Thesis (Ph.D.). / Title from title page of PDF (viewed 2008/01/30). Written for the Division of Experimental Medicine. Includes bibliographical references.

Insulin receptor (IR) exists in two isoforms (IR-A and IR-B), which differ in the tissue distribution and probably also in their function, i.e. in their response to insulin binding. It is supposed that IR-A activates mainly mitogenic processes and that IR-B triggers mainly metabolic effects resulting in the uptake of glucose by muscle and fat cells. Insulin can also weakly bind to the receptor for IGF-1 (IGF-1R), a growth factor involved in the regulation of growth and development. Insulin derivatives selectively binding only to one of the receptors would be interesting for the study of the receptors but also potentially for the treatment of diseases such as diabetes or cancer. Here we used our experience in the structure-activity studies of insulin for the design, synthesis and biological characterization of 4 new insulin derivatives in order to modify their selectivity towards the individual receptors. We systematically modified insulin by amidation of the C-terminus of its B-chain or by prolongation of the B-chain by 1-3 carboxyamidated glycine residues. Binding affinities of all new analogues for IR-A and IR-B were determined and for some of the analogues binding affinities for IGF-1R as well. Finally, abilities of analogues to activate autophosphorylation of intracellular subunits of IR-A and...

Submitted by Tatiana Silva (tsilva@icict.fiocruz.br) on 2012-12-27T17:57:17Z No. of bitstreams: 1 luciana_s_rodrigues_ioc_bcm_0026_2010.pdf: 8319063 bytes, checksum: 23ce271789168f4d9723b9a7df6ffe11 (MD5) / Made available in DSpace on 2012-12-27T17:57:17Z (GMT). No. of bitstreams: 1 luciana_s_rodrigues_ioc_bcm_0026_2010.pdf: 8319063 bytes, checksum: 23ce271789168f4d9723b9a7df6ffe11 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil / A lesão neural é uma das principais consequências da hanseníase e responsável pela instal ação de deformidades e incapacidades físicas, al ém de contribuir para o esti gma da doença. O dano ao nervo é exacerbado com o desenvolvimento de episódi os reaci onais (Ti po I e Ti po II) e está correlaci onado à resposta imunol ógi ca desenvolvida pel o indivíduo, contra o Mycobacterium leprae – agente eti ol ógico da hanseníase que apresenta especi al tropismo por macrófagos e células de Schwann (CS) nos nervos periféri cos. Os fatores de crescimento semelhante à Insulina (IGFs) são hormôni os pept ídi cos implicados no metabolismo, indução de proliferação, inibição de apoptose e diferenciação de diferentes ti pos cel ulares. Evi dências da li teratura apontam também propri edades imunomoduladoras e anti -inflamatóri as do IGF-I. O objetivo do presente estudo vi sa a invest i gação da parti cipação do si stema IGF na infecção pel o M. leprae. Ini cialmente, verificamos o efei to anti -apoptóti co da bactéri a sobre CS humanas primárias e da linhagem ST88-14 cul t ivadas em condições livres de soro pel a inibição da at ivação de caspase-3. Demonstramos, ainda, através de ensai os de imunoci toquímica, que o bacil o é capaz de induzir a proliferação da CS, tal efei to provavelmente mediado pel a indução de IGF-I, confi rmado pel a técnica de RT-PCR quant i tativo e pel a detecção da proteína em sobrenadantes de cul tura através de ensai o imunoenzimát i co. Na segunda etapa do trabalho, avaliamos a part i cipação do IGF-I ci rculante na evol ução natural da hanseníase. Utilizando ELISA quimi oluminescente, quantificamos os níveis de IGF-I, da principal proteína ligadora de IGF (IGFBP-3) e TNF-a no soro de indivíduos sadi os e pacientes que desenvolveram ou não quadros reacionais ao l ongo do tratamento. No caso dos pacientes reaci onais, a dosagem de IGF-I, IGFBP-3 e TNF-a fo i realizada em duas etapas: i ) no momento do diagnóstico e ii ) durante o aparecimento da reação, antes do tratamento específico. Ini cialmente, numa comparação