Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Susan Goodman & Carl Dawson, Dept. of English. Includes bibliographical references.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-12-04T21:15:20Z No. of bitstreams: 1 AngelicaFernandesGurgelDeSousa_DISSERT.pdf: 4154120 bytes, checksum: 9b7e3179ef04b1aafa2fdd770a6124d4 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-12-08T23:03:33Z (GMT) No. of bitstreams: 1 AngelicaFernandesGurgelDeSousa_DISSERT.pdf: 4154120 bytes, checksum: 9b7e3179ef04b1aafa2fdd770a6124d4 (MD5) / Made available in DSpace on 2017-12-08T23:03:33Z (GMT). No. of bitstreams: 1 AngelicaFernandesGurgelDeSousa_DISSERT.pdf: 4154120 bytes, checksum: 9b7e3179ef04b1aafa2fdd770a6124d4 (MD5) Previous issue date: 2017-05-26 / Os problemas relacionados com defeitos ?sseos continuam a motivar a busca de terapias mais eficazes. Assim, a combina??o de biomateriais, c?lulas-tronco e mol?culas bioativas fazem parte das ferramentas usadas pela medicina regenerativa para alcan?ar esse objetivo. Diversos estudos j? mostraram o potencial osteog?nico dos polissacar?deos sulfatados (PSs) extra?dos de macroalgas marinhas. Entre eles, o fucoidan, isolado da alga marrom Fucus vesiculosus, ? o mais estudado, sendo j? comercializado por algumas empresas. As algas verdes tamb?m s?o fonte de PSs, mas estas s?o ainda pouco exploradas para aplica??es na regenera??o ?ssea. Em geral, as aplica??es cl?nicas dos PSs extra?dos de algas ainda s?o limitadas devido ? escassez de estudos sobre os seus efeitos, como por exemplo, o potencial genot?xico ? desconhecido na maioria dos casos. Assim, neste trabalho avaliamos a atividade osteog?nica 1) do Fucoidan de F. vesiculosus (um extrato comercializado pela Sigma), 2) das amostras ricas em PSs obtidas do subfracionamento do fucoidan comercial, e 3) do extrato rico em PSs extra?dos da alga verde Caulerpa sertularioides usando como modelo c?lulas-tronco humanas isoladas da geleia de Wharton de cord?es umbilicais (CTMH-GW). Estudou-se tamb?m o potencial genot?xico dos extratos totais de F. vesiculosus e C. sertularioides e posteriormente, da subfra??o do fucoidan que apresentou maior potencial osteog?nico, utilizando do ensaio de micron?cleo com bloqueio da citocinese (CBMN) na linhagem CHO-K1. Os ensaios de redu??o do MTT evidenciaram que as amostras ricas em PSs n?o apresentaram citotoxicidade significativa ao longo de 72 h, at? 10 ?g.mL-1. Os ensaios de atividade da fosfatase alcalina (ALP) e de mineraliza??o sugerem que as amostras ricas em PSs possuem atividades osteog?nicas diferentes: a subfra??o do fucoidan FUC 0.5 (5 ?g.mL-1) foi a que apresentou melhor resultado, aumentando 124% a atividade da ALP em rela??o ao controle positivo (c?lulas mantidas em meio osteog?nico). J? o extrato total de C. sertularioides (5 ?g.mL-1) aumentou 192% a atividade da ALP. Adicionalmente, todas as amostras testadas induziram o ac?mulo de c?lcio na matriz extracelular. Quanto ? genotoxicidade, os resultados do ensaio CBMN sugerem que, nas condi??es testadas, estas amostras n?o s?o genot?xicas, indicando que podem ser uma alternativa para terapias de regenera??o ?ssea. / Problems related with bone defects continue to motivate the search for more effective therapies. Thus, the combination of biomaterials, stem cells and bioactive molecules is part of the tools used by regenerative medicine to achieve this goal. Several studies have already shown the osteogenic potential of sulfated polysaccharides (SPs) extracted from marine macroalgae. Among them, fucoidan, isolated from brown algae Fucus vesiculosus, is the most studied, and is already commercialized by some companies. Green algae are also a source of SPs, but these are even more unexploited in the scope of bone regeneration. In general, the clinical applications of SPs from algae are very limited, because studies on their effects are scarce, for example, their genotoxic effects are unknown in most cases. Thus, in this work, we evaluated the osteogenic activity of Fucoidan from F. vesiculosus (an extract commercialized by Sigma), 