Existem evidências que o hormônio adrenocorticotrópico (ACTH) tem um papel importante no equilíbrio entre proliferação e morte celular na glândula supra-renal. As proteínas dos genes de resposta primária, proteínas da família Fos e Jun são componentes do fator de transcrição AP1, que dependendo de sua composição, pode estar relacionado com proliferação, diferenciação ou morte celular. Nesse trabalho utilizamos culturas de células primárias de adrenal de ratos para avaliar por imunocitoquímica e por imunoblotting, os efeitos do ACTH e do FGF2, na expressão das proteínas c-Fos, FosB, Fra1, Fra2, c-Jun, JunB e JunD. Os resultados mostram que tratamentos com ACTH e FGF2 modificam o padrão de expressão dessas proteínas. O ACTH induz aumento consistente da expressão de JunB, o que sugere uma composição de AP1 formada por JunB/c-Fos ou FosB. Tratamentos com FGF2, indicam a formação de um complexo c-Jun/c-Fos, FosB e Fra2. Esses resultados estão de acordo com os efeitos biológicos observados da ação do ACTH e do FGF2, como, inibição e proliferação celular nessas células. / There are evidences that in vivo the adrenocorticotropic hormone (ACTH) displays an important role in the balance of proliferation and cellular death in the adrenal gland. The early response gene proteins, Fos and Jun family, are components of the transcription factor AP-1 that, depending on its composition, could be related with proliferation, differentiation or cellular death. In this work we have been used adrenocortex cells of rat primary cultures, to evaluate, by immunocytochemistry and immunoblotting, the effects of ACTH and FGF2, in the expression of c-Fos, FosB, Fra1, Fra2, c-Jun, JunB and JunD proteins, and in such wise as to predict the composition of AP1 complex. The results showed that ACTH and FGF2 treatments modify the expression pattern of these proteins, inducing consistent and expressive increase of JunB expression in the ACTH-treated cells, suggesting an AP1 composition with JunB/c-Fos or FosB. FGF2 treatments indicate the composition of c-Jun/c-Fos or FosB or Fra-2 complexes. These results are in agreement with the biological effects observed in rat adrenal primary culture treated with ACTH and FGF2, with inhibition and cellular proliferation.

Existem evidências que o hormônio adrenocorticotrópico (ACTH) tem um papel importante no equilíbrio entre proliferação e morte celular na glândula supra-renal. As proteínas dos genes de resposta primária, proteínas da família Fos e Jun são componentes do fator de transcrição AP1, que dependendo de sua composição, pode estar relacionado com proliferação, diferenciação ou morte celular. Nesse trabalho utilizamos culturas de células primárias de adrenal de ratos para avaliar por imunocitoquímica e por imunoblotting, os efeitos do ACTH e do FGF2, na expressão das proteínas c-Fos, FosB, Fra1, Fra2, c-Jun, JunB e JunD. Os resultados mostram que tratamentos com ACTH e FGF2 modificam o padrão de expressão dessas proteínas. O ACTH induz aumento consistente da expressão de JunB, o que sugere uma composição de AP1 formada por JunB/c-Fos ou FosB. Tratamentos com FGF2, indicam a formação de um complexo c-Jun/c-Fos, FosB e Fra2. Esses resultados estão de acordo com os efeitos biológicos observados da ação do ACTH e do FGF2, como, inibição e proliferação celular nessas células. / There are evidences that in vivo the adrenocorticotropic hormone (ACTH) displays an important role in the balance of proliferation and cellular death in the adrenal gland. The early response gene proteins, Fos and Jun family, are components of the transcription factor AP-1 that, depending on its composition, could be related with proliferation, differentiation or cellular death. In this work we have been used adrenocortex cells of rat primary cultures, to evaluate, by immunocytochemistry and immunoblotting, the effects of ACTH and FGF2, in the expression of c-Fos, FosB, Fra1, Fra2, c-Jun, JunB and JunD proteins, and in such wise as to predict the composition of AP1 complex. The results showed that ACTH and FGF2 treatments modify the expression pattern of these proteins, inducing consistent and expressive increase of JunB expression in the ACTH-treated cells, suggesting an AP1 composition with JunB/c-Fos or FosB. FGF2 treatments indicate the composition of c-Jun/c-Fos or FosB or Fra-2 complexes. These results are in agreement with the biological effects observed in rat adrenal primary culture treated with ACTH and FGF2, with inhibition and cellular proliferation.

