Orientador: Carmen Verissima Ferreira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T15:16:22Z (GMT). No. of bitstreams: 1 ChavesNeto_AntonioHernandes_D.pdf: 8240508 bytes, checksum: 293949f75ca619cdc0fa61d29d1703ba (MD5) Previous issue date: 2009 / Resumo: Riboflavina (Rb - Vitamina B2) é o precursor das flavocoenzimas essenciais flavina mononucleotídeo (FMN) e flavina adenina dinucleotídeo (FAD). Estas coenzimas participam de processos enzimáticos dependentes das reações de transferências de elétrons, que ocorrem nas vias de produção de energia, biossíntese, desintoxicação e sequestro de elétrons. O aumento dietético da riboflavina e piridoxina foi associado com maiores densidades minerais em mulheres e homens idosos. Fotoderivados da riboflavina demonstraram efeitos citotóxicos em células cancerosas de próstata e leucemias, entretanto, o efeito direto da Rb e seus fotoderivados em osteoblastos não foram examinados. Neste trabalho os efeitos biológicos da Rb e riboflavina irradiada (IRb) foram investigados na linhagem de pré-osteoblastos MC3T3-E1, um modelo bem aceito de osteogênese in vitro caracterizado pela indução de genes específicos associados com o fenótipo osteoblástico quando tratados com ácido ascórbico e ß-glicerofosfato. A viabilidade celular foi avaliada através da redução do MTT, da incorporação do corante vermelho neutro e do conteúdo de ácidos nucléicos. Marcadores de diferenciação osteoblástica foram analisados através do RT-PCR semi-quantitativo (osteopontina e osteocalcina) e através de análises colorimétricas de atividade da fosfatase alcalina (FAL) e síntese de colágeno pela coloração de picrosirius. As atividades das metaloproteinases (MMP) -9 e -2 foram avaliadas pela zimografia de gelatina. Microarranjos de peptídeos com subtratos específicos para quinases e imunoblotting foram usados para identificar os efeitos na sinalização celular. As atividades de enzimas antioxidantes (superóxido dismutase, catalase, glutationa peroxidase e glutationa S-transferase) foram determinadas em lisados celulares usando métodos espectrofotométricos. As atividades das caspases-8, -9 e -3 foram analisadas através de métodos colorimétricos. Na primeira análise Rb e IRb causaram a parada do ciclo celular na fase G0/G1 e também a inibição da quinase AKT, um mediador da proliferação. Flavinas causaram a diferenciação de pré-osteoblastos, evidenciada pelo aumento da expressão de osteocalcina, osteopontina e BMP-2. Atividades mais elevadas de MMP-9 e MMP-2 também foram observadas. A capacidade das flavinas em engatilhar a diferenciação de osteoblastos foi reforçada pelo aumento da conexina 43, diminuição da caveolina-1 e repressão da sinalização Notch. Na segunda análise, nós encontramos que as interações entre Rb, em sua forma irradiada e não-irradiada, e indutores osteogênicos (ácido ascórbico e ß-glicerofosfato) afetaram significativamente a proliferação de osteoblastos, a atividade de FAL, biossíntese de colágeno, expressão de osteocalcina e osteopontina, a atividade das MMP-2 e MMP-9 e a expressão de fatores osteoclastogênicos (RANKL e osteoprotegerina). Nós também encontramos que os efeitos das flavinas em osteoblastos nesta segunda etapa foram independentes das suas propriedades antioxidantes. A atividade biológica da combinação de indutores osteogênicas com Rb e seus fotoprodutos foi associada com a ativação de diferentes vias de sinalização (AKT, FAK, CaMKII), caspases -8, -9 e -3 e aumento da expressão e/ou estabilização de fatores de transcrição osteoblásticos (Runx2 e ß-catenin). Este estudo nos trouxe fortes evidências que altas concentrações de Rb e IRb geraram um microambiente osteogênico através da modulação de diferentes vias de sinalização, além de promover um efeitos aditivo durante a diferenciação das células pré-osteoblasticas MC3T3 induzida por ácido ascórbico e ß- glicerofosfato. Em resumo, este estudo aponta para uma potencial aplicação da Rb e seus fotoprodutos no desenvolvimento do fenótipo osteoblástico e, consequentemente, uma alternativa terapêutica coadjuvante para osteoporose. / Abstract: Riboflavin (Rb-Vitamin B2) is the precursor of essential flavocoenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These coenzymes participate in numerous enzymatic processes dependent on electron transfer reactions that occur in energyproducing, biosynthetic, and detoxifying and electron-scavenging pathways. Increase dietary riboflavin and pyridoxine intake has been associated with higher bone mineral density in elderly men and women. Photoderivatives of riboflavin have been shown strong activity in haematological malignancy and prostate cancer cells, however, the direct effect of Rb and its photoderivatives on osteoblast has not been examined. In this work, the biologic effects of Rb and irradiated riboflavin (IRb) were investigated in the MC3T3-E1 pre-osteoblastic cell line, a well-accepted model of osteogenesis in vitro characterized for the induction of specific genes associated with the osteoblastic phenotype when treated with ascorbic acid and ß-glycerophosphate. Cell viability was assessed by MTT reduction, neutral red uptake and nucleic acids content. Osteoblastic differentiation markers were analyzed by semiquantitative RT-PCR (osteopontin and osteocalcin), alkaline phosphatase (ALP) activity measured colorimetrically and collagen synthesis by Sirius red staining. Metalloproteinases (MMP) -9 and -2 activities were assayed by gelatin zymography. Peptide microarray of substrate specificity to kinases and immunoblotting were used to identify the effects on signal transduction pathways. Antioxidant enzyme activities (superoxide dismutase, catalase, glutathione peroxidase and glutathione Stransferase) were determined in cellular lysate using spectrophotometric methods. Caspase-8, -9 and -3 activation were measured by a colorimetric assay. In the first analysis Rb and IRb caused cell cycle arrest at G0/G1 phase and accordingly inhibited AKT kinase, a proliferation mediator. Flavins caused differentiation of preosteoblast cells as evidenced by increase of osteocalcin, osteopontin and BMP2 expressions. In addition, higher MMP-9 and -2 activities were observed. Importantly, the capacity of flavins to trigger osteoblasts differentiation was also reinforced by upregulation of connexin 43, down regulation of caveolin-1 and negative modulation of Notch cascade. In the second analysis, we found that the interaction between Rb and IRb and osteogenic inductors (ascorbic acid and ß-glycerophosphate) significantly affected the osteoblast proliferation, alkaline phosphatase activity, collagen biosynthesis, osteopontin and osteocalcin mRNA expression, MMP-2 and MMP-9 activities and the expression of osteoclastogenesis factors (RANKL and OPG). We also showed that the effects of flavins in osteoblasts cells were independent on flavins antioxidant property. The biological activity of the combination of osteogenic medium with riboflavin and its photoderivatives was associated with the activation of different signaling pathways (AKT, FAK, CaMKII), caspases -8, -9 and -3, and up-regulation and/or stabilization of osteoblastic transcription factors (Runx2 and ß-catenin). This study brought out strong evidences that high concentration of Rb and IRb generates an osteogenic microenvironment through modulating different mediators of signaling pathways, besides of the additive effect of riboflavin and its photoproducts during the ascorbate and ß-glycerophosphateinduced osteoblast differentiation of MC3T3-E1 cells. In summary, this study pointed out the potential application of Rb and its photoproducts in osteoblasts phenotype development and, consequently, it is possible use as an alternative therapeutic adjuvant of osteoporosis. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

