Orientador: Nicola Amanda Conran Zorzetto / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T05:53:22Z (GMT). No. of bitstreams: 1 Silveira_AngelicaAparecidaAntoniellis_M.pdf: 1268408 bytes, checksum: 22c750391706cfa07cb1fd66d9e8a9c1 (MD5) Previous issue date: 2012 / Resumo: Durante a resposta inflamatória, os neutrófilos e outros leucócitos aderem ao endotélio, deixando os vasos sanguíneos e movimentando-se ativamente em direção ao foco inflamatório. A migração dos neutrófilos para sítios inflamatórios depende de uma série de eventos adesivos e quimiotáticos, resultantes da ativação de moléculas de adesão como as selectinas e integrinas e receptores de quimiocinas. Devido às suas propriedades, os neutrófilos podem ser ativados por proteínas de sinalização intracelular, as Rho GTPases, que auxiliam os neutrófilos a desempenhar esta função por interferirem em mudanças no citoesqueleto. Estas proteínas também estão envolvidas na adesão e proliferação celular. Os neutrófilos são capazes de sintetizar óxido nítrico (NO), sendo que esta produção de NO é um importante componente da resposta imune inata durante a inflamação. Estudos demonstraram que os neutrófilos têm papel indutor na geração de inflamação e esforços visando compreender o mecanismo adesivo destas células nos processos inflamatórios podem ser um ponto chave para intervenções farmacológicas em doenças que são caracterizadas por inflamação vascular com consequente obstrução de fluxo sanguíneo. Diante disso, este estudo objetivou avaliar o papel da via do NO e das Rho GTPases no mecanismo pelo qual os estímulos inflamatórios aumentam a adesão de neutrófilos. Também foi avaliado os efeitos da sinvastatina na modulação das propriedades adesivas de neutrófilos, como ferramenta para auxiliar no estudo do envolvimento das Rho GTPases, Rac1 e RhoA, no processo adesivo destas células. Este mecanismo foi estudado a partir de neutrófilos isolados do sangue periférico por ensaios de adesão estática e em fluxo e citometria de fluxo. Além disso, foi analisada a expressão gênica das Rho GTPases, Rac1 e RhoA, através de PCR em tempo real. Sob potente estímulo de TNF-?, as propriedades adesivas dos neutrófilos aumentam significativamente. Inibidores de NO sintase e doadores de NO não alteraram as propriedades adesivas de neutrófilos quando estimulados com TNF-?. Não observamos grande diferença quanto à adesão e expressão das moléculas de adesão na superfície dos neutrófilos usando inibidor de Rac1, porém o composto Y-27632, inibidor de ROCK (Rho-associated coiled coil forming protein serine/threonine kinase), proteína efetora de RhoA, mostrou aumentar a adesão dos neutrófilos sob condições basais. O uso da sinvastatina modulou as propriedades adesivas e a expressão de Mac-1 nos neutrófilos na presença de um estímulo inflamatório, apoiando evidências de seu uso como anti-inflamatório. Em destaque foi observado que o Y-27632 reverteu o efeito da sinvastatina sob estímulo de TNF-? e que o mevalonato e os isoprenóides intermediários da via do colesterol, GGPP e FPP, não foram capazes de reverter o efeito da sinvastatina. Dados indicam que a via de sinalização dependente em NO-GMPc aparentemente não modula as propriedades adesivas dos neutrófilos sob condições inflamatórias. Por outro lado, resultados indicam que as Rho GTPases parecem estar envolvidas na regulação das propriedades adesivas dos neutrófilos sob condições inflamatórias. O envolvimento de ROCK na adesão celular ainda não está completamente compreendido, mas de acordo com nossos resultados podemos sugerir a hipótese de que esta enzima efetora tenha um papel na inducão de adesão dos neutrófilos na presença de um estímulo inflamatório. A sinvastatina foi capaz de inibir as propriedades adesivas de neutrófilos quando ativados indicando mais uma utilidade desta classe de drogas no tratamento de doenças inflamatórias. O papel das Rho GTPases nas propriedades adesivas dos neutrófilos sob condições inflamatórios ainda precisa ser melhor elucidado / Abstract: During the inflammatory response, neutrophils and other leukocytes adhere to the endothelium leaving the blood vessels and actively moving towards the inflammatory focus. The migration of neutrophils to inflammatory sites depends on a variety of chemotactic and