O objetivo deste estudo foi investigar o papel do fator de crescimento derivado de plaquetas-BB (PDGF-BB) na concentração de 300ng/ml na taxa de proliferação e adesão de células derivadas da granulação óssea humana a fragmentos radiculares periodontalmente comprometidos. Na primeira etapa do estudo, foi estabelecida cultura primária de células da granulação óssea de dois pacientes adultos, sistemicamente saudáveis, não fumantes. Após a expansão celular, as células foram caracterizadas para determinação do fenótipo por meio de ensaios de viabilidade celular, MTT, ensaio de atividade de fosfatase alcalina, ensaio de mineralização e caracterização imunohistoquímica por meio de citometria de fluxo (segunda etapa). Na terceira etapa do estudo, os efeitos da adição de PDGF-BB recombinante humano na concentração de 300ng/ml na taxa de proliferação e adesão de células derivadas da granulação óssea a superfícies radiculares periodontalmente comprometidas foram investigados. A taxa de proliferação celular estimulada pelo PDGF-BB (grupo teste) ou pelo meio de cultura (grupo controle) foi investigada por meio de contagem de células viáveis nos frascos de cultura após 1, 3, 5 e 7 dias do cultivo celular. Foram obtidos 30 fragmentos dentários a partir de dentes extraídos por razões periodontais. Os fragmentos foram raspados com curetas Gracey e condicionados com solução em gel de EDTA a 24% durante 3 minutos, lavados com solução de soro fisiológico, secos e posicionados em placas de 24 poços. Foram incubadas sobre os fragmentos tratados 1x104 células GO por 24 horas, seguido por fixação e preparo para análise por microscopia eletrônica de varredura (MEV). O número de células aderidas sobre os fragmentos foi analisado nas fotomicrografias. O padrão de crescimento das células GO foi compatível com células ósseas, com modificação do padrão do crescimento com o aumento do número de passagens. Houve atividade de fosfatase alcalina em meio osteogênico e convencional, com pico máximo aos 7 dias e atividade de mineralização estimulada ou não por meio osteogênico, com pico máximo aos 21 dias. A análise por meio de citometria de fluxo demonstrou que as células GO não expressaram CD105 e CD166 na 14a passagem, indicando sua diferenciação celular avançada nesse período. A adição de rhPDGF-BB resultou em mudança na taxa de proliferação celular, observando-se pico máximo de crescimento aos 7 dias, com diferenças estatisticamente significantes (p < 0.005; ANOVA post hoc Tukey) em relação aos períodos de 1, 3 e 5 dias. O ensaio de MTT demonstrou maior viabilidade celular no período de 48 hs, comparativamente aos períodos de 24 e 72 horas, quando a densidade óptica celular diminuiu de forma significativa (p< 0.05; Friedmann pósteste Dunn). No ensaio de adesão celular, pode-se observar que a adição de rhPDGFBB aumentou significativamente o número de células aderidas aos fragmentos dentários (p< 0.05; teste t não pareado com correção Welch), com alteração da morfologia celular. Esses resultados sugerem que as células GO tem características compatíveis com linhagem de células osteoblásticas, de fenótipo mais diferenciado após a 12a passagem. A adição de rhPDGF-BB (300ng/ml) resulta em aumento da taxa de proliferação das células GO e do número de células aderidas a fragmentos radiculares, indicando que, nesta concentração, o fator de crescimento é citocompatível, favorecendo a proliferação e adesão celular. / The goal of this study was to investigate the effects of recombinant human platelet derived growth factor (rhPDGF-BB) at the concentration of 300ng/ml in the proliferation and adhesion of human bone granulation cells to periodontally diseased root fragments. At the first stage of the study, the granulation tissue existent in healing sockets (21 days after its creation) was collected from two systemically healthy nonsmoking adults to the establishment of primary culture. The in vitro properties of bone granulation (BG) cell lineage were characterized by cell viability, MTT, alkaline phosphatase activity and mineralization assays. The effects of culture medium (control) and rhPGDF-BB 300ng/ml (test) in the proliferation and adhesion of BG cells were investigated. The rate of BG cells proliferation was investigated by the number of viable cells present at 1, 3, 5 and 7 days after platting. Thirty root fragments were obtained from teeth extracted for periodontal reasons. Root fragments were scaled and root planed, conditioned with EDTA 24% for 3 minutes, rinsed in saline solution, air-dryed and positioned in 24-well plates. Each fragment was seeded with 104 BG cells, fixated after 24 hours and prepared for analysis in SEM. The number of cells adhered to the fragments was analysed in photomicrographies. BG cells growth pattern was compatible with osteogenic cell lineage, showing modification with the increasing number of cell passage. GO cells expressed alkaline phosphatase activity in conventional and osteogenic culture medium, with maximum peak at 7 days, as well as mineralization activity stimulated or not by osteogenic or non-osteogenic culture medium, with maximum peak at 21 days. The analysis by flow cytometer showed that BG cells have not expressed CD105 and CD106 at the 14th passage, indicating its advanced cell differentiation. The addition of rhPDGF-BB resulted in modification of proliferation rate, with maximum peak observed at 7 days, significantly different from 1-, 3- and 5-day periods (p< 0.005; ANOVA post hoc Tukey). MTT assay showed greater cell viability after 48 hours than after 24 and 72 hours, when optical density has significantly diminished (p< 0.05; Friedmann post hoc Dunn). At cell adhesion assay, it could be observed that the adhesion of rhPDGF-BB has significantly increased the number of cells adhered to root fragments (p< 0.05; unpaired t test with Welchs correction), and alterations in cell morphology. These results suggest that BG cells present in vitro characteristics compatible with osteoblastic cell lineages, with a more differentiated phenotype after the 12th passage. The addition of rhPDGF-BB (300 ng/ml) results in increase of the rate of BG cell proliferation and in the number of cells adhered to root fragments, indicating that, at this concentration, the growth factor is compatible with BG cells and favors cells proliferation and adhesion.

