation in vitro de cellules souches musculaires, mais aussi pour l'étude de la myogenèse sur des microenvironnements contrôlés 2D et dans les matrices 3D. Dans ce travail, nous avons utilisé la technique d'assemblage couche par couche (LbL, layer-by-layer) pour deux buts. Le premier a été de développer de nouveaux films biomimétiques possédant des propriétés biochimiques et mécaniques parfaitement contrôlées, pour étudier les interrelations entre ces deux paramètres sur les processus cellulaires. En plus, nous avons associé ces films biomimetiques aux substrats avec la topographie contrôlée, afin de guider la formation du tissu. Dans un second temps, nous avons utilisé la technique LbL pour organiser les cellules en structures 3D. Nous avons ainsi crée des microtissus d'épaisseur contrôlée, qui pourraient être utilisés en tant que modèles de tissus artificiels pour les applications thérapeutiques ou pour les évaluations de médicament en industrie pharmaceutique. / Tissue engineering approach consists in combining cells, engineering and biomaterials to improve the biological functions of damaged tissues or to replace them. Production of “artificial tissues” is still challenging and requires collaboration of scientists from different domains like cell biology, chemistry, materials and polymer science. Skeletal muscle tissue engineering holds promise for the replacement of muscle due to an injury and for the treatment of muscle diseases, such as muscle dystrophies or paralysis, but is also required for pharmaceutical assays. To this end, materials with tunable mechanical and biochemical properties for myoblast expansion and differentiation in vitro, as well as for the studies of myogenesis on controlled 2D microenvironments or in 3D scaffolds, are crucially needed. In this work, we use layer-by-layer (LbL) assemblies for two goals. The first consisted in the development of multifunctional biomimetic thin films for the control of skeletal muscle cell fate on 2D substrates. We use LbL films made of polypeptides, which can be stiffened by chemical cross-linking and can be specifically functionalized by grafting of biomimetic peptides onto their surface. In addition, we combined the peptide-grafted films with substrate microtopography. Such approach is promising for the development or multifunctional materials that combine the different stimuli present in in vivo ECM, among them physical and biochemical cues, but also microtopography. In the second part, we use LbL assemblies for the construction of 3D skeletal muscle microtissues. This allows to rapidly build 3D muscle tissues and is promising for the in vitro construction of physiologically relevant skeletal muscle tissue models.

Submitted by LARISSA MENDES DE LÁZARI null (la_mendes@hotmail.com) on 2016-04-15T18:28:04Z No. of bitstreams: 1 TeseDoutorado_LarissaMendes_2016_Biotecnologia.pdf: 8907863 bytes, checksum: 3f7981847cc9e559ac7b11f4d8bfd245 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-04-18T19:50:29Z (GMT) No. of bitstreams: 1 lazari_lm_dr_araiq_par.pdf: 1543856 bytes, checksum: 1cc6d6cd934ed8b9a35d1931133a26cd (MD5) / Made available in DSpace on 2016-04-18T19:50:29Z (GMT). No. of bitstreams: 1 lazari_lm_dr_araiq_par.pdf: 1543856 bytes, checksum: 1cc6d6cd934ed8b9a35d1931133a26cd (MD5) Previous issue date: 2016-03-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O desenvolvimento de biomateriais que promovam a reparação de tecidos lesionados tem sido objeto de intensa investigação. Em relação à reparação do tecido ósseo, as cerâmicas são materiais muito pesquisados em função de sua ampla possibilidade de uso, inclusive na confecção de pastas cimentícias moldáveis. A sílica mesoporosa apresenta elevada área de superfície específica (~1000 m2.g-1) e tamanho de poros usualmente em torno de 2-30 nm, atraindo atenção para aplicações como importantes carreadores de fármacos e proteínas. O peptídeo de crescimento osteogênico (OGP) é um tetradecapeptídeo endógeno, cuja forma ativa atua como agente anabólico e estimulador hematopoiético, promovendo a diferenciação osteoblástica. Desta forma, o objetivo deste trabalho foi desenvolver