O parasita da malária Plasmodium falciparum percebe o ambiente em que se encontra, elaborando respostas celulares adequadas, que envolvem secreção de proteínas, crescimento e diferenciação celular. Fatores relacionados com a geração de segundos-mensageiros e proteínas efetoras da sinalização celular estão descritos na literatura. Porém, a função de receptores responsáveis pela percepção de estímulos extracelulares no parasita é um tema pouco explorado. A identificação in silico de receptores de sete domínios transmembrânicos putativos no genoma de P. falciparum possibilitou a exploração da função dos mesmos. A tese caracteriza funcionalmente dois receptores, PFSR10 e PFSR25. A expressão proteica dos receptores foi demonstrada em fases eritrocíticas de P. falciparum. Os receptores possuem candidatos a parceiros moleculares que executam diversas funções celulares, entre elas invasão do eritrócito, endocitose e exocitose. Os receptores foram transfectados em células de mamíferos e, através de ensaios de dinâmica de cálcio de high-throughput, sugere-se que PFSR10 codifique um receptor que participa na percepção de ATP extracelular e que PFSR25 codifique um sensor de KCl. O trabalho também sugere que KCl modula cálcio citosólico em P. falciparum e que parasitas nocaute para PFSR25 são incapazes de modular cálcio citosólico em resposta a KCl. / The malaria parasite P. falciparum perceives its milieu and elaborates adequate intracellular responses, that involve protein secretion, growth and cell differentiation. Factors related to second messengers generation and effectors of cell signaling are described in the literature. However, the function of receptors responsible for stimulus perception remains elusive. The in silico identification of putative seven transmembrane receptors in the Plasmodium falciparum genome allowed the exploration of their function. In the thesis, two putative receptors were characterized, PFSR10 and PFSR25. The proteic expression of the receptors was demonstrated in erythrocytic stages of P. falciparum. The receptors have putative interaction partners that participate in cellular functions such as invasion, exocytosis and endocytosis.The receptors were transfected in mammalian cells and, through high-throughput calcium dynamics assays, it is suggested that PFSR10 codes for a receptor that participates in extracellular ATP perception and that PFSR25 codes for a KCl sensor. It is also suggested that KCl modulates cytosolic calcium in response to KCl and that knockout parasites for PFSR25 are incapable of modulating cytosolic calcium in response to KCl.

Orientadores: Patrícia Maria Bergamo Favaro, Sara Teresinha Olalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T08:01:05Z (GMT). No. of bitstreams: 1 Pereira_JoaoKleberNovais_M.pdf: 3579832 bytes, checksum: 2f21cc28ac6161522a1ab6a958a04389 (MD5) Previous issue date: 2014 / Resumo: As neoplasias linfoides constituem um grupo heterogêneo de doenças originadas por alterações genéticas das células progenitoras hematopoéticas de origem linfoide, levando à proliferação clonal desordenada de células B ou T, e ao desenvolvimento de leucemias linfoides e linfomas. É notória a participação de diferentes vias de sinalização envolvidas tanto no desenvolvimento como na manutenção das neoplasias hematológicas. A ativação constitutiva da via de sinalização PI3K/AKT/mTOR é bem descrita na leucemia linfoide aguda de células T (LLA-T) e recentemente identificou-se, em modelos animais, que a atividade da PI3K coopera com o desenvolvimento do linfoma de Burkitt (LB). Deste modo, o papel da via PI3K/AKT/mTOR no crescimento e sobrevivência celular, duas características importantes da leucemogênese, transformou-a em um potencial alvo farmacológico. Seguindo essa perspectiva, o presente trabalho procurou avaliar o potencial terapêutico de NVP-BKM120, um pan-inibidor de PI3K de classe I, em linhagens celulares de LLA-T (Jurkat e MOLT-4) e LB (Daudi e NAMALWA). NVP-BKM120 foi capaz de diminuir a viabilidade celular e capacidade clonogênica dessas células. Foi observada uma parada na fase G2/M do ciclo celular, com subsequente diminuição de Ciclina B1 e aumento da apoptose pelas vias intrínseca e extrínseca, nas linhagens Jurkat, MOLT-4 e NAMALWA. Também foi observada diminuição da fosforilação da AKT e dos alvos downstream ao mTORC1, P70S6K e 4EBP1, e aumento da razão BAX:BCL2. Houve um aumento da produção de AVOs nas linhagens celulares após o tratamento com a droga, o que