Mulheres com risco de falência ovariana prematura, assim como aquelas diagnosticadas com câncer que desejam preservar sua fertilidade, têm como opção a criopreservação do tecido ovariano. Esse tecido seria destinado, dependendo do caso, ao reimplante posterior ou para o cultivo in vitro de folículos ovarianos isolados do tecido criopreservado. Nesse contexto, os folículos primordiais são uma população importante tanto por serem mais resistentes ao processo de criopreservação, como por representarem cerca de 90% da população total folicular. Porém o uso destes folículos para procedimentos de Reprodução Assistida ainda é bastante limitado, pois os mecanismos responsáveis pelo seu processo de ativação ainda não são completamente conhecidos. A via de sinalização fosfatidilinositol 3- quinase (PI3K) foi recentemente identificada como determinante para o controle da ativação dos folículos primordiais. Portanto o objetivo deste estudo foi identificar e localizar os fatores componentes da via: supressores (FOXO3a e PTEN) e ativadores (Akt e Phospho-Akt). O que ofereceria uma valiosa ferramenta para elucidar os mecanismos envolvidos na ativação do pool de reserva folicular e permitiria o desenvolvimento de sistemas de cultivo folicular que atuassem diretamente nestes mecanismos. Sendo assim, foi realizado um estudo transversal com amostras de tecido ovariano humano, que foram submetidas à reação imunohistoquímica dos fatores previamente citados. Foram incluídas na casuística 40 pacientes, com idade média de 27,7 anos ± 7,26. Foi realizada uma análise comparativa da expressão dessas proteínas entre os folículos primordiais e primários. Foi encontrada diferença significativa para a proteína Akt, (p<0,05) em que os folículos primordiais (oócito e células da granulosa) manifestaram mais expressão da proteína Akt em comparação aos folículos primários. Também foi encontrada diferença significativa para a proteína phospho-Akt, porém apenas nas células da granulosa, em que houve maior expressão em folículos primordiais comparados aos primários. Enquanto ambos os estágios tiveram marcação negativa para o PTEN e FOXO3a na maioria dos folículos analisados. Sendo assim, neste estudo não foi possível identificar dentre as proteínas escolhidas uma que tivesse expressão claramente característica de uma ou de outra fase folicular, não sendo possível inferir que a atividade de qualquer uma das proteínas fosse estritamente ligada à ativação dos folículo primordiais. / Women at risk of premature ovarian failure, as well as those diagnosed with cancer who wish to preserve their fertility, have, as option, the ovarian tissue cryopreservation. This tissue would be destined, depending on the case, to posterior reimplantation, or for the in vitro culture of ovarian follicles isolated from the cryopreserved tissue. In this context, primordial follicles are an important population of cells. As they are more resistant to the cryopreservation process and they represent about 90% of the whole follicular population. However, the use of these follicles for Assisted Reproduction procedures is still quite limited, since the mechanisms responsible for its activation process are not fully understood. The phosphatidylinositol 3-kinase (PI3K) signaling pathway has recently been identified as determinant for the control of primordial follicle activation. Therefore, the aim of this study was to identify and localize the components of this pathway: suppressors (FOXO3a and PTEN) and activators (Akt and phospho-Akt). This would offer a valuable tool to elucidate the mechanisms involved in the activation of the follicular reserve pool and would allow the development of in vitro culture protocols that would act directly in these mechanisms. Thus, a cross-sectional study with samples of human ovarian tissue was performed. These samples were submitted to the immunohistochemical reaction of the previously mentioned factors. Forty patients were included in the study, with a mean age of 27.7 ± 7.26. A comparative analysis of the expression of these proteins was performed between primordial and primary follicles. A significant difference was found for the Akt protein (p<0.05) in which the primordial follicles (oocyte and granulosa cells) showed more Akt expression than primary follicles. Another significant difference was found for the phosphor-Akt protein, but only for the granulosa cells, where there was a greater expression in primordial follicles compared to the primary ones. While both stages were negatively stained for PTEN and FOXO3a in most of the follicles analyzed. Thus, in this study it was not possible to identify among the selected proteins one that had clearly characteristic expression of one or the other follicular phase, and it was not possible to infer that the activity of any of the proteins was strictly linked to the activation of the primordial follicles.

