Orientador: Ricardo Della Coletta / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T21:07:52Z (GMT). No. of bitstreams: 1 Bufalino_Andreia_D.pdf: 2552060 bytes, checksum: 9156e1c08764322bea090809a2b166d5 (MD5) Previous issue date: 2013 / Resumo: Ativina A é um membro da família dos fatores de crescimento transformante-? e sua expressão têm sido associados ao desenvolvimento tumoral. Contudo, sua expressão, função e mecanismos de regulação, por exemplo, via folistatina, no carcinoma espinocelular (CEC) oral é parcialmente conhecida. Diante disto, nosso estudo teve como objetivo avaliar a participação de ativina A na tumorigênese dos CECs orais. Para alcançar este objetivo, a expressão imuno-histoquímica de ativina A foi analisada em 115 amostras de CEC oral e sua expressão foi correlacionada com características clínico-patológicas e de sobrevida. In vitro a influência de ativina A sobre os principais eventos biológicos relacionados à tumorigênese oral foi verificada por 3 abordagens: 1) exposição da linhagem celular HaCAT a ativina A recombinante nas concentrações de 0, 1, 10 e 100 ng/ml, 2) tratamento das linhagens tumorais LN2 com folistatina recombinante nas concentrações de 0, 1, 10 e 100 ng/ml e 3) silenciamento estável da expressão de ativina A na linhagem LN2 com RNA de interferência (shINHBA). A expressão aumentada de ativina A em CECs orais foi significantemente correlacionada com a presença de metástases regionais (estádio N, p=0,034), tumores classificados como pobremente diferenciados (p=0,013) e demonstrou ser preditiva de um menor período de sobrevida livre de doença em 5 anos (HR: 1,74; 95% CI: 1,39-2,97; p=0,016). Os resultados dos estudos in vitro revelaram que ativina A apresenta um efeito pleotrópico no controle dos principais eventos associados à tumorigênese oral. A exposição das células HaCAT a ativina A resultou em um significante bloqueio da morte celular por apoptose e necrose, promoveu alteração do padrão de expressão dos marcadores da transição epitélio mesenquimal (TEM), aumentou a adesão celular aos componentes da matriz extracelular (MEC) e induziu a invasão e migração celular. Por outro lado, o tratamento das células LN2 com folistatina foi capaz de induzir significantemente a apoptose e a morte celular por necrose, reduzir a proliferação celular, alterar o padrão de expressão dos marcadores da TEM, além de reduzir a adesão celular aos componentes da MEC e os potenciais invasivo e migratório. O bloqueio de ativina A com a transdução estável de shINHBA na linhagem tumoral LN2 promoveu significantemente a apoptose e a morte por necrose, alterou a expressão dos marcadores da TEM de maneira similar aos efeitos da folistatina e reduziu a proliferação, invasão, migração e motilidade celular, que foi avaliada por formação de filopódios e lamelipódios. Interessantemente, o bloqueio com shINHBA significantemente facilitou a adesão das células LN2 aos componentes da MEC, diferente do que foi observado no tratamento com folistatina. Em conclusão, os resultados deste estudo sugerem que a ativina A regula eventos biológicos essenciais para a tumorigênese oral e é um fator prognóstico independente de sobrevida livre de doença em pacientes com CECs orais / Abstract: Activin A is a member of the transforming growth factor-? family and its deregulated expression has been described in different cancers. However, its expression, function and regulatory mechanisms, particularly via follistatin, in oral squamous cell carcinoma (OSCC) are partially known. The aim of the present study was to evaluate the role of activin A in the promotion of oral tumorigenesis. To achieve this goal, immunohistochemical expression of activin A was analyzed in 115 samples of OSCCs and its expression was correlated with clinicopathological features and outcome. In vitro, the influence of activin A on oral tumorigenesis was determined by 3 different approaches: 1) exposition of HaCaT cells to recombinant activin A in different concentrations (0, 1, 10 and 100 ng/ml), 2) treatment of LN2 tumor cells with recombinant follistatin in different concentrations (0, 1, 10 and 100 ng/ml) and 3) stable knockdown of activin A expression in the LN2 tumor cells by using interferencing RNA (shINHBA). Increased activin an expression in OSCCs was significantly correlated with the presence of regional metastases (stage N, p=0.034), poorly differentiated tumors (p=0.013), and shown to be predictive of a shortened disease-free survival (HR: 1.74, 95% CI: 1.39-2.97, p=0.016). In vitro studies showed a pleotropic effect of activin an on control of key events associated with oral tumorigenesis. Activin A resulted in a reduction of cell death by apoptosis and necrosis, promoted changes in the expression of epithelial-mesenchymal transition (EMT) markers, increased cell adhesion to extracellular matrix (ECM) components and induced cell invasion and migration in HaCAT cells. On the other hand, the inhibition of activin a using follistatin induced apoptosis and cell death by necrosis, reduced cell proliferation, changed the expression of EMT markers, reduced cell adhesion to ECM components and reduced the cell invasion and migration in LN2 cells. The activin a knockdown with shINHBA stable transduction in the tumor cell line LN2 significantly promoted death by apoptosis and necrosis changed the expression of EMT markers, and decreased cell proliferation, invasion, migration and motility evaluated by lamellipodia and filopodia formation. Interestingly, knockdown with shINHBA significantly promoted the adhesion of cells to ECM components in LN2 cells, different to the results observed in the treatment with follistatin. In conclusion, our results suggest that activin A regulate biological events essential for oral tumorigenesis, and is an independent prognostic factor for disease-free survival in patients with OSCCs / Doutorado / Patologia / Doutora em Estomatopatologia