2) SPs-rich samples obtained from subfractionation of commercial fucoidan, and 3) SPs-enriched extract from green algae Caulerpa Sertularioides, using human mesenchymal stem cells isolated from the Wharton jelly of umbilical cords (CTMH-GW) as cell model. It was also studied the genotoxic potential of the total extracts of F. vesiculosus and C. sertularioides, using the cytokinesis block micronucleus assay (CBMN) in the line CHO-K1. The genotoxicity of fucoidan?s subfraction that presented greater osteogenic potential was also evaluated. The MTT reduction assays showed that SPs-enriched samples did not show significant cytotoxicity over 72 h, up to 10 ?g.mL-1. Alkaline phosphatase (ALP) activity and mineralization assays suggest that SPs-enriched samples have different osteogenic activities: fucoidan subfraction FUC 0.5 (5 ?g.mL-1) showed the best result, increasing 124% the ALP activity in relation to the positive control (cells maintained in osteogenic medium). The total extract of C. sertularioides (5 ?g.mL-1) increased the ALP activity by 192%. In addition, all samples tested induced calcium accumulation in the extracellular matrix. Concerning to genotoxicity, CBMN assay results suggest that, under the tested conditions, these samples are not genotoxic, indicating their potential as alternative bone regeneration therapy.

Resumo:o presente trabalho teve por objetivo isolar células mesenquimais da geléia de Wharton do cordão umbilical canino e avaliar, in vitro, o efeito do plasma rico em plaquetas (PRP) e da matriz óssea desmineralizada (MOD) na diferenciação osteogênica destas células na ausência de meio osteoindutor. As células foram isoladas e caracterizadas como células mesenquimais indiferenciadas com fenótipo semelhante a de fibroblastos e fenótipo característico confirmado pela citometria de fluxo com CD44+, CD105+, CD271+, CD34- e CD45-; além da habilidade de diferenciação em adipócitos, condrócitos e osteócitos. Com exceção do grupo controle (células mesenquimais), em todos os grupos, G-PRP (células mesenquimais e PRP), G-MOD (células mesenquimais e MOD) e G-PRP+MOD (células mesenquimais e PRP + MOD), houve diferenciação osteoblástica com produção de matriz óssea mineralizada, confirmada pela coloração vermelho de Alizarina. A dosagem de fosfatase alcalina (FA) aumentou aos 14 dias em todos os grupos, com redução temporal subsequente. Proteínas ósseas foram expressas na reação em cadeia da polimerase em tempo real e puderam confirmar a diferenciação óssea em todos os grupos. A osteopontina teve sua maior expressão no G-PRP aos sete dias, seguido do grupo G-PRP+MOD... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract:This study aimed to isolate MSCs from canine Wharton's jelly umbilical cord and evaluate in vitro the effect of PRP and the MOD in osteogenic differentiation of these cells in the absence of osteogenic medium. Cells were isolated and characterized as undifferentiated mesenchymal cells with a phenotype fibroblasts-like and characteristic phenotype of confirmed by flow cytometry with CD44 +, CD105 +, CD271 +, CD34-and CD45-, plus the ability to differentiate into adipocytes, chondrocytes and osteocytes. Except for the control group (MSCs), in all groups, G-PRP (PRP and MSCs), G-DBM (DBM and MSCs) and G-PRP + DBM (MSCs and PRP + DBM) was differentiation osteoblastic with production of mineralized bone matrix confirmed by Alizarin red staining. The measurement of alkaline phosphatase (AP) increased up to 14 days in all groups. Bone proteins were expressed in the polymerase chain reaction in real time and were able to confirm the differentiation bone in all groups. Osteopontin had its greatest expression in G- PRP to seven days followed by G-PRP + DBM, at other times the G-PRP + DBM had higher expression than the other groups. The osteocalcin and Runx2 were expressed more intensely in the G-PRP + DBM at the end of the experiment. It can be concluded that the PRP and DBM are capable of promoting osteogenesis mesenchymal cells in Wharton's jelly of the umbilical cord with canine, with advantage in the presence of DBM / Orientador: Alexandre Lima de Andrade / Banca: Rita Cássia Menegati Dornelles / Banca: Márcio Mateus Beloti / Doutor