sugere ativação da autofagia. Portanto, este estudo demonstra a capacidade antitumoral de NVP-BKM120 contra linhagens celulares de LLA-T e LB. Nossos resultados sugerem que a diminuição da proliferação celular, após o tratamento com a droga, seja devido à redução da Ciclina B1 e que o aumento da razão BAX:BCL2 seja um dos mecanismos envolvidos na indução da apoptose / Abstract: The lymphoid neoplasms are a heterogeneous group of diseases caused by genetic alterations that were originated from hematopoietic progenitor cells of lymphoid origin, leading to uncontrolled clonal proliferation of B or T cells, and to the development of lymphoid leukemias and lymphomas. These findings emphasize the involvement of different signaling pathways involved in both the development and the maintenance of hematological malignancies. Constitutive activation of the PI3K /AKT/mTOR signaling pathway is well described for acute lymphoblastic leukemia T cells (T-ALL), recently been identified in animal models, that the activity of PI3K cooperates with the development of Burkitt's lymphoma (LB).Thus, the role of the PI3K / AKT / mTOR pathway in cell growth and survival, two important features of leukemogenesis, morphed into a potential drug target. Following this perspective, the present study aimed to evaluate the therapeutic potential of NVP-BKM120, a pan-PI3K inhibitor class I in cell lines of T-ALL (Jurkat and MOLT-4) and LB (Daudi and NAMALWA). NVP-BKM120 was able to decrease cell viability and clonogenic capacity of these cells. A blocked phase was observed in G2/M phase of the cell cycle with subsequent reduction of Cyclin B1 and increased apoptosis by the intrinsic and extrinsic pathways in the lines Jurkat and MOLT-4 NAMALWA. It was also observed, decreased phosphorylation of AKT and of the downstream targets mTORC1, p70S6K and 4EBP1, and an increase of BAX:BCL2 ratio. There was an increase in AVOs production in the cell lines after treatment with the drug, suggesting activation of autophagy. Therefore, this study demonstrates the antitumor ability of NVP-BKM120 against cell lines of T-ALL and LB. Our results suggest that the decrease in cell proliferation after treatment with the drug, is due to the reduction of Cyclin B1 and the increase of the BAX:BCL2 ratio is one of the mechanisms that are involved in the induction of apoptosis / Mestrado / Fisiopatologia Médica / Mestre em Ciências

Abstract Neurofibromatosis type 1 syndrome (NF1) is caused by mutations of the NF1 gene. The NF1 protein (neurofibromin) contains a domain which is related to the GTPase-activating protein (GAP) and accelerates the switch of active Ras-GTP to inactive Ras-GDP. The NF1 protein has been referred to as a tumor suppressor, since the cells of malignant schwannomas of NF1 patients may display a loss of heterozygosity of the NF1 gene. Psoriasis is characterized by hyperproliferation of the epidermis and by down-regulated levels of NF1 mRNA and protein. Ca2+ is an universal signal transduction element modulating cell growth and differentiation. Many cell types coordinate their activities by transmitting waves of elevated intracellular calcium levels from cell to cell. The propagation of calcium waves had not been studied previously in human keratinocytes. Thus, the aim of the present study was to find out which pathways may play a role in Ca2+ signaling at different extracellular calcium concentrations in NF1 and and psoriatic keratinocytes versus normal control keratinocytes. The results demonstrated that NF1 and psoriatic keratinocytes have a tendency to form cultures characterized by altered Ca2+-mediated cell signaling compared to normal keratinocytes. Specifically, the main route of calcium-mediated signaling was gap-junctional in normal keratinocytes. In contrast, ATP-mediated calcium signaling predominated and capacitative calcium influx was defective in NF1 and psoriatic keratinocytes. The results of the present study suggest that mutations of the NF1 tumor suppressor gene or lowered levels of NF1 protein/mRNA may eventually lead to altered intercellular communication.