La physiopathologie de l'hypertension artérielle pulmonaire (HTAP) implique de multiples mécanismes. Elle est caractérisée, entre autres, par la réduction de la production des facteurs vasodilatateurs, l'augmentation exagérée des facteurs vasoconstricteurs et des facteurs de croissance qui conduisent à la vasoconstruction, la prolifération et le remodelage vasculaire pulmonaire. Les cellules endothéliales pulmonaires secrètent des facteurs paracrines qui contribuent à l'hyperplasie des cellules musculaires lisses pendant la progression de l'hypertension artérielle pulmonaire. Le FGF2 produit par les cellules endothéliales et stocké dans la matrice extracellulaire, est l'un de ces facteurs très bien documenté pour entraîner une prolifération des CML. Cette étude montre que l'inhibition de l'expression pulmonaire du bFGF par l'injection répétée du SiRNA chez les rats traités à la MCT, est corrélée à l'amélioration des paramètres hémodynamiques, du remodelage vasculaire et de l'hypertrophie cardiaque droite, en traitement préventif et curatif. Chez les sujets atteints d'hypertension pulmonaire le niveau de la sérotonine circulant est très élevé. La biosynthèse de la sérotonine dépend de la tryptophane hydroxylase. Nous avons étudié l'impact des variations génétiques de la Tph1 et de la Tph2 sur le développement de l'HTAP chez les souris. Cette étude a montré que la déficience en Tph1 (Tph périphérique) protège les souris contre l'hypoxie alors que les souches de souris porteuses du polymorphisme C1473G au niveau du gène Tph2 (Tph centrale) montrent des phénotypes d'HTAP différents pendant l'hypoxie. En plus de l'importance de la TPH, l'expression du 5-HTT est aussi déterminant dans l'HTAP. Les souris SM22 surexprimant le 5-HTT dans les CML à un niveau proche de celui des CML des patients atteints d'hypertension artérielle pulmonaire développent spontanément une HTAP qui s'aggrave avec l'âge. Du fait de la baisse considérable de l'activité de ces deux enzymes, le NO synthase et la prostacycline synthase, au niveau des cellules endothéliales chez les patients HTAP, le taux de l'AMPc et du GMPc baisse. Les PDEs hydrolysent les deux messagers des agents vasodilatateurs prostacycline et oxyde nitrique, et l'utilisation des inhibiteurs de phosphodiestérases augmentent la concentration intracellulaire de GMPc et AMPc et causent la vasodilatation pulmonaire. Le traitements des rats HTAP avec l'inhibiteur du PDE4 (Roflumilast) avec deux doses 1,5 et 0,5mg/Kg/jour, a montré une régression de l'HTAP, baisse de l'hypertrophie cardiaque et du remodelage vasculaire des rats traités à la monocrotaline ou mises en hypoxie aigüe (10% O2), chez qui un traitement préventif et curatif a été effectué. / The physiopatholy of pulmonary arterial hypertension (PAH) implies multiple mechanisms. It is characterized by the reduction of the production of vasodilatators factors, the overproduction of vasoconstrictor factors and growth factors which lead to the pulmonary vasoconstriction, and the pulmonary vascular remodeling. The pulmonary endothelial cells release paracrine factors which contribute to the hyperplasy of the smooth muscule cells (SMC) during the progression of PAH. The FGF2 produced by the endothelial cells and stored in the extracellular matrix is reproted to be involved in the SMC proliferation. This study shows that inhibition of the pulmonary expression of FGF2 by the repeated injection of SiRNA in the rats treated with monocrotaline (MCT), is correlated with the improvment of the hemodynamic parameters, vascular remodeling and right ventricular hypertrophy, in both, a preventive and a curative treatment. In pateints with PAH, it has been shown that the circulating level of serotonin is very high as compared to control patients. The biosynthesis of serotonin depends on tryptophan hydroxylase. Therefore, we studied the impact of the genetic variations of Tph1 and Tph2 on the development of PAH in mice. This study showed that deficiency in Tph1 (peripheral Tph) protects mice against the hypoxia-induced PAH severity during hypoxia. In addition to the importance of the Tph, the serotonin transporter (5-HTT) expression is also a determining factor in the development of PAH. Mice overexpressing 5-HTT (SM22-5-HTT+ mice) specifically in SMC develop spontaneously PAH in normoxia, which worsens with age. We observed a significant decrease int he rate of cAMP and cGMP produced in the endothelial cells from patients with PAH, which has been reported to be due to low activities of NO synthase and prostacyclin synthase. Phosphodiesterases (PDE)hydrolize the two messengers (cAMP and cGMP), and the use of specific inhibitors of PDE increases the intracellular concentration of cGMP and cAMP and causes pulmonary vasodilatation. A daily oral administration of Roflumilast (1,5 and 0,5 mg/kg/day) a specific inhibitor of PDE4, was effective in preventing and in reversin the PAH in both experimental model of PAH, the MCT and chronic hypoxia (10%O2,2 weeks)