um cimento ósseo injetável, reabsorvível e bioativo, com sílica mesoporosa e peptídeo de crescimento osteogênico. O peptídeo foi sintetizado pelo método em fase sólida, purificado por Cromatografia Líquida (HPLC) e caracterizado por Espectrometria de Massas. O material mesoporoso foi sintetizado pela metodologia sol-gel e sua porosidade confirmada por Adsorção-dessorção de N2, Espalhamento de Raios X à Baixo Ângulo e Microscopia Eletrônica de Transmissão. Os cimentos foram preparados a partir de sulfato de cálcio (CaS), fosfato de cálcio (CaP) e aluminato de cálcio (CaAl), sem e com sílica mesoporosa, e analisados quanto suas características físico-químicas. Os experimentos in vitro foram realizados para avaliar o potencial citotóxico e genotóxico dos cimentos em cultura de células de ovário (CHO-K1) e análise da viabilidade celular e formação da matriz mineralizada em células pré-osteosblásticas (MC3T3-E1). O estudo in vivo foi realizado em defeitos ósseos críticos em calvária de ratos e analisado quanto à formação de tecido ósseo, por histomorfometria, e densidade do tecido neoformado, por imagens de raios X. As análises dos cimentos demostraram que a presença de partículas de sílica mesoporosa promoveu diferentes comportamentos físico-químicos quando comparados aos cimentos sem sílica, como maior razão líquido/pó e, consequentemente, maior porosidade e menor resistência mecânica à compressão. Os cimentos CaP e CaAl, sem e com sílica, mostraram bioatividade in vitro quando imersos em solução que simula o fluido corpóreo (Simulated Body Fluid - SBF). No estudo de liberação do peptídeo OGP, incorporado nas partículas de sílica mesoporosa pré-misturada aos cimentos, o cimento CaS apresentou maior velocidade de liberação em relação aos cimentos estudados, com 80% do conteúdo peptídico liberado em 24 horas. Em relação à viabilidade celular, os cimentos CaS, com e sem sílica, não foram citotóxicos, mas os cimentos CaP e CaAl, apresentaram citotoxicidade; todavia esse comportamento não comprometeu a proliferação celular e nos ensaios de avaliação da mutagenicidade, os cimentos não promoveram dano celular significante. Os testes envolvendo células MC3T3-E1 mostraram que a viabilidade celular e a capacidade de formação da matriz mineralizada foram independentes da presença do peptídeo OGP, sendo mais sensível à presença de sílica e ao tempo de tratamento com os meios condicionados. Os resultados do teste in vivo, com os cimentos CaP, com e sem sílica mesoporosa e peptídeo OGP, demostraram que esses se degradaram e promoveram maior formação óssea durante os primeiros 15 dias pós-cirúrgico, com aproximadamente 30% do defeito preenchido por tecido neoformado, assim como maior densidade nas margens dos defeitos quando comparados com o controle. No entanto, a presença do peptídeo OGP foi significante somente nos primeiros 30 dias pós-cirúrgico de análise e não houve diferença estatística com o cimento com sílica e sem peptídeo. Além do mais, não houve diferença entre os grupos experimentais e o controle nos períodos mais tardios de análise. De acordo com os resultados obtidos, conclui-se que, dentre os cimentos estudados, aqueles com partículas de sílica mesoporosa e peptídeo OGP são os mais promissores para o reparo do tecido ósseo, principalmente nos períodos iniciais de cicatrização, devido ao seu potencial osteogênico. / The development of biomaterials that promote repair of injured tissues has been the subject of intense research. Regarding the bone tissue repair, ceramics are one of the most researched biomaterials groups due to its wide possibility of use, including cement pastes with good moldability. The mesoporous silica has high specific surface area (~1000 m2.g-1) and pore size usually around 2-30 nm attracting attention for its applications as drugs and proteins carriers. The osteogenic growth peptide (OGP) is an endogenous tetradecapeptide, whose active form acts as an anabolic agent and hematopoietic stimulator, promoting osteoblast differentiation. Thus, the aim of this study was to develop an injectable bone cement, resorbable and bioactive, mesoporous silica and osteogenic