No description available.

Résumé : Bien que l’hypoxie soit un puissant inducteur de l’angiogenèse, l’activation des facteurs de croissance est perturbée en hyperglycémie au niveau du pied et du cœur. Cette perturbation entraîne la perte de prolifération et de migration chez les cellules endothéliales, musculaires lisses vasculaires et péricytes empêchant la formation de nouveaux vaisseaux qui mènera à l’amputation des membres inférieurs chez les patients diabétiques. Une étude a démontré qu’une augmentation de la protéine tyrosine phosphatase Src homology-2 domain-containing phosphatase-1 (SHP-1) en condition hyperglycémique chez les péricytes entraînait l’inhibition de la signalisation du PDGF-BB, ce qui résultait en le développement d’une rétinopathie diabétique. Nous avons alors soulevé l’hypothèse que l’expression de SHP-1 dans les cellules musculaires lisses vasculaires affecte la prolifération et la migration cellulaire par l’inhibition de la signalisation de l’insuline et du PDGF-BB en condition diabétique. Nos expérimentations ont été effectuées principalement à l’aide d’une culture primaire de cellules musculaires lisses primaires provenant d’aortes bovines. Comparativement aux concentrations normales de glucose (NG : 5,6 mM), l’exposition à des concentrations élevées de glucose (HG : 25 mM) pendant 48 h a résulté en l’inhibition de la prolifération cellulaire par l’insuline et le PDGF-BB autant en normoxie (20% O2) qu’en hypoxie (24 dernières heures à 1% O2). Lors des essais de migration cellulaire, aucun effet de l’insuline n’a été observé alors que la migration par le PDGF-BB fut inhibée en HG autant en normoxie qu’en hypoxie. L’exposition en HG à mener à l’inhibition de la signalisation de la voie PI3K/Akt de l’insuline et du PDGF-BB en hypoxie. Aucune variation de l’expression de SHP-1 n’a été observée mais son activité phosphatase en hypoxie était fortement inhibée en NG contrairement en HG où on observait une augmentation de cette activité. Finalement, une association a été constatée entre SHP-1 et la sous-unité bêta du récepteur au PDGF. En conclusion, nous avons démontré que l’augmentation de l’activité phosphatase de SHP-1 en hypoxie cause l’inhibition des voies de l’insuline et du PDGF-BB réduisant les processus angiogéniques des cellules musculaires lisses vasculaires dans la maladie des artères périphériques. / Abstract : Even though hypoxia is a strong angiogenic inducer, pro-angiogenic factor signaling pathways in peripheral limb and heart are altered by hyperglycemia. This disruption leads to loss of endothelial cells, vascular smooth muscle cells and pericytes proliferation and migration preventing new blood vessel formation which results in amputation of lower extremities in diabetic patients. A study has shown that increase expression of the protein tyrosine phosphatase Src homology-2 domain-containing phosphatase-1 (SHP-1) in hyperglycemic condition in pericytes caused PDGF-BB signaling inhibition resulting in the development of diabetic retinopathy. Our hypothesis is that SHP-1 expression in vascular smooth muscle cells inhibits cell proliferation and migration induced by insulin and PDGF-BB in diabetic condition. Our experiments were performed using primary culture of vascular smooth muscle cells (SMC) from bovine aortas. As compared to normal glucose concentrations (NG:5,6 mM), high glucose level (HG: 25 mM) exposure for 48h inhibited SMC proliferation induced by insulin and PDGF-BB in both normoxia (20% O2) or hypoxia (1% O2 for the last 24h). During cell migration assays, no effect of insulin was observed while PDGF-BB action of SMC migration was reduced in HG in both normal and low oxygen concentrations. HG exposure lead to inhibition of insulin- and PDGF-BB-stimulated PI3K/Akt signaling pathway in hypoxia. No variation of SHP-1 expression was observed in HG condition. However, SHP-1 phosphatase activity was elevated in HG condition during hypoxia as compared to NG concentrations. Finally, our data showed an association between SHP-1 and the PDGF receptor beta subunit. In conclusion, our results demonstrated that the increase of SHP-1 phosphatase activity in hyperglycemia and hypoxia environment caused inhibition of insulin and PDGF-BB signaling pathways reducing angiogenic processes in vascular smooth muscle cells contributing to peripheral arterial disease in diabetes.

