As células tronco mesenquimais de tecido adiposo (MSCs-TA) são células progenitoras que residem entre adipócitos e contribuem para o turnover do tecido adiposo. Gangliosídios são glicoensfigolipídios localizados na membrana das células, envolvidos na regulação do crescimento celular, interação de superfície, sinalização transmembrana e diferenciação celular. O gangliosídio neural GD2 foi relatado como um marcador de superfície de células tronco mesenquimais de medula óssea e cordão umbilical, mas existem poucos dados sobre a expressão do GD2 em MSCs-TA indiferenciadas e nas diferenciadas para adipócito ou osteoblasto. Nosso principal objetivo foi estudar a expressão de gangliosídios nas MSCs-TA, em especial o GD2, durante a diferenciação adipogênica e osteogênica. Para isso, as MSCs-TA foram isoladas de lipoaspirado humano, cultivadas e induzidas para diferenciação adipogênica e osteogênica. As análises foram feitas por HPTLC, microscopia confocal, citometria de fluxo e PCR em tempo real. Por HPTLC, as MSCs-TA indiferenciadas e MSCs-TA diferenciadas para adipócitos e osteoblasto mostraram aumento do perfil de gangliosídios complexos. A microscopia confocal evidenciou os gangliosídios GM3, GM1 e GD2 na superfície das células e, por citometria de fluxo, identificamos uma subpopulação de células GD2 positivas nas MSCs-TA e MSCs-TA diferenciadas para adipócito ou osteoblasto. Entretanto, o percentual de células GD2 positivas decresceu com a diferenciação. A expressão do mRNA da GD2 sintase aumentou na diferenciação adipogênica e diminui na diferenciação osteogênica. O GD2 é um substrato para a biosíntese de gangliosídios complexos e o aumento da expressão da GD2 sintase pode estar relacionado com o aumento de gangliosídios complexos que ocorre durante a diferenciação adipogênica. / Mesenchymal Stem Cells from Adipose Tissue (MSCs-TA) are progenitor cells that reside between adipocytes, and contribute to the turnover of adipose tissue. Gangliosides are glycosphingolipids localized in cell membrane, involved in cell growth regulation, surface interaction, transmembrane signaling and differentiation. The neural ganglioside GD2 has been reported as surface marker for MSCs from bone marrow and umbilical cord, but sparse data exist about the expression of GD2 in MSCs-TA and during the differentiation to adipocytes and osteoblast. Our aim was to study the expression of glangliosides, in special of GD2 in MSCs-TA and during the adipogenic and osteogenic differentiation. Thus MSCs-TA were isolated from lipoaspirate, cultured and induced to adipogenic and osteogenic differentiation. Then, we examined the gangliosides expression by HPTLC, confocal microscopy, flow citometry and real-time PCR. By HPTLC, the MSCs-TA and MSCs-TA differentiated into adipocytes and osteoblast demonstrate an increased complex gangliosides profile. The confocal microscopy showed the presence of GM3, GM1, and GD2 on the cell surface. By the flow cytometry, we identified a GD2 positive subpopulation in MSCs-TA and in MSCs-TA differentiated to adipocytes and osteoblast. However, the percentage of GD2 positive cells decreased with the differentiation. The expression of GD2 synthase mRNA increased during the adipogenic differentiation and decreased in osteogenic differentiation. GD2 is a substrate for the complex gangliosides biosynthesis, and the increase in GD2 synthase expression could be related with the increase in complex gangliosides that occurs during the adipogenic differentiation.