Les cellules stromales/souches mésenchymateuses de la gelée de Wharton humaines (CSM-WJ) représentent une source abondante et intéressante de cellules souches pour des applications en ingénierie cellulaire et tissulaire. Leur origine fœtale leur confère des caractéristiques spécifiques par rapport aux cellules stromales/souches mésenchymateuses isolées à partir de moelle osseuse humaine (CSM-MO). Tout d'abord, le but de ce travail est d'optimiser les conditions de culture des CSM-GW pour leur utilisation clinique ultérieure. Nous nous concentrons sur l'influence de la concentration en oxygène lors de l'expansion en monocouche de P1 à P7 sur plusieurs paramètres permettant de caractériser les CSM. Les résultats obtenus sont comparés à ceux obtenus avec les CSM-MO. Notre travail a montré des différences entre les deux sources cellulaires en termes de prolifération et de différenciation adipocytaire. D’après nos résultats, l'hypoxie, au cours de l'expansion, est un paramètre important à prendre en compte en ce qui concerne la prolifération et le potentiel de différenciation chondrocytaire. L'influence des facteurs obstétricaux sur les caractéristiques des CSM-GW est également explorée. Cette étude se situant également dans le cadre de l’ingénierie tissulaire du cartilage, la seconde phase du projet consiste à induire la différenciation des cellules en chondrocytes en ensemençant ces dernières dans un biomatériau à base d’alginate et d’acide hyaluronique, et sur une cinétique de 28 jours. Les résultats obtenus sont comparés à ceux obtenus avec les CSM-MO. Après 4 semaines de culture, les CSM-GW sont capables de s'adapter à leur environnement et d’exprimer des gènes et des protéines matriciels spécifiques du cartilage tels que le collagène de type 2, qui se trouve plus exprimé après différenciation à partir des CSM-GW qu’à partir de CSM-MO / Mesenchymal Stromal/Stem Cells from human Wharton’s jelly (WJ-MSC) are an abundant and interesting source of stem cells for applications in cell and tissue engineering. Their fetal origin confers specific characteristics compared to Mesenchymal Stromal/Stem Cells isolated from human bone marrow (BM-MSC). First, the aim of this work is to optimize WJ-MSC culture conditions for their subsequent clinical use. We focus on the influence of oxygen concentration during monolayer expansion on several parameters to characterize MSC. The results are compared to those obtained with BM-MSC. Our work distinguishes WJ-MSC from BM-MSC in terms of proliferation and adipogenic differentiation. Considering our results, hypoxia during cell expansion is an important parameter to take into account regarding proliferation potential but also chondrogenic differentiation potential. The influence of obstetric factors on WJ-MSC characteristics is also explored. In cartilage tissue engineering context, the second phase of the project is to induce cell differentiation into chondrocytes by seeding them in Alginate/Hyaluronic Acid hydrogel scaffold, and during 28 days. The results obtained are compared to those obtained with BM-MSC. After 4 weeks of culture, WJ-MSC are able to adapt to their environment and express specific cartilage-Related genes and matrix proteins such as type 2 collagen, which is found more expressed after differentiation fromWJ-MSC, than from BM-MSC