entre paci entes que não desenvolveram reação, verificamos que 81% e 72% dos paci entes lepromatosos (LL) apresentaram níveis de IGF-I e IGFBP-3, respectivamente, abaixo do normal por i dade, diferentemente dos paci entes com outras formas clínicas. Dentre os pacientes reaci onais, 93% e 86% do grupo BL também apresentou níveis de IGF-I e IGFBP-3, respectivamente, abaixo do normal por i dade, diferentemente do grupo BL não-reaci onal , que apresentou níveis de IGFs similares aos indivíduos sadi os. Durante o desenvolvimento dos epi sódi os reaci onais, houve uma queda dos níveis de IGF-I, IGFBP-3 e da rel ação IGF/TNF-a no grupo LL com reação ti po II. Já no grupo de paci entes BL, observamos um aumento dos níveis de IGF-I e IGFBP-3, como uma tentativa de alcançar os níveis normais. Nossos dados sugerem a xv parti cipação destes fatores de crescimento endócrinos na ínt ima relação entre bacil o e a CS, como uma estratégi a de obtenção e manutenção de um nicho favorável de mul t iplicação e, ainda, os revelam como potenci ais candidatos a bi omarcadores dos episódi os reaci onais na hanseníase. / Neural injury is a maj or consequence of l eprosy and responsible for the disabili t ies installat i on, beyond to contribute to the st i gma of the disease. The nerve damage is exacerbated by the devel opment or l eprosy react i ons (Type I and Type II) and is correl ated to the immune response against Mycobacterium leprae – et i ol ogi c agent of leprosy that has especial tropism for macrophages and Schwann cells (SC) in peri pheral nerves. Insulin-like growth factors (IGFs) are pept i de hormones involved in metabolism, proliferati on induct i on, apoptosis inhibi t i on and cell different i at i on. Evidence from the li t erature also indicate immunomodulatory and ant i -inflammatory properti es of IGF-I. The aim of this study is to investi gate the involvement of IGF system in the M. leprae infect i on. Ini t ially, we verified the ant i -apoptoti c effect of the bacteria on human primary CS and ST88-14 lineage growing in serum-f ree condi t i ons by inhibi t ing caspase-3 activat i on. It was also demonstrated by immunocytochemistry, that the bacill us is able to induce the SC proliferat i on, this effect i s probably mediated by induct i on of IGF-I, as verified by quant i tative RT-PCR and confirmed by protein detecti on supernatants using immunoenzimat i c assay (ELISA). On the second phase we evaluate the parti cipat i on of ci rculating IGFs in the natural course of leprosy. Through chemi ol uminescent ELISA, we quantified the IGF-I, the main IGF binding protein (IGFBP-3) and TNF-a serum levels in heal t hy individuals and pat i ents who devel oped or not reacti onal states during the treatment. In the case of react i onal pat i ents, the IGF-I, IGFBP-3 and TNF-a was performed in two steps: i ) at the diagnosis of leprosy and ii) during react i onal episode, pri or to specific treatment. Ini t i ally, a comparison of nonreact i onal pat i ents, we found that 81% and 72% of lepromatous l eprosy (LL) showed IGF-I and IGFBP-3 levels, respectively, bel ow normal for age, unlike pat i ents wi th other clinical forms. Among the reacti onal pat i ents, 93% and 86% of BL group also showed IGF-I and IGFBP-3 levels, respectively, bel ow normal for age, unlike the nonreact i onal BL group, wi ch showed similar levels of IGFs to heal thy individuals. During the devel opment of reacti onal episodes, there was a decrease in the levels of IGF-I, IGFBP-3 and the IGF/TNF-a rat i o in the LL group wi t h type II reacti on. In the BL group undergoing type II reacti on, the IGF-I and IGFBP-e levels increased, as an attempt to reach normal levels. Our data suggest the involvement of these growth factors in the relat i onship between bacilli and CS as a strategy for obtaining and maintaining a favorable niche for mul t iplicat i on, and also reveal the IGFs as potencial candidates for bi omarkers of react i onal episodes in leprosy.