Current usage of human embryonic stem cells (hES) and induced pluripotent stem cells (iPS) in clinical therapies and personalized medicine are limited as a result of ethical, technical and medical problems that arise from isolation and generation of these cells. Isolation of hES cells faces ethical problems associated with their derivation from human pre-implantation embryos. The most controversial aspect of hES cell isolation targets the generation of autologous hES cell lines which requires the transfer of a somatic-cell nucleus from the patient to an enucleated oocyte. While already established embryonic stem cell lines from IVF embryos can be used in a similar manner, lack of genetic identity can cause therapy rejection from the host, and prevent their use in personalized medicine. Induced pluripotent stem cells on the other hand, are generated from somatic cells that have been reprogrammed in vitro to behave like stem cells. While these cells can potentially be used for personalized medicine without the risk of rejection by the host system, derivation methods prevent their therapeutic use. The most efficient method used to generate iPS cells involves usage of viral particles which can result in viral DNA being integrated in the host cell’s genome and render these cells non-compliant for clinical therapies. Other methods not involving viral particles exist as well, but the reprogramming efficiency is too low and technical problems with generating large enough numbers of cells prevent these methods from being feasible approaches for clinical therapies. Direct reprogramming of a differentiated cell into a developmentally more plastic cell would offer alternatives to applications in regenerative medicine that currently depend on either embryonic stem cells (ES), adult stem cells or iPS cells. We hypothesize that Xenopus laevis egg cytoplasmic extract contains critical factors needed for reprogramming that may allow for non-viral, chemically defined derivation of human induced pluripotent/multipotent cells which can be maintained by addition of exogenous FGF2. In this thesis we investigated a new method for generation of multipotent cells through determining the ability of select fractions of Xenopus laevis egg extract to induce multipotency in already differentiated cells. We were able to identify select fractions from the extract that in combination with exogenously added FGF2 can reprogram and maintain the reprogrammed cells in an undifferentiated state. The findings of this work also determined that Xenopus laevis egg extract mRNA is required for achieving full reprogramming. The body of work presented in this thesis showed the ability of FGF2 isoforms to bind and activate select FGF receptor tyrosine kinases, act as extracellular mitogenic factors to support growth of hES cells in an undifferentiated state as well bind to nuclear DNA and affect expression of endogenous genes. Moreover, we showed that all FGF2 isoforms can induce expression of stem cell specific proteins in human dermal fibroblasts as well as extend lifespan of human dermal fibroblasts in vitro. In this work we identified HECW1, the gene coding for E3 ubiquitin ligase NEDL1, as a novel nuclear target for all FGF2 isoforms and showed that overexpression of recombinant FGF2 isoforms in human dermal fibroblasts can down regulate expression of HECW1 gene.