adhesive events resulting from the activation of adhesion molecules such as selectins and integrins and chemokine receptors. Due to its properties, neutrophils may be activated by small intracellular signaling proteins, the Rho GTPases, which help neutrophils to fulfill this function by interfering in cytoskeletal changes. These proteins are also involved in cell adhesion and proliferation. The neutrophils are able to synthesize nitric oxide (NO), and this production of NO is an important component of innate immunity during inflammation. Studies have shown that neutrophils play a role in inducing inflammation and generation of efforts to understand the adhesive mechanism exerted by neutrophils in inflammatory processes may be a key point for pharmacological interventions for diseases that are characterized by vascular inflammation with consequent obstruction of blood flow. Thus, this study aimed to evaluate the role of the NO pathway and the Rho GTPases in the mechanism by which inflammatory stimuli increases neutrophil adhesion. We also assessed the effects of simvastatin on neutrophil adhesive properties as a tool to aid in studying the involvement of Rho GTPases, RhoA and Rac1 in these mechansims. Neutrophils were isolated from peripheral blood and aspects of adhesion studied by static and flow adhesion assays as well as flow cytometry. In addition, we analyzed the gene expression of Rho GTPases, Rac1 and RhoA by real time - PCR. Following a strong stimulation with TNF-?, the adhesive properties of neutrophils increase significantly. NO synthase inhibitors and NO donors did not modify the adhesive properties of neutrophils when stimulated with TNF-?. We did not observe any significant differences in the adhesion of neutrophils and the expression of adhesion molecules on their surface in the presence of a Rac1 inhibitor. However, an inhibitor of ROCK (Rho-associated coiled coil forming protein serine/threonine kinase, an efector protin for the RhoA), Y-27632, was shown to increase the adhesion of neutrophils under basal conditions. The use of simvastatin decreased adhesive properties and modulated the expression of Mac-1 of neutrophils in the presence of an inflammatory stimulus, supporting the use of this class of drugs as anti-inflammatory agents. Importantly, the attenuating effects of simvastatin on TNF-? stimulated neutrophil adhesion were reversed by Y-27632, whereas the cholesterol pathway intermediates, mevalonate, and the isoprenoids, GGPP FPP, were unable to reverse the effects of this drug. Data indicate that the NO-cGMP signaling pathway does not appear to modulate the adhesive properties of neutrophils under inflammatory conditions. Moreover, results suggest that Rho GTPases may be involved in the regulat ion of the adhesive properties of neutrophils. The involvement of ROCK in cellular adhesion is not yet fully understood but, according to our findings, it may be hypothesized that this protein effector has a role in the induction of neutrophil adhesion. Simvastatin was able to inhibit the adhesive properties of neutrophils when activated, indicating another use of this class of drugs for the treatment of inflammatory diseases. The role of Rho GTPases in the adhesive properties of neutrophils under inflammatory conditions should be further elucidated / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

The function of GTP-binding proteins (G-proteins) in diverse intracellular pathways depends on their ability to switch between two forms, a GDP-bound (inactive) form and a GTP bound (active) form in a highly regulated GTPase cycle. The inactivation step of this cycle is regulated by GTPase-activating proteins (GAPs) which increase the intrinsic rate of hydrolysis of bound GTP; the activation step is regulated by a diverse family of GDP/GTP exchange factors (GEFs). A unique model system, which consists of the 13 kDa GEF Mss4 and the monomeric G protein Rab3A involved in presynaptic neurotransmission, was chosen to study the mechanism of G-protein regulation. Structure of Rab3A at high resolution The 2.0 Å crystal structure of Rab3A, bound to a non-hydrolyzable GTP-analog (GppNHp), enables a detailed description of the structural determinants that stabilize the active conformation and regulate