Orientador: Maria Julia Marques / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T07:19:08Z (GMT). No. of bitstreams: 1 Moreira_DrielendeOliveira_M.pdf: 2075338 bytes, checksum: 00bb5917e4f176b73c58453340cb3a5e (MD5) Previous issue date: 2012 / Resumo: A Distrofia muscular de Duchenne (DMD) é uma doença caracterizada pela fraqueza muscular progressiva que leva à insuficiência respiratória e cardíaca, resultando em morte por volta dos 30 anos de idade. No camundongo mdx, modelo experimental da DMD, os músculos diafragma e cardíaco são severamente afetados apresentando fibrose semelhante à observada na patologia humana. O objetivo deste trabalho foi investigar os efeitos do tratamento a longo prazo com suramina, uma droga anti-fibrótica, nos músculos diafragma e cardíaco de camundongos mdx idosos. Camundongos mdx (n=20; 8 meses de idade) receberam injeções intraperitoneais de suramina (60 mg/kg), durante 3 meses. Controles mdx (n=20; 8 meses) e C57BL/10 (n=18; 8 meses) foram injetados com solução salina. Os camundongos da linhagem C57BL/10 expressam distrofina e são utilizados como controle da linhagem mdx. A suramina diminuiu os níveis de CK e atenuou a perda da força muscular. No músculo diafragma, a suramina reduziu a área de fibrose e a mionecrose. No músculo cardíaco, houve redução da fibrose, da inflamação e melhora significativa de parâmetros funcionais cardíacos (amplitude das ondas P, Q, R e S do eletrocardiograma). Sugere-se que a suramina possa ser potencialmente útil nas distrofinopatias, atenuando a miopatia nos músculos mais afetados, o coração e o diafragma, nos estágios tardios da doença / Abstract: Duchenne muscular dystrophy (DMD) is a disease characterized by progressive muscle weakness leading to respiratory and cardiac failure, resulting in death around 30 years of age. In the mdx mice model of DMD, diaphragm and cardiac muscles are severely affected in the later stages of the disease, showing intense fibrosis similar to that observed in human pathology. The aim of the present study was to investigate the effects of long-term treatment with suramin, an anti-fibrotic agent, in the diaphragm and cardiac muscles of the mdx mice. Mdx mice (n=20; 8 months of age) received intraperitoneal injections of suramin (60 mg/kg) for 3 months. Mdx controls (n=20; 8 months) and C57BL/10 (n=18; 8 months old) were injected with saline. C57BL/10 mice express dystrophin and are the control strain for the mdx mice. Suramin decreased CK levels and reduced the loss of muscle strength. Suramin reduced fibrosis and myonecrosis in diaphragm. In the cardiac muscle, suramin decreased fibrosis, inflammation and improved cardiac functional parameters (P, Q, R and S waves of the electrocardiogram). It is suggested that suramin may be a potential therapy for distrophinopaties, attenuating the dystrophic phenotype of the most affected cardiac and diaphragm muscles of the mdx mice, during later stages of the disease / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