<p>Glioblastoma multiforme (GBM) is the most common form of primary brain tumor in humans. Its aggressive and infiltrative growth into the brain, and, at best, only partial sensitivity to radiotherapy and chemotherapy, renders it extremely difficult to treat and survival remains dismal. </p><p>Growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), and their corresponding receptors are seen in glioma tissue, suggesting the presence of autocrine stimulatory loops. <i>PDGFB</i> and a mutated EGF receptor were also identified as cellular homologues of two oncoviruses, thereby indicating a role in tumorigenesis. This thesis presents a brain tumor model in mice, developed using a <i>PDGFB</i> coding retrovirus to induce overexpression of PDGF-B in neonatal mouse brain. Immature tumors, with histological characteristics of primary brain tumors developed at relatively high frequencies. Mice injected with a non-coding retrovirus did not develop tumors, indicating the crucial role of PDGF stimulation in this system. Tumor cells were also shown to continue to depend on PDGF stimulation when cultured <i>in vitro</i>. </p><p>In human glioblastomas, growth factor receptor signaling is present in conjunction with defects in cell cycle arrest pathways. When the <i>PDGFB</i>-virus model was used with <i>p53</i> or <i>Ink4a-Arf</i> deficient mice, tumors arose with shorter latency and higher frequency. Loss of <i>p53</i> or <i>Ink4a-Arf</i> seemed to facilitate signaling through the PI3K/Akt pathway. Thus, a functional role for the co-existence of p53 loss of function and PDGF signaling in a subset of gliomas is presented. </p><p>Human GBM samples were collected and analyzed with respect to expression and activation of the EGFR and PDGFRα. Most tumors expressed the both receptors at moderate to high levels, but high activation of either receptor seemed mutually exclusive.</p>

Glioblastoma multiforme (GBM) is the most common form of primary brain tumor in humans. Its aggressive and infiltrative growth into the brain, and, at best, only partial sensitivity to radiotherapy and chemotherapy, renders it extremely difficult to treat and survival remains dismal. Growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), and their corresponding receptors are seen in glioma tissue, suggesting the presence of autocrine stimulatory loops. PDGFB and a mutated EGF receptor were also identified as cellular homologues of two oncoviruses, thereby indicating a role in tumorigenesis. This thesis presents a brain tumor model in mice, developed using a PDGFB coding retrovirus to induce overexpression of PDGF-B in neonatal mouse brain. Immature tumors, with histological characteristics of primary brain tumors developed at relatively high frequencies. Mice injected with a non-coding retrovirus did not develop tumors, indicating the crucial role of PDGF stimulation in this system. Tumor cells were also shown to continue to depend on PDGF stimulation when cultured in vitro. In human glioblastomas, growth factor receptor signaling is present in conjunction with defects in cell cycle arrest pathways. When the PDGFB-virus model was used with p53 or Ink4a-Arf deficient mice, tumors arose with shorter latency and higher frequency. Loss of p53 or Ink4a-Arf seemed to facilitate signaling through the PI3K/Akt pathway. Thus, a functional role for the co-existence of p53 loss of function and PDGF signaling in a subset of gliomas is presented. Human GBM samples were collected and analyzed with respect to expression and activation of the EGFR and PDGFRα. Most tumors expressed the both receptors at moderate to high levels, but high activation of either receptor seemed mutually exclusive.