Made available in DSpace on 2015-04-09T12:28:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-08-30Bitstream added on 2015-04-09T12:48:01Z : No. of bitstreams: 1 000814146.pdf: 660875 bytes, checksum: 9336b38c8a05d65357c828a9a2773a9b (MD5) / This study aimed to isolate MSCs from canine Wharton's jelly umbilical cord and evaluate in vitro the effect of PRP and the MOD in osteogenic differentiation of these cells in the absence of osteogenic medium. Cells were isolated and characterized as undifferentiated mesenchymal cells with a phenotype fibroblasts-like and characteristic phenotype of confirmed by flow cytometry with CD44 +, CD105 +, CD271 +, CD34-and CD45-, plus the ability to differentiate into adipocytes, chondrocytes and osteocytes. Except for the control group (MSCs), in all groups, G-PRP (PRP and MSCs), G-DBM (DBM and MSCs) and G-PRP + DBM (MSCs and PRP + DBM) was differentiation osteoblastic with production of mineralized bone matrix confirmed by Alizarin red staining. The measurement of alkaline phosphatase (AP) increased up to 14 days in all groups. Bone proteins were expressed in the polymerase chain reaction in real time and were able to confirm the differentiation bone in all groups. Osteopontin had its greatest expression in G- PRP to seven days followed by G-PRP + DBM, at other times the G-PRP + DBM had higher expression than the other groups. The osteocalcin and Runx2 were expressed more intensely in the G-PRP + DBM at the end of the experiment. It can be concluded that the PRP and DBM are capable of promoting osteogenesis mesenchymal cells in Wharton's jelly of the umbilical cord with canine, with advantage in the presence of DBM

Made available in DSpace on 2014-06-12T15:54:28Z (GMT). No. of bitstreams: 2 arquivo6489_1.pdf: 1208262 bytes, checksum: c1b40f981f11c014c773e3922d544d81 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A caracterização de canais iônicos em células tronco é importante pelo fato destas estruturas atuarem no controle da proliferação e diferenciação destas células. Há poucos trabalhos focados nos canais iônicos presentes nas membranas plasmáticas das células tronco e nenhum deles trata das células tronco mesenquimais da geléia de Wharton do cordão umbilical humano (whMSC). O cordão umbilical é uma fonte importante de células tronco para tão esperada terapia celular. Estes fatos motivaram o estudo dos canais iônicos das whMSC como objetivo maior do presente trabalho. Foi utilizada a técnica de patch-clamp na configuração whole-cell e a atuação de inibidores específicos para identificar os canais iônicos presentes nas whMSC. Os inibidores escolhidos foram: tetraetilamonio (TEA) e 4-aminopiridina (4-AP) como inibidores de canais de potássio dependentes de voltagem e de Ca2+, Glibenclamida (GB) um inibidor de canais de potássio dependentes de ATP, canais aniônicos dependentes de volume celular e CFTR e 5-nitro-2-(3-phenylpropylamino) ácido benzóico (NPPB) um potente bloqueador dos canais de cloreto, mas que também é capaz de bloquear os canais de potássio dependentes de Ca2+ de condutância intermediária. Os resultados mostraram que as correntes iônicas ativadas por voltagem das whMSC podem ser bloqueadas consideravelmente por TEA (10 mM), 4-AP (5 mM) e NPPB (100 μM) indicando a presença dos canais de K+ dependentes de voltagem e de Ca2+ e de canais de Cl- dependentes de voltagem conforme descrito para células tronco de outras origens. Utilizando GB (100 μM) observamos um aumento (estimulação) significativo da corrente. Este é um resultado inédito, pois estudos anteriores demonstram que o GB atua inibindo a corrente iônica transmembrana. Em todos os casos, com todos os inibidores testados, os efeitos foram reversíveis. Estes resultados demonstram que as membranas citoplasmáticas das whMSC possuem canais de K+ dependentes de voltagem e de Ca2+ e canais de Cl- dependentes de voltagem. O surpreendente efeito da glibenclamida indica a presença de canais iônicos desconhecidos nas membranas das whMSC

L’ingénierie du tissu osseux est un domaine qui représente un enjeu majeur dans le cadre de la médecine régénératrice. Trois composants sont généralement décrits dans le cadre de l’ingénierie tissulaire : un biomatériau pour pallier le volume de tissu défectueux, une source de cellules progénitrices qui seront responsables de la synthèse des composants tissulaires ainsi que des facteurs de croissance ou des signaux issus des propriétés physico-chimiques du biomatériau afin de guider la prolifération et la différenciation cellulaire. Le but de cette étude a été de synthétiser des substrats phospho-calciques à l’aide de la technique de pulvérisation simultanée