Made available in DSpace on 2014-06-11T19:22:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-05-04Bitstream added on 2014-06-13T19:28:18Z : No. of bitstreams: 1 leme_jaca_me_rcla.pdf: 614425 bytes, checksum: bf0825069c6f13243a70965f900b9372 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Para investigar os efeitos do treinamento físico aeróbio nas concentrações de IGF-I em ratos diabéticos, ratos wistar foram distribuídos em quatro grupos: controle sedentário, controle treinado, diabético sedentário e diabético treinado. O diabetes foi induzido por aloxana (32 mg/kg) e o protocolo de treinamento consistiu de natação 1 hora/dia, 5 dias/semana, durante 8 semanas e suportando 5% do peso corporal. Durante o período experimental foram registrados semanalmente o peso, ingestão de água e comida. Na 7ª semana foram determinadas a glicemia e insulinemia em jejum para cálculo do índice Homa. Ao final deste período, os ratos foram sacrificados e o sangue foi coletado para determinação da glicose, insulina, albumina, triglicerídeos séricos e hematócrito. Amostras dos músculos gastrocnêmio e sóleo foram coletadas para determinação do glicogênio. Amostras do miocárdio foram utilizadas para determinar glicogênio e triglicerídeos e do fígado para determinar as concentrações de glicogênio, triglicerídeos, proteína, DNA e IGF-I. O diabetes aumentou a glicemia em jejum e em estado alimentado, triglicerídemia, glicogênio e triglicerídeos cardíacos além do DNA hepático. A doença ainda reduziu insulinemia em jejum e em estado alimentado, razão proteína/DNA hepática e concentrações de IGF-I no sangue e fígado. O protocolo de treinamento físico reduziu nos animais diabéticos a glicemia em jejum e em estado alimentado, trigliceridemia, glicogênio e triglicerídeos cardíaco além do DNA hepático. O treinamento, por outro lado, aumentou glicogênio muscular e recuperou a razão proteína/DNA hepático e IGF-I hepático e sérico nos animais diabéticos. Em conclusão, treinamento físico moderado melhora as condições metabólicas e endócrinas, particularmente no eixo GH-IGF, em ratos diabéticos. / To investigate the influence of aerobic physical training on IGF-I concentrations in diabetic rats, male wistar rats were allocated into four groups: sedentary control, trained control, sedentary diabetic and trained diabetic. Diabetes was induced by Alloxan (32 mg/kg b.w.) and training protocol consisted of swimming 1hour/day, 5 days/week, during 8 weeks, supporting 5% b.w. During the experimental period, rats weight, water and food ingestion were weekly colected. In the 7th week, blood was collected for glicose and insulin in fasting to Homa index determination. At the end of this period, rats were sacrificed and blood was collected for determinations of serum glucose, insulin, albumin, triglycerides, IGF-I and hematocrit. Gastrocnemius and soleus muscle samples were collected for glycogen determination. Samples of myocardium were used to determine glycogen and triglyceride contents and of liver to determine glycogen, triglyceride, protein, DNA and IGF-I concentrations. Diabetes increased fasting and fed state glycemia, triglyceridemia, cardiac glycogen and triglyceride younder hepatic DNA. The disease still reduced fasting and fed state insulinemia, hepatic ratio protein/DNA and IGF-I concentrations in blood and liver. Physical training protocol was able to reduce fasting and fed state serum glucose, triglyceridemia, cardiac glycogen and trygliceride and hepatic 92 DNA, to increase gastrocnemius muscle glycogen and to recover hepatic ratio protein/DNA and blood and hepatic IGF-I concentrations in diabetic animals. In conclusion, moderate physical training improved the metabolic and endocrine conditions, particulary in GH-IGF axis, in diabetic rats.

During pregnancy, increased uterine blood-flow and efficient placental perfusion is essential for a successful outcome. Despite the essential role of these vascular beds, data on the physiological mechanisms involved in the maintenance of a high-flow/low resistance circulation within the uterus and placenta are limited. The need to fully understand the regulation of blood-flow within the uterine and feto-placental circulations is further highlighted by pathological pregnancies which are characterised by vascular dysfunction within these circulations. Oestrogen and insulin-like growth factor-I (IGF-I) levels increase during pregnancy and correlate with increased uterine blood flow. In vivo and in vitro studies of other vascular beds show that both 17-β oestradiol and IGF-I act as vasodilators. However, surprisingly little is known of their vaso-active effects on human uterine and placental arteries. The aims of the studies described within this thesis, were to investigate, ex vivo, the possible roles of oestrogen and IGF-I in regulating human placental and uterine vascular beds in vivo. Placental chorionic plate arteries and myometrial demonstrated acute vasodilation in response to oestrogen. Vascular bed differences in ER-responsiveness were observed; vasodilation within myometrial arteries was elicited by both oestrogen receptors, ERα and ERβ, although activation of the latter receptor generated a greater response. In contrast, oestrogen-dependent acute vasodilation of placental arteries was via ERβ alone. Furthermore, species differences, between human and rat arteries, were demonstrated in terms of ER-responsiveness. The predominant ER receptor within human arteries studied was ERβ, whilst rat arteries demonstrated a predominantly ERα-mediated mechanism of oestrogen-induced vasodilation. The data presented suggests that within the uterine vascular bed, oestrogen-induced vasodilation involves both an endothelium-dependent and –independent mechanism of action, whilst within the placenta, oestrogen-mediated vasodilation is endothelial-independent. Indeed, data suggests that oestrogen influences the level of intracellular calcium of vascular smooth cells to induce vasodilation of placental arteries.IGF-I did not have a vaso-active effect on chorionic plate arteries isolated from the placenta. However, uterine myometrial arteries exhibited reduced vaso-reactivity in the presence of IGF-I, demonstrated by a depressed response to the vasoconstrictor, U46619. Collectively, these data contribute towards a further understanding of the regulatory mechanisms of the uterine circulation, by identifying oestrogen and IGF-I as possible regulators of the uterine vasculature during pregnancy. Additionally, oestrogen may also have a role in controlling the feto-placental circulation. In the future, targeting ERb may offer a therapeutic strategy for increasing uterine/placental perfusion in pregnancies complicated by vascular dysfunction.

