Amylin, a 37 amino acid peptide secreted from pancreatic beta cells upon stimulation by meal/glucose, belongs to the family of the calcitonin or calcitonin gene-related peptide (CGRP) and shares up to 50% homology with CGRP, which is a well-documented pain-related peptide. The amylin receptor is composed of a calcitonin receptor (CTR) and receptor activity modifying proteins (RAMPs). Numerous studies have shown that amylin plays an important role in glucose homeostasis and food intake. Few studies have been conducted with respect to the effect of amylin in the central or peripheral neuraxis. In this thesis, immunohistochemical study revealed a dense network of amylin-immunoreactive (irAMY) cell processes in the superficial dorsal horn of the mice. Numerous dorsal root ganglion and trigeminal ganglion cells expressed moderate to strong irAMY. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed amylin receptor mRNA in the mouse spinal cord, brain stem, cortex, hypothalamus and hippocampus. The nociceptive or antinociceptive effects of amylin were evaluated in the tail flick and acetic acid-induced writhing test. Amylin (1-10 µg, i.t.) reduced the number of writhing in a dose-dependent manner. Pretreatment of the mice with the amylin receptor antagonist salmon calcitonin (8-32) [sCT(8-32)]or AC187 by i.t. antagonized the effect of amylin on acetic acid-induced writhing test. Locomotor activity was not significantly modified by amylin injected either i.p. (0.01-1 mg/kg) or i.t. (1-10 µg). Measurement of c-fos mRNA by RT-PCR or proteins by Western blot showed that the levels were up-regulated in the spinal cord of mice in acetic acid-induced visceral pain model and the increase was attenuated by pretreatment with amylin. Pretreatment of sCT[8-32] or AC187 significantly reversed the effect of amylin on c-fos expression in the spinal cord. As the neuronal response to amylin is closely dependent on the molecular property of amylin receptor, the localization, internalization and regulation of the calcitonin and amylin receptor were examined in the second part of the thesis. Immunofluorescence microscopy demonstrated the surface expression of CTRa, and intracellular distribution of RAMP1. Moreover, co-expression of CTRa translocated the RAMP1 to the cell surface and generated the amylin receptor phenotype. Both immunocytochemistry and on cell western analysis showed the internalization of CTRa and amylin receptor (CTRa/RAMP1) stimulated by different agonists, which was partially ß-arrestin dependent. Moreover, RAMP1 did not change the surface expression pattern of CTRa, but co-localized with the receptor with and without agonist treatment. sCT and amylin activated the ERK1/2 in HEK293 cells stably expressing amylin receptors, indicating the involvement of MAPK in amylin receptor signaling cascade. Collectively, these results led us to conclude that 1) irAMY is expressed in dorsal root ganglion neurons with their cell processes projecting to the superficial layers of the dorsal horn, and that the peptide by interacting with amylin receptors in the spinal cord may be antinociceptive; 2) RAMP1 does not change the pattern of CTR cell-surface localization and internalization, but receptor phenotype, presumably through a direct or indirect effect on the ligand-binding site; 3) amylin internalizes the amylin receptor (CTRa/RAMP1 complex); which is partially ß-arrestin dependent. Our studies extend the current knowledge of amylin on the spinal cord and new insight on the cellular and molecular mechanism underlying the antinociceptive effect of amylin. Also we demonstrate for the first time agonist induced-internalization of CTR/RAMP complex and its possible regulation pathway. / Pharmacology

G protein-coupled receptors (GPCRs) are composed of seven transmembrane domains and play an essential role in regulating physiological functions and mediating responses to environmental stimuli, biogenic amines, neurotransmitters, peptides, lipids, and hormones. The calcitonin-like diuretic hormone 31 (DH31) is known to elicit natriuresis from the Malpighian tubules (MTs) of mosquitoes Anopheles gambiae and Aedes aegypti upon blood feeding. However, the contribution of DH31 cognate receptor, calcitonin receptor 1 (GPRCAL1), has not been evaluated with respect to postprandial fluid regulation or myostimulatory activity in blood feeding insects. Thus, this dissertation has investigated potential roles of AedaeGPRCAL1 in the regulation of fluid homeostasis and hindgut muscle contraction in female A. aegypti mosquito. The full length cDNA encoding AedaeGPRCAL1 was cloned and sequenced. The