GTPase activity within the Rab family. Although the overall structure is similar to that of GppNHp-bound Ras and other GTPases, localized but significant differences are observed in the vicinity of the conformational switch regions and the α3/β5 loop. The active conformation is stabilized primarily by extensive hydrophobic contacts between the switch I and II regions. Novel interactions with the γ phosphate, mediated by serine residues in the P-loop and switch I region, impose stereochemical constraints on the mechanism of GTP hydrolysis and provide a structural explanation for the broad range of GTPase activities within the Rab family. Residues implicated in interactions with effectors and regulatory factors map to a common face of the protein. The asymmetric distribution of charged and non-polar residues suggests a plausible orientation with respect to vesicle membranes that would position predominantly hydrophobic surfaces to interact with membrane-associated effectors and regulatory factors. Thus, the structure of Rab3A establishes a framework for understanding the molecular mechanisms underlying the function of Rab proteins in vesicle trafficking. High resolution structure of Mss4 and structure-based mutagenesis Activation of monomeric Rab GTPases, which function as ubiquitous regulators of intracellular membrane trafficking, requires the catalytic action of guanine nucleotide exchange factors. Mss4, an evolutionarily conserved Rab exchange factor, promotes nucleotide release from exocytic but not endocytic Rab GTPases. Chapter III describes the results of a high resolution crystallographic and mutational analysis of Mss4. The 1.65 Å crystal structure of Mss4 reveals a network of direct and water mediated interactions that stabilize a partially exposed structural sub-domain derived from four highly conserved but non-consecutive sequence elements. The conserved sub-domain contains the invariant cysteine residues required for Zn2+ binding as well as the residues implicated in the interaction with Rab GTPases. A strictly conserved DΦΦ motif, consisting of an invariant aspartic acid residue (Asp73) followed by two bulky hydrophobic residues (Met74 and Phe75), encodes a prominently exposed 310 helical turn in which the backbone is well ordered but the side chains of the conserved residues are highly exposed and do not engage in intramolecular interactions. Substitution of any of these residues with alariine dramatically impairs exchange activity towards Rab3A, indicating that the DΦΦ motif is a critical element of the exchange machinery. In particular, mutation of Phe75 results in a defect as severe as that observed for mutation of Asp96, which is located near the zinc binding site at the opposite end of Rab interaction epitope. Despite severe defects, however, none of the mutant proteins is catalytically dead. Taken together, the results suggest a concerted mechanism in which distal elements of the conserved Rab interaction epitope cooperatively facilitate GDP release. The basis for selective recognition of exocytic Rab family GTPases by Mss4 Rab3A is involved in Ca2+ -dependent exocytosis and neurotransmitter release. Mss4, an evolutionarily conserved Rab exchange factor, promotes nucleotide release from exocytic RabGTPase (Rab1, Rab3A, Rab8, and Rab10, Sec4 and Ypt1) but not endocytic Rab GTPases (Rab2, Rab4, Rab5, Rab6, Rab9 and Rab11). To understand the basis for selective recognition of exocytic Rab family GTPases by Mss4, a structure based mutagenesis study of Rab3A was conducted. Three residues in Rab3A (Phe51, Val61 and Thr89) were found to be critical for interaction with Mss4. Phe51 is located at the N- terminus of the switch region, adjacent to the Mg2+ and nucleotide binding site. Val61 in the β2 strand and Thr89 in the switch II region flank a triad of hydrophobic residues that is conserved in the Rab family. These residues comprise critical determinants underlying the broad specificity of Mss4 for exocytic Rab family proteins. In addition to determining the high resolution crystal structures of Rab3A and Mss4, the experiments described above identify critical structural determinants for the exchange activity of Mss4 and provide insight into the selective recognition of Mss4 by exocytic Rab GTPases.