d’espèces réactives et de caractériser les différentes propriétés physico-chimiques des substrats obtenus. Nous avons pu démontrer la possibilité d’inclure des molécules organiques (chitosane et acide hyaluronique) à la phase minérale avec cette technique. Nous avons aussi montré la possibilité de faire varier des propriétés telles la rugosité (entre 300 et 700 nm), l’élasticité (entre 2 à 6 GPa), la composition chimique (phosphate octacalcique ou phosphate dicalcique dihydraté) et la bioactivité (précipitation des phosphates de calcium à la surface des substrats) avec la technique de pulvérisation. Par ailleurs, des cellules souches issues de la gelée de Wharton de cordons ombilicaux humains ont été isolées, puis caractérisées sur le plan génique et protéique. Ces cellules étant candidates pour l’utilisation en ingénierie tissulaire osseuse, nous nous sommes intéressés à plusieurs types de marqueurs dont les marqueurs mésenchymateux et les cytokines immuno-modulatrices.La dernière partie de cette thèse a concerné l’association des cellules souches isolées à partir de la gelée de Wharton aux substrats phospho-calciques obtenus à l’aide de la technique de pulvérisation. Nous avons pu démontrer que les cellules adhéraient sur ces substrats et s’organisaient en structures nodulaires au sein desquelles a été observée une couche de cellules sécrétrices entourant des fibres de collagène, des formations cristallines faîtes de phosphates de calcium et des cellules dont la morphologie rappelait celle des ostéocytes. Des variations dans l’expression de marqueurs ostéoblastiques ont aussi été observées, et ce en l’absence de facteurs solubles ostéogéniques dans le milieu de culture. En conclusion, les substrats phospho-calciques obtenus avec la technique de pulvérisation sont capables d’induire la différenciation de cellules souches issues du cordon ombilical en ostéoblastes. Ce modèle se révèle être prometteur pour la mise en place de thérapies en vue de la régénération du tissu osseux. / Bone tissue engineering is a major issue within regenerative medicine. There are three main components in the field of tissue engineering: a scaffold providing a structure for tissue development, a source of stem cells for tissue formation and growth factors or physical stimuli from the biomaterial to direct growth and differentiation of cells. The purpose of this study was to synthesize calcium phosphate substrates by simultaneous spraying of interacting species and to carry out the physico-chemical characterization of the built substrates. We showed that the spraying technique allows the inclusion of organic molecules such as chitosan and hyaluronic acid. The spraying technique allows several physio-chemical characteristics to be varied, rugosity (300 – 700 nm), elasticity (2 – 6 GPa), chemical composition (octacalcium phosphate or dicalcium phosphate dehydrate), but also studied the bioactivity of the substrates (calcium phosphate from the culture medium precipitates at thesurface of the substrates). In another hand, our aim was to isolate stem cells from human umbilical cords’ Wharton’s Jelly and to carry out their genic and proteic characterization by focusing on mesenchymal markers and immunomodulating cytokines, knowing that these cells are candidates for a use in bone regeneration therapy.The last purpose of our study was to evaluate the potential of Wharton’s jelly stem cells to adhere and proliferate onto the sprayed substrates, and also the formation of nodules. The ultrastructural analysis of nodules formed by Wharton’s jelly stem cells showed a layer of secretory cells surrounding collagen fibers, calcium phosphate crystals and cells with a similar morphology to that of osteocytes. Osteoblastic markers appeared to be regulated in cells cultured without osteogenic supplements. To conclude, sprayed calcium phosphate substrates seem to induce osteoblastic differentiation of Wharton’s jelly stem cells through the substrate’s physico-chemical properties. Our model appears as promising for further bone regenerative therapies.