The Smad proteins transduce the signal of transforming growth factor-β (TGF-β) and related factors from the cell surface to the nucleus. Following C-terminal phosphorylation by a corresponding receptor kinase, the R-Smad proteins form heteromeric complexes with Smad4. These complexes translocate into the nucleus, bind specific transcriptional activators and DNA, ultimately modulating gene expression. Though studied through a variety of means, the stoichiometry of the R-Smad/Smad4 complex is unclear. We investigated the stoichiometry of the phosphorylation-induced R-Smad/Smad4 complex by using acidic amino acid substitutions to simulate phosphorylation. Size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry analysis revealed that the R-Smad/Smad4 complex is a heterotrimer consisting of two R-Smad subunits and one Smad4 subunit. In addition, a specific mechanism for phosphorylation-induced R-Smad/Smad4 complex formation was studied. Although it had been previously established that part of the mechanism through which phosphorylation induces Smad oligomerization is through relieving MH1-domain mediated autoinhibition of the MH2 (oligomerization) domain, it is also evident that phosphorylation serves to energetically drive Smad complex formation. Through mutational and size exclusion chromatography analysis, we established that phosphorylation induces oligomerization of the Smads by creating an electrostatic interaction between the phosphorylated C-terminal tail of one R-Smad subunit in a Smad trimer with a basic surface on an adjacent R-Smad or Smad4 subunit. The basic surface is defined largely by the L3 loop, a region that had previously been implicated in R-Smad interaction with the receptor kinase. Furthermore, the Smad MH2 domain shares a similar protein fold with the phosphoserine and phosphothreonine-binding FHA domains from proteins like Rad53 and Chk2. Taken together, these results suggest that the Smad MH2 domain may be a distinct phospho serine-binding domain, which utilizes a common basic surface to bind the receptor kinase and other Smads, and takes advantage of phosphorylation-induced allosteric changes dissociate from the receptor kinase and oligomerize with other Smads. Finally, the structural basis for the preferential formation of the R-Smad/Smad4 heterotrimeric complex over the R-Smad homotrimeric complex was explored through X-ray crystallography and isothermal titration calorimetry. Crystal structures of the Smad2/Smad4 and Smad3/Smad4 complexes revealed that specific residue differences in Smad4 compared to R-Smads resulted in highly favorable electrostatic interactions that explain the preference for the interaction with Smad4.

During cell communication, cells produce secreted signals termed morphogens, which traffic through the tissue until they are received by target, responding cells. Using the fruit fly Drosophila melanogaster as a model organism, I have studied transforming growth factor-beta (TGF-beta) signal from the secreting to the receiving cells in the developing wing epithelial cells and at the neuromuscular junctions. Cell culture studies have suggested that cells modulate morphogenetic signaling by expressing the receptors and secreting the ligand in spatially defined areas of the cell. Indeed, I have found that TGF-beta ligands, receptors and R-Smads show a polarized distribution both in the epithelial cells and at the synapses. My results indicate that the cellular junctions define a signaling domain within the plasma membrane, to which TGF-beta signaling machinery is targeted. In the context of epithelial cells, the junctions play a role in TGF-beta signaling regulation through their component beta-cat. A complex forms between beta-cat and the R-Smad Mad, but the mechanism by which beta-cat modulates signaling is not yet understood. At the synapse, the sub-cellular localization of TGF-beta pathway components indicates the occurrence of an anterograde signal. Moreover, my results suggest a scenario in which TGF-beta signaling is coupled with synaptic activity: quanta of growth factor, released upon neurostimulation together with neurotransmitter quanta, could modulate therefore the development and the function of the synapse.

The function of transforming growth factor β (TGF-β) in cancer is notoriously complex. Initially TGF-β limits tumorigenesis, but at later stages in tumor progression TGF-β promotes the malignant spread of tumor cells. Past studies to understand the pro-metastasis utility of TGF-β centered upon its ability to regulate protein-coding genes. Recently, a small class of non-coding RNAs known as microRNAs (miRNAs) emerged as novel posttranscriptional regulators of gene expression. The significance of miRNA function in cellular processes from embryonic development to the maintenance of homeostasis in adult tissues is becoming increasingly clear. Also apparent is the strong association between aberrant miRNA expression and human diseases, such as cancer. The contribution of miRNAs to TGF-β-mediated cellular responses remains an open question. Thus, I became interested if miRNAs offered an additional layer of regulation in TGF-β signaling through which this cytokine exerts its pro-metastasis function. To address this inquiry, in the first part of this dissertation I investigated whether miRNAs influenced the ability of TGF-β to induce cellular responses directly involved with carcinoma metastasis, such as epithelial-mesenchymal transition (EMT). Here, I identified two miRNAs, miR-21 and miR-31, that are upregulated during EMT in LIM 1863 organoids, a colon carcinoma model of EMT driven by TGF-β. We performed in vitro studies to characterize the function of miR-21 and miR-31 and found that these two miRNAs positively impact the induction of EMT, migration and invasion by TGF-β. Furthermore, we uncovered TIAM1 (T lymphoma and metastasis gene 1) as a novel target of both miR-21 and miR-31 and show that downregulation of TIAM1 is critical for the pro-migration and pro-invasion activities of miR-21 and miR-31. Together these findings reveal miR-21 and miR-31 as downstream effectors of TGF-β signaling by facilitating EMT, migration and invasion of colon carcinoma cells. How TGF-β regulates miR-21 and miR-31 became important questions and thus the focus of the second part of this thesis. Interestingly, I found that TGF-β and TNF-α synergize to increase miR-21 and miR-31 levels in LIM 1863 organoids and that the synthesis of new factors induced by TGF-β/TNF-α are required for this upregulation. Moreover, I report that regulation of miR-21 by TGF-β/TNF-α occurs at multiple levels of biogenesis. More specifically data provided here show that Smad4 binds to the promoter of miR-21 to upregulate its expression thereby specifying miR-21 as a typical TGF-β target gene. This mechanism is different from one recently observed in smooth muscle cells in which TGF-β did not stimulate miR-21 transcription, but interestingly, Smad4 enhanced the Drosha-mediated processing of the miR-21 precursor. These two mechanisms suggest that TGF-β regulation of miR-21 is contextual and highlight the complexity of TGF-β signaling. As a whole, my findings establish important roles for miR-21 and miR-31 in TGF-β-mediated cellular responses that facilitate the pro-metastasis utility of TGF-β in colon cancer. Also, I describe a novel mechanism by which TGF-β/TNF-α signaling elevates the level of miR-21 and miR-31. Future studies that identify additional targets of miR-21 and miR-31 may offer further insight into the molecular mechanisms underlying cellular regulation by TGF-β. This information will be vital for the design of therapeutic interventions for colon cancer patients.

