<p>The Rho GTPases, which are related to the Ras family of proto-oncogenes, have been found to have important roles in regulating the morphogenic and migratory properties of eukaryotic cells. In addition, these proteins have been shown to regulate aspects of cell signaling, cell growth, cell division and cell survival. The Rho GTPases cycle between inactive GDP-bound and active GTP-bound states. In resting cells, Rho GTPases are sequestered in the cytoplasm by forming an inactive complex with guanine dissociation inhibitors (GDIs), and are, thus, unable to exchange guanine nucleotides. Rho GTPases exchange guanine nucleotides at slow rates <i>in vivo</i>, and these reactions can be catalyzed by two different classes of proteins. Upon cell activation, guanine exchange factors stimulate the exchange of GTP for GDP and thereby activate the Rho GTPases, whereas the GTPase activating proteins turn off the Rho GTPase by stimulating their inherent GTP-hydrolysis activity. The active Rho GTPase associates with so-called effector proteins, which in turn mediate a plethora of responses.</p><p>In recent years a great number of Rho GTPase effectors have been identified. The Cdc42-interacting protein 4 (CIP4) is one such protein, and this thesis has focused on elucidating the role of this protein in Rho GTPase regulated activities resulting in changes in the organization of the actin filament system. Changes in actin dynamics are required for many cellular activities, such as cell migration, cytokinesis and membrane-trafficking. CIP4 is a member of the Pombe Cdc15 homology (PCH) family of proteins. Many PCH proteins been proposed to cooperate with so-called formin homology proteins to induce changes in actin dynamics resulting in cytokinesis. We show that CIP4 interacts with the diaphanous-related formin DAAM1 (Disheveled associated activator of morphogenesis 1). DAAM1 appeared to influence both changes in actin dynamics and microtubule dynamics, possibly by integrating signals from CIP4, Src and the Rho GTPases Rac, Cdc42</p><p>The RhoGAP domain-containing protein RICH-1 (Rho GAP interacting with CIP4 homologoues-1) was isolated in a yeast two hybrid screen for proteins binding to CIP4. RICH-1 was shown to down-regulate the Rho GTPases Cdc42 and Rac1. In addition to the RhoGAP domain, RICH-1 possesses a proline-rich motif which confers binding to a variety of Src homology 3 (SH3) domain-containing proteins including CIP4, FBP17, Src, Abl and CIN85. Furthermore, RICH-1 exhibits a BIN/amphiphysin/Rvsp (BAR) domain which associates with membrane lipids, and in addition this domain was shown to deform liposomes in an in vitro assay, which is thought to mimic the deformation of cellular lipid bilayers, for example the invagination of the plasma membrane during endocytosis. Our results suggest a role for RICH-1 in intracellular membrane-trafficking events. RICH-1 was in addition shown to interact with the SH3 domains of two BAR domain-containing proteins, endophilin A1 and amphiphysin, which induce deformation of the plasma membrane during the specialized clathrin-mediated endocytosis. In conclusion, our data supports the notion that RhoGAPs are multi-functional proteins, fulfilling not only the role as downregulators of Rho GTPase activity, but also as signal transducers of numerous vital cellular processes.</p>

The Rho GTPases, which are related to the Ras family of proto-oncogenes, have been found to have important roles in regulating the morphogenic and migratory properties of eukaryotic cells. In addition, these proteins have been shown to regulate aspects of cell signaling, cell growth, cell division and cell survival. The Rho GTPases cycle between inactive GDP-bound and active GTP-bound states. In resting cells, Rho GTPases are sequestered in the cytoplasm by forming an inactive complex with guanine dissociation inhibitors (GDIs), and are, thus, unable to exchange guanine nucleotides. Rho GTPases exchange guanine nucleotides at slow rates in vivo, and these reactions can be catalyzed by two different classes of proteins. Upon cell activation, guanine exchange factors stimulate the exchange of GTP for GDP and thereby activate the Rho GTPases, whereas the GTPase activating proteins turn off the Rho GTPase by stimulating their inherent GTP-hydrolysis activity. The active Rho GTPase associates with so-called effector proteins, which in turn mediate a plethora of responses. In recent years a great number of Rho GTPase effectors have been identified. The Cdc42-interacting protein 4 (CIP4) is one such protein, and this thesis has focused on elucidating the role of this protein in Rho GTPase regulated activities resulting in changes in the organization of the actin filament system. Changes in actin dynamics are required for many cellular activities, such as cell migration, cytokinesis and