Submitted by Verena Bastos (verena@uefs.br) on 2015-08-05T22:08:06Z No. of bitstreams: 1 Disserta??o Mona Oliveira vers?o final Maio2003 PPGbiotec.pdf: 4793209 bytes, checksum: 5e62150796f4527c8d492f92de5041e2 (MD5) / Made available in DSpace on 2015-08-05T22:08:06Z (GMT). No. of bitstreams: 1 Disserta??o Mona Oliveira vers?o final Maio2003 PPGbiotec.pdf: 4793209 bytes, checksum: 5e62150796f4527c8d492f92de5041e2 (MD5) Previous issue date: 2013-05-08 / Funda??o de Amparo ? Pesquisa do Estado da Bahia - FAPEB / Glioblastomas (GBMs) are the most common and aggressive primary tumors of the CNS. The survival of patients with this diagnosis remains very low, with poor prognosis even after surgical therapy associated with radiotherapy and chemotherapy. The present work carried out a screening for 24 synthetic ?alkaloid-like? to determine their effects on cell viability of quimioresistentes human (Gl-15 and U251) glioblastoma cells and murine (C6) glioma cells. Among the alkaloids tested (100?M) RLB87 was the most cytotoxic for transformed cells, inhibiting the viability in 75.0% 97.2% 76.6% of GL-15, U251 and C6 cells, respectively, after 72 h exposure, and it did not show toxicity to normal glial cells. It was also observed that RLB87 promoted apoptosis, 24 and 72 h after treatment, in a time-dependent manner. Moreover RLB87 also inhibited cell migration and proliferation with cells arrest at G0/G1 phase, since 24 h after treatment. Additionally, the cytotoxicity of four RLB87 analogues was tested in view to elucidate important aspects in chemical structure, required for its activity. We observed positive correlation between cytotoxic effect and isomeric of phenyl functions, with ester function and also lipophilicity. These finding suggest the ?alkaloid-like? RLB87 as a promising anticancer agent as well as a prototype for new agents for treatment of malignant and recurrent gliomas. / Glioblastomas (GBMs) s?o os tumores prim?rios mais comuns e agressivos do SNC. A sobrevida dos pacientes com esse diagn?stico continua muito baixa, tendo progn?stico ruim mesmo ap?s terapia cir?rgica seguida de radio e quimioterapia. No presente trabalho, foi realizada a prospec??o de 24 mol?culas de s?ntese, alkaloids-like, para determina??o de seus efeitos sobre a viabilidade de c?lulas quimioresistentes de glioblastoma humano (GL-15 e U251) e murina (C6). Entre os compostos testados ? (100JM), RLB87 foi o mais citot?xico para c?lulas transformadas, inibindo a viabilidade em 75,0%, 97,2%, 76,6% da GL-15, U251 e C6, respectivamente e o mesmo n?o apresentou toxicidade para c?lulas gliais normais. Observou-se, que o RLB87 promoveu apoptose 24 e 72 h ap?s o tratamento de forma tempo-dependente. O RLB87 igualmente inibiu a prolifera??o celular com acumulo na fase G0/G1 do ciclo celular ap?s 24 h. A migra??o das c?lulas de glioma foi tamb?m inibida ap?s tratamento com RLB87. Adicionalmente 4 an?logos do RLB87 foram tamb?m avaliados, elucidando aspectos importantes na estrutura qu?mica, requeridos para sua atividade, que possuem correla??o positiva com regioisomeria das fenilas, presen?a da fun??o ?ster e lipofilicidade. O RLB87 ? apresentado como promissor agente antineopl?sico assim como um prot?tipo para novos agentes terap?uticos para o tratamento de gliomas malignos e recidivados.