No description available.

Abstract Background – The heart cell is a prime example of a system, in which numerous interconnected regulatory mechanisms affect the dynamic balance of cellular function. The function of the system emerges from the interactions of its components rather than from their individual properties. Thus, it is a challenging task to understand the causal relations within such a system, based on the analysis of experimental results. Facing this complexity, the systems biological approach has gained interest during recent years, since with using it we can make an effort to observe, quantitatively and simultaneously, multiple components and their interdependencies in biological networks. Methods and aims – One of the most important tools in systems biology is mathematical modelling. In this thesis, novel model components have been developed and existing components integrated to describe mathematically the calcium dynamics in cardiac myocytes with improved physiological accuracy. Special attention was paid to both the activity-dependent and automatic regulation of the dynamics. This enabled the quantitative analysis of the regulation’s role in both physiological and pathophysiological conditions. Results – Validation of the novel model components that describe the calcium transport mechanisms indicates that the developed schemes are accurate and applicable also beyond the normal physiological state of the cardiac myocyte. Results also highlight the importance of autoregulation of calcium dynamics in the excitation-contraction coupling. Furthermore, the analysis indicates that the CaMK-dependent regulation of the calcium uptake to and release from the sarcoplasmic reticulum calcium stores could have substantial roles as downstream effectors in beta-adrenergic stimulation. Conclusions – Results emphasize mathematical modelling as a valuable complement to experiments in understanding causal relations within complex biological systems such as the cardiac myocytes. That is, rigorous data integration with mathematical models can provide significant insight to the quantitative role of both the individual model components and the interconnected regulatory loops. This is especially true for the analysis of genetically engineered animal models, in which the intended modification is always accompanied by compensatory changes that can mask to a varying degree the actual phenomenon of interest.

The Salvador/Warts/Hippo (Hippo) pathway defines a novel signalling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis and cancer development in mammals. The Hippo pathway was initially utilised in D. melanogaster, where the Expanded protein acts in the Hippo signalling cascade to control organ size. Willin is the proposed human orthologue of Expanded and the aim of this thesis is to investigate whether willin can activate the mammalian Hippo signalling pathway. Ectopic willin expression causes an increase in phosphorylation of the core Hippo signalling pathway components MST1/2, LATS1 and YAP, an effect which can be antagonised by ezrin. In MCF10A cells, willin over-expression antagonises a YAP-induced epithelial-to-mesenchymal transition via the N- terminal FERM (Four-point-one Ezrin Radixin Moesin) domain of willin. Preliminary results show that willin is expressed within the sciatic nerve of rat and mice, and within the neuromast cells in the zebrafish; suggesting that willin and the Hippo pathway may play a vital role in the developmental regulation within the peripheral nervous system. To conclude, willin influences Hippo signalling activity by activating the core Hippo pathway kinase cassette in mammalian cells.

Self-renewal of mouse embryonic stem (mES) cells is dependent upon the presence of leukemia inhibitory factor (LIF). LIF induces tyrosine phosphorylation and nuclear translocation of STAT3 (signal transducer and activator of transcription 3) which is thought to promote self-renewal by inducing key target genes. The molecular chaperone heat shock protein 90 (Hsp90) is involved in signal transduction pathways and regulates STAT3 activity in different cell types. However, the role of Hsp90 in regulating STAT3 activity in mES cells has not previously been investigated. The aim of this study was to investigate if Hsp90 interacts with STAT3 in mES cells and to determine if this interaction is important for the maintenance of self-renewal. It was found that when mES cells were cultured for 24.0 hours in the absence of LIF, the expression levels of total STAT3, tyrosine-phosphorylated STAT3 (pYSTAT3), and the pluripotency marker, Nanog, were down regulated. However, the expression level of Hsp90 was found to be slightly up-regulated over the same period. Significantly, it was found that the amount of STAT3 in differentiating mES cells available for binding to Hsp90 was decreased upon down-regulation of STAT3 by LIF withdrawal. Therefore, STAT3-Hsp90 interactions in mES cells were dependent on the presence of LIF, which suggested that the reduction in STAT3-Hsp90 interaction may have resulted from the low levels of STAT3. Despite a dramatic reduction in the expression levels of pYSTAT3 upon 24.0 hours of culture of mES cells in the presence of the STAT3 tyrosine phosphorylation inhibitor, cucurbitanin I, there was no obvious reduction in the levels of total STAT3, Oct-3/4 or Nanog. These results suggested that the levels of unphosphorylated STAT3 rather than pYSTAT3, maybe more important in the maintenance of mES cells self-renewal.