receptor expression in the MTs and hindgut from female mosquito was analyzed by western blot and immunohistochemistry using anti-AedaeGPRCAL1 affinity purified antibodies, and subsequently its role in fluid transport and hindgut contraction was evaluated by RNA interference (RNAi). The mosquitoes that underwent knock-down of the AedaeGPRcal1 exhibited up to 57% lower rate of MT fluid secretion in presence of Aedae-DH31 in the in vitro assay and a ~30% reduction in the fluid excreted from live females upon blood feeding. The receptor was immunolocalized in principal cells, predominantly towards the distal end of MTs. Analyses of receptor signal probability indicate the receptor is expressed in a gradient-like fashion along the length of the MTs. A striking discovery was the fact that not all principal cells express the receptor, contrary to previous belief. Immunolocalization revealed the AedaeGPRCAL1 is expressed in hindgut circular and longitudinal muscles. The application of DH31 increased the frequency of hindgut contractions in all female mosquitoes, those injected with AedaeGPRcal1 dsRNA and controls, as compared to their basal contraction rate, but the percent change in frequency of hindgut contraction from AedaeGPRcal1 knock-down females was about 2-fold lower than the controls after application of Aedae-DH31. To my knowledge, this is first evidence of RNAi-induced phenotypes in any invertebrate that allowed the quantification of the contribution of single family B GPCR to fluid loss and muscle contractility.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-12-10T09:47:46Z No. of bitstreams: 2 Dissertação - Allison Filipe Lopes Martins - 2015.pdf: 3205167 bytes, checksum: 5c24397e18241a8a809af753bb233428 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-12-10T10:03:57Z (GMT) No. of bitstreams: 2 Dissertação - Allison Filipe Lopes Martins - 2015.pdf: 3205167 bytes, checksum: 5c24397e18241a8a809af753bb233428 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2015-12-10T10:03:57Z (GMT). No. of bitstreams: 2 Dissertação - Allison Filipe Lopes Martins - 2015.pdf: 3205167 bytes, checksum: 5c24397e18241a8a809af753bb233428 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-03-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The Central Giant Cell Lesion (CGCL) is an intraosseous lesion that can be classified into non aggressive and aggressive. Due to the aesthetic and functional defects of surgical treatment of CGCL, therapies with drugs have been reported, such as glucocorticoid injections and calcitonin. The studies reported in the literature support the use of these drugs through the investigation of the presence of glucocorticoid receptors (RGC) and calcitonin (RCT) in CGCL; however there is no consensus if all lesions express these receptors and if there is any difference between non aggressive and aggressive lesion. In addition, there are no studies that evaluated the bone formation potential through the investigation of Osteocalcin (OC) in aggressive and non-aggressive lesions. The aim of this study was to compare, using immunohistochemistry, the GR and CTR and osteocalcin protein (OC) expression in non aggressive (n = 20) and aggressive (n = 11) CGCL, and the correlation between the OC expression and these receptors determined in both groups of lesions. The number of mononuclear cells in mitosis (MOC), and the number of multinucleated giant cells (MGC) were also investigated using immunohistochemical techniques (hematoxylin and eosin). Our results show that all the cases express the GR and CTR and that there is no difference in the expression of these receptors or the number of mitosis between non aggressive and aggressive lesions. The OC expression was rare and higher in non aggressive lesions, however, not statistically significant (p> 0.05). There was a correlation between the CTR expression in MOC and MGC (r = 0.45; p <0.01). Considering the different variants of CGCL, there was a correlation between CTR expression in MOC and MGC in non aggressive lesions (r = 0.66; p <0.01) and between the CTR and OC expression in MGC (r = 0.718; p = 0.01). There was a higher number of MGC in aggressive lesions (p = 0.01). The results indicate that all cases express GR and CTR and that there are no differences between non aggressive and aggressive CGCL lesions of these receptors expression, these results strengthens CGCL treatment with glucocorticoids and calcitonin. Aggressive lesions have a higher number of MGC. The CGCL express glucocorticoid and calcitonin receptors and this finding give biological basis to the CGCL treatment with intralesional