Made available in DSpace on 2014-12-17T14:52:48Z (GMT). No. of bitstreams: 1 VanessaD_DISSERT.pdf: 1754024 bytes, checksum: 58967ac36bad99001221e5b5ac07ba66 (MD5) Previous issue date: 2010-02-22 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / This study searches to supply an existing gap in the empirical research about the performance of controllers of Micron and Small Companies (MSCs) in the context of corporative education (EC) through the Method of Support of Decision in Corporative Education (MSDCE). In the context of MCSs, this is a pioneering work, for other works related to this thematic searched in literature are limited to conjecture the possibility of its implantation via cooperation between companies; moreover, they do not apply any type of diagnosis method or viability. The object of study is an association of supermarkets of the city of Natal/RN, composed by 16 stores, of what 14 had been searched. The main objective of this work was to apply the first stage of the MSDCE and to verify the possibility of implanting the Corporative Education in the searched MSCs. As a result, it was obtained the profile of the companies and the validation of the above-mentioned Method. The phase of diagnosis conceived through the implantation of the first stage occurred through visits to the stores, interviews, application of questionnaires and place observation. The first stage - strategical analysis for professional education - was divided in two phases: analysis of the current corporative situation and available identification of the involved difficulties and resources. The implantation of the first stage of the MSDCE in the Association of Supermarkets Parceiros da Economia demanded a mapping of abilities and demonstrated how the education management works, the main difficulties and limitations of the MCSs of the supermerket branch of Natal/RN. Beyond the technological aspects, it was verified that cultural and educational aspects need to be worked to reach efficiency in the process of implantation of a corporative education program / Este estudo busca suprir uma lacuna existente na pesquisa emp?rica sobre a atua??o de dirigentes de Micro e Pequenas Empresas (MPEs) no contexto de Educa??o Corporativa (EC), utilizando para isso, o M?todo de Apoio a Decis?o em Educa??o Corporativa (MADEC). No contexto de MPEs, ? um trabalho pioneiro, pois os demais trabalhos relacionados a essa tem?tica e pesquisados na literatura limitam-se a conjecturar a possibilidade de implanta??o via coopera??o entre empresas; al?m disso, n?o aplicam qualquer tipo de m?todo de diagn?stico ou viabilidade. O objeto de estudo ? uma associa??o de supermercados da cidade de Natal/RN composta por 16 lojas, das quais 14 foram pesquisadas. O principal objetivo desse trabalho foi aplicar a primeira etapa do MADEC e verificar a possibilidade de implanta??o de Educa??o Corporativa nas MPEs pesquisadas. Como resultado, obteve-se o perfil das empresas e a valida??o do M?todo supracitado. A fase de diagn?stico concebida atrav?s da implanta??o da primeira etapa ocorreu atrav?s de visitas realizadas ?s lojas, entrevistas, aplica??o de question?rios e observa??o no local. A primeira etapa - an?lise estrat?gica para educa??o profissional - foi dividida em duas fases: an?lise da situa??o corporativa atual e identifica??o das dificuldades envolvidas e recursos dispon?veis. A implanta??o da primeira etapa do MADEC na Associa??o de Supermercados Parceiros da Economia exigiu um mapeamento de compet?ncias e demonstrou como funciona a gest?o de educa??o, as principais dificuldades e limita??es das MPEs do ramo supermercadista de Natal/RN. Al?m dos aspectos tecnol?gicos, verificou-se que existem aspectos culturais e educacionais que precisam ser trabalhados para alcan?ar efici?ncia no processo de implanta??o de um programa de educa??o corporativa