growth peptide. The peptide was synthesized by the solid phase method, purified by High Performande Liquid Chromatography (HPLC) and characterized by Mass Spectrometry. The mesoporous materials were synthesized by sol-gel method and its porosity confirmed by Adsorption-desorption of N2, Small-angle X-ray Scattering and Transmission Electronic Microscopy. Cements were prepared from calcium sulfate (CaS), calcium phosphate (CaP) and calcium aluminate (CaAl), without and with mesoporous silica, and analyzed for its physicochemical characteristics. In vitro experiments were carried out to evaluate the cements cytotoxic and genotoxic potential in CHO-K1 hamster ovary cell line and analysis of the mineralized matrix formation in MC3T3-E1 osteosblastics cell line. The in vivo study was performed in critical defects in rat calvaria, and it has analized as formation of bone tissue by histomorphometry and density of newly formed tissue by X-ray images. The cements analysis have shown that the presence of mesoporous silica particles promoted different physico-chemical behavior when compared to those without silica, such as higher ratio Liquid/Powder, higher porosity and, hence, decreases the mechanical resistance. CaP and CaAl cements showed bioactivity in vitro when immersed in Simulated Body Fluid solution. Concerning OGP liberation, the CaS cement showed the fastest release in the OGP-mesoporous silica-loaded cement release studies, releasing 80% peptide loaded in 24 hours. Regarding cell viability, CaS cements with and without silica, were not cytotoxic, but the CaP and CaAl cement showed cytotoxicity; however this behavior did not affect cell proliferation. And in mutagenicity tests, the cements did not promote significant cell damage. The tests involving MC3T3-E1 cells showed that cell viability and mineralized matrix formation capacity is independent of the OGP peptide presence and it is more sensitive to the presence of silica and the treatment time with the conditioned culture media. The test in vivo, with CaP cements, with and without mesoporous silica and OGP, demonstrated that these cements have degraded and promoted increased bone formation during the first 15 postoperative days, with approximately 30% of the defect filled by newly formed tissue as well as higher density on the defects borders when compared to the control. However, the presence of OGP peptide was significant only during the first 30 days postoperative, but there was no statistical difference with silica cement and without this peptide. Furthermore, there was no difference between experimental groups and the control in the later study periods. According to the results, it is concluded that, among the cements studied, those with mesoporous silica particles and OGP peptide are the most promising for bone tissue repair, especially in the initial stages of healing due to its osteogenic potential. / FAPESP: 2012/21735-6 / CAPES: 0224-13-8

Made available in DSpace on 2014-12-17T14:03:37Z (GMT). No. of bitstreams: 1 MariliaSNS_TESE.pdf: 2303324 bytes, checksum: d055f1a32ee94b3ffe5f389dbf9dffe0 (MD5) Previous issue date: 2014-04-08 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Compounds derived from fungi has been the subject of many studies in order to broaden the knowledge of their bioactive potential. Polysaccharides from Caripia montagnei have been described to possess anti-inflammatory and antioxidant properties. In this study, glucans extracted from Caripia montagnei mushroom were chemically characterized and their effects evaluated at different doses and intervals of treatment. It was also described their action on colonic injury in the model of colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS), and its action on cells of the human colon carcinoma (HT-29). Compounds extracted of C. montagnei contain high level of carbohydrates (96%), low content of phenolic compounds (1.5%) and low contamination with proteins (2.5%). The (FT-IR) and (NMR) analysis showed that polysaccharides from this species of mushroom are composed of &#945;- and &#946;-glucans. The colonic damage was evaluated by