No description available.

This work sought to investigate the clinical phenotypes and molecular basis of cardio-facio-cutaneous syndrome (CFC), a germline disorder of the Ras-MAPK pathway, like Noonan syndrome (NS) and neurofibromatosis type I, caused by mutations in genes encoding proteins that act within this signal transduction pathway. CFC is most commonly due to mutation in BRAF, and less commonly MAP2K1, MAP2K2 or KRAS. A proportion of patients currently have no mutation identified. Mutations and clinical features of patients with a molecular diagnosis of CFC were investigated, which demonstrated a wide range of causative mutations, and some unclassified variants. Both known and novel clinical features of CFC were identified. A strong association between severe contractures and the p.(Tyr130Cys) mutation in MAP2K1 was found, which has not previously been reported. In contrast to the large number of patients with a confirmed molecular diagnosis, several with a highly suggestive clinical phenotype have been found to have no mutationin any of the known CFC genes. The molecular basis of these presentations was investigated by conventional Sanger sequencing of candidate genes. Fourteen patients with the p.(Ser2Gly) mutation in SHOC2 were identified, with clinical presentations consistent with CFC, NS or CS. Target enrichment and massively parallel sequencing of selected genes was undertaken in ten patients. Mutations in known genes were identified in four patients (including the positive control). Candidate causative variants in novel genes were suggested in two further patients, one of which was confirmed on Sanger sequencing. Whole exome sequencing of patient-parent trios was also undertaken to identify de novo variants. Three trios were analysed, and in one patient with a clinical diagnosis of CFC, a frameshift mutation in NF1 was identified, which was confirmed by Sanger sequencing to be present and de novo. The molecular effects of CFC-associated mutations in BRAF on Ras-MAPK pathway signalling were studied in cell culture systems, using Western blotting for ERK1/2 phosphorylation, in vitro kinase assays and luciferase assays, to assess activity of downstream targets of the Ras-MAPK pathway. Altered pathway activity was demonstrated for novel variants that had not previously been characterised at the molecular level, which was in keeping with the findings of the effects of previously studied mutations. The cardiac phenotype in animal models of CFC, CS and NS/CFC was explored using expression microarrays to identify potentially important genes and pathways in the pathogenesis of hypertrophic cardiomyopathy (a progressive but potentially treatable disease feature) in these conditions. A signature of increased expression of Myh7, the embryonic form of myosin, was identified in the heart of the mouse model of CFC due to a B-Raf mutation at four weeks postnatal age, but comparative analysis suggested significant differences in either the mechanisms causing cardiac phenotypes, or the timescales over which these may exert their effects, in the three models. In summary, the most significant findings of this work were that SHOC2 mutation is a frequent cause of a severe NCFC presentation, and massively parallel sequencing can be an effective means of molecular investigation of this group of disorders. Novel features of CFC syndrome that were identified include severe contractures in association with p.(Tyr130Cys) mutations in MAP2K1. The analysis of mouse models of the NCFCs was hampered by heterogeneity within the expression microarray results, and low levels of expression of the H-Ras mutant allele in the mouse model of Costellosyndrome.