The dynamic processes that occur at specific times and locations in cells and/or whole organisms during cellular division, migration, morphogenesis and development are critical. When these molecular events are not properly regulated, disease states can develop. Tools that can allow us to better understand the specific events that, when misregulated, result in disease development can also allow us to determine better ways to combat such misregulation. Specifically, tools that could allow us to better visualize cellular processes or those that allow us to control cellular functioning in a spatiotemporal manner could present great insight into the detailed inner workings of cells and/or whole organisms. Where chemistry and biology intersect presents a powerful starting point for the development of such tools. The first half of this thesis addresses tools to allow the better visualization of cellular events, in particular the intriguing process of bioluminescence and the work that has been done to better understand and optimize its utilization, particularly in living organisms. The novel work presented here details a parallel approach to improve our ability to observe cellular functioning specifically by improving bioluminescence imaging through the generation and characterization of mutant luciferase proteins that can better utilize novel small molecule luciferin substrates. The second half of this thesis discusses methods that have been developed to better control cellular events through the control of protein activity, specifically a family of proteins called the Rho GTPases. This family’s activation at specific times and locations is essential to proper cellular function and exemplifies the need for spatiotemporal control. Described are methods to control the activation states of the Rho GTPases to probe their cellular roles in a temporal and spatial manner using photosensitive small molecules. Taken together, the findings described herein demonstrate the application of chemistry to allow for the better observation and control of cellular processes, toward the ultimate goal of improving our understanding of the regulatory processes involved in the control of key factors leading to disease states.

No description available.

Os tumores hipofisários representam cerca de 10% a 15% das neoplasias intracranianas. Embora a etiopatogenia ainda não seja plenamente caracterizada, muitos mecanismos moleculares envolvidos na tumorigênese hipofisária já foram desvendados. Utilizandose da metodologia de arranjos de cDNA contendo aproximadamente 20.000 genes, nosso grupo recentemente comparou a expressão de duas condições distintas: um pool de quatro adenomas hipofisários clinicamente não funcionantes e a metástase de um carcinoma hipofisário não funcionante. Vários genes mostraram-se diferencialmente expressos, entre eles, CRABP1 (cellular retinoic acid binding protein 1), CRABP2 (cellular retinoic acid binding protein 2), GRP (gastrin-releasing peptide) e RERG (RAS-like, estrogen-regulated, growth inhibitor). Este estudo visou avaliar a expressão desses quatro genes em uma série de 59 adenomas hipofisários (30 adenomas clinicamente não funcionantes, 13 somatotrofinomas, 8 corticotrofinomas e 8 prolactinomas), comparando cada grupo tumoral com um conjunto de tecidos hipofisários normais. Enquanto os prolactinomas demonstraram expressão reduzida do RNAm dos genes CRABP1 e CRABP2 quando comparados ao grupo de tecidos normais, os somatotrofinomas apresentaram expressão reduzida apenas do RNAm de CRABP2. Os adenomas clinicamente não funcionantes, por sua vez, demonstraram menor expressão do RNAm de GRP e maior expressão do RNAm de RERG quando comparados ao grupo de hipófises normais. Portanto, observou-se que tanto o gene CRABP1 quanto os genes CRABP2, GRP e RERG apresentaram diferenças na expressão do transcrito entre os grupos de adenomas de hipófise, contudo, seu papel na tumorigênese hipofisária permanece a ser investigado. / Pituitary tumors account for approximately 10%-15% of the intracranial neoplasms. Although the pathogenesis is not fully characterized, many molecular mechanisms involved in pituitary tumorigenesis have been unraveled. Using the methodology of cDNA microarray containing approximately 20000 genes, our group recently compared the expression of two distinct conditions: a pool of four clinically nonfunctioning pituitary adenomas and a spinal cord metastasis of a nonfunctioning pituitary carcinoma. Several genes were shown to be differentially expressed, among them, CRABP1 (cellular retinoic acid binding protein 1), CRABP2 (cellular retinoic acid binding protein 2), GRP (gastrin-releasing peptide) and RERG (RAS-like, estrogen-regulated, growth inhibitor). This study aimed to evaluate the expression of these four genes in a series of 59 pituitary adenomas (30 nonfunctioning, 13 GH-secreting, 8 ACTH-secreting and 8 PRL-secreting adenomas), comparing each tumor group with a set of normal pituitary tissues. While PRL-secreting adenomas showed lower expression of CRABP1 and CRABP2 mRNA when compared with normal tissues, GH-secreting adenomas had only lower expression of CRABP2 mRNA. Clinically nonfunctioning adenomas showed lower expression of GRP mRNA and higher expression of RERG mRNA when compared with the normal pituitary glands. Therefore, it was observed that not only the CRABP1 gene but also the CRABP2, GRP and RERG genes showed differences in transcript expression between the groups of pituitary adenomas. However, their role in pituitary tumorigenesis remains to be investigated.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Also available online. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 187 pages. Includes vita. Includes bibliographical references.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 106-117.