macroscopic, histological, biochemical and immunologic analyses. The results showed a reduction of colonic lesions in all groups treated with the glucans of Caripia montagnei (GCM). GCM significantly reduced the levels of IL-6 (50 and 75 mg/kg, p < 0.05), a major inflammatory cytokine. Biochemical analyses showed that such glucans acted on reducing levels of alkaline phosphatase (75 mg/kg, p < 0.01), nitric oxide (p < 0.001), and myeloperoxidase (p < 0.001). These results were confirmed microscopically by the reduction of cellular infiltration. The increase of catalase activity suggest a protective effect of GCM on colonic tissue, confirming their anti-inflammatory potential. GCM displayed cytostatic activity against HT-29 cells, causing accumulation of cells in G1 phase, blocking the cycle cell progression. Those glucans also showed ability to modulate the adhesion of HT-29 cells to Matrigel? and reduced the oxidative stress. The antiproliferative activity against HT-29 cells displayed by GCM (p <0.001) can be attributed to its cytostatic activity and induction of apoptosis by GCM / Compostos derivados de fungos tem sido alvo de muitos estudos a fim de desenvolver o conhecimento acerca de seu potencial bioativo. Polissacar?deos de Caripia montagnei j? foram descritos por possu?rem propriedades anti-inflamat?ria e antioxidante. Neste estudo, os polissacar?deos extra?dos do fungo Caripia montagnei foram caracterizados quimicamente e seus efeitos sobre as les?es intestinais foram avaliados em diferentes intervalos de tratamento no modelo de colite induzida por ?cido 2,4,6 - trinitrobenzenossulf?nico (TNBS), verificou-se ainda sua a??o sobre c?lulas do carcinoma de c?lon humano, HT-29. Na an?lise realizada no extrato obtido de C. montagnei foi verificado que este ? formado principalmente, por carboidratos (96%) apresentando um baixo teor de compostos fen?licos (1,5%) e baixa contamina??o prot?ica (2,5%). As an?lises por espectroscopia de infra vermelho (FT-IR) e resson?ncia magn?tica nuclear (RMN) mostraram que os polissacar?deos desta esp?cie de fungo s?o &#945; e &#946; -glucanas. O dano col?nico foi avaliado por an?lises macrosc?picas, histol?gicas, bioqu?micas e imunol?gicas. Os resultados mostraram a redu??o das les?es no c?lon em todos os grupos tratados com as glucanas (GCM). GCM reduziram significativamente os n?veis de IL-6 (50 e 75 mg/Kg, p < 0,05), uma importante citocina inflamat?ria. As an?lises bioqu?micas mostraram que essas glucanas atuaram na redu??o dos n?veis de fosfatase alcalina (75 mg/Kg, p < 0,01), ?xido n?trico (p < 0,001) e mieloperoxidase (p < 0,001). Estes resultados foram confirmados pela redu??o da infiltra??o celular observado microscopicamente. O aumento da atividade da catalase, sugere um efeito protetor de GCM no tecido do c?lon, o que confirma o seu potencial anti-inflamat?rio. GCM mostraram atividade citost?tica sobre as c?lulas HT-29, causando ac?mulo de c?lulas na fase G1 e impedindo, assim, a progress?o do ciclo celular. As glucanas deste estudo tamb?m mostraram habilidade em modular a ades?o de c?lulas HT-29 ao Matrigel? e reduzir o estresse oxidativo nessas c?lulas. A atividade antiproliferativa contra c?lulas HT-29 exibida por GCM pode ser atribu?da ? sua a??o citost?tica ou indu??o da apoptose por essas glucanas

Os papilomavírus são pequenos vírus de DNA dupla-fita, não envelopados, mucoepiteliotrópicos, capazes de infectar inúmeros vertebrados superiores de maneira espécie-específica. A infecção por estes vírus está associada a uma série de desordens proliferativas que levam desde de a formação de verrugas comuns até a do carcinoma invasivo. Aproximadamente 200 tipos de papilomavírus humano (HPVs) foram identificados, sendo que cerca de 40 deles infectam o trato genital. Dentre estes, os chamados HPVs de alto-risco estão associados etiologicamente ao carcinoma de colo de útero, enquanto que os de baixo-risco estão relacionados às lesões epiteliais benignas. A infecção por HPVs de alto-risco é muito comum, no entanto, a maioria destas é transitória e somente uma pequena proporção de mulheres desenvolvem o carcinoma. Entretanto, algumas mulheres são incapazes