membrane-trafficking. CIP4 is a member of the Pombe Cdc15 homology (PCH) family of proteins. Many PCH proteins been proposed to cooperate with so-called formin homology proteins to induce changes in actin dynamics resulting in cytokinesis. We show that CIP4 interacts with the diaphanous-related formin DAAM1 (Disheveled associated activator of morphogenesis 1). DAAM1 appeared to influence both changes in actin dynamics and microtubule dynamics, possibly by integrating signals from CIP4, Src and the Rho GTPases Rac, Cdc42 The RhoGAP domain-containing protein RICH-1 (Rho GAP interacting with CIP4 homologoues-1) was isolated in a yeast two hybrid screen for proteins binding to CIP4. RICH-1 was shown to down-regulate the Rho GTPases Cdc42 and Rac1. In addition to the RhoGAP domain, RICH-1 possesses a proline-rich motif which confers binding to a variety of Src homology 3 (SH3) domain-containing proteins including CIP4, FBP17, Src, Abl and CIN85. Furthermore, RICH-1 exhibits a BIN/amphiphysin/Rvsp (BAR) domain which associates with membrane lipids, and in addition this domain was shown to deform liposomes in an in vitro assay, which is thought to mimic the deformation of cellular lipid bilayers, for example the invagination of the plasma membrane during endocytosis. Our results suggest a role for RICH-1 in intracellular membrane-trafficking events. RICH-1 was in addition shown to interact with the SH3 domains of two BAR domain-containing proteins, endophilin A1 and amphiphysin, which induce deformation of the plasma membrane during the specialized clathrin-mediated endocytosis. In conclusion, our data supports the notion that RhoGAPs are multi-functional proteins, fulfilling not only the role as downregulators of Rho GTPase activity, but also as signal transducers of numerous vital cellular processes.

Orientadores: Patrícia Maria Bergamo Favaro, Sara Teresinha Olalla Saad / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T08:21:12Z (GMT). No. of bitstreams: 1 Lopes_MatheusRodrigues_M.pdf: 3896765 bytes, checksum: d056b823ef7d5306e932f125ae5b6eaa (MD5) Previous issue date: 2012 / Resumo: As síndromes mielodisplásicas (SMD) são um grupo heterogêneo de doenças caracterizadas por hematopoese ineficaz e risco de progressão para leucemia mieloide aguda (LMA). SMD de baixo de risco é caracterizada por um aumento de apoptose na medula óssea e alterações clínicas com perfil autoimune, enquanto que na SMD de alto risco há uma evasão imune, baixa apoptose e danos secundários ao DNA, contribuindo para a progressão para LMA. Essas evidências, junto com os dados de terapia imunossupressora em pacientes com SMD, sugerem o papel do sistema imune na progressão desta doença. Entretanto, o papel do sistema imune não é claro, e estudos que abordem o perfil das células T são importantes para o melhor entendimento da patogênese da SMD. Formin-like 1 (FMNL1) pertence à família de proteínas formina, indispensáveis para muitos processos fundamentais actina-dependentes. FMNL1 é restritamente expressa em células derivadas de linhagem hematopoética e superexpressa em células neoplásicas hematopoéticas malignas. Recentemente, foi descrito que FMNL1 está envolvida no processo de citotoxicidade de células CD8+. Desse modo, estudar a expressão de FMNL1 tanto nos linfócitos como nas células da MO dos pacientes com SMD, poderia contribuir para o melhor entendimento do papel dessa nova proteína neste modelo de neoplasia hematológica. No presente estudo, foi observada uma diminuição significativa na contagem absoluta de linfócitos periféricos no grupo SMD, após ajuste para idade, quando comparada com o grupo de doadores saudáveis (controle). Entretanto, houve um aumento da frequência de células CD3+, resultante do aumento significativo das subpopulações de células CD3+CD4+ no grupo de alto risco e CD3+CD8+ no grupo de baixo risco, de acordo com as classificações FAB e WHO. A razão CD4:CD8 encontrou-se aumentada no grupo de alto risco comparado com o de baixo risco. Dependência transfusional foi correlacionada positivamente com a porcentagem de CD3+CD4+, enquanto que a idade dos pacientes correlacionou-se de forma negativa com a porcentagem de CD3+ e CD3+CD8+. Os níveis de expressão de FOXP3, nas células CD3+ de sangue periférico, foram significativamente menores no grupo de baixo risco quando comparado com o grupo controle, e esse padrão se repetiu para a expressão de IL10. A quantificação dos transcritos de IL10 correlacionou-se negativamente com a porcentagem de células CD3+CD8+. Em conclusão, evidenciamos que pacientes com SMD apresentaram um menor número de linfócitos, porém com a frequências das células T CD3+, CD3+CD4+ e CD3+CD8+ aumentadas. Os pacientes de baixo risco apresentaram uma diminuição da expressão de FOXP3 e de IL10, quadro característico de um microambiente apoptótico e inflamatório. Já no grupo de alto