Glucose transporter 3 (GLUT3), while first found in human fetal muscle, is predominantly expressed in brain and neural tissue. By several independent techniques we have previously shown that GLUT3 is expressed in human skeletal muscle cells. The structure of the human GLUT3 gene has not been previously reported nor has there been any evaluation of the 5′-untranslated region (UTR). To this end, we have cloned and sequenced the human GLUT3 gene. Insulin-like growth factor-1 (IGF-1) increased endogenous Glut3 protein in cultured L6 myotubes, and similarly stimulated luciferase activity in a construct of the human GLUT3 5′-UTR linked to a luciferase reporter gene. Actinomycin D, an inhibitor of mRNA synthesis, prevented IGF-1 stimulation of Glut3 protein. Transfection of L6 cells with Sp1 increased Glut3 and augmented IGF-1 stimulation of Glut3 expression. Knockdown of Glut3 expression in cultured L6 muscle cells using small interference RNA (siRNA) specific for Glut3 significantly reduced myocyte glucose uptake. DNAse footprinting and gel shift assays showed Sp1 specifically bound to the human GLUT3 5′-UTR. Substitution mutants of the human GLUT3 5′-UTR luciferase construct indicated that only one of three Sp1 site clusters was involved in IGF-1 action. These data, using both a human GLUT3 5′-UTR construct and L6 cells' endogenous promoter, suggest that IGF-1 plays a role in maintaining muscle GLUT3 expression and basal glucose uptake via the transcriptional factor Sp1.

Spinal Muscular Atrophy (SMA) is a debilitating neurodegenerative disease resulting in death of the lower motor neurons, muscle atrophy, and in severe cases death. Due to mutations or deletions in the Survival Motor Neuron 1 (SMN1) gene, levels of functional SMN protein product are decreased. While SMA was previously described as a motor neuron exclusive disorder, recent evidence suggests that many tissue and cell types throughout the body are affected. The objective of our study was to outline the effects of varying levels of SMN depletion on two genes of interest, namely Insulin-like growth factor 1 (Igf-1) and Tumor suppressor protein 53 (Trp53) in multiple tissues throughout disease course. The severe Smn2B/- and mild Smn2B/-; SMN2+/- mouse models of SMA were utilized in our studies to determine the levels of mRNA expression and subsequent protein output for these two genes. We employed RT-qPCR, western blot, and ELISA experimental methods. In Smn2B/- mice, Igf-1 mRNA was substantially decreased in symptomatic liver tissue. This was accompanied by widespread decrease in IGF-1 protein in peripheral tissues. Interestingly, this depletion effect on Igf-1 was not observed in the mild mouse model. Our analysis also showed that Trp53 mRNA was dramatically increased within tibialis anterior skeletal muscle of symptomatic Smn2B/- mice, alongside an upregulation of factors involved in p53 mediated apoptosis. Once again, this effect was not observed in the mild Smn2B/-; SMN2+/- mouse model. Overall, we have demonstrated that the extent of SMN depletion, determines whether the expression of Igf-1 and Trp53 is perturbed, suggesting that disease severity is an important factor in what pathways are affected. Finally, we show that alterations in gene expression patterns or subsequent protein levels act in a tissue-specific fashion. More investigation is encouraged to highlight IGF-1’s role as a potential SMN-independent therapeutic for SMA.

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