de eliminar esta infecção, levando a persistência viral e o conseqüente desenvolvimento da neoplasia. Para que a infecção pelo HPV persista é necessário um mecanismo de escape ao sistema imune do hospedeiro. O mecanismo de escape à resposta imune inata parece ser característico da infecção pelo HPV, pois o ciclo infeccioso deste vírus não promove inflamação. A infecção por HPV promove a liberação de citocinas, tal como o fator de necrose tumoral (TNF). Esta citocina possui um potente efeito citostático em queratinócitos normais e imortalizados com HPV, enquanto que em queratinóctos imortalizados com HPV18 este efeito não é observado. Do mesmo modo, observamos que a expressão do oncogene E6 de HPV16 ou 18 é suficiente para promover resistência ao efeito antiproliferativo do TNF em culturas em monocamada e organotípica. A expressão aumentada e contínua destes ocogenes é sabidamente o principal evento favorável ao desenvolvimento do câncer de colo de útero. Estas proteínas são essenciais na indução da transformação celular, visto que interferem na regulação do ciclo celular e apoptose. O produto dos genes E6 e E7 se liga ao produto dos genes supressores de tumor p53 e pRb, respectivamente, levando a sua degradação pela via de proteólise dependente de ubiquitina. As bases moleculares desta resistência ao TNF ainda são pouco conhecidas. Neste estudo, comparamos o efeito desta citocina em queratinócitos normais e que expressam E6 ou E7. Observamos através de cDNA Microarray a expressão de um grupo de genes, entre eles TCN1, DEK, HMGB2, INHBA, MCM2, MCM5 e MMP9, com expressão diferencial entre as células sensíveis e as resistentes ao TNF. / Papillomaviruses are small, non-enveloped, epitheliotropic, double-stranded DNA viruses that infect mucosal and cutaneous epithelia in a wide variety of higher vertebrates in a species-specific manner. Papillomavirus infections are associated to a series of proliferative disorders that range from common warts to invasive carcinomas. Almost 200 types of human papillomaviruses (HPVs) have been identified and approximately 40 of them infect the genital tract. Only the so-called high-risk HPV types mediate human carcinogenesis, whereas the low-risk HPVs have been linked to benign epithelial lesions. High-risk genital HPV infection is very common, and the majority of individuals clear their infection with time. However, a proportion of women cannot effectively clear the virus, and the persistence of a high-risk HPV is the major risk factor for the development of anogenital malignancies. To persist, HPV must escape the host immune system. Effective evasion of innate immune recognition seems to be the hallmark of HPV infections, since the infectious cycle is one in which viral replication and virion release is not associated with inflammation. Furthermore, HPV infections promote cytokine release, as tumor necrosis factor-alpha (TNF). This cytokine has a potent cytostatic effect on normal and HPV16 immortalized keratinocytes, while it does not affect HPV18 immortalized keratinocytes proliferation. In addition, we have observed that expression of HPV 16 or 18 E7 oncogene is sufficient to overcome TNF antiproliferative effect in monolayer and organotypic cell cultures. The increased and sustained expression of HPV oncogenes, E6 and E7, is the main contributor to the development of cervical cancer. Both E6 and E7 proteins are essential to induce and maintain cellular transformation, due to their interference with cell-cycle and apoptosis regulation. The most manifest function of the E6 protein is to promote the degradation of p53, while E7 is known to bind to and promote the proteasomal degradation of the retinoblastoma tumor suppressor gene product, pRb, and its family members. The molecular basis of TNF resistance is not well understood. In this study we compared the effect of TNF between normal and HPV16 E6 or E7 expressing keratinocytes. We observed by cDNA Microarray the differential expression of a common set of genes in TNF-sensitive cell lines, including TCN1, DEK, HMGB2, INHBA, MCM2, MCM5 and MMP9, that differs from those modulated in TNF-resistant cells.