Thesis (Ph.D.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed ). Includes bibliographical references.

Os tumores hipofisários representam cerca de 10% a 15% das neoplasias intracranianas. Embora a etiopatogenia ainda não seja plenamente caracterizada, muitos mecanismos moleculares envolvidos na tumorigênese hipofisária já foram desvendados. Utilizandose da metodologia de arranjos de cDNA contendo aproximadamente 20.000 genes, nosso grupo recentemente comparou a expressão de duas condições distintas: um pool de quatro adenomas hipofisários clinicamente não funcionantes e a metástase de um carcinoma hipofisário não funcionante. Vários genes mostraram-se diferencialmente expressos, entre eles, CRABP1 (cellular retinoic acid binding protein 1), CRABP2 (cellular retinoic acid binding protein 2), GRP (gastrin-releasing peptide) e RERG (RAS-like, estrogen-regulated, growth inhibitor). Este estudo visou avaliar a expressão desses quatro genes em uma série de 59 adenomas hipofisários (30 adenomas clinicamente não funcionantes, 13 somatotrofinomas, 8 corticotrofinomas e 8 prolactinomas), comparando cada grupo tumoral com um conjunto de tecidos hipofisários normais. Enquanto os prolactinomas demonstraram expressão reduzida do RNAm dos genes CRABP1 e CRABP2 quando comparados ao grupo de tecidos normais, os somatotrofinomas apresentaram expressão reduzida apenas do RNAm de CRABP2. Os adenomas clinicamente não funcionantes, por sua vez, demonstraram menor expressão do RNAm de GRP e maior expressão do RNAm de RERG quando comparados ao grupo de hipófises normais. Portanto, observou-se que tanto o gene CRABP1 quanto os genes CRABP2, GRP e RERG apresentaram diferenças na expressão do transcrito entre os grupos de adenomas de hipófise, contudo, seu papel na tumorigênese hipofisária permanece a ser investigado. / Pituitary tumors account for approximately 10%-15% of the intracranial neoplasms. Although the pathogenesis is not fully characterized, many molecular mechanisms involved in pituitary tumorigenesis have been unraveled. Using the methodology of cDNA microarray containing approximately 20000 genes, our group recently compared the expression of two distinct conditions: a pool of four clinically nonfunctioning pituitary adenomas and a spinal cord metastasis of a nonfunctioning pituitary carcinoma. Several genes were shown to be differentially expressed, among them, CRABP1 (cellular retinoic acid binding protein 1), CRABP2 (cellular retinoic acid binding protein 2), GRP (gastrin-releasing peptide) and RERG (RAS-like, estrogen-regulated, growth inhibitor). This study aimed to evaluate the expression of these four genes in a series of 59 pituitary adenomas (30 nonfunctioning, 13 GH-secreting, 8 ACTH-secreting and 8 PRL-secreting adenomas), comparing each tumor group with a set of normal pituitary tissues. While PRL-secreting adenomas showed lower expression of CRABP1 and CRABP2 mRNA when compared with normal tissues, GH-secreting adenomas had only lower expression of CRABP2 mRNA. Clinically nonfunctioning adenomas showed lower expression of GRP mRNA and higher expression of RERG mRNA when compared with the normal pituitary glands. Therefore, it was observed that not only the CRABP1 gene but also the CRABP2, GRP and RERG genes showed differences in transcript expression between the groups of pituitary adenomas. However, their role in pituitary tumorigenesis remains to be investigated.