Les protéines Vestigial-like constituent une famille de cofacteurs de transcription contenant un domaine très conservé, appelé Tondu, qui permet l’interaction avec les facteurs de transcription de la famille TEAD. L’état de l’art des connaissances actuelles sur cette famille, en termes de répertoire, de structure et de fonction des gènes dans les différents groupes d’animaux, a fait l’objet d’une revue. Durant la thèse, a été étudiée la fonction de deux gènes vestigial, vestigial-like 3 et vestigial-like 4, dans le modèle amphibien xénope. Ce choix découle d’une part, des travaux antérieurs de notre laboratoire qui a caractérisé la famille des gènes vestigial chez le xénope et d’autre part des avantages de ce modèle expérimental qui permet les analyses cellulaires et moléculaires. Les approches de gain et perte de fonction indiquent que vestigial-like 3 est plus particulièrement impliqué dans la migration des cellules de la crête neurale. Vestigial-like 4 a un rôle dans la neurogenèse précoce et la formation de la crête neurale. / Vestigial-like proteins belong to a transcription co factors family with a conserved domain, called tondu, which allows their interaction with TEAD family transcription factors. The state of the art on the current knowledge about this family in terms of gene repertory, structure and functions in different animals has given rise to a review. PhD work has focused on vestigial-like 3 and vestigial-like 4 genes functions in the Xenopus amphibian. This choice stemmed from the laboratory previous works that has described vestigial like gene family in Xenopus, and from the Xenopus model advantages that allows cellular and molecular analysis. Gain and loss of function approaches indicate that vestigial-like 3 is especially implicated in neural crest cells migration. Vestigial-like 4 plays a role in early neurogenesis and neural crest formation.

1 v. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1994

xxiii, 222 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Insulin-like growth factor-I circulates at high concentrations in blood, mainly complexed with IGF-binding proteins. The main objective of the thesis is to determine the general role played by plasma IGF-binding proteins in the regulation of IGF transfer from blood to tissues. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 1994

The insulin-like growth factor (IGF) system is composed of IGF, IGF binding proteins (IGFBP-1 to -10) and the acid labile subunit (ALS). IGF exists as two isoforms, IGF-I and IGF-II. IGF-I is the major circulatory form and is primarily secreted by the liver. It functions to regulate proliferation and differentiation in a number of different cell types and elicits an insulin-like metabolic effect. As well as being regulated at levels of transcription and translation, IGF-I activities are also regulated through formation of complexes in circulation. IGF complexes form as binary complexes, such as the IGFBP-1 complex, and ternary complexes containing IGF-I, IGFBP-3 and ALS. Binary and ternary IGF complexes function to maintain stable pools of bioactive IGF-I. They also function to increase IGF half-life and sequester IGF in the bloodstream. <p> ALS and IGFBP-1 are well characterized and exist as 85 kDa and 32 kDa proteins, respectively. They are expressed primarily in liver hepatocytes. Circulating ALS binds the IGF-I-IGFBP-3 complex and increases IGF half-life from 10 min in the IGFBP-3 binary complex to 10-15 hr in the ternary complex. IGFBP-1 binds IGF-I and increases the half-life from 10 min to 30 min. The ternary complex is the predominant IGF-I binding protein complex found in circulation. The IGFBP-1 complex represents only a small fraction of circulating IGF complexes. <p> In this thesis ALS and IGFBP-1 regulation were investigated in terms of expression related to metabolic modulators and streptozotocin (STZ)-induced diabetes. Results from rat studies showed a decreased liver ALS gene expression in STZ-induced diabetic rats. STZ-treatment in rats mimics type-I diabetes with no change in secreted insulin with increase of circulatory glucose. The administration of insulin into the STZ-induced diabetic rats brought ALS levels to that of the untreated controls. ALS expression was positively regulated by insulin in H4IIE hepatoma cells. Growth hormone (GH), glucose, dexamethasone also positively regulated ALS gene expression while cAMP (2-b-cAMP) acted as a negative regulator in H4IIE cells. HepG2 cells expressing constitutively active protein kinase B (PKB) (HepG2-PKB-CA) increased ALS gene expression to levels 20% higher then parental HepG2. Insulin treatment of these cells unexpectedly increased ALS levels in both parental and PKB-CA HepG2. This may have indicated a partial regulatory role of the mitogen activated protein (MAP) kinase pathway as PKB was thought to be over-expressed therefore rendering the insulin signal redundant. Inhibition of the phosphoinositol-3 (PI-3) kinase and MAP kinase pathways through wortmannin and PD98059 incubation, respectively, suggested a possible interplay or crosstalk between the two pathways in insulin signaling. PKB is known to be activated through the PI-3 kinase pathway. Results suggested possibility that PKB may interact through the MAP kinase pathway in regulation of ALS gene expression. The activity of cAMP on ALS gene expression may occur through interaction with the PI-3 kinase pathway as inhibition enhanced the negative effect of cAMP on ALS expression. <p> The secretion of IGFBP-1 was positively regulated by glucose and GH and negatively regulated by insulin in H4IIE cells. HepG2-PKB-CA cells showed significantly lower IGFBP-1 secretion as compared to parental HepG2 cells. The involvement of the PI-3 and MAP kinase pathways in the modulator-mediated effect on IGFBP-1 secretion were. As observed for ALS expression, the effect of insulin on IGFBP-1 secretion may also be affected through interplay or crosstalk between the PI-3 kinase and MAP kinase pathways. Glucose and GH effected IGFBP-1 expression and secretion independent of these pathways although glucose expression may interact in some way through the PI-3 kinase pathway. Our investigation of hepatic regulation of IGFBP-1 secretion and ALS gene expression has shown regulatory roles for the metabolic hormones tested, especially insulin. Mechanisms of cell signaling have also been approached with the use of pathway inhibiters and HepG2-PKB-CA cells. Much work is yet to be done to fully understand the effects of insulin and other hormones on the secretion and expression of IGFBP-1 and ALS.