risco, a expressão de FOXP3 e de IL10 aumenta em relação ao grupo de baixo risco. É interessante ressaltar que nos pacientes com SMD houve uma correlação entre o aumento da expressão de IL10 e a diminuição das células T CD3+CD8+, sugerindo a contribuição das Tregs na progressão da doença através da produção de IL10. A análise da expressão de FMNL1 em células CD3+ de sangue periférico não denotou diferenças significativas entre os pacientes com SMD e o grupo controle. Entretanto observou-se uma correlação positiva entre a expressão de FMNL1 e o número de células CD3+CD4+ e ambos com a dependência transfusional. Quanto à expressão de FMNL1 em amostras de MO, houve uma expressão significativamente menor nos pacientes com SMD quando comparado com as células de doadores normais, além de uma correlação negativa entre FMNL1 e número de citopenias. Usando modelos de linhagens celulares hematopoéticas para a diferenciação, observou-se um aumento significativo na expressão gênica e protéica de FMNL1 durante a diferenciação megacariocítica. Esses resultados sugerem a participação de FMNL1 na ativação de linfócitos CD4+ no sangue periférico e na diferenciação hematopoética na medula óssea / Abstract: Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders characterized by ineffective hematopoiesis and risk of progression towards acute myeloid leukemia. Low-risk MDS is characterized by increased apoptosis in the bone marrow (BM), with a clinical autoimmune profile, whereas in high-risk MDS an immune evasion, low apoptosis and secondary DNA damage occurs, contributing to the progression of AML. This evidence, together with the data of immunosuppressive therapy in patients with MDS, suggests a role of the immune system in the progression of this disease. However, this role of the immune system is remains unclear, and studies that address the profile of T cells are important for a better understanding of the pathogenesis of MDS. Formin-like 1 (FMNL1) belongs to the family of proteins formina indispensable for many fundamental processes in actin-dependent. FMNL1 is strictly expressed in hematopoietic lineage derived cells, and overexpressed in malignant hematopoietic neoplastic cells. FMNL1 has recently been reported to be involved in the cytotoxicity of CD8+ cells. Thus, studies on the expression of FMNL1, both in lymphocytes and BM cells of MDS patients, could contribute to a better understanding of the role of this protein in this new model of hematologic malignancy. In the present study, we observed a significant decrease in absolute peripheral lymphocyte counts in the MDS group, after adjusting for age, compared with the healthy donor group (control). However, there was an increased frequency of CD3+, resulting in a significant increase of the CD3+CD4+ subpopulation in high risk and CD3+CD8+ in MDS low risk, according to FAB and WHO classifications. CD4:CD8 ratio was increased in the high risk when compared to the low risk group. Transfusion dependence was positively correlated with the percentage of CD3+CD4+, whereas the age of patients correlated negatively with the percentage of CD3+ and CD3+CD8+. The expression levels of FOXP3, in peripheral blood CD3+ cells, was significantly lower in the low risk group compared to controls and this pattern was repeated for the expression of IL10. Interestingly, IL10 transcripts correlated negatively with the percentage of CD3+CD8+. In conclusion, we found that patients with MDS had a lower lymphocyte number, however presented an increased frequency of CD3+ and CD3+CD8+ T cells. Our low risk patients showed a decreased expression of FOXP3 and IL10, characteristic of apoptotic and inflammatory microenvironment. In the high risk group, the expression of FOXP3 and IL10 was normal. Interestingly, there was a correlation between increasing expression of IL10 and reduction of CD3+CD8+ T cells in patients, suggesting the contribution of Treg in disease progression due to IL10 production. Analysis of FMNL1expression in CD3+ cells of peripheral blood showed no significant differences between patients with MDS and the control group. However, there was a positive correlation between FMNL1 expression and the number of CD3+CD4+, and both were transfusion dependence. FMNL1 expression in BM samples was significantly lower in MDS patients when compared with cells from normal donors, and there was a negative correlation between FMNL1 and number of cytopenias. Using models of hematopoietic cell lineages for differentiation, we observed an increase in gene and protein expression of FMNL1 during megakaryocytic and granulocytic differentiation. These results suggest the participation of FMNL1 in the activation of CD4+ lymphocytes in peripheral blood and bone marrow hematopoietic differentiation / Mestrado / Fisiopatologia Médica / Mestre em Ciências

No description available.