No description available.

This thesis is focused on neural stem cell (NSC) and glioma biology. I discuss how NSCs interact with extracellular matrix (ECM) proteins in the stem cell niche, and investigate the consequences of deregulated Platelet-derived growth factor (PDGF) signaling for embryonic NSCs in transgenic mice. Furthermore I present cell cultures of human glioblastoma multiforme (GBM) that models human disease, taking into account the heterogeneity of GBM. Finally, interactions between brain tumors and mast cells are studied using the glioma cultures. In paper I, the importance of NSC interactions with the ECM in the stem cell niche during development is discussed. Contacts between NSCs and the ECM in the subventricular zone (SVZ) are emerging as important regulatory mechanisms. We show that early postnatal neural stem and progenitor cells (NSPC) attach to collagen I, and that the adhesion is explained by higher expression of collagen receptor integrins compared to adult NSPC. Further, blood vessels in the SVZ express collagen I, indicating a possible functional relationship. Growth factors, e.g. PDGF, regulate NSC proliferation and differentiation. Aberrant activation of growth factor signaling pathways also plays a role in brain tumor formation. Paper II demonstrates that transgenic mice expressing PDGF-B at high levels in embryonic NSCs displayed mild neurological defects but no hyperplasia or brain tumors. This suggests that a high level of PDGF is not sufficient to induce brain tumors from NSCs without further mutations. Paper III presents a novel panel of human glioma stem cell (GSC) lines from GBM that display NSC markers in vitro and form secondary orthotopic tumors in vivo. GBM has recently been categorized in molecular subclasses and we demonstrate, for the first time, that these subclasses can be retained in vitro by stem cell culture conditions. We have thus generated models for research and drug development aiming at a focused treatment depending on GBM subtype. Interactions with the immune system are integral parts of tumorigenesis. Mast cells are found in glioma and in paper IV we demonstrate that the grade-dependent infiltration of mast cells is in part mediated by macrophage migration inhibitory factor and phosphorylation of STAT5.

No description available.

Glioma is the most frequent primary tumor of the central nervous system. By using the RCAS/tv-a mouse glioma model, we have studied mechanisms controlling glioma development and the effect of cell of origin on these processes. SOX5 was identified as a brain tumor locus in a retroviral insertional mutagenesis screen of PDGF-B induced mouse gliomas. Here we found that SOX5 could suppress PDGFB-induced glioma development particularly in Ink4a-/- mice. Analysis of putative PDGF-B signaling pathways revealed that the underlying mechanism could involve the activation of AKT and p27, which caused an acute cellular senescence. When cultured in a highly selective serum free medium, glioma-initiating cells could be isolated from mouse GBMs and their self-renewal and proliferation was independent on exogenous EGF and FGF2. Addition of serum into the medium induced aberrant differentiation that was reversible. Specific depletion of viral PDGF-B demonstrated that PDGF-B was necessary for stemness and tumorigenicity of GICs by preventing them to differentiate. Subsequently, by applying the same culture conditions, GICs of APC, NSC and OPC origins were isolated from mouse GBMs. GICs derived from NSCs exhibited higher self-renewal, faster proliferation and more potent tumorigenicity than those of APC or OPC origin. Furthermore, addition of 5% serum significantly inhibited the proliferation of APC- and OPC-derived GICs, but did not in NSC-derived GICs. Transcriptome analysis revealed that GICs of the same cell of origin displayed distinct expression profiles. In the last study, we showed that OPCs could serve as the origin for astrocytic glioma. Results from immunostainings revealed that these tumors might belong to a different molecular subtype than the oligodendroglial tumors induced in OPCs. We also found differences in tumorigenic potential between OPCs in neonatal and adult mice, which suggest that developmental age of the cell of origin is important for its susceptibility to oncogenic transformation.