The gut hormone glucagon-like peptide-2 (GLP-2) has numerous beneficial effects on the intestinal epithelium, including increased mucosal growth and proliferation. GLP-2 is also necessary for the adaptive intestinal re-growth that occurs upon re-feeding after fasting. Although insulin-like growth factor (IGF)-1 and the IGF-1 receptor are known to be required for GLP-2-induced crypt-cell proliferation, the precise cellular localization of the IGF-1 receptor through which the intestinotrophic actions of GLP-2 are mediated remains unknown. I hypothesized that small intestinal growth responses to GLP-2 occur through an intestinal epithelial IGF-1 receptor-dependent pathway, through the use of an inducible, intestinal epithelial-specific IGF-1 receptor knockout (IE-igf1rKO) mouse. Intestinal growth and proliferative responses were examined in IE-igf1rKO and control mice following treatment with GLP-2, as well as in animals that were fasted and re-fed to induce GLP-2-dependent adaptation. In Chapter 3, it was demonstrated that IE-igf1rKO mice, as compared to control littermates, had normal small intestinal weight, morphometric parameters, proliferative index and differentiated epithelial cell lineage distribution. Administration of GLP-2 for 30 minutes increased nuclear translocation of !-catenin in non-Paneth crypt-cells, and stimulated the crypt-cell proliferative marker c-Myc 90 minutes following GLP-2 treatment, in control littermates but not in IE-igf1rKO mice. In Chapter 4, adaptive re-growth was studied by fasting IE-igf1rKO and control animals for 24 hours, or by fasting and then re-feeding mice for 24 hours. Small intestinal weight, crypt depth, villus height and crypt-cell proliferation were decreased in both control and IE-igf1rKO mice after 24 hour fasting. While re-feeding in control mice restored all of these parameters, re-fed IE-igf1rKO mice displayed abrogated adaptive re-growth of the crypt-villus axis as well as reduced crypt-cell proliferation. In Chapter 5, control mice responded to chronic GLP-2 with increased small intestinal weight, mucosal cross-sectional area, crypt depth, villus height and crypt-cell proliferation. However, the GLP-2-induced increase in crypt-cell proliferation was absent in IE-igf1rKO mice, in association with impaired growth of the crypt-villus axis. Taken together, these results indicate that the proliferative responses of the intestinal epithelium to exogenous GLP-2 administration and during conditions of GLP-2-dependent adaptive re-growth are dependent on the intestinal epithelial IGF-1 receptor.