Mesenchymal stem cells (MSCs) derived from human adipose tissue and umbilical cord (Wharton’s jelly, UCB) represent a useful source of adult stem cells for cellular therapy and tissue engineering. The biggest concern with the use of MSCs therapeutically relates to their isolation and growth/manipulation ex vivo. This study aimed to establish methods for the routine isolation and characterization of MSCs from human tissues. The objectives were (1) to show that MSCs could be isolated from different human tissues, namely adipose tissue, Wharton’s jelly, and UCB; (2) to confirm the MSC phenotypic profile over at least 10 passages; and (3) to show the multilineage differentiation capacity of the isolated cells. The minimal criteria as defined by the International Society for Cellular Therapy (ISCT) were used to determine whether MSCs were successfully isolated from various human tissues. Two different techniques involving enzymatic digestion or explant cultures were utilized, and compared for isolating MSCs from Wharton’s jelly. Umbilical cord blood has been suggested as another source of MSCs. However, we were unable to grow MSCs from UCB. Proliferation kinetics of isolated MSCs revealed that cords, either from digested cords or cord pieces had a mean PDT from passage 1 to 4 that was approximately 3 fold lower than for the ASCs. Mesenchymal stem cells from adipose tissue and Wharton’s jelly expressed the classical MSC phenotype (CD73+, CD90+, CD105+, CD34-, and CD45-). The cells from Wharton’s jelly showed a more uniform MSC profile over passages, with higher levels of marker expression when compared to ASCs. Variability in phenotype was observed in early ASC passages, whereas WJ-MSCs seemed to attain the MSC phenotype as early as passage 0 for both isolation techniques. Low levels of CD34 positive cells remained in the ASCs. Oil red O staining was used for identifying the lipid droplets in adipogenic differentiation cultures. A colorimetric assay as well as image analysis was used to quantify the differentiation. For the cord samples, both assays produced positive results. Histological examination, however, revealed that the cords did not form lipid droplets. The ASCs showed a statistically significantly greater differentiation capacity into adipocytes compared with the cords (pooled digested and pieces data). Alizarin red S staining was used for identifying calcium deposition during matrix mineralization in osteogenic differentiation cultures. No significant differences in osteogenic differentiation were observed between ASCs and WJ-MSCs. Chondrogenic differentiation was observed for both MSC sources by positive staining of glycosaminoglycans using toluidine blue O. The main findings of the study showed that MSCs, according to the ISCT guidelines, were successfully harvested from adipose tissue. However, due to the lack of adipogenic differentiation of WJ-derived cells, they did not meet the ISCT guidelines to be classified as MSCs, and were referred to as MSC-like cells. Regardless of the isolation technique used, Wharton’s jelly yielded cells with similar proliferation capacity, phenotype, and differentiation capacity. This study did, however, reveal that biological differences do exist between stem cells from different sources. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Immunology / unrestricted

No description available.

Acute myeloid leukemia (AML), a blood malignancy resulting in abnormal hematopoiesis, is associated with alterations in the bone marrow environment (BME). Current treatments for this heterogeneous disease, mainly targeting the leukemic cells, are largely unsuccessful for the majority of AML subtypes. By better understanding the mechanisms by which the BME contributes to leukemogenesis, it may be possible to introduce more effective treatments for AML. Mesenchymal stromal/stem cells (MSCs) are essential cellular components of the BME/hematopoietic niche and have been shown to support normal hematopoiesis. As a critical component, they may have several roles in altering the BME, thus providing an excellent model for studying the BME in-vitro. Several studies have characterized AML-derived MSCs (AML-MSCs). However, their exact role in altering BME remains unclear. Here, we investigated the MSCs' potential role in BME alteration by investigating the genetic profiles of previously characterized AMLMSCs (n=29) and healthy donor MSCs (HD-MSCs) (n=8). We identified that among 7565 common genes, 21 genes were significantly differentially expressed in AMLMSCs. The CD248 gene was identified among these significantly upregulated genes in AML/HD-MSCs (n=29). Focusing on AML-MSCs derived from high-risk patients (HR), CD248 protein was investigated and validated using HR AML-MSCs (n=11) and HD-MSCs (n=4). Interestingly, it was highly abundant in HR samples at the intracellular and cell-surface levels. CD248 is an MSC marker and has a biological significance potentially on their function. To better understand its potential role in MSC, CD248 was knocked down (KD) in HD-MSCs using siRNA (siCD248-MSCs). Functional capacity, the ability of HD-MSCs and siCD248-MSCs to differentiate into cell types that form the BME (adipocytes and osteocytes), and their ability to promote the growth of HL60 human leukemia cell line were assessed. Posttransfection functional assessments showed that siCD248-MSCs had a reduced adipogenic but not osteogenic potential via differentiation assays. Quantitative validation of the adipogenesis pathway by qRT-PCR confirmed the reduction. KD CD248 increased SIRT2 expression and potentially led to adipogenesis inhibition. However, co-culture experiments showed no effect of HD-MSCs or siCD248-MSCs on HL60 proliferation. Together these data showed that CD248 is a potential player in adipogenesis, essential to MSC’s functionality. Thus, it could serve as a prognostic marker and target for AML therapy.