A leading cause of morbidity and mortality in the developed world is cardiovascular disease (CVD). Like many other disease processes the etiology of CVD has origins in both genetic and environmental factors. These factors affect the development of the heart and vasculature and how they respond to physiological and pathological stress. Abnormal heart development can lead to cardiac pathologies that manifest in a shift from normal cardiac geometry and physiology to what is called pathological cardiac remodeling. Often though, pathological remodeling can result from cardiovascular stress even when heart development is normal. Growth factors are essential mediators of cardiac development and physiology and a good number of clinical and experimental studies have implicated growth factors and their signaling effectors as potential therapeutic targets for pathological cardiac remodeling. Of those is Fibroblast Growth Factor 2 (FGF2) which is a potent inducer of fibroblast and cardiomyocyte proliferation in vitro. FGF2 is made in high molecular weight and low molecular weight isoforms (Hi FGF2 and Lo FGF2, respectively). It has already been demonstrated that, in the context of the heart, FGF2 modulates cardiac hypertrophy, cardiac fibrosis and mediates protection against cardiac injury. However, the isoform specific role of FGF2 in cardiac development, physiology and pathological remodeling has not been disclosed, and in this dissertation I address the hypothesis that FGF2 has isoform-specific function in cardiac physiology and remodeling. To test this hypothesis I used mice that are either deficient in Hi FGF2 (Hi KO) or Lo FGF2 (Lo KO) and subjected them to echocardiographic analysis and isoproterenol (Iso) treatment and compared them to wildtype (WT) cohorts. At baseline echocardiographic measurements, female Lo KO hearts are smaller and present with increased peak E-wave velocity, a diminutive A wave, and shortened mitral-flow deceleration time consistent with a restricted filling pattern and myocardial stiffness. Conversely, male Lo KO hearts present with a lower E wave and a higher A-wave velocity and a prolonged isovolumic-relaxation time consistent with impaired left ventricular (LV) relaxation. Female Hi KO hearts display no significant deviation from WT, while male Hi KO hearts exhibit increased systolic function. Hence, a deficiency in Lo FGF2 results in a shift from normal diastolic parameters and geometric measurements which is gender specific. Conversely, a deficiency in Hi FGF2 produces a phenotype in male hearts only. Histological and gravimetric analysis of Lo KO and Hi KO hearts post-Iso treatment reveals that female Lo KO hearts remain smaller even though their cardiomyocytes are hypertrophied while female Hi KO hearts present with a blunted hypertrophic response indicating a hypoplastic myocardium. Male Lo KO hearts present with an exacerbated fibrotic response and increased alpha-smooth muscle actin protein expression while Hi KO hearts exhibit a resistance to the fibrotic response and an induction of atrial natriuretic factor protein expression. Thus, in female hearts Hi FGF2 mediate cardiac hypertrophy while in male hearts Lo FGF2 and Hi FGF2 display an antithetical role in cardiac fibrosis where Lo FGF2 is protective while Hi FGF2 is damaging. Hence, cardiac remodeling following catecholamine overactivation is modulated by FGF2 in isoform- and gender-specific manners. In conclusion, the results presented here provide novel evidence on the interaction of gender and endogenous FGF2 isoforms as modulators of cardiac development, physiology and remodeling. Lo FGF2 signaling is necessary in the male heart for normal myocardial relaxation and for amelioration of the fibrotic response induced by beta-adrenergic stress, while in female hearts Lo FGF2 is necessary for normal cardiac growth and normal myocardial compliance. Hi FGF2 is necessary only in female hearts for mediating the hypertrophic response. Hence, I demonstrate that Lo FGF2 and Hi FGF2 have non-redundant roles in cardiac physiology and remodeling which are gender-specific.

In our Laboratory of Developmental Biology there was established a long term culture derived from Xenopus tropicalis testes. It contains pre-Sertoli cells mostly. They compose a feeder layer allowing cultivation of stem cells, revealing the morphology of spermatogonial stem cells. This diploma thesis was focused on a preparation of two growth factors, FGF2 (fibroblast growth factor 2) and GDNF (glial cell line-derived neurotrophic factor), with the subsequent characterization of their influence at cell culture mentioned above. Factors were selected on the basis of the microenvironmental niche theory, according which FGF2 and GDNF are the most important factors for spermatogonial stem cells proliferation and self-renewal. FGF2 recombinant factor was gained using the expression plasmid pET-15b. Its characterization in the testicular culture brought surprising result. Even a low concentration of FGF2 factor (2.5ng/ml) caused cell detaching and dying. Similar result was previously shown in differentiating osteoblast culture only. More experiments need to be done to prove if apoptose take place and why do testicular cells act this way. Key words: Xenopus tropicalis, FGF2, GDNF, SSC, pre-Seroli cells

No description available.

No description available.

The human proteome is exponentially derived from a limited number of genes via alternative splicing, where one gene gives rise to multiple proteins. Alternatively spliced gene products, although crucial for normal physiology, are also linked to an increasing number of pathologies. Consequently, a growing focus is currently being placed on elucidating the extrinsic cues and ensuing signaling mechanisms which direct changes in gene splicing to yield functionally distinct proteins. Of note is the dysregulation of the vascular endothelial growth factor (VEGF) receptor, Flt1 and its soluble splice variants, sFlt1_v1 and sFlt1_v2, in the pregnancy-related disorder, preeclampsia. Preeclampsia is characterized by proteinuria and hypertension and is responsible for almost 600,000 maternal and fetal yearly deaths, worldwide. Here, we examined the impact of endothelial mitogens VEGF and FGF2 (fibroblast growth factor 2), both of which are upregulated in preeclampsia, on Flt1 transcript variants in umbilical vein endothelial cells. We tested the hypothesis that VEGF modulates the expression of Flt1 variants via the signaling kinase Akt and its impact on SR proteins. VEGF was observed to induce expression of overall Flt1 mRNA, principally as variants Flt1 and sFlt1_v1. Conversely, FGF2 induced a shift in splicing toward sFlt1_v2 without significant increase in overall Flt1. Based on inhibitor studies, the VEGF and FGF2 signals were transduced via ERK, but with the involvement of different upstream components. We mapped predicted SR protein binding to Flt1 pre-mRNA and identified two candidate proteins, SRSF2 and SRSF3, that may be involved in VEGF- or FGF2-induced Flt1 pre-mRNA splicing. Examination of SRSF2 and SRSF3 relative mRNA expression levels, following inhibition of VEGF- and FGF2-activated kinases, indicates that FGF2 significantly downregulates SRSF3 mRNA levels via PKC-independent activation of ERK. Additionally, our data suggest that FGF2 may impact Flt1 and sFlt1_v1 via SR protein kinases Akt and SRPK, while conversely regulating sFlt1_v2 levels via Clk. We did not find evidence of VEGF-induced Flt1 variant splicing via SR protein kinase activation or SRSF2 and SRSF3 mRNA levels. Thus, VEGF and FGF2 signals were tranduced via related but distinct mechanisms to differentially influence Flt1 pre-mRNA splicing. These findings implicate VEGF and FGF2 and their related intracellular signaling mechanisms in soluble Flt1 regulation. / Ph. D.