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The tumor microenvironment is a key modulator in cancer progression and has become a novel target in cancer therapy. An increase in hyaluronan (HA) accumulation and metabolism can be found in advancing tumor progression and are often associated with aggressive malignancy, drug resistance and poor prognosis. Wound-healing related myofibroblasts or activated cancer-associated fibroblasts (CAF) are assumed to be the major sources of HA. Both cell types are capable to synthesize new matrix components as well as reorganize the extracellular matrix. However, to which extent myofibroblasts and CAF perform these actions are still unclear. In this work, we investigated the matrix remodeling and HA production potential in normal human dermal fibroblasts (NHFB) and CAF in the absence and presence of transforming growth factor beta -1 (TGF-β1), with TGF-β1 being a major factor of regulating fibroblast differentiation. Three-dimensional (3D) collagen matrix was utilized to mimic the extracellular matrix of the tumor microenvironment. We found that CAF appeared to response insensitively towards TGF-β1 in terms of cell proliferation and matrix remodeling when compared to NHFB. In regards of HA production, we found that both cell types were capable to produce matrix bound HA, rather than a soluble counterpart, in response to TGF-β1. However, activated CAF demonstrated higher HA production when compared to myofibroblasts. The average molecular weight of produced HA was found in the range of 480 kDa for both cells. By analyzing gene expression of HA metabolizing enzymes, namely hyaluronan synthase (HAS1-3) and hyaluronidase (HYAL1-3) isoforms, we found expression of specific isoforms in dependence of TGF-β1 present in both cells. In addition, HAS2 and HYAL1 are highly expressed in CAF, which might contribute to a higher production and degradation of HA in CAF matrix. Overall, our results suggested a distinct behavior of NHFB and CAF in 3D collagen matrices in the presence of TGF-β1 in terms of matrix remodeling and HA production pointing to a specific impact on tumor modulation.

Transforming growth factor beta 3 (TGF3) promotes tenogenic differentiation and may enhance tendon regeneration in vivo. This study aimed to apply TGF3 absorbed in decellularized equine superficial digital flexor tendon scaffolds, and to investigate the bioactivity of scaffold-associated TGF3 in an in vitro model. TGF3 could effectively be loaded onto tendon scaffolds so that at least 88% of the applied TGF3 were not detected in the rinsing fluid of the TGF3-loaded scaffolds. Equine adipose tissue-derived multipotent mesenchymal stromal cells (MSC) were then seeded on scaffolds loaded with 300 ng TGF3 to assess its bioactivity. Both scaffold-associated TGF3 and TGF3 dissolved in the cell culture medium, the latter serving as control group, promoted elongation of cell shapes and scaffold contraction (p < 0.05). Furthermore, scaffold-associated and dissolved TGF3 affected MSC musculoskeletal gene expression in a similar manner, with an upregulation of tenascin c and downregulation of other matrix molecules, most markedly decorin (p < 0.05). These results demonstrate that the bioactivity of scaold-associated TGF3 is preserved, thus TGF3 application via absorption in decellularized tendon scaffolds is a feasible approach.

No description available.

No description available.