No description available.

No description available.

No description available.

Le traitement du cancer à l’aide d’une exposition aux radiations ionisantes (RI) peut mener au développement de plusieurs effets secondaires importants, dont un retard de réparation et de régénération des tissus. Les mécanismes responsables de ces effets demeurent largement inconnus encore aujourd’hui, ce qui a pour effet de limiter le développement d’approches thérapeutiques. À l’aide d’un modèle de guérison de plaie cutanée chez la souris, nous avons cherché à déterminer les mécanismes par lesquels l’exposition aux RI limite la régénération de la peau. Nos résultats démontrent que l’induction de la "stromal-derived growth factor 1α" (SDF-1α), une cytokine normalement surexprimée dans les tissus hypoxiques, est sévèrement diminuée dans les plaies de souris irradiées versus non-irradiées. Ce défaut corrèle avec un retard de guérison des plaies et est encore évident plusieurs mois suivant l’exposition aux RI, suggérant qu’il y a une altération permanente de la capacité de la peau à se réparer. Parce que SDF-1α est secrété principalement par les fibroblastes du derme, nous avons évalué le potentiel des cellules stromales multipotentes (MSCs), qui sont reconnues pour secréter des niveaux élevés de SDF-1α, à accélérer la régénération de la peau chez les souris irradiées. L’injection de MSCs en périphéries des plaies a mené à une accélération remarquable de la guérison de la peau chez les souris exposées aux RI. Les actions des MSCs étaient principalement paracrines, dû au fait que les cellules n’ont pas migré à l’extérieur de leur site d’injection et ne se sont pas différentiées en kératinocytes. L’inhibition spécifique de l’expression de SDF-1α a mené à une réduction drastique de l’efficacité des MSCs à accélérer la fermeture de plaie indiquant que la sécrétion de SDF-1α par les MSCs est largement responsable de leur effet bénéfique. Nous avons découvert aussi qu’un des mécanismes par lequel SDF-1α accélère la guérison de plaie implique l’augmentation de la vascularisation au niveau de la peau blessée. Les résultats présentés dans ce mémoire démontrent collectivement que SDF-1α est une importante cytokine dérégulée au niveau des plaies cutanées irradiées, et que le déclin du potentiel de régénération des tissus qui est observé suivant une exposition au RI peut être renversé, s’il est possible de restaurer le microenvironnement de la blessure avec un support stromal adéquat. / Cancer treatment using ionizing radiation (IR) may lead to significant side effects, like delayed tissue repair and regeneration. The mechanisms mediating these defects remain largely unknown at present, thus limiting the development of therapeutic approaches. Using a wound healing model, we investigate the mechanisms by which IR exposure limits skin regeneration. Our results show that induction of the stromal-derived growth factor 1α (SDF-1α), a cytokine normally overexpressed in hypoxic tissues, is severely impaired in the wounded skin of irradiated, compared to non-irradiated, mice. This defect is correlated with delayed healing, and is evident for several months following exposure to IR, suggesting permanent impairment of skin repair. Because SDF-1α is secreted mainly by dermal fibroblasts, we evaluated the potential of multipotent stromal cells (MSCs), which secrete high levels of SDF-1α, to improve skin regeneration in irradiated mice. Injection of MSCs into the wound margin led to remarkable enhancement of skin healing in mice exposed to IR. The MSC actions were mainly paracrine, as the cells did not migrate away from the injection site or differentiate into keratinocytes. Specific knockdown of SDF-1α expression led to drastically reduced efficiency of MSCs in improving wound closure, indicating that SDF-1α secretion by MSCs is largely responsible for their beneficial action. We also found that one mechanism by which SDF-1α enhances wound closure likely involves increased skin vascularization. Findings presented in this thesis collectively indicate that SDF-1α is an important deregulated cytokine in irradiated wounded skin, and that the decline in tissue regeneration potential following IR can be reversed, given adequate microenvironmental support.