The House of Mirth's main character, Lily Bart, is charaterized a fated character, incapable of exerting free will. With the help of Lawrence Selden and Gerty Farish, she realizes that, for the most part, she has lived and acted according to what others expect of her.

Le choc septique est actuellement la dixième cause de mortalité à travers le monde à égalité avec les infarctus du myocarde. Sa physiopathologie extrêmement complexe, entrelaçant un état pro-inflammatoire et anti-inflammatoire, rend caduque l’action des thérapeutiques conventionnelles. En ce sens, les recherches s’orientent vers les thérapeutiques innovantes et notamment les cellules souches/stromales mésenchymateuses (CSM). En effet, les études murines ont mis en évidence que les CSM étaient en mesure, notamment par leurs actions paracrines, d’améliorer la survie, la défaillance d’organes mais également la bactériémie de souris soumises à un choc septique. Cependant, les propriétés des CSM varient en fonction du tissu dont elles sont issues et particulièrement selon qu’elles proviennent de tissus fœtaux (cordon ombilical, placenta, liquide amniotique) ou adultes (moelle osseuse, tissu adipeux...). Ainsi, notre premier objectif a été de comparer, dans un modèle murin de choc septique, l’action des CSM issues de la moelle osseuse (MO) à celle des CSM issues de la gelée de Wharton (GW) du cordon ombilical. Cette étude murine a permis de mettre en évidence une action quelque peu différente, entre les CSM-GW et les CSM-MO, sur la physiopathologie du choc septique sans que pour autant, l’une des deux sources de CSM, ne se dégage significativement de l’autre en termes d’efficacité. Cependant, en raison de leur importante capacité de prolifération et de l’accessibilité du tissu source, les CSM-GW apparaissent comme étant nettement plus avantageuses que les CSM-MO. En conséquence, notre deuxième objectif a été d’évaluer l’action des CSM-GW dans un modèle porcin de péritonite afin de se rapprocher un peu plus près de la clinique humaine. Cette étude, menée en double aveugle et en présence continue d’un médecin réanimateur expérimenté, a permis de mettre en évidence que les CSM-GW, produites en grade clinique et utilisées juste après décongélation, étaient en mesure d’améliorer la survie, les paramètres hémodynamiques ainsi que les défaillances d’organes, selon un mécanisme d’action différent de celui rapporté par les études murines / Septic shock, equal to the myocardial infraction, is currently the tenth cause of death in the world. The pathophysiological complexity of this syndrome, with a simultaneous pro and anti- inflammatory state, results in the failure of conventional treatments. In this sense, research is focusing on innovative therapeutics agent, including mesenchymal stem cells (MSC). Indeed, murine studies of septic shock showed that MSC improve organ injuries, bacteremia and survival by notably a paracrine mechanism. However, MSC properties vary according to the source tissue, especially if they are derived from a fetal tissue (Wharton’s jelly (WJ), placenta amniotic fluid) or an adult tissue (bone marrow (BM), adipose tissue...). Our first objective was to compare, in a septic shock murine model, the effect of BM-MSC with that of WJ-MSC. Although some differences were observed, the same efficiency was demonstrated between these two sources. However, WJ-MSC present large advantages in comparison to BM-MSC due to their important proliferation capacities and potential quantities of umbilical cord donation. Consequently, our second objective was to investigate the effect of WJ-MSC administration in a relevant pig model of peritonitis in order to better mimic a clinical approach in humans. This study, conducted in double-blind and in presence of an experimented intensivist, showed that WJ-MSC produced in clinical grade and used immediately after thawing, improve survival, hemodynamic parameters and organ injuries by another action than that described in murine studies

No description available.