glucocorticoid and calcitonin either in non aggressive and aggressive cases. It was also identified osteocalcin positive cells, that may be related to bone repair, it is believed that these cells may also serve as a therapeutic target. / A Lesão Central de Células Gigantes (LCCG) é uma lesão intraóssea que pode ser classificada em não agressiva e agressiva. Devido aos defeitos estéticos e funcionais do tratamento cirúrgico da LCCG, terapias medicamentosas tem sido relatadas, como injeções de glicocorticoide e calcitonina. Há na literatura estudos que suportam o uso desses medicamentos através da investigação da presença de receptores de glicocorticoides (RGC) e de calcitonina (RCT) em LCCG. No entanto não existe consenso se todas as LCCG expressam esses receptores e se existe alguma diferença entre lesões agressivas e não agressivas. Além disso, não existem estudos sobre a avaliação do potencial de formação óssea através da Osteocalcina (OC) em lesões agressivas e não agressivas. O propósito deste estudo foi avaliar comparativamente, por meio de imunohistoquímica, a expressão de RGC e RCT e da OC em LCCG não agressivas (n= 20) e agressivas (n= 11) e a correlação entre a expressão da OC e desses receptores nos dois grupos de lesões estudados. O número de mitoses nas células mononucleares e o número de células gigantes multinucleadas também foram investigados, utilizando técnica histoquímica (hematoxilina e eosina). Nossos resultados mostram que todos os casos analisados expressam o RGC e RCT e que não existe diferença na expressão do RGC, RCT ou do número de mitoses entre lesões não agressivas e agressivas. A expressão de OC em células mononucleares foi rara e maior em lesões não agressivas, no entanto, sem diferenças estatisticamente significantes (p>0,05). Houve correlação entre a expressão do RCT em células mononucleares e células gigantes multinucleadas (r=0,45; p<0,01). Considerando as diferentes variantes foi verificada correlação do RCT entre o componente mononuclear e as células gigantes multinucleadas nas lesões não agressivas (r=0,66; p<0,01) e entre a expressão de OC e RCT em células gigantes multinucleadas (r= 0,718; p=0,01). Houve maior número de células gigantes em lesões agressivas (p= 0,01). Os resultados indicam que todos os casos expressam RGC e RCT e que não há diferenças entre lesões agressivas e não agressivas de LCCG quanto à expressão desses receptores, fortalecendo a recomendação o tratamento da LCCG com o uso de glicocorticoide e calcitonina. Lesões agressivas apresentam maior número de CGM. As células da LCCG expressam o RGC e RCT e esse achado pode fornecer bases biológicas para o tratamento com injeções intralesionais de glicocorticoides e o uso de calcitonina, seja em lesões não agressivas ou agressivas. Adicionalmente, foram identificadas células expressando OC, que podem estar relacionadas ao reparo ósseo, acredita-se que essa linhagem celular também pode se tornar um alvo terapêutico.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A LesÃo Central de CÃlulas Gigantes dos maxilares (LCCG) à intra-Ãssea, nÃo tem predileÃÃo por sexo, classifica-se em agressivas e nÃo-agressivas, histologicamente consistem tecido fi-broso e celularizado fusiforme associado a cÃlulas gigantes multinucleadas (CGM), focos de hemorragia e neovascularizaÃÃo, tendo na cirurgia seu habitual tratamento. Novas abordagens terapÃuticas foram propostas, sendo a principal delas o uso de corticÃides intralesionais. Este trabalho analisa retrospectivamente 21 pacientes portadores de LCCG que foram tratados por hexacetonido de triancinolona intralesional, atravÃs do seguinte protocolo: injeÃÃo de hexace-tonido de triancinolona 20mg/ml diluÃdo na soluÃÃo anestÃsica de lidocaÃna 2%/epinefrina 1:200.000 numa proporÃÃo de 1:1; infiltrando 1ml de soluÃÃo para cada 1cm3 de lesÃo, totali-zando 06 aplicaÃÃes em intervalos quinzenais. Estabeleceu-se 04 critÃrios clÃnicos para classi-ficar a resposta ao tratamento: 1- estabilizaÃÃo ou regressÃo clÃnica da lesÃo 2- ausÃncia de sintomas 3- aumento da densidade nos controles radiogrÃficos 4- aumento da resistÃncia a infiltraÃÃo intralesional da droga, bem como, fez-se uma anÃlise imunohistoquÃmica quanto à expressÃo dos Receptores de corticÃides (GCR) e Calcitonina (CTR), Cox-2, proteÃna p16 e amplificaÃÃo gÃnica da Ciclina D1 por CISH, comparando quanto a agressividade e a resposta terapÃutica a corticoterapia intralesional. Dos 21 pacientes incluÃdos neste estudo, 11 eram homens e 10 mulheres, 09 tinham lesÃo em maxila, 12 em mandÃbula. Dez eram lesÃes agres-sivas e 11 nÃo-agressivas, 15 (71,4%) apresentaram uma boa resposta ao