Le traitement du cancer à l’aide d’une exposition aux radiations ionisantes (RI) peut mener au développement de plusieurs effets secondaires importants, dont un retard de réparation et de régénération des tissus. Les mécanismes responsables de ces effets demeurent largement inconnus encore aujourd’hui, ce qui a pour effet de limiter le développement d’approches thérapeutiques. À l’aide d’un modèle de guérison de plaie cutanée chez la souris, nous avons cherché à déterminer les mécanismes par lesquels l’exposition aux RI limite la régénération de la peau. Nos résultats démontrent que l’induction de la "stromal-derived growth factor 1α" (SDF-1α), une cytokine normalement surexprimée dans les tissus hypoxiques, est sévèrement diminuée dans les plaies de souris irradiées versus non-irradiées. Ce défaut corrèle avec un retard de guérison des plaies et est encore évident plusieurs mois suivant l’exposition aux RI, suggérant qu’il y a une altération permanente de la capacité de la peau à se réparer. Parce que SDF-1α est secrété principalement par les fibroblastes du derme, nous avons évalué le potentiel des cellules stromales multipotentes (MSCs), qui sont reconnues pour secréter des niveaux élevés de SDF-1α, à accélérer la régénération de la peau chez les souris irradiées. L’injection de MSCs en périphéries des plaies a mené à une accélération remarquable de la guérison de la peau chez les souris exposées aux RI. Les actions des MSCs étaient principalement paracrines, dû au fait que les cellules n’ont pas migré à l’extérieur de leur site d’injection et ne se sont pas différentiées en kératinocytes. L’inhibition spécifique de l’expression de SDF-1α a mené à une réduction drastique de l’efficacité des MSCs à accélérer la fermeture de plaie indiquant que la sécrétion de SDF-1α par les MSCs est largement responsable de leur effet bénéfique. Nous avons découvert aussi qu’un des mécanismes par lequel SDF-1α accélère la guérison de plaie implique l’augmentation de la vascularisation au niveau de la peau blessée. Les résultats présentés dans ce mémoire démontrent collectivement que SDF-1α est une importante cytokine dérégulée au niveau des plaies cutanées irradiées, et que le déclin du potentiel de régénération des tissus qui est observé suivant une exposition au RI peut être renversé, s’il est possible de restaurer le microenvironnement de la blessure avec un support stromal adéquat. / Cancer treatment using ionizing radiation (IR) may lead to significant side effects, like delayed tissue repair and regeneration. The mechanisms mediating these defects remain largely unknown at present, thus limiting the development of therapeutic approaches. Using a wound healing model, we investigate the mechanisms by which IR exposure limits skin regeneration. Our results show that induction of the stromal-derived growth factor 1α (SDF-1α), a cytokine normally overexpressed in hypoxic tissues, is severely impaired in the wounded skin of irradiated, compared to non-irradiated, mice. This defect is correlated with delayed healing, and is evident for several months following exposure to IR, suggesting permanent impairment of skin repair. Because SDF-1α is secreted mainly by dermal fibroblasts, we evaluated the potential of multipotent stromal cells (MSCs), which secrete high levels of SDF-1α, to improve skin regeneration in irradiated mice. Injection of MSCs into the wound margin led to remarkable enhancement of skin healing in mice exposed to IR. The MSC actions were mainly paracrine, as the cells did not migrate away from the injection site or differentiate into keratinocytes. Specific knockdown of SDF-1α expression led to drastically reduced efficiency of MSCs in improving wound closure, indicating that SDF-1α secretion by MSCs is largely responsible for their beneficial action. We also found that one mechanism by which SDF-1α enhances wound closure likely involves increased skin vascularization. Findings presented in this thesis collectively indicate that SDF-1α is an important deregulated cytokine in irradiated wounded skin, and that the decline in tissue regeneration potential following IR can be reversed, given adequate microenvironmental support.