The gut hormone glucagon-like peptide-2 (GLP-2) has numerous beneficial effects on the intestinal epithelium, including increased mucosal growth and proliferation. GLP-2 is also necessary for the adaptive intestinal re-growth that occurs upon re-feeding after fasting. Although insulin-like growth factor (IGF)-1 and the IGF-1 receptor are known to be required for GLP-2-induced crypt-cell proliferation, the precise cellular localization of the IGF-1 receptor through which the intestinotrophic actions of GLP-2 are mediated remains unknown. I hypothesized that small intestinal growth responses to GLP-2 occur through an intestinal epithelial IGF-1 receptor-dependent pathway, through the use of an inducible, intestinal epithelial-specific IGF-1 receptor knockout (IE-igf1rKO) mouse. Intestinal growth and proliferative responses were examined in IE-igf1rKO and control mice following treatment with GLP-2, as well as in animals that were fasted and re-fed to induce GLP-2-dependent adaptation. In Chapter 3, it was demonstrated that IE-igf1rKO mice, as compared to control littermates, had normal small intestinal weight, morphometric parameters, proliferative index and differentiated epithelial cell lineage distribution. Administration of GLP-2 for 30 minutes increased nuclear translocation of !-catenin in non-Paneth crypt-cells, and stimulated the crypt-cell proliferative marker c-Myc 90 minutes following GLP-2 treatment, in control littermates but not in IE-igf1rKO mice. In Chapter 4, adaptive re-growth was studied by fasting IE-igf1rKO and control animals for 24 hours, or by fasting and then re-feeding mice for 24 hours. Small intestinal weight, crypt depth, villus height and crypt-cell proliferation were decreased in both control and IE-igf1rKO mice after 24 hour fasting. While re-feeding in control mice restored all of these parameters, re-fed IE-igf1rKO mice displayed abrogated adaptive re-growth of the crypt-villus axis as well as reduced crypt-cell proliferation. In Chapter 5, control mice responded to chronic GLP-2 with increased small intestinal weight, mucosal cross-sectional area, crypt depth, villus height and crypt-cell proliferation. However, the GLP-2-induced increase in crypt-cell proliferation was absent in IE-igf1rKO mice, in association with impaired growth of the crypt-villus axis. Taken together, these results indicate that the proliferative responses of the intestinal epithelium to exogenous GLP-2 administration and during conditions of GLP-2-dependent adaptive re-growth are dependent on the intestinal epithelial IGF-1 receptor.