tratamento, 04(19%) moderada e 02(9,1%) negativa. Das 11 nÃo agressivas, 10(90,9%) apresentaram boa resposta e 01 (9,1%) resposta moderada, das 10 agressivas 05(50%), 03(30%) e 02(20%) apresentaram boa, moderada e negativa resposta respectivamente, nenhuma apresentou recidiva apÃs o tra-tamento, com preservaÃÃo que variou entre 04 a 08 anos. Os achados histopatolÃgicos mos-traram uma reduÃÃo da densidade e do tamanho das CG, e um estroma fibro-colagenoso das lesÃes. Dentre os marcadores pesquisados, apenas GCR em CG antes do tratamento mostrou significÃncia estatÃstica (p<0,004) com relaÃÃo a uma boa resposta terapÃutica. O CTR ex-pressou-se em cÃlulas gigantes e mononucleares de forma variada. A p16 apresentou-se ex-pressa em 30% da amostra, COX2 nÃo apresentou expressÃo na lesÃo e 33% da amostra apre-sentou amplificaÃÃo gÃnica da ciclina D1. NÃo mostraram significÃncia estatÃstica nem quanto à agressividade, nem quanto resposta ao tratamento, nenhum dos marcadores, exceto o GCR. O estudo mostrou que a corticoterapia intralesional à efetiva e segura para o tratamento das LCCG, com tendÃncia a melhor resposta nas lesÃes nÃo-agressivas do que nas agressivas. Mostrou ainda que a marcaÃÃo para GCR em CG demonstrou ser um parÃmetro confiÃvel para prever a resposta à terapÃutica com a corticoterapia intralesional e que 33% das LCCG tÃm comportamento neoplÃsico pela amplificaÃÃo gÃnica da ciclina D1. / Central Giant Cells Lesion (CGCL) of the jaws is an intra-bone lesion with no predilection for sex and clinically divided into aggressive and non-aggressive subtypes. Histological, it shows as fibrous tissue with fusiform cells, as well as multinucleated giant cells (GC) clusters, he-morrhagic foci and neovascularization. Surgery is the regular treatment option. As new the-rapeutic approaches have been proposed, intralesional glucocorticoid injection is the main option. This paper assesses retrospectively 21 patients presenting CGCL, treated with intrale-sional triamcinolone hexacetonide by using the following protocol: intralesional injection of triamcinolone hexacetonide 20mg/mL, diluted in a solution of lidocain 2% plus epinephrine 1:200000, at a 1:1 proportion; 1mL of this final solution for each 1cm3 of lesion volume was the injected, with a total of 06 injections, one in every 15 days. Four clinical criteria were sta-bilished to evaluate treatment outcome: 1- Clinical regression or stabilization of the lesion; 2- Absence of symptoms; 3- Raising in density on radiographic controls; 4-Increased resistence when injecting the drug intralesionally. It was also performed immunohistochemical assess-ment for glucocorticoid receptor (GCR) expression, calcitonin receptor (CTR) expression, COX-2 expression, p16 expression and Ciclin D1 gene amplification by CISH, making com-parisons related to aggressivity and to therapeutic outcome. Eleven out of 21 patients of this study were women, and 10 were men. Nine of the patients had lesion located in the maxilla, 12 in the mandible. Ten patients showed aggressive lesions and 11 non-aggressive lesions. Fifteen patients showed good treatment outcome, four patients showed moderate outcome, and two patients showed negative answer to the treatment. Among the 11 patients with non-aggressive lesions, ten showed good outcome and the other, moderate outcome. Among the ten aggressive lesions, five patients showed good outcome, three patients showed moderate outcome and the remaining two patients showed negative answer to the treatment. None of them showed reicidive in a four to eight years follow-up period. Morphologic analysis found positive correlation between volume density of GC/mm2 and lesion aggressiveness, as well as significant reduction in number of GC/mm2 after treatment. Among the markers, only GCR in GC showed statistical relevance associated to the treatment. CTR was espresse in GC and in mononuclear cells in a varying way; p16 was expressed in 30% of the sample; COX-2 was not expressed at all in lesion samples and 33% of the sample showed gene amplification in Ciclin D1. None of the markers showed any statistical significant difference related to aggres-siveness nor to treatment outcome, except for GCR. The study showed the feasibility of the adopted treatment, with tendency to better outcomes in non-aggressive lesion, if compared to the aggressive ones. It also showed evidence pointing to GCR expression in GC as a reliable parameter to predict therapeutic responsiveness to glucocorticoids; and it showed that 33% of CGCL have neoplastic behaviour by Ciclin D1 gene amplification.

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