Nas últimas décadas tem aumentado o interesse pelos enxertos de pele humana radioesterilizadas, para aplicação principalmente em queimaduras extensas e profundas. Isto se deve ao fato destes enxertos apresentarem rápida aderência e menor potencial antigênico, em comparação com os demais tratamentos utilizados. A proposta deste estudo foi avaliar a histoarquitetura do enxerto de pele humana irradiada com doses de 25 kGy, 50 kGy e não irradiada, durante o processo de reparação tecidual, em camundongos Nude submetidos a enxertia de pele na região dorsal. Três grupos de animais receberam enxertos de pele humana irradiada (25 kGy e 50 kGy) e não irradiada e foram eutanasiados no 3º, 7º e 21º dia após a realização da cirurgia. Após os procedimentos histológicos de rotina, as amostras de tecido foram coradas com hematoxilina e eosina (HE) para a quantificação de queratinócitos, fibroblastos, células de defesa e vasos sanguíneos e a reação de imunofluorescência (IF) foi realizada para a determinação da expressão de colágeno do tipo I humano e do colágeno dos tipos I e III de camundongo. A quantificação, tanto das células quanto dos tipos de colágeno foi realizada por análise de imagem, utilizando o programa Image-Pro PLus 6.0. Os resultados histológicos demostraram que a pele humana irradiação, quando enxertada, influencia o aumento do número de células no local de cicatrização ao longo do tempo, principalmente na dose de 25 kGy, além de proporcionar uma melhor dispersão destas células. No 21º dia, os três grupos de animais com enxertia de pele humana tiveram parte do enxerto incorporado no processo de cicatrização. O grupo não irradiado apresentou maior incorporação do enxerto (43%), porém menor produção de colágeno do tipo III de camundongo (22%). Já os grupos com enxertia de pele irradiada apresentaram menor incorporação do enxerto (6 e 15%), mas com maior produção de colágeno do tipo III de camundongo (35% e 28%, para 25 kGy e 50 kGy, respectivamente). Com este estudo pôde-se concluir que o grupo irradiado a 25 kGy, apresenta maior proliferação celular e formação de vasos,além de melhor remodelamento da região de cicatrização. / Over the last few years it has increased the interest in the human skin grafts radio sterilized for application mainly in extensive and deep burns. Because these grafts quickly grip and present antigenic lower potential, compared with other treatments used. The purpose of this study was to evaluate the histoarchitecture of human skin grafts irradiated with doses 25 kGy, 50 kGy and non-irradiated during the pepair tissue process in nude mice submitted by skin grafting in the dorsal region. Three groups of animals received irradiated human skin grafts (25 kGy and 50 kGy) and non-irradiated and were euthanized on the 3rd, 7th and 21th day after the surgery. Indeed, routine histologic procedures, tissue samples were stained with hematoxylin and eosin (HE) for quantification of keratinocytes, fibroblasts, immune cells and blood vessels and immunofluorescence (IF) was performed to determine the expression human collagen type I and collagen type I and III mouse. Therefore, quantification of both the cells and the collagen types was performed by image analysis using Image-Pro Plus 6.0 software. Histologic results demonstrated at a dose of 25 kGy that human skin irradiation when grafted influences the increase in the number of cells in wound site over time and it provides better dispersion of these cells. In addition, on the 21st day, three groups of animals with human skin graft were embedded part of the graft in the healing process. On the other hand, the group not irradiated showed greater incorporation of the graft (43 %), but less production of collagen type III mouse (22 %). Since the groups irradiated skin graft showed lower graft incorporation (6 and 15%), but with greater production of collagen type III mice (35 % and 28 % to 25 kGy and 50 kGy, respectively). In conclusion, this study presented that the group irradiated to 25 kGy and it has a higher cell proliferation and vessel formation, and better remodeling of the healing area.