O objetivo desta tese é estudar o papel de FGF2 no controle do ciclo celular em células de mamíferos. Nosso principal modelo, a linhagem Y1, é derivada de um tumor funcional de córtex de camundongo que possui o proto-oncogene c-ki-ras amplificado, tendo consequentemente a super-expressão da proteína c-Ki-Ras na forma ativa (c-Ki-Ras-GTP). Células Y1 sincronizadas na interface G0/G1 do ciclo celular são prontamente responsivas a tratamentos de FGF2 (fator de crescimento de fibroblasto-2) sendo este capaz de ativar toda a progressão G0/G1 → S do ciclo celular, mas surpreendentemente, sob estas mesmas condições, FGF2 induz em cultura e in vivo morte celular nesta linhagem, bloqueando o progresso no ciclo após a entrada na fase S. Sob condições induzidas de baixo c-Ki-Ras-GTP, células Y1 respondem à FGF2 com um aumento na proliferação, mostrando que a indução de morte nesta linhagem está diretamente relacionado com os níveis de Ki-Ras-GTP. Além disso, a população de células Y1 é heterogênea, caracterizada por uma maioria de células FGF2-sensíveis e uma minoria de células que são selecionadas positivamente na presença de FGF2. Estas células FGF2-resistentes exibem uma resposta proliferativa a FGF2 e apresentam traços fenotípicos próximos aos observados em uma célula normal, embora o mecanismo de resistência independa da redução dos níveis de Ki-Ras-GTP. Semelhantemente, linhagens normais de fibroblastos 3T3 exibem uma resposta mitogênica a FGF2 que é substituída por uma resposta de morte após sua transformação com o oncogene EJ-ras. Nosso conjunto de resultados associados a uma nova análise dos dados bibliográficos permite-nos sugerir um novo efeito biológico de FGF2: proteção adicional contra o surgimento de tumores originados por oncogene. / The purpose of this work is to study the role of FGF2 (fibroblast growth factor-2) in the cell cycle control of mammalian cells. Our model of study is the lineage Y1, derived from a murine adrenocortical functional tumor, which presents the proto-oncogene c-ki-ras amplified and, as a consequence, exhibits enhanced expression of the c-Ki-Ras protein in its active forms (c-Ki-Ras-GTP). Arrested Y1 cells in the G0/G1 interface of the cell cycle are promptly responsive to FGF2 treatments, responding with progression through G0/G1 → S, but surprisingly, under the same conditions, FGF2 elicits a strong death response in cultured or in vivo cells, blocking the progress in the cell cycle after S phase entry. Under low c-Ki-Ras-GTP conditions, Y1 cells respond to FGF2 with enhanced proliferation, showing that death induction is related to c-Ki-Ras-GTP levels. Moreover, the Y1 population is heterogeneous, with a majority of FGF2-sensitive cells, and a minority of cells that can be positively selected in the presence of FGF2. These FGF2-resistant cells exhibit a proliferative response to FGF2 and phenotypic traits close to those observed in normal cells, even though the mechanisms of resistance are independent of c-Ki-Ras-GTP decrease. Comparable to that, normal lineages 3T3 display a mitogenic response to FGF2 that is substituted by a death response after their transformation with the oncogene EJ-Ras. The collection of our results associated with a review in the bibliography lead us to suggest a new biological effect of FGF2: enhanced protection against tumors originated by oncogenes.