The insulin-like growth factor (IGF) system is composed of IGF, IGF binding proteins (IGFBP-1 to -10) and the acid labile subunit (ALS). IGF exists as two isoforms, IGF-I and IGF-II. IGF-I is the major circulatory form and is primarily secreted by the liver. It functions to regulate proliferation and differentiation in a number of different cell types and elicits an insulin-like metabolic effect. As well as being regulated at levels of transcription and translation, IGF-I activities are also regulated through formation of complexes in circulation. IGF complexes form as binary complexes, such as the IGFBP-1 complex, and ternary complexes containing IGF-I, IGFBP-3 and ALS. Binary and ternary IGF complexes function to maintain stable pools of bioactive IGF-I. They also function to increase IGF half-life and sequester IGF in the bloodstream. <p> ALS and IGFBP-1 are well characterized and exist as 85 kDa and 32 kDa proteins, respectively. They are expressed primarily in liver hepatocytes. Circulating ALS binds the IGF-I-IGFBP-3 complex and increases IGF half-life from 10 min in the IGFBP-3 binary complex to 10-15 hr in the ternary complex. IGFBP-1 binds IGF-I and increases the half-life from 10 min to 30 min. The ternary complex is the predominant IGF-I binding protein complex found in circulation. The IGFBP-1 complex represents only a small fraction of circulating IGF complexes. <p> In this thesis ALS and IGFBP-1 regulation were investigated in terms of expression related to metabolic modulators and streptozotocin (STZ)-induced diabetes. Results from rat studies showed a decreased liver ALS gene expression in STZ-induced diabetic rats. STZ-treatment in rats mimics type-I diabetes with no change in secreted insulin with increase of circulatory glucose. The administration of insulin into the STZ-induced diabetic rats brought ALS levels to that of the untreated controls. ALS expression was positively regulated by insulin in H4IIE hepatoma cells. Growth hormone (GH), glucose, dexamethasone also positively regulated ALS gene expression while cAMP (2-b-cAMP) acted as a negative regulator in H4IIE cells. HepG2 cells expressing constitutively active protein kinase B (PKB) (HepG2-PKB-CA) increased ALS gene expression to levels 20% higher then parental HepG2. Insulin treatment of these cells unexpectedly increased ALS levels in both parental and PKB-CA HepG2. This may have indicated a partial regulatory role of the mitogen activated protein (MAP) kinase pathway as PKB was thought to be over-expressed therefore rendering the insulin signal redundant. Inhibition of the phosphoinositol-3 (PI-3) kinase and MAP kinase pathways through wortmannin and PD98059 incubation, respectively, suggested a possible interplay or crosstalk between the two pathways in insulin signaling. PKB is known to be activated through the PI-3 kinase pathway. Results suggested possibility that PKB may interact through the MAP kinase pathway in regulation of ALS gene expression. The activity of cAMP on ALS gene expression may occur through interaction with the PI-3 kinase pathway as inhibition enhanced the negative effect of cAMP on ALS expression. <p> The secretion of IGFBP-1 was positively regulated by glucose and GH and negatively regulated by insulin in H4IIE cells. HepG2-PKB-CA cells showed significantly lower IGFBP-1 secretion as compared to parental HepG2 cells. The involvement of the PI-3 and MAP kinase pathways in the modulator-mediated effect on IGFBP-1 secretion were. As observed for ALS expression, the effect of insulin on IGFBP-1 secretion may also be affected through interplay or crosstalk between the PI-3 kinase and MAP kinase pathways. Glucose and GH effected IGFBP-1 expression and secretion independent of these pathways although glucose expression may interact in some way through the PI-3 kinase pathway. Our investigation of hepatic regulation of IGFBP-1 secretion and ALS gene expression has shown regulatory roles for the metabolic hormones tested, especially insulin. Mechanisms of cell signaling have also been approached with the use of pathway inhibiters and HepG2-PKB-CA cells. Much work is yet to be done to fully understand the effects of insulin and other hormones on the secretion and expression of IGFBP-1 and ALS.

<p>Peptide drugs are an exciting class of pharmaceuticals currently in development for the treatment of a variety of diseases; however, their main drawback is a short half-life, which dictates multiple and frequent injections. We have developed two novel peptide delivery approaches -Protease Operated Depots (PODs) and GLP-1-ELP depots- to provide sustained and tunable release of a peptide drug from an injectable s.c. depot. </p><p>We demonstrate proof-of-concept of these delivery systems, by fusion of monomer or protease cleavable oligomers of glucagon-like peptide-1 (GLP-1), a type-2 diabetes peptide drug, and a thermally responsive, depot-forming elastin-like-polypeptide (ELP) that undergoes thermally triggered inverse phase transition below body temperature, thereby forming an injectable depot. Utilizing a novel system we designed for repetitive gene synthesis, various GLP-1 polymers were designed and tested as potential therapeutic payload for PODs. By attachment to various ELPs, designed to transition above or below body temperature, we created both depot forming GLP-ELP fusions and soluble control. All fusion constructs maintained alpha helical content and were shown to be resistant to proteolytic degradation. In vitro activated PODs and GLP-ELP fusions were able to activate the GLP-1 receptor and remarkably, a single injection of both GLP-1 PODs and GLP-ELP fusions were able to reduce blood glucose levels in mice for up to 5 days, 120 times longer than an injection of the native peptide drug. These findings suggest that ELP based peptide depots may offer a modular, genetically encoded alternative to various synthetic peptide delivery schemes for sustained delivery of peptide therapeutics.</p> / Dissertation

Thesis (Ph.D.)